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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.


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)


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.


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.


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


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


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


(garlic ) on MRS media

40


(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



46



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


C.B& precipitated


stability

55


stability

56

19 Zone of inhibition of pH 59


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


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.


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,


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.


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:


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


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


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


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).


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.


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


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


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:

http://en.wikipedia.org/wiki/Salmonella

http://en.wikipedia.org/wiki/Genus

http://en.wikipedia.org/wiki/Bacterium

http://en.wikipedia.org/wiki/Gram-negative

http://en.wikipedia.org/wiki/Flagella


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

http://en.wikipedia.org/wiki/Fungemia

http://en.wikipedia.org/wiki/Genital

http://en.wiktionary.org/wiki/oral

http://en.wikipedia.org/wiki/Opportunistic_infection

http://en.wikipedia.org/wiki/Hypha

http://en.wikipedia.org/wiki/Yeast

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Diploid

http://en.wikipedia.org/wiki/Pathogen

http://en.wikipedia.org/wiki/Vitamin_K

http://en.wikipedia.org/wiki/Gut_(zoology)

http://en.wikipedia.org/wiki/Human_flora

http://en.wikipedia.org/wiki/Product_recall

http://en.wikipedia.org/wiki/Human

http://en.wikipedia.org/wiki/Foodborne_illness

http://en.wikipedia.org/wiki/Serotype

http://en.wikipedia.org/wiki/Strain_(biology)

http://en.wikipedia.org/wiki/Warm-blooded

http://en.wikipedia.org/wiki/Gastrointestinal_tract


http://en.wikipedia.org/wiki/Bacillus_(shape)


http://en.wikipedia.org/wiki/16S_rRNA


http://en.wikipedia.org/wiki/Pseudomonas




http://en.wikipedia.org/wiki/Facultative_anaerobic

http://en.wikipedia.org/wiki/Fermentation_(biochemistry)

http://en.wikipedia.org/wiki/Lactose

http://en.wikipedia.org/wiki/Bacterial_capsule


http://en.wikipedia.org/wiki/Motility

http://en.wikipedia.org/wiki/Facultative_anaerobic_organism

http://en.wikipedia.org/wiki/Endospore


http://en.wikipedia.org/wiki/Dysentery

http://en.wikipedia.org/wiki/Shigellosis

http://en.wikipedia.org/wiki/Wikipedia:Citing_sources

http://en.wikipedia.org/wiki/Shigella


http://en.wikipedia.org/wiki/Bacteria


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

http://en.wikipedia.org/wiki/Nosocomial_infection

http://en.wikipedia.org/wiki/Biofilms

http://en.wikipedia.org/wiki/Bone_marrow

http://en.wikipedia.org/wiki/Chemotherapy

http://en.wikipedia.org/wiki/AIDS

http://en.wikipedia.org/wiki/Immunodeficiency

http://en.wikipedia.org/wiki/Death

http://en.wikipedia.org/wiki/Morbidity

http://en.wikipedia.org/wiki/Fungemia

http://en.wikipedia.org/wiki/Genital

http://en.wiktionary.org/wiki/oral

http://en.wikipedia.org/wiki/Opportunistic_infection

http://en.wikipedia.org/wiki/Hypha

http://en.wikipedia.org/wiki/Yeast

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Diploid

http://en.wikipedia.org/wiki/Nosocomial_infection

http://en.wikipedia.org/wiki/Biofilms

http://en.wikipedia.org/wiki/Bone_marrow

http://en.wikipedia.org/wiki/Chemotherapy

http://en.wikipedia.org/wiki/AIDS

http://en.wikipedia.org/wiki/Immunodeficiency

http://en.wikipedia.org/wiki/Death

http://en.wikipedia.org/wiki/Morbidity


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


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


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


CHAPTER 3

Aims and Scope of Study

Production of the Bacteriocin from Natural Lactic Acid fermented

vegetables.

Purification, Biochemical, Stability of the Bacteriocin.


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),


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.


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.


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)



Iodine 1.0g

Potassium Iodine 3.0

Table 4

Saffranin ( Counter Stain)


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


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


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


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


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


a. Salmonella typhi




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


a. Salmonella typhi



d. Candida

e. Bacillus Cerus


Then form well, inoculated the sample in well & keep it for

incubation in incubator at 37ºc for 24 hrs.

CHAPTER 4


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.


Fig 1:Incoculated in minimal media


Fig 2: Incoculated in minimal media


Fig 3:growth of bacteriocin(tomato) on MRS media


Fig 4: Growth of bacteriocin(garlic) on MRS broth

Fig 5: Growth of bacteriocin(cucumber) on MRS broth


Fig 6: Pure culture of cumcumber

Fig 7:Pure culture of garlic and tomato


Fig 8: Pure culure of tomato


Fig 9: Gram staining of bactreiocin (cumcumber) Lactobacillus acidophillus


Fig 10: Gram staining of bactreiocin (tomato) Bacillus spp


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,


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.


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


Fig 11: inhibition zone of antimicriobal activity


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


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


Fig: urease test

Fig 13: indole test


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.


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


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


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.


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


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.


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


Fig 19: effect of pH and zone of inhibtion


Fig 20: effect of pH and zone of inhibtion


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.


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.


C HAPTER 7

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