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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 418132 14 pageshttpdxdoiorg1011552013418132

Research ArticleTechnological Aptitude and Applications ofLeuconostoc mesenteroides Bioactive Strains Isolated fromAlgerian Raw Camel Milk

Zineb Benmechernene1 Hanane Fatma Chentouf1 Bellil Yahia1 Ghazi Fatima1

Marcos Quintela-Baluja2 Pilar Calo-Mata2 and Jorge Barros-Velaacutezquez2

1 Laboratory of Applied Microbiology Department of Biology Faculty of Sciences Oran University BP 16Es-Senia 31100 Oran Algeria

2 Department of Analytical Chemistry Nutrition and Food Science School of Veterinary SciencesCollege of BiotechnologyUniversity of Santiago de Compostela Rua Carballo Calero sn Campus Lugo 27002 Lugo Spain

Correspondence should be addressed to Zineb Benmechernene b zinebhotmailcom

Received 14 June 2013 Revised 21 September 2013 Accepted 23 October 2013

Academic Editor Denis Groleau

Copyright copy 2013 Zineb Benmechernene et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Two strains (B7 andZ8) of the Leuconostocmesenteroides subspeciesmesenteroides thatwere isolated fromAlgerian camelmilk froman initial pool of 13 strains and demonstrated a high ability to inhibit the growth of Listeria spp were selected and characterised atthe phenotypic and genotypic levels Probiotic profiling and inhibition spectra against food borne pathogens inmixed cultures werealso investigatedThebacteriocin produced byLmesenteroides strain B7was identified as leucocin B by specific PCR In vitro studiesdemonstrated that both Leuconostoc mesenteroides strains exhibited amarked probiotic profile showing high survival at low pH (2-3 and 4) in the presence of 05 1 and 2 of bile salts and at pH 3 in the presence of 3mgmL pepsin Susceptibility testing againstantimicrobial agents was also performed for both strains When tested in a mixed culture with Listeria innocua Listeria ivanovii orStaphylococcus aureus strain B7 reduced the numbers of these species by 187 178 and 138 log units respectively Consequentlythese two strains were found to possess good probiotic properties in vitro and a high capacity for Listeria spp inhibition in mixedcultures Therefore these strains have a favourable technological aptitude and a potential application as novel probiotic starters

1 Introduction

Camel milk has antimicrobial activity and a good conser-vation aptitude Barbour et al [1] reported that camel milkinhibits some pathogenic bacteria because of several protec-tive proteins found in the milk including lysozymes lactop-eroxidase lactoferrin immunoglobulin and vitamin C Forthese reasons Yagil et al [2] support that pasteurisation is notessential for camel milk if the camels are in good healthCamel milk composition is less stable than milk from otheranimals These variations can be caused by many factorssuch as geographical lactation alimentary conditions andthe breed and age of the camel [3] Lactic acid bacteria(LAB) from cow and goat milk have been well studied forboth antimicrobial activity and bacteriocin production [4ndash6]

However few studies have been conducted on the isolationand characterisation of LAB from camel milk [7ndash9] or on theantimicrobial activity [10ndash12]

Several LAB species produce a wide variety of antimicro-bials that can be used for food preservation In a number ofinstances the inhibitory activity of LAB could be attributed tometabolic end products such as hydrogen peroxide diacetyland organic acids and bacteriocin [13] Currently LABinclude (13) different bacterial genera Lactobacillus Leu-conostoc Lactococcus Enterococcus Streptococcus Pediococ-cus Carnobacterium Oenococcus Weissella Aerococcus Tet-ragenococcus Vagococcus and Bifidobacterium These bacte-ria are used primarily as starters in fermented food productswhere they can develop certain organoleptic characteristicsand increase the time of conservation [14ndash17]

2 BioMed Research International

LAB exhibit probiotic properties because these bacteriaare normal flora in gastrointestinal tract [18] and have noharmful effects [19] Moreover the addition of antagonis-tic bacterial preparations as protective cultures is anotherapproach for biopreservation [20] In addition possiblemicrobial interactions either beneficial (cooperation) orunfavourable (inhibition) can be achieved in mixed straincultures These mixed cultures are commonly used as startercultures in dairy manufacturing because of the acid produc-tion growth rate proteolytic activity bacteriocin productionand sensitivity aroma production and phage sensitivityof these cultures [21] However these technologies can belimiting for commercial application because of the negativeimpacts or the low reproducibility percentage of the response[22] For these reasons the characterisation of LAB as pro-biotic cultures should respond to several criteria such as theability to survive the specific conditions of the gastrointestinaltract including low pH proteolytic enzymes and bile saltconcentrations [23]

Because these bacteria are meant to be used as protectivecultures the inhibition pattern safety and functional andtechnological properties of these bacteria should also beexamined

Few studies have reported the high potential of usingLeuconostoc as a probiotic strain Kekkonen et al [24] showedthat the use of Leuconostoc as probiotic strain in inducingcytokines was better than that of the probiotic Lactobacillusstrain that is presently in clinical use In addition Allamehet al [18] reported for the first time the use of Leuconostocmesenteroides isolated from the intestine of snakehead fishas a potentially new probiotic for aquaculture systems forthe development of fish production Moreover Leuconostoclactis has been shown to have a potential for use as a singlestarter culture in Khanoon-jeen production and could reducethe microbial risk and fermentation processing time [25]Additionally Leuconostoc strains have potential as protectivecultures for vacuum packed meat products [26] and thebacteriocins of Leuconostoc can be used as protective agentsin combination with another starter culture in fermentedmeat [27] However the use of Leuconostoc mesenteroidesisolated from camel milk as either a probiotic strain or a pro-tective culture has never been reportedTherefore we isolatedand identified Leuconostoc strains from camel milk as newprobiotic or protective cultures for the first time

2 Materials and Methods

21 Raw Camel Milk Sampling Thirteen Leuconostoc strainswere isolated from four different camel milk samples whichwere collected from two different Saharan areas (Naama andBechar) in SouthWestern AlgeriaThe first two samples werecollected from two camels (Camelus dromados) in Naamathat differed in age (10 and 15 years old) and colour (greyand black) but had the same lactation period (March 2011)The diet of the camels was based on natural Saharan plantssuch as Drinn (Arstide pungens) Samples from Bechar werecollected from brown camels aged less than 10 years in thearid Abadla region in the period 2009ndash2011 Sampling wasperformed under aseptic conditions bywashing the teats with

warm water containing 2 bleach and collecting the milkin sterile bottles after hand washing with diluted alcoholSamples were maintained at 4∘C and were transported iniceboxes to the laboratory for analysis

22 Leuconostoc Isolation The bioactive Leuconostoc consid-ered in this work were isolated from the raw camel milkas described above All strains were stored at minus80∘C andwere maintained in reconstituted skimmed milk containing30 (wv) glycerol All strains were cultured in MRS broth(Liofilchem Teramo Italy) at 30∘C for 24 h and were thenseeded onto MRS agar (Liofilchem) to obtain single coloniesThe wild type and reference Leuconostoc strains used in thisstudy belonged to the collection of our laboratory

Based on the results obtained from the inhibitory assaystwo bioactive leuconostocs strains (B7 and Z8) were selectedand subjected to phenotypic and morphological character-isation based on the following criteria CO

2production

growth at different temperatures (4∘C 15∘C 30∘C 37∘C and45∘C) growth at different pH (48 and 68) and growth atdifferent NaCl concentrations (3 and 65) Additionallyall strains were subjected to the following biochemical teststo differentiate between Leuconostoc and lactobacilli dextranproduction on MSE medium [28] arginine hydrolysis onM16BCP medium (Oxoid Ltd London UK) and citric aciddegradation on Kempler and McKay solid medium Carbo-hydrate fermentation was tested on MRS supplemented withbromocresol purple as a pH indicator using the followingsugars to differentiate the subspecies of Leuconostocs arabi-nosemaltose rhamnose esculinmanitol sorbitol galactoselactose fructose glucose sucrose and xylose All strainswerephenotypically identified as belonging to the Leuconostocgenus based on the following criteria ovoid shape Gram-positivity catalase negativity vancomycin-resistance pro-duction of gas from glucose lack of arginine hydrolysis andfermentation profiles

23 Genetic Identification of Bioactive Leuconostoc Strains Afragment of the 16S rRNA gene of the two bioactive strainswas amplified by PCR using the universal primer pair p8FPL(forward 51015840-AGTTTGATCCTGGCTCAG-31015840) and p806R(reverse 51015840-GGACTACCAGGGTATCTAAT-31015840) [29] Theassays comprised 100 ng of template DNA 25 120583L of a mas-ter mix (BioMix Bioline London UK) (this included thereaction buffer dNTPs and magnesium chloride) Taq DNApolymerase 25 pmol of each oligonucleotide primer anddouble-distilled water to achieve a final volume of 50 120583LAmplification conditions were as follows denaturing at 94∘Cfor 7min 35 cycles of denaturation (94∘C for 60 sec)annealing (55∘C for 60 sec) extension (72∘C for 60 sec) anda final extension at 72∘C for 15min The PCR was performedas described by Bohme et al [30]

The two PCR products were then sequenced using thesame primers used for PCR The sequences were analysedwith Chromas software (Griffith University QueenslandAustralia) and aligned using Clustal-X software [31 32]Following alignment these sequences were identified bysearching for sequence homology among published referencesequences using the web BLAST tool (National Center for

BioMed Research International 3

Biotechnology Information (NCBI httpblastncbinlmnihgov) [33] Homologies higher than 99 with respect to astrain type were considered acceptable identifications

24 Genetic Identification of Bacteriocin Produced by Leu-conostocmesenteroides Strains Thebacteriocins produced byL mesenteroides strains were identified by PCR using theprimers described by Xiraphi et al [34] The detection ofthe following bacteriocins mestenterocin B mesenterocin Yleucoccin A leucoccin B and leucocin A-TAF was carriedout using the following primers mesB mesY lcnA lcnBand lcnA-TAF respectively The reaction conditions were asdescribed by Xiraphi et al [34]The nucleotide sequencing ofthe bacteriocin gene was performed as described above

25 Probiotic Evaluation of Bioactive L mesenteroides Strains

251 Inhibition Spectra of L mesenteroides Strains againstIndicator Microorganisms Preliminarily all strains weretested for the ability to produce antimicrobial substancesusing the direct method described by Fleming et al [35]Inhibitory activity was investigated using the following indi-cator bacteria Staphylococcus aureus 43300 (Centre Hospi-talier Universitaire CHU Oran Algeria) Listeria innocua(ATCC 33090) and Listeria ivanovii (ATCC 19119)

Alliquots of 80120583L from 18 h cultures of 107 CFUmLminus1Leuconostoc strains were spotted onto MRS agar using mul-tipoint inoculators and were incubated at 30∘C for 24 h[36] Following the incubation a semisolid Mueller Hinton(Oxoid) medium containing 100 120583L of 107 CFUmLminus1 of theindicator culture was poured as an overlay All plates werethen incubated at 37∘C for 24 h and examined for inhibitionzone formation Inhibition was considered positive when thewidth of the clear inhibition halos was ge05 cm

252 Detection of the Proteinaceous Nature of the InhibitoryAgent The proteinaceous nature of the inhibitory substancewas detected using an indirect method In this method aLeuconostoc strain was incubated for 18 h in MRS broth at30∘C and was then centrifuged at 8000 rpm for 10 minutesThen 100 120583L of the supernatant (FCS) was inoculated onwells formed on solid MRS medium that were seeded byindicator strains and the wells were incubated for 24 h to 48 hat 37∘C Colonies surrounded by a clear zone with a diametergreater than 2mm in the layer of the indicator culture wereconsidered positive Several factors were eliminated to con-firm the proteinous nature of the inhibitory substance suchas lactic acid using a buffered medium and the eliminationof the effect of hydrogen peroxide by using indicator strainswith catalase enzyme such as S aureus Listeria innocuaand L ivanovii In addition proteolytic enzymes (trypsin-chymotrypsin) and heat treatments of the supernatant atdifferent temperatures (75∘C 80∘C and 100∘C) were used toidentify the proteinaceous nature of the inhibitory substance

253 pH Tolerance Bacterial cells from overnight MRScultures were collected by centrifugation and were washedwith sterile phosphate buffer saline pH 8 Centrifugation andwashing procedures were repeated three times The bacterial

cells were resuspended in sterile PBS adjusted to pH 2 3 or 4andwere incubated at 37∘C for 3 hThe viable bacterial countswere then determined in MRS agar

254 Bile Salt Tolerance and Bile Salt Hydrolyses Bacterialcells from overnight MRS cultures were harvested by cen-trifugation washed and resuspended in PBS (pH 8) supple-mented with 05 10 or 20 (wv) oxgall (Oxoid LtdEngland) Viable cell counts were determined in MRS agarafter 4 h at 37∘C

For the bile salt hydrolysis assay overnight bacterial cul-tures of each L mesenteroides strain were streaked on MRSagar supplemented with 05 (wv) oxgall and incubated for24 and 48 h at 37∘C The bacterial hydrolysis of the bile saltwas visualised as altered colony morphology compared withthe control MRS plates

255 Resistance to Pepsin Bacterial cells from overnightMRS cultures were collected washed and resuspended inPBS buffer (pH 2 and 3) supplemented with 3mgmL ofpepsin The resistance of the L mesenteroides strains wasdetermined by counting the initial viable cells in MRS agarafter 3 h incubation at 37∘C

256 Haemolytic Activity An overnight culture of the Lmesenteroides strains was streaked in triplicates on Columbiaagar plates containing 5 (wv) human blood and incubatedfor 48 h at 30∘C Blood agar plates were examined for signsof 120573-haemolysis (clear zones around colonies) 120572-haemolysis(green zones around colonies) and 120574-haemolysis (no zonesaround colonies)

257 Antibiotic Sensitivity Test The antibiotic susceptibilityof the two strains of L mesenteroides (B7 and Z8) was testedthree times against 13 antibiotics using Bio-Rad discs (6mm)L mesenteroides strains were cultured in MRS broth at 30∘Cfor 18 h and were then adjusted to a 05 McFarland scale andsmeared homogeneously on MRS culture plate Antibioticdiscs were placed on the plates and incubated for 24 h at 37∘CThe antibiotics included gentamycin (GM 10 120583g) strepto-mycin (S 10 120583g) amoxicillin (AMX 25 120583g) tetracycline (TE30 120583g) chloramphenicol (C 30120583g) ampicillin (AM 10 120583g)erythromycin (E 15120583g) cephalotin (CEF 30 120583g) lincomycin(L 15 120583g) Neomycin (N 30 120583g) kanamycin (K 30 120583g)penicillin (P 6120583g) and vancomycin (VA 30 120583g)

The inhibitory circles emerging after 24 h of incubationwere measured Activity was assessed as sensitive (le21mm)intermediate (16ndash20mm) and resistant (ge15mm) as previ-ously described by Liasi et al [37]

26 Acidity and Growth Kinetics in Pure and Mixed Cul-tures To study the growth kinetics of L mesenteroides inpure cultures and cultures mixed with pathogens strains (Linnocua L ivanovii and S aureus) strain B7was selected andinoculated by streaking on solidMRSmedium and incubatedat 30∘C for 18 h After incubation a colony was inoculatedintoMRS liquid andwas incubated at 30∘C for 18 hoursThen100mL of the 18 h culture was inoculated into 10mL of


skimmed milk containing 03 yeast extract and was incu-bated at 30∘C for 18 h [38]

The bacterial populationmeasurement with the indicatorstrains in pure and mixed cultures was performed by count-ing in Nutrient Agar medium (Oxoid) to differentiatebetween the colonies of Leuconostoc and Listeria spp wherelatter appear larger the Baird Parker medium (Oxoid) tocount S aureus and MRS to count the Leuconostoc strain

Strain B7 which is the most efficient producer of antimi-crobial substances and three indicator strains that is Linnocua ATCC 33090 L ivanovii ATCC 19119 and S aureusATCC 43300 were routinely subcultured in 10mL of skimmilk with 03 yeast extract (Oxoid) that had an initialconcentration of 107 CFUmL for L innocua ATCC 33090107 CFUmL for L ivanoviiATCC 19119 and 107UFCmL forS aureus 43300 The three strains were inoculated separatelyinto 100mL of skim milk for monitoring pure cultures andthe mixed culture was prepared by mixing a culture of theindicator strains with the test strain B7 at a concentration of107 CFU The cultures were divided into tubes and incubatedat 30∘C for 24 h Every three hours the samples were asep-tically withdrawn from tubes to determine the pH titrableacidity and the growth rate This experiment was repeatedthree times [4]

27 Statistical Analysis Several statistical models have beenproposed to estimate growth parameters from the curvesobtained by the counting methods An ANOVA and rangetests were used to evaluate the difference between the averageof pH acidity and bacterial load and were represented by log119873 and 120583max The significance of the variation in the resultsof the antimicrobial activity was evaluated by two factorsrepeatability and reproducibility [39 40]

3 Results

31 Isolation Selection and Identification of LeuconostocIsolates fromRawCamelMilk A total of thirteen Leuconostocstrains were isolated from camel milk The isolates exhibitedovoid shape and were associated in short pairs andor chainsAll isolates were Gram-positive catalase negative citratepositive able to produce CO

2from glucose able to produce

dextran from sucrose and unable to hydrolyse arginineAccording to the antibacterial test B7 and Z8 showed moreinhibition zones than the other strains Therefore these twostrains were selected for probiotic profiling and behaviouralstudies in the presence of food borne pathogens The resultsobtained from physiological fermentation profiling (Table 1)and 16 srRNA as a molecular technique (data not shown)identified B7 and Z8 as L mesenteroides and revealed 99homology with other sequences from the reference strainsdeposited in the GenBank according to the BLAST tool

32 Genetic Identification of the Bacteriocin Specific primersfor mesenterocin Y mesB and Leucocin A LcnB andLcnA-TAF were tested on the extracted DNA As shown inFigure 1 the specific primer for leucocin B produced a faintPCR product of the expected molecular weight Sequenc-ing and alignment with respect to sequences deposited in

M 1817161514131211109876543211000900800700600500400300200

100

Figure 1The amplification of bacteriocins produced by Leuconostocmesenteroides B7 strain with LcnB primers lane M MW markerlane 2 Leuconostoc mesenteroides B7B lane 5 Leuconostoc mesen-teroides B71015840B and lane 8 Leuconostoc mesenteroides RB

GenBank database confirmed matching with respect to abacteriocin (mesY mesC mesD mesE mesF mesH andmesB) from L mesenteroides (data not shown)

33 Antimicrobial Activity of Leuconostoc Isolates B7 andZ8 strains exhibited inhibitory activity against severalpathogenic bacteria including S aureus 43300 L innocua(ATCC 33090) and L ivanovii (ATCC 19119) The inhibitionzones were measured and the results indicated that theinhibition intensity and range varied depending on the Leu-conostoc species assayed (Figure 2) Furthermore to inves-tigate whether the cause of the inhibition was due to theprotein substance buffered supernatants adjusted to pH 68were treated with proteolytic enzymes which led to thedisappearance of the inhibition zones (Figure 2) This resultindicated that inhibition was caused by a proteinaceouscompound

However inhibition remained after heating the bacterialsupernatants to a temperature of 100∘C (data not shown)which indicated that the causative inhibitory agent is heatresistant These results agree with previously reported results[41ndash45]

34 pH Tolerance The viable cell counts of the two L mesen-teroides strains after a 3 h exposure to low pH are shownin Table 2 Strain B7 was viable at all pH levels The resultsshowed a decrease of 2117 at pH 2 but increases of 049and 506 at pH 3 and 4 respectively The L mesenteroidesstrain Z8 was not viable at pH 2 but marked increasesof 749 and 243were observed at pH 3 and 4 respectivelyThe highest viability was observed at pH 4

35 Bile Salt Tolerance and Bile Salt Hydrolysis The resultsof bile salt tolerance assay are shown in Table 3 The two Lmesenteroides strains were able to grow in the presence of05 1 and 2 oxgall The reduction ranges after a 4 hexposure were 1821ndash2127 and 192ndash1353 for strainsB7 and Z8 respectively The highest resistance was observed


Z8 B3

B5 B7

Z8 B1 B2

B5 B7Z6

Z8 B3

B5B7

(a1)

(a2)

(a3)

Z8 B1 B2

B5 B7Z6

B3

Z8 B1 B2

B5 B7Z6

Z8 B2B1

B3B7B5

(b1)

(b2)

(b3)

Figure 2 The inhibition spectra of Leuconostoc mesenteroides strains against indicator microorganisms B1 B2 B5 B7 and B3 Leuconostocmesenteroides isolated from camel milk sample 1 Z6 and Z8 Leuconostoc mesenteroides isolated from camel milk sample 2 (a1) Inhibitionof Listeria innocua (ATCC 33090) by Leuconostoc mesenteroides using a direct method (a2) Inhibition of Listeria ivanovii (ATCC 19119) byLeuconostocmesenteroides using a directmethod (a3) Inhibition of Staphylococcus aureus by Leuconostocmesenteroides using a directmethod(b1) Antibacterial activity of Leuconostoc mesenteroides versus Listeria innocua (ATCC 33090) using a buffered medium (b2) Antibacterialactivity of Leuconostocmesenteroides versus Listeria ivanovii (ATCC 19119) using a bufferedmedium (b3) Antibacterial activity of Leuconostocversus Listeria ivanovii (ATCC 19119) using a buffered medium treated by chymotrypsin


Table1Ph

ysiologicaltestsandferm

entatio

nprofi

lingof

bioactiveL

euconosto

cstrains

isolated

from

camelmilk

Strains

Catalase

Growth

inthep

resence

ofNaC

lpH

Growth

atFerm

entatio

nprofi

le

365

48684∘C

15∘

C30∘

C37∘

C45∘

CArabino

seMaltose

Rham

nose

Esculin

Manito

lSorbito

lGalactose

LactoseFructose

Glucose

SucroseXy

lose

Z8minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

++

B7minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

+minus


Table 2 The effect of low pH on the viability of Leuconostoc mesenteroides strains

pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h

Ln B7 836 plusmn 029 659 816 820 829 plusmn 014 787Ln Z8 799 0 814 plusmn 0007 875 plusmn 0007 862 plusmn 0007 883 plusmn 0007

All results are expressed as log CFUmL Values in the same row followed by a different letter are significantly different (119875 lt 005)Ln B7 refer to Leuconostoc mesenteroides B7Ln Z8 refer to Leuconostoc mesenteroides Z8

Table 3 The effect of oxgall concentration on the viability of Leuconostoc mesenteroides strains

05 1 20 h 4 h 0 h 4 h 0 h 4 h

Ln B7 845 plusmn 0007 686 plusmn 014 865 plusmn 019 681 plusmn 018 815 plusmn 0007 666Ln Z8 835 plusmn 0056 722 plusmn 016 830 plusmn 0056 792 plusmn 0056 833 plusmn 0007 817 plusmn 023


Table 4 The effect of pepsin on the viability of Leuconostoc mesen-teroides strains

Pepsin (pH2) Pepsin (pH3)0 h 3 h 0 h 3 h

Ln B7 718 0 771 751Ln Z8 881 0 872 plusmn 0071 825All results are expressed as log CFUmL Values in the same row followed bya different letter are significantly different (119875 lt 005)Ln B7 refer to Leuconostoc mesenteroides B7Ln Z8 refer to Leuconostoc mesenteroides Z8

in B7 Neither L mesenteroides strain was able to hydrolysebile salt

36 Resistance to Pepsin Neither strain was able to surviveat pH 2 when 3mgmL of pepsin was added However aremarkable resistance was observed at pH 3 when 3mgmLof pepsin was added Strain B7 decreased by 26 and strainZ8 decreased by 54 The results are shown in Table 4

37 Haemolytic Activity Neither of the Leuconostoc mesen-teroides strainswas able to hydrolyse human blood indicatingthat these strains are nonhaemolytic bacteria

38 Antibiotic Sensitivity Test Thediameters of the inhibitionzones (inmm) of the antibiotic tested against Lmesenteroidesstrains are shown in Table 5 The two strains were resistantto kanamycin streptomycin tetracycline and vancomycinand were sensitive to amoxicillin ampicillin cephalotinchloramphenicol erythromycin lincomycin and penicillinA moderate resistance was observed against gentamycin andneomycin

39 Kinetic Monitoring of pH Evolution and Acidity Theevolution of pH in pure and mixed cultures can be observedin Figures 3 and 4 A significant pH decrease was observedin the mixed cultures after 72 h for the three indicator strainsThus L innocua L ivanovii and S aureus pure cultures were

less acidifying in milk medium as compared to the mixedcultures Accordingly significant pH decreases from 641 plusmn001 to 373 plusmn 024 for L innocua from 627 plusmn 003 to 387 plusmn 0for L ivanovii and from 637plusmn000 to 384plusmn003 for S aureuswere determined

310 The Growth Kinetics of Pathogenic Indicator Strains PureCultures and Cultures Mixed with a Leuconostoc Strain Themaximum growth rate ldquo120583maxrdquo was estimated using themodel described by Baranyi and Roberts [39] Significantreductions of the listerial load after the addition of the B7protective culture were observed as shown by a regression inthe G time The largest bacterial regression was attributed toListeria innocua ATCC 33090 (Figure 5) A lower decrease inthe staphylococcal load was also observed after the additionof strain B7

The maximum growth rate (120583max) of the control Linnocua ATCC 33090 culture was 0243 The 120583max in thepresence of strain B7 was 0148 which is a growth delay of1092min compared to the control (Figure 5)Listeria ivanoviiATCC 19119 exhibited a 120583max of 0219 in pure culture and0168 in mixed culture which is a growth delay of 576minThe 120583max of S aureus ATCC 43300 was 0338 for the controland 0293 in the presence of strain B7 with a growth delay of31min

Following the study of the growth kinetics culture B7showed relatively a slow growth Bioprotective strains pro-moted their own growth to control pathogens by inhibitingpathogenic growth The reductions in the listerial loads wereapproximately 187 and 178 log units for L innocua and Livanovii respectively A reduction of approximately 138 logunits was observed for S aureus which was a smaller reduc-tion than those observed for Listeria strains

4 Discussion

TwoLeuconostoc strains (B7 andZ8) isolated fromcamelmilkwere characterised by their genetic profile probiotic profile


0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10

Leuconostoc mesenteroides B7Listeria innocuaLn mesenteroides B7 L innocua

(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)

Leuconostoc mesenteroides B7Staphylococcus aureusLn mesenteroides B7 S aureus

(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10

Leuconostoc mesenteroides B7Listeria ivanoviiLn mesenteroides B7 L ivanovii

(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)

Ln mesenteroides B7 L innocuaLn mesenteroides B7 L ivanoviiLn mesenteroides B7 S aureus

(d)

Figure 3 Monitoring the pH of pure and mixed cultures of B7 strain and indicator pathogens (a) pH variation of Leuconostoc mesenteroidesB7 and Listeria innocua strains in both pure and mixed cultures (b) pH variation of Leuconostoc mesenteroides B7 and Staphylococcus aureusstrains in both pure andmixed cultures (c) pH variation of Leuconostoc mesenteroides B7 and Listeria ivanovii strains in both pure andmixedcultures (d) pH variation comparison of Leuconostoc mesenteroides B7 Listeria innocua Staphylococcus aureus and Listeria ivanovii strainsin mixed culture

and behaviour against food-borne pathogens in mixed cul-ture The antimicrobial activities exhibited by these strainswere sensitive to proteolytic enzymes but were heat stabletherefore the antimicrobial activitymay be due to heat-stableprotein or peptides

In the present study the genes responsible for the produc-tion of bacteriocin were detected using LcnB primers Inter-estingly L mesenteroides B7 showed the expected molecularweight for a leucocinB suggesting that these strains should beexamined on the genetic and functional levelsThemolecularcharacterisation by the 16s rRNA gene was in agreement withthe phenotypic characterisation The strains exhibited high

similarity among themselves and with sequences from thereference strains in GenBank

Probiotic foods should maintain the viability of theprobiotic bacteria during the preparation and shelf life of theproducts and during the transit through the gastrointestinaltract to exert their beneficial effects [44] Selecting potentialprobiotic strains that can effectively perform in the gastroin-testinal (GI) tract is a significant challenge [45] Thereforewe characterised the probiotic profiles of two strains of Lmesenteroides (B7 and Z8) Acid and bile tolerance weretwo fundamental properties that indicate the ability of thesemicroorganisms to survive through the host GI tract [46]


0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70

Leuconostoc mesenteroides B7Listeria ivanovii

Acid

ity [D

]∘

Ln mesenteroides B7 L ivanovii

(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Leuconostoc mesenteroides B7Staphylococcus aureus

Acid

ity [D

]∘

Ln mesenteroides B7 S aureus

(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)

Leuconostoc mesenteroides B7Listeria innocua

Acid

ity [D

]∘

Ln mesenteroides B7 L innocua

(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)

Figure 4The acidity kinetics of LeuconostocmesenteroidesB7 strain and indicator pathogens in pure andmixed cultures (a) Acidity variationexpressed in dornic degree for Leuconostoc mesenteroides B7 and Listeria ivanovii in both pure and mixed cultures (b) Acidity variationexpressed in dornic degree for Leuconostoc mesenteroides B7 and Staphylococcus aureus in both pure andmixed cultures (c) Acidity variationexpressed in dornic degree for Leuconostoc mesenteroides B7 and Listeria innocua in both pure and mixed cultures (d) Comparison ofacidity variation expressed in dornic degree for Leuconostoc mesenteroides B7 Listeria ivanovii Staphylococcus aureus and Listeria innocuarespectively in mixed culture

Argyri et al [47] found no resistance to low pH for 16 Lmesenteroides strains from a total of 17 One strain was able toresist exposure to pH 25 but the viable counts ofmost strainswere less than 1 logCFUmL after 3 h Our results showed agood viability for the two L mesenteroides when exposed tothe acidic condition of the stomach (pH 3 and 4) The viablecount of strain B7 in pH 2 was 659 log CFUmL but strainZ8 showed no viability

The presence of bile salt in the small intestine is anotherchallenge for probiotic bacteria The two L mesenteroidesstrains survived well in the presence of different concentra-tions of bile salt (05 1 and 2 (wv)) with some loss in via-bility A recent study showed that bile salt affected the growthrate and ability of isolated L mesenteroides subsp mesen-teroides [18] Surono [48] found that L mesenteroides subspmesenteroides IS-27526 had a poor survival rate of 437logCFUmL in the presence of 03 oxgall (wv) Allameh

et al [18] showed the tolerance of L mesenteroides subspmesenteroides after 2 4 and 8 h incubation periods in pres-ence of 00 015 and 03 of bile salt respectivelyThe resultsof this study showed not only viability but also proliferationin all three concentrations for all incubation periods

A probiotic needs to survive conditions such as low pHpepsin and pancreatin activity and bile while adhering toepithelial cells and competitively excluding pathogens [49]In our study L mesenteroides strain B7 was viable at pH 2but neither strain showed viability at pH 2 when 3mgmL ofpepsin was added However both strains were viable at pH 3and 4 with pepsin supplementation A similar study by Seoet al [50] showed that L mesenteroides YML003 exhibited ahigher survival of 1sdot7sdot105 CFUmL after exposure to artificialgastric juices with an initial cell number of 2sdot5sdot108 CFUmL

The absence of haemolytic activity and antibiotic resis-tance are considered safety prerequisites for the selection of


0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11

Leuconostoc mesenteroides B7

Ln mesenteroides B7 L innocuaListeria innocua

Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9

Time (hours)Ln mesenteroides B7 L innocuaLn mesenteroides B7 L ivanoviiLn mesenteroides B7 S aureus

Log N

(d)

Figure 5 The growth kinetics of Leuconostoc mesenteroides B7 strain and indicator pathogens in pure and mixed cultures (a) Graphicalrepresentation of the growth kinetics expressed in log119873 of Leuconostoc mesenteroides B7 and Listeria ivanovii in pure andmixed cultures (b)Graphical representation of the growth kinetics expressed in log 119873 of Leuconostoc mesenteroides B7 and Staphylococcus aureus in pure andmixed cultures (c) Graphical representation of the growth kinetics expressed in log119873 of Leuconostoc mesenteroides B7 and Listeria innocuain pure and mixed cultures (d) Graphical representation of the growth kinetics expressed in log119873 of Leuconostoc mesenteroides B7 Listeriaivanovii Staphylococcus aureus and Listeria innocua respectively in mixed culture

a probiotic strain [51] No zones were detected around thecolonies of the two L mesenteroides strains when grown inColumbia human blood agar suggesting that there was no 120574-haemolytic activity in vitro The lack of 120574-haemolytic activityis a desirable trait in probiotic bacteria Several authors haveshown similar results [18ndash47]

In addition the two strains were resistant to kanamycinstreptomycin tetracycline and vancomycin but were sensi-tive to amoxicillin ampicillin cephalotin chloramphenicolerythromycin lincomycin and penicillin Moderate resis-tance was observed against gentamycin and neomycin Our

results agree with those obtained in previous studies theobserved sensitivity to ampicillin cephalotin erythromycinlincomycin and penicillin and resistance to vancomycin aresimilar to results obtained by Zarour et al [52] and sensitivityto chloramphenicol and ampicillin was also observed byAllameh et al [18] All studies showed resistance to van-comycin in L mesenteroides strains Vancomycin resistanceis a general intrinsic feature that is linked to the presence ofa pentadepsipeptide with a C terminal-lactate instead of a d-alanine in the peptidoglycan [53] Few reports are availableon other antibiotics


Table 5 Antibiotic susceptibility of Leuconostoc mesenteroides strains

Antibiotics Symbol 120583gdisc Clear zone diameter (mm)Z B7 Z8

Amoxicillin AMX 25 25 S 25 SAmpicillin AM 10 23 S 24 SCephalotin CEF 30 23 S 24 SChloramphenicol C 30 28 S 27 SErythromycin E 15 27 S 28 SGentamycin GM 10 18 I 18 IKanamycin K 30 12 R 13 RLincomycin L 15 25 S 25 SNeomycin N 30 16 I 16 IPenicillin P 6 23 S 24 SStreptomycin S 10 14 R 15 RTetracycline TE 30 14 R 14 RVancomycin VA 30 00 R 00 RR resistance I intermediate and S sensitive

Table 6 Statistical analysis of the growth kinetics (variance analysis)

Variance analysisL ivanovii L innocua St aureus L ivanovii L innocua St aureus

Test number 119875 2 2 2 Repeatability variance 1205902119903

0091 0144 0161Result number119873 20 20 20 cv repeatability 120 217 117Standard deviation 0098 0049 0091 Reproducibility variance 1205902

119877

022 0441 2673Variance 0009 0002 0008 cv reproducibility 342 754 1352

Mean (Log119873)800 8065 822SD Var and mean at 9 h of incubation

Our in vitro studies demonstrated that the two L mesen-teroides strains had good probiotic profiles These strainsexhibited high viability at low pH levels both in the presenceof 2 of bile salt and in the presence of pepsin These strainshave acceptable susceptibility antibiotic profiles and arenonhaemolytic bacteria L mesenteroides strains B7 and Z8could be ideal probiotic candidates

These interactions can be the stimulation of one or moremicroorganism or may correspond to the inhibition ofgrowth or metabolic activity Inhibition may occur throughthe production of inhibitory substances or when one of thetwomicroorganisms is inhibited by another Inhibition couldalso be induced by reciprocal competition [54 55]Thereforeto study the behaviour of these two strains against food-bornepathogens a kinetic profile of these strains was measured inboth pure and mixed cultures

Monitoring the pH and acidity showed a significant vari-ability between pure and mixed culture a result that can beexplained by the production of organic acids (lactic and aceticacids) Therefore we can conclude that the incubation timepositively influenced the performance of the L mesenteroidesstrains Consequently we can see that the amount of acidproduced varies depending on the life stage of the bacterium

The growth curve analysis in the mixed cultures showeda significant reduction of pathogen bacteria growth after 9 hof incubation during the late exponential phase of growthwhich can explain the inhibition of the B7 strain towards Lis-teria and Staphylococcus through the production of inhibitorysubstances such as bacteriocins Similar results were reportedby Lacroix and Millette [56] The antimicrobial activity ofbacteriocin-producing LAB against pathogens was explainedby the production of bacteriocins in broth cultures whichwasestimated to be maximal after 9 hours of incubation wherethe maximum number of bacteria had been attained in theearly stationary phase of growth

Study of the antimicrobial activity against L ivanoviiATCC 19119 showed a small variation of repeatability (givinga lower limit of the variability of results) which indicates aninternal dispersion close to the results in homogeneous coef-ficients of repeatability (154 171 and 176) (Table 6)Conversely the pathogen strains showed variability in theirgrowth rates meaning that the observed potential showedan inhomogeneous distribution indicated by a coefficient ofreproducibility of 348 (Table 6) The estimation of thisactivity against S aureus ATCC 43300 displayed a minorinsignificant variability in the coefficients of repeatability


(160 265 and 082) (Table 6) which is explained by alower antistaphylococcal capacity than the antilisterial capac-ity of L mesenteroides B7

5 Conclusion

In conclusion the results of this study showed that the twostrains of Leuconostoc mesenteroides (B7 and Z8) were foundto possess good probiotic properties in vitro Moreoverthe kinetic studies showed that these two strains especiallyB7 can be used as protective cultures to inhibit pathogenicbacteria growth in food Therefore these strains are goodcandidates for further investigation with in vivo studies toelucidate their potential health benefits and in fermentationstudies to assess their technological characteristics for appli-cations as novel probiotic starters

Acknowledgment

This work was funded by Project A1033813311 funded by theSpanish Agency for International Cooperation and Develop-ment

References

[1] E K Barbour N H Nabbut W M Frerisch and H M Al-Nakhli ldquoInhibition of pathogenic bacteria by camelrsquos milk rela-tion to whey lysozyme and stage of lactationrdquo Journal of FoodProtection vol 47 no 11 pp 838ndash840 1984

[2] R Yagil O Zagorski and C Van Creveld ldquoScience and camelrsquosmilk productionrdquo in Actes du Colloque Dromadaires et cha-meaux animaux laitier Nouakchott Mauritanie Octobre 1994

[3] M Khaskheli M A Arain S Chaudhary A H Soomro and TA Qureshi ldquoPhysico-chemical quality of camel milkrdquo Journalof Agriculture Social Science vol 1 no 2 pp 164ndash166 2005

[4] B Guessas M Hadadji N Saidi and M Kihal ldquoInhibition ofStaphylococcus aureus growth in milk by lactic acid bacteriardquoDirassat vol 32 no 5 pp 53ndash60 2005

[5] H Labioui L Elmoualdi M El yachioui and M OuhssineldquoSelection de souches de bacteries Lactiques antibacteriennesrdquoBulletin de la Societe de pharmacie de Bordeaux vol 144 no 3-4pp 237ndash250 2005

[6] AMezaini N E Chihib A Dilmi Bouras N Nedjar-Arroumeand J PHornez ldquoAntibacterial activity of some lactic acid bacte-ria isolated from an algerian dairy productrdquo Journal of Environ-mental and Public Health vol 2009 Article ID 678495 6 pages2009

[7] N Benkerroum A Boughdadi N Bennani and K HidaneldquoMicrobiological quality assessment of Moroccan camelrsquos milkand identification of predominating lactic acid bacteriardquoWorldJournal of Microbiology and Biotechnology vol 19 no 6 pp645ndash648 2003

[8] O Hassaıne H Zadi-Karam and N E Karam ldquoTechnologi-cally important properties of lactic acid bacteria isolated fromraw milk of three breeds of Algerian dromedary (Camelusdromedarius)rdquo African Journal of Biotechnology vol 6 no 14pp 1720ndash1727 2007

[9] K Khedid M Faid A Mokhtari A Soulaymani and A Zine-dine ldquoCharacterization of lactic acid bacteria isolated from the

one humped camel milk produced inMoroccordquoMicrobiologicalResearch vol 164 no 1 pp 81ndash91 2009

[10] E I el Agamy R Ruppanner A Ismail C P Champagne andR Assaf ldquoAntibacterial and antiviral activity of camel milk pro-tective proteinsrdquo Journal of Dairy Research vol 59 no 2 pp169ndash175 1992

[11] N Benkerroum M Mekkaoui N Bennani and K HidaneldquoAntimicrobial activity of camelrsquos milk against pathogenicstrains of Escherichia coli and Listeria monocytogenesrdquo Inter-national Journal of Dairy Technology vol 57 no 1 pp 39ndash432004

[12] E O Khay M Idaomar L M P Castro P F Bernardez N SSenhaji and J Abrini ldquoAntimicrobial activities of the bacterioc-in-like substances produced by lactic acid bacteria isolated fromMoroccan dromedary milkrdquo African Journal of Biotechnologyvol 10 no 51 pp 10447ndash10455 2011

[13] M A Daeschel ldquoAntibacterial substances from lactic acid bac-teria for use as food preservativesrdquo Food Technology vol 43 pp164ndash167 1989

[14] T Abee L Krockel and C Hill ldquoBacteriocins modes of actionand potentials in food preservation and control of food poison-ingrdquo International Journal of Food Microbiology vol 28 no 2pp 169ndash185 1995

[15] J Hugenholtz and M Kleerebezem ldquoMetabolic engineeringof lactic acid bacteria overview of the approaches and resultsof pathway rerouting involved in food fermentationsrdquo CurrentOpinion in Biotechnology vol 10 no 5 pp 492ndash497 1999

[16] A Badis D Guetarni B Moussa Boudjema D E Henni andM Kihal ldquoIdentification and technological properties of lacticacid bacteria isolated from raw goatmilk of fourAlgerian racesrdquoFood Microbiology vol 21 no 5 pp 579ndash588 2004

[17] H Drici C Gilbert M Kihal and D Atlan ldquoAtypical citrate-fermenting Lactococcus lactis strains isolated from dromedaryrsquosmilkrdquo Journal of Applied Microbiology vol 108 no 2 pp 647ndash657 2010

[18] S K Allameh H Daud F M Yusoff C R Saad and A IderisldquoIsolation identification and characterization of Leuconostocmesenteroides as a new probiotic from intestine of snakeheadfish (Channa Striatus)rdquo African Journal of Biotechnology vol 11no 16 pp 3810ndash3816 2012

[19] E Ringoslash and F J Gatesoupe ldquoLactic acid bacteria in fish areviewrdquo Aquaculture vol 160 no 3-4 pp 177ndash203 1998

[20] S Torriani G Zapparoli and F Dellaglio ldquoUse of PCR-basedmethods for rapid differentiation of Lactobacillus delbrueckiisubsp bulgaricus and L delbrueckii subsp lactisrdquo Applied andEnvironmentalMicrobiology vol 65 no 10 pp 4351ndash4356 1999

[21] P Bellengier J Richard and C Foucaud ldquoNutritional require-ments of Leuconostoc mesenteroides subsp mesenteroides andsubsp dextranicum for growth in milkrdquo Journal of DairyResearch vol 64 no 1 pp 95ndash103 1997

[22] S Rodgers ldquoNovel applications of live bacteria in food servicesprobiotics and protective culturesrdquo Trends in Food Science andTechnology vol 19 no 4 pp 188ndash197 2008

[23] S V Hosseini S Arlindo K Bohme C Fernandez-No P Calo-Mata and J Barros-Velazquez ldquoMolecular and probiotic char-acterization of bacteriocin-producing Enterococcus faeciumstrains isolated from nonfermented animal foodsrdquo Journal ofApplied Microbiology vol 107 no 4 pp 1392ndash1403 2009

[24] R A Kekkonen E Kajasto M Miettinen V Veckman R Kor-pela and I Julkunen ldquoProbiotic Leuconostoc mesenteroidesssp cremoris and Streptococcus thermophilus induce IL-12 and


IFN-120574 productionrdquo World Journal of Gastroenterology vol 14no 8 pp 1192ndash1203 2008

[25] S Supannikar and T Sudsai ldquoPotential use of leuconostoc lactisisolated from fermented broken rice as a single starter culturefor small-scale production of thai fermented rice noodlerdquoin Proceedings of the 21st International ICFMH SymposiumEvolving Microbial Food Quality and Safety IUMSICFMHAberdeen Scotland September 2008

[26] B B Budde T Hornbaeligk T Jacobsen V Barkholt and A GKoch ldquoLeuconostoc carnosum 4010 has the potential for use asa protective culture for vacuum-packedmeats culture isolationbacteriocin identification and meat application experimentsrdquoInternational Journal of FoodMicrobiology vol 83 no 2 pp 171ndash184 2003

[27] E H Drosinos M Mataragas N Xiraphi G Moschonas FGaitis and J Metaxopoulos ldquoCharacterization of the microbialflora from a traditional Greek fermented sausagerdquoMeat Sciencevol 69 no 2 pp 307ndash317 2005

[28] J V Mayeux W W E Sandine and P R Elliker ldquoA selectivemedium for detecting Leuconostoc organisms in mixed strainstarter culturesrdquo Journal of Dairy Science vol 45 pp 655ndash6561962

[29] D A Relman T M Schmidt R P MacDermott and S FalkowldquoIdentification of the uncultured bacillus of Whipplersquos diseaserdquoNew England Journal of Medicine vol 327 no 5 pp 293ndash3011992

[30] K Bohme I C Fernandez-No J M Gallardo B Canas and PCalo-Mata ldquoSafety assessment of fresh and processed seafoodproducts by MALDI-TOF mass fingerprintingrdquo Food and Bio-process Technology vol 4 no 6 pp 907ndash918 2011

[31] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[32] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[33] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[34] N Xiraphi M Georgalaki K Rantsiou L Cocolin E Tsakali-dou and E H Drosinos ldquoPurification and characterization of abacteriocin produced by Leuconostoc mesenteroides E131rdquoMeatScience vol 80 no 2 pp 194ndash203 2008

[35] H P Fleming J L Etchells and R N Costilow ldquoMicrobial inhi-bition by an isolate of Pediococcus from cucumber brinesrdquoJournal of Applied Microbiology vol 30 no 6 pp 1040ndash10421975

[36] S F Barefoot and T R Klaenhammer ldquoDetection and activityof lactacin B a bacteriocin produced by Lactobacillus aci-dophilusrdquo Applied and Environmental Microbiology vol 45 no6 pp 1808ndash1815 1983

[37] S A Liasi T I Azmi M D Hassan M Shuhaimi M Ros-farizan and A B Ariff ldquoAntimicrobial activity and anti-bioticsensitivity of three isolates of lactic acid bacteria from fer-mented fish productrdquoMalaysian Journal of Microbiology vol 5no 1 pp 33ndash37 2009

[38] M Kihal H Prevost D E Henni Z Benmechernene andC Divies ldquoCarbon dioxide production by leuconostoc mesen-teroıdes grown in single and mixed culture with Lactococcus

lactis in skim milkrdquo Scientific Research and Essays vol 4 no11 pp 1348ndash1353 2009

[39] J Baranyi and T A Roberts ldquoA dynamic approach to predictingbacterial growth in foodrdquo International Journal of Food Micro-biology vol 23 no 3-4 pp 277ndash294 1994

[40] P Dalgaard ldquoModelling of microbial activity and prediction ofshelf life for packed fresh fishrdquo International Journal of FoodMicrobiology vol 26 no 3 pp 305ndash317 1995

[41] M Lachance Purification et caracterisation drsquoune bacteriocineproduite par Lactococcus lactis ssp lactis mjc15 Memoire maıtredes sciences [MS thesis] Universite Laval 2000

[42] S Arlindo P Calo C Franco M Prado A Cepeda and JBarros-Velazquez ldquoSingle nucleotide polymorphism analysis ofthe enterocin P structural gene of Enterococcus faecium strainsisolated from nonfermented animal foodsrdquoMolecular Nutritionand Food Research vol 50 no 12 pp 1229ndash1238 2006

[43] C A Campos O Rodrıguez P Calo-Mata M Prado and JBarros-Velazquez ldquoPreliminary characterization of bacteri-ocins from Lactococcus lactis Enterococcus faecium and Ente-rococcus mundtii strains isolated from turbot (Psetta maxima)rdquoFood Research International vol 39 no 3 pp 356ndash364 2006

[44] I Sumeri L Arike K Adamberg and T Paalme ldquoSingle biore-actor gastrointestinal tract simulator for study of survival ofprobiotic bacteriardquoAppliedMicrobiology and Biotechnology vol80 no 2 pp 317ndash324 2008

[45] P Prommadee GWunwiboon L Kees andN Sunee ldquoCharac-terization of Lactobacillus johnsoniiKUNN19-2 and PediococcuspentosaceusKUNNE6-1 isolated from thai-style fermented pork(Nham) for their probiotic properties in the gastrointestinaltract and immunomodulationrdquo Kasetsart Journal Natural Sci-ence vol 46 no 3 pp 440ndash450 2012

[46] S Erkkila and E Petaja ldquoScreening of commercial meat startercultures at low pH and in the presence of bile salts for potentialprobiotic userdquoMeat Science vol 55 no 3 pp 297ndash300 2000

[47] A A Argyri G Zoumpopoulou G Kimon-Andreas et alldquoSelection of potential probiotic lactic acid bacteria fromfermented olives by in vitro testsrdquo Food Microbiology vol 33no 1 pp 282ndash291 2013

[48] I S Surono ldquoIn vitro probiotic properties of indigenousdadih lactic acid bacteriardquoAsian-Australasian Journal of AnimalSciences vol 16 no 5 pp 726ndash731 2003

[49] P A Maragkoudakis G Zoumpopoulou C Miaris G Kalant-zopoulos B Pot and E Tsakalidou ldquoProbiotic potential of Lac-tobacillus strains isolated from dairy productsrdquo InternationalDairy Journal vol 16 no 3 pp 189ndash199 2006

[50] B J Seo I A Rather V J R Kumar et al ldquoEvaluation of Leu-conostoc mesenteroidesYML003 as a probiotic against low-pathogenic avian influenza (H9N2) virus in chickensrdquo Journalof Applied Microbiology vol 113 no 1 pp 163ndash171 2012

[51] FAOWHO Joint FAOWHOWorkingGroupReport onDraftingGuidelines for the Evaluation of Probiotics in Food FAOWHOLondon Canada 2002

[52] K Zarour Z Benmechernene M Hadadji B Moussa-Boud-jemaa J E Henni and M Kihal ldquoCaracterisation microbi-ologique et technologique des especes de Leuconostoc mesen-teroıdes isolees du lait cru de chevre et de chamelle drsquoAlgerierdquoNature amp Technologie Revue vol 8 pp 39ndash47 2012

[53] D Hemme and C Foucaud-Scheunemann ldquoLeuconostoc char-acteristics use in dairy technology and prospects in functionalfoodsrdquo International Dairy Journal vol 14 no 6 pp 467ndash4942004


[54] O Cholet Etude de lrsquoecosysteme fromager par une approchebiochimique et moleculaire [PhD thesis] Institut NationalAgronomique Paris-Grignon Ecole Doctorale ABIES UMR deGenie et Microbiologie des Procedes Alimentaires 2006

[55] V Monnet E Latrille C Beal and G Corrieu ldquoCroissance etproprieties fonctionnelles des bacteries lactiquesrdquo in Bacterieslactiques de la genetique aux ferments G Corrieu and F MLuquet Eds pp 512ndash592 Tec amp Doc Paris 2008

[56] M Lacroix and M Millette ldquoAntimicrobial activity of bacteri-ocin-producing lactic acid bacteriardquo US 20110236359 A1 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


LAB exhibit probiotic properties because these bacteriaare normal flora in gastrointestinal tract [18] and have noharmful effects [19] Moreover the addition of antagonis-tic bacterial preparations as protective cultures is anotherapproach for biopreservation [20] In addition possiblemicrobial interactions either beneficial (cooperation) orunfavourable (inhibition) can be achieved in mixed straincultures These mixed cultures are commonly used as startercultures in dairy manufacturing because of the acid produc-tion growth rate proteolytic activity bacteriocin productionand sensitivity aroma production and phage sensitivityof these cultures [21] However these technologies can belimiting for commercial application because of the negativeimpacts or the low reproducibility percentage of the response[22] For these reasons the characterisation of LAB as pro-biotic cultures should respond to several criteria such as theability to survive the specific conditions of the gastrointestinaltract including low pH proteolytic enzymes and bile saltconcentrations [23]

Because these bacteria are meant to be used as protectivecultures the inhibition pattern safety and functional andtechnological properties of these bacteria should also beexamined

Few studies have reported the high potential of usingLeuconostoc as a probiotic strain Kekkonen et al [24] showedthat the use of Leuconostoc as probiotic strain in inducingcytokines was better than that of the probiotic Lactobacillusstrain that is presently in clinical use In addition Allamehet al [18] reported for the first time the use of Leuconostocmesenteroides isolated from the intestine of snakehead fishas a potentially new probiotic for aquaculture systems forthe development of fish production Moreover Leuconostoclactis has been shown to have a potential for use as a singlestarter culture in Khanoon-jeen production and could reducethe microbial risk and fermentation processing time [25]Additionally Leuconostoc strains have potential as protectivecultures for vacuum packed meat products [26] and thebacteriocins of Leuconostoc can be used as protective agentsin combination with another starter culture in fermentedmeat [27] However the use of Leuconostoc mesenteroidesisolated from camel milk as either a probiotic strain or a pro-tective culture has never been reportedTherefore we isolatedand identified Leuconostoc strains from camel milk as newprobiotic or protective cultures for the first time

2 Materials and Methods

21 Raw Camel Milk Sampling Thirteen Leuconostoc strainswere isolated from four different camel milk samples whichwere collected from two different Saharan areas (Naama andBechar) in SouthWestern AlgeriaThe first two samples werecollected from two camels (Camelus dromados) in Naamathat differed in age (10 and 15 years old) and colour (greyand black) but had the same lactation period (March 2011)The diet of the camels was based on natural Saharan plantssuch as Drinn (Arstide pungens) Samples from Bechar werecollected from brown camels aged less than 10 years in thearid Abadla region in the period 2009ndash2011 Sampling wasperformed under aseptic conditions bywashing the teats with

warm water containing 2 bleach and collecting the milkin sterile bottles after hand washing with diluted alcoholSamples were maintained at 4∘C and were transported iniceboxes to the laboratory for analysis

22 Leuconostoc Isolation The bioactive Leuconostoc consid-ered in this work were isolated from the raw camel milkas described above All strains were stored at minus80∘C andwere maintained in reconstituted skimmed milk containing30 (wv) glycerol All strains were cultured in MRS broth(Liofilchem Teramo Italy) at 30∘C for 24 h and were thenseeded onto MRS agar (Liofilchem) to obtain single coloniesThe wild type and reference Leuconostoc strains used in thisstudy belonged to the collection of our laboratory

Based on the results obtained from the inhibitory assaystwo bioactive leuconostocs strains (B7 and Z8) were selectedand subjected to phenotypic and morphological character-isation based on the following criteria CO

2production

growth at different temperatures (4∘C 15∘C 30∘C 37∘C and45∘C) growth at different pH (48 and 68) and growth atdifferent NaCl concentrations (3 and 65) Additionallyall strains were subjected to the following biochemical teststo differentiate between Leuconostoc and lactobacilli dextranproduction on MSE medium [28] arginine hydrolysis onM16BCP medium (Oxoid Ltd London UK) and citric aciddegradation on Kempler and McKay solid medium Carbo-hydrate fermentation was tested on MRS supplemented withbromocresol purple as a pH indicator using the followingsugars to differentiate the subspecies of Leuconostocs arabi-nosemaltose rhamnose esculinmanitol sorbitol galactoselactose fructose glucose sucrose and xylose All strainswerephenotypically identified as belonging to the Leuconostocgenus based on the following criteria ovoid shape Gram-positivity catalase negativity vancomycin-resistance pro-duction of gas from glucose lack of arginine hydrolysis andfermentation profiles

23 Genetic Identification of Bioactive Leuconostoc Strains Afragment of the 16S rRNA gene of the two bioactive strainswas amplified by PCR using the universal primer pair p8FPL(forward 51015840-AGTTTGATCCTGGCTCAG-31015840) and p806R(reverse 51015840-GGACTACCAGGGTATCTAAT-31015840) [29] Theassays comprised 100 ng of template DNA 25 120583L of a mas-ter mix (BioMix Bioline London UK) (this included thereaction buffer dNTPs and magnesium chloride) Taq DNApolymerase 25 pmol of each oligonucleotide primer anddouble-distilled water to achieve a final volume of 50 120583LAmplification conditions were as follows denaturing at 94∘Cfor 7min 35 cycles of denaturation (94∘C for 60 sec)annealing (55∘C for 60 sec) extension (72∘C for 60 sec) anda final extension at 72∘C for 15min The PCR was performedas described by Bohme et al [30]

The two PCR products were then sequenced using thesame primers used for PCR The sequences were analysedwith Chromas software (Griffith University QueenslandAustralia) and aligned using Clustal-X software [31 32]Following alignment these sequences were identified bysearching for sequence homology among published referencesequences using the web BLAST tool (National Center for






















3 Results





M 1817161514131211109876543211000900800700600500400300200

100








Z8 B3

B5 B7

Z8 B1 B2

B5 B7Z6

Z8 B3

B5B7

(a1)

(a2)

(a3)

Z8 B1 B2

B5 B7Z6

B3

Z8 B1 B2

B5 B7Z6

Z8 B2B1

B3B7B5

(b1)

(b2)

(b3)



Table1Ph


entatio

nprofi

lingof

bioactiveL

euconosto

cstrains

isolated

from

camelmilk

Strains

Catalase

Growth

inthep

resence

ofNaC

lpH

Growth

atFerm

entatio

nprofi

le

365

48684∘C

15∘

C30∘

C37∘

C45∘

CArabino

seMaltose

Rham

nose

Esculin

Manito

lSorbito

lGalactose

LactoseFructose

Glucose

SucroseXy

lose

Z8minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

++

B7minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

+minus



pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h




05 1 20 h 4 h 0 h 4 h 0 h 4 h















4 Discussion



0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















3 Results





M 1817161514131211109876543211000900800700600500400300200

100








Z8 B3

B5 B7

Z8 B1 B2

B5 B7Z6

Z8 B3

B5B7

(a1)

(a2)

(a3)

Z8 B1 B2

B5 B7Z6

B3

Z8 B1 B2

B5 B7Z6

Z8 B2B1

B3B7B5

(b1)

(b2)

(b3)



Table1Ph


entatio

nprofi

lingof

bioactiveL

euconosto

cstrains

isolated

from

camelmilk

Strains

Catalase

Growth

inthep

resence

ofNaC

lpH

Growth

atFerm

entatio

nprofi

le

365

48684∘C

15∘

C30∘

C37∘

C45∘

CArabino

seMaltose

Rham

nose

Esculin

Manito

lSorbito

lGalactose

LactoseFructose

Glucose

SucroseXy

lose

Z8minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

++

B7minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

+minus



pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h




05 1 20 h 4 h 0 h 4 h 0 h 4 h















4 Discussion



0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Results





M 1817161514131211109876543211000900800700600500400300200

100








Z8 B3

B5 B7

Z8 B1 B2

B5 B7Z6

Z8 B3

B5B7

(a1)

(a2)

(a3)

Z8 B1 B2

B5 B7Z6

B3

Z8 B1 B2

B5 B7Z6

Z8 B2B1

B3B7B5

(b1)

(b2)

(b3)



Table1Ph


entatio

nprofi

lingof

bioactiveL

euconosto

cstrains

isolated

from

camelmilk

Strains

Catalase

Growth

inthep

resence

ofNaC

lpH

Growth

atFerm

entatio

nprofi

le

365

48684∘C

15∘

C30∘

C37∘

C45∘

CArabino

seMaltose

Rham

nose

Esculin

Manito

lSorbito

lGalactose

LactoseFructose

Glucose

SucroseXy

lose

Z8minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

++

B7minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

+minus



pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h




05 1 20 h 4 h 0 h 4 h 0 h 4 h















4 Discussion



0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Z8 B3

B5 B7

Z8 B1 B2

B5 B7Z6

Z8 B3

B5B7

(a1)

(a2)

(a3)

Z8 B1 B2

B5 B7Z6

B3

Z8 B1 B2

B5 B7Z6

Z8 B2B1

B3B7B5

(b1)

(b2)

(b3)



Table1Ph


entatio

nprofi

lingof

bioactiveL

euconosto

cstrains

isolated

from

camelmilk

Strains

Catalase

Growth

inthep

resence

ofNaC

lpH

Growth

atFerm

entatio

nprofi

le

365

48684∘C

15∘

C30∘

C37∘

C45∘

CArabino

seMaltose

Rham

nose

Esculin

Manito

lSorbito

lGalactose

LactoseFructose

Glucose

SucroseXy

lose

Z8minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

++

B7minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

+minus



pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h




05 1 20 h 4 h 0 h 4 h 0 h 4 h















4 Discussion



0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Ph


entatio

nprofi

lingof

bioactiveL

euconosto

cstrains

isolated

from

camelmilk

Strains

Catalase

Growth

inthep

resence

ofNaC

lpH

Growth

atFerm

entatio

nprofi

le

365

48684∘C

15∘

C30∘

C37∘

C45∘

CArabino

seMaltose

Rham

nose

Esculin

Manito

lSorbito

lGalactose

LactoseFructose

Glucose

SucroseXy

lose

Z8minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

++

B7minus

+minusminus

+minus

++

+minus

minus+

minusminus

minusminus

++

++

+minus



pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h




05 1 20 h 4 h 0 h 4 h 0 h 4 h















4 Discussion



0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



pH2 pH3 pH40 h 3 h 0 h 3 h 0 h 3 h




05 1 20 h 4 h 0 h 4 h 0 h 4 h















4 Discussion



0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 10 20 30 40 50 60 70 80

35

40

45

50

55

60

65pH

Time (hours)minus10


(a)

0 10 20 30 40 50 60 70 8035

40

45

50

55

60

65

pH

Time (hours)


(b)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70

pH

Time (hours)minus10


(c)

0 10 20 30 40 50 60 70 8030

35

40

45

50

55

60

65

70pH

Time (hours)


(d)







0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 10 20 60 800

10

20

30

40

50

60

70

80

50Time (hours)

70


Acid

ity [D

]∘


(a)

0 10 20 50 60 70 800

1020304050607080

Time (hours)


Acid

ity [D

]∘


(b)

0 10 20 50 60 70 80

100

20304050607080

Time (hours)


Acid

ity [D

]∘


(c)

0 10 20 50 60 70 800

1020304050607080

Time (hours)

Acid

ity [D

]∘


(d)








0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(a)

0 10 20 30 40 50 60 70 80

6

7

8

9

10

11

Time (hours)


Log N

(b)

0 10 20 30 40 50 60 70 80Time (hours)

7

8

9

10

11



Log N

(c)

0 10 20 30 40 50 60 70 806

7

8

9


Log N

(d)













119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









119877










5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusion


Acknowledgment


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Technological Aptitude and Applications ...downloads.hindawi.com/journals/bmri/2013/418132.pdf · Technological Aptitude and Applications of Leuconostoc mesenteroides