Production of Bacteriocin from isolates Natural Lactic acid
Production, Purification, Stability and Efficacy of
Bacteriocin from Isolates of Natural Lactic Acid
Fermentation of Vegetables
By
Uma B. ShindeMITCON Biopharma, Pune.
Under the Guidance of
Miss Priya Bhande
MITCON Biopharma, Pune.
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Production of Bacteriocin from isolates Natural Lactic acid
CERTIFICATE
This is to certify that Miss. Uma Bhagwat Shinde student of Industrial
Biotechnology has successfully completed her project work entitled
“Production, Purification, Stability and Efficacy of Bacteriocin from Isolates of Natural Lactic Acid Fermentation of Vegetables”
during 19Dec 2011 to 19 feb 2012 at MITCON biopharma centre, Pune
Miss Priya Bhande
(project guide)
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Production of Bacteriocin from isolates Natural Lactic acid
ACKNOWLEDGEMENT
It gives me immense pleasure to express my deep sincere gratitude to my guide Miss. Priya Bhande for her suggestions, guidance, encouragement and support throughout the period of project.
I am grateful to our head of department Mr. Kulkarni sir, for giving me opportunity to join this esteemed institute and extending all laboratory facilities.
I am grateful to Miss Angha and Miss Neha for the positive support in my whole project work and the guidance for handling the instruments and in technical work and for all help, understanding me and my problems, supporting me in all possible way throughout my work.
I am thankful to our lab assistants Mr. Amit and Mr. Sandeep for the co-operation and providing all necessary things in all possible way.
I wish to extend my thanks to my family, especially my parents, my elder brother and my elder sister without whose love and support I could not be here.
Last but not least my final thanks to my all lovely room mates my all friends in MITCON, and specially my friend Aditi Ambekar. They understood mi and tried to help mi in all possible way they could. Thank you very much for being by my side, believing in me and putting up with me in all my good and bad times.
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Production of Bacteriocin from isolates Natural Lactic acid
Student Introduction
Name of the Student: Shinde Uma Bhagwat
Registration No.: BTN -33/2008
Name of the Training Program: Hands on Training
Name of the Training Organization: MITCON Institute
Duration of the Training: Dec to Feb
Name of the College: K. K. Wagh College of Agricultural Biotechnology, Nashik.
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Production of Bacteriocin from isolates Natural Lactic acid
Index
Sr No. Title Page No
1 Introduction & objectives 9 to 17
2 Review of the Literature 18 to 25
3 Aim and Scope of the
Work
26
4 Material and Methods 27 to 37
5 Result and Discussion 38 to 61
6 summary 62
7 Reference 63
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Production of Bacteriocin from isolates Natural Lactic acid
List of Tables
Sr. no Table no Page no
1 Media composition 29
2 Composition of crystal
voilet
30
3 Composition of gram
iodine
30
4 Composition of saffrain 31
5 Composition of Nutrient
agar
32
6 Preparation of acetone 33
7 Preparation of sodium
phosphate buffer
34
8 Inhibition zone of
antimicrobial activity
40
9 Observation of
biochemical test on
bacteriocin
48
10 Observation of sugar test
on bacteriocin
49
11 Zone of inhibition of
crude bacteriocin and
precipitated
52 to 53
12 Zone of inhibition of heat
stability
55
13 Zone of inhibition of
effect of pH
57
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Production of Bacteriocin from isolates Natural Lactic acid
List of Figures
Sr.no Figures Page no
1 Tomato,cucumber
inoculated Minimal media
37
2 Garlic inoculated Minimal
media
38
3 Growth of bacteriocin
(tomato) on MRS media
39
4 Growth of bacteriocin
(garlic ) on MRS media
40
5 Growth of bacteriocin
(cucumber) on MRS
media
41
6 Pure culture of cucumber 41
7 Pure culture of garlic and
tomato
42
8 Gram staining of
bacteriocin(cucumber)
43
9 Gram staining of
bacteriocin(tomato)
44
10 Inhibition zone of
antimicrobial activity
46
11 Inhibition zone of
antimicrobial activity
47
12 Citrate test utilization 49
13 Urease test 50
14 Indole test 50
15 Methyl red test 51
16 Zone of inhibition of 53
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Production of Bacteriocin from isolates Natural Lactic acid
C.B& precipitated
17 Zone of inhibition of heat
stability
55
18 Zone of inhibition of heat
stability
56
19 Zone of inhibition of pH 59
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Production of Bacteriocin from isolates Natural Lactic acid
List of Abbreviations
1 G.S Gram staining
2 GPB Gram phosphate broth
3 E.coli Escherichia coil
4 MRT Methly red test
5 MRS De Mann Rogose Sharpe
6 LAB Lactic acid bacteria
7 Nacl Sodium chloride
8 TSS Triple sugar test
9 N.A. Nutrient agar
10 Y.E Yeast extract
11 PBS Phosphate buffered saline
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Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 1
Introduction
Natural fermentation is one of the oldest means of preservation. Lactic acid bacteria
(LAB) subject the vegetables to a fermentation process. The vegetables when
fermented, it develops a pleasant sour taste and it is rich in vitamin and minerals.
Fermented vegetables are important for healthy nutrition.
In spite of the introduction of modern technologies and safety concepts, the
reported number of food borne illnesses and intoxications are on the rise. Many of
the ready-to-eat and novel food products represent new food systems with respect to
health and spoilage risks.
In the production of food, it is crucial to take proper measures for ensuring
its safety and stability during the shelf life. Food preservation is carried out to
maintain the quality of raw material and physicochemical properties as well as
functional quality of the product. Whilst providing safe and stable products.
Preservation of vegetables by lactic acid fermentation is an ancient practice
involving lactic acid bacteria (LAB), which predominantly product lactic acid
besides certain compound such as bacteriocin, which has antimicrobial activity
against other group of microorganisms. The antimicrobial activity of bacteriocin
produced by LAB has been detected in foods such as dairy products, meat, barley,
sourdough, fermented vegetables, etc. therefore, the strains of lactic acid bacteria
have also potential to act as a biopreservative or natural food preservative.
Preservation aims at either to eliminate or reduce the outgrowth potential of
spoilage and pathogenic organisms in foods. Lesser the severity of preservative
treatment, lower is the product damage, meeting both the food industry as well as
consumer interests for high quality products with improved organoleptic and
nutritional quality while maintaining microbial safety.
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Production of Bacteriocin from isolates Natural Lactic acid
The extensive use of antibiotics in food preservation has increased the risk of
development of resistance in human and animals. Consequently, there has been an
interest with the use of natural antimicrobials like bacteriocins. The exploration of
naturally occurring antimicrobials in food preservation receives increasing attention
due to consumer’s awareness of natural products. So these naturally synthesized
bacteriocins present high potential to be applied in hurdle technology.
Lactic acid bacteria have long been used as commercial vegetables,
fermented beverages for their contribution to flavor and aroma development and
spoilage retardation (Gilliland, 1986, Kleerebezen and Huqennotz, 2003).
The use of the LAB is a natural method for extending the shelf life of food.
The use of bacteriocin ro the microorganisms that produce them is attractive to the
food industry in the face of increasing consumer demand for natural products and
the growing concern about food-borne disease. It has Also necessitated the need to
exploit the biologically derived antimicrobial substances produces by LAB in
natural or inoculated fermentation the bacteriocin produced by the strains isolated
from natural fermented vegetables has neither been characterized nor checked for its
efficiency in various food products.
The use of bacteriocin is among the new approach, which may further
contribute to reduce risks of food borne disease, outbreaks and increased food
quality. Bacteriocins have attracted as potential substitutes for antibiotics to cure or
prevent bacterial infections. Research work accomplished over the last few decades
has focused on the detection, characterization and purification of bacteriocins.
The term Bacteriocin was first coined by Jacob et al. in 1953.bacteriocins are
naturally synthesized microbial substance produces by gram positive and gram
negative bacteria. Besides other antimicrobial compound, bacteriocins of lactic acid
bacteria gained worldwide attention.
The bacteriocin produced inhibited food spoilage and pathogenic bacteria
such as Salmonella typhi, Shigella dysenteriae, Klebsiella pneumonia,
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Production of Bacteriocin from isolates Natural Lactic acid
Pseuadomonas Fluroscence, E-coli(Escherichia coli), Candida and Bacillus Cerus
which are recalcitrant to traditional food preservatives method.
Lactic acid bacterial strains are known to produce various types of
bacteriocins, which have bactericidal effects against gram positive bacteria including
food borne pathogens.
Organic vegetables are becoming increasingly popular among modern
consumers, LAB isolated from the same type of vegetables or products in which
they will be subsequently used as biocontrol agents, may have the greatest success
chance in controlling pathogenic bacteria (Vescovo, et al., 1996 Breidt and Fleming,
1997). This may be applied interest as biopreservative of minimally processed
organic vegetables. They also find application in various industries like brewing,
pharmaceuticals and healthcare. Nisin, a bacteriocin produced Lactobacillus
acidophilus is the only commercially used bacteriocin, which is generally
recognized as safe.
The present goal was to investigate LAB with potent antimicrobial activity
within the microbial ecosystem and finally to select interesting strains that have the
role of biopreservation
Bacteriocins:
Natural antimicrobials as novel means of food preservation.
Bacteriocins of LAB are used as the novel method of natural food preservation.
An ideal natural food preservative should fulfill a number of criteria, such as
a) Acceptable low toxicity
b) Stability to processing and storage.
c) Efficacy at low concentration.
d) No deleterious effect on the food and
e) Economic viability, while most bacteriocins fulfill these criteria.
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Production of Bacteriocin from isolates Natural Lactic acid
The bacteriocins produced by LAB offer several desirable properties
that make them suitable for food preservation:
i. Are generally regarded as safe substances,
ii. Are not active and non toxic on eukaryotic cells,
iii. Become inactivated by digestive proteases, having little influence on
the gut microbiota.
iv. Are usually pH and heat tolerant,
v. They have a relatively broad antimicrobial spectrum against many food
borne pathogenic and spoilage bacteria.
vi. They show a bactericidal mode of action, usually acting on the bacterial
cytoplasmic membrane.
These bacteriocins are not like therapeutic antibiotics that can
potentially illicit allergic reactions in humans (Cleveland et al., 2001) and
also bacteriocins are proteinecious, ribosomaly synthesized where as
antibiotics are not. There are several large categories of bacteriocins which
are only phenomenologically related. Both gram positive and gram negative
bacteria produce Bacteriocins. These include the colicins, the microcins, and
the bacteriocins from Archaea. The bacteriocins from E. coli are called
colicins (formerly called ‘colicins,’ meaning ‘coli killers’). They are the
longest studied bacteriocins. Colicins produced by E. coli, are often proteins
larger than 20kDa that inhibit the closely related genera. In contrast, gram
positive bacteria produce bacteriocins smaller that 6 kDa. Bacteriocins of
LAB are generally regarded as safe. Most of the bacteriocins from LAB are
cationic, hydrophobic (or) amphophilic molecules composed of 20 to 60
aminoacids residues (Nesand holo, 2000).
Methods of Classification:Methods of classification include:
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Production of Bacteriocin from isolates Natural Lactic acid
i. Method of killing (pore forming, dnase, nuclease, murein production
inhibition, etc),
ii. Genetics (large plasmids, small plasmids, chromosomla),
iii. Molecular weight and chemistry (large protein, polypeptide, with/without
sugar moiety, containing atypical amino acids like lanthinonine) and
iv. Method of production (ribosomal, post ribosomal modifications, non-
ribosomal).
Bacteriocins are classified into four general classes. They are Class I,
Class IIa/b/c and Class III, Class IV.
Class I bacteriocin (lantibiotic):Lantibiotics are a family of membrane active peptids that conatin the
unusual thio-ether amino acid (lanthionine) as well as other modified amino
acid, such as dehydrated serine and lanthionine. They are small composed of
one or two peptides of opproximately 3kDa. Their distinguishing feature is
the presence of post- translationally modified residues.the best example in
this group is nisinproduce by lactococcus lactic subsp. Lactis. Class I is
being further subdivide into Ia and Ib. classIa bacteriocin, which include
nisin, consist of cationic and hydrophobic peptidies that form pores in the
target membranes and have a flexible structure compared to the more rigid
ones of classIb. ClassIb bacteriocins, which are globular in nature, have no
net negative charge.
Nisin:
Nisin is the star of bacteriocins. It is the only bacteriocin allowed as a
food additive (E234, 1983), recognized as safe (FAO/WHO, 1968) and
accepted by the American Food and Drug Administration (1988). Its
structure has been solved, revealing modified amino acids and lanthionine
rings, and its chemical and enzymatic syntheses have been successful.
Killing activities of nisin include formation of membrane pores, blockage of
cell wall biosynthesis, release of cell wall hydrolases, and prevention of
spore outgrowth. The structural gene ( nisA, nisZ, nis Q, and nsuA), and the
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Production of Bacteriocin from isolates Natural Lactic acid
genes (nisBTCIPRKFEG) require for its synthesis, regulation, and slef-
protection have been cloned, sequenced, and functionally characterized. The
regulation includes auto induction by nisin. The nisin inducible promoters
have been used for protein production and for bioassays to quantify nisin in
food. Properties of nisn have been improved by genetic engineering.
Class II bacteriocin:
The class II bacteriocin is small, heat-stable, bioactive protins. These
are generally unmodified peptides of <5 kDa. They are also further subdivide
into classIIa and classIIb. The action of class IIa bacteriocin seems to involve
disruption of mannose transport into target cells. ClassIIb bacteriocin form
pores in the membranes of target cells and disrupt the proton gradient of
target cells. Class IIa includes pediocin-like bacteriocin having antilisterial
activity. Other bacteriocins can be grouped together as Class IIc. These have
a wide range of effects on membrane permaeability, cell wall formation and
pheromone actions of target cells.
Class III bacteriocins:
Large, heat-labile protein bacteriocins of large molecular weight
include Helviticin-J, Lactacins A and B which are generally > 30kDa like
Enterolysin produced by Enterococcus faecium.
Class IV bacteriocins:
This group is consisting of complex bacteriocins that require
carbohydrate or lipid moieties for activity.
Bacteriocins isolated from different Lactobacillus species
Substance Producing species
Acidolin L. acidophilus
Lactacin B L. acidophilus
Lactacin F L. acidophilus
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Production of Bacteriocin from isolates Natural Lactic acid
Factors affecting Bacteriocin production:
Bacteriocins are economically important, so it has to be produced in
large amounts and preferably by growing the strains in media containing
food grade ingredients (Halami et al, 2005; Yang and Ray, 1994). Production
of bacteriocin in a simple medium can be increased by growing the cells at
an optimum pH and supplementing with nutrients specific for a species or
strain. Growing cells under optimum environment for 16 hours facilitates
high bacteriocin production.
Productions of the bacteriocins:
Class I and class II a bacteriocins are usually very stable acidic pH.
For example Rodriguez et al (2002) found that pediocin-PA-was perfectly
stable after 2ld of storage at 15c at 4to6; However half of the activity was
lost at pH7 Larsen and others (1993) found that Bavaricin A was very stable
at pH 2 to 9.7. But storage of Bavaricin A at pH 12.5 for 4 hours resulted in
the complete loss of activity. In addition, bacteriocins from these two classes
are heat stable at acidic pH. As PH increases the heat stability decreases. In
general, bacteriocins are usually sensitive to proteolyytic enzymes, such as
tyrpsin, due to their Proteinaceous nature.
Extraction of bacteriocin:
Extraction of bacteriocin in a large, pure, and concentrated from
(Bibek et al, 1992) is important to study its physical, chemical and
antimicrobial properties and to apply in food systems. Muriana and
Luchansky (1993) have summarized the method to concentrate the proteins
from the culture filterate it involves vaccum concentration, solvent
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Production of Bacteriocin from isolates Natural Lactic acid
precipitation, acid precipitation. Ammonium sulphate precipitation is the
most common and effective method for the extraction of the concentrated
protein. Yang et al (1992) have also reported a cell adsorption method, which
allowed bacteriocin in the broth to be absorbed to the producer cells at an
optimum pH.
Identification of LAB:
Classical and molecular techniques were involved in the
identification of bacteriocinogenic LAB. Traditionally, LAB have been
identified and grouped by conventional techniques such as morphological,
physiological and biochemical tests (Bergey’s manual 1986’s edition and
Sharpe, 1979).
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Production of Bacteriocin from isolates Natural Lactic acid
Objective
The aim of the work was to evaluate the potential use of Bacteriocin
produced by Organisms isolated after Natural Lactic Acid fermentation of
vegetables.
1. The production, identification, purification of Bacteriocin.
2. Checking the stability and efficiency of bacteriocin produced.
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Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 2
Review of Literature
The LAB comprise a clade of Gram positive, catalase negative, acid
tolerant, rod shape that are associated by their common metabolic and
physiological characteristics. They are widely distributed in nature. They are
isolated fr4om fermented vegetables and lactic products produce lactic acid.
This trait has historically linked LAB with food fermentations as
acidification inhibits the growth of spoilage agents. Proteinaceous
bacteriocins are produced by several LAB strains and provide an additional
hurdle for spoilage and pathogenic microorganisms. The industrial
importance of the LAB is further evidenced by their generally regarded as
safe (GRAS) status, due to their ubiquitous appearance in food and their
contribution to the healthy microflora of human mucosal surfaces.
LAB can be divided into two groups based upon the products produced from
the fermentation of glucose they are.
Homofermenters :
The fermentation of one mole of glucose yields two mole of lactic
acid, generation a net of 2 ATP per mole of glucose metabolized
C6H12O6 2CH3CHOHCOOH
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Production of Bacteriocin from isolates Natural Lactic acid
Glucose lactic acid
Heterofermenters:
The fermentation of 1 mole of glucose yields 1 mole each of lactic
acid, ethanol and carbon dioxide. One mole of ATP is generated per mole of
glucose.
C6H12O6 CH3CHOHCOOH + C2H2OH + CO2
Glucose Lactic acid Ethyl Alcohol
Classification of Lactic acid bacteria based in type of fermentation
Homofermenters Facultative
homofermenters
Obligate heterofermenters
Lactobacillus acidophilus Lactobacillus
coryniformis
Lactobacillus cellobiosus
Lactobacillus lactis Lactobacillus curvatus Lactobacillus confusus
Lactobacillus delbrueckii Lactobacillus plantarum Lactobacillus coprophillus
Lactobacillus leichmannii Lactobacillus sake Lactobacillus
fermentatum
Lactobacillus bovis ---- Lactobacillus sanfrancisco
Lactobacilli are gram positive and vary in morphology from
long that can be either hetero-or homofermentative. They are widespread and
can be isolated from many vegetables sources. Lactobacilli are more tolerant
to acid than the other genera of lactic acid bacteria and this property makes
them important in the final phases of many food fermentations when other
organisms are inhibited by the low pH.
Probiotic application of LAB
The comcept of probiotics was in use in the early 1900s, response
however, the term was coined in 1965 by Lilly and Stillwell. Probiotic is a
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Production of Bacteriocin from isolates Natural Lactic acid
preparation of live microorganisms, which when applies to man or animal,
beneficially affects the host by improving the properties of the indigenous
microbiota (Havenaar et al, 1992).
LABs are regarded as a major group of probiotic bacteria (Collins
at al., 1998; Schrenmeir and de Vrese, 2001). The probiotic concept has been
defined by Fuller (1989) to mean “ a live microbial feed supplement which
beneficially affects the host animal by improving its intestinal microbial
balance”, Salminen et al, (1999) proposed the probiotics are microbial cell
preparations or components of microbial cells that have a beneficial effect on
the health and well-being of the host. Several lactobacilli, lactococci and
bifidobacteria are held to be health-benegiting bacteria (Rolfe, 2000; Tuohy
et al., 2003), but little is known about the probiotic mechanisms of gut micro
biota (Gibson and Fuller, 2000).
LAB constitute an integral part of the healthy gastrointestinal (GI)
micro ecology and are involved in the host metabolism (Fernandes et al.,
1987). Fermentation has been specified as a mechanism of Probiotics
(Gibson and Fuller, 2000; Metchnikoff, 1908). LAB along with other gut
micro biota ferment various substrates like lactose, biogenic amines and
allergic compounds into short-chain fatty acids and other organic acids and
gases (Gibson and Fuller, 2000; Jay, 2000).
Reaction of organisms on the pathogens.
1. Salmonella typhi:
A rod-shaped flagellated, facultative anaerobic, Gram-negative bacterium, and a member of the genus Salmonella.
The given salmonella typhi was inoculated in sterile saline. This 1 ml saline was spread on Nutrient agar media and kept for 5 min in laminar air flow.The obtained inhibition forming organisms were streaked on the media, and kept in incubator at 37ºc for 24 hours.
2. Shigella dysenteriae:
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Production of Bacteriocin from isolates Natural Lactic acid
Shigella dysenteriae is a species of the rod-shaped bacterial genus Shigella.[1][page needed] Shigella can cause shigellosis (bacillary dysentery). Shigellae are Gram-negative, non-spore-forming, facultatively anaerobic, non-motile bacteria.[2]
This strain also spread alike salmonella, and organism forming inhibition zone was streaked and kept for incubation.
3. Klebsiella pneumoniae:
Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines.[
Same procedure as like salmonella and shigella is done and kept for incubation.
4. Pseudomonas Fluroscence:
Pseudomonas fluorescens is a common Gram-negative, rod-shaped bacterium.[1] It belongs to the Pseudomonas genus; 16S rRNA analysis has placed P. fluorescens in the P. fluorescens group within the genus,[2] to which it lends its name.
The inhibiting organism is streaked on plate on which suspension of pseudomonas is spread,and incubated overnight.
5. E-coli: ( Escherichia coli)
Escherichia coli (commonly abbreviated E. coli) is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls.[2][3] The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2,[4] and by preventing the establishment of pathogenic bacteria within the intestine.[5
The pathogenic strain of E-coli was spread on nutrient agar and the inhibition forming organism was streaked and incubated overnight.
6. Candida:
Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans.[3][4] Systemic fungal infections (fungemias) including
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Production of Bacteriocin from isolates Natural Lactic acid
those by C. albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). C. albicans biofilms may form on the surface of implantable medical devices. In addition, hospital-acquired infections by C. albicans have become a cause of major health concerns.
Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans.[3][4] Systemic fungal infections (fungemias) including those by C. albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). C. albicans biofilms may form on the surface of implantable medical devices. In addition, hospital-acquired infections by C. albicans have become a cause of major health concerns.
The given strain of Candida was spread on nutrient agar and streaked with the same organism streaked in all above plates. And incubated at 37º c for 24 hours.
Biochemical tests:
Principle:
According to the physical characters, the biochemical tests are done. When we come to know the gram’s nature, motility and colony structure, we can conclude the biochemical tests referring to the Bergey’s manual.
The presence of the particular enzyme in a micro organism can be tested by incorporating a specific substrate in a medium, (if necessary), and then detecting the products formed or even checking the disappearance of the substrate from the medium.
These biochemical tests employ various media (having different substrate) which when inoculated with a particular species of bacteria will follow a specific metabolic pathway to hydrolyze the substrate available to them. Some of the routine biochemical tests used for determining metabolic activities of bacteria can be broadly classified as
Utilization of carbohydrates and acids Utilization nitrogenous compounds Decomposition of large molecules Miscellaneous tests
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Production of Bacteriocin from isolates Natural Lactic acid
According to the physical characters, as the bacteria are gram negative, the biochemical tests for the obtained bacteria are mentioned below:
a) Carbohydrate fermentation (Sugar utilization) test -
Principle:
Sugars are metabolized through different metabolic pathways (Depending type of species and aerobic and anaerobic environment) to form various acids like pyruvate, lactase, succinate, formate, acetate ect. These acids so formed may further break down to gases (formic hydrogenlyase will split formic acid to H2 and CO2 ).
Due to acid formation, the PH of the medium is lowered and phenol red indicator is being faint pink to colorless. Gas formation is demonstrated by the use of Derham’s tube (a small tube inverted in the sugar solution.) which collect gas.
b) Methyl Red test-
Principle:
Only mixed acid fomenters ( e.g. Escherichia coli) produces sufficient quantity of acids during initial phase of incubation (PH below than 4.4) which can be detected by methyl red indicator. This is because the fact the medium glucose phosphate broth is strongly buffered, hence minute quantities of acids if produced, will not permit the PH of the medium to drop down. Moreover, methyl red is a PH indicator having ranges between 6.2 (yellow) to 4.4 (red), so the PH at which Methyl red detects acid is considerably lower than the PH for other indicators used in bacteriological medium.
c) Indole Production Test:
Principle:
Indole, a benzyl pyrrole, is one of the metabolic degradation products of the amino acid tryptophan. Organisms that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvate and ammonia.
Indole so produced react with the aldehyde group of a weakly acid alcoholic solution of Þ-dimethylaminobenzaldehyde (Kovac’s reagent)in
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Production of Bacteriocin from isolates Natural Lactic acid
presence of heat to form pink colored rose-indole complex. The reaction can also occur without heat, if the reagent is prepared with HCL.
Indole is a substance which reduces surface tension and hence it is concentrated in the surface layer of the medium. Moreover, because indole is soluble in organic compounds. It is recommended that chloroform or xylene be added prior to adding Ehrlich’s reagent. This serves two purposes, firstly it extracts indole from whole of the medium and secondly it forms a separate layer above the medium. As a result, reagent reacts with the indole extracted in the xylene and forms a pink color. Organic solvents like chloroform, ether, and light petroleum can be used instead of xylene. This step is not necessary with kovac’s reagent because the amyl alcohol is used for the diluent is capable of extracting sufficient indole from the aqueous medium to produce a positive reaction.
d) Vogous Proskauer (VP) test-
Principle:
In presence of alkali and air (vigorous shaking) acetoin is oxidized to diacetyl which reacts with guanidine nucleus of arginine present in proteins present in proteins of peptone to produce pink color. At times a pinch of creatine is added to provide an additional source of guanidine nucleus and thus accelerate pink color formation. Test is made sensitive by adding α-naphthol,which serves as catalyst.
e)Citrate utilization test:
Principle:
The test determines the ability of the bacteria to use citrate as sole of carbon and energy. This ability depends on the presence of a citrate permease that facilitates transport of citrate into the bacterium. Once inside the cell, citrate is converted to pyruvate and CO2 . Citrate agar slant contain sodium citrate as the sole source of carbon, ammonium phosphate as a sole source of nitrogen, and bromothymol blue as a PH indicator [PH 6 (yellow)-PH 7.6 (blue)]. This test is done on slant since o2 is necessary for citrate utilization. When bacteria oxidise citrate, they remove it from the medium and liberate CO2. This CO2 combines with sodium (Supplied by sodium citrate and water to form sodium carbonate – an alkaline product. Similarly,bacteria that utilize citrate can also extract nitrogen from the ammonium salt, with the production of ammonia, which is convertwd to
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Production of Bacteriocin from isolates Natural Lactic acid
ammonium hydroxide (NH4OH). These alkaline products raise PH, and turn pH indicator to a blue color and represents a positive citrate test
f)Urea Hydrolysis Test (Urease Test):
Principle:
A strongly buffered medium in which urea is only nitrogen source is used for the test. Urease is an enzyme possessed by many species of micro organisms that can hydrolyze urea.
The ammonia so produced reacts in solution to form ammonium carbonate, resulting in alkalinization and an increase in pH of medium. This is indicated by change in color of the indicator phenol red (pH 6.8-8.4 yellow to purple red).
Due to high buffering capacity of the medium, only those organisms possessing vigours urease activity (proteus vulgaris) can given test positive.
f) Catalase test :
Principle:
Catalase is an enzyme that splits up hydrogen peroxide into oxygen and water. Chemically catalase is a hemoprotein, similar in structure to hemoglobin.
Catalase is present, often in high concentrations in the majority of aerobic organisms but is absent from most obligate anaerobes. Thus when H2O2 is added externally in the medium,catalase activity results in the production of molecular gaseous oxygen. Catalase activity can be tested either by slide test or tube test.
g) Oxidase test:
Principle:
Gordon and McLeod (1) introduced oxidase test for identifying Gonococci based upon the ability of certain bacteria to produce indophenol blue from the oxidation of dimethyl-p-phenylenediamine and α-naphthol. Gaby and Hadley (2) introduced amore sensitive method by using N,N-dimethyl-p-phenylenediamine oxalate where all Staphylococci were oxidase negative. In presence of the enzyme cytochrome oxidase (gram-negative bacteria) the N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol react to indophenol blue
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Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 3
Aims and Scope of Study
Production of the Bacteriocin from Natural Lactic Acid fermented
vegetables.
Purification, Biochemical, Stability of the Bacteriocin.
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Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 4
Materials and Methods
Fermented Vegetables
Vegetables like a Cucumber, Tomato, and Garlic were washed,
peeled and grated/sliced. The sliced of Cucumber, Tomato, Garlic were
inoculated in minimal salt media and Micro flora were isolated from there
samples.
Composition of Minimal Salt 1000ml
30g Na2HPo4
15g NH5Po4
5g NH4Cl
25g NaCl
15g CaCl2
I was taken 3 Bottle in 1 flask 100 ml minimal salt media.
In each flask I inoculate the in flask and keep at room temperature.
All this procedure I had done luminal air flow.
Pathogenic Bacterial Cultures.
I was taken Standard Bacterial Culture 7 standard Bacterial Culture
were taken to sub – culturing of standard bacterial culture. By using N.A
o Standard Bacterial Culture
Salmonella typhi
Shigella dysenteriae
Klebsiella pneumonia
Pseuadomonas Fluroscence,
E-coli(Escherichia coli),
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Production of Bacteriocin from isolates Natural Lactic acid
Candida
Bacillus Cerus
I were take 7 test tubes and prepare N.A
o Composition of Nutrient Agar 100ml
Peptone – 1gm
Yeast extra – 1gm
NaCl – 05gm
Agar – 2gm
Standard bacterial cultures were procured from MITCON Institute
Pune, were used in bacteriocin screening procedure and all cultures were sub
- culture and were used.
Autoclave the test tubes and N.A than inoculate in test tubes and
prepare the slant streaked the standard bacterial culture and keep it for
incubation at 370c in incubator.
Isolation and Identification of Bacteriocin producing Bacteria
The Bacteriocin procedure from naturally fermented Cucumber,
Tomato and Garlic were isolated by pour plate method technique. By using
MRS Agar (de Mann Rogosa and Sharpe).
Bacteriological Media
The media used for the work were MRS (de Mann Rogosa and
Sharpe) broth and agar were purchased from Himedia, India.
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Production of Bacteriocin from isolates Natural Lactic acid
Media Composition
MRS (de Mann Rogosa and Sharpe) Medium
Table 1
Ingredient Amount(g/l)
Protease Peptone 10.00
Yeast extract 5.00
Beef extract 10.00
Dextrose 20.00
Polysorbate 80 1.00
Ammonium Citrate 2.00
Sodium Acetate 5.00
Magnesium Sulphate 0.10
Manganese Sulphate 0.05
Dipotassium Phosphate 2.00
MRS Agar Medium
MRS Agar Medium was prepare by adding 2gm in 100ml to the broth
and boiled to dissolve the agar.
The requisite amount of the medium were taken is Conical flask,
plugged with cotton and autoclaved.
Pour the plate and from Cucumber 1ml, Tomato 1ml and Garlic 1ml
sample were taken were spreading, streaking method were done and
inoculated for 24 – 48 hr at 320c.
After incubation typical colonies were isolated and pure culture is
formed in N.A by streaking method.
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Production of Bacteriocin from isolates Natural Lactic acid
The isolated were differentiated on their Morphological and
Biochemical Test, staining is done to identify the M.O.
The native isolate of LAB were subjected to species level
identification by various microbial, biochemical test, according as described
in Bergey’s manual of Bacteriology.
Study of physical properties
The study of physical character includes colony characters and gram
staining i.e. Morphology of the organism
The gram staining of the organism is done as well as the characters of
the formed colonies are noted down.
o Microbiological tests(Morphological Tests)
Gram Staining
Material
Table 2:
Crystal Voilet
Ingredients Amount g/100ml
Crystal Voilet 2.0
Ethyl Alcohol 20.0
Ammonium Oxalate 0.8
D/W 80.0ml
Crystal voilet and ammonium oxalate were dissolved separately in
ethyl alcohol and d/w respectively and the two solution were then mixed.
The prepared stain was filtered and stored in a clean, dry glass stoppered
bottle.
Table 3:
Grams Iodine (Mordant)
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Production of Bacteriocin from isolates Natural Lactic acid
Ingredients Amount g/100ml
Iodine 1.0g
Potassium Iodine 3.0
Table 4
Saffranin ( Counter Stain)
Ingredients Amount g/100ml
Saffranin 2.5g
Ethyl Alcohol 100ml
Methods (Hucker’s Modification)
Prepare a heat fixed smear of the culture
Cover the smear with crystal voilet stain for 1min
Add gram’s iodine to wash off crystal voilet stain and cover it with
iodine till smear turns coffee brown in color (Approx. 1min)
Rinse the slide in the running water.
Add decolorizing solution drop wise at the upper end of slide held in
inclined position, till voilet color fails to come out from the smear, for
normal smear 10 – 15 sec are enough.
Rinse the smell with water
Counterstained with Saffranin for 45 – 60min
Rinse with the tap water, drain, blot, air dry and examine under electro
microscope
The morphology of cell is record.
For the identification of the bacteria some common tests are done for
identification of the specific bacteria. Physical characters studied first
accordingly the chemical tests are done.
Screening of Isolates for Antimicrobial Activity
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Production of Bacteriocin from isolates Natural Lactic acid
Antimicrobial activity of the bacterial isolates against all the
pathogenic microorganism was determined by well diffusion method (13 -
16) under the aerobic conditions.
MaterialTable 5:
Nutrient agar media composition
Ingredients Amount gm/l
Peptone 1 gm
Yest extract 0.3 gm
Nacl 0.5 gm
Agar 2 gm
Method
N.A. medium was prepare & the requisite amount of the medium
wear taken in conical flask plugged with cotton and autoclaved.
Then the bacterial culture wear grown in MRS broth and dilution
approx.
The N.A. pour in plate& make media solidify then the lawn of pathogen
is formed with the help of spreader.
There wear seven pathogen are used.
a. Salmonella typhi
b. Shigella dysenteriae
c. Klebsiella pneumonia
d. Pseuadomonas Fluroscence,
e. E-coli(Escherichia coli),
f. Candida
g. Bacillus Cerus
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Production of Bacteriocin from isolates Natural Lactic acid
Before preparing lawn prepare the saline and inoculate the pathogen
saline and lawn with the help of spreader and form well and isolated stain
were placed into each well. After all this procedure plates were kept in
incubator for incubation at 370c for 24 hr.
The antimicrobial activity was determined by measuring the diameter
of the inhibition zone around the well.
Partial purification of Bacteriocin
Isolated strain which were isolated from vegetables by the screening
of isolates for antimicrobial activity which showing widest zone of inhibition
against the target microorganisms having the maximum antimicrobial zone
where used for partial purification of Bacteriocin.
The maximum antimicrobial zone was grown in MRS broth at 370c
for 24hrs.
The isolated strain were inoculated in MRS broth and used.
After incubation, the broth was centrifuged at 5000x g for 10min. I
was taken 4 centrifuge test tubes for centrifugation. After centrifugation, the
cells were separated out and only superannuated was used as a crude
bacteriocin.
Material
Table 5:
Preparation of Acetone
Ingredient Ml
Acetone 80ml
Distilled Water 20ml
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Production of Bacteriocin from isolates Natural Lactic acid
The acetones were added to the supernatant. I were taken 4
test tubes in equal amount of Acetone were added in supernatant.
After that it was kept undisturbed at 40c overnight.
Next day it removes from freezer. Precipitates formed were
collected by centrifugation at 5000 *g for 5min and again centrifuge
at 5000*g for 5min.
Preparation of Sodium Phosphate buffer
Table 7:
Ingredient Ml/Gm
Sodium Phosphate O.335 gm
Distilled Water 100 ml
PH 6
And after preparation of sodium phosphate buffer with
PH=6.0
The comparison with crude bacteriocin and precipitate bacteriocin.
Again i preparing the NA. 5 pathogen were used against the strain.5
pathogen
There wear five pathogen are used.
a. Salmonella typhi
b. Klebsiella pneumonia
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus substils
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Production of Bacteriocin from isolates Natural Lactic acid
N.A wear taken in conical falsk & plugged with cotton & autoclave ,
petri plate wear also autoclave.
Then pour in petri plate then keep for solidification after that I wear
prepar lawn of pathogen with help of spreader. after that wells wear formed.
then by 200 µl micropippte the crude bacteriocin & precipitation were
incoculated in well & it keep for incubation at 37ºc at incubator.
Next day i wear observe the inhibition zone of different fraction was
recorded in comparision with the crude bacteriocin.
Characterization of bacteriocin
Heat stabilityProtocol
Taken 4 test tube in each test tube 5 ml of sample. Two test tube is
heated with 68ºc for 10 to 20 mins respectively & two remaining test tube
wear heated at 121ºc for 15 mins.heat treated with bacteriocin samples wear
assy by antimicrobial activity.
Material
Nutrient agar, pathogen, bacteriocin.
Method
Prepare the nutrient agar then autoclave the N.A. & Petri plate then
pour the N.A. in Petri plate make media solidify ,then prepare a lawn of
pathogen
There wear five pathogen are used.
a. Salmonella typhi
b. Klebsiella pneumonia
c. E-coli(Escherichia coli),
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Production of Bacteriocin from isolates Natural Lactic acid
d. Candida
e. Bacillus Cerus
Then form well, inoculated the sample in well & keep it for
incubation in incubator at 37ºc for 24 hrs.
Effect of PH
Protocol
Taken 8 test tube in each test tube purified bacteriocin wear taken in
each test tube with different PH 4,5,6,8.activity checked by antimicrobial
activity.
Material
PH meter, N.A.,bacteriocin,pathogen.
Method
Prepare the nutrient agar then autoclave the N.A. & Petri plate
then pour the N.A. in Petri plate make media solidify ,then prepare a
lawn of pathogen
There wear five pathogen are used.
a. Salmonella typhi
b. Klebsiella pneumonia
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus Cerus
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Production of Bacteriocin from isolates Natural Lactic acid
Then form well, inoculated the sample in well & keep it for
incubation in incubator at 37ºc for 24 hrs.
CHAPTER 4
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Production of Bacteriocin from isolates Natural Lactic acid
RESULT & DISCSUSSION
SCREENING OF BACTRRIOCIN ISOLATES FROM
VARIOUS SAMPLE
Samples like cucumber, tomato, garlic were collected from pune
market and they were subjected for the presence of bacteriocin were isolated.
MRS medium was used as the selective medium to isolate the
LAB .the isolate were screen by overlaying the freshly grown indicator strain
of lactobacillus few of the isolates showed bacteriocin against the standard
bacterial cultural which shown by the prominent zone of inhibition with (fig)
different in zone diameter. the cultural were pricked and inoculated into
MRS broth to reconfirm and purify the isolates.
The inoculated colonies were once again pour plated in N.A & make
the colonies purified.
The bacteriocingenic isolate were obtain from the cucumber, garlic,
tomato.
Apart from cucumber samples tomato samples, tomato sample had
bacteriocin strains. i did not get bacteriocin from garlic. only two strains
were slected for further analysis.
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 1:Incoculated in minimal media
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 2: Incoculated in minimal media
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 3:growth of bacteriocin(tomato) on MRS media
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 4: Growth of bacteriocin(garlic) on MRS broth
Fig 5: Growth of bacteriocin(cucumber) on MRS broth
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 6: Pure culture of cumcumber
Fig 7:Pure culture of garlic and tomato
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 9: Gram staining of bactreiocin (cumcumber) Lactobacillus acidophillus
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 10: Gram staining of bactreiocin (tomato) Bacillus spp
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Production of Bacteriocin from isolates Natural Lactic acid
Charaterization of bacteriocin LABThe isolate were subjected to microbiological biochemical
physiological examination based on” Bergey’s manul” of classification.
Microbiological characterizationThe morphological examination of the isolate were identified through
the gram staining, which show the isolate were gram positive,rod shaped of
cucumber and tomato gram postive short rod shaped & by observing the
electron microscopic showed they are Lactobicillus acidophlis from
cucumber & Bacillus spp from tomato.morphological examination shows
that the isolate were LAB.the result are shown in fig.
Antimicrobial activity of the purified isolates various pathogenic
strian
The neutralize cluture filterate checked for the antimicrobial activity
against various strains like lactobacillus acidophillus inhibitory action
against various strains with different rates are tabulated in table.
The seven pathogen strains wear used
a. Salmonella typhi
b. Shigella dysenteriae
a. Klebsiella pneumonia
b. Pseuadomonas Fluroscence,
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus subtilus
They show the antimicrobial activity against this strain like
Lactobacillus spp from cucumber & bacillus spp from tomato
the result are present in the table.
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Production of Bacteriocin from isolates Natural Lactic acid
The LAB isolate from the vegetables products by Marjon
H.J. Bennik et al 1997 also showed broad range of inhibitory
action.
Table 1: inhibition zone of antimicribal activity
Sr.no Specis Lactobacillus
acidophilus
dimeter of
zone
Bacillus
spp dimeter
of zone
1 E.coli(Escherichia
coli
1 cm 0.6cm
2 Shigella
dysenteriae
0.2 cm -
3 Candida o.6 cm 0.4
4 Salmonella typhi 0.8 cm 0.6 cm
5 Bacillus subtilus 0.5 cm -
6 Klebsiella
pneumonia
0.6 cm 0.5 cm
Fig 10: inhibition zone of antimicriobal activity
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 11: inhibition zone of antimicriobal activity
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Production of Bacteriocin from isolates Natural Lactic acid
Biochemical chacterization:Based on biochemical tests, all the isolates were identified as
belonging to lactic acid bacteria (LAB) group, which was identified. the
isolating lactobacillus acidophilus, Bacillus spp giving maximum
antimicrobial activity was gram positive, long rod shaped, negative for
catalase and oxidase is positive, having circular and white colonies on the
MRS media. the strain was also positive test for glucose, sucrose,
maltose,indole test, methyl red test, citrate, urease, oxdiase and there is no
change for mannitol, rhammnose, lactose, vogos proskrous test.
: Observations for the biochemical tests on inhibition forming bacteria.:
Table 2
Sr. No. Test name Observations on bacteriocin cucumber
Observations on
bacteriocin tomato
1. Indole test No change occured Ring of orange color
2. Methyl red test Ring of red color on the surface
Ring of red colour on the surface
3. Vogos proskrous test No color change No color change
4. Citrate test Blue color Blue color
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Production of Bacteriocin from isolates Natural Lactic acid
5. Urease test Color changed Pink color
6. Oxidase test Paper showed no color change Paper showed voilet color
7. Catalase test No effervesces of O2 Bubbles are observed
Fig 12:Cirtrate test
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Production of Bacteriocin from isolates Natural Lactic acid
Fig: urease test
Fig 13: indole test
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 14: Methly red test
Carbohydrate fermentationCarbohydrate fermentation is carried out to identify the isolate is
herofermenters or homofermenters.the culture were incoculated into the
MRS broth durhams tube (to detect the gas production)the culture utilize the
glucose and produced acid and gas the indicates that hetrofermenters the
result shown in table.
Sugar utilization test
The sugar utilization test was carried out to test the ability of the
isolate to utilize various sugar. the isolate to utilize various sugar Like
lactose, sucrose, maltose, raffinose , arabinose individually and allow to
grow are shown in table.
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Production of Bacteriocin from isolates Natural Lactic acid
Observations for the sugar (Utilization of carbohydrate)
Table 3
Sr No. Test name
(sugars used)
Observations on organism lactobacillus acidophillus
Observation on organism inhibiting bacillus spp
1. Glucose Faint pink color Faint orange
2. Sucrose No change Orange color
3. Maltose Red orange color No change
4. Mannitol Orange color Orange color
5. Rhamnose No change No change
6. Lactose No change No change
Partial purification of bacteriocin
An increase in antimicrobial activity after partial purification of crude
bacteriocin by ammonium sulphate precipitation took place (fig).the fraction
with the highest bacteriocin activity precipited with 10 to 20% by
ammonium sulphate.
The antimicrobial activity increase from 0.8 to 1.4 cm the precipated
bacteriocin shows more antimicrobial activity.
Observation of zone of inhibition
Table 4:
Sr.no Species Crude bactreiocin Precipated
1 Salomenella typhi 0.6 cm 0.4 cm
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Production of Bacteriocin from isolates Natural Lactic acid
2 Bacillus substils 0.4 cm 0.6 cm
3 E.coli 0.5 0.7
Fig 15: zone of inhibition of crude bacteriocin and precipated
Fig 16: Zone of inhibition of crude bacteriocin and precipated
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 17 :Observation of zone of inhibition of crude bacteriocin and precipitates.
Characterization of bacteriocin
Heat stability
Paritially purified bacteriocin was found to be stable at 68ºc for upto
10 mins at 100ºc it could not show antimicrobial activity at 100ºc temp.
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Production of Bacteriocin from isolates Natural Lactic acid
However, after incubation for 15 mins at 121ºc they complete loss of
activity took place. the compared to the earlier report on bacteriocin, residual
activity was lower in our study than report of earlier.
Furthermore tolerance of bacteriocin to heat is known to depends on
the stages of purification, PH, presence of culture medium other protective
components, etc. the antimicrobial activity in our finding too.
Observation table inhibition zone diameter
Table 5: Heat stability on different temp
Sr.no temperature Min E.coli B.substils
1 68 10 0.3 cm 0.4 cm
2 121 15 0.3 cm 0.2 cm
Fig 17: Heat stability on different temp
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 18: Heat stability on different temp.
Effect of PH
The partially purified bacteriocin shows maximum activity against
the target microorganism at PH 4 to 5. But the activity of the bacteriocin
gradually continuosly decreased. at PH 8 the antimicrobial activity was
drastically reduced to more than 2.5 time. Thus , the bacteriocin was found
active over a wide PH range with the highest activity at low PH range of 4 to
5 earlier. Earlier, the bacteriocin produced by a newely isolated
Lactobacillus acidophilus active the PH range at 4 to 6 another bacteriocin
produced by bacillus spp was active over. zone of inhibition shown in table.
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Production of Bacteriocin from isolates Natural Lactic acid
Table 6 : effect of pH and zone of inhibtion
Sr.no PH E.coli Salo
monella
1 4 o.4 cm 0.2 cm
2 6 0.3 cm -
3 8 - 0.2 cm
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 19: effect of pH and zone of inhibtion
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Production of Bacteriocin from isolates Natural Lactic acid
Fig 20: effect of pH and zone of inhibtion
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Production of Bacteriocin from isolates Natural Lactic acid
Conclusion
The study revealed that bacteriocin from Lactobacillus sp. Isolate from natural lactic acid fermentation of vegetables possesses a wide spectrum of inhibitory activity against Escherichia coli , bacillus substlis, candida, shigilla, salomenella & klebcilla
Therefore, it has a potential for application as a biopreservation in different food products as such or in combination with other preservation methods. Since lactic acid fermentation is employed mostly for development of products, especially for flavour and taste of the fermented products, the production of bacteriocin in such products assumes more significance as biopreservative apart from imparting probiotic effect to the product.
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Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 6
Summary
LAB were isolated from fermented product as vegetables. Lactic acid bacteria (LAB) have been used for centuries in the preparation, processing and preservation of food & beverages.
LAB have long been employed in fermentation as a preservation techniques because of the ability of LAB to inhibit the growth of undesired bacteria due to the production of antimicrobial substance such as bacteriocin. The industrial importance of the LAB is further evidence by their generally regard as safe (GRAS) status, due to their ubiquitous appearance in food and their contribution to the healthy microflora of human mucosal surface .though many gram positive and gram negative bacteria were able to produce bacteriocin, the bacteriocin produced by the LAB is widely used because of it’s GRAS status
.From various vegetables produce, the bacteriocin LAB were isolated
bacteriocin were subjected to microbial, biochemical and characterization of bacteriocin. They were tentatively identified as Lactobacillus acidophilus and bacillus spp they produce the bacteriocin. Their wide range of the antimicriobial property indicates that it is a wide spectrum bacteriocin.
The bacteriocin is a particular importance to the food industry and also to other pathogens E Coli, Shigella, Candida, Salmonella, Klebsieva, pseudomonas and Bacillus Substis. They are inhibitory towards both Gram negative and Gram positive organisms, so they are said to be broad spectrum bacteri-ocin.
The bacteriocin is treated with heat at 680C for 10min and 1210c for 15min, the results indicate that all the bacteriocins were 680C stable even at autoclaving temperature. Hence it can persist and exhibit its inhibitory action in the cooked foods. Therefore, it can be used as an effective preservative.
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Production of Bacteriocin from isolates Natural Lactic acid
C HAPTER 7
Reference
1.R Ananthnarayn and c k Panikar’s textbook of microbiology sixth edition.
2.Experimental Microbiology by J patel Volume 1 and 2
3.Farkas-Himsley H (1980). "Bacteriocins--are they broad-spectrum antibiotics?". J. Antimicrob. Chemother. 6 (4): 424–4. doi:10.1093/jac/6.4.424. PMID 7430010.
5. Cotter PD, Hill C, Ross RP (2006). "What's in a name? Class distinction for bacteriocins". Nature Reviews Microbiology 4 (2). doi:10.1038/nrmicro1273-c2. is author reply to comment on article :Cotter PD, Hill C, Ross RP (2005). "Bacteriocins: developing innate immunity for food". Nature Reviews Microbiology 3 (?): 777–88.doi:10.1038/nrmicro1273. PMID 16205711.
6. HENG, C. K. N., WESCOMBE, P. A., BURTON, J. P., JACK, R. W.,& TAGG, J. R. (2007). The diversity of bacteriocins in Gram-positive bacteria. In: Bacteriocins: Ecology
7. Z. Yildirim, M.G. Johnson, Detection and characterizationof a bacteriocin produced by Lactococcus lactis subsp.
cremoris Lett. Appl. Microbiol. 26 (1998)
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J. DeOliveira, Isolation of bacteriocin producing lactic acid
bacteriafrom meat and meat products and its spectrum of
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9.Characterization of the heat stable bacteriocin produced by lactic acid bacteria isolated from dairy products and veg Reg no 06PY11, department of microbiology.
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