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D A L I A Č I Ž E I K I E N Ė
S U M M A R Y O F D O C T O R A L D I S S E R T A T I O N
K a u n a s2 0 1 5
B I O P R O D U C T S O F B A C T E R I O C I N S
P R O D U C I N G L A C T I C A C I D B A C T E R I A T H E I R
A N T I M I C R O B I A L A N D P H Y T A S E A C T I V I T I E S
A N D A P P L I C A T I O N S
T E C H N O L O G I C A L S C I E N C E S C H E M I C A L
E N G I N E E R I N G ( 0 5 T )
KAUNAS UNIVERSITY OF TECHNOLOGY
Dalia Čižeikienė
BIOPRODUCTS OF BACTERIOCINS PRODUCING LACTIC
ACID BACTERIA THEIR ANTIMICROBIAL AND PHYTASE
ACTIVITIES AND APPLICATIONS
Summary of Doctoral Dissertation
Technological Sciences Chemical Engineering (05T)
2015 Kaunas
The study was carried out at Kaunas University of Technology Faculty of
Chemical Technology Department of Food Science and Technology during the
years 2010-2014 The research part was performed at the University of
Copenhagen Faculty of Science Department of Food Science Food
Microbiology section and this part of work was partly supported by project
Promotion of Student Scientific Activities (VP1-31-ŠMM-01-V-02-003) from
the Research Council of Lithuania The part of research was performed at
Lithuanian Institute of Agriculture Department of plant pathology and
protection Kėdainiai distr and at Institute of Botany Laboratory of
Biodeterioration Research Vilnius The part of research was funded by a grant
(project 31V-46 BIOEKOTECH) from Lithuanian Agency for Science
Innovation and Technology and by a grant (project SVE-092011 BIOFITAS)
from the Research Council of Lithuania
Scientific Supervisor
Prof Dr Habil Gražina JUODEIKIENĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T)
Dissertation Defense Board of Chemical Engineering Science Field
Prof Dr Jolanta LIESIENĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T) ndash Chairperson
Dr Ina JASUTIENĖ (Kaunas University of Technology Technological
Sciences Chemical Engineering ndash 05T)
Assoc Prof Dr Rima KLIMAVIČIŪTĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T)
Prof Dr Mindaugas MALAKAUSKAS (Academy of Veterinary of
Lithuanian University of Health Sciences Agricultural Sciences Veterinary
Medicine ndash 02A)
Assoc Prof Dr Vytautas RUZGAS (Lithuanian Research Centre for
Agriculture and Forestry Institute of Agriculture Agricultural Sciences
Agronomy ndash 01A)
The official defense of the dissertation will be held at an open meeting of the
Board of Chemical Engineering science field at 1 pm on March 6 2015 in the
Dissertation Defense Hall at the Central Building of Kaunas University of
Technology
Address K Donelaičio St 73-403 LT-44029 Kaunas Lithuania Telephone
+370 37 300042 fax +370 37 324144 e-mail doktoranturaktult
The send-out date of the Summary of the Dissertation is February 6 2015 The
Dissertation is available on the internet httpktuedu and at the library of
Kaunas University of Technology (K Donelaičio St 20 Kaunas)
KAUNO TECHNOLOGIJOS UNIVERSITETAS
Dalia Čižeikienė
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES
BAKTERIJŲ BIOPRODUKTAI JŲ ANTIMIKROBINIS IR
FITAZINIS AKTYVUMAS BEI TAIKYMAS
Daktaro disertacijos santrauka
Technologijos mokslai chemijos inžinerija (05T)
2015 Kaunas
Disertacija rengta 2010ndash2014 metais Kauno technologijos universitete
Cheminės technologijos fakultete Maisto mokslo ir technologijos katedroje
Dalis tyrimų atlikta Kopenhagos universitete Gamtos mokslų fakultete Maisto
mokslo katedroje (University of Copenhagen Faculty of Science Department of
Food Science Food Microbiology section) Danija Botanikos institute
Biodestruktorių tyrimo laboratorijoje Vilnius ir LAMMC Žemdirbystės
institute Augalų patologijos ir apsaugos skyriuje Kėdainių r Mokslinius
tyrimus rėmė Lietuvos mokslo taryba (doktorantų stažuotės užsienio mokslo
centruose VP1-31-ŠMM-01-V-02-003 projektas SVE-092011 BIOFITAS) ir
Mokslo inovacijų ir technologijų agentūra (projekto Nr 31V-46 BIOEKOTECH)
Mokslinė vadovė
Prof habil dr Gražina JUODEIKIENĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Chemijos inžinerijos mokslo krypties disertacijos gynimo taryba
Prof dr Jolanta LIESIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T) ndash pirmininkė
Dr Ina JASUTIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T)
Doc dr Rima KLIMAVIČIŪTĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Prof dr Mindaugas MALAKAUSKAS (Lietuvos sveikatos mokslų
universiteto Veterinarijos akademija žemės ūkio mokslai veterinarija ndash
02A)
Doc dr Vytautas RUZGAS (Lietuvos agrarinių ir miškų mokslų centro
Žemdirbystės institutas žemės ūkio mokslai agronomija ndash 01A)
Disertacija bus ginama viešame chemijos inžinerijos mokslo krypties tarybos
posėdyje 2015 m kovo 6 d 13 val Kauno technologijos universiteto centrinių
rūmų Disertacijų gynimo salėje
Adresas K Donelaičio g 73-403 LT-44029 Kaunas Lietuva
Tel +370 37 300042 faksas +370 37 324144 el paštas doktoranturaktult
Daktaro disertacijos santrauka išsiųsta 2015 m vasario 6 d
Su disertacija galima susipažinti internete httpktuedu ir Kauno technologijos
universiteto bibliotekoje (K Donelaičio g 20 Kaunas)
5
ABBREVIATIONS
BLIS bacteriocin like inhibitory substances
CFU colony forming units
CDM chemically defined media
CDM+P chemically defined media containing inorganic phosphorus
CDM-P phosphate free chemically defined media
CDM+Phy chemically defined media supplemented with phytic acid
dipotassium salt as phosphate source
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
LAB lactic acid bacteria
LSD Fishers least significant difference
mMRS modified de Man Rogosa and Sharpe media
MRS de Man Rogosa and Sharpe media
PCR
PDA
polymerase chain reaction
potato dextrose agar
pfu plaque forming units
rep-PCR repetitive sequence based polymerase chain reaction
TTA total titratable acidity
U units
List of microorganisms genera
A ndash Aspergillus
Alt ndash Alternaria
Aure ndash Aureobasidium
B ndash Bacillus
C ndash Candida
Coch ndash Cochliobolus
D ndash Debaryomyces
E ndash Escherichia
Ent ndash Enterococcus
F ndash Fusarium
G ndash Geotrichum
Y ndash Yersinia
K ndash Kluyveromyces
L ndash Lactobacillus
Lact ndash Lactococcus
Leuc ndash Leuconostoc
List ndash Listeria
P ndash Pediococcus
Penic ndash Penicillium
Pi ndash Pichia
Ps ndash Pseudomonas
R ndash Rhodotorula
Rh ndash Rhizopus
S ndash Salmonella
Sc ndash Saccharomyces
Sach ndash Saccharomycopsis
St ndash Staphylococcus
Z ndash Zygosaccharomyces
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
KAUNAS UNIVERSITY OF TECHNOLOGY
Dalia Čižeikienė
BIOPRODUCTS OF BACTERIOCINS PRODUCING LACTIC
ACID BACTERIA THEIR ANTIMICROBIAL AND PHYTASE
ACTIVITIES AND APPLICATIONS
Summary of Doctoral Dissertation
Technological Sciences Chemical Engineering (05T)
2015 Kaunas
The study was carried out at Kaunas University of Technology Faculty of
Chemical Technology Department of Food Science and Technology during the
years 2010-2014 The research part was performed at the University of
Copenhagen Faculty of Science Department of Food Science Food
Microbiology section and this part of work was partly supported by project
Promotion of Student Scientific Activities (VP1-31-ŠMM-01-V-02-003) from
the Research Council of Lithuania The part of research was performed at
Lithuanian Institute of Agriculture Department of plant pathology and
protection Kėdainiai distr and at Institute of Botany Laboratory of
Biodeterioration Research Vilnius The part of research was funded by a grant
(project 31V-46 BIOEKOTECH) from Lithuanian Agency for Science
Innovation and Technology and by a grant (project SVE-092011 BIOFITAS)
from the Research Council of Lithuania
Scientific Supervisor
Prof Dr Habil Gražina JUODEIKIENĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T)
Dissertation Defense Board of Chemical Engineering Science Field
Prof Dr Jolanta LIESIENĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T) ndash Chairperson
Dr Ina JASUTIENĖ (Kaunas University of Technology Technological
Sciences Chemical Engineering ndash 05T)
Assoc Prof Dr Rima KLIMAVIČIŪTĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T)
Prof Dr Mindaugas MALAKAUSKAS (Academy of Veterinary of
Lithuanian University of Health Sciences Agricultural Sciences Veterinary
Medicine ndash 02A)
Assoc Prof Dr Vytautas RUZGAS (Lithuanian Research Centre for
Agriculture and Forestry Institute of Agriculture Agricultural Sciences
Agronomy ndash 01A)
The official defense of the dissertation will be held at an open meeting of the
Board of Chemical Engineering science field at 1 pm on March 6 2015 in the
Dissertation Defense Hall at the Central Building of Kaunas University of
Technology
Address K Donelaičio St 73-403 LT-44029 Kaunas Lithuania Telephone
+370 37 300042 fax +370 37 324144 e-mail doktoranturaktult
The send-out date of the Summary of the Dissertation is February 6 2015 The
Dissertation is available on the internet httpktuedu and at the library of
Kaunas University of Technology (K Donelaičio St 20 Kaunas)
KAUNO TECHNOLOGIJOS UNIVERSITETAS
Dalia Čižeikienė
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES
BAKTERIJŲ BIOPRODUKTAI JŲ ANTIMIKROBINIS IR
FITAZINIS AKTYVUMAS BEI TAIKYMAS
Daktaro disertacijos santrauka
Technologijos mokslai chemijos inžinerija (05T)
2015 Kaunas
Disertacija rengta 2010ndash2014 metais Kauno technologijos universitete
Cheminės technologijos fakultete Maisto mokslo ir technologijos katedroje
Dalis tyrimų atlikta Kopenhagos universitete Gamtos mokslų fakultete Maisto
mokslo katedroje (University of Copenhagen Faculty of Science Department of
Food Science Food Microbiology section) Danija Botanikos institute
Biodestruktorių tyrimo laboratorijoje Vilnius ir LAMMC Žemdirbystės
institute Augalų patologijos ir apsaugos skyriuje Kėdainių r Mokslinius
tyrimus rėmė Lietuvos mokslo taryba (doktorantų stažuotės užsienio mokslo
centruose VP1-31-ŠMM-01-V-02-003 projektas SVE-092011 BIOFITAS) ir
Mokslo inovacijų ir technologijų agentūra (projekto Nr 31V-46 BIOEKOTECH)
Mokslinė vadovė
Prof habil dr Gražina JUODEIKIENĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Chemijos inžinerijos mokslo krypties disertacijos gynimo taryba
Prof dr Jolanta LIESIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T) ndash pirmininkė
Dr Ina JASUTIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T)
Doc dr Rima KLIMAVIČIŪTĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Prof dr Mindaugas MALAKAUSKAS (Lietuvos sveikatos mokslų
universiteto Veterinarijos akademija žemės ūkio mokslai veterinarija ndash
02A)
Doc dr Vytautas RUZGAS (Lietuvos agrarinių ir miškų mokslų centro
Žemdirbystės institutas žemės ūkio mokslai agronomija ndash 01A)
Disertacija bus ginama viešame chemijos inžinerijos mokslo krypties tarybos
posėdyje 2015 m kovo 6 d 13 val Kauno technologijos universiteto centrinių
rūmų Disertacijų gynimo salėje
Adresas K Donelaičio g 73-403 LT-44029 Kaunas Lietuva
Tel +370 37 300042 faksas +370 37 324144 el paštas doktoranturaktult
Daktaro disertacijos santrauka išsiųsta 2015 m vasario 6 d
Su disertacija galima susipažinti internete httpktuedu ir Kauno technologijos
universiteto bibliotekoje (K Donelaičio g 20 Kaunas)
5
ABBREVIATIONS
BLIS bacteriocin like inhibitory substances
CFU colony forming units
CDM chemically defined media
CDM+P chemically defined media containing inorganic phosphorus
CDM-P phosphate free chemically defined media
CDM+Phy chemically defined media supplemented with phytic acid
dipotassium salt as phosphate source
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
LAB lactic acid bacteria
LSD Fishers least significant difference
mMRS modified de Man Rogosa and Sharpe media
MRS de Man Rogosa and Sharpe media
PCR
PDA
polymerase chain reaction
potato dextrose agar
pfu plaque forming units
rep-PCR repetitive sequence based polymerase chain reaction
TTA total titratable acidity
U units
List of microorganisms genera
A ndash Aspergillus
Alt ndash Alternaria
Aure ndash Aureobasidium
B ndash Bacillus
C ndash Candida
Coch ndash Cochliobolus
D ndash Debaryomyces
E ndash Escherichia
Ent ndash Enterococcus
F ndash Fusarium
G ndash Geotrichum
Y ndash Yersinia
K ndash Kluyveromyces
L ndash Lactobacillus
Lact ndash Lactococcus
Leuc ndash Leuconostoc
List ndash Listeria
P ndash Pediococcus
Penic ndash Penicillium
Pi ndash Pichia
Ps ndash Pseudomonas
R ndash Rhodotorula
Rh ndash Rhizopus
S ndash Salmonella
Sc ndash Saccharomyces
Sach ndash Saccharomycopsis
St ndash Staphylococcus
Z ndash Zygosaccharomyces
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
The study was carried out at Kaunas University of Technology Faculty of
Chemical Technology Department of Food Science and Technology during the
years 2010-2014 The research part was performed at the University of
Copenhagen Faculty of Science Department of Food Science Food
Microbiology section and this part of work was partly supported by project
Promotion of Student Scientific Activities (VP1-31-ŠMM-01-V-02-003) from
the Research Council of Lithuania The part of research was performed at
Lithuanian Institute of Agriculture Department of plant pathology and
protection Kėdainiai distr and at Institute of Botany Laboratory of
Biodeterioration Research Vilnius The part of research was funded by a grant
(project 31V-46 BIOEKOTECH) from Lithuanian Agency for Science
Innovation and Technology and by a grant (project SVE-092011 BIOFITAS)
from the Research Council of Lithuania
Scientific Supervisor
Prof Dr Habil Gražina JUODEIKIENĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T)
Dissertation Defense Board of Chemical Engineering Science Field
Prof Dr Jolanta LIESIENĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T) ndash Chairperson
Dr Ina JASUTIENĖ (Kaunas University of Technology Technological
Sciences Chemical Engineering ndash 05T)
Assoc Prof Dr Rima KLIMAVIČIŪTĖ (Kaunas University of Technology
Technological Sciences Chemical Engineering ndash 05T)
Prof Dr Mindaugas MALAKAUSKAS (Academy of Veterinary of
Lithuanian University of Health Sciences Agricultural Sciences Veterinary
Medicine ndash 02A)
Assoc Prof Dr Vytautas RUZGAS (Lithuanian Research Centre for
Agriculture and Forestry Institute of Agriculture Agricultural Sciences
Agronomy ndash 01A)
The official defense of the dissertation will be held at an open meeting of the
Board of Chemical Engineering science field at 1 pm on March 6 2015 in the
Dissertation Defense Hall at the Central Building of Kaunas University of
Technology
Address K Donelaičio St 73-403 LT-44029 Kaunas Lithuania Telephone
+370 37 300042 fax +370 37 324144 e-mail doktoranturaktult
The send-out date of the Summary of the Dissertation is February 6 2015 The
Dissertation is available on the internet httpktuedu and at the library of
Kaunas University of Technology (K Donelaičio St 20 Kaunas)
KAUNO TECHNOLOGIJOS UNIVERSITETAS
Dalia Čižeikienė
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES
BAKTERIJŲ BIOPRODUKTAI JŲ ANTIMIKROBINIS IR
FITAZINIS AKTYVUMAS BEI TAIKYMAS
Daktaro disertacijos santrauka
Technologijos mokslai chemijos inžinerija (05T)
2015 Kaunas
Disertacija rengta 2010ndash2014 metais Kauno technologijos universitete
Cheminės technologijos fakultete Maisto mokslo ir technologijos katedroje
Dalis tyrimų atlikta Kopenhagos universitete Gamtos mokslų fakultete Maisto
mokslo katedroje (University of Copenhagen Faculty of Science Department of
Food Science Food Microbiology section) Danija Botanikos institute
Biodestruktorių tyrimo laboratorijoje Vilnius ir LAMMC Žemdirbystės
institute Augalų patologijos ir apsaugos skyriuje Kėdainių r Mokslinius
tyrimus rėmė Lietuvos mokslo taryba (doktorantų stažuotės užsienio mokslo
centruose VP1-31-ŠMM-01-V-02-003 projektas SVE-092011 BIOFITAS) ir
Mokslo inovacijų ir technologijų agentūra (projekto Nr 31V-46 BIOEKOTECH)
Mokslinė vadovė
Prof habil dr Gražina JUODEIKIENĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Chemijos inžinerijos mokslo krypties disertacijos gynimo taryba
Prof dr Jolanta LIESIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T) ndash pirmininkė
Dr Ina JASUTIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T)
Doc dr Rima KLIMAVIČIŪTĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Prof dr Mindaugas MALAKAUSKAS (Lietuvos sveikatos mokslų
universiteto Veterinarijos akademija žemės ūkio mokslai veterinarija ndash
02A)
Doc dr Vytautas RUZGAS (Lietuvos agrarinių ir miškų mokslų centro
Žemdirbystės institutas žemės ūkio mokslai agronomija ndash 01A)
Disertacija bus ginama viešame chemijos inžinerijos mokslo krypties tarybos
posėdyje 2015 m kovo 6 d 13 val Kauno technologijos universiteto centrinių
rūmų Disertacijų gynimo salėje
Adresas K Donelaičio g 73-403 LT-44029 Kaunas Lietuva
Tel +370 37 300042 faksas +370 37 324144 el paštas doktoranturaktult
Daktaro disertacijos santrauka išsiųsta 2015 m vasario 6 d
Su disertacija galima susipažinti internete httpktuedu ir Kauno technologijos
universiteto bibliotekoje (K Donelaičio g 20 Kaunas)
5
ABBREVIATIONS
BLIS bacteriocin like inhibitory substances
CFU colony forming units
CDM chemically defined media
CDM+P chemically defined media containing inorganic phosphorus
CDM-P phosphate free chemically defined media
CDM+Phy chemically defined media supplemented with phytic acid
dipotassium salt as phosphate source
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
LAB lactic acid bacteria
LSD Fishers least significant difference
mMRS modified de Man Rogosa and Sharpe media
MRS de Man Rogosa and Sharpe media
PCR
PDA
polymerase chain reaction
potato dextrose agar
pfu plaque forming units
rep-PCR repetitive sequence based polymerase chain reaction
TTA total titratable acidity
U units
List of microorganisms genera
A ndash Aspergillus
Alt ndash Alternaria
Aure ndash Aureobasidium
B ndash Bacillus
C ndash Candida
Coch ndash Cochliobolus
D ndash Debaryomyces
E ndash Escherichia
Ent ndash Enterococcus
F ndash Fusarium
G ndash Geotrichum
Y ndash Yersinia
K ndash Kluyveromyces
L ndash Lactobacillus
Lact ndash Lactococcus
Leuc ndash Leuconostoc
List ndash Listeria
P ndash Pediococcus
Penic ndash Penicillium
Pi ndash Pichia
Ps ndash Pseudomonas
R ndash Rhodotorula
Rh ndash Rhizopus
S ndash Salmonella
Sc ndash Saccharomyces
Sach ndash Saccharomycopsis
St ndash Staphylococcus
Z ndash Zygosaccharomyces
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
KAUNO TECHNOLOGIJOS UNIVERSITETAS
Dalia Čižeikienė
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES
BAKTERIJŲ BIOPRODUKTAI JŲ ANTIMIKROBINIS IR
FITAZINIS AKTYVUMAS BEI TAIKYMAS
Daktaro disertacijos santrauka
Technologijos mokslai chemijos inžinerija (05T)
2015 Kaunas
Disertacija rengta 2010ndash2014 metais Kauno technologijos universitete
Cheminės technologijos fakultete Maisto mokslo ir technologijos katedroje
Dalis tyrimų atlikta Kopenhagos universitete Gamtos mokslų fakultete Maisto
mokslo katedroje (University of Copenhagen Faculty of Science Department of
Food Science Food Microbiology section) Danija Botanikos institute
Biodestruktorių tyrimo laboratorijoje Vilnius ir LAMMC Žemdirbystės
institute Augalų patologijos ir apsaugos skyriuje Kėdainių r Mokslinius
tyrimus rėmė Lietuvos mokslo taryba (doktorantų stažuotės užsienio mokslo
centruose VP1-31-ŠMM-01-V-02-003 projektas SVE-092011 BIOFITAS) ir
Mokslo inovacijų ir technologijų agentūra (projekto Nr 31V-46 BIOEKOTECH)
Mokslinė vadovė
Prof habil dr Gražina JUODEIKIENĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Chemijos inžinerijos mokslo krypties disertacijos gynimo taryba
Prof dr Jolanta LIESIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T) ndash pirmininkė
Dr Ina JASUTIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T)
Doc dr Rima KLIMAVIČIŪTĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Prof dr Mindaugas MALAKAUSKAS (Lietuvos sveikatos mokslų
universiteto Veterinarijos akademija žemės ūkio mokslai veterinarija ndash
02A)
Doc dr Vytautas RUZGAS (Lietuvos agrarinių ir miškų mokslų centro
Žemdirbystės institutas žemės ūkio mokslai agronomija ndash 01A)
Disertacija bus ginama viešame chemijos inžinerijos mokslo krypties tarybos
posėdyje 2015 m kovo 6 d 13 val Kauno technologijos universiteto centrinių
rūmų Disertacijų gynimo salėje
Adresas K Donelaičio g 73-403 LT-44029 Kaunas Lietuva
Tel +370 37 300042 faksas +370 37 324144 el paštas doktoranturaktult
Daktaro disertacijos santrauka išsiųsta 2015 m vasario 6 d
Su disertacija galima susipažinti internete httpktuedu ir Kauno technologijos
universiteto bibliotekoje (K Donelaičio g 20 Kaunas)
5
ABBREVIATIONS
BLIS bacteriocin like inhibitory substances
CFU colony forming units
CDM chemically defined media
CDM+P chemically defined media containing inorganic phosphorus
CDM-P phosphate free chemically defined media
CDM+Phy chemically defined media supplemented with phytic acid
dipotassium salt as phosphate source
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
LAB lactic acid bacteria
LSD Fishers least significant difference
mMRS modified de Man Rogosa and Sharpe media
MRS de Man Rogosa and Sharpe media
PCR
PDA
polymerase chain reaction
potato dextrose agar
pfu plaque forming units
rep-PCR repetitive sequence based polymerase chain reaction
TTA total titratable acidity
U units
List of microorganisms genera
A ndash Aspergillus
Alt ndash Alternaria
Aure ndash Aureobasidium
B ndash Bacillus
C ndash Candida
Coch ndash Cochliobolus
D ndash Debaryomyces
E ndash Escherichia
Ent ndash Enterococcus
F ndash Fusarium
G ndash Geotrichum
Y ndash Yersinia
K ndash Kluyveromyces
L ndash Lactobacillus
Lact ndash Lactococcus
Leuc ndash Leuconostoc
List ndash Listeria
P ndash Pediococcus
Penic ndash Penicillium
Pi ndash Pichia
Ps ndash Pseudomonas
R ndash Rhodotorula
Rh ndash Rhizopus
S ndash Salmonella
Sc ndash Saccharomyces
Sach ndash Saccharomycopsis
St ndash Staphylococcus
Z ndash Zygosaccharomyces
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
Disertacija rengta 2010ndash2014 metais Kauno technologijos universitete
Cheminės technologijos fakultete Maisto mokslo ir technologijos katedroje
Dalis tyrimų atlikta Kopenhagos universitete Gamtos mokslų fakultete Maisto
mokslo katedroje (University of Copenhagen Faculty of Science Department of
Food Science Food Microbiology section) Danija Botanikos institute
Biodestruktorių tyrimo laboratorijoje Vilnius ir LAMMC Žemdirbystės
institute Augalų patologijos ir apsaugos skyriuje Kėdainių r Mokslinius
tyrimus rėmė Lietuvos mokslo taryba (doktorantų stažuotės užsienio mokslo
centruose VP1-31-ŠMM-01-V-02-003 projektas SVE-092011 BIOFITAS) ir
Mokslo inovacijų ir technologijų agentūra (projekto Nr 31V-46 BIOEKOTECH)
Mokslinė vadovė
Prof habil dr Gražina JUODEIKIENĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Chemijos inžinerijos mokslo krypties disertacijos gynimo taryba
Prof dr Jolanta LIESIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T) ndash pirmininkė
Dr Ina JASUTIENĖ (Kauno technologijos universitetas technologijos
mokslai chemijos inžinerija ndash 05T)
Doc dr Rima KLIMAVIČIŪTĖ (Kauno technologijos universitetas
technologijos mokslai chemijos inžinerija ndash 05T)
Prof dr Mindaugas MALAKAUSKAS (Lietuvos sveikatos mokslų
universiteto Veterinarijos akademija žemės ūkio mokslai veterinarija ndash
02A)
Doc dr Vytautas RUZGAS (Lietuvos agrarinių ir miškų mokslų centro
Žemdirbystės institutas žemės ūkio mokslai agronomija ndash 01A)
Disertacija bus ginama viešame chemijos inžinerijos mokslo krypties tarybos
posėdyje 2015 m kovo 6 d 13 val Kauno technologijos universiteto centrinių
rūmų Disertacijų gynimo salėje
Adresas K Donelaičio g 73-403 LT-44029 Kaunas Lietuva
Tel +370 37 300042 faksas +370 37 324144 el paštas doktoranturaktult
Daktaro disertacijos santrauka išsiųsta 2015 m vasario 6 d
Su disertacija galima susipažinti internete httpktuedu ir Kauno technologijos
universiteto bibliotekoje (K Donelaičio g 20 Kaunas)
5
ABBREVIATIONS
BLIS bacteriocin like inhibitory substances
CFU colony forming units
CDM chemically defined media
CDM+P chemically defined media containing inorganic phosphorus
CDM-P phosphate free chemically defined media
CDM+Phy chemically defined media supplemented with phytic acid
dipotassium salt as phosphate source
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
LAB lactic acid bacteria
LSD Fishers least significant difference
mMRS modified de Man Rogosa and Sharpe media
MRS de Man Rogosa and Sharpe media
PCR
PDA
polymerase chain reaction
potato dextrose agar
pfu plaque forming units
rep-PCR repetitive sequence based polymerase chain reaction
TTA total titratable acidity
U units
List of microorganisms genera
A ndash Aspergillus
Alt ndash Alternaria
Aure ndash Aureobasidium
B ndash Bacillus
C ndash Candida
Coch ndash Cochliobolus
D ndash Debaryomyces
E ndash Escherichia
Ent ndash Enterococcus
F ndash Fusarium
G ndash Geotrichum
Y ndash Yersinia
K ndash Kluyveromyces
L ndash Lactobacillus
Lact ndash Lactococcus
Leuc ndash Leuconostoc
List ndash Listeria
P ndash Pediococcus
Penic ndash Penicillium
Pi ndash Pichia
Ps ndash Pseudomonas
R ndash Rhodotorula
Rh ndash Rhizopus
S ndash Salmonella
Sc ndash Saccharomyces
Sach ndash Saccharomycopsis
St ndash Staphylococcus
Z ndash Zygosaccharomyces
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
5
ABBREVIATIONS
BLIS bacteriocin like inhibitory substances
CFU colony forming units
CDM chemically defined media
CDM+P chemically defined media containing inorganic phosphorus
CDM-P phosphate free chemically defined media
CDM+Phy chemically defined media supplemented with phytic acid
dipotassium salt as phosphate source
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
LAB lactic acid bacteria
LSD Fishers least significant difference
mMRS modified de Man Rogosa and Sharpe media
MRS de Man Rogosa and Sharpe media
PCR
PDA
polymerase chain reaction
potato dextrose agar
pfu plaque forming units
rep-PCR repetitive sequence based polymerase chain reaction
TTA total titratable acidity
U units
List of microorganisms genera
A ndash Aspergillus
Alt ndash Alternaria
Aure ndash Aureobasidium
B ndash Bacillus
C ndash Candida
Coch ndash Cochliobolus
D ndash Debaryomyces
E ndash Escherichia
Ent ndash Enterococcus
F ndash Fusarium
G ndash Geotrichum
Y ndash Yersinia
K ndash Kluyveromyces
L ndash Lactobacillus
Lact ndash Lactococcus
Leuc ndash Leuconostoc
List ndash Listeria
P ndash Pediococcus
Penic ndash Penicillium
Pi ndash Pichia
Ps ndash Pseudomonas
R ndash Rhodotorula
Rh ndash Rhizopus
S ndash Salmonella
Sc ndash Saccharomyces
Sach ndash Saccharomycopsis
St ndash Staphylococcus
Z ndash Zygosaccharomyces
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
6
1 Introduction
Motivation for the research Grain products including daily bread products
are the basis of nutritional pyramid and the main source of fibres and minerals
such as iron calcium magnesium selenium zinc as well as folic acid iodine B
vitamins group On the other hand the intake of minerals from cereal productsrsquo
fibres is limited in the human and animal bodies The phytic acid in their
composition tends to form phytate complexes with minerals that are insoluble at
physiological pH and therefore minerals and phosphate are unavailable for
absorption in the human intestine This problem is especially relevant in the
production of wheat baked goods which use speeded up production processes
and during the fermentation minerals are not released from the phytates
complexes Due to the lack of minerals in the daily diet and insufficient intake
from grain products the human body experiences specific metabolic disorders
that cause various ailments and even diseases
When assessing the potential nutritional problems the search for new
biotechnological tools to improve the intake of minerals from grain products is
relevant One of the natural and most available tools to improve the quality of
wheat baked goods could be the microorganisms with phytase activity Recently
scientists have been interested in both yeast and lactic acid bacteria (LAB) with
phytase activity However there seems to be no high phytase-active LAB
available for the bread industry today Therefore the LAB with high phytase
activity extraction from fermented products and their further application for the
development of new bioproducts is relevant when solving the problem of mineral
intake More and more attention is brought to the possibilities to use LAB for
this purpose whose synthesized bacteriocins can ensure the stability of
fermented products suppress the growth of bread diseases causing
microorganisms such as Bacillus subtilis and fungi
Baked goods microbiological failure problem remained relevant in the bread
production technology especially in summer which is caused by Bacillus
subtilis and fungi which spread during the warm season Recently scientists
have been interested in LAB which produces substances similar to bacteriocins
whose antimicrobial effect occurs against various bacteria and even food
pathogens eg Listeria Salmonella Escherichia genera belonging bacteria
Natural preservative effect-having supplements ndash LAB fermented bioproducts
that have both antimicrobial and phytase activities could be one of the ways to
slow down the microbiological spoilage of wheat bread and at the same time
increase the nutritional value of wheat bakery products enriched with fibre
Kaunas University of Technology Food Science and Technology
Department previously for the first time assessed the microflora of industrial
Lithuanian sourdough and spontaneous rye sourdough and distinguished five
LAB that produce bacteriocins Lactobacillus sakei KTU05-6 Pediococcus
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
7
acidilactici KTU05-7 P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains Initial test results of the antimicrobial properties of microorganisms
showed the perspective of ongoing work and gave the basis for expanding the
scope for the use of antimicrobial preparations not only in the bread industry but
in other fields as well especially for reduction of biological pollution of the
grain seeds in organic farming The bioproducts for the processing of grain seeds
in organic farming should be not only effective but also cheap By setting such
goal the search of new media for the cultivation of LAB is relevant by paying
attention to by-products of food industry which could reduce the price of the
bioproduct production
When studying the use possibilities of LAB in the fermentation processes and
creation of new bioproducts it is necessary to pay attention to the inhibitors of
the fermentation ndash bacteriophages Bacteriophages attacking the LAB are main
factor responsible for fermentation failures thus creating significant economic
losses worldwide Phage infections can cause serious problems in fermentation
processes because the viral particles can reduce or even stop the activity of
fermentative bacteria Although bacteriophages are considered as a main factor
responsible for fermentation failures very few studies have dealt with lactic acid
bacteria isolated from sourdough Still is no data about phages influence on LAB
population in sourdough during continuously fermentation
The aim of the thesis To determinate the antimicrobial and phytase
activities of LAB isolated from sourdough and to assess the use opportunities of
bacteriocins producing LAB in order to reduce biological pollution of summer
wheat grain seeds in the organic farming and in order to increase the nutritional
value and safety of bakery products
The following tasks were set to achieve the aim of the thesis 1 To determinate antimicrobial activity of bacteriocins producing LAB and
their metabolites against the microorganism of grain seeds and causing
food spoilage
2 To evaluate the nutrients of media and fermentation conditions influence
on antimicrobial activity of bacteriocins like inhibitory substances
3 To isolate select and genetically identify LAB with phytase activity from
industrial sourdough and assess their phytase activity (intracellular and
extracellular) as well as evaluate phytase activity of LAB that produce
bacteriocins
4 To assess by imitating the environment pH of the digestive tract the
usability of selected LAB with phytase activity in order to increase the
biologically available minerals from whole-grain wheat bread
5 To isolate and characterise bacteriophages from bread sourdough and
evaluate the influence of them on LAB population during the sourdough
production
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
8
6 To evaluate the impact of temperature on populations of LAB species in
bread sourdough
7 To evaluate the impact of LAB bioproducts on the biological pollution
reduction of summer wheat grain seeds and their wellness increasing
Novelty of the study For the first time the antimicrobial activity of
bacteriocins producing LAB (L sakei KTU05-6 P acidilactici KTU05-7 P
pentosaceus KTU05-8 KTU05-9 and KTU05-10) isolated from Lithuanian
sourdough was evaluated against various bacteria belonging to Bacillus
Pseudomonas Escherichia Enterococcus Salmonella Staphylococcus
Micrococcus Listeria Yersinia genera and against fungi belonging to
Aspergillus Penicillium Alternaria Fusarium Candida Pichia Rhodotorula
and other genera and the features of the impact
LAB that produce bacteriocins and have high phytase activity were selected
for increasing the nutritional value of wheat bakery goods with higher fibre
content and under in vitro conditions their usability for increasing the intake of
minerals was proved
The usability of food processing wastes such as wheat bran molasses spent
brewers grains and spent distillers grains in the production of L-lactic acid was
assessed
For the first time the LAB bioproducts were tested for the summer wheat
grain seeds as biological fertilizers for Fusarium Alternaria genera belonging
fungi and Cochliobolus sativus fungi infection reduction and grains wellness
increasing and was set their positive impact
The practical value of the work Bioproducts of LAB for processing
organic summer wheat grain seeds was developed The new bioproducts allows
reducing on the grain surface Fusarium Alternaria Cochliobolus sativus
infection the diseases of root and the lesion of seeds by Fusarium genera
belonging fungi and Cochliobolus sativus fungi In addition it was found that
food processing waste (wheat bran molasses spent brewers grains and spent
distillers grains) might be besides potato juice a suitable media for the
multiplication of LAB and the formation of lactic acid (including L(+) isomer)
that has antimicrobial properties
In order to increase the nutritional value of whole grain wheat bread and slow
down the microbiological failure bioproducts with antimicrobial and phytase
activity were developed by using selected LAB with high phytase activity and
antimicrobial properties and for their reproduction ndash whole grain wheat flour
It was found that LAB population in bread sourdough during longer
fermentation was changed due to bacteriophage infection and temperature
changes in bakery therefore more appropriate often prepare new industrial
sourdough
Structure and content of the dissertation The thesis is written in
Lithuanian language It consists of the list of abbreviations introduction
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
9
literature review materials and methods results and discussion conclusions
references and list of publications relevant to the subject of the dissertation The
dissertation has 137 pages 24 tables and 20 figures The list of references
includes 300 bibliographic sources
Approval and publications of the results of the study The results of the
research have been published in 4 scientific articles in the journals indexed by
Thomson Reuters (WOS) and in 1 international conferences proceedings book
The results of the research have also been presented in the 16 international
scientific conferences and in 5 papers of Lithuania scientific conferences
Defended claims of the dissertation
The use of lactic acid bacteria with phytase and antimicrobial activities in
whole grain wheat baked goods increase the mineral bioavailability and
prevention of microbiological spoilage of bakery goods
The LAB bioproducts decrease fungi infection on summer wheat grain
seeds surface the diseases of root and the lesion of seeds by Fusarium
spp and Cochliobolus sativus fungi
2 RESEARCH OBJECTS AND METHODS
21 RESEARCH OBJECTS
Microorganisms L sakei KTU05-6 P acidilactici KTU05-7 and P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 strains previously isolated
from spontaneous Lithuanian rye sourdoughs and selected because of their
preliminary inhibiting properties were obtained from the culture collection of
Kaunas University of Technology Department of Food Science and Technology
L sakei was propagated at 30 oC P acidilactici and P pentosaceus KTU05-10
at 35 oC and P pentosaceus KTU05-8 and KTU05-9 strains at 25
oC in MRS
media for 18 h and used for further experiments
Indicator microorganisms used in this study for the evaluation of the
antimicrobial activity of the LAB were isolated from various vegetables meat
fats grains and water as well as outpatient pathological material and belong to
Bacillus Pseudomonas Listeria Escherichia Salmonella Staphylococcus
Enterococcus Micrococcus Yersinia Rhizopus Penicillium Aspergillus
Alternaria Fusarium Aureobasidium Candida Debaryomyces Geotrichum
Kluyveromyces Pichia Rhodotorula Saccharomycopsis Zygosaccharomyces
Saccharomyces and Yarrowia genera obtained from the Institute of Botany the
Food Institute and Vilnius University Hospital Santariskiu Klinikos
Sourdoughs Nine sourdoughs were obtained from three Lithuanian bakeries
for the screening and isolation of phytase positive LAB Two liquid sourdoughs
(wheat and rye) from a Danish bakery (propagated at approximately 18 oC ndash a
typical winter temperature at the bakery) were collected during the month (40
samples in total) for the screening of bacteriophages and evaluation of LAB
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
10
species population Moreover six weeks the same sourdoughs were propagated
according to the bakeries original recipes at 25 oC (this temperature is reached at
the bakeries in summer time) and collected (60 samples in total)
Fermented bioproducts with phytase positive LAB for bread making P
pentosaceus KTU05-8 KTU05-9 and KTU05-10 LAB strains were used for
bioproduct preparation for bread making The LAB cells used for bioproduct
preparation were grown in MRS media for 18 h The LAB cells washed three
times and re-suspended in distilled sterile water A four-stage fermentation
process was applied for the production of the fermented bioproduct (Table 21)
Table 21 Preparation of fermented bioproducts
Ingredients and technological parameters I stage II stage III stage IV stage
LAB cells re-suspended in the water mL 5 mdash mdash mdash
Fermented bioproduct g mdash 355 905 2005
Whole grain wheat flour g 10 20 35 70 Distilled water g 20 40 70 140
Fermentation time h 3-4 20 24 24
Contaminated seeds Seeds of spring wheat cultivars lsquoGranaryrsquo
(contaminated with Fusarium spp and the dominant species being F culmorum)
and lsquoTrisorsquo (naturally contaminated with Cochliobolus sativus Alternaria and
Fusarium spp) were obtained from an experiment field of the Institute of
Agriculture
Media for bioproduct preparation Food processing wastes namely wheat
bran spent distillers grains spent brewers grains and molasses were mixed with
water at the ratios of 13 14 13 and 110 respectively and autoclaved Potato
media was prepared from potato juice supplemented with 5 of extruded rice
02 gL of MgSO47H2O 01 gL of MnSO44H2O and 2 gL of cisteine-
HClH2O 2 of LAB inoculum was used for fermentation
Laboratory baking Bread samples containing 10 20 and 30 of the
fermented bioproduct (at ~65 moisture) were prepared using whole grain
wheat flour (Ustukiu Malunas Ltd Lithuania) The baking formulation for
whole grain bread was as follows (in weight bases) whole-grain wheat flour -
100 fermented bioproduct - 10 20 or 30 salt - 15 Bread prepared
without the LAB was analysed as a control sample Bread samples were baked in
an automatic AFKGERMATIC BM-2 oven (Germany) according to the
following program mixing I - 10 min at 18-22 oC proofing I - 25 min at 25
oC
mixing II - 15 min at 30 oC proofing II - 35 min at 32
oC proofing III - 70 min
at 38 oC baking - 55 min at 121
oC The bread samples were analysed after 24 h
of baking
22 METHODS
Determination of antimicrobial activity The antimicrobial activity of LAB
metabolites (supernatants) supernatants that was neutralized to pH 65 (for
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
11
bacteriocin like inhibitory substances analyse (BLIS)) and LAB cells was
evaluated using agar well diffusion assay method Proteinase K sensitivity test
was carried out to confirm that the antimicrobial compounds are BLIS
Isolation and enumeration of LAB The LAB were isolated from the
sourdough on modified MRS (mMRS) media under anaerobic conditions
Screening of phytase positive LAB and determination of phytase activity
The LAB strains isolated from the sourdough were screened for phytase activity
using three different Chemically Defined Media (CDM) supplemented with 074
gL phytic acid dipotassium salt (CDM+Phy) as the only phosphorus source
phosphate containing media (CDM+P) and phosphate free media (CDM-P)
Volumetric and specific activities of intra and extracellular phytase were
determined One unit of phytase activity was defined as the amount of phytase
that liberates 1 nmolmL of inorganic phosphate per minute from a 3 mM
potassium phytate solution at pH 55 and 30 degC
DNA extraction molecular typing and identification DNA was extracted
by using GenEluteTM
Bacterial Genomic kit (Sigma-Aldrich Broendby
Denmark) and following the manufacturerrsquos instructions for gram positive
bacteria The isolated LAB were grouped according to their repetitive DNA
sequence-based polymerase chain reaction (rep-PCR) fingerprint patterns
Initially the rep-PCR reactions were carried out with repetitive primer pair Rep-
1R-Dt (5rsquo-III NCGNCGNCATCNGGC-3rsquo)Rep2-Dt (5rsquo-NCGNCTTATCNGGC
CTAC-3rsquo) in a thermal cycler under standard procedures Electrophoretic
patterns were compared using BioNumerics version 45 (Applied Maths NV
Sint-Martens-Latem Belgium) pattern analysis software package For cluster
analysis the similarity among digitized profiles was calculated on the basis of
Dicersquos coefficient
The extracted DNA was used for the amplification of 16S rDNA gene using
primers 16S-27F (5rsquo-AGAGTTTGATCMTGGCTCAG-3rsquo) and 16S-1492R (5rsquo-
GGTTACCTTGTTACGACTT-3rsquo) by Macrogen Inc Wageningen The
sequences were assembled by using CLC Main Workbench 571 (CLC bio AS
Aarhus) and compared to the sequences reported in GenBank by using BLASTn
(Basic Local Alignment Search Toolnucleotide) algorithm
The analysis of mineral elements The amounts of calcium zinc ferrum
and manganese were determined using Atomic Absorption Spectrophotometer
(Spectr AA-Plus) according to AACC method 40-70 whereas phosphorus was
determined spectrometrically using the molybdate method The fractionation of
soluble compounds in bread was examined in accordance with the following
procedure 10 g of bread samples was mixed with 50 mL of deionized water and extracted for 30 min then centrifuged (3000 g 5 min) The pellets were mixed
with 50 mL 09 NaCl (pH 20) extracted for 30 min then centrifuged The
pellets were then washed with 50 mL 09 NaCl (pH 70) for 30 min and
centrifuged The pellets washed again with 30 mL of water then centrifuged All
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
12
supernatant fractions were merged and dried at 105 oC and washed in a muffle
oven at 500plusmn25 oC All treatments of the samples were made in triplicate
The effect of inoculum level temperature and media nutrients on BLIS
activity LAB growths in MRS and mMRS media were monitored as turbidity at
600 nm using a spectrophotometer during 48 h moreover BLIS antimicrobial
activities against B subtilis during 48 h were evaluated using well diffusion agar
method The effect of inoculums at levels 001 and 2 temperature at 20 25
30 and 35 oC and pH range at 45 55 and 65 on BLIS production was
investigated The influence of nutrients on BLIS activity was examined when
sole mMRS media component was changed One unit was defined as the
reciprocal of the highest dilution showing the inhibition of the indicator lawn and
was expressed in activity units per millilitre (UmL)
Lactic acid determination Lactic acid and lactic acid DL isomers were
determined by using enzymatic test K-DLATE 0811 (Megazyme International
Ireland Limited) and following the manufacturerrsquos instructions
Tests on the impact of the LAB bioproduct on grain wellness 10 20 and
30 mL of the LAB bioproduct (LAB propagated in MRS media for 24 h) per 100
g were applied on spring wheat seeds lsquoGranaryrsquo in the 2011 years experiment
LAB bioproducts prepared using MRS and potato juice media with additives
were used at a dose of 20 mL per 100 g lsquoTrisorsquo seeds in the 2012 years
experiment LAB efficacy against pathogenic fungi in controlled conditions was
tested by using the agar plate method Seeds treated with the bioproduct were
placed onto Petri dishes (10 seeds per plate) with potato dextrose agar (PDA) and
incubated in the dark for 7 days at 15 20 25 oC and for 20 days at 10 and 15
oC
(120 seeds per treatment - 12 plates were tested) The fungi were examined with
a Nikon Eclipse microscope (NIKON Instruments Inc USA) at magnifications
of 100 200 and 400 The significance of the differences between the treatment
means was determined according to the least significant difference (Fisherrsquos
LSD) at 005 and 001 probability level The data were processed using ANOVA
software from SELEKCIJA package
The evaluation of fermented bioproduct and bread quality The total
titratable acidity (TTA) value was expressed in mL of 01 M NaOH solution per
10 g of sample to obtain pH=85 The LAB count was evaluated according to
standard procedures After baking the bread samples (artificially contaminated
by adding 5104 spores of B subtilis per gram of dough into the mixing bowl)
were stored at 23 and 30 oC in polythene bags For 6 days the samples were
being evaluated by sensory examination for the presence of a typical unpleasant
sweet fruity odour which characterizes the first stage of ropiness
Isolation and purification of bacteriophages Bacteriophages were isolated
from the sourdough by using double agar overlay plaque assay method The
isolated phages were propagated in small (10 mL) and large (500 mL) scales
concentrated using 1 M NaCl and 10 of Polyethylene glycol 6000 and
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
13
purified using two-step CsCl gradient Electron microscopy was performed on
the purified phages to investigate phagesrsquo morphology (FEI Eindhoven
Netherlands) Phage DNA was isolated using standard procedures and sequenced
by Illumina MiSeq 2x250 PE
Denaturing gradient gel electrophoresis (DGGE) The LAB DNA was
extracted from the sourdough by using Sigma GenElutetrade Bacterial Genomic Kit
and according to the manufacturerrsquos recommendations The PCR reactions were
carried out with primer pair PRBA338fGC PRUN518r
3 RESULTS AND DISCUSSION
31 LAB antimicrobial activities
LAB antimicrobial activities against bacteria LAB during fermentation
produce antimicrobial compounds active against varies pathogens For
expanding the possibilities of the application of LAB that produce bacteriocins in
the other food production is important to evaluate the antimicrobial activity
spectrum of the LAB
This study evaluated the antimicrobial activities of LAB metabolites
(supernatants) LAB produced BLIS and LAB cells The metabolites produced
by the LAB strains showed different antimicrobial activities against the indicator
microorganisms (Table 31) Supernatants of all five LAB strains effectively
inhibited the growth of gram positive B subtilis strains B thuringiensis and
gram negative S typhimurium Ps gladioli pv aliicola 31 Ps cepacia 11 Ps
fluorescens biovar V 33 Ps marginalis 35 Ps fluorescens biovar V 12 Ps
facilis 41 Ps aureofaciens biovar III 241 Ps marginalis 42 Ps cichorii
731 and Ps fluorescens biovar III 21111 strains in varying degrees (the
diameters of the inhibition zones varied from 65plusmn06 to 225plusmn06 mm) The
highest antimicrobial activity was shown by P pentosaceus KTU05-10 strain
against Ps pseudoalcaligenes 11 and Ps cepacia 72 P acidilactici inhibited
the growth of List monocytogenes 11 and B cereus P acidilactici and L sakei
showed antimicrobial activity against E coli 110 whereas E coli ATCC25922
was inhibited by P acidilactici L sakei and P pentosaceus KTU05-10
BLIS produced by L sakei showed wide ranging antimicrobial activities
against gram positive and gram negative strains BLIS produced by L sakei
inhibited both B subtilis substrains (the diameters of the inhibition zones were
158plusmn04 and 135plusmn05 mm respectively) Among gram negative bacteria 7 out
of 13 Pseudomonas spp strains were inhibited by BLIS produced by L sakei
(the diameters of the inhibition zones varied from 73plusmn04 to 135plusmn05 mm
respectively) and E coli (the diameter of the inhibition zone was 158plusmn04 mm)
BLIS produced by P acidilactici and P pentosaceus KTU05-8 and KTU05-9
strains were only active against gram positive B subtilis whereas BLIS
produced by P pentosaceus KTU05-10 additionally inhibited Ps fluorescens
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
14
biovar V33 and showed bacteriostatic activity against S enteritidis and E coli
BLIS produced by P pentosaceus KTU05-8 and L sakei were found to have
poor bacteriostatic activity against the growth of Listeria spp and Ps aeroginosa
V 33 Moreover LAB produced BLIS show antimicrobial activity against B
subtilis spores (the diameters of the inhibition zones varied from 128plusmn04 to
195plusmn05 mm and depended on LAB strain)
In summary the results can be proposed that antimicrobial activity of LAB
and their produced BLIS depended on LAB strain L sakei and P pentosaceus
KTU05-10 produce BLIS that have broad antimicrobial activity spectrum and
inhibit gram positive and gram negative bacteria meanwhile P acidilactici P
pentosaceus KTU05-8 and KTU05-9 strains produce BLIS with narrow
spectrum of antimicrobial activity only against gram negative bacteria
Antimicrobial activities of the LAB supernatants against fungi and yeast
The LAB showed fungistatic activities against Penic expansum A versicolor A
fumigatus Penic chrysogenum F avenaceum A versicolor F solani and A
niger All tested LAB supernatants and their neutralized supernatants showed
very good fungicide inhibition of mycelial growth and sporulation with large
clear zones around the punched well against F culmorum and F poae The
growth of Penic verrucosum Alt alternata Penic cyclopium A tereus and Rh
stolonifer was not suppressed by the LAB supernatants
Supernatants of L sakei P pentosaceus KTU05-8 and KTU05-9 strains as
well as neutralized supernatants were active against C parapsilosis (the
diameters of the fungistatic inhibition zones varied from 100plusmn0 to 120plusmn08
mm) Besides L sakei and P pentosaceus KTU05-10 supernatants showed
fungistatic activities against D hansenii (the diameters of the inhibition zones
ranged from 80plusmn0 to 85plusmn0 mm respectively) L sakei P pentosaceus KTU05-8
and KTU05-10 supernatants and neutralized supernatants were active against Pi
farinosa (the diameters of the fungistatic inhibition zones varied from 98plusmn05 to
111plusmn10 mm) P pentosaceus KTU05-9 supernatants and neutralized
supernatants were active against C pelliculosa (the diameters of the fungistatic
inhibition zones were 115plusmn13 and 113plusmn10 mm) The growth of Pi
membranifaciens was suppressed by L sakei supernatants (the diameter of the
fungistatic inhibition zone was 100plusmn0 mm)
Meanwhile the LAB cells did not show antimicrobial activity against the
tested microorganisms The LAB did not suppress the growth of Aure pullalans
C kruisii D vanrijiae G fermentants K lodderae Pi fermentans R rubra
Sc cerevisiae K marxianus var lactis LAB cultivated together did not increase
antimicrobial activity with the exception of P pentosaceus KTU05-8 KTU05-9
and KTU05-10 strains mix which showed fungistatic activity against Alt
alternata (the diameter of the inhibition zone was 100plusmn14 mm)
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
Table 31 Inhibition of the growth of several bacteria by the LAB supernatants (100 microl)
Indicator microorganism
The diameters of the inhibition zones on agar plate mm
L sakei P acidilactici P pentosaceus
KTU05-8
P pentosaceus
KTU05-9
P pentosaceus
KTU05-10
Gram
positive
B thuringiensis 11 142plusmn04 173plusmn05 147plusmn05 172plusmn04 157plusmn05
B subtilis subsp subtilis 175plusmn06 188plusmn05 215plusmn06 215plusmn06 225plusmn06
B subtilis subsp spizizenii 158plusmn04 165plusmn06 185plusmn06 185plusmn06 185plusmn06
List monocytogenes 11 ndash 17plusmn0 ndash ndash ndash
B cereus ATCC 10876 ndash 95plusmn06 ndash ndash ndash
St aureus ATCC 25923 ndash ndash ndash ndash ndash Micrococcus spp 19plusmn14 245plusmn07 235plusmn35 22plusmn57 25plusmn0
Ent faecalis ndash 13plusmn14 ndash ndash 11plusmn14
St aureus 14plusmn0 12plusmn0 145plusmn21 13plusmn07 145plusmn07
B macerans 145plusmn07 13plusmn0 145plusmn07 135plusmn07 12plusmn0
Listeria spp 10plusmn0 13plusmn14 125plusmn07 15plusmn14 10plusmn14
Gram
negative
Ps gladioli pv aliicola 31 16plusmn0 153plusmn05 143plusmn05 145plusmn05 165plusmn05
Ps cepacia 11 17plusmn0 142plusmn04 147plusmn08 155plusmn05 150plusmn06
Ps fluorescens biovar V33 145plusmn05 148plusmn10 152plusmn04 147plusmn05 140plusmn0
Ps marginalis 35 175plusmn05 128plusmn04 158plusmn08 130plusmn09 122plusmn04 Ps fluorescens biovar V12 102plusmn04 122plusmn04 137plusmn05 98plusmn04 103plusmn05
Ps facilis 41 13plusmn0 132plusmn04 125plusmn05 117plusmn05 133plusmn05
Ps aureofaciens biovar III241 142plusmn10 148plusmn08 138plusmn04 160plusmn06 170plusmn06
Ps marginalis 42 87plusmn05 115plusmn05 102plusmn04 8plusmn0 105plusmn05
Ps cichorii 731 122plusmn04 115plusmn05 92plusmn04 123plusmn05 122plusmn08
Ps fluorescens biovar III21111 123plusmn05 145plusmn05 127plusmn05 167plusmn05 130plusmn06
Ps pseudoalcaligenes 11 ndash ndash ndash 11plusmn0 240plusmn07
Ps cepacia 72 153plusmn05 15plusmn0 ndash 27plusmn0 275plusmn07
Ps fluorescens biovar I ndash ndash ndash ndash 12plusmn0 E coli 110 13plusmn0 143plusmn06 ndash ndash ndash
E coli ATCC25922 7plusmn0 83plusmn05 ndash ndash 88plusmn05
S typhimurium ATCC 14028 85plusmn06 78plusmn05 95plusmn06 8plusmn08 65plusmn06
Ps aeroginosa 145plusmn07 15plusmn0 155plusmn07 15plusmn28 15plusmn28
Y enterocolitica 145plusmn07 135plusmn07 135plusmn07 15plusmn14 14plusmn0
S enteritidis 14plusmn0 10plusmn0 ndash 10plusmn0 11plusmn14
E coli 105plusmn07 145plusmn07 11plusmn0 105plusmn07 145plusmn07
bacteriostatic activity (bacteria growth was partly suppressed and delayed) daggerantimicrobial activity including wall diameter (6 mm) (minus) no inhibition
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
16
The results confirm that the LAB metabolites show broad range spectrum of
antimicrobial activity against bacteria and fungi especially F culmorum and F
poae and therefore could be used in varies food production to prevent the food
spoilage and increase safety Was proved the appropriateness to use this LAB
not only in bread production but also for other foods especially in organic
farming for grain seeds biological pollution reduction where major problems are
caused by Fusarium spp fungi
32 Phytase activity of the LAB
Whole-grain foods play an important role in human diet as they are relatively
rich in minerals however the absorption of those minerals in human gut can be
very low due to high content of the mineral binding phytate No LAB with
identified high phytase activity is available for the bread industry today
Therefore the objective of this study was to isolate and identify phytase-active
LAB which could be used as starter to increase the mineral bioavailability in
whole-meal bread fermented in short time
The LAB were isolated from Lithuanian sourdough for phytase-active LAB
screening Additionally five LAB strains with antimicrobial properties (L sakei
P acidilactici and P pentosaceus KTU05-10 KTU05-8 KTU05-9) previously
isolated from Lithuanian sourdoughs were screened for phytase activity on
CDM+P CDM+Phy and CDM-P agar plates (Fig 31) According to the ability
of LAB to degrade phytate and use the phosphorus for growth was selected 16
LAB strains for identification and phytase activity determination The phytase
activities were determined in the LAB that produce BLIS as well
Fig 31 Growth of the LAB a ndash P acidilactici b ndash P pentosaceus KTU05-8 c ndash P
pentosaceus KTU05-9 d ndash P pentosaceus KTU05-10 on agar media CDM+P (top left)
CDM+FIT (top right) and CDM-P (bottom) plates after 4 days
Volumetric and specific activities were measured for both extra and
intracellular phytase for the 21 strains (Table 32) The extracellular volumetric
and specific phytase activities differed both among the species and among the
strains within the same species The highest extracellular volumetric activity was
observed in two strains of L frumenti (21 and 25) and one strain of P
pentosaceus (KTU05-9) with a value of 54-60 UmL The highest extracellular
specific activity was observed in four strains of P pentosaceus (23-30
Umiddot10310
10 CFU) Extracellular volumetric and specific activities differed among
a d c b
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
17
the four L fermentum strains from 3 to 46 UmL and from 1 to 10 Umiddot10310
10
CFU respectively Both (volumetric and specific) extracellular activities were
highest in L fermentum 51 in comparison to other L fermentum strains
Intracellular volumetric and specific activities also differed among the species
The highest intracellular activities of all strains were observed in L fermentum
72 isolated from rye sourdough with volumetric activity of 61 UmL and
specific activity of 117 U10310
10 CFU followed by L reuteri 81 two other
strains of L fermentum (61 and 52) two L frumenti strains (21 and 25) and
P pentosaceus KTU05-9
Table 32 Volumetric (UmL) and specific (Umiddot1031010 CFU) phytase activities of the
LAB
Species Strains
Phytase activitiesa
Extracellular Intracellular
UmL Umiddot1031010 CFU UmL Umiddot1031010 CFU
L sakei KTU05-6 25plusmn0 24plusmn3 14plusmn1 072plusmn007
P acidilactici KTU05-7 11plusmn1 13plusmn3 17plusmn0 105plusmn011
P pentosaceus KTU05-10 21plusmn4 23plusmn4 12plusmn2 070plusmn004
KTU05-8 32plusmn1 30plusmn3 8plusmn3 034plusmn010
KTU05-9 54plusmn6 25plusmn4 28plusmn3 068plusmn005
12 43plusmn2 28plusmn3 7plusmn3 019plusmn007
L frumenti 21 56plusmn8 13plusmn2 13plusmn2 029plusmn005
25 60plusmn3 16plusmn1 16plusmn1 032plusmn004
L reuteri 23 6plusmn1 12plusmn4 13plusmn2 154plusmn036
81 19plusmn2 7plusmn1 61plusmn4 117plusmn012
L fermentum 51 46plusmn7 10plusmn3 10plusmn3 018plusmn004
52 3plusmn0 1plusmn0 35plusmn10 071plusmn026
61 11plusmn3 3plusmn1 52plusmn6 083plusmn002
72 16plusmn9 6plusmn3 61plusmn9 124plusmn025 a Results are expressed as the mean of three replicated measurements and standard error of the mean (plusmnSEM)
Phytase activity (U) is the amount of enzyme required to liberate 1 nmol of phosphate per min under the assay
conditions
LAB show 33 times higher extracellular phytase activity in comparison with
intracellular phytase activity The extracellular phytase activity depended on
LAB strain and was observed 23-30 U10310
10 CFU (for P pentosaceus strains)
and 13 U10310
10 CFU (for P acidilactici KTU05-6) The similar phytase
activity 28 U10310
10 CFU show P pentosaceus 12 strain isolated from
industrial sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU) The most
prominent strain for extracellular phytase production would be bacteriocins
producing P pentosaceus KTU05-8 with a specific phytase activity of 30
U10310
10 CFU
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
18
33 Influence of the growth factors on BLIS activity against B subtilis
The analysis shows that LAB cells inocula have a significant effect on BLIS
production An increase of LAB inocula from 001 to 2 increased BLIS
produced inhibition zones against B subtilis (Fig 32) In our case detectable
levels of BLIS were recorded after 6 h of growth in MRS and mMRS media
indicating that the peptides are primary metabolites The highest activities of
BLIS against B subtilis were observed during 15-30 h of cultivation The
analysis of the effect of temperature showed that the highest activities of BLIS
against B subtilis were measured when cultivating P pentosaceus KTU05-10 P
acidilactici and L sakei at 30 oC (320 220 and 310 respectively) and P
pentosaceus KTU05-9 and P pentosaceus KTU05-8 at 25 oC (270 and 250
AUmL respectively)
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhi
biti
on z
one
mm
Time ha
0
2
4
6
8
10
12
14
0 3 6 9 12 15 18 21 24 27 30 33 36 42 48
Inhib
itio
n z
one
mm
Time hb
Fig 32 Antimicrobial activities of BLIS against B subtilis expressed in mm (without well
diameter) when LAB inoculum was 001 (a) and 2 (b) ndash L sakei diams ndash P
acidilactici times ndash P pentosaceus KTU05-8 + ndash P pentosaceus KTU05-9 ndash ndash P
pentosaceus KTU05-10
Antimicrobial activities of BLIS against B subtilis were at 310 270 250 and
200 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively in mMRS media whereas in MRS media the
activities were significantly lower (260 220 200 and 160 UmL respectively)
The results show that the composition of the fermentation media has a significant
impact on the activities of BLIS of the tested LAB When using tryptone (20
gL) as a sole nitrogen source BLIS activities were found to be higher (310 280
260 and 220 UmL for P pentosaceus KTU05-10 KTU05-9 KTU05-8 and P
acidilactici KTU05-7 respectively) as compared with the same amount of
peptone yeast or meat extracts Moreover significantly lower activities were
observed when using 15 gL and lower amounts of tryptone as a sole nitrogen
source Higher activities of the BLIS were noticed when using glucose (20 gL)
as a sole carbon source as compared with maltose or fructose A 2 gL cysteine
hydrochloride supplement significantly increased the antimicrobial activity of all
tested BLIS produced by the LAB MnSO44H2O concentration increased to 02
gL significantly enhanced the activities of BLIS produced by P pentosaceus
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
19
KTU05-8 KTU05-9 and KTU05-10 strains (respectively 360 320 and 300
UmL) MgSO47H2O concentration increased to 05 gL enhanced the
concentration of BLIS produced by P pentosaceus tested strains When
increasing the amounts of some K and Na salts in the media by 4 times and
more BLIS activities decreased by 13 Folic acid thiamine hydrochloride
cyanocobalamin and DL-68-thioctic acid had no significant effect on BLIS
activities
34 Food processing wastes for LAB cultivation and biotechnological
production of lactic acid
The bioproducts for the processing of grain seeds in organic farming should
be not only effective but also cheap By setting such goal the search of new
media for the cultivation of LAB is relevant by paying attention to by-products
of food industry which could reduce the price of the bioproduct production The
aim of this part of the study was to evaluate the LAB propagation possibilities in
by-products media and lactic acid production (including L(+) isomer) Wastes
from the food production industry namely wheat bran spent distillers grains
spent brewers grains and molasses as well as potato juice media fermented with
bacteriocin producing LAB showed good suitability for LAB propagation and
lactic acid biotechnological production A bran substrate system proved to be
stable and very successful in ensuring LAB viability (Fig 33) The amount of
LAB in the wheat bran bioproduct ranged from 102 log10 to 108 log10 CFUg
The propagation of the LAB in other waste media (spent distillers grains and
molasses) was lower Lactic acid production depends on the LAB strain and the
fermentation media (Fig 34)
5
6
7
8
9
10
11
P pentosaceus
KTU05-10
P pentosaceus
KTU05-9
P pentosaceus
KTU05-8
P acidilactici L sakei
Via
bil
ity o
f L
AB
lo
g1
0C
FU
g
wheat bran molasses spent brewers grains spent distillers grains
Fig 33 LAB viability in food processing wastes medium after 48 h of fermentation
P pentosaceus KTU05-9 strain produced the highest amount of lactic acid
in the wastes from the food production industry such as wheat bran spent
brewers grains and molasses (781 629 and 656 gkg respectively) In spent
brewerrsquos grains media the highest amount of lactic acid was produced by P
acidilactici P pentosaceus KTU05-8 and KTU05-9 strains (665 744 and
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
20
629 gkg respectively) Lactic acid production in potato juice media and potato
juice media supplemented with extruded rice was lower and varied from 31 to
243 gkg L(+) lactic acid production mainly depended on the fermentation
media in wheat bran spent distillers grains spent brewers grains and molasses
L(+) lactic acid varied from 9968 to 9997 whereas in potato media D(-)
isomer varied from 43 to 522 of the total content of lactic acid Meanwhile in
potato juice media with additives the content of D(-) isomer decreased and was
observed from 4 to 23 and depended on LAB strain therefore could be used
for grain seeds processing in organic farming
0
10
20
30
40
50
60
70
80
90
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pp
8
Pp
9
Pp
10
Pa
Ls
Pa
Pp
10
Ls
Pa
Pp
10
Wheat bran Molasses Spent brewers
grains
Spent distillers
grains
Potato
juice
Potato
juice
Lacti
c a
cid
gk
g D (-)
L (+)
Fig 34 Biotechnological production of lactic acid using different LAB Ls ndash L sakei Pa
ndash P acidilactici Pp8 ndash P pentosaceus KTU05-8 Pp9 ndash P pentosaceus KTU05-9 Pp10 ndash
P pentosaceus KTU05-10 strains in fermentation of food processing waste and potato
juice ( with additives of MgSO47H2O MnSO44H2O and cysteine-HClH2O)
35 Potato juice media for bioproduct preparation
The aim of this part of the research was to evaluate the possibility of using
potato juice media with additives for LAB bioproduct production for summer
wheat grain seeds wellness Extruded rice MgSO47H2O and MnSO44H2O salts
and cysteine additives were used to improve LAB propagation in the media The
changes in bioproductrsquos TTA during 96 h of fermentation are shown in Fig 35a
In all cases the TTA of the bioproduct increased during the fermentation
Extruded rice Mg2+
Mn2+
and cysteine additives had a positive influence on the
increased TTA of the bioproduct The highest TTA was observed when 20 and
25 of extruded rice was used (278 and 260 respectively) after 72 h of
fermentation LAB growth kinetics during 90 h of fermentation while using 1 3
5 and 10 of extruded rice Mg2+
Mn2+
and cysteine additives and cultivating
at 35 oC is shown in Fig 35b The highest LAB amount was found after 48 h of
fermentation when 5 and 10 of extruded rice Mg2+
Mn2+
and cysteine
additives were used
This study confirms that potato juice and extruded rice media are suitable for
LAB propagation and bioproduct production
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
21
0
5
10
15
20
25
30
35
0 1 3 5 5 10 10 15 20 25
TT
A
Extruded rice additives a
24 h 48 h 72 h 96 h
0
10
20
30
40
50
60
70
0 24 48 72 96
CF
Ux 1
08g
Fermentation time hb
1
3
5
5
10
10
Fig 35 The influence of extruded rice (1 3 5 10 15 20 and 25 ) and other additives
(MgSO47H2O MnSO44H2O and cysteine-HClH2O) in potato juice media on
bioproductrsquos TTA (a) and LAB viability (b) during fermentation
36 Temperature influence on the dominant microbial population in
sourdough in summer and winter time in the bakery
The temperature in bakery during different seasons is not constant therefore
the LAB population may change during sourdough propagation The aim of this
part of the research was to evaluate the temperature influence on the LAB
population in sourdough during different seasons
The characterization of the dominant microbial population of the two Danish
sourdoughs (rye and wheat) was performed using PCR-DGGE technique at
approximately 18 oC (a typical temperature for a winter period in bakery) and at
25 oC (this temperature is reached at the bakery in summer time) According to
the results of the PCR-DGGE analysis the dominant microbial population
changed in the sourdoughs when the fermentation temperature was increased up
to 25 oC The dominant micro flora was additionally investigated by rep-PCR
and based on this LAB species were identified by 16S ribosomal gene
sequencing Based on the sequencing results two main species L sanfranciensis
and L curvatus were identified from the wheat sourdough (propagated at
approximately 18 oC) from the first day of sourdough fermentation the
population of LAB were 931 and 69 respectively Meanwhile after 6 weeks
of wheat sourdough propagation at increased temperature up to 25 oC new LAB
species L crustorum and L paraalimentarius were identified from the wheat
sourdough whereas L curvatus was not found However L sanfranciscensis
remains dominant species (936 ) Meanwhile in rye sourdough high changes
of LAB population was found Three main LAB species - L sanfranciscensis L
crustorum and L plantarumpentosus were dominant in rye sourdough on the
first day of fermentation at approximately 18 oC (respectively 781 156 and 63
of LAB population) Meanwhile after 6 weeks of sourdough propagation at 25 oC the species population decreased to 148 74 and 37 respectively and new
species L paraalimentarius L spicheri L fabifermentans L brevis and L
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
22
plantarumpentosus paraplantarum (respectively 370 185 148 37 and 37
) were found
The results confirm that temperature changes during seasons can influence
the LAB population in sourdough during sourdough propagation in bakeries
37 Bacteriophages in sourdough
Bacteriophages attacking the LAB are main factor responsible for
fermentation failures thus creating significant economic losses worldwide
Phage infections can cause serious problems in fermentation processes because
the viral particles can reduce or even stop the activity of fermentative bacteria The aim of this part of the work was to isolate and characterize the
bacteriophages from rye and wheat bread sourdough and to evaluate their
influence on LAB population during sourdough fermentation
Four bacteriophages attacking two different LAB species namely L
sanfranciensis and L crustorum were isolated from the Danish sourdoughs
Phages ФMR29 ФMW15 and ФW2 which are able to attack L sanfranciensis
are members of Siphoviridae family and belong to B1-morphotype Phage
ФMR29 and ФW2 were forming large plaques (~1-15 mm in diameter)
whereas phages ФMW15 and ФMW15 were forming micro plaques of ~02 mm
in diameter The phages attacking L sanfranciensis MR29 and W2 strains
developed in 8 and 10 days respectively solely in the rye sourdough and their
concentration varied from 103-10
6 pfug to 10
2-10
3 pfug respectively The
phages attacking L sanfranciensis W15 strain were found in detectable levels
during 10 and 14 days of wheat and rye sourdough fermentation respectively in
the range of 102-10
6 pfug The phages attacking L sanfranciensis W19 were
found in the wheat sourdough in detectable levels during a 3 days period of
sourdough fermentation in the range of 102-10
3 pfug Host range of the phages is
presented in Table 33 L sakei P acidilactici P pentosaceus KTU05-8
KTU05-9 and KTU05-10 strains previously isolated from rye sourdough with
wide range of antimicrobial activity and high phytase activity was not sensitive
to phages isolated from bread sourdough
Table 33 Host range of the phages
LAB strain Bacteriophages
ФMR29 ФMW15 ФW2 ФW19
L sanfranciensis MR29 + - - -
L sanfranciensis MW15 - + - +
L sanfranciensis W2 + + + +
L crustorum W19 - - - +
Phages ФMR29 ФMW15 and ФW2 were propagated in large scale and
purified using CsCl gradient centrifugation for electron microscopic analysis
(Fig 36) and isolation of phage DNA Many empty heads of phages ФMR29
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
23
and ФMW15 were observed (Fig 36b and c) because of sensitivity and low
stability under concentration and purification conditions The length of the tails
of bacteriophages ФMR29 and ФMW15 was 360 nm Meanwhile bacteriophage
ФW2 had a tail of 190 nm and an extra thin tail of 160 nm According to rep-
PCR L sanfranciscensis MR29 and MW15 strains which were sensitive to
phage ФMR29 and ФMW15 respectively were the dominant LAB in the
sourdough whereas after 10 week of fermentation these strains were not found
between the LAB isolates Schematic representation of the genomic comparison
of sequenced phages confirms that these phages are different (Fig 37) Were
identified five putative complete genome sequences responsible for phages DNA
packaging phages replication lysogenity morphogenesis and lysis
The research confirms that bacteriophages in sourdough can influence LAB
population and because of this sourdough properties may change during long
fermentation
Fig 36 Bacteriophages a ndash ФW2 b ndash ФMR29 c ndashФMW15 isolated from the
sourdoughs
a
c
b
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
24
Fig 37 Schematic representation of the genomic comparison of sequenced phages
Genomes are indicated with thick black lines Arrows represent putative Complete
Genome Sequences The colour of the arrows indicates the putative function of the gene
product Orange shading connecting two genomes corresponds to the similarity level
obtained from tblastx comparison
38 LAB bioproducts for the wellness of summer wheat grains
LAB bioproducts prepared using MRS media The LAB bioproducts at
different temperatures showed different antimicrobial activity against Fusarium
spp on the grains surface L sakei and P pentosaceus KTU05-10 bioproducts
were most effective at 25 oC while P acidilactici showed the best efficacy at 15
oC P pentosaceus KTU05-8 gave similar efficacy at both 15 and 20
oC P
pentosaceus KTU05-9 slightly reduced Fusarium spp infection at all
temperatures but the reduction was insignificant P pentosaceus KTU05-10 gave
the best control against Fusarium spp and significantly controlled Fusarium
infection level regardless of the temperature LAB bioproducts applied at
different doses (concentrations) significantly reduced Fusarium infection of
spring wheat seeds after one week of incubation at 20 oC Significantly better
antifungal activity was recorded after LAB application at 20 and 30 mL per 100
g as compared with a dose of 10 mL per 100 g LAB efficacy at 20 and 30 mL
doses was similar no significant differences between these two doses for all
LAB were established Given the results of this test a 20 mL dose can be used in
future experiments (Table 34)
The application of bioproducts prepared using L sakei P acidilactici and P
pentosaceus KTU05-10 showed significant positive effect on seeds germination
after three days of germination (seeds germination increased 60 113 and 73
respectively) Bioproduct prepared using P acidilactici significantly increased
the seedling height up to 55 after 6 days of germination Other LAB
bioproduct increased seedling height from 7 to 26 after 6 days of germination
LAB bioproducts increased root length up to 28 after 6 days of germination
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
25
Table 34 LAB bioproduct influence on the level of Fusarium spp infection on spring
wheat seeds cv lsquoGranaryrsquo at different temperatures and bioproduct doses
Fusarium spp infected seed
LAB strain used for the production of bioproduct
Temperature oC
(10 mL bioproduct used)
LAB bioproduct dose per
100 g seeds mL (20 oC temperature applied)
15 20 25 10 20 30
Control 742 883 908 842 842 842
L sakei KTU05-6 608 567 367 350 150 175
P acidilactici KTU05-7 383 625 567 325 183 192 P pentosaceus KTU05-8 483 492 958 300 183 133
P pentosaceus KTU05-9 650 742 792 333 242 225
P pentosaceus KTU05-10 517 508 450 358 175 225
LSD05 843 5961 14601 6428 4545 11133
LSD01 11236 7945 19461 8567 6058 14839
and ndash substantial differences compared with the control at 95 and 99 probability level
LAB bioproducts prepared using MRS and newly prepared potato juice
media and selected LAB mixtures LAB bioproducts showed high antifungal
activity against Coch sativus Fusarium and Alternaria spp on spring wheat cv
lsquoTrisorsquo seeds All tested bioproducts decreased Coch sativus Fusarium and
Alternaria spp fungi on spring wheat lsquoTrisorsquo at 10 and 15 ordmC (Table 35) in
varies degree The highest Fusarium spp decreasing (after 20 days of
experiment) was achieved applying bioproduct prepared using potato juice media
and mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10
strains (Fusarium spp contaminated seeds decreased up to 73 ) Whereas the
highest Coch sativus infection decreasing was achieved applying bioproduct
prepared using potato juice media and mixture of LAB P acidilactici amp P
pentosaceus KTU05-10 as well as using MRS media and P pentosaceus
KTU05-10 strain (Coch sativus contaminated seeds in both case decreased up to
63 ) whereas applying bioproduct prepared using potato juice media and
mixture of LAB L sakei amp P acidilactici amp P pentosaceus KTU05-10 for seed
decontamination Coch sativus infection decreasing was achieved up to 25 P
pentosaceus KTU05-10 strain alone and in mixtures with other LAB propagated
in MRS media showed significant effect against Alternaria spp on spring wheat
seeds (up to 50 ) Whereas bioproducts prepared using potato juice media and
P pentosaceus KTU05-10 strain and mixture of strains L sakei amp P
acidilactici amp P pentosaceus KTU05-10 show less antimicrobial activity against
Alternaria spp (respectively up to 27 and 21 ) Bioproduct prepared using
potato juice media and P acidilactici amp P pentosaceus KTU05-10 strains that
show strong antimicrobial activity against Coch sativus did not show
antimicrobial activity against Alternaria spp
The influence of the bioproduct of LAB on summer wheat grains seedling
disease was not found whereas bioproduct of P pentosaceus KTU05-10 and
mixture of P acidilactici amp P pentosaceus KTU05-10 strains and mixture of L
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
26
sakei amp P acidilactici amp P pentosaceus KTU05-10 strains significantly
decreased roots prevalence (Table 36) Bioproducts of P pentosaceus KTU05-
10 and L sakei amp P acidilactici amp P pentosaceus KTU05-10 mixture
significantly reduced Cochliobolus sativus infection on the seed surface
Table 35 LAB bioproducts (20 mL100 g seeds) antifungal activity against Fusarium and
Alternaria spp and Cochliobolus sativus on the seeds of spring wheat cv lsquoTrisorsquo at
different incubation temperatures
LAB strain used for
the production of
bioproduct
After 5 days After 10 days After 15 days 20 days
10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC 15 ordmC 10 ordmC
Fusarium spp infected seed
Control 00 108 167 567 175 567 183
Pp10a 00 08 42 125 58 125 58
Pab amp Pp10 00 00 58 75 83 75 100 Lsc amp Pa amp Pp10 08 08 42 150 58 150 67
Pp10 00 00 42 350 133 383 158
Pa amp Pp10 00 08 50 100 100 125 108 Ls amp Pa amp Pp10 00 08 25 67 50 100 50
LSD05 436 747 1151 793 1284 877
LSD01 597 1023 1577 1086 1759 1201
Cochliobolus sativus infected seed
Control 58 200 108 233 183 250 200 Pp10 00 33 08 92 67 108 75
Pa amp Pp10 00 17 33 33 100 67 100
Ls amp Pa amp Pp10 00 17 00 83 75 108 92 Pp10 00 17 17 67 67 150 100
Pa amp Pp10 00 33 08 133 58 158 75
Ls amp Pa amp Pp10 00 08 17 192 83 200 150
LSD05 236 642 469 674 739 629 746
LSD01 323 879 643 924 1012 861 1022
Alternaria spp infected seed
Control 225 533 600 683 633 750 675
Pp10 00 92 200 258 275 308 300
Pa amp Pp10 17 92 175 300 267 350 300
Ls amp Pa amp Pp10 08 92 200 267 300 333 342
Pp10 17 108 325 375 408 517 492 Pa amp Pp10 25 450 558 650 667 683 700
Ls amp Pa amp Pp10 25 200 383 417 508 467 533
LSD05 7031 1234 1334 1489 1043 1400 1343 LSD01 9633 1690 1827 2039 1429 1918 1840
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
27
Table 36 The influence of LAB bioproducts (20 mL100 g seeds) on summer wheat
lsquoTrisorsquo root and seedling diseases and seed violation by Fusarium spp and Coch sativus
fungi
LAB strain used for
the production of bioproduct
The prevalence of
sprout
The prevalence of
roots Damaged seeds
damaged
sprout
disease
index
damaged
roots
disease
index
Fusarium
spp
Coch
sativus
Control 585 238 730 274 75 300 Pp10a 516 202 361 129 85 115
Pab amp Pp10 576 216 487 179 40 170
Lsc amp Pa amp Pp10 673 253 623 216 110 115
Pp10 575 200 391 130 10 160
Pa amp Pp10 770 282 710 248 30 275
Ls amp Pa amp Pp10 457 173 391 135 35 150
LSD05 2500 1055 2273 924 1084 1327
LSD01 3425 1446 3114 1266 1486 1819
a ndash P pentosaceus KTU05-10 b ndash P acidilactici and c ndash L sakei strains
and ndash substantial differences compared with the control at 95 and 99 probability level
Bioproduct when LAB were propagated in new potato juice media
39 The impact of phytase producing LAB bioproducts on the solubility and
bioavailability of minerals from whole grain wheat bread
The bioavailability of minerals in whole grain wheat bread was evaluated by
imitating the pH conditions of human gastrointestinal tract The content of
soluble minerals (ferrum zinc manganese calcium and phosphorus) in whole
grain wheat bread was observed higher when 10 20 and 30 of the fermented
bioproduct prepared with P pentosaceus KTU05-8 KTU05-9 and KTU05-10
strains was used as compared with bread samples that were prepared with
sourdough without phytase positive LAB (Fig 38)
The concentration of soluble calcium compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
39 68 and 285 higher respectively when 10 of the LAB bioproduct was
used 120 102 and 278 higher respectively when 20 of the LAB
bioproduct was used and 124 2691 and 260 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without phytase positive LAB The concentration of soluble ferrum compounds
in bread prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and
KTU05-8 strains was 68 578 and 646 higher respectively when 10 of the
LAB bioproduct was used 450 740 and 1002 higher respectively when 20
of the LAB bioproduct was used and 508 687 and 825 higher when 30
of the LAB bioproduct was used as compared with bread that was prepared with
sourdough without phytase positive LAB The concentration of soluble zinc
compounds in bread prepared with bioproduct of P pentosaceus KTU05-10
KTU05-9 and KTU05-8 strains was 127 285 and 452 higher respectively
when 10 of the LAB bioproduct was used 166 236 and 520 higher
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
28
respectively when 20 of the LAB bioproduct was used and 168 367 and 419
higher when 30 of the LAB bioproduct was used as compared with bread
that was prepared with sourdough without selected LAB
0
2
4
6
8
10
12
14
05-8 05-9 05-10 K
Ca
mg1
00
g
a
0
05
1
15
2
25
05-8 05-9 05-10 K
Fe
mg1
00
g
b
00
02
04
06
08
05-8 05-9 05-10 K
Zn m
g1
00
g
c
0
02
04
06
08
1
05-8 05-9 05-10 K
Mn m
g1
00
g
d
0
20
40
60
80
100
120
140
160
05-8 05-9 05-10 K
P m
g1
00
g
e
The concentration of soluble manganese compounds in bread prepared with
bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8 strains was
132 447 and 263 higher respectively when 10 of the LAB bioproduct
was used 151 384 and 186 higher respectively when 20 of the LAB
bioproduct was used and 146 422 and 156 higher when 30 of the LAB
bioproduct was used as compared with bread that was prepared with sourdough
without selected LAB The concentration of soluble phosphorus in bread
Fig 38 The content of the soluble forms of minerals a ndash Ca b ndash Fe c ndash Zn d ndash Mn e ndash P in whole grain wheat bread prepared with 10 ( ) 20 ( ) and 30 ( ) of fermented bioproduct using phytase positive LAB (05-8 ndash P pentosaceus KTU05-8 05-9 ndash P pentosaceus KTU05-9 05-10 ndash P pentosaceus KTU05-10 K ndash control without selected LAB)
The average values indicates significant
differences between the control sample and tested
sample assessed according to the Dunkan criteria
(p le 005)
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
29
prepared with bioproduct of P pentosaceus KTU05-10 KTU05-9 and KTU05-8
strains was 162 295 and 87 higher respectively when 10 of the LAB
bioproduct was used 111 214 and 205 higher respectively when 20 of
the LAB bioproduct was used and 160 305 and 259 higher when 30 of
the LAB bioproduct was used as compared with bread that was prepared with
sourdough without the LAB
310 The impact of bioproducts of BLIS producing LAB on the safety of
whole grain wheat bread
Anti-rope activity of LAB in baked bread The addition of bioproduct
prepared with P pentosaceus KTU05-9 as a starter had a significant effect on
reducing the bacterial spoilage of bread as compared with bread samples
prepared without the LAB sourdough The level of B subtilis inoculums of 5104
CFUg caused rope spoilage in the control bread sample after 1 and 15 days of
storage at 30 and 23 degC respectively (Table 37) whereas bread prepared with 20
P pentosaceus KTU05-9 as a starter did not show visual roping until 4 and 6
days of storage at 30 degC and 23 degC respectively
Table 37 The effect of P pentosaceus KTU05-9 as a starter on bread roping on
artificially contaminated bread during 8 days of storage
Amount of sourdough used for bread making Time until first visual roping days
23 degC 30 degC
10 2 2
15 3 2
20 6 4
0 (control) 15 1
Inhibition of fungi by the LAB on a bread surface P acidilactici KTU05-
7 P pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on a
bread surface inhibited the growth of fungi during 8 days of storage (Table 38)
whereas on the control bread sample fungi colonies were detected in large
quantities after 8 days of storage
Table 38 The effect of the LAB suspension on bread surface moulding after 8 days of
storage
LAB strain Mould intensity
P acidilactici KTU05-7 ndash L sakei KTU05-6 +
P pentosaceus KTU05-8 ndash
P pentosaceus KTU05-9 + P pentosaceus KTU05-10 ndash
Control ++
Mould colony description (ndash) no mould (+) scarce and small (++) large
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
30
4 CONCLUSIONS
1 Assessing bacteriocins producing lactic acid bacteria (LAB) and their
produced metabolites antimicrobial activity against varies food borne
pathogens was found
11 The LAB cells did not show antimicrobial activity against tested fungi
whereas LAB produced metabolites (LAB supernatants and neutralized
LAB supernatants) show antifungal activity in varies degree LAB
supernatants show fungicidal activity against Fusarium culmorum and
Fusarium poae whereas antimicrobial activity of neutralized LAB
supernatants observed fungicidal and fungistatic and was observed
lower
12 The LAB cells did not show antimicrobial activity against tested
bacteria whereas LAB supernatants show high antimicrobial activity
against pathogenic bacteria belonging to Listeria monocytogenes
Escherichia coli Salmonella typhimurium and other species as well as
bacteria belonging to Bacillus and Pseudomonas genera Antimicrobial
activity of LAB produced bacteriocins like inhibitory substances
(BLIS) depended on LAB strain Lactobacillus sakei KTU05-6 and
Pediococcus pentosaceus KTU05-10 produced BLIS inhibited gram
positive and gram negative bacteria meanwhile Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 and KTU05-9 strains
produced BLIS show antimicrobial activity only against gram negative
bacteria
2 The highest antimicrobial activities of BLIS against B subtilis were
observed during 15-30 h of LAB cultivation and depended on LAB
strain media pH and cultivation temperature The highest BLIS activity
observed at 30 oC for P pentosaceus KTU05-10 P acidilactici and L
sakei strains whereas the highest BLIS activity of P pentosaceus
KTU05-8 and KTU05-9 strains were observed at 25 oC The highest
BLIS activity was produced in media with pH 65 Antimicrobial
activity of BLIS increased when as azote source for LAB cultivation 20
gL of triptone was used as carbon source ndash 20 gL of glucose was
used Increased amounts of MgSO47H2O up to 02 gL MnSO44H2O
up to 01 gl and cysteine-HClH2O up to 2 gL increased BLIS activity
against B subtilis
3 BLIS producing LAB show 33 times higher extracellular phytase
activity in comparison with intracellular phytase activity The
extracellular phytase activity depended on LAB strain and was observed
23-30 U10310
10 CFU (for P pentosaceus strains) and 13 U10
310
10
CFU (for P acidilactici KTU05-7) The similar phytase activity 28
U10310
10 CFU show P pentosaceus 12 strain isolated from industrial
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
31
sourdough whereas other LAB strains isolated from industrial
sourdough show lower phytase activity (1-16 U10310
10 CFU)
4 In vitro studies in simulated gastrointestinal tract media pH provide
that bioproducts prepared with LAB that produce BLIS used in whole
grain wheat bread preparation increased minerals such as ferrum zinc
manganese calcium and phosphorus biological bioavailability average
30 The LAB bioproducts used in whole grain flour wheat bread
suppressed the microbiological spoilage of bread
5 Four bacteriophages ФMR29 ФW2 ФW19 and ФMW15 attacking
LAB that belongs to Lactobacillus sanfranciscensis and Lactobacillus
crustorum species were isolated from bread sourdough Bacteriophages
ФMW15 ФMR29 and ФW2 attacking bacteria strains that belongs to
L sanfranciscensis species are members of family Siphoviridae and
belong to B1-morphotype Whereas LAB that produce BLIS and show
phytase activity were not inhibited by isolated phages The research
confirms that bacteriophages infections could change LAB population
6 Was found the LAB population changes in sourdough after the
sourdough propagation temperature was increased from 18 oC (a typical
temperature for a winter period in bakery) to 25 oC (this temperature is
reached at the bakery in summer time) typical sourdough microflora
such as L sanfranciscensis L crustorum decreased and developed new
bacteria species - L brevis L fabifermentans L spicheri L
paraalimentarius The LAB population stability during temperature
changes depended on the used flour type for sourdough preparation as
well and higher changes of the LAB population observed in rye
sourdough in comparison with wheat sourdough
7 LAB bioproducts prepared using potato juice media reduced seed-borne
pathogens from spring wheat seeds (Fusarium spp decreased up to 73
Alternaria spp decreased ndash 27 and Cochliobolus sativus ndash 63 )
LAB bioproducts decreased root diseases up to 46 and decreased
seed violation by Fusarium and Cochliobolus sativus fungi respectively
87 and 50 It has been shown that food by-products (wheat bran
spent distillers grains spent brewers grains and molasses) are also a
good media for the multiplication of LAB and formation of lactic acid
(including L(+) isomer) that has antimicrobial properties
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
32
LIST OF PUBLICATIONS
Publications on the dissertation topic in the main list of the Institute
for Scientific Information (Thomson Reuters Web of Knowledge)
1 Supronienė Skaidrė Semaškienė Roma Juodeikienė Gražina
Mankevičienė Audronė Čižeikienė Dalia Vidmantienė Daiva
Bašinskienė Loreta Sakalauskas Simonas Seed treatment with lactic
acid bacteria against seed-borne pathogens of spring wheat Biocontrol
Science and Technology 2015 Vol 25 no 2 p 144-154 (ISI Web of
Science IF 0731)
2 Juodeikienė Gražina Vidmantienė Daiva Bašinskienė Loreta
Černauskas Darius Bartkienė Elena Čižeikienė Dalia Green metrics
for sustainability of biobased lactic acid from starchy biomass vs
chemical synthesis Catalysis Today 2014 Vol 239 no 1 p 11-16
(ISI Web of Science IF 3309)
3 Čižeikienė Dalia Juodeikienė Gražina Paškevičius Algimantas
Bartkienė Elena Antimicrobial activity of lactic acid bacteria against
pathogenic and spoilage microorganism isolated from food and their
control in wheat bread Food Control ISSN 0956-7135 2013 Vol 31
iss 2 p 539-545 (ISI Web of Science IF 2819)
4 Digaitienė Agnė Hansen Ase Juodeikienė Gražina Eidukonytė
Dalia Josephsen J Lactic acid bacteria isolated from rye sourdoughs
produce bacteriocin-like inhibitory substances active against Bacillus
subtilis and fungi Journal of Applied Microbiology ISSN 1364-5072
2012 Vol 112 iss 5 p 732ndash742 (ISI Web of Science IF 2196)
In the other publications from the main list of the Institute for
Scientific Information (Thomson Reuters Web of Knowledge)
1 Stimbirys Artūras Bartkienė Elena Šiugždaitė Jūratė Augenienė
Dovilė Juodeikienė Gražina Vidmantienė Daiva Maruška Audrius
Stankevičius Mantas Čižeikienė Dalia Safety and quality parameters
of ready-to-cook minced pork meat products supplemented with
Helianthus tuberosus L tubers fermented by BLIS producing lactic acid
bacteria Journal of Food Science and Technology Published online
on June 2 2014 DOI 101007s13197-014-1328-4 (ISI Web of
Science IF 2024)
2 Juodeikienė Gražina Čižeikienė Dalia Češkevičiūtė Vaida
Vidmantienė Daiva Bašinskienė Loreta Akuneca Ieva Stankevičius
Mantas Maruška Audrius Bartkienė Elena Ragažinskienė Ona
Petrauskas Algimantas Solid-state fermentation of Silybum marianum
L seeds used as additive to increase the nutritional value of wheat bread
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
33
Food Technology and Biotechnology ISSN 1330-9862 2013 Vol
51 no 4 p 528-538 (ISI Web of Science IF 0977)
3 Bartkienė Elena Juodeikienė Gražina Vidmantienė Daiva Zdunczyk
Zenon Zdunczyk Przemyslaw Juskiewicz Jerzy Čižeikienė Dalia
Matusevičius Paulius Influence of diets to Wistar rats supplemented
with soya flaxseed and lupine products treated by lactofermentation to
improve their gut health International Journal of Food Sciences and
Nutrition ISSN 0963-7486 2013 Vol 64 no 6 p 730-739 (ISI Web
of Science IF 1202)
4 Juodeikienė Gražina Šalomskienė Joana Eidukonytė Dalia
Vidmantienė Daiva Narbutaitė Vilma Vaičiulytė-Funk Lina The
impact of novel fermented products containing extruded wheat material
on the quality of wheat bread Food Technology and Biotechnology
ISSN 1330-9862 2011 Vol 49 no 4 p 502-510 (ISI Web of Science
IF 1195)
Publications referred in Database from the List approved by
Institute of Science Information (Thomson Reuters Web of
Knowledge) [Proceedings et cetera]
1 Čižeikienė Dalia Juodeikienė Gražina Vidmantienė Daiva
Bašinskienė Loreta Supronienė Skaidrė Semaškienė Roma
Mankevičienė Audronė Bartkienė Elena Antimicrobial activity of
lactic acid bacteria and their application on wheat seeds decontamination
Proceedings of the 7th international congress Flour-Bread 13 9th
Croatian Congress of cereal technologists BrašnondashKruh 13 Opatija
Croatia October 16-18 2013 p 50-58 ISSN 1848-2562
Publications referred in International Database from the List
approved by Science Council of Lithuania
1 Bartkienė Elena Vidmantienė Daiva Juodeikienė Gražina Čižeikienė
Dalia Bašinskienė Loreta Valatkevičienė Žydronė Alternatyvaus
plikinio fermentuoto skirtingomis pieno rūgšties bakterijomis įtaka
pusruginės duonos kokybei ir saugai Maisto chemija ir technologija
ISSN 1392-0227 2012 T 46 Nr 2 p 5-13
2 Juodeikiene Grazina Bartkiene Elena Viskelis Pranas Urbonaviciene
Dalia Eidukonyte Dalia Bobinas Ceslovas Fermentation processes
using lactic acid bacteria producing bacteriocins for preservation and
improving functional properties of food products Advances in Applied
Biotechnology Ed by M Petre Rijeka InTech ISBN 9789533078205
2012 p 63-100
3 Mankevičienė Audronė Supronienė Skaidrė Semaškienė Roma
Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
34
Vidmantienė Daiva Grigiškis Saulius Ančenko Olga Naujas
antimikrobinis pieno rūgšties bakterijų bioproduktas ekologiškai
auginamų grūdų sveikatingumui didinti Agrariniai ir miškininkystės
mokslai naujausi tyrimų rezultatai ir inovatyvūs sprendimai Kėdainiai
Lietuvos agrarinių ir miškų mokslų centras ISSN 2029-6878 2014 nr
4 p 95-98
Papers of international scientific conferences
1 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Selina Marina Application of
biotools for recycling of agro-industrial wastebyproducts to lactic acid
8th International congress of food technologists biotechnologists and
nutritionists 21-24 October 2014 Opatija Croatia book of abstracts
University of Zagreb ISBN 9789539972552 p 34
2 Juodeikienė Gražina Čižeikienė Dalia Černauskas Darius
Vidmantienė Daiva Bašinskienė Loreta Supronienė Skaidrė
Semaškienė Roma Mankevičienė Audronė Novel antifungal lactic
acid bacteria bioproducts for health of cereal grains 7th International
Congress Flour - Bread13 9th Croatian Congress of Cereal
Technologists Brašno-Kruh13 Opatija Croatia October 16-18 2013
book of abstracts Faculty of Food Technology Osijek University of
Osijek ICC - International Association for Cereal Science and
Technology Osijek University of Osijek ISSN 1848-2554 2013 p
21
3 Juodeikienė Gražina Bašinskienė Loreta Bartkienė Elena
Vidmantienė Daiva Čižeikienė Dalia Černauskas Darius Enzymatic
hidrolysis of agro-industrial wastes to C6ndashC5 sugars for
biotechnological production of lactic acid UBIOCHEM IV
Utilization of Biomass for Sustainable Fuels amp Chemicals 4th
International Workshop of COST Action CM0903 October 14-16
2013 Valencia Spain book of abstracts Valencia Institute of
Chemical Technology 2013 p 52
4 Matusevičius Paulius Juodeikienė Gražina Čižeikienė Dalia
Češkevičiūtė Vaida Vidmantienė Daiva Bašinskienė Loreta
Akunceva Ieva Stankevičius Mantas Maruška Audrius Bartkienė
Elena Ragažinskienė Ona The advantages of solid state fermentation
to developed products of Silybum marianum L seeds with higher
nutrition value and safety for wheat bread European Biotechnology
Congress 2013 Vol24 Supp 1 July 2013 Pages S58 Poster
presentations Current Opinion in Biotechnology 24S (2013) S48ndash
S143
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
35
5 Čižeikienė Dalia Kot Witold Muhammed Musemma K Neve
Horst Hansen Lars H Soslashrensen Soslashren J Heller Knut J
Juodeikienė Gražina Nielsen Dennis S Vogensen Finn K Isolation
and characterization of bacteriophages from Danish sourdough
Conference and exhibitionldquo Bacteriophages 2013 bacteriophages in
medicine food and biotechnology 10-12 September 2013 Sant Hildas
college Oxford UK p 24
6 Juodeikienė Gražina Čižeikienė Dalia Bašinskienė Loreta
Antimicrobial activities of BLIS producing lactic acid bacteria against
undesirable microorganisms in food industry COST FAO804
Workshop 6-8 February 2013 Rostock Germany abstract Rostock
COST European Cooperation in Science and Technology 2013 p 12
7 Juodeikienė Gražina Čižeikienė Dalia Maruška Audrius Sigitas
Bartkienė Elena Bašinskienė Loreta Ragažinskienė Ona
Vidmantienė Daiva bdquoSolid state fermentation of the winter savory
(Satureja montana L) plant changes of chemical composition and
antimicrobial activity 61st International Congress and Annual
Meeting of the Society of Medicinal Plant and Natural Product
Research Planta Medica Stuttgard George Thieme Verlag ISSN
0032-0943 2013 Vol 79 PI50 p 1192
8 Čižeikienė Dalia Juodeikienė Gražina Growth factors influence for
lactic acid bacteria produced BLIS Foodbalt - 2013 8th Baltic
conference on food science and technology Food health and well-
being May 23-24 2013 Tallinn Estonia conference program and
abstracts Tallinn University of Technology Tallinn Tallinn
University of Technology ISBN 978-963-642-517-3 2013 p 19
9 Stankevičius Mantas Akuneca Ieva Drevinskas Tomas Kaškonienė
Vilma Mickienė Rūta Bimbiraitė-Survilienė Kristina Juodeikienė
Gražina Čižeikienė Dalia Bartkienė Elena Ragažinskienė Ona
Maruška Audrius Increased biological value and safer food product
development using solid state fermentation of plant material with
bacteriocins-producing lactic acid bacteria comparative phytochemical
analysis of fermented and non-fermented samples CECE 2013 10th
International Interdisciplinary Meeting on Bioanalysis University of
Peacutecs Peacutecs Hungary 2013 April 25-27 2013 Peacutecs Hungary program
and abstract book ISBN 978-963-642-517-3 p 51
10 Juodeikienė Gražina Bartkienė Elena Vidmantienė Daiva
Bašinskienė Loreta Eidukonytė Dalia The use of combined
fermentation for increasing efficiency of bioethanol production from
Jerusalem artichoke COST Action CM0903 Utilization of biomass
for sustainable fuels and chemical (UBIOCHEM) Sustainable
production of fuels energy materials and chemicals from biomass
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
36
UBIOCHEM-III workshop November 1-3 2012 Thessaloniki Greece
book of abstracts Chemical Process and Energy Resources Institute
Centre for Research and Technology Hellas Aristotle University of
Thessaloniki COST Action CM0903 2012 p 72
11 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Antimicrobial activity of Pediococcus acidilactici against undesirable
microorganism in the food industry Foodbalt-2012 Innovate and
healthy food for consumers 7th Baltic Conference on Food Science
and Technology Kaunas May 17-18 conference program and
abstracts Kaunas University of Technology Department of Food
Technology Kaunas Technologija ISBN 9786090204153 2012 p
60
12 Juodeikienė Gražina Češkevičiūtė Vaida Eidukonytė Dalia
Vidmantienė Daiva Bašinskienė Loreta Maruška Audrius Sigitas
Ragažinskienė Ona Kaškonienė Vilma Bartkienė Elena Petrauskas
Algimantas The advantages of solid state fermentation to develop plant
products with a higher nutritional value and safety Foodbalt-2012
Innovate and healthy food for consumers 7th Baltic Conference on
Food Science and Technology Kaunas May 17-18 conference
program and abstracts Kaunas University of Technology Department
of Food Technology Kaunas Technologija ISBN 9786090204153
2012 p 61-62
13 Juodeikienė Gražina Mankevičienė Audronė Eidukonytė Dalia
Vidmantienė Daiva Černauskas Darius Bašinskienė Loreta
Supronienė Skaidrė Semaškienė Roma Novel antifungal bio-product
for the health of cereal grains Foodbalt-2012 Innovate and healthy
food for consumers 7th Baltic Conference on Food Science and
Technology Kaunas May 17-18 conference program and abstracts
Kaunas University of Technology Department of Food Technology
Kaunas Technologija ISBN 9786090204153 2012 p 63
14 Eidukonytė Dalia Juodeikienė Gražina Paškevičius Algimantas
Lactobacillus sakei as an Alternative for Synthetic Preservatives against
Foodborne Pathogens European Symposium on Food Safety 21-23
May 2012 Warsaw Poland programme Des Moines International
Association for Food Protection 2012
15 Eidukonytė Dalia Nuobarienė Lina Vogensen Finn K Juodeikienė
Gražina Hansen Ase S Screening of lactic acid bacteria isolated from
Lithuanian sourdoughs for phytase activity 6th International
Congress Flour - Bread 11 8th Croatian Congress of Cereal
Technologists Opatija Croatia October 12-14 2011 book of abstracts
organised by Faculty of Food Technology Osijek University of Osijek
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
37
ICC-International Association for Cereal Science and Technology
Osijek University of Osijek ISSN 1848-2554 2011 p 108
16 Eidukonytė Dalia Juodeikienė Gražina Hansen Ase S Nuobarienė
Lina Vogensen Finn K Phytase activity of lactic acid bacteria strains
isolated from Lithuanian sourdoughs FOODBALT - 2011 6th Baltic
Conference on Food Science and Technology Innovations for Food
Science and Production May 5ndash6 2011 Jelgava Latvia abstract
book Latvia University of Agriculture Jelgava LLU ISBN
9789984480466 2011 p 84
Papers of Lithuania scientific conferences
1 Čižeikienė Dalia Juodeikienė Gražina Vogensen Finn Bakteriofagų
išskirtų iš duonos raugų charakteristika jų ir temperatūros pokyčių įtaka
pieno rūgšties bakterijų populiacijai rauguose Doktorantų stažuotės
užsienio mokslo centruose 2012-2013 konferencijos pranešimų
santraukos Vilnius Lietuvos mokslo taryba 2013 ISBN 978-9955-
613-64-0 p 31-33
2 Skabeikytė Erika Bartkienė Elena Juodeikienė Gražina
Vidmantienė Daiva Eidukonytė Dalia Bašinskienė Loreta Maruška
Audrius Ragažinskienė Ona Bakteriocinus produkuojančių pieno
rūgšties bakterijų taikymas kietafazei augalinių produktų fermentacijai
Jaunieji mokslininkai - žemės ūkio pažangai jaunųjų mokslininkų
konferencijos pranešimų tezės ISBN 978-9955-786-79-5 2012 p 53-
54
3 Češkevičiūtė Vaida Eidukonytė Dalia Juodeikienė Gražina Tikrojo
margainio L sakei fermentacijos sąlygų įtaka augalinio produkto
mikrobiologinės taršoms mažinimui Chemija ir cheminė technologija
studentų mokslinės konferencijos pranešimų medžiaga ISBN
9789955186519 2012 p 29-31
4 Eidukonytė Dalia Paškevičius Algimantas Juodeikienė Gražina
Pieno rūgšties bakterijų antimikrobinis aktyvumas prieš patogenines
bakterijas ir mikroskopinius grybus Mokslas - sveikatai [elektroninis
išteklius] V nacionalinė doktorantų mokslinė konferencija 2012 m
balandžio 11 d Kaunas [Lietuva] konferencijos tezių rinkinys
Kaunas Lietuvos sveikatos mokslų universiteto Leidybos namai 2012
ISBN (CD) 978-9955-15-232-3 p75
5 Marcinkevičiūtė Greta Mankevičienė Audronė Supronienė Skaidrė
Eidukonytė Dalia Maistui naudojamų vasarinių kviečių grūdų taršos
mažinimas ekologinėmis priemonėmis Studentų mokslinė praktika
2012 konferencijos pranešimų santraukos D 2 Vilnius Lietuvos
mokslo taryba ISBN 978-9955-786-79-5 2012 p 119-121
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
38
BAKTERIOCINUS GAMINANČIŲ PIENO RŪGŠTIES BAKTERIJŲ
BIOPRODUKTAI JŲ ANTIMIKROBINIS IR FITAZINIS AKTYVUMAS
BEI TAIKYMAS
SANTRAUKA
Temos aktualumas Grūdų produktai įskaitant kasdien vartojamus duonos
kepinius yra mitybinės piramidės pagrindas ir pagrindinis skaidulinių ir
mineralinių medžiagų pvz geležies kalcio magnio seleno cinko taip pat ir
folio rūgšties jodo B grupės vitaminų šaltinis Tačiau mineralinių medžiagų
pasisavinamumas žmonių ir gyvūnų organizme iš grūdų produktų skaidulinių
medžiagų yra ribotas Jų sudėtyje esanti fito rūgštis linkusi su skaidulinių
medžiagų mineralinėmis medžiagomis sudaryti organizmo neįsisavinamus fitatų
kompleksus Ši problema ypač aktuali viso grūdo dalių kvietinių kepinių
gamyboje kur naudojami pagreitinti tešlos gamybos būdai ir fermentacijos metu
mineralinės medžiagos nėra išlaisvinamos iš fitatų kompleksų Dėl mineralinių
elementų trūkumo kasdieninėje mityboje ir nepakankamo jų įsisavinimo iš grūdų
produktų žmogaus organizme atsiranda specifiniai medžiagų apykaitos
sutrikimai sukeliantys įvairius negalavimus ir netgi susirgimus
Įvertinant šias mitybos problemas aktuali naujų biotechnologinių priemonių
leidžiančių padidinti mineralinių medžiagų pasisavinamumą iš grūdų produktų
paieška Viena iš natūralių ir dažniausiai galimų naudoti kvietinių kepinių
kokybei pagerinti priemonių galėtų būti fitaziniu aktyvumu pasižymintys
mikroorganizmai Pastaruoju metu daugelyje šalių mokslininkų susidomėjimo
sulaukė tiek mielės tiek pieno rūgšties bakterijos (PRB) pasižyminčios fitaziniu
aktyvumu Literatūroje sutinkamos prieštaringos nuomonės dėl PRB fitazinio
aktyvumo Šiuo metu nėra išskirtos PRB pasižyminčios dideliu fitaziniu
aktyvumu kurias būtų galima panaudoti duonos gamyboje Todėl jų paieška ir
išskyrimas iš fermentuotų produktų bei tolesnis pritaikymas vystant naujus
bioproduktus yra aktualus sprendžiant mineralinių medžiagų pasisavinamumo
problemą Vis plačiau nagrinėjamos galimybės šiam tikslui panaudoti
antimikrobinėmis savybėmis pasižyminčias PRB kurių sintetinami bakteriocinai
gali slopinti duonos ligas sukeliančių mikroorganizmų pvz Bacillus subtilis ir
mikroskopinių grybų augimą
Duonos gamybos technologijoje ypač vasaros laikotarpiu aktuali išlieka
kepinių mikrobiologinio gedimo kurį sukelia šiltuoju metų laikotarpiu kepyklose
plintantys Bacillus subtilis ir mikroskopiniai grybai problema Pastaruoju metu
didelio mokslininkų susidomėjimo sulaukė ir į bakteriocinus panašias medžiagas
(BPM) gaminančios PRB kurių antimikrobinis poveikis pasireiškia prieš įvairias
bakterijas ir net maisto patogenus pvz Listeria Salmonella Escherichia
gentims priklausančias bakterijas Natūralūs priedai ndash PRB fermentuoti
bioproduktai pasižymintys tiek antimikrobiniu tiek ir fitaziniu aktyvumu ndash
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
39
galėtų būti vienas iš būdų sulėtinti kvietinės duonos mikrobiologinį gedimą ir tuo
pačiu padidinti kvietinių kepinių praturtintų skaidulinėmis medžiagomis
maistinę vertę
KTU Maisto mokslo ir technologijos katedroje jau anksčiau buvo įvertinta
lietuviškų pramoninių duonos raugų bei spontaninių ruginių raugų mikroflora ir
išskirtos iš jų penkios bakteriocinus gaminančios PRB Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10 padermės Pirminiai šių mikroorganizmų antimikrobinių
savybių tyrimo rezultatai parodė vykdomų darbų perspektyvumą ir tikslingumą
plėsti antimikrobinių preparatų taikymą ne tik duonos pramonėje bet ir kitose
srityse ypač ekologinėje žemdirbystėje mažinti grūdų sėklos biologinę taršą
Todėl aktuali naujų terpių skirtų PRB kultivuoti paieška atkreipiant dėmesį į
šalutinius maisto pramonės produktus galinčius atpiginti bioproduktų gamybą
Tiriant PRB panaudojimo galimybes fermentacijos procesuose ir naujiems
bioproduktams kurti būtina ištirti fermentacijos inhibitorius bakteriofagus kurie
patekę į fermentacijos terpę gali suardyti jiems jautrias PRB ląsteles ir
fermentacijos procesą pakreipti nepageidaujama linkme todėl būtų gaunamas
nekokybiškas produktas Nors žinoma kad bakteriofagai yra atsakingi už
fermentacijos proceso nesėkmes tačiau mažai duomenų yra apie duonos raugų
PRB atakuojančius bakteriofagus Iki šiol nėra duomenų ir apie bakteriofagų
įtaką PRB populiacijai ilgalaikės duonos raugų gamybos metu
Disertacinio darbo tikslas Nustatyti pieno rūgšties bakterijų išskirtų iš
duonos raugų antimikrobinį ir fitazinį aktyvumą bei įvertinti bakteriocinus
gaminančių pieno rūgšties bakterijų panaudojimo galimybes ekologinėje
žemdirbystėje vasarinių kviečių grūdų sėklos biologinės taršos mažinimui ir viso
grūdo dalių kvietinių kepinių maistinės vertės ir saugos didinimui
Darbo tikslui pasiekti suformuluoti šie uždaviniai 1 Nustatyti bakteriocinus gaminančių PRB ir jų metabolizmo produktų
antimikrobinį aktyvumą prieš grūdų sėklų ir maisto produktų gedimą
sukeliančius mikroorganizmus
2 Įvertinti terpės komponentų ir fermentacijos sąlygų įtaką
antimikrobinėmis savybėmis pasižyminčių į bakteriocinus panašių
medžiagų aktyvumui
3 Išskirti iš pramoninių duonos raugų atrinkti ir genetiškai identifikuoti
fitaziniu aktyvumu pasižyminčias PRB ir įvertinti jų fitazinį aktyvumą
(intraląstelinį ir ekstraląstelinį) o taip pat nustatyti bakteriocinus
gaminančių PRB fitazinį aktyvumą
4 Įvertinti imituojant virškinamojo trakto terpės pH vertes atrinktų
fitaziniu aktyvumu pasižyminčių PRB bioproduktų panaudojimo
galimybes viso grūdo dalių kvietinių kepinių biologiškai pasisavinamų
mineralinių medžiagų padidinimui ir mikrobiologinio gedimo lėtinimui
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
40
5 Išskirti ir charakterizuoti bakteriofagus iš duonos raugų ir įvertinti jų
poveikį PRB populiacijai bioproduktų fermentacijos metu
6 Nustatyti temperatūros įtaką PRB rūšių populiacijos pokyčiams duonos
rauguose
7 Ištirti PRB bioproduktų įtaką vasarinių kviečių grūdų sėklos biologinės
taršos mažinimui ir sveikumo didinimui
Mokslinis darbo naujumas Pirmą kartą įvertintas iš lietuviškų ruginių
duonos raugų išskirtų bakteriocinus gaminančių PRB (Lactobacillus sakei
KTU05-6 Pediococcus acidilactici KTU05-7 P pentosaceus KTU05-8
KTU05-9 ir KTU05-10) metabolizmo produktų antimikrobinis aktyvumas prieš
įvairias bakterijas priklausančias Bacillus Pseudomonas Escherichia
Enterococcus Salmonella Staphylococcus Micrococcus Listeria Yersinia
gentims ir mikroskopinius grybus priklausančius Aspergillus Penicillium
Alternaria Fusarium Candida Pichia Rhodotorula ir kitoms gentims bei šio
poveikio ypatumai
Kvietinių kepinių su padidintu skaidulinių medžiagų kiekiu maistinės vertės
didinimui atrinktos didžiausiu fitaziniu aktyvumu pasižyminčios bakteriocinus
gaminančios PRB ir in vitro sąlygomis įrodytos jų panaudojimo galimybės
mineralinių medžiagų pasisavinamumo didinimui
Ištirtos L(+) pieno rūgšties gamybos galimybės iš maisto gamybos šalutinių
produktų (sėlenų melasos salyklojų ir žlaugtų)
Pirmą kartą vasarinių kviečių grūdų sėklų biologinės taršos Fusarium
Alternaria genčių ir Cochliobolus sativus rūšies mikroskopinių grybų mažinimui
bei grūdų sveikumo didinimui išbandyti PRB bioproduktai ir nustatytas jų
teigiamas poveikis
Praktinė darbo vertė Sukurtas naujas PRB bioproduktas leidžiantis grūdų
sėklų paviršiuje sumažinti Fusarium Alternaria Cochliobolus sativus infekcijas
šaknų ligotumą bei sėklų pažeidimą Fusarium genties ir Cochliobolus sativus
grybais Įrodyta kad maisto gamybos šalutiniai produktai ndash kviečių sėlenos
salyklojai melasa žlaugtai taip pat galėtų būti (ne tik bulvių sultys) tinkama
terpė PRB dauginimui ir antimikrobinėmis savybėmis pasižyminčios pieno
rūgšties (įskaitant L(+) izomerą) susidarymui
Sukurti antimikrobiniu ir fitaziniu aktyvumu pasižymintys bioproduktai
kvietinių viso grūdo dalių kepinių maistinės vertės didinimui ir mikrobiologinio
gedimo lėtinimui panaudojant atrinktas PRB o jų dauginimui ndash viso grūdo dalių
kvietinių miltų terpę
Nustatyta kad PRB sudėtis duonos rauguose laikui bėgant keičiasi dėl fagų
infekcijos ir temperatūros pokyčių kepyklose todėl tikslinga dažniau ruošti naują
gamybinį raugą
Ginamieji disertacijos teiginiai
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
41
Antimikrobiniu ir fitaziniu aktyvumu pasižyminčių PRB panaudojimas
viso grūdo dalių kvietinių kepinių gamyboje didina mineralinių medžiagų
pasisavinamumą ir lėtina kepinių mikrobiologinį gedimą
PRB bioproduktai mažina mikroskopinių grybų infekciją vasarinių
kviečių grūdų sėklų paviršiuje daigų pašaknio ligotumą bei sėklų
pažeidimą Fusarium spp ir Cochliobolus sativus grybais
IŠVADOS
1 Ištyrus bakteriocinus gaminančių pieno rūgšties bakterijų (PRB) ir jų
metabolizmo produktų antimikrobinį aktyvumą nustatyta
11PRB ląstelės neslopino mikroskopinių grybų o susidarę fermentacijos
metabolizmo produktai (MP) ir neutralizuoti metabolizmo produktai
(NMP) pasižymėjo savitu antigrybiniu poveikiu MP išsiskyrė fungicidiniu
poveikiu daugiausiai slopindami Fusarium culmorum ir Fusarium poae
vystymąsi o NMP veikimas buvo silpnesnis nei MP ir jis buvo
fungicidinis bei fungistatinis
12PRB ląstelės nepasižymėjo antibakteriniu aktyvumu tačiau išryškėjo
stiprus jų MP antibakterinis poveikis maisto produktų gedimą
sukeliantiems mikroorganizmams įskaitant Listeria monocytogenes
Escherichia coli Salmonella typhimurium rūšies Bacillus ir Pseudomonas
genties patogenus Į bakteriocinus panašių medžiagų (BPM)
antimikrobinis poveikis priklausė nuo PRB rūšies Lactobacillus sakei
KTU05-6 ir Pediococcus pentosaceus KTU05-10 gaminamos BPM
slopino tiek gramteigiamas tiek gramneigiamas bakterijas o Pediococcus
acidilactici KTU05-7 P pentosaceus KTU05-8 ir KTU05-9 padermių
sintetinamos BPM slopino tik gramteigiamas
2 Didžiausias PRB BPM antibakterinis poveikis prieš Bacillus subtilis
pasireiškė 15ndash30 val laikotarpyje ir priklausė nuo PRB padermės terpės
pH verčių ir kultivavimo temperatūros Optimaliausia temperatūra P
pentosaceus KTU05-10 P acidilactici ir L sakei BPM susidarymui yra
30 oC o P pentosaceus KTU05-8 ir KTU05-9 padermių BPM ndash 25
oC
Tinkamiausia terpės pH vertė yra 65 BPM antimikrobinį aktyvumą
didino azoto šaltiniu PRB kultivavimui naudojant 20 gl triptono o anglies
šaltiniu ndash 20 gl gliukozės Padidinti iki 02 gl MgSO47H2O ir iki 01 gl
MnSO44H2O o taip pat iki 2 gl cisteino-HClH2O priedai didino BPM
aktyvumą prieš B subtilis
3 Tirtos bakteriocinus gaminančios PRB pasižymėjo vidutiniškai 33 kartus
didesniu ekstraląsteliniu fitaziniu aktyvumu nei intraląsteliniu ir
priklausomai nuo PRB padermės kito ribose nuo 23ndash30 AV10310
10 KSV
(P pentosaceus rūšies bakterijos) iki 13 AV10310
10 KSV (P acidilactici
KTU05-7) Šiems aktyvumams prilygo kai kurių iš pramoninių raugų
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
42
išskirtų PRB (P pentosaceus 12) aktyvumai (28 AV10310
10 KSV)
tačiau kitų PRB jie buvo daug mažesni (nuo 1 iki 16 AV10310
10 KSV)
4 In vitro tyrimai atlikti imituojant virškinamojo trakto terpės pH vertes
įrodė kad fitaziniu aktyvumu pasižyminčių bakteriocinus gaminančių
PRB bioproduktai padidino tirpių geležies cinko mangano kalcio ir
fosforo junginių kiekį viso grūdo dalių kvietiniuose kepiniuose
vidutiniškai 30 PRB bioproduktai pritaikyti viso grūdo dalių kvietinių
kepinių gamyboje sulėtino kepinių mikrobiologinį gedimą
5 Iš duonos raugų išskirti keturi bakteriofagai ФMR29 ФMW15 ФW2 ir
ФW19 infekuojantys Lactobacillus sanfranciscensis ir Lactobacillus
crustorum rūšims priklausančias PRB Bakteriofagai ФMW15 ФMR29
ФW2 lizuojantys L sanfranciscensis rūšiai priklausančias PRB priklauso
Siphoviridae šeimai ir pagal morfologiją priskiriami B1 tipui Tirtosios
bakteriocinus gaminančios ir fitaziniu aktyvumu pasižyminčios PRB
nebuvo jautrios išskirtiems bakteriofagams Tyrimai patvirtino kad
bakteriofagų infekcija gali pakeisti raugų PRB populiaciją
6 Nustatyta kad pakeitus fermentacijos temperatūrą iš 18 oC (būdinga
žiemos laikotarpiui kepyklose) į 25 oC (būdinga vasaros laikotarpiui
kepyklose) ruginiuose rauguose pasikeitė PRB populiacija sumažėjo
tipinė raugų mikroflora ndash L sanfranciscensis L crustorum ndash ir atsirado
naujos PRB rūšys ndash L brevis L fabifermentans L spicheri L
paraalimentarius PRB populiacijos pokyčiai priklausė ne tik nuo
fermentacijos temperatūros bet ir nuo naudotos raugų ruošimui žaliavos ir
labiau kito raugų gamybai naudojant ruginę žaliavą nei kvietinę
7 Sukurti PRB bioproduktai iš bulvių sulčių sumažino vasarinių kviečių
grūdų sėklos biologinę taršą Fusarium genties mikroskopinių grybų
sumažėjo 73 Alternaria genties ndash 27 o Cochliobolus sativus ndash 63
Bioproduktai sumažino šaknų ligotumą 46 bei sėklų pažeidimą
Fusarium genties ir Cochliobolus sativus grybais atitinkamai 87 ir 50
Įrodyta kad maisto gamybos šalutiniai produktai (kviečių sėlenos
salyklojai melasa žlaugtai) yra tinkama terpė PRB dauginimui ir
antimikrobinėmis savybėmis pasižyminčios pieno rūgšties įskaitant jos
L(+) izomerą susidarymui
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
43
Information about the Author Dalia Čižeikienė (Eidukonytė) was born 1985 05 12 in Varėna Lithuania
1992 ndash 2004 Ryto Secondary School Varėna Lithuania
2004 ndash 2008 Bachelors degree studies at Kaunas University of Technology
Faculty of Chemical Technology Bachelor of Science in Chemical
Engineering
2008 ndash 2010 Masterrsquos degree studies at Kaunas University of Technology
Faculty of Chemical Technology Master of Science in Chemical
Engineering
2009-09 ndash 2010-01 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
2010 ndash 2014 Doctoral degree studies at Kaunas University of Technology
Faculty of Chemical Technology
2013-02 ndash 2013-06 Scientific mission at University of Copenhagen
(Copenhagen Denmark)
Acknowledgements
I wish to express my gratitude to my scientific supervisor Prof habil Dr
Gražina Juodeikienė for the idea and the subject for this doctoral dissertation
valuable scientifical consultations and discussions her help in preparing
publications her trust encouragement and support during my doctoral studies at
Kaunas University of Technology to Assoc Prof Aringse Solvej Hansen and Assoc
Prof Finn Kvist Vogensen for the opportunity to do an internship Their kind
support guidance and friendship have been of great value to me on an academic
as well as a personal level Colleagues from the Department of Food Science and
Technology (Kaunas University of Technology) and Department of Food
Science (University of Copenhagen) ndash for friendly and creative atmosphere and
cheerful mood during the working hours Finally thanks to my family for their
moral support encouragement understanding and patience
UDK 6023579864+6646657966](0433)
SL 344 2015-01-26 25 leidyb apsk 1 Tiražas 70 egz Užsakymas 20 Išleido
leidykla bdquoTechnologijardquo K Donelaičio g 73 44029 Kaunas Spausdino leidyklos
bdquoTechnologijardquo spaustuvė Studentų g 54 51424 Kaunas
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