STUDIES ON PROBIOTICS AND ANTIMICROBIAL PROPERTIES OF...

STUDIES ON PROBIOTICS AND ANTIMICROBIAL

PROPERTIES OF LACTIC ACID BACTERIA ISOLATED FROM

MARINE FISH, SHRIMP AND SOUTH INDIAN FERMENTED

FOODS

Thesis Submitted to Pondicherry University for the Degree of

DOCTOR OF PHILOSOPHY

By

R. SATISH KUMAR, M.Sc.,

Department of Biotechnology

School of Life Sciences

Pondicherry University

Pondicherry 605014

INDIA

December 2011

PONDICHERRY UNIVERSITY

DEPARTMENT OF BIOTECHNOLOGY

SCHOOL OF LIFE SCIENCES

PONDICHERRY-605014

INDIA

DR. V. ARUL

Associate Professor

CERTIFICATE

Certified that this thesis entitled “Studies on probiotics and antimicrobial properties of lactic

acid bacteria isolated from marine fish, shrimp, and South Indian fermented foods” is a

record of research work done by the candidate Mr. R. Satish Kumar during the period of his

study in the Department of Biotechnology, School of Life Sciences, Pondicherry University,

under my supervision and that it has not previously formed the basis of the award of any degree,

diploma, associateship or fellowship.

Pondicherry

Date: (V. ARUL)

Phone: 91-413-2654429 (Off.) Fax: 91-413-2655715/2655265

91-413-2357492 (Res.) Email: [email protected]

DECLARATION

I hereby declare that the work presented in this thesis has been carried out by me under the

guidance of Dr. V. Arul, Associate Professor, Department of Biotechnology, School of Life

Sciences, Pondicherry University, Pondicherry, and this work has not been submitted elsewhere

for any other degree.

Pondicherry

Date: R. SATISH KUMAR

DEDICATED TO MY

BELOVED PARENTS

CONTENTS

S.NO. CHAPTERS PAGE NO.

1 GENERAL INTRODUCTION 1

2 REVIEW OF LITERATURE 5

3 MATERIALS AND METHODS 29

4 ISOLATION AND SCREENING OF LACTIC ACID BACTERIA

FROM SOUTH INDIAN FERMENTED FOODS

59

5 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN

PRODUCED BY STREPTOCOCCUS PHOCAE PI80

71


PRODUCED BY ENTEROCOCCUS FAECIUM MC13

88


PRODUCED BY LACTOBACILLUS PLANTARUM AS1

101

8 IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA

STRAINS FOR PROBIOTIC CHARACTERISTICS

114

9 LACTOBACILLUS PLANTARUM AS1 BINDS TO CULTURED

HUMAN INTESTINAL CELL LINE HT-29 AND INHIBITS CELL

ATTACHMENT BY ENTEROVIRULENT BACTERIUM VIBRIO

PARAHAEMOLYTICUS

126

10 LACTOBACILLUS PLANTARUM AS1 ISOLATED FROM SOUTH

INDIAN FERMENTED FOOD KALLAPPAM SUPPRESS 1, 2-

DIMETHYL HYDRAZINE (DMH) INDUCED COLORECTAL

CANCER IN MALE WISTAR RATS

140

11 SUMMARY 158

12 REFERENCES 161

13 PUBLICATIONS 200

ACKNOWLEDGEMENTS

Foremost, I would like to express my heartfelt gratitude to my supervisor Dr. V. Arul, Associate

Professor, Department of Biotechnology for the continuous support of my Ph.D., study and

research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped

me throughout my research and writing thesis.

Besides my supervisor, I would like to thank my doctoral committee members: Prof. Priya

Davidar and Dr. N. Arumugham, for their encouragement, insightful comments, and hard

questions.

I express my sincere gratitude to Prof. N. Sakthivel, Head, Department of Biotechnology for

extending lab facilities and valuable suggestions.

My special thanks go to Dr. C. Thirunavukarasu, Assistant Professor, Department of

Biochemistry and Molecular Biology, for his suggestions and guidance in animal studies.

I am thankful to Prof. S. Jayachandran, Dr. Sudhakar, Dr. Hanna Rachel Vasanthi, Dr.

Prashant, Dr. Arun Kumar Dhayalan, Dr. Venkateswara Sarma, and Mr. V.

Balasubramanian who have been influential in all my academic pursuits.

I thank my fellow labmates: Dr. P. Kanmani, Mr. N. Yuvaraj, Mr. K.A. Paari, Mr. V.

Pattukumar and Mr. Venkatesh, for the stimulating discussions, immense co-operation,

emotional support, and for all the fun we had in the last four years.

I genuinely thank my seniors, Dr. A. Gopalakannan, Dr. S. Isaac Kirubakaran, Dr.

Krishnaveni, Dr. P. Ravindra Naik, Dr. S. Vaithinathan, Dr. N. Badrinarayanan and

colleagues Mr. G. Raman, Mr. Jean Cletus, Mr. Kennedy, Mr. Saranathan, Mr. Abhijith,

Mr. Aezaj, Mr. Wasim, Mr. Abid, Mr. Anand, Mr. Suresh, Mr. Manoj, Ms. M. Revathi,

Ms. P. Lalitha, Ms. Moushmi Priya, Ms. Asha, Ms. Pathma, Ms. Veena, Ms. Gokulapriya,

and Ms. Sonia for their immense co-operation and valuable support.

I also thank our office staffs, Mr. Ramalingam, Mr. C. Balakrishnan, Mr. Vadivel, Ms.

Sarala, Ms. Vinothini, Mr. Meiappan, and Ms. Muthammal for their co-operation and help.

I gratefully acknowledge the monetary support given by Indian Council of Medical Research,

New Delhi in the form of Junior/Senior Research Fellowship and Department of

Biotechnology, New Delhi for project funding.

I would like to thank Central Instrumentation Facility (CIF), Pondicherry University for

providing assistance in using necessary instruments.

I wish to thank my brother Mr. Senthil Kumar, sisters Ms. Kavita, Ms. Krishna Veni and their

kids Harshitha, Siddhanth for providing a loving environment for me.

I wish to thank my parents, Ms. Subbulakshmi Ramraj and Mr. K.S. Ramraj for showering

with love and care throughout my life. To them I dedicate this thesis.

Finally, I would like to thank everybody who was important to the successful realization of

thesis, as well as expressing my apology that I could not mention personally one by one.

Date: R. Satish Kumar

LIST OF ABBREVIATIONS

CFU Colony Forming Units

AU Arbitrary Unit

CFCS Cell Free Culture Supernatant

cm Centimeter

DNA Deoxyribose Nucleic Acid

dNTP 2’ deoxynucleotide 5’ triphosphate

EDTA Ethylene Diamine Tetra Acetic acid

MRS de Man Rogosa and Sharpe

SDS Sodium Dodecyl Sulfate

PBS Phosphate Buffered Saline

rpm Revolution Per Minute

TEMED N, N , N - Tetramethylethylenediamine

APS Ammonium Per Sulphate

PCR Polymerase Chain Reaction

UV Ultra Violet

bp Base pairs

g/l Gram per liter

h Hours

kDa Kilo Dalton

V Volt

μl Microliter

μM Micromolar

mg/l Milligram per liter

min Minutes

ml Milliliter

mM Millimolar

mm Millimeter

nm Nanometer

OD Optical Density

w/v Weight per volume

v/v Volume per volume

N Normality

s Second

CHAPTER 1

GENERAL INTRODUCTION

CHAPTER 2

REVIEW OF LITERATURE

CHAPTER 3

MATERIALS AND METHODS

CHAPTER 4

ISOLATION AND SCREENING OF LACTIC ACID

BACTERIA FROM SOUTH INDIAN FERMENTED

FOODS

CHAPTER 5

PURIFICATION AND CHARACTERIZATION OF

BACTERIOCIN PRODUCED BY

STREPTOCOCCUS PHOCAE PI80

CHAPTER 6


BACTERIOCIN PRODUCED BY ENTEROCOCCUS

FAECIUM MC13

CHAPTER 7


BACTERIOCIN PRODUCED BY LACTOBACILLUS

PLANTARUM AS1

CHAPTER 8

IN VITRO CHARACTERIZATION OF LACTIC

ACID BACTERIA STRAINS FOR PROBIOTIC

CHARACTERISTICS

CHAPTER 9

LACTOBACILLUS PLANTARUM AS1 BINDS TO

CULTURED HUMAN INTESTINAL CELL LINE

HT-29 AND INHIBITS CELL ATTACHMENT BY

ENTEROVIRULENT BACTERIUM VIBRIO

PARAHAEMOLYTICUS

CHAPTER 10

LACTOBACILLUS PLANTARUM AS1 ISOLATED

FROM SOUTH INDIAN FERMENTED FOOD

KALLAPPAM SUPPRESS 1, 2-DIMETHYL

HYDRAZINE (DMH) INDUCED COLORECTAL

CANCER IN MALE WISTAR RATS

1

CHAPTER 1

INTRODUCTION

The word ―probiotics‖ was earlier used as an antonym of the word ―antibiotics‖. It is derived

from Greek and termed as ―for life‖ (Hamilton-Miller et al., 2003; Vasiljevic & Shah, 2008).

The concept of probiotics was first proposed by Elie Metchnikoff, a Noble Laureate of the year

1908. He was astonished by the exceptionally long life-span of Bulgarian peasants and

subsequently studied their lifestyle as well as diet. Bulgarian peasants used to consume large

amounts of fermented milk. Metchnikoff postulated that pathogens inside the human gut produce

toxic compounds that steadily weaken the body. Lactic acid bacteria present in the sour milk

successfully defended the body against enteropathogens (Vasiljevic & Shah, 2008). Many

eminent researchers‘ defined probiotics by the manner they observed them. Mostly cited

definition was that of Fuller‘s (1992), who defined them as ―a live microbial feed supplement,

which beneficially affects the host animal by improving its intestinal microbial balance‖.

However, this definition was more applicable to animals than to humans. Another widely used

definition was ―probiotics are mono or mixed cultures of live microorganisms that might

beneficially affect the host by improving the characteristics of indigenous microflora‖ (Holzapfel

et al., 1998). FAO/WHO defined it more precisely as ―live microorganisms which when

administered in adequate amounts confer a health benefit on the host‖ (FAO/WHO, 2002).

Certain strains of bacteria have been discovered over the years to have probiotic properties,

mainly consisting of lactic acid producing bacteria (Lactobacilli, Streptococci, Enterococci,

Lactococci, Bifidobacteria), Bacillus and yeast Saccharomyces and fungi such as Aspergillus.

They chiefly harbor gut of animals, air, water, food, soil etc. These probiotic groups find wide

application nowadays due to valuable products synthesized by them and special antimicrobial,

antioxidant properties. Probiotic cultures are exploited commercially as a tool for the

2

development of novel functional products. It has been estimated that there were approximately

70 probiotic containing products marketed in the world (Shah, 2004). Probiotic organisms are

available commercially in milk, sour milk, fruit juices, ice-cream, oat-based products, Luneber,

Olifus, Bogarde, Progurt etc. The consumption of probiotic based functional dairy products

across West Europe, United States and Japan increased by 12% since 2005 (Zenith International,

2007). Probiotic products are becoming popular in Japan, as more than 53 types of probiotic

containing products have hit the market recently (Vasiljevic & Shah, 2008).

Lactic acid bacteria (LAB) produce a number of antimicrobial substances including organic

acids, hydrogen peroxide, bacteriocins, and bacteriocin-like substances. Bacteriocins or

bacteriocin-like substances are peptides or proteins, which exhibit inhibitory activity against

sensitive strains of bacteria. Bacteriocins confer important defence systems against other

microorganisms. Bacteriocins differ from usual antibiotics in that they are ribosomally

synthesized while antibiotics are generally secondary metabolites (Rodriguez et al., 2002). Also,

antibiotics inhibit microorganisms by diverse mechanism of inhibition such as peptidoglycan

synthesis inhibition, protein synthesis inhibition, and blocking promoters‘ etc., but bacteriocin

generally act over the cell membrane to make pores thus leaking the metal ions K+

, Na+

and

other cellular contents. Although bacteriocins may be produced by Gram-positive and Gram-

negative bacteria, those from LAB are of particular interest due to their potential use in the food

industry as natural safe food preservatives (O ‗Sullivan et al., 2002).

In the present study we explored for certain potential probiotic strains which could be employed

to preclude fish and shrimp pathogens. Shrimp and fish culture is grown as a million dollar

industry and rearing of shrimps and fish in culture system becomes popularized throughout the

world especially in Southeast Asia. Worldwide, major economic losses in cultured shrimp and

3

fish result from a relatively small number of opportunistic pathogenic bacteria (Toranzo et al.,

2005). Vibrio is one of the most important pathogen recognized in larval cultures, provoking a

high mortality (Austin & Zhang, 2006; Paillard et al., 2004). Many pathogenic bacteria become

resistant to antibiotics and hence farmers resort to dump more antibiotics. The residues of

antibiotics are found in most of the seafoods (Aoki, 1975). At the same time, use of prophylactic

antibiotics is detrimental to aquatic and terrestrial environments, animal and human health

(Cabello, 2006; Zhou et al., 2009). That‘s why authorities such as the European Authority have

chosen to limit antibiotic use as a curative situation. In this context, scientific communities have

proposed friendly alternatives such as vaccines (Kurath, 2008), antibiotic substitutes (Dorrington

& Gomez-Chiarri, 2008) or use of probiotics (Kesarcodi et al., 2008). Bacteriocinogenic

bacterial strains appear to be an excellent candidate for an affable alternative since bacteriocin

would be used as an antibiotic substitute (Joerger, 2003), whereas bacteria would be a potential

probiotic (Gillor et al., 2008). Most of the candidate probiont used commercially in aquaculture

are heterolactic i.e., strains isolated from source other than host animal. In our lab, attempt was

made to use homolactic strains i.e., lactic acid bacteria (LAB) isolated from the host gut (fish

and shrimp) and applied for control of diseases during its culturing. We studied the probiotic

properties of LAB strains Streptococcus phocae PI80 and Enterococcus faecium MC13 isolated

from the gut of Penaeus indicus and Mugil cephalus respectively and also characterized its

antimicrobial protein. In vivo studies to evaluate their potential against shrimp and fish pathogens

were carried out by other researchers in the laboratory (Swain et al., 2009; Pattukumar et al.,

2010; Gopalakannan & Arul, 2011).

Furthermore, we isolated some LAB strains from the south Indian fermented foods. Fermented

foods were consumed in many countries since it has disease prevention capacity and improves

4

health. We exploited certain South Indian traditional fermented foods such as Koozh, Kallappam,

and Mor Kuzhambu to isolate LAB strains. Our aim was to employ the isolated LAB against

human diseases and pathogens. From the literature survey it was evident that microbiota

influence important host activities, including the local immune response and several intestinal

metabolic traits (Servin, 2004; Round & Mazmanian, 2009). Probiotics are also known to exert

other health advantages such as improving lactose intolerance, increasing humoral immune

responses, biotransformation of isoflavone phytoestrogen to improve post-menopausal

symptoms, bioconversion of bioactive peptides for antihypertension, and reducing serum

cholesterol level (Liong, 2007). But for therapeutic purposes, probiotics should have certain

features: to be of human origin, safe for the host, and genetically stable (Holzapfelm et al.,

1998). Furthermore, it is important that probiotics, in order to be active, survive passage through

the gastrointestinal (GI) tract irrespective of gastric acids, pancreatic enzymes, and bile acids so

that they can reach the ileum and colon and colonize the intestinal mucosa (Reid et al., 2003).

We pined to investigate all such properties of the potential probiotic strain Lactobacillus

plantarum AS1 before applying it towards colorectal cancer treatment in rodent model.

Colorectal cancer was chosen for this study since it represents a major public health problem

accounting for over 1 million cases and about half a million death worldwide (Chau &

Cunningham, 2006). Diet interventions and natural bioactive supplements have now been

extensively studied to reduce the risks of colon cancer, as a cause of prevention instead of cure.

Postulated mechanisms include reduction in the activity of several cancer causing agents,

desmutagenic and anti-carcinogenic properties (Collins & Gibson, 1999).We also intended to

study the efficacy of L. plantarum AS1 against human pathogen V. parahaemolyticus using HT-

29 cell lines as simulating intestinal epithelial cells.

5

CHAPTER 2

REVIEW OF LITERATURE

2.1. Lactic acid bacteria

Lactic acid bacteria (LAB) are group of Gram-positive bacteria that are devoid of cytochromes

and preferring anaerobic conditions, fastidious, acid-tolerant and strictly fermentative. They are

usually non-motile and non-sporulating bacteria that produce lactic acid. This bacterial group

contains both rods (Lactobacilli and Carnobacteria) and cocci (Streptococci). Different species

of lactic acid bacteria (such as Streptococcus, Leuconostoc, Pediococcus, Aerococcus,

Enterococcus, Vagococcus, Lactobacillus, Carnobacterium) have adapted to grow under widely

different environmental conditions. They are found in the gastrointestinal tract of various

animals, dairy products, seafood products, soil and on some plant surfaces (Ring & Gatesoupe,

1998). Although lactic acid bacteria are not dominant in the normal intestinal microbiota, several

trials have been undertaken to induce an artificial dominance of lactic acid bacteria (Verschuere

et al., 2000). Based on their carbohydrate metabolism LAB are divided into two distinct groups.

The homo-fermentative group utilizes the Embden-Meyerhof-Parnas (glycolytic) pathway to

transform a carbon source chiefly into lactic acid. Hetero-fermentative bacteria produce

equimolar amounts of lactate, CO2, ethanol or acetate from glucose exploiting phosphoketolase

pathway. Homo-fermentative group consist of Lactococcus, Pediococcus, Enterococcus,

Streptococcus. Hetero-fermentative group include Leuconostoc, Weisella (Vasiljevik & Shah,

2008).

2.2. Probiotics

The word ‗probiotics‘ originates from the Greek word ‗for life‘, and is currently used to name

bacteria associated with beneficial effects for humans and animals. According to WHO

6

guidelines probiotics defined as ‗live organisms which when administered in adequate amounts

confer a health benefit on the host‘. Probiotics bacteria were first studied by Elie Metchnikoff, a

Noble laureate of 1908 in the field of medicine. He theorized that proteolytic microbes in the

colon produce toxic substances responsible for the aging process and proposed that consumption

of fermented milk would coat the colon with LABs, decreasing intestinal pH, suppressing

proteolytic bacteria and thus leading to slowing of the aging process (Gordon, 2008).

Metchnikoff and his followers‘ ingested milk fermented with Bulgarian Bacillus and reported

health benefits (Vaughan, 1965). To qualify as a probiotic, certain criteria need to be met by a

bacterium: a bacterial strain must be identified completely, be harmless for ingestion, adhere to

mucosal membrane, able to colonize the gut epithelium, stable when stored, must survive the

acid and bile salt concentration persisting in upper GI tract (Verna & Lucak, 2010). Researchers

have studied and used probiotics in a variety of medical conditions. Bowe & Logan, (2011)

discussed the possibility of probiotics to cure acne vulgaris although there was no suitable trial

conducted up to now. Rerksuppaphol & Rerksuppaphol, (2010) tested Lactobacillus acidophilus

and Bifidobacterium bifidum against acute diarrhea in infants and children aged 2 months to 7

years. Probiotics shortened duration of diarrhea (34.1 and 34.8 h as against 58 h with placebo)

and also reduced the number of stools (7.3 and 8 vs 15.9 with placebo). Probiotics are also

helpful in preventing intestinal barrier dysfunction in acute pancreatitis (Lutgendorff et al.,

2009). Probiotic pre-treatment diminished acute pancreatitis induced increase in E. coli passage

(Probiotics 57.4 vs. placebo 223), Cr-EDTA flux (16.7 vs. 32.1 cm/s 10-6

), apoptosis, lipid

peroxidation (0.42 vs. 1.62 pmol MDA/mg protein). Ouwehand et al., (2009) reported efficacy

of their strain L. acidophilus NCFM in the alleviation of allergic rhinitis. Probiotic strain

Lactobacillus fermentum VR1-033PCC diminished atopic dermatitis in fifty-six children aged 6-

7

18 months and reduced the cases by 54% as compared to only 30% in placebo group. Probiotic

bacteria were also studied for their beneficial effect on autoimmune Encephalomyelitis (Lavasani

et al., 2010), childhood constipation (Bekkali et al., 2007), hypertension (Huey-Shilye et al.,

2009) and found them to be effective.

2.3. Antimicrobial peptide Bacteriocin produced by lactic acid bacteria

Bacteriocins are biologically active protein molecules with a bactericidal mode of action (Tagg

et al., 1976). Bacteriocin may serve as anti-competitors enabling the invasion of strain into an

established microbial community (Margaret & John, 2002). Bacteriocins of Gram positive

bacteria are diverse and their production is not necessarily the lethal event as in the case of Gram

negative bacteria. Some Gram positive bacteria have evolved a bacteriocin specific transport

system whereas others employ sec-dependent export pathway (Margaret & John, 2002). Among

Gram positive bacteria the lactic acid bacteria (LAB) are particularly prolific in bacteriocin

production. Based upon the mass, structure and characteristics, bacteriocins have been divided

into three (Klaenhammer, 1988): Class I bacteriocins are otherwise known as lantibiotics since

they contain post-translationally modified amino acid such as lanthionine and β-

methyllanthionine (Guder et al., 2000). It is further divided into A and B subgroups based on

structure and mode of inhibition (Jung & Sahl, 1991). Type A inhibits bacterial species by

depolarizing the cytoplasmic membrane. They usually range from 21 to 38 amino acids and

larger than type B lantibiotics. Nisin is a type A lantibiotic who is best studied and commercially

exploited bacteriocin. Type B lantibiotics inhibit bacteria by suppressing their enzymes e.g.

mersacidin, it interferes with cell wall biosynthesis (Brotz et al., 1995).

8

Class II LAB bacteriocins are small (30-60 amino acids), heat stable, non-lanthionine containing

peptides (Jung & Sahl, 1991). They have three subgroups a, b and c. Class IIa members share

conserved amino-terminal sequence (YGNGVXaac) and inhibitory activity towards food borne

pathogen Listeria. Example includes Pediocin ACH, Sakacin A, Leucocin A. Class IIb

bacteriocins inhibit target cells by forming pore in the membrane. Example include Lacticin F

and Lactococcin G. Class IIc bacteriocins are sec-dependent e.g. Acidocin B (Leer et al., 1995).

Class III consist of large, heat-labile bacteriocin such as Helveticins J and V (Joerger &

Klaenhammer, 1986; Vaughan et al., 1992). An additional class of bacteriocin is known recently

that require lipid or carbohydrate moiety for their activity e.g. Leuconocin S (Bruno &

Montiville, 1993) and Lactocin (Uprati & Hindsdill, 1975).

2.3.1. Bacteriocin produced by Streptococcus sp.

Most Streptococcus strains are reported to be pathogenic. Subsequently, bacteriocins isolated

from Streptococcus are mostly pathogenic Streptococci (Nes et al., 2007). Also, those

bacteriocins that have been characterized were originated from few species i.e., Streptococcus

salivarius, S. pyogenes, S. macedonicus, S. mutans, S. bovis, S. uberis, S. thermophilus, S. rattus,

S. phocae (Table 1). Most Streptococci bacteriocins were characterized to be lantibiotics among

that cationic type A-lantibiotics are prevalent (Nes et al., 1997). S. salivarius bacteriocin was

first among Streptococci lantibiotic characterized that effectively inhibited human pathogen

Streptococcus pyogenes (Ross et al., 1993). Mutacins are peptide bacteriocins produced by

Streptococcus mutans. Atleast three different lantibiotics namely mutacin I, II and III were

reported from some S. mutans isolates. Mutacin II showed structural resemblance to the lacticin

481 group of type A lantibiotics (Krull et al., 2000). S. mutans UA140 was reported to produce a

9

two peptide class II bacteriocin (mutacin IV). Nisin is most prominent lantibiotic bacteriocin that

was reported from Lactococcus lactis. Recently, nisin U produced by Streptococcus uberis

revealed 78% identity to Nisin A, subsequently considered to be a nisin variant (Wirawan et al.,

2006). Whitford et al., (2001) described a lantibiotic bovicin 255 produced by a Streptococcus

bovis isolated from cow rumen, whereas a class II non-lantibiotic, bovicin HJ50 was reported

from similar source by Xiao and co-workers (Xiao et al., 2004). In another study, two different

peptides bacteriocins named BHT-A and BHT-B were reported from Streptococcus rattus

(Hyink et al., 2005). During S. pyogenes screening 10% of the strains were found to produce

bacteriocins. Consequently, a lantibiotic ‗Streptin‘ was produced to homogeneity that showed a

molecular mass of 2.42 kDa (Wescombe & Tagg, 2003). Streptococcal bacteriocins were

reported from fermented foods. Macedocin is a lantibiotic from S. macedonicus, isolated from

artisan cheese (Georgalaki et al., 2002). Thermophilin 13, a two-peptide class IIb bacteriocin

produced by S. thermophilus, isolated from yoghurt (Marciset et al., 1997). An anti-listerial

bacteriocin phocaecin PI80 was produced by S. phocae PI80, isolated from the gut of Penaeus

indicus (Satish & Arul, 2009).

2.3.2. Bacteriocins produced by Enterococcus faecium

Enterococcus faecium is a Gram-positive, homo-fermentative, lactic acid bacteria that is natural

inhabitant of the gastrointestinal tract. Nevertheless, they are also found in fermented foods and

are frequently isolated from starter cultures and cheese producers (Galvez et al., 1998). E.

faecium bacteriocins have gained attentions in recent years as they could be isolated easily from

several fermented foods and because many of them are active against food-borne pathogens such

as Listeria monocytogenes (Giraffa, 1995). E. faecium T136 was isolated from Spanish dry

10

fermented sausages that produced enterocin A and B. They were active against a wide range of

Gram-positive bacteria, including Listeria and Staphylococci. N-terminal amino acid sequencing

revealed the similarity of enterocin A with pediocin family of bacteriocins whereas enterocin B

showed strong homology to carnobacteriocin A (Casaus et al., 1997). Enterocin P inhibited most

of food-borne Gram-positive pathogenic bacteria, such as L. monocytogenes, S. aureus,

Clostridium perfringens and C. botulinum. It withstood high temperature treatment (121°C for

15 min) as well as wide exposure to pH (2.0 – 11.0), freeze thawing, lyophilization and long-

term storage at 4 and 20°C (Cintas et al., 1997). Enterocin L50, initially referred to as pediocin

L50 is a plasmid encoded broad-spectrum bacteriocin produced by E. faecium L50. It showed

similarity to small group of cytolytic peptides secreted by certain Staphylococci (Cintas et al.,

1998). Enterocin A, enterocin B and enterocin P like bacteriocins were reported from E. faecium

JCM 5804. They inhibited the growth of Clostridium spp., L. monocytogenes, and vancomycin

resistant Enterococcus (Park et al., 2003). Elotmani et al., (2002), Characterized anti-L.

monocytogenes bacteriocin from E. faecium isolated from Raib, a Morrocon tradition fermented

milk. In a similar manner, another research group reported anti-L. monocytogenes bacteriocin-

like inhibitory substance from E. faecium UQ31 (Alvarado et al., 2005). Biochemical and

genetic characterization of enterocin A was performed by Aymerich et al., (1996) and reported

that enterocin A leader sequence contain 18 amino acid residues. This belongs to the double-

glycine leaders which are found among most other small non-lantibiotics bacteriocins, some

lantibiotics and colicin V. Similarly, genetic characterization of enterocin I from E. faecium 6T1a

was performed and it was reported that enterocin I does not belongs to the pediocin family of

bacteriocins (Floriano et al., 1998). E. faecium EK13 produces enterocin A and possess probiotic

properties and proved as a candidate for rabbit probiotics (Laukova et al., 2006).

11

2.3.3. Bacteriocins produced by Lactobacillus plantarum

Lactobacillus plantarum are isolated from various sources but more prominently from fermented

food products. Todorov et al., (2007), isolated L. plantarum AMA-K from naturally fermented

milk and characterized its bacteriocin AMA-K. Bacteriocin production is stimulated by the

presence of Listeria innocua. L. plantarum AMA-K grows in milk and produces only 800 AU/ml

after 24 h. Similarly, bacteriocin ST8KF produced by a kefir isolate L. plantarum ST8KF was

characterized by Powell et al., (2007). Bac ST8KF was heat resistant (121°C for 20 min), and

showed bacteriostatic mode of activity. Leal et al., (1998), studied bacteriocin production and

competitiveness of L. plantarum LPC010 in olive juice broth. Todorov et al., (2011), for the first

time isolated bacteriocinogenic L. plantarum ST16Pa from papaya (Carica papaya). It was

active against Pseudomonas, Streptococcus, Staphylococcus, Listeria spp. In another report, L.

plantarum NCIM 2084 was attempted to grow in a simple glucose broth to produce bacteriocin.

Its bacteriocin Planatricin LP84 exhibited a bactericidal and lytic effect against Bacillus cereus

F4810 and Escherichia coli D21 (Suma et al., 1998). A bacteriocin produced by L. plantarum

ATCC8014 showed inhibition of Staphylococcus aureus, E. coli, L. innocuo, P. aeruginosa. Its

apparent molecular weight based on SDS-PAGE analysis was 122 kDa (Lash et al., 2005). Hata

et al., (2010) characterized Plantaricin ASM1 from L. plantarum A-1. It showed stability in a

wide pH range, heat and finally concluded as potential food bio-preservative. Xie et al., (2011)

reported an anti-listerial Pediocin LB-B1 produced by L. plantarum LB-B1, isolated from

Koumiss, a fermented dairy product from China. The gene cluster encoding Pediocin LB-B1

showed 99.8% homology with the operon encoding Pediocin PA-1, suggesting that the two

bacteriocins are identical.

12

2.4. Probiotics in aquaculture

Bacteria are found in every small corner of the aquatic environment. Aquatic animal egg is the

first stage of their life-cycle that could be exposed to bacteria. Therefore, a relatively dense,

nonpathogenic, and diverse adherent microbiota present on the eggs would probably be an

effective barrier against the formation of a colony by pathogens on fish eggs. In addition, the

establishment of a normal gut microbiota may be regarded as complementary to the

establishment of the digestive system, and under normal conditions it serves as a barrier against

invading pathogens (Farzanfar, 2006). Larvae may ingest substantial amounts of bacteria. It is

obvious that the egg microbiota will affect the primary colonization of the larvae (Verschuere et

al., 2000). Kennedy et al., (1998) used probiotic bacteria in the culture of marine fish larvae.

They identified and used probionts for the culture of common snook, red drum, spotted sea trout

and striped mullet. They then observed that the application of probiotic bacteria to larval fish

tanks (from egg through transformation) increased survival, size uniformity, and growth rate.

The periodic addition of bacteria to the tanks altered the microbial communities of both tanks

and fish. In addition, they noticed that the fish eggs incubated with probiotic bacteria were less

likely to develop bacterial overgrowth and die than those incubated without probiotic bacteria.

Carnevali et al., (2004) isolated Lactobacillus fructivorans (AS17B) from sea bream (Sparus

aurata) gut, and then administered it during sea bream development using Brachinons plicatilis

and/or Artemia salina and dry feed as vectors. At the end of the experiments, they found a

significantly decreased larvae and fry mortality in their treated groups. Previously, Gildberg et

al., (1997) had analyzed the effect of a probiotic of lactic acid bacteria in the feed of Atlantic cod

fry (Gadus morha) on growth and survival rates. In their study, a dry feed containing lactic acid

bacteria (Carnobacterium divergens) that had been isolated from adult intestines was given to

13

cod fry. After 3 weeks of feeding the fry, they were exposed to a virulent strain of Vibrio

anguillarum. The number of death was recorded during a further 3 weeks of feeding with feed

supplemented with lactic acid bacteria. A certain improvement in disease resistance was

obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in

surviving fish given feed supplemented with lactic acid bacteria. Lara-Flores et al., (2003) used

two probiotic bacteria and the yeast, Saccharomyces cerevisiae as growth promoters in Nile

tilapia (Oreochromis niloticus) fry. The results of this study indicated that the fry subjected to

diets with a probiotic supplement exhibited greater growth than those fed with the control diet. In

addition, they suggested that the yeast is an appropriate growth-stimulating additive in tilapia

cultivation. Gopalakannan & Arul, (2011) isolated Enterococcus faecium MC13 from the gut of

grey mullet Mugil cephalus and studied their protective effect on Cyprinus carpio after

challenging with pathogen Aeromonas hydrophila. In their study, fish group treated with

probiotic showed reduced mortality (22%) and also fish were healthy but untreated fish group

resulted in 100% mortality.

2.5. Indian traditional fermented foods as source of lactic acid bacteria

Traditional fermented foods are popular products since early history that have formed an integral

part of the diet and it can be prepared in the household or in cottage industry using relatively

simple techniques and equipment (Aidoo et al., 2006). Fermentation was evolved as a

preservation technique during lean periods and prevention technique to counter spoilage of food

products. It is one of the oldest and most economical methods for producing and preserving

foods. In addition to preservation, fermented foods can also have the added benefits of enhancing

flavor, increased digestibility, improving nutritional and pharmacological values (Jeyaram et al.,

14

2009). Lactic acid bacteria perform an essential role in the preservation and production of

wholesome fermented foods. Homo-fermentative and the hetero-fermentative lactic acid bacteria

are generally fastidious on artificial media but they grow readily in most food substrates and

lower the pH rapidly to a point where other competing organisms are no longer able to grow.

Leuconostocs and lactic Streptococci generally lower the pH to 4.0-4.5 and some of the

Lactobacilli and Pediococci to about 3.5 (Steinkraus, 1983). Lactic acid bacteria (LAB)

comprise large part of probiotic microflora. There are many LAB strains that have obtained

―generally regarded as safe‖ (GRAS) status and used commonly in commercial food products for

human consumption. Probiotics are mono or mixed cultures of live microorganisms that might

beneficially affect the host by improving the characteristics of indigenous microflora (Holzapfel

et al., 1998). Lactic acid bacterial genera consist of Lactobacillus, Lactococcus, Enterococcus,

Streptococcus, Pediococcus, Leuconostoc, Wesiella etc.

India is traditionally rich in fermented foods. In the Indian subcontinent, making use of

fermented food using local food crops and other biological resources are very common. But the

nature of the products and base material vary from region to region (Sekar & Mariappan, 2007).

Fermented foods like idli and dahi were described as early as 700 BC. At present there are

hundreds of fermented foods with different base materials and preparation methodology. Each

fermented food is associated with unique group of microflora which increases the level of

proteins, vitamins, essential amino acids and fatty acids. However, fermented foods are still

produced traditionally by spontaneous fermentation and only limited knowledge has been

obtained regarding the microflora of these products (Jeyaram et al., 2009). Based upon the basic

ingredients used, fermented foods have been divided into 7 major types (Sekar & Mariappan,

2007): (i) cereal based (with/without pulses) fermented foods, (ii) cereal/pulse and butter milk

15

based fermented food, (iii) cereal based fermented sweets and snacks, (iv) milk based fermented

foods, (v) vegetable, bamboo shoot and unripe fruits based fermented foods, (vi) meat based

fermented foods, and (vii) pulse based fermented foods.

2.5.1. Cereal based (with/without pulses) fermented foods

Cereal based fermented foods are considered as staple foods in their respective regions. Cereals

such as rice (Oryza sativum), ragi flour (Eleusine coracana), wheat flour (Triticum spp.) and

barley flour (Hordeum vulgare) are predominantly used and pulses such as black gram dhal, red

gram, green gram dhal are used. These cereals and legumes are cultivated in India since Indus

valley civilization (9000-5500 BC) period (Samanta et al., 2011). They are considered as

effective substrates for the production of probiotic-incorporated functional food, as they can be

used as a source of non-digestible carbohydrates which stimulate the growth of Lactobacilli and

Bifidobacteria. They contain water soluble fibres like β-glucan, arabinoxylan, galacto-

oligosaccharides and fructooligosaccharides, which are prebiotics (Swennen et al., 2006).

Cereals and legumes are fermented by several groups of bacteria in the large intestine, yielding a

variety of fermentation products, particularly short-chain fatty acids (SCFA). The resulting

SCFA are known to provide an acidic environment in the large intestine, which stimulates the

proliferation of probiotic cultures (Roopashri & Vardaraj, 2009; Macfarlane et al., 2006). Mostly

batter is prepared from these basic ingredients and batter is left overnight at room temperature

for fermentation, occasionally sodium bicarbonate is added to provide anaerobic conditions for

the growth of yeast and lactic acid bacteria. During the preparation of Kallappam fermented

toddy is added as additional source of LAB. Fermented batter is either prepared as steamed cakes

(idli) or pan cakes (dosa, appam) before it gets too soured. Predominant microflora isolated from

16

batter of these foods include: Weissella paramesenteroides, Lactobacillus fermentum, L.

plantarum, Streptococcus faecalis, Pediococcus acidilactici, P. cerevisiae, Leuconostoc

mesenteroides (Table 1). L. plantarum AS1 isolated from south Indian fermented food

Kallappam successfully prevented colonization of enterovirulent bacterium Vibrio

parahaemolyticus in HT-29 cell line (Satish et al., 2011).

2.5.2. Cereal/pulse and butter milk based fermented food

Buttermilk is an additional source of lactic acid bacteria in this type of fermented foods.

Although there are very few dishes reported in this category, these are very popularly consumed

in most part of India. Wiesella paramesenteroides isolated from Mor kuzhambhu showed

antibacterial activities towards food borne pathogens Salmonella typhi and Listeria

monocytogenes (Satish et al., 2010). Blandino et al., (2003) reported non-lactic acid bacteria

Bacillus sp., Micrococcus sp. in rabdi (Table 1).

2.5.3. Cereal based fermented sweets and snacks

These foods are consumed mostly during festival times and other special occasions. Wheat, rice

and barley flours are predominantly used cereals. Sugar or salt is added compulsorily in all food

items and this selects only those microbes which can survive low water activity. Pathogens are

discouraged and it allows growth of sugar/salt tolerant yeast and lactic acid bacteria. Fermented

sweets and snacks are popularly consumed throughout India, consequently many reports are

available for this category but only few reports are available on microflora isolation. L.

fermentum, L. buchneri, L. plantarum, L. acidophilus, L. mesenteroides, Lactococcus lactis,

Streptococcus lactis and S. faecalis were isolated from this class of fermented foods (Table 1).

17

2.5.4. Milk based fermented foods

Milk and milk based products are consumed most popularly due to their nutritive value. For the

same reason milk is easily spoiled by pathogenic microorganisms, hence fermentation of milk

using lactic acid bacteria is preferred for prevention. Lactic acid bacteria convert milk sugar

lactose into lactic acid and produces antibacterial substance bacteriocin to suppress spoilage

bacteria. Dahi or curd is most popular traditional Indian fermented product prepared by

fermentation of milk by lactic acid bacteria. Dahi differs from yogurt in its use of mixed starters

of mesophilic lactococci. A principle flavour-inducing metabolite is diacetyl, which is

appreciated more by people of South Asian origin compared to the acetaldehyde flavour in

yogurt (Yadav et al., 2007b). Yak (Bos grunniens; now Poephagus grunniens) is one of a few

domesticated animals capable of surviving in extreme environmental conditions. It is mainly

found in the highlands of the Nepalese Himalayas, India (Kashmir and Arunachal Pradesh),

China (Tibetan highlands), Mongolia and Bhutan. The composition of yak milk is 16.9–17.7 g/l

dry matter, 49–53 g/l protein, 55–72 g/l fat, 45–50 g/l lactose and 8–9 g/l minerals (Prashant et

al., 2009). Yak milk is processed into a number of dairy products such as fermented milk

(Kurut), cheese (Chhurpi), Chhur churpen, Churkham, Chhu, Philuk, Shyow and Maa. The

chemical composition of yak cheese is around 68.2% of total solid (TS), 49.4% of butterfat on a

dry matter basis and 1.37% of salt. It is largely consumed in the Himalayan highland and its

industrial production is not yet standardized (Prashant et al., 2009).

LAB species isolated from fermented milk products include Streptococcus cremoris, S. lactis, S.

thermophilus, Lactobacillus bulgaricus, L. acidophilus, L. helveticus, L. cremoris, L. plantarum,

L. curvatus, L. fermentum, L. paracasei subsp. pseudoplantarum, L. alimentarius, L. kefir, L.

hilgardii, Enterococcus faecium, Leuconostoc mesenteroides, L. farciminis, L. brevis,

18

Lactococcus lactis subsp. cremoris, L. casei subsp. casei and L. bifermentans (Table 1). There

are reports that LAB isolated from dahi can be used to cure intestinal disease such as diarrhea

(Agarwal & Bhasin, 2002), intake of dahi has anti-cholesteremic (Sinha & Sinha, 2000), anti-

carcinogenic (Arvind et al., 2010), anti-diabetic (Yadav et al., 2007a), angiotensin-converting

enzyme inhibition effect (Harun-ur-Rashid et al., 2007) and anti-atopic dermatitis effect

(Watanabe et al., 2009). Mitra et al., 2007, isolated Lactococcus lactis from dahi which

produced nisin like (Nisin Z) bacteriocin that inhibited important food pathogens Listeria

monocytogenes and Staphylococcus aureus.

2.5.5. Vegetable, bamboo shoot and unripe fruits based fermented foods

The lactic acid fermentation of vegetables, applied as a preservation method for the production

of finished and half-finished products, is considered as an important technology because of its

capability to improve the nutritive value, palatability, acceptability, microbial quality and shelf

life of the fermented product (Kingston et al., 2010). Moreover, this is a remarkable procedure to

store the perishable vegetable in absence of cold-storage or refrigeration, where majority of rural

people cannot afford canned or frozen foods. Certain fermented vegetable products (gundruk,

sinki, iniziangsang) are said to be good appetizers and the ethnic people use these foods for

remedies from indigestion (Tamang & Tamang, 2009). Fermented bamboo shoot (BSs) products

are consumed as a traditional food by ethnic people of North-Eastern states of India (Tamang et

al., 2009). In India, BSs are harvested annually in Sikkim (26.2 t), Meghalaya (435 t) and

Mizoram (426.8 t). Bamboo shoots are low in fat and cholesterol, but very high in potassium,

carbohydrates and dietary fibres. Many nutritious and active materials (vitamins and amino

acids) and antioxidants (flavones, phenols and steroids) can be extracted from bamboo shoots

19

(Choudhury et al., 2011). LABs are the dominant microorganisms in ethnic fermented vegetables

and bamboo shoot products (Tamang et al., 2009). Pediococcus pentasaceous, Lactobacillus

cellubiosus, L. plantarum, L. fermentum, L. brevis, L. mesenteroides, Lactococcus lactis,

Enterococcus faecium, and P. acidilactici are predominant LAB species found in fermented

vegetables (Table 1). Tamang et al., (2009) determined the functional properties of lactic acid

bacteria isolated from ethnic fermented vegetables (gundruk, sinki, khalpi and inziangsang) of

the Himalayas. LAB strains showed strong acidification and coagulation activities. They showed

antimicrobial activity, particularly a strain L. plantarum isolated from inziangsang, a fermented

leafy vegetable product, was inhibitory towards Staphylococcus aureus and Pseudomonas

aeruginosa. LAB strains showed various enzymatic activities such as alkaline phosphatase,

esterase, lipase, leucine arylamidase, valine arylamidase, cysteine-arylamidase, acid phosphatase,

napthol-AS-B1-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-

glucosidase, N-acetyl-β-glucosaminidase and also degraded oligosaccharides. Some strains of L.

plantarum showed more than 70% hydrophobicity and adherence to the mucus secreting HT-29

MTX cells. L. plantarum isolated from ayurvedic medicinal food Kanji or Kanjika is a potential

source of Vitamin B12 (Madhu et al., 2010). During fermentation of radish tap root product sinki

L. plantarum utilizes mannitol to remove the bitter flavor from finished product (Tamang &

Sarkar, 1993). Bamboo shoot based fermented foods contain Lactobacillus plantarum, L. brevis,

L. corniformis, L. delbrueckii, L. fermentum, Leuconostoc fallax, Lactococcus lactis, L.

mesenteroides, Enterococcus durans, Streptococcus lactis, L. casei and Tetragenococcus

halophilus as predominant LAB species, they also showed functional probiotic properties

(Tamang et al., 2009)

20

2.5.6. Meat based fermented foods

Meat is highly susceptible to microbial spoilage. Drying, smoking or fermentation of meat is

critical steps in the traditional processing of meat (Oki et al., 2011). In India, North-Eastern

region people ferment meat of yak, goat, pig, fish and crab to preserve for longer period.

Kargyong is an ethnic sausage-like fermented product prepared from yak, beef and pork meat.

Three varieties of Kargyong are prepared and consumed: yak kargyong (prepared from yak

meat), lang kargyong (prepared from beef) and faak kargyong (prepared from pork). Yak

kargyong is a popular fermented sausage in Sikkim, Ladak, Tibet, Arunachal Pradesh and

Bhutan in the Himalayas (Rai et al., 2010). Fermented fish products are important dietary

components in the protein deficient far East especially in southeast Asia. Preservation of fish by

salt is an age old technology. This method of preservation still enjoys popularity in many

developing countries owning to its simplicity and low cost of processing. When fatty fishes are

salted there is usually a certain degree of fermentation involved. Fermentation of fish is brought

about by autocatalytic enzymes from fish and microorganisms in the presence of high salt

concentration (Majumdar & Basu, 2010). Lactococcus lactis subsp. cremoris, Lactococcus

plantarum, Enterococcus faecium, Lactobacillus fructosus, L. amylophilus, L. corneformis subsp.

torquens are predominant LAB species reported in fermented fish. Lactobacillus sake, L.

curvatus, L. divergens, L. carnis, L. sanfrancisco, Leuconostoc mesenteroides, E. faecium, L.

plantarum, L. brevis, Pediococcus pentosaceous are reported in fermented meat products of

Eastern Himalayas (Table 1). These LAB showed inhibitory activity towards Klebsiella

pnemoniae which is a contaminant of stored meat. Also, they demonstrated probiotic characters

such as enzymes production and hydrophobicity (Rai et al., 2010).

21

Table 1: Lactic acid bacteria isolated from Indian fermented foods

Fermented food Usual

Composition/Ingredi

ents

Place of

Origin/Us

age

Lactic acid Bacteria Isolated Reference

Cereal Based (with/without pulses) Fermented Foods

Koozhu Eleusine coracana

(ragi) flour, boiled

rice, non-fat yoghurt

Tamil

Nadu

Weissella paramesenteroides,

Lactobacillus fermentum

Satish et al.,

2010

Pazhaiya soru Rice, curd and salt Tamil

Nadu

Streptococcus faecalis,

Pediococcus acidilactici

Ramakrishnan,

1977; 1979

Idli Rice, black gram

dhal, table salt,

fenugreek seeds

South

India

Leuconostoc mesenteroides, S.

faecalis, P. cerevisiae

Mukherjee et al.,

1965, Steinkraus

et al., 1967

Dosa Rice, black gram dhal

(either raw or

parboiled rice), table

salt

South

India

L. mesenteroides, S. faecalis Labana, &

Kawatra, 1986,

Chavan, &

Kadam, 1989;

Steinkraus, 1996

Adai Dosa Boiled rice, Bengal

gram, red gram, black

gram, green gram

South

India

Pediococcus sp.,

Streptococcus sp.,

Leuconostoc sp.

Chavan, &

Kadam, 1989

Kallappam

Boiled or raw rice,

coconut toddy

South

India

L. fermentum, L. plantarum

Satish et al.,

2010

Dhokla Bengal gram dhal,

rice and leafy

vegetables

North

India

L. fermentum, L.

mesenteroides, S. faecalis

Ramakrishnan et

al., 1976;

Blandino et al.,

2003, Roy et al.,

2007

Ambali Ragi (Millet) flour

and rice

India L. fermentum, L.

mesenteroides, S. faecalis

Ramakrishnan,

1977; 1979

Cereal/pulse and butter milk based fermented food

Rabdi (Rabadi) Flour of Barley, Pearl

millet, corn or

soybean and country

buttermilk

Rajasthan Bacillus sp., Micrococcus sp. Blandino et al.,

2003

Mor Kuzhambhu Butter milk, gram

flour, vegetables,

spices

Tamil

Nadu

Weissella paramesenteroides Satish et al.,

2010

Cereal based fermented sweets and snacks

Jilebi Wheat, sugar and

curd

South

India

L. fermentum, S. lactis, L.

buchneri, S. faecalis

Ramakrishnan,

1977, Prakash et

al., 2004

Bhaturu or

Indigenous bread

Wheat and starter

material Khameer/

Malera

Himachal

Pradesh

L. plantarum, L. acidophilus,

L. mesenteroides,

Lactococcus lactis

Tamang, 1998,

Thakur et al.,

2004; Kanwar et

22

al., 2007

Milk based fermented foods

Curd (Dahi,

Thayir)

Milk India S. cremoris, S. lactis,

S.thermophilus,

L. bulgaricus, L. acidophilus,

L. helveticus, L. cremoris,

Pediococcus pentosaceous, P.

acidilactici, W. cibara, W.

paramesenteroides, L.

fermentum, L. plantarum,

Lactobacillus delbrueckii

subsp. indicus

Srinivasan &

Banerjee, 1946;

Steinkraus, 1996;

Patil et al., 2010;

Davies, 1940;

Dellaglio et al.,

2005

Chhurpi or Durkha

or churapi

Yak milk is preferred

for making this

cheese although any

other fresh milk may

be used

Arunachal

Pradesh

L. plantarum, L.curvatus, L.

fermentum, L. paracasei

subsp. pseudoplantarum, L.

alimentarius, L. kefir, L.

hilgardii, Enterococcus

faecium and Leuconostoc

mesenteroides, L. helveticus

Tamang &

Sarkar, 1988;

Tamang, 1998,

Tamang et al.,

2000; Singh et

al., 2007a;

Tiwari, &

Mahanta, 2007;

Prashant et al.,

2009

Chhu Yak or cow milk Sikkim L. farciminis, L. brevis, L.

alimentarius, Lactococcus

lactis subsp. cremoris

Dewan &

Tamang 2007b

Philu or Philuk Cow or yak milk Sikkim L. casei subsp. casei,

L.bifermentans

and Enterococcus faecium

Dewan &

Tamang 2007a

Shyow Cow/yak milk Sikkim L.bifermentans, L. paracasei

subsp. pseudoplantarum

Dewan &

Tamang 2007a

Mohi Cow milk Sikkim L. alimentarius, Lactococcus

lactis subsp. lactis, L. lactis

subsp. cremoris

Dewan &

Tamang 2007a

Somar Cow milk Sikkim L. paracasei subsp.

pseudoplantarum

Dewan &

Tamang 2007a

Khadi Buttermilk/curd Gujrat Pediococcus sp. Sukumar &

Ghosh 2010

Vegetable, Bamboo shoot and unripe fruits based fermented foods

Gundruk Leaves of

mustard/radish/

cauliflower

Arunachal

Pradesh

Pediococcus pentasaceous, L.

fermentum, L. casei, L. casei

subsp pseudoplantarum, L.

plantarum

Dahal et al.,

2005; Singh et

al., 2007b;

Tamang &

Tamang, 2009

Sinki Radish root Northeast

India

L. casei, L. brevis, L.

plantarum, Leuconostoc

fallax, L. fermentum

Tamang &

Sarkar, 1993;

Singh et al.,

23

2007b

Sauerkraut or

Sauerkohi

Cabbage India L. mesenteroides, L.

plantarum

Steinkraus, 1996

Soibum or Soijim Bamboo shoots Manipur,

Nagaland

Lactobacillus plantarum, L.

brevis, L. corniformis, L.

delbrueckii, Leuconostoc

fallax, L. lactis, L.

mesenteroides, Enterococcus

durans,

Streptococcus lactis, Bacillus

subtilis, B. licheniformis, B.

coagulans

Tamang &

Tamang,

2009; Jayaram et

al., 2009

Soidon Bamboo shoots Manipur Lactobacillus brevis,

Leuconostoc fallax, L. lactis,

L. plantarum, Carnobacterium

sp., E. faecium

Tamang &

Tamang, 2009;

Jeyaram et al.

2010

Hiring Bamboo shoots Northeast

India

L. plantarum, Lactococcus

lactis

Singh et al.,

2007b; Tamang

& Tamang, 2009

Ekung Bamboo shoots Manipur Lactobacillus plantarum, L.

brevis, L.

casei, Tetragenococcus

halophilus

Singh et al.,

2007b

Eup Bamboo shoots Arunachal

Pradesh

L. plantarum , L. fermentum Tamang &

Tamang, 2009

Mesu Bamboo shoots Darjeeling

hills and

Sikkim

L. plantarum, L. brevis, L.

curvatus, Leuconostoc

citreum, Pediococcus

pentosaceus

Tamang &

Tamang, 2009

Khalpi Cucumber Sikkim L. brevis, L. plantarum Tamang, 1998;

Tamang &

Tamang, 2010

Goyang Wild plant magane-

saag (Cardamine

macrophylla Willd.)

leaves

Darjeeling

hills and

Sikkim

L. plantarum, L. brevis,

Lactococcus lactis,

Enterococcus faecium,

Pediococcus pentosaceus

Tamang &

Tamang, 2009

Inziangsang Mustard leaves Nagaland,

Manipur

Lactobacillus plantarum, L.

brevis, Pediococcus

acidilactici

Tamang &

Tamang, 2009

Kanji Carrot or beet root,

rice, mustard

North

India

L. pentosus, L.

paraplantarum, L. plantarum

Reddy et al.,

2007; Madhu et

al.,2010;

Kingston et al.

2010

Meat based fermented foods

Ngari Puntius sophore Manipur Enterococcus faecium, L. Thapa et al.,

24

(‗Phoubu‘) Fish and Assam fructosus, L. amylophilus, L. plantarum

2004; Jeyaram et

al., 2009

Hentak Esomus danricus

(Fish), petioles of

Alocasia macrorhiza

Manipur Lactococcus lactis sub sp. cremoris, L. plantarum,

Enterococcus faecium, L.

fructosus, L. amylophilus,

Thapa et al.,

2004; Jeyaram et

al., 2009

Tungtap Danio sp. (Fish) Meghalaya Lactococcus lactis sub sp. cremoris, L. plantarum,

Enterococcus faecium, L.

fructosus, L. corneformis sub

sp. torquens,

Thapa et al.,

2004, Murugkar

&

Subbulakshmi,

2006

Lang kargyong Meat of cattle Eastern

Himalayas

Lactobacillus sake, L. curvatus,

L. divergens, L. carnis, L.

sanfrancisco, Leuconostoc

mesenteroides, E. faecium

Rai et al., 2010

Yak kargyong Meat of yak Eastern

Himalayas

L. plantarum, L. sake, L.casei,

L. curvatus, L. carnis, L.

divergens, L. sanfrancisco, Leu.

mesenteroides, E. faecium

Rai et al., 2010

Faak kargyong Meat of pig Eastern

Himalayas

L. brevis, L. plantarum, L.

carnis, L. mesenteroides Rai et al., 2010

Kheuri Yak/beef meat Sikkim Not reported Rai et al., 2009

Lang satchu Red meat of beef Sikkim L. casei, L. carnis, Pediococcus pentosaceous

Rai et al., 2010

Yak satchu Red meat of yak Sikkim E. faecium, P. pentosaceous Rai et al. 2010

Suka Ko Masu Red meat of buffalo

or goat

Darjeeling

hills and

Sikkim

L. plantarum, L. carnis, E.

faecium Rai et al., 2010

Chilu Yak/beef/lamb meat Sikkim Not reported Rai et al., 2009

Chartayshya Red meat of cattle Western

Himalayas

Enterococcus hirae,

Pediococcus pentosaceous,

Weissella cibaria

Rai et al., 2009;

Oki et al., 2011

Geema Red meat of cattle Western

Himalayas

Enterococcus durans, E. hirae,

Leuconostoc mesenteroides, L.

citreum, Pediococcus

pentosaceous

Oki et al., 2011

Arjia Red meat of cattle Western

Himalayas

Enterococcus hirae, E. faecalis,

Pediococcus pentosaceous Oki et al., 2011

Pulse Based Fermented Foods

Kinema Soybeans Darjeeling

Sikkim

Enterococcus faecium

Kiers et al.,

2000; Sarkar et

al., 2002,

Tamang, 2003;

Singh et al.,

2007b

Tungrymbai Soybeans Meghalaya Enterococcus faecium Dike, & Odunfa,

2003, Murughar,

25

2.5.7. Pulse based fermented foods

Black gram, soyabean, Bengal gram, red gram and green gram are most commonly used pulses

in this type of fermented foods. Soyabean (Glycine max) is a summer leguminous crop, grown

under rain fed conditions in upland terraces as a sole crop as well as mixed crop with rice and

maize up to an elevation of 1,500 m in North-East India. Due to increased Mongolian population,

they consume different fermented foods of soyabean as a tradition. Food researchers have

documented number of soyabean based Indian fermented foods (Tamang et al., 2009).

Fermented soyabean food is an economical source of plant protein as compared to animal and

milk products on the basis of protein cost per kg, which is easily accessible to rural poor of

North-East region. A remarkable increase in free amino acids, mineral contents, vitamin-B

complex and antioxidant activity were reported during kinema fermentation (Sarkar et al., 1997;

Tamang & Nikkuni, 1998; Tamang et al., 2009). Increase in carotene and folic acid has been

& Subblakshmi,

2006; Tamang et

al.,

2009; Sohliya et

al., 2009

Wadi Black gram and oil Punjab,

West

Bengal

L. mesenteroides, L.

fermentum

Batra & Millner,

1974; Sandhu et

al., 1986;

Sandhu & Soni

1989; Aidoo et

al., 2006

Wari Black bean and

soybean

Uttar

Pradesh

Lactobacillus bulgaricus

Streptococcus thermophilus

Tewary &

Muller, 1989,

Tewary &

Muller, 1992;

Kulkarni et al.,

1997

Masyaura Blackgram or

greengram, Colocosia

tuber, ashgourd or

radish

Darjeeling

hills and

Sikkim

Pediococcus pentosaceous,

Pediococcus acidilactic, and

Lactobacillus sp.

Dahal et al.,

2005; Dahal et

al., 2003.

26

reported in tungrymbai (Murungkar & Subbulakshmi, 2006). LAB reported includes

Enterococcus faecium, L. mesenteroides, L. fermentum, Lactobacillus bulgaricus, Streptococcus

thermophilus, Pediococcus pentosaceous and P. acidilactici (Table 1).

2.6. Adherence of lactic acid bacteria to intestinal cell line and inhibition of pathogen

adherence

HT-29 and Caco-2 cells are human intestinal cell lines expressing morphologic and physiologic

characteristics of normal human enterocytes and these have been exploited to elucidate the

mechanisms mediating enteropathogen adhesion. More recently, these cell lines were used to

select and subsequently assess lactic acid bacteria on the basis of their adhesion properties

(Dunne et al., 2001). Nevertheless, they have also been used to study inhibition of pathogen

adhesion by lactic acid bacteria. Spurbeck & Arvidson, (2010) studied the inhibitory effect of

lactic acid bacteria strain Lactobacillus jensenii over pathogenic bacterium Neisseria gonorrhoea

during adherence to epithelial cells. Inhibitory protein produced by Lactobacillus jensenii blocks

gonococcal binding to extracellular matrix component. Similarly, probiotics attenuated

Campylobacter jejuni association with and internalization into E12 cells and translocation to the

basolateral medium of transwells (Alemka et al., 2010). Also, the probiotic agents L. plantarum

299v and L. rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing

pathogenic E. coli to HT-29 cells (Mack et al., 1999). In another experiment, Lactobaillus

rhamnosus GG reduced morphological changes and diminished the number of A/E lesions

induced in response to EHEC 0157:H7 infection. With probiotic pre-treatment there was

corresponding attenuation of EHEC-induced drop in electrical resistance and the increase in

barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayer against

EHEC-induced redistribution of the Claudin-I and Zo-I tight junction proteins (Johnson-Henry et

27

al., 2008). Coconnier et al., (1992) with the help of scanning electron microscope determined

that L. acidophilus BG2F04 interacted with the well-defined apical microvilli of Caco-2 cells

with cell damage and with mucus secreted by the sub-population of HT-29MTX-cells.

Adlerberth et al., (1996) defined a mannose specific adherence mechanism in L. plantarum

conferring binding to the human colonic cell line HT-29.

2.7. Protective Roles of Probiotics on Colon Cancer

CRC is the second most common cause of mortality from malignant disease in Europe with 1,90,

000 new cases per year. Prognosis for advanced CRC is poor (Sant et al., 1995) and hence

prevention is required to control the incidence of the disease. Many studies confirm the

involvement of the endogenous microflora in the onset of colon cancer. This makes it reasonable

to think that changing the intestinal microflora could influence tumour development. Many

studies confirm the involvement of the endogenous microflora in the onset of colon cancer. This

makes it reasonable to think that changing the intestinal microflora could influence tumour

development. In one such experiment, Le Leu et al., (2005) used symbiotic combination of

resistant starch and probiotic bacteria to treat experimentally induced colon cancer. They used

Sprague-Dawley rats as their animal model and fed them semipurified diet containing resistant

starch, L. acidophilus and Bifidobacterium lactis (1 x 1010

CFU/g). The symbiotic combination

significantly facilitated the apoptotic response to a genotoxic carcinogen in the distal colon of

rats. In another such experiment, Femia et al., 2002 demonstrated the anti-tumorigenic activity of

the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus

rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in male

F344 rats. Similarly, Gallaher & Khil, (1999) used synbiotic combination of Bifidobacterium and

oligofructose that reduced aberrant crypt number in five of six experimental rats. More work on

28

Bifidobacterium strains employed to treat colorectal cancer was undertaken. Challa et al., (1997)

conducted anticancer trial on male Fisher 344 rats using Bifidobacterium longum. Feeding of B.

longum reduced the number of aberrant crypt foci to 143 ± 9 as against untreated carcinogen

control with 187 ± 9 aberrant crypts. Other noteworthy work on anti-colorectal cancer property

of Bifidobacterium longum was performed by Singh et al., (1997) and Reddy & Revenson,

(1993). Randomized trial of dietary fiber and Lactobacillus casei administration for prevention

of colorectal cancer was performed by Ishikawa et al., (2005).They reported that the occurrence

rate of tumors with a grade of moderate atypia or higher was significantly lower in group

administered L. casei. But no significant difference in the development of new colorectal tumor

was observed with administration of L. casei. McIntosh et al., (1999) fed rats with Lactobacillus

acidophilus (Delvo Pro LA-1), Lactobacillus rhamnosus (GG), Bidobacterium animalis (CSCC

1941) and Streptococcus thermophilus (DD145) and strains were examined for their influence on

colon cancer. There was 25% reduced colon cancer in the L. acidophilus treated rats compared to

untreated DMH control.

29

CHAPTER 3

MATERIALS AND METHODS

3.1. Materials

All bacterial culture media and standard enzymes were purchased from HiMedia, India. PCR

reagents and molecular weight markers were purchased from Bangalore Genei, India. Other

molecular biology chemicals and cell culture reagents were purchased from Sigma, USA. Protein

gel chromatography matrixes were purchased from GE healthcare, Sweden. HPLC grade

solvents were obtained from Merck, Germany. Reverse phase HPLC column was purchased

from phenomenex, USA. Dialysis bag was obtained from Spectrumlab, USA. Cell culture plates

and flasks were purchased from Tarsons, India. HT-29 cell line was procured from National

Centre for Cell Sciences, India. Male Wistar rats were purchased from King‘s Institute, Chennai,

India.

3.1.1. Instruments

Autoclave York Scientific Instruments, India.

Fermenter Super Tech Instruments, India

Deep freezer (-70 °C) Forma Scientific, Inc., USA

Deionized water system Milli-Q, Millipore Corporation, USA

Weighing balance Sartorius AG, Germany

pH meter STL Instruments, India

Water bath Matri Instruments, India

High speed centrifuge Sigma, USA

Table top centrifuge Remi Laboratories, India

30

UV-visible spectrophotometer Hitachi, Japan

Gel electrophoresis Amersham Bioscience, UK

Gel documentation system Bio-Rad Laboratories, Inc., USA

Ice flaker Scottman, Italy

Incubators Scigenics, India

Orbital shaker Scigenics, India

Laminar flow hoods Kirloskar, India

Fluorescent microscope Nikon, Japan

PCR thermal cycler Eppendorf, Germany

Freeze dryer Virtis, USA; Savant, USA

HPLC Shimadzu, Japan

Protein purification system GE Healthcare, Sweden

CO2 Incubator Therma Scientific, Inc., USA

Gel rocker Bangalore Genei, India

Scanning electron microscope Hitachi, Japan

MALDI-TOF mass spectrometer Bruker, USA

Ultra-filtration Watson Marlow, USA

Semidry blotter Axygen, UK

UV-transilluminator Fotodyne Inc., USA

31

3.1.2. Bacterial strains

Bacterial strains Source

Vibrio parahaemolyticus 451 MTCC, Chandigarh, India

Vibrio vulnificus1145 MTCC, Chandigarh, India

Vibrio fischeri1738 MTCC, Chandigarh, India

Vibrio anguillarum MTCC, Chandigarh, India

Escherichia coli DH5α MTCC, Chandigarh, India

Lactobacillus acidophilus 447 MTCC, Chandigarh, India

Lactobacillus rhamnosus 1408 MTCC, Chandigarh, India

Salmonella typhi 734 MTCC, Chandigarh, India

Listeria monocytogenes 1143 MTCC, Chandigarh, India

Proteus vulgaris 426 MTCC, Chandigarh, India

V. harveyi Hatchery water

Aeromonas hydrophila 646 MTCC, Chandigarh, India

Aeromonas salmonicida 1945 MTCC, Chandigarh, India

Pseudomonas aeroginosa Spoilage food

Bacillus cereus MTCC, Chandigarh, India

32

3.1. 3. Microbiological Media

3.1.3.1. MRS agar

Proteose peptone 10.0 g

Beef extract 10.0 g

Yeast extract 5.0 g

Dextrose 20.0 g

Polysorbate 80 1.0 g

Ammonium citrate 2.0 g

Sodium acetate 5.0 g

Magnesium sulphate 0.05 g


Dipotassium phosphate 2.0 g

Distilled water 1000 ml

pH 6.5

3.1.3.2. Tryptone Soy Agar

Casein enzymatic hydrolysate 17.0 g

Papaic digest of soyabean meal 3.0 g

Sodium chloride 5.0 g


Dextrose 2.5 g

Agar 15.0 g


33

pH 7.5

3.1.3.3. Brain heart infusion broth

Calf brain 200 g

Beef heart infusion 250 g

Protease peptone 10.0 g

Dextrose 2.0 g


Disodium phosphate 2.5 g


pH 7.5

3.1.3.4. Sea Water Agar

Peptic digest of animal tissue 5.0 g

Yeast extract 5.0 g

Beef extract 3.0 g

Agar 15.0 g

pH 7.0 ± 0.2

3.1.3.5. Simmon’s citrate agar


Ammonium dihydrogen phosphate 1.0 g


34

Sodium citrate 2.0 g


Bromothymol blue 0.08 g

Agar 15.0 g


3.1.3.6. Nitrate agar

Peptone 5.0 gm

Beef extract 3.0 gm

Potassium nitrate 1.0 gm

Agar 12.0 gm


pH 7.0

3.1.3.7. Tryptone broth

Tryptone 20 g



pH 7.4

3.1.3.8. MR-VP broth

Peptone 5.0 g

Dipotassium hydrogen phosphate 5 g

35

Glucose, 10% solution (sterilized separately) 50 ml


3.1.3.9. Phenol red agar base

Protease peptone 10.0 g

Beef extract 1.0 g


Agar 15 g

Phenol red 0.025 g


pH 7.5

3.1.4. Antibiotics Concentration (µg/disc)

Chloramphenicol 30

Amoxycillin 10

Vancomycin 30

Gentamycin 10

Oxytetracycline 30

Erythromycin 10

Amikacin 30

Penicillin 10

Cefpodoxime 10

36

3.1.5. Reagents

3.1.5.1. Alkaline phosphatase reagents

Carbonate-bicarbonate buffer 0.1 M, pH-10.0

Disodium phenyl phosphate 0.1 M

Magnesium chloride 0.1 M

Sodium carbonate 15%

Folin‘s phenol reagent 1.0 ml

Standard: 100 mg of phenol was dissolved in 100 ml of distilled water

3.1.5.2. Acid phosphatase reagents

Citrate buffer 0.1 M, pH- 4.3

Disodium phenyl phosphate 0.1 M

Neomycin 30

Methicillin 30

Novobiocin 30

Kanamycin 30

Rifampicin 5

Tetracycline 30

Ampicillin 10

Polymyxin 300

Sulphafurazole 300

Ciproflaxin 5

37

Sodium carbonate 15%

Folin‘s phenol reagent 1.0 ml

Standard: 100 mg of phenol was dissolved in 100 ml of distilled water.

3.1.5.3. Lipid peroxide assay reagents

3.1.5.3.1. Trichloroacetic acid (TCA)

15 ml of TCA from 100% was made up to 100 ml with distilled water.

3.1.5.3.2. Thiobarbituric acid (TBA)

0.375 g of TBA was dissolved in 100 ml of distilled water.

3.1.5.3.3. Hydrochloric acid

1.08 ml of HCl was made up to 50 ml with distilled water.

3.1.5.3.4. TCA: TBA: HCl (1:1:1)

Equal volume of above three solutions was mixed to make 1:1:1 ratio of TCA: TBA: HCl.

3.1.5.4. Superoxide dismutase assay reagents

3.1.5.4.1. Tris-HCl buffer (50 mM) containing EDTA (1 mM; pH-8.2)

605 mg of Tris-HCl was dissolved in 100 ml of distilled water. To this 0.0371 g of EDTA was

added and pH adjusted to 8.2.

3.1.5.4.2. Pyrogallol (0.2 mM) in 50 ml of HCl (10 mM)

1.26 mg of pyrogallol was dissolved in 50 ml of 10 mM HCl.

3.1.5.4.2. HCl (10 mM)

41.6 µl of concentrated HCl was made up to 50 ml with distilled water.

38

3.1.5.5. GST assay

1-chloro-2,4- Dinitrobenzene 1 mM

EDTA 1 mM

Glutathione reduced 1 mM

Potassium phosphate buffer 0.1 M, pH- 6.5

3.1.5.6. Catalase assay reagents

Potassium phosphate buffer 0.01 M, pH- 6.5

Hydrogen peroxide 0.2 M

5% dichromate-acetic acid 1:3 v/v

3.1.5.7. Antioxidant activity assay (TBA method)

Linoleic acid emulsion 20 ml

Ferrous sulphate 0.01%

Hydrogen peroxide 0.56 mM

TCA 4%

TBA 0.8%

Butylated hydroxytoluene 0.4%

Chloroform 2 ml

3.1.5.8. α, α-Diphenyl-β-Picrylhydrazyl (DPPH) radical scavenging assay

DPPH solution 0.2 mM

39

3.1.5.9. Lowry’s reagent

Solution A

Sodium carbonate 2.0 g

Sodium hydroxide 0.4 g


Solution B

Copper sulphate 0.5 g

Sodium potassium tartarate 1 g


50 parts of solution A were mixed with 1 parts of solution B.

3.1.6. Tricine SDS-PAGE Stock solutions

3.1.6.1. Stacking Acrylamide

Acrylamide 48.0 g

Bis-Acrylamide 1.5 g

Water 100 ml

3.1.6.2. Separating Acrylamide

Acrylamide 46.5 g

Bis-Acrylamide 1.5 g

Water 100 ml

40

3.1.6.3. Gel Buffer

Tris 3.0 M

SDS 3.0 g

Water 100 ml

pH 8.45

3.1.6.4. Separating Acrylamide (10%)

Separating acrylamide 1.22 ml

Gel Buffer 2.0 ml

Glycerol (50%) 2.0 ml

Water 0.78 ml

Ammonium per sulphate (10%) 75.0 µl

TEMED 7.5 µl

3.1.6.5. Stacking Acrylamide

Separating acrylamide 0.25 ml

Gel Buffer 0.75 ml

Water 2.0 ml

Ammonium per sulphate (10%) 20.0 µl

TEMED 2.0 µl

3.1.6.6. Sample buffer

Tris (0.5M) 5.0 ml

41

SDS (20%) 4.0 ml

2-mercaptoethanol 1.0 ml

Glycerol (50%) 4.0 ml

Bromophenol blue 0.004 g

Water 6.0 ml

pH 6.8

3.1.6.7. Anode buffer (+) stock (10X)

Tris 200 mM

pH 8.9

3.1.6.8. Cathode buffer (–) stock (10X)

Tris 100 mM

Tricine 100 mM

SDS 1%

pH 8.25

3.1.6.9. Silver Staining

3.1.6.9.1. Fixing solution

Ethanol 50%

Acetic acid (CH3 COOH) 12%

Formaldehyde (HCHO) 0.1M

The volume was made up to 100 ml using water.

42

3.1.6.9.2. Washing solution

Ethanol 50%

3.1.6.9.3. Sodium thiosulphate (Na2S2O3) fixing solution

Na2S2O3 0.01 g

Water 100 ml

3.1.6.9.4. Silver nitrate (Ag NO3) solution

Ag NO3 0.2 g

Formaldehyde 160 µl

Water 100 ml

3.1.6.9.5. Sodium thiosulphate developing solution

Na2S2O3 0.01 g

Sodium carbonate (Na2 CO3) 3.0 g

Formaldehyde 40.0 µl

Water 100 ml

3.1.6.9.6. Neutralizing solution

Citric acid (2.3M) 5.0 ml

43

3.1.7. Genomic DNA isolation

3.1.7.1. GTE stock solution (Lysis buffer)

Glucose 50 mM

Tris 25 mM

EDTA 10 mM

Water 100 ml

pH 8.0

3.1.7.2. SDS stock solution (20%)

SDS 20.0 g

Water 100 ml

SDS was mixed with water at warm condition to proper dissolving.

3.1.7.3. EDTA stock solution (0.5M)

EDTA 14.61 g

Water 100 ml

To proper dissolving, the EDTA was mixed with water at warm condition or autoclaving.

3.1.7.4. 10 mM Tris (pH 7.6)

Tris 0.12 g

Water 100 ml

3.1.7.5. Sodium acetate (3M)

Sodium acetate 10.2 g

44


3.1.7.6. Tris-Saturated Phenol

To prepare buffered phenol, distilled phenol is equilibrated first with equal volume of 1M

Tris-HCl (pH 8.0) and then with equal volume of 0.1M Tris-Cl (pH 7.5), 8-hydroxyquinoline is

added to a final concentration of 0.1% and stored at 4oC in dark bottle.

3.1.7.7. Phenol: Chloroform

Buffered phenol and Chloroform were mixed in the ratio 24:1 and stored in a brown

bottle at 4C along with 0.1 M Tris-Cl in aqueous phase.

3.1.7.8. RNase

RNase (10 mg/ml) was dissolved in 10 mM Tris-Cl (pH-7.5) and kept in boiling water

bath for 15 min and cooled. It is stored at -20oC.

3.1.7.9. TE buffer

1M Tris HCl (pH 8.0) 1.0 ml

0.5M EDTA (pH 8.0) 0.2 ml


3.1.8. Agarose gel electrophoresis

3.1.8.1. TAE Buffer (50X)

Tris-base 242 g

Glacial acetic acid 57.1 ml

45

0.5 M EDTA (pH8.0) 100 ml

The final volume was made up to 1000 ml using distilled water.

3.1.8.2. Ethidium bromide (EtBr) stock solution

Ethidium bromide 10.0 mg

Distilled water 1.0 ml

3.1.8.3. Gel loading dye (6X)

Bromophenol blue 0.25 g

Xylene cyanol 0.25 g

Glycerol 30 ml


3.1.9. PCR mixture for 16s rDNA sequencing

Water 7.5 µl

Buffer 3 µl

dNTP 2.5 µl

Primer 2 µl

Taq DNA polymerase 5 µl

Template DNA 5 µl

3.1.9.1. Phosphate buffered saline (PBS)

Sodium dihydrogen phosphate 1.4 g

46

Disodium hydrogen phosphate 0.02 g

NaCl 0.8 g

KCl 0.02 g

Water 100 ml

pH 7.0

3.2. Methods

3.2.1. Agar well diffusion method

Agar well diffusion method described by Lyon & Glatz, (1993) was used for the analysis of

bacteriocin activity. The wells of 6 mm were made using well borer and bottom of the wells were

sealed with a few drops of MRS agar media. 100 µl of culture free supernatant was added to the

wells and kept at 4oC. After 2 h of incubation, the agar base was loosened from edge of the petri

dish with spatula and filliped into the petri dish lid. 10 ml of soft agar containing indicator strains

were overlaid on the agar base. After 24 h of incubation period, zone of inhibition was measured

and tabulated.

3.2.2. Assay of bacteriocin activity

The bacteriocin activity of the culture free supernatants was determined by agar well diffusion

method. To the wells, 100 µl of twofold serially diluted supernatants were added and incubated

at 4oC. After 2 h, the agar base was loosened from edge of the petri dish with spatula and the

agar media was flipped into the petri dish lid which was covered with soft agar containing

indicator strains. After 24 h of incubation, zone of inhibition was measured. The bacteriocin

47

activity was expressed in arbitrary units (AU/ml), calculated as ab × 100, whereas ―a‖ represents

the dilution factors and ―b‖ the last dilution that produces an inhibition zone of minimum 2 mm

in diameter. One Arbitrary unit (AU) of antimicrobial or bacteriocin activity was defined as the

reciprocal of the highest two fold dilution that showing a clear zone of growth inhibition and

activity was expressed per ml after multiplication by 100 (Todorov & Dicks, 2005).

3.2.3. Tricine SDS-PAGE (Schägger & Jagow, 1987)

The gel of 1 mm thickness was casted using a protein gel casting apparatus. The stacking gel was

of 5% and separating gel was of 10%. The separating gel was poured between the sealed clean

glass plates. Distilled water was layered on the top of the resolving gel gently to remove the air

bubbles and give an even surface on the top. After complete polymerization of the resolving gel,

the water on the top is blotted out and the stacking gel was poured onto the resolving gel up to

the top. A suitable comb was inserted into the cassette and polymerization was allowed to take

place. The gel cassette was then removed from the casting unit and placed on the electrophoresis

platform carefully so that no air bubble is formed at the bottom of the gel in the lower tank. Then

the comb is removed and upper tank is also filled with 1X buffer. Then the samples are loaded

into the wells and run at 50 V. After electrophoresis is over, the gel was carefully removed from

the plates and stained using silver staining technique.

3.2.3. Silver Staining Procedure (Morrissey, 1981)

The polyacrylamide gel was fixed overnight and washed twice with distilled water. The gel was

immersed into 50% ethanol for 20 min and 100 ml of 0.01% sodium thiosulphate solution was

transferred into it. This was washed twice and treated with 0.2% silver nitrate and kept in dark

48

for 20 min. After washing, 100 ml of developing solution was added and rinsed until bands

develop. The reaction is stopped using distilled water.

3.2.4. Molecular biology assay

3.2.4.1.Total genomic DNA isolation (Marmur, 1961)

Lactic acid bacteria strains were grown in MRS broth at 37 °C. After 12 h of incubation, 1.5 ml

of cultured broth was taken and centrifuged at 8,000 g for 6min. The pellets was resuspended

with 330 µl of GTE solution and incubated at room temperature for 30 min. The pinch amount of

lysozyme was added to the same solution and incubated at 37 °C for 1 h. 10 µl of 20% SDS was

added and incubated at 37 °C for overnight. RNase (0.1 mg/ml) was added to the solution to

remove the RNA from solution and it was kept at 37 °C. After 3 h of incubation 17 µl of EDTA

(0.5M) was mixed and incubated at 50oC for 10 min. Proteinase K (10 µl) was added and

incubated at 37 °C for 3 h. After incubation, 200 µl of phenol: chloroform (24:1) was added,

mixed slowly and centrifuged at 16,000 g for 15 min. After centrifugation, the aqueous phase

layer was collected and mixed with equal volume of isopropanol. It was slowly shacked up,

down until to see the pool of DNA and centrifuged at 16,000 g for 15 min. The DNA pellet was

washed with 1 ml of 95% ethanol and centrifuged at 16,000 g for 15 min. After centrifugation,

the pellet was air dried and dissolved in 40 µl of 1X TE buffer. It was confirmed by running the

agarose gel electrophoresis.

3.2.4.2. Agarose gel electrophoresis

The isolated DNA sample was separated on 0.8% agarose gel. 1X TAE buffer was prepared by

appropriate concentration of

STUDIES ON PROBIOTICS AND ANTIMICROBIAL PROPERTIES OF...

Documents

Transcript of STUDIES ON PROBIOTICS AND ANTIMICROBIAL PROPERTIES OF...