pdfs.semanticscholar.org · 80 dr nivedita sharma professor department of basic sciences...

ISOLATION OF LACTIC ACID BACTERIA AND TO STUDY THEIR POTENTIAL AS

PROBIOTICS

ThesisThesisThesisThesis

by

SHWETA HANDA

Submitted in partial fulfilment of the requirements for the degree of

MASTER OF SCIENCEMASTER OF SCIENCEMASTER OF SCIENCEMASTER OF SCIENCE

MICROBIOLOGY

COLLEGE OF FORESTRY Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni,

Solan - 173 230 (H.P.), INDIA

2012

80

Dr Nivedita Sharma Professor

Department of Basic Sciences (Microbiology Section) College of Forestry Dr Y S Parmar University of Horticulture and

Forestry, Nauni-Solan – 173 230 (HP)

CERTIFICATE-I This is to certify that the thesis entitled, “Isolation of lactic acid

bacteria and to study their potential as probiotics”, submitted in

partial fulfillment of the requirements for the award of degree of MASTER

OF SCIENCE MICROBIOLOGY to Dr Yashwant Singh Parmar University

of Horticulture and Forestry, Nauni, Solan (H.P.) is a bonafide record of

research work carried out by Ms Shweta Handa (F-2010-29-M) under my

guidance and supervision. No part of this thesis has been submitted for

any other degree or diploma.

The assistance and help received during the course of

investigation has been fully acknowledged. Place: Nauni-Solan (Nivedita Sharma) Dated: 22.11.2012 Chairperson Advisory Committee

81

CERTIFICATE-II

This is to certify that the thesis entitled, “Isolation of lactic acid

bacteria and to study their potential as probiotics”, submitted by Ms

Shweta Handa (F-2010-29-M) to Dr Yashwant Singh Parmar University

of Horticulture and Forestry, Nauni, Solan (H.P.), in partial fulfillment of

the requirements for the award of degree of MASTER OF SCIENCE

MICROBIOLOGY has been approved by the Student’s Advisory

Committee after an oral examination of the same in collaboration with the

internal examiner.

Dr Nivedita Sharma Chairperson

Advisory Committee

Internal Examiner

Members of Advisory Committee

Dr Mohinder Kaur Dr R.K. Gupta Professor Professor

Department of Basic Sciences Department of Basic Sciences

Dr Neerja Rana Assistant Professor Department of Basic Sciences

Dean’s Nominee

Professor and Head

Department of Basic Sciences

Dean College of Forestry

82

CERTIFICATE-III

This is to certify that all the mistakes and errors pointed out

by the external examiner have been incorporated in the thesis

entitled, “Isolation of lactic acid bacteria and to study their potential

as probiotics” submitted to Dr Y S Parmar University of Horticulture

and Forestry, Nauni, Solan (H.P.) by Ms Shweta Handa (F-2010-29-

M) in partial fulfillment of the requirements for the award of degree of

MASTER OF SCIENCE MICROBIOLOGY.

________________________________

Dr Nivedita Sharma Chairperson

Advisory Committee

________________________________

Professor and Head Department of Basic Sciences

Dr Y S Parmar UHF, Nauni, Solan (H.P.)

83

ACKNOWLEDGEMENTS

With limitless humility, I am deeply indebted to God for patience, perseverance and

diligence which were bestowed on my body and soul to achieve important milestone of academic

career. I am thankful to the Almighty, whose eternal presence grows even stronger in my soul with

every passing time. At the end of my thesis, I would like to thank all those people who made this an

unforgettable experience for me. Eventually, it is a pleasant task to express my thanks to all those

who contributed in many ways to the success of this study.

At this moment of accomplishment, first of all I would like to offer my heartfelt gratitude

towards Dr (Mrs) Nivedita Sharma, Professor, Department of Basic Sciences and Chairperson of

my advisory committee for her guidance, support and encouragement. Under her guidance, I

successfully overcame many difficulties and learned a lot. I hope that I could be as lively,

enthusiastic, and energetic as her and to someday be able to command an audience as well as she

can.

My Sincere thanks to Dr C. K. Shirkot Professors & Head, Department of Basic Sciences, Dr Mohinder Kaur, Dr R K Gupta, Dr Neerja Rana, worthy members of my advisory committee who helped me to complete this manuscript with their valuable suggestions.

My sincere thanks and gratitude to Dr A.K. Sharma, Former Head, Department of

Basic Sciences for his necessary help during his work tenure.

I would like to pay high regards to my parents for their sincere encouragement and

inspiration throughout my research work and lifting me uphill this phase of life. I would not have

made it this far without them. I owe everything to them. My sibling Shreya and Akash for their

advice and support. Words are inadequate to express my gratitude to my best friend Sonam for her

affection and care.

Special thanks to the newest addition to my family, Ashish, my fiance who has been a true

and great supporter even when I was irritable and depressed.

Heart felt and special thanks to my seniors Neha, Sanjeev, Nisha, Divya, Richa,

Anupama, Geetanjali, Sushma, Pallavi, Shruti, Hitender Sharma, Shweta Sharma for their

unconditional help. Thanks are due to Bhawna, Manorma, Rashmi, Parul, Balkar and Smriti.

Besides this, thanks to all who have knowingly and unknowingly helped me in the successful

completion of this project.

I am highly thankful to academic staff and laboratory staff of Department of Basic

Sciences for their help and cooperation.

Financial assistance rendered by DBT is duly acknowledged.

The painstaking efforts of Sh. Sohan Lal and Sh. Ashok Kumar, DPT Computers,

Nauni, in preparing this manuscript are highly acknowledged.

Needless to say, errors and omissions are solely mine.

( Shweta Handa )

84

CCOONNTTEENNTTSS

CHAPTER TITLE PAGE(S)

1. INTRODUCTION 1-4

2. REVIEW OF LITERATURE 5-62

3. MATERIALS AND METHODS 63-78

4. RESULTS AND DISCUSSION 79-128

5. SUMMARY 129-132

6. REFERENCES 133-150

ABSTRACT 151

APPENDICES I-III

85

LLIISSTT OOFF TTAABBLLEESS Table Title Page(s)

Review of literature

1. Fermented foods from round the world (Sahlin, 1999) 5

2. Requirements of probiotics (Salminen et al., 1998a) 16

3. Various special therapeutic or prophylactic properties of specific probiotics (Parvez et al., 2006)

21-22

4. Commercially used probiotics 22

5. Microorganisms applied in probiotic products (Yavuzdurm, 2007)

24

6. Antimicrobial peptides of Lactic acid bacteria 47

Results and Discussion

1. Isolation of Lactic Acid bacteria from different food sources showing their morphological characteristics

81

2. Biochemical characteristics of isolated Lactic acid bacteria and their tentative identification

83

3. Preliminary Screening of isolated LAB on the basis of antagonistic pattern against test indicators by bit/disc method

86

4. Preliminary screening of isolated Lactic acid bacteria on the basis of percent survival in the presence of bile salt

89

5. Preliminary screening of isolated Lactic acid bacteria on the basis of percent survival in acidic pH

91

5a. Final screening of six lactic acid bacteria with high probiotic potential

93

5b. Total microbial profile of food sources of finally screened LAB having high probiotic potential

94

6. Genotyping of finally screened lactic acid bacterial isolate F3

96


97


97


98

86

Table Title Page(s)


99


101

12. Estimation of *autoaggregation of screened LAB’s 102-103

13. Expression of adhesion by screened LAB’s to different hydrocarbons

105-106

14. Potential of screened LAB’s for acidity tolerance 111-112

15. Detection of Antibiotic sensitivity for screened LAB’s 114

16. Extended inhibitory spectrum of screened Lactic Acid Bacteria by Bit method

117

17. Extended inhibitory spectrum of screened Lactic Acid Bacteria by Well Diffusion method

119

18. Effect of different enzymes on the activity of supernatant of LAB’s against test indicator

122

19. Cumulative probiotic effect of screened LAB’s 125

20. Compatibility of screened LAB isolates 127

87

LLIISSTT OOFF PPLLAATTEESS

Plates Title Between page(s)

1a. Inhibitory spectrum of screened Lactic acid bacteria by Bit/Disk method

86-87

1b. Inhibitory spectrum of screened Lactic acid bacteria by Bit/Disk method

86-87

2. Morphology of isolate F3 94-95

3. Genomic DNA and PCR product of isolate F3 94-95











14a. Inhibitory spectrum of screened Lactic acid bacteria by well diffusion method

116-117

14b. Inhibitory spectrum of screened Lactic acid bacteria by well diffusion method

118-119

15. Effect of different enzymes on the activity of six screened LAB’s supernatant against test indicator

122-123

88

LLIISSTT OOFF FFIIGGUURREE

Figure Title Between page(s)

Review of Literature

1. Glucose utilization metabolic pathways of LAB 11

2. Guidelines for evaluation of candidate probiotic strains (ICMR and DBT, 2011)

13

3. Various health benefits from probiotic consumption (Parvez et al., 2006)

17

Results and Discussion

1a Differentiation of isolated LAB’s on the basis of their form

82-83

1b Morphology of isolated LAB’s on the basis of their elevation

82-83

1c Morphology of isolated LAB’s on the basis of colour 82-83

2a Gram’s reaction of isolated LAB’s 84-85

2b Morphology of isolated LAB’s on the basis of their shape

84-85

2c Catalase test of isolated LAB’s 84-85

2d Mode of growth conditions of isolated LAB’s 84-85

3 Antagonistic potential of Lactic acid bacteria against test indicators

86-87

4. Percent survival of Lactic acid bacteria in the presence of bile salt

88-89

5. Percent survival of Lactic acid bacteria in the presence of acidic pH

92-93

6. Phylogenetic tree of Lactobacillus fermentum F3 94-95

7. Phylogenetic tree of Lactobacillus sp. F8 96-97

8. Phylogenetic tree of Lactobacillus crustorum F11 96-97

9. Phylogenetic tree of Lactobacillus acidophilus F14 98-99

89

Figure Title Between page(s)

10. Phylogenetic tree of Lactobacillus delbreuckii subsp. bulgaricus F18

98-99

11. Phylogenetic tree of Lactobacillus plantarum F22 100-101

12. Comparison of the autoaggregation ability of screened LAB cells resuspended in buffer after growing in MRS broth

102-103

13. Comparison of the hydrophobicity of screened LAB cells resuspended in buffer after growing in MRS broth

106-107

14. Relationship between auto-aggregation (%) ability and hydrophobicity (%) of screened six isolates – L.

fermentum F3, ∆– Lactobacillus sp. F8, O – L. crustorum F11, – L. acidophillus F14, – L. delbrueckii subsp. Bulgaricus F18, – L. plantarum F22

106-107

15. Acidity tolerance range of Lactobacillus fermentum F3 112-113

16. Acidity tolerance range of Lactobacillus sp. F8 112-113

17. Acidity tolerance range of Lactobacillus crustorum F11 112-113

18. Acidity tolerance range of Lactobacillus acidophilus F14 112-113

19. Acidity tolerance range of Lactobacillus delbrueckii subsp. bulgaricus F18

112-113

20. Acidity tolerance range of Lactobacillus plantarum F22 112-113

21. Inhibitory spectrum of Lactobacillus fermentum F3 during its growth phase against three different test indicators

120-121

22. Inhibitory spectrum of Lactobacillus sp. F8 during its growth phase against three different test indicators

120-121

23 Inhibitory spectrum of Lactobacillus crustorum F11 during its growth phase against three different test indicators

120-121

24. Inhibitory spectrum of Lactobacillus acidophilus F14 during its growth phase against three different test indicators

120-121

25. nhibitory spectrum of Lactobacillus delbreuckii subsp. bulgaricus F18 during its growth phase against three different test indicators

120-121

26. Inhibitory spectrum of Lactobacillus plantarum F22 during its growth phase against three different test indicators

120-121

90

LIST OF ABBREVIATIONS α - alpha β - beta oC - Degree centigrade % - Per cent & - And µg - Microgram µl - Microlitre Bp - Base pair cfu - Colony forming units cm - Centimeter C - Control DNA - Deoxyribonucleic Acid dNTPs - deoxyribonucleotide triphosphate ER - Enzyme reaction FAO - Food and Agriculture Organization Fig. - Figure g - Gram g/l - Gram per litre h - Hour i.e - That is KMS - Potassium metabisulphite LAB - Lactic acid bacteria l - Litre M - Molar mg - Milligram min - Minutes ml - Millilitre mm - Millimeter MRS - De Man Rogosa sharpe agar N - Normal nm - Nanometer OD - Optical density ppm - Parts Per Million psi - Per square inch PCR - Polymerase Chain Reaction RNA - Ribonucleic Acid rDNA - Ribosomal DNA rRNA - Ribosomal RNA rpm - Rotations per minute sp. - Species temp - Temperature UV - Ultra violet v/v - volume/volume viz. - Visually w/v - weight/volume WHO - World Health Organization

Chapter-1

INTRODUCTION

"Let food be thy medicine and medicine be thy food" as Hippocrates said,

is the principle of today (Suvarna and Boby, 2005). Probiotics are one of the

functional foods that link diet and health. Probiotic terms derived from Greek

words Pro (favor) and bios (life). Probiotics "For Life" are living, health-

promoting microbial food ingredients that have a beneficial effect on humans

(Chuayana et al., 2003). Probiotics are described as ‘live microorganisms which,

when administered in adequate numbers, confer a health benefit on the host’

FAO/WHO, (2002).

The concept of probiotics have been first proposed by Nobel Prize winner

Russian scientist Elie Metchnikoff, who suggested that the long life of Bulgarian

peasants resulted from utilization of fermented milk products thus owing the

credit to fermenting lactobacilli for positively influencing the gut microflora and

consequently reducing toxic metabolic activities there (Chuayana et al., 2003;

Tannock, 2005).

Probiotics are beneficial bacteria in that they favourably alter the

intestinal microflora balance, inhibition of undesirable bacteria (El-Nagger,

2004), promote good digestion, boost immune function and increase resistance to

infection (Collado et al., 2007a). Other physiological benefits of probiotics

include removal of carcinogens, lowering of cholesterol, immune stimulating and

allergy lowering effect, synthesis and enhancing the bioavailability of nutrients,

alleviation of lactose intolerance (Parvez et al., 2006), neutralization of toxins,

increase of the immune response (Ghafoor et al., 2005), anti-mutagenic and anti-

carcinogenic activities (Boutron-Ruault, 2007; Davis and Milner, 2009; Baldwin

et al., 2010), reduction of cholesterol levels (Park et al., 2008), control of

diarrhoea (Dylewski et al., 2010; Gao et al., 2010), alleviation of lactose

intolerance (Guarner et al., 2005), inflammatory bowel diseases (Matthes et al.,

2

2010). They are also a source of vitamins, especially of the B group (Crittenden

et al., 2003).

In India, a wide variety of traditional fermented foods made from

ingredients like milk, cereals, pulses and vegetables have been developed for the

benefit of human health from ancient times. The state of Himachal Pradesh is

also well known for their culture and taste. Different region of Himachal Pradesh

known for their typical beverages like chhang that is known throughout the

Himalayan region and Angoori of Kinnaur district. Many of the fermented

products are well known as house hold items while a few are prepared at cottage

scale. Mostly these foods are cereal-based (wheat/barley/buckwheat/ragi) but

some legume (black gram) and milk-based fermented foods are also common.

Some of the products like Bhaturu, Siddu, Chilra, Marchu, Manna, Dosha,

Pinni/Bagpinni, Seera, etc. are unique to Himachal Pradesh. A large variety of

fermented foods is prepared either daily, during special occasions or for

consumption during journey. Traditional starter cultures like 'Malera' and 'Treh'

are used as inocula in making these fermented foods. However, the natural

fermentation (without the addition of inoculum, as microorganisms present in the

raw materials carry out fermentation) is used in the production of Seera,

Sepubari, Bari etc. The primary microorganisms responsible in bringing about the

desirable attributes in the final products are those belonging to Lactic acid

bacteria (LAB). LAB’s are regarded as the major group of probiotic bacteria

(Collins et al., 1998). Since traditional fermented food items are least explored,

rich repositories of rare/novel LAB strains with immense potential of various

health beneficiaries. Thus, there is a high probability of these food items along

with other fermented food sources yielding highly desirable LAB’s upon

isolation.

Lactic acid bacteria (LAB) form a phylogenetically diverse group, widely

distributed in nature and defined as Gram-positive, non-sporulating, and catalase-

negative, devoid of cytochromes, of anaerobic habit but aerotolerant, fastidious,

acid tolerant and strictly fermentative bacteria that secrete lactic acid as their

major end product of sugar fermentation (Pelinescu et al., 2009).

3

Mankind had exploited lactic acid bacteria (LAB) for the production of

fermented foods because of their ability to produce desirable changes in taste,

flavor and texture as well as to inhibit pathogenic and spoilage microbes. Since

they are involved in numerous food fermentations for millennia, it is assumed

that most representatives of this group do not pose any health risk to man and are

designated as GRAS (generally recognized as safe) organisms (Holzapfel et al.,

1995). Different antimicrobials, such as lactic acid, acetic acid, hydrogen

peroxide, carbon dioxide and bacteriocins produced by these bacteria, can inhibit

pathogenic and spoilage microorganisms, extending the shelf-life and enhancing

the safety of food products (Yukeskdag and Aslim, 2010). One important

attribute of LAB is their ability to produce antimicrobial compound called

bacteriocin. Bacteriocins are proteinaceous compound showing inhibition

towards sensitive strains produced by both Gram-positive and Gram-negative

bacteria (Nomoto, 2005). They have the potential to be used in the food industry

and pharmaceutical industries to substitute for chemical preservation (Gao et al.,

2010).

Research on lactic acid bacteria (LAB) has advanced greatly since the last

decade due to its important roles in many diverse areas of food biotechnology,

nutrition, health and safety. Health promoting skills of these microbes was first

documented by Elie Metchnikoff in his book “Prolongation of Life”

(Metchnikoff, 1908). Some standard fermented food items, e.g., yogurt,

sauerkraut and cheese contain probiotics in the form of live lactic acid bacteria

and thus behave as probiotics.

Therefore, the present work entitled “Isolation of lactic acid bacteria and

to study their potential as probiotics” is taken for study with the following

objectives:

i) To isolate, screen and identify hyperbacteriocin producing lactic acid

bacteria and to detect their antimicrobial pattern.

ii) To explore probiotic potential of selected LAB’s.

Chapter-2

REVIEW OF LITERATURE

Fermented foods rich in fermenting microbe’s viz., lactic acid bacteria,

yeasts and other bacteria have played an important role in human health for

hundreds of years. Societies known for their long lives have always eaten some

form of fermented food.

Table 1. Fermented foods from round the world (Sahlin, 1999)

Food Ingredients Main species present Country

Beer Barley Yeast, Lactic acid bacteria World wide Cheese Milk Lactic acid bacteria, mold World wide Dadih Milk Lactic acid bacteria Indonesia

Dawadawa Locust beans Bacillus, Staphylococcus West Africa Gari Cassava Leucononstoc, Alcaligenes,

Cornnebacterium, Lactobacillus

Nigeria

Idli/dossa Rice, black gram L. mesenteroids, E. faecalis, yeast India Injera Tef L. mesenteroids, P. cerevisiae, S.

cerevisiae, L. plantarum

Ethiopia

I-sushi Fish Lactic acid bacteria, yeast Japan Kaanga piro Maize Lactic acid bacteria New Zealand

Kefir Milk Streptococcus, Lactobacillus,

Leucononstoc sp., Candida kefyr,

Kluyveromyces fragilis

Eastern Europe

Kenkey Maize, Sorghum Lactic acid bacteria Ghana Kimchi Milk L. mesenteroids, L. brevis, L.

plantarum

Korea

Koko Maize, Sorghum Lactic acid bacteria Ghana Leavened bread Wheat Yeast Europe, North

America Lambic beer Barley Yeast, Lactic acid bacteria Belgium

Mahewu Maize L. lactis, Lactobacillus sp. South Africa Nam

Pork, rice, garlic, salt P. cerevisiae, L. plantarum, L. brevis Thailand

Nono Milk Lactic acid bacteria Nigeria Ogi Maize, sorghum, millet L. plantarum, Acetobacter, yeast Nigeria

Palm wine Palm sap Yeast and Lactic acid bacteria World wide Poi Taro Lactic acid bacteria Hawaii Puto Rice L. mesenteroids, E. faecalis Philippines

Salami Meat Lactic acid bacteria World wide Saueurkraut Cabbage Lactic acid bacteria Europe, North

America Sorghum beer Sorghum Lactic acid bacteria South Africa

Sourdough bread Wheat, rye Lactic acid bacteria Europe, North America

Soy souce, miso Soy beans Lactic acid bacteria, mold South East Asia Tempeh Soy beans Mold, yeast and bacteria Indonesia Trahanas Milk and wheat Lactic acid bacteria Greece Yogurt Milk Lactic acid bacteria World wide

6

All around the world, fermented foods and beverages are part of the

human diet. In some places they make up a minor 5% of the daily intake, while in

others their role can be substantial as 40% as shown in Table 1. Using native

knowledge of locally available raw materials from the plant or animal sources,

people across the globe produce this type of food and drink either naturally or

adding starter cultures that contain microorganisms. Microorganisms transform

these raw material both biochemically (i.e., the nutrients) and organolaptically

(i.e., the taste/texture/odour) into edible products that are culturally acceptable to

the maker and consumer (Tamang, 2010).

Fermented foods can be fried, boiled or candied, or consumed in curries,

stews, side dishes, pickle, confectionery, salads, soups and desserts. They can be

in form of pastes, seasonings, condiments, masticators, and even colorants.

Fermented drinks can be either alcoholic (such as beer and wine) or non-

alcoholic, like butter milk, certain teas, or things that contain vinegar.

However, though most fermented foods have health-promoting benefits;

their global consumption is declining as traditional food systems give way to the

influence of a western and fast foods.

Definition of fermented food

Campbell-Platt (1987) has defined fermented foods as those foods which

have been subjected to the action of micro-organisms or enzymes so that

desirable biochemical changes cause significant modification to the food.

However, to the microbiologist, the term “fermentation” describes a form of

energy-yielding microbial metabolism in which an organic substrate, usually a

carbohydrate, is incompletely oxidised, and an organic carbohydrate acts as the

electron acceptor (Adams, 1990). This definition means that processes involving

ethanol production by yeasts or organic acids by lactic acid bacteria are

considered as fermentations, but not the production of fish sauces in Southeast

Asia, that still has not been shown to have a significant role for microorganisms,

and not the Tempe production since the metabolism of the fungi is not

fermentative according to Adams definition. Whichever definition used, foods

7

submitted to the influence of lactic acid producing microorganisms is considered

a fermented food.

Microflora in fermented foods

By tradition, lactic acid bacteria (LAB) are the most commonly used

microorganisms for preservation of foods. Their importance is associated mainly

with their safe metabolic activity while growing in foods utilising available sugar

for the production of organic acids and other metabolites. Their common

occurrence in foods and feeds coupled with their long-lived use contributes to

their natural acceptance as GRAS (Generally Recognised As Safe) for human

consumption (Aguirre & Collins, 1993). However, there are many kinds of

fermented foods in which the dominating processes and end products are

contributed by a mixture of endogenous enzymes and other microorganisms like

yeast and mould. Very often, a mixed culture originating from the native

microflora of the raw materials is in action in most of the food fermentation

processes. However, in an industrial scale a particular defined starter culture,

which has been developed under controlled conditions, is of first preference so

that the qualities of the finished product could be consistently maintained day

after day. Moreover, modern methods of gene-technology make it possible for the

microbiologists to design and develop starter cultures with specific qualities.

Nutritional value of fermented foods

Generally, a significant increase in the soluble fraction of a food is

observed during fermentation. The quantity as well as quality of the food proteins

as expressed by biological value, and often the content of water soluble vitamins

is generally increased, while the antinutritional factors show a decline during

fermentation (Paredes-López & Harry, 1988). Fermentation results in a lower

proportion of dry matter in the food and the concentrations of vitamins, minerals

and protein appear to increase when measured on a dry weight basis (Adams,

1990). Single as well as mixed culture fermentation of pearl millet flour with

yeast and lactobacilli significantly increased the total amount of soluble sugars,

reducing and non-reducing sugar content, with a simultaneous decrease in its

8

starch content (Khetarpaul & Chauhan, 1990). Combination of cooking and

fermentation improved the nutrient quality of all tested sorghum seeds and

reduced the content of antinutritional factors to a safe level in comparison with

other methods of processing (Obizoba & Atii, 1991).

2.1 LACTIC ACID BACTERIA

2.1.1. Historical background of lactic acid bacteria

Lactic acid-producing fermentation is an old invention. Many different

cultures in various parts of the world have used fermentation to improve the

storage qualities and nutritive value of perishable foods such as milk, vegetables,

meat fish and cereals. The organisms that produce this type of fermentation,

lactic acid bacteria, have had an important role in preserving foods. In developed

world, lactic acid bacteria are mainly associated with fermented dairy products

such as cheese, buttermilk, and yogurt. The use of dairy starter cultures has

become an industry during this century.

The concept of the group name ‘lactic acid bacteria’ was created for

bacteria causing fermentation and coagulation of milk, and defines as those

which produce lactic acid from lactose. The family name Lactobacteriaceae was

applied by (Orla-Jensen, 1919) to a physiological group of bacteria producing

lactic acid alone or acetic and lactic acids, alcohol and carbon dioxide. Today,

lactic acid bacteria are regarded as synonymous by and large with the family

Lactobacteriaceae (Breed et al., 1957).

Since the days of Russian scientist Metchnikoff, lactic acid bacteria have

also been associated with beneficial health effects. Today, an increasing number

of health food and so- called functional foods as well as pharmaceutical

preparation are promoted with health claims based on the characteristics of

certain strains of lactic acid bacteria. Most of these strains, however, have not

been thoroughly studied, and consequently the claims are not well substantiated.

Moreover, health benefits are judged mainly using subjective criteria.

Additionally the specific bacterial strains used in the studies are often poorly

identified. Most information about the health effects of lactic acid bacteria is thus

9

anecdotal. There is clear need for critical study of the effect on health of strain

selection and the quality of fermented foods and their ingredients.

Lactic acid bacteria are a group of Gram-positive bacteria united by a

constellation of morphological, metabolic, and physiological characteristics.

They are non-sporing, carbohydrate- fermenting lactic acid producers, acid

tolerant of non-aerobic habitat and catalase negative. Typically they are non-

motile and do not reduce nitrite. They are subdivided into four genera

Streptococcus, Leuconstoc, Pediococcus, and Lactobacillus. Recent taxonomic

revisions suggest that lactic acid bacteria group could be comprised of genera

Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus,

Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, and Vagococcus.

Originally, bifidobacteria were included in the genus Lactobacillus and the

organism was referred to as Lactobacillus bifidus. Although the classification of

lactic acid bacteria into different genera is mainly based on the characteristics

used by Orla-Jensen, (1919); however, confusion was still prevalent when his

monograph appeared. This work has had a large impact on the systematic of

lactic acid bacteria, and, although revised to some extent, it is still valid and the

basis of classification remarkably unchanged. The classification of lactic acid

bacteria into different genera is largely based on morphology, mode of glucose

fermentation, growth at different temperatures, and configuration of the lactic

acid produced, ability to grow at high salt concentrations, and acid or alkaline

tolerance. Even some of the newly described genera of lactic acid bacteria,

additional characteristics such as fatty acid composition and motility are used as

the basis of classification. Lactic acid bacteria is an important group of bacteria

being placed in group 19 with important biochemical characters that is gram’s

reaction, catalase negative, carbohydrate utilizing, casein hydrolysis as

authenticated in Bergey’s manual of Determinative Bacteriology (7th Edn).

The term lactic acid bacteria were used synonymously with “milk souring

organisms.” Important progress in the classification of these bacteria was made

when the similarity between milk-souring bacteria and other lactic-acid

10

producing bacteria of other habitats was recognized (Axelsson, 1993). Lactic acid

bacteria are generally associated with habitats rich in nutrients, such as various

food products (milk, meat, vegetables), but some are also members of the normal

flora of the mouth, intestine, and vagina of mammals. The genera that, in most

respects, fit the general description of the typical lactic acid bacteria are (as they

appear in the latest Bergey’s Manual from 1986) Aerococcus, Lactobacillus,

Leuconostoc, Pediococcus, and Streptococcus. The genera Lactobacillus,

Leuconostoc, and Pediococcus have largely remained unchanged, but some rod-

shaped lactic acid bacteria, previously included in Lactobacillus, is now forming

the genus Carnobacterium (Collins et al., 1987).

2.1.2. Classification at genus level

The basis for the classification of lactic acid bacteria in different genera

has essentially remained unchanged since the work of Orla-Jensen, (1919).

Although their morphology is regarded as questionable as a key character in

bacterial taxonomy given by Woese, (1987), it is still very important in the

current descriptions of the lactic acid bacteria genera. Thus lactic acid bacteria

can be divided into rods (Lactobacillus and Carnobacterium) and cocci (all other

genera).

An important characteristic used in the differentiation of the lactic acid

bacteria genera is the mode of glucose fermentation under standard conditions,

i.e., non limiting concentrations of glucose and growth factors (amino acids,

vitamins and nucleic acid precursors) and limited oxygen availability. Under

these conditions, lactic acid bacteria can be divided into two groups:

homofermentative, which convert glucose almost quantitatively to lactic acid, and

heterofermentative, which ferment glucose to lactic acid, ethanol/acetic acid, and

CO2 (Sharpe, 1979). In practice, a test for gas production from glucose will

distinguish between the groups (Sharpe, 1979). More detailed glucose metabolic

pathways are shown in Fig. 1. Leuconostocs and a subgroup of Lactobacillus are

heterofermentative; all other lactic acid bacteria are homofermentative.

11

Heterofermentative LAB Homofermentative LAB

Glucose Glucose–6–phosphate 6-phosphogluconate fructose-6-phosphate CO2 D-ribulose-5-phosphate Fructose-1,6-diphosphate Xylulose-5-phosphate Glyceraldehydes Dehydroxy Phosphate acetone Phosphate Acetyl phosphate P.enol pyruvate pyruvate pyruvate acetate ethanol LDH Lactic acid Fig 1. Glucose utilization metabolic pathways of LAB

Growth at certain temperatures is mainly used to distinguish between

some of the cocci. Enterococci grow at, 10oC and 45oC, lactococci and vagococci

at 10oC, but not at 45oC. Streptococci do not grow at 100C, while growth at 45oC

in dependent on the species (Axelsson, 1993). Salt tolerance (6.5% NaCl) may

also be used to distinguish among enterococci, lactococci/vagococci, and

streptococci, although variable reactions can be found among streptococci

(Mundt, 1986). Extreme salt tolerance (18% NaCl) is confined to genus

Tetragenococcus. Tolerances to acid and/or alkaline conditions are also useful

characteristics. Enterococci are characterised by growth at both high and low pH.

The formation of the different isomeric forms of lactic acid during fermentation

12

of glucose can be used to distinguish between Leuconostoc and most

heterofermentative lactobacilli, as the former produce only D- lactic acid and the

latter a racemate (DL-lactic acid).

2.2. PROBIOTICS One manner in which modulation of the gut microbiota composition has

been attempted is through the use of live microbial dietary additions, as

probiotics. The word probiotic is translated from the Greek meaning ‘for life’. An

early definition was given by Parker, (1974): ‘Organisms and substances which

contribute to intestinal microbial balance.’ However, this was subsequently

refined by Fuller, (1989) as: ‘a live microbial feed supplement which beneficially

affects the host animal by improving its intestinal microbial balance.’ This latter

version is the most widely used definition and has gained widespread scientific

acceptability. A probiotic would therefore incorporate living micro-organisms,

seen as beneficial for gut health, into diet.

Probiotics has a long history. In fact, the first records of intake of bacterial

drinks by humans are over 2000 years old. However, at the beginning of this

century probiotics were first put onto a scientific basis by the work of

Metchnikoff at the Pasteur Institute in Paris. Metchnikoff, (1907) observed

longevity in Bulgarian peasants and associated this with their elevated intake of

soured milks. During these studies, he hypothesized that the normal gut

Microflora could exert adverse effects on the host and that consumption of

certain bacteria could reverse this effect. Metchnikoff refined the treatment by

using pure cultures of what is now called Lactobacillus delbruckeii subsp.

bulgaricus, which, with Streptococcus salivarius subsp. thermophilus, is used to

ferment milk in the production of traditional yoghurt. Subsequent research has

been directed towards the use of intestinal isolates of bacteria as probiotics

(Fernandes et al., 1987). Over the years many species of micro-organisms have

been used. They mainly consist of lactic acid producing bacteria (lactobacilli,

streptococci, enterococci, lactococci, bifidobacteria) but also Bacillus spp. and

fungi such as Saccharomyces spp. and Aspergillus spp.

13

Despite the very widespread use of probiotics, the approach may have

some difficulties. The bacteria used are usually anaerobic and do not relish

extremes of temperature. To be effective, probiotic must be amenable to

preparation in a viable form at a large scale. During use and under storage the

probiotic should remain viable and stable, and be able to survive in the intestinal

ecosystem, and be able to survive in the ecosystem, and the host animal should

gain beneficially from harbouring the probiotic. It is therefore proposed that the

exogenous bacteria reach the intestine in an intact and viable form, and establish

therein and exert their advantageous properties. In order to do so, microbes must

overcome a number of physical and chemical barriers in the gastrointestinal tract.

These include gastric acidity and bile acid secrection. Moreover, on reaching the

colon the probiotics may be in some sort of stressed state that would probably

compromise chances of survival.

Fig 2. Guidelines for evaluation of candidate probiotic strains (ICMR

and DBT, 2011)

14

2.2.2. Mechanism of Probiotics

Probiotic microorganisms are considered to support the host health.

However, the support mechanisms have not been explained (Holzapfel et al.,

1998). There are studies on how probiotics work. So, many mechanisms from

these studies are trying to explain how probiotics could protect the host from the

intestinal disorders. These mechanisms listed below briefly (Çakır 2003,

Salminen et al., 1999).

1. Production of inhibitory substances: Production of some organic acids,

hydrogen peroxide and bacteriocins which are inhibitory to both gram-

positive and gram-negative bacteria.

2. Blocking of adhesion sites: Probiotics and pathogenic bacteria are in a

competition. Probiotics inhibit the pathogens by adhering to the intestinal

epithelial surfaces by blocking the adhesion sites.

3. Competition for nutrients: Despite of the lack of studies in vivo,

probiotics inhibit the pathogens by consuming the nutrients which

pathogens need.

4. Stimulating of immunity: Stimulating of specific and nonspecific

immunity may be one possible mechanism of probiotics to protect the host

from intestinal disease. This mechanism is not well documented, but it is

thought that specific cell wall components or cell layers may act as

adjuvants and increase humoral immune response.

5. Degradation of toxin receptor: Because of the degredation of toxin

receptor on the intestinal mucosa, it was shown that S. boulardii protects

the host against C. difficile intestinal disease. Some other offered

mechanisms are suppression of toxin production, reduction of gut pH,

attenuation of virulence (Fooks, et al., 1999).

2.2.3. Properties required for probiotics being effective in nutritional and

therapeutic settings

A probiotic can be used exogenously or endogenously to enhance

nutritional status and/or the health of the host. In the case of exogenous use,

microorganisms are most commonly used to ferment various foods and by this

15

process can preserve and make nutrients bioavailable. In addition,

microorganisms can metabolize sugars, such as lactose in yoghurt, making this

food more acceptable for consumption by individuals suffering from lactose

intolerance. However, the most interesting properties that probiotics acting

exogenously can have are the production of substances that may be antibiotics,

anticarcinogens or have other pharmaceutical properties. The properties required

for exogenously derived benefits from probiotics are the ability to grow in the

food or the media in which the organism is placed, and the specific metabolic

properties which result in the potential beneficial effects stated above. The

selection of organisms that can be helpful therapeutically and nutritionally would

be based on specific properties that are desired.

This can be achieved by either classical biological selection techniques or

genetic engineering. Probiotics that are ingested by the host and exert their

favorable properties by virtue of residing in the gastrointestinal tract have to have

certain properties in order to exert an effect.

2.2.4. Requirements for probiotics It is of high importance that the probiotic strain can survive the location

where it is presumed to be active. For a longer and perhaps higher activity, it is

necessary that the strain can proliferate and colonize at this specific location.

Probably only host-specific microbial strains are able to compete with the

indigenous microflora and to colonize the niches. Besides, the probiotic strain

must be tolerated by the immune system and not provoke the formation of

antibodies against the probiotic strain. So, the host must be immune-tolerant to

the probiotic. On the other hand, the probiotic strain can act as an adjuvant and

stimulate the immune system against pathogenic microorganisms. It goes without

saying that a probiotic has to be harmless to the host: there must be no local or

general pathogenic, allergic or mutagenic/carcinogenic reactions provoked by the

microorganism itself, its fermentation products or its cell components after

decrease of the bacteria.

For the maintenance of its favorable properties the strain must be

genetically stable. For the production of probiotics it is important that the

16

microorganisms multiply rapidly and densely on relatively cheap nutrients and

that they remain viable during processing and storage. Besides the specific

beneficial property, these general requirements must be considered in developing

new probiotics, but also for determining the scientific value of a claimed

probiotic. A number of these requirements can be screened during in vitro

experiments. It is advised of the drawing up of a decision-tree for the minimal

requirements which can be tested in vitro, such as culture conditions and viability

of the probiotic strains during processing and storage; sensitivity to low pH

values, gastric juice, bile, pancreas, intestinal juice and intestinal or respiratory

mucus; adherence to isolated cells or cell cultures and interactions with other

(pathogenic) microorganisms. If these in vitro experiments are successful, further

research can be performed during in vivo experiments in animals or humans.

Requirements of probiotics that are important for their use in humans are

presented in Table 2.

Table 2. Requirements of probiotics (Salminen et al., 1998a)

• Survival of the environmental conditions on the location where it must be active

• Proliferation and/or colonisation on the location where it is active • No immune reaction against the probiotic strain • No pathogenic, toxic, allergic, mutagenic or carcinogenic reaction by the

probiotic strain itself, its fermentation products or its cell components after decrease of the bacteria

• Genetically stable, no plasmid transfer • Easy and reproducible production • Viable during processing and storage

2.2.1 The Effects of Probiotics on Health

There are lots of studies on searching the health benefits of fermented

foods and probiotics. However, in most of these studies researchers did not use

sufficient test subjects or they use microorganisms were not identified definitely

(Çakır, 2003). So, while a number of reported effects have been only partially

established, some can be regarded as well-established and clinically well

documented for specific strains. These health-related effects can be considered as

17

in the below (Çakır 2003, Scherezenmeir and De Vrese 2001, Dunne, et al. 2001,

Dugas, et al. 1999).

− Managing lactose intolerance.

− Improving immune system.

− Prevention of colon cancer.

− Reduction of cholesterol and triacylglycerol plasma concentrations (weak

evidence).

− Lowering blood pressure.

− Reducing inflammation.

− Reduction of allergic symptoms.

− Beneficial effects on mineral metabolism, particularly bone density and

− stability.

− Reduction of Helicobacter pylori infection.

− Suppression of pathogenic microorganisms (antimicrobial effect).

− Prevention of osteoporosis.

− Prevention of urogenital infections.

Fig 3. Various health benefits from probiotic consumption (Parvez et al.,

2006)

18

2.2.1.1. Lactose Intolerance

Most of human commonly non-Caucasians become lactose intolerant after

weaning. These lactose intolerant people cannot metabolize lactose due to the

lack of essential enzyme β-galactosidase. When they consume milk or lactose-

containing products, symptoms including abdominal pain, bloating, flatulence,

cramping and diarrhoea ensue. If lactose passes through from the small intestine,

it is converted to gas and acid in the large intestine by the colonic microflora.

Also the presence of breath hydrogen is a signal for lactose maldigestion. The

studies provide that the addition of certain starter cultures to milk products,

allows the lactose intolerant people to consume those products without the usual

rise of breath hydrogen or associated symptoms (Fooks, et al. 1999, Scheinbach

1998, Quewand and Salminen 1998, Lin, et al. 1991). The beneficial effects of

probiotics on lactose intolerance are explained by two ways. One of them is

lower lactose concentration in the fermented foods due to the high lactase activity

of bacterial preparations used in the production. The other one is; increased

lactase active lactase enzyme enters the small intestine with the fermented

product or with the viable probiotic bacteria (Salminen et al., 2004).

When the yogurt is compared with milk, cause the lactose is converted to

lactic acid and the yogurt consist of bacterial β-galactosidase enzyme; it is

suitable end beneficial to consume by lactose intolerants. Furthermore, the LAB

which is used to produce yogurt, Lactobacillus bulgaricus and Streptococcus

thermophilus, are not resistant to gastric acidity. Hence, the products with

probiotic bacteria are more efficient for lactose intolerant human. It is thought

that the major factor improves the digestibility by the hydrolyses of lactose is the

bacterial enzyme β-galactosidase. Another factor is the slower gastric emptying

of semi-solid milk products such as yogurt. So the β-galactosidase activity of

probiotic strains and other lactic acid bacteria used in dairy products is really

important. β-galactosidase activity within probiotics varies in a huge range. It has

to be considered both the enzyme activity of probiotic strain and the activity left

in the final product for their use in lactose intolerant subjects (Salminen et al.,

2004).

19

2.2.1.2. Immune System and Probiotics

The effects of immune system are promising. However, the mechanism is

not well understood. Human studies have shown that probiotic bacteria can have

positive effects on the immune system of their hosts (Mombelli and Gismondo

2000). Several researchers have studied on the effects of probiotics on immune

system stimulation. Some in vitro and in vivo searches have been carried out in

mice and some with human. Data indicate that oral bacteriotherapy and living

bacteria feeding in fermented milks supported the immune system against some

pathogens (Scheinbach 1998, Dugas, et al. 1999). Probiotics affect the immune

system in different ways such as; producing cytokines, stimulating macrophages,

increasing secretory IgA concentrations (Çakır 2003, Scheinbach 1998, Dugas, et

al. 1999). Some of these effects are related to adhesion while some of them are

not (Quwehand et al., 1999).

2.2.1.3. Diarrhea

Diarrhea is many causes and many types so it is difficult to evaluate the

effects of probiotics on diarrhea. But there are lots of searchs and evidence that

probiotics have beneficial effects on some types of dierrhea. Diarrhea is a severe

reason of children death in the worldwide and rotavirus is its common cause

(Scheinbach, 1998). In the treatment of rotavirus dierrhea, Lactobacillus GG is

reported really effective. The best documented probiotic effect is shortened

duration of rotavirus diarrhea using Lactobacillus GG. Also Lactobacillus

acidophilus LB1, Bifidobacterium lactis and Lactobacillus reuterii are reported

to have beneficial effects on shortening the diarrhea (Salminen et al., 2004).

Probiotics which are able to restore and replace the normal flora should be

used. Also they should be used in high risk patients such as old, hospitalised or

immunocompromised. Studies with Saccharomyces boulardii proved that

Clostridium difficile concentration is decreased in the presence of Saccharomyces

boulardii (Gismondo et al., 1999).

2.2.1.4. Cancer

Epidemiological studies point out that if the consumption of saturated fats

increases in the diet, the occurrence of colon cancer increases in Western World.

20

Bacterial enzymes (β-glucornidase, nitroreductase and azoreductase) convert

precarcinogens to active carcinogens in the colon. It is thought that probiotics

could reduce the risk of cancer by decreasing the bacterial enzymes activity

(Fooks et al., 1999, Scheinbach 1998). The exact mechanism for the anti-tumour

action is not known, some suggestions have been proposed by McIntosh which

are given as follows:

1. Carcinogen/procarcinogen are suppressed by binding, blocking or

removal.

2. Suppressing the growth of bacteria with enzyme activities that may

convert the procarcinogens to carcinogens.

3. Changing the intestinal pH thus altering microflora activity and bile

solubility.

4. Altering colonic transit time to remove fecal mutagens more efficiently.

5. Stimulating the immune system.

2.2.1.5. Cholesterol Reduction

Lots of researchers proposed that probiotics have cholesterol reduction

effects. However, the mechanism of this effect could not been explained

definitely. There are two hypotheses trying to explain the mechanism. One of

them is that bacteria may bind or incorporate cholesterol directly into the cell

membrane. The other one is, bile salt hydrolysis enzymes deconjugate the bile

salts which are more likely to be exerted resulting in increased cholesterol

breakdown (Çakır 2003, Scheinbach 1998, Prakash and Jones, 2004).

A study on the reduction of cholesterol was showed that Lactobacillus

reuteri CRL 1098 decreased total cholesterol by 38% when it is given to mice for

7 days in the rate of 104 cells/day. This dose of Lactobacillus reuteri caused a

40% reduction in triglycerides and a 20% increase in the ratio of high density

lipoprotein to low density lipoprotein without bacterial translocation of the native

microflora into the spleen and liver as cited by Kaur et al. (2002).

21

Table 3. Various special therapeutic or prophylactic properties of specific

probiotics (Parvez et al., 2006)

Microflora Associated actions Reference

Bifidobacteria species Reduced incidence of neonatal necrotizing enterocolitis

Caplan and Jilling (2000)

Enterococcus faecium Decreased duration of acute diarrhoea from gastroenteritis

Marteau et al. (2001)

Lactobacillus strains Administration of multiple organisms, predominantly Lactobacillus strains shown to be effective in ameliorating pouchitis Lactose digestion improved, decreased diarrhoea and symptoms of intolerance in lactose intolerant individuals, children with diarrhoea, and in individuals with short-bowel syndrome Microbial interference therapy – the use of nonpathogenic bacteria to eliminate pathogens and as an adjunct to antibiotics Improved mucosal immune function, mucin secretion and prevention of disease

Vanderhoof (2000)


Bengmark (2000)

Lactobacillus

acidophilus

Significant decrease of diarrhoea in patients receiving pelvic irradiation Decreased polyps, adenomas and colon cancer in experimental animals Prevented urogenital infection with subsequent exposure to three ropathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa Lowered serum cholesterol levels


Gorbach et al. (1987)

Sanders and Klaenhammer

(2001)

Ouwehand et al. (2002)

Lactobacillus

plantarum

Reduced incidence of diarrhoea in daycare centres when administered to only half of the children Especially effective in reducing inflammation in inflammatory bowel; e.g., enterocolitis in rats, small bowel bacterial overgrowth in children, pouchitis Reduced pain and constipation of irritable bowel syndrome Reduced bloating, flatulence, and pain in irritable bowel syndrome in controlled trial. Positive effect on immunity in HIV+ children

Vanderhoof (2000)

Schultz and Sartor (2000);

Vanderhoof (2000)

Vanderhoof (2000)

Nobaek et al. (2000)

Walker (2000)

Lactobacillus reuteri Shortened the duration of acute gastroenteritis Shortened acute diarrhoea


Shornikova et al. (1997a,

1997b)

Lactobacillus

rhamnosus

Enhanced cellular immunity in healthy adults in controlled trial

Tomioka et al. (1992)

Lactobacillus

salivarius

Suppressed and eradicated Helicobacter pylori in tissue cultures and animal models by lactic acid secretion

Aiba et al. (1998)

Bacteroides species Chronic colitis, gastritis, arthritis (increased bacterial urease activity in chronic juvenile

Vanderhoof (2000)

22

arthritis) Saccharomyces

boulardii (yeast) Reduced recurrence of Clostridium difficile diarrhoea Effects on C. difficile and Klebsiella oxytoca resulted in decreased risk and/or shortened duration of antibiotic-associated diarrhoea Shortened the duration of acute gastroenteritis Decreased only functional diarrhoea, but not any other symptoms of irritable bowel syndrome

Pochapin (2000)




Table 4. Commercially used probiotics:

Strain Country Company

Lactobacillus rhamnosus Finland Valio Dairy, Helsinki Lactobacillus johnsonii Lal Switzerland Nestle, Lausanne Lactobacillus casei Shirota Japan Yakult, Tokyo

Lactobacillus acidophilus NCFM USA Rhodia, Madison L. casei CRL-43i Gilliland (La-Mo) USA Chr. Hansens, Wisconsin

Lactobacillus reuteri SD 2112 USA BioGaia, North Carolina Lactobacillus plantarum 299V Sweden Probi, Lund

L. casei DN 014001, Lactobacillus

delbreukii subsp bulgaricus 2038 France Danone

Streptococcus thermophiles 1131 Lactobacillus acidophilus SBT-2062

Japan Meiji milk products, Tokyo

Bifidobacterium longum SBT-2928 Japan Snow brand milk products, Tokyo

Saccharomyces boulardii USA Biocodex, Seattle B. longum BB536 Japan Moringa milk industry

Bifidobacterium breve Yakult Japan Yakult, Tokyo

2.2.2 Commercial status of probiotics in India:

Currently, some of the pharma preparations of probiotics are used as

prescription drugs. The perception that fermented milk or dahi is beneficial has

already been widespread across India because, traditionally, these products have

been used since Vedic times for the treatment of diverse conditions such as skin

allergies, stomach upsets, especially diarrhoea. Probiotic food concept has just

come into limelight with introduction of some of dairy products. However,

presently Indian market is at infancy stage. Leading players in India, who are

engaged in Probiotic Dairy products are Mother Dairy, Amul, Nestle, Britannia

and Yakult-Danone. Presently, Mother Dairy’s dahi and lassi are available with

23

brand name B-active in National Capital region which has received a very good

consumer response due to its acceptable body and texture and having appropriate

acidity level. B-active probiotic dahi is available in different pack sizes to suit the

different customer requirements. Amul has also entered in to the probiotic

segment with the introduction of ice-cream and dahi. Nestle has launched Active

Plus dahi and Yakult-Danone, a probiotic drink Yakult.

Probiotic foods are becoming increasingly popular. A number of health

benefits have been claimed for Bifidobacterium sp. and therefore inclusion of

these organisms in the diet is considered to be important in maintaining good

health. Probiotics have anticarcinogenic properties, a specific probiotic effect,

which are of three types: (1) elimination of procarcinogens; (2) modulation of

procarcinogenic enzymes; and (3) tumour suppression (Wollowski et al., 2001).

Furthermore, consumption of these organisms is an ideal method to re-establish

the balance in the intestinal flora after antibiotic treatment (Gibson et al., 1995).

There is a growing agreement relating to the beneficial aspects of specific dairy

products such as fermented milk and yoghurt and of bacterial cultures that

ferment the dairy products in human and animal nutrition. Experimental and

epidemiological studies provide evidence that fermented milk and bacterial

cultures that are routinely used to ferment the milk reduce the risk of certain

types of cancer and inhibit the growth of certain tumours and tumour cells

(Boutron-Ruault, 2007).

Rolfe, (2000) has reported that, many health promoting effects have been

attributed to certain Bifidobacterium sp. These include reduction of ammonia

levels, stimulation of the immune system, and alleviation of lactose intolerance

and prevention of gastrointestinal disorders (O’Sullivan, 1996). Several probiotic

bacteria have been introduced in the market and the range of products in which

probiotic bacteria are added is increasing. However, many of the prophylactic and

therapeutic properties of these foods containing bifidobacteria are a matter of

speculation because there are inherent difficulties in obtaining definitive evidence

for proposed effects of ingesting bifidobacteria.

24

Table 5. Microorganisms applied in probiotic products (Yavuzdurm, 2007)

Lactobacillus species Bifidobacterium

species

Others

L. acidophilus

L. rhamnosus L. gasseri

L. casei

L. reuteri

L. delbrueckii subsp.

bulgaricus

L. crispatus

L. plantarum

L. salivarus

L. johnsonii

L. gallinarum

L. plantarum

L. fermentum

L. helveticus

B. bifidum

B. animalis

B. breve

B. infantis

B. longum

B. lactis

B. adolascentis

Enterococcus faecalis

Enterococcus faecium

Streptococcus salivarus subsp. thermophilus Lactococcus lactis subsp. lactis Lactococcus lactis subsp. cremoris Propionibacterium

freudenreichii

Pediococcus acidilactici

Saccharomyces boulardii

Leuoconostoc mesenteroides

2.2.6. Viability of probiotic organisms Microorganisms introduced orally have to, at least, transiently survive in

the stomach and small intestine. Although this appears to be a rather minimal

requirement, many bacteria including the yoghurt-producing bacteria

Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus

often do not survive to reach the lower small intestine. The reason for this

appears to be low pH of the stomach. In fasting individuals, the pH of the

stomach is between 1.0 and 2.0 and most microorganisms, including lactobacilli,

can only survive from 30 seconds to several minutes under these conditions

(Dunne et al., 2001). Therefore, in order for a probiotic to be effective, even the

selection of strains that can survive in acid at pH 3.0 for sometime would have to

be introduced in a buffered system such as milk, yoghurt or other food.

Hamid and Abulfazl, (2012) isolated potentially probiotic Lactobacilli

from traditional cheese presented in Iran, Tabriz market. In total, 50 samples of

traditional cheese were collected randomly from Tabriz market. After enrichment

in MRS broth, the bacterial flora was screened for its acid and bile resistance.

Then, the single colony was picked up from MRS agar for the evaluation and

25

molecular identification. The modified ARDRA technique was exploited for the

identification of isolates of lactobacillus at the level of species. After ARDRA,

two isolates were sequenced. The isolated lactobacilli belonged to Lactobacillus

fermentum and Lactobacillus plantarum. The results showed that the traditional

Tabriz cheese contained the superior strain of probiotic lactobacilli.

Lactic acid bacteria (LAB) strains from the intestinal tissue of African

catfish Clarias griepinus were successfully screened, characterized and

identified. The isolates (S1#8, S1#9, S1#10, S1#18, S1#19 S1#20 and S1#21)

shared common morphological characteristics of LAB such as non-sporulating,

Gram positive cocci or cocco-bacilli shape. These were the dominating

morphology found in the catfish, as compared to other types of LABs reported to

be found in freshwater fishes. All of the 35 isolates have the ability to utilize

lactose as part of their metabolism process and showed negative reactions

towards catalase test. These isolates were also tested for antimicrobial activities

using disc diffusion assay against indicator Salmonella typhimurium and

Escherichia coli. Based on the partial 16S rRNA sequences, the selected LAB

isolates belonged to a member of Lactococcus lactis with 98% DNA similarity.

This strain can be used as probiotic in aquaculture feeding (Hamid et al., 2012).

Different lactic acid bacteria generally found in fruits, cultivated in

Pakistan were studied using microscopic analysis as well as the technique of

ribotyping by Naeem et al. (2012). Following the culture enrichment method

randomly selected fruit samples were analyzed. The initial identification was

based on conventional morphological and biochemical analysis while the final

confirmation was done by utilizing advanced molecular tools (i.e., 16S rRNA

gene amplifications). Prior to all these manipulations the growth conditions were

carefully optimized for the respective strains. The surveillance of all strains after

experiencing an acid shock mimicking the harsh acidic surroundings of

gastrointestinal tracts reflects their probiotic potential. The results of antibiotic

sensitivity test demonstrated an unusual high rate of kanamycin and oxacillin

resistance among isolated LAB strains. Hence it can be hypothesized that use of

these antibiotics against pathogens would not disrupt the natural microbiota of

26

gastrointestinal tract which mainly consist of lactic acid bacteria. The study

finally led us to conclude that Lactobacillus plantarum was the most abundant

type of lactic acid bacteria distributed in most of the fruit samples studied, on the

other hand Leuconostoc mesenteroide subsp. mesenteroides showed sporadic

occurrence. The presence of such bacteria in raw fruits satisfies the nutritive and

microbial profile of fruits to be a healthy food.

Twenty lactic acid bacterial strains were isolated and identified by

phenotypic method from samples of probiotic dairy products as fermented milk

drinks, yoghurts and infant milk powder. These strains were grouped at species

level as Lactobacillus casi, Lactobacillus acidophilus, Lactobacillus plantarum

and Bifidobacteria species. These strains were further tested for the presence of

functional traits useful for probiotic applications, such as resistance to acidic

condition at pH 2, bile salt hydrolytic activity and ability for adhesion to Caco-2

cells as well as ability to inhibit the adhesion of Escherichia coli, Salmonella

typhimurium and Shigella flexneri to caco-2 cells. Our obtained results showed

that most of tested strains exhibited characteristics suggesting that they would

survive in the gastrointestinal tract and also had the capability for adhesion to

caco-2 cells. Greater variability was observed for the other traits analyzed. These

data suggest that these probiotic strains had characteristic and differential

functions traits. Therefore, results from our present study are expected to

encourage people to consume more probiotic dairy products, as it was revealed

that these products contain some probiotic lactic acid bacteria which play a major

role for the beneficial health effects of consumers (Dardir, 2012).

Sieladie et al. (2011) isolated total of one hundred and seven colonies of

lactobacilli from thirty-two samples of raw cow milk were screened for their

probiotic use. 15 isolates of lactobacilli were selected for acid and bile tolerance.

Almost all the acid and bile tolerant isolates of lactobacilli were sensitive to eight

of the nine antibiotics tested. None of the assayed strains showed hemolytic and

gelatinase activity. In addition, isolate 29V showed strong antimicrobial activities

against the used indicator pathogens. All isolates expressed bile salt hydrolase

activity and had ability to assimilate cholesterol in vitro. The 15 selected isolates

27

were identify to species level as Lactobacillus plantarum using API 50CH Kits.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)

was carried out to discriminate between three new best probiotic strains of

Lactobacillus plantarum. According to these results, the Lactobacillus strains

associated with dominant microflora that people from Mbororo’s tribe in the

western highlands of Cameroon use to manufacture fermented milk contain new

potentially probiotic strains with antimicrobial and cholesterol-lowering

properties.

Different strains were isolated from the fermented milk in Vellore and

were subjected to preliminary screening and 45 isolates were obtained and it was

characterized and examined for the presence of probiotic properties like

cholesterol assimilation, exopolysaccride production and antibiotic resistance.

The cholesterol assimilation ranged from 28-83%, which is significantly highest

and observed for the first time, and exopolysaccride varied from 16-89%.

Further, resistance to 8 commonly used antibiotics $-lactans (penicillin,

ampicilin), gram positive spectrum (vanomycin), broad spectrum (rifampin,

trimethoprim) and aminogycosides (kanamycin, streptomycin, and bacitracin)

was assessed by disk diffusion method. Among the selected 45 strains, 20, 20, 60,

70, 90 and 100% were found to be exhibiting a significant degree of resistance to

kanamycin, trimetroprim, rifampicin, kanamycin, amphicilin and penicillin

respectively. However, all strains were resistant to penicillin and 90% were

resistant to ampicillin. Usually all Lactobacillus and Bifidobacterium strains were

susceptible to $-lactum antibiotics but our isolates showed resistance which is

contrary and new information to the previous investigation. Based on the above

characters 7 isolates were considered to be best for probiotic applications

(Lavanya et al., 2011).

Mahrous et al. (2011), studied the effect of two probiotic lactic acid

bacteria strains for their ability to assimilation of Cholesterol In Vivo (in previous

work; In Vitro) and their effect on feeding mice on general health indicators,

hematological parameters and their ability to reduce the cholesterol level in blood

serum; the results showed that no adverse effect on the hematological parameters

28

and the probiotic strains (Lactobacillus acidophilus P106, Lactobacillus

acidophilus P110) effect on reduce the level of cholesterol in the blood serum

especially Lactobacillus acidophilus P106. Pelinescu et al. (2011) characterized

the probiotic effects of three Lactococcus lactis strains isolated from breast fed

infant feces (resistance to pH variation and taurocholic acid sodium salt

concentrations, antimicrobial activity, ability to metabolize lactose and

cholesterol, the ability to adhere to eukaryotic cells and competition with

enteropathogenic Salmonella enteritidis, Shigella flexnerii and enteropathogenic

Escherichia coli, immunomodulatory effect). The tested strains showed an

increased resistance to a wide pH range of 3.0 to 8.0, as well as to thauroglicolate

concentrations, from 0.5 to 3%, these features representing great advantages for

the survival of these bacteria, once introduced in the gastrointestinal tract. Strains

reduced the serum cholesterol with an average of 45%. The three strains lacked

mutual inhibition and high inhibitory activity of pathogenic or potentially

pathogenic strains growth, demonstrating their potential use in the treatment of

pediatric gastro-intestinal disorders, as an alternative or in association with

antibiotics. The immunomodulatory studies demonstrated that the probiotic

culture fractions are modulating the expression of the most important cytokines in

the development of the anti-infectious immunity against enteric pathogens,

expressed by the stimulation of TNF-alpha and INF gamma pro-inflammatory

cytokines and the inhibition of IL-6 and IL-8 cytokines, known to be implicated

in the occurrence of lesional effects upon the infected host.

Probiotic potentials of two bacterial isolates from 20 different curd

samples were identified as Lactobacillus spp. by the determination of

morphological, cultural, physiological and biochemical characteristics. The

antibacterial potential against diarrhoegenic bacterial pathogens was also

examined. The reference strain used was Lactobacillus acidophilus, MTCC 447.

The percentage survivability of the strains at pH 3.5, was found to be satisfactory

(>90%). Bile salt resistance (0.3% sodium thioglycollate) was found to be

between 80.41% and 83.2%. The pH decrease of the strains with time showed

slow acidification activity. The lactic acid production of the strains ranges from

1.83 ± 0.12 to 3.93 ± 0.07 g. The strains were β-galactosidase producer and were

29

resistant to principal antibiotics tested. But the absence of plasmids showed that

they are intrinsically resistant or chromosome encoded. Strains showed maximum

inhibition zone against Vibrio cholerae O139 (13.67 ± 0.57 to 15.33 ± 0.57 mm)

in comparison to other diarrhoeagenic bacteria. Only 10% of the examined curd

samples had probiotic bacteria. Isolated strains of Lactobacillus spp. showed

satisfactory probiotic potentials in comparison with reference strains and with

antibacterial activity against diarrhoeagenic pathogens and thus maybe useful in

the management of diarrhoea and also in functional food industry (Shruthy et al.,

2011).

Ebrahimi et al. (2011) isolated and identified new potential probiotic

lactobacilli from traditional Iran dairy products. The isolates were screened for

their probiotic potential activities, including acid and bile resistance, antagonistic

activity and cholesterol removal. Screening of acid and bile tolerant strains from

14 different samples led to the identification of 20 isolates of Lactobacillus spp.

Most promising strains which assimilated more than 75% of the cholesterol in the

medium and/or exhibited high inhibition value against pathogen identified based

on 16S rDNA sequence. Lactobacillus plantarum, Lactobacillus brevis,

Lactobacillus casei isolated from yogurt and cheese exhibited both high

cholesterol assimilation and antagonist activity. These results suggested that,

Iranian indigenous lactobacilli have potential as probiotics, and they might also

be good candidates to be used as probiotic carrier or functional foods. Tambekar

and Bhutada, (2010) isolated 110 isolates from 120 milk samples (40 each from

buffalo, cow, and goat) were analyzed and isolates were identified as Lactic Acid

Bacteria (LAB). Out of these 11 isolates were identified as prominent probiotics,

among them 3 isolates were excellent probiotics. These excellent probiotics were

compared for their probiotic potential with commercial probiotic preparations

such as Sporlac powder, LactoBacil plus, P-Biotics kid, Gastroline, Pre-Pro kid

and standard probiotic bacterial strains Lactobacillus plantarum (MTCC 2621)

and Lactobacillus rhamnosus (MTCC 1048). The isolated LAB exhibited

excellent probiotic characteristics than commercial probiotic preparations and

standard probiotic bacterial strains. Study suggested that use of these probiotic

30

bacteria from milk of domestic animals can be help to prevent or control the

intestinal infections and contributes health benefits to consumers.

Hoque et al. (2010) isolated Lactobacillus spp. from two regional

yoghurts in Bangladesh, which were identified on the basis of their colony

morphologies and some biochemical tests. It was observed that isolated

Lactobacillus spp. were resistance to inhibitory substances like phenol (0.4%),

NaCl (1-9%) and bile acid (0.05-0.3%). Additionally, good growths were

observed in the presence of 1% NaCl and 0.3% bile acid. The isolated

Lactobacillus spp. did show good survival abilities in acidic (pH 2.5) and alkaline

(pH 8.5) conditions, while, their maximum growth was observed at pH 5.0 for

lactobacilli isolated from Bogra yoghurt and at pH 6.5 for lactobacilli isolated

from yoghurt of Khulna region of Bangladesh. Isolated lactobacilli were able to

produce organic acid in skim milk which was determined by titrimetric method.

The Lactobacillus spp. also did show good survival abilities in simulated gastric

juice at pH 2.22 and pH 6.6 (Control). Their susceptibility to selected nine

antibiotics was determined in terms of minimum inhibition concentration (MIC).

The MICs results showed that, Lactobacillus spp. Isolated from Bogra yoghurt

were sensitive to amoxicillin, moderately sensitive to gentamycin, clindamycin,

azithromicin and resistant to kanamycin, nalidixic acid, metronidazol, cefradine

and tetracyclin. On the other hand, Lactobacillus spp. isolated from yoghurt of

Khulna region were sensitive to gentamicin, clindamicin and resistant to

amoxicillin, tetraciclin, kanamicin, nalidixic acid, metronidazol, azithromicin and

cefradine. In conclusion, most of the results from the present experiments showed

that, there were variations in probiotic properties of the isolated Lactobacillus

spp. from different regions.

Selective isolation of LAB performed using de Man Rogosa Sharpe

medium. LAB isolates that potential as probiotics was screened. Selection was

based on its ability to suppress the growth of pathogenic bacteria, bacterial

resistance to acidic conditions and bacterial resistance to bile salts (bile). Further

characterization and identification conducted to determine the species. The results

showed that two of the ten isolates potential to be developed as probiotic bacteria

31

that have the ability to inhibit several pathogenic bacteria such as Eschericia coli,

Bacillus cereus and Staphylococus aureus, able to grow at acidic condition and

bile tolerance during the incubation for 24 hour. Based on the API test kit, the

both of isolate identified as members of the species Lactobacillus paracasei ssp.

paracasei (Sarkano et al., 2010).

The antagonistic activity of two Bacillus strains isolated from the

gastrointestinal tract of giant freshwater prawns against Aeromonas hydrophila

was evaluated in vitro by Sansawat and Thirabunyanon, (2009). The

characterisation of the novel probiotic strains of these bacilli was also performed.

Bacillus subtilis P33 and 72 were found to have high inhibition activities against

the growth of Aeromonas hydrophila by two assay methods: paper disc and well

diffusion. Probiotic properties, namely acid and bile salt tolerance,

autoaggregation, coaggregation, hydrophobicity and adhesion to Caco-2 cells,

were further analysed. Survival rates in model gastrointestinal tract condition,

viz. pH 2.5 for 3 h and 0.3% bile salt for 24 h, were shown to be more than 95%

and 90% respectively. The ability of B. subtilis strains of P33 and P72 to adhere

to epithelial cells of the host animal was measured by percentage autoaggregation

(35.7 and 42.2%), coaggregation (11.1 and 11.6%), hydrophobicity in n-

hexadencane (25.6 and 30.0%), xylene (32.2 and 36.1%), toluene (30.3 and

31.6%), and adhesion to Caco-2 cells (4.21 and 3.23 log cfu/ml respectively).

These results indicate that both strains of Bacillus subtilis P33 and P72 can be

considered to be good novel probiotic candidates for use in the prawn aquaculture

industry.

Iñiguez-Palomares et al. (2007), selected Lactobacillus strains isolated

from small intestine of piglets based on the characteristics of resistance to low pH

and bile salts, surface properties and antagonistic effect against Escherichia coli

K88. To identify Lactobacillus species, a fragment of 16S rRNA gene of the

strains was sequenced. Low pH, bile salts resistance and antagonistic activity

were quantified by viable count in plates. Surface properties were measured using

a spectrophotometer at 600 nm. Sixty-two Lactobacillus strains were isolated

from small intestine of piglets. Species Lactobacillus salivarius, Lactobacillus

32

reuteri and Lactobacillus mucosae were identified. We found that 20

Lactobacillus strains resisted low pH and bile salt, 8 of them were adherents and

they inhibited in vitro the growth of E. coli K88. In conclusion, our results

showed that 8 strains have potential probiotic value, according resistance to

gastrointestinal tract, surface properties and antagonistic characteristics. L.

salivarius was the species that fulfilled the criterion to be identified as a possible

probiotic microorganism.

Mourad and Nour-Eddine, (2006) evaluated some probiotic traits of

Lactobacillus plantarum strains previously isolated from fermented olives. 11

strains were tested for their in vitro antibiotics susceptibility, tolerance to bile,

resistance to low pH values, acidifying activity, proteolytic activity, haemolytic

activity, lactic acid and exopolysaccharide production. Collectively, the strains

were susceptible to the most of antibiotics tested, showed the survivability (11 ±

2.2 to 65 ± 1.8%) at high bile salt concentration (2% oxgall) and resistance at pH

2. Most strains have showed fast (1.035 ± 0.29 to 0.912 ± 0.21 mmol/l±sd of

lactic acid) or medium (0.556 ± 0.29 to 0.692 ± 0.18 mmol/l±sd) acidification

activity with a good proteolytic activity (1.49± 0.25 and 5.25± 0.11 mg l-1

tyrosine at 72 h). None of the strains produced exopolysaccharides or

haemolysin.

Nowroozi et al. (2004) isolated Lactic acid bacteria from sausage. Each

isolate of lactobacillus species was identified by biochemical tests and comparing

their sugar fermentation pattern. Antibacterial activities were done by an agar

spot, well diffusion and blank disk method. Enzyme sensitivity of supernatant

fluid and concentrated cell free culture after treatment with α-amylase, lysozyme

and trypsin was determined. The isolated bacteria were L. plantarum, L.

delbruekii, L. acidophilus, L. brevis. The isolated bacteria had strong activity

against indicator strains. The antibacterial activity was stable at 100oC for 10 min

and at 56oC for 30 min, but activity was lost after autoclaving. The maximum

production of plantaricin was obtained at 25 - 30oC at pH 6.5. Because,

lactobacilli that used to process sausage fermentation are producing antimicrobial

activity with heat stability bacteriocin, so, these bacteria may be considered to be

33

a healthy probiotic diet. Lactobacilli originally isolated from meat products are

the best condidates as probiotic bacteria to improve the microbiological safety of

these foods.

2.3. FERMENTED FOOD ITEMS AS A SOURCE OF POTENTIAL

PROBIOTIC STRAINS

Grosu-Tudor and Zamfir, (2012) isolated six LAB strains Romanian

fermented vegetables were selected: Leuconostoc citreum 344 and Lactobacillus

brevis 183 (showing an antibacterial activity against Listeria monocytogenes

ATCC 1911), Leuconostoc mesenteroides 348 and Lactobacillus brevis 312 (with

an antibacterial activity against Eschrichia coli ATCC 25922) and Lactobacillus

plantarum 327 and Lactobacillus brevis 380 (with an antibacterial activity

against Salmonella enterica ATCC 14028) (unpublished results). The cultures

were preserved in MRS broth (de Man et al., 1960) and stored at -75°C in the

presence of 25% (v/v) of glycerol as cryoprotectant. Prior to use, the strains were

subcultured twice in MRS broth and incubated overnight at 37°C.

Fresh Polygonum minus (Kesum) plant was obtained from the Herbal

Garden of University Putra Malaysia (UPM) and placed in a sterile plastic bag.

Then the leaves of Kesum were washed with sterile distilled water and chopped

into small pieces (5 mm). One-gram of samples that have been cut, were

suspended in 9 mL of 0.85% (w/v) sterilized NaCl solution by shaking for several

minutes. From the suspensions, 1 ml was inoculated in de Man, Rogosa and

Sharpe (MRS) broth (Merck, Darmstadt, Germany) and incubated for 3 days at

30oC and 37oC. After that, aliquots of the culture were diluted 10 fold up to 106

with MRS broth and 100 µL was spread on MRS agar (Merck, Darmstadt,

Germany). The plates were incubated for 3 days at 30oC and 37oC. Bacterial

colonies that grew on the plates were picked and streaked on new MRS agar

(Merck, Darmstadt, Germany) plates to get pure culture. Isolated bacteria were

stored at -80oC in MRS broth containing 15 % (v/v) glycerol (Baradaran et al.,

2012). Ten grams of butter was homogenized with 90 mL sterile NaCl solution

(0.85%, w/v) to a homogenous suspension and then a tenfold serial dilution in

NaCl solution (0.85%, w/v) was carried out by Bettache et al. (2012). For

34

isolation of lactic acid bacteria, acidified MRS pH 5.4 incubated anaerobically for

72 h at 37°C was used for isolation of lactobacilli. MSE incubated aerobically for

48 h at 35°C was used for isolation of leuconostocs. M17 agar incubated

aerobically for 48 h at 30°C for the isolation of lactococci.

Twenty samples of raw seafood such as shrimp (Lito-vannamei) and

green mussel (Perna viridis) and 32 samples of Thai fermented seafood products

such as kung-jom (fermented shrimp), pla-jom (fermented fish) and hoi-dong

(lowly salted fermented mussel) were purchased at local markets in Bangkok and

Prachinburi, Thailand by Nanasombat et al. (2012). Total viable LAB counts in

each sample were analyzed by spread plating the serially diluted samples onto

MRS agar (deMan Rogosa Sharpe, Difco, pH 7.2±0.2) supplemented with 0.5%

calcium carbonate. After incubation at 37°C for 48 h, colonies with clear zones

were counted. Some of these colonies were selected and purified on MRS agar.

Fleck et al. (2012) isolated strains of lactobacilli from sausages produced under

industrial conditions without the addition of starter cultures. The lactic acid

bacteria count was determined by microbiological analysis (MRS agar;

Lactobacillus agar according to De Man, Rogosa and Sharpe; manufacturer

Merck, Germany). The isolates confirmed as gram-positive and catalase-negative

bacilli were biochemically identified using the API 50 CHL (BioMerieux) test

and the computer program APILAB Plus. All the isolates with fermentative

profile confirmed by the API test as excellent (ID>99%; 50 isolates) were

considered as lactobacilli and were subjected to further analyses. The reference

strain was L. brevis, American Type Culture Collection, ATCC B287 (Oxoid).

A total of 331 lactic acid bacteria (LAB) were isolated from kantong

production sites in two villages in the Northern region of Ghana. Samples were

taken aseptically from various stages (0h, 24h, 48h of fermentation and final

product) of kantong production, serially diluted and cultured anaerobically on

MRS agar at 30oC for 48 h. They were phenotypically identified on the basis of

colony and cell morphology, in addition to Gram, oxidase and catalase reactions

at the UDS/DANIDA MICROBIOLOGY LAB, Navrongo Ghana. These isolates

then underwent genotypic tests. They were maintained at 4oC on De Man,

35

Rogosa and Sharpe (MRS) agar (Merck, Darmstadt, Germany) (Kpikpi et al.,

2010).

Ashiraf et al. (2009) collected contents from crop, gizzard, ileum and

caecum of 20 adult healthy chicks for the isolation of lactobacilli. These contents

were mixed homogeneously in sterilized phosphate buffer saline (PBS)

separately. Similarly, 20 conventional yogurt samples were procured from the

local market in sterile plastic bags and homogenized by dissolving in 100 ml of

sterilized phosphate buffer saline. Samples from both sources were diluted

serially 10 - fold in PBS and then inoculated on deMan Rogosa and Sharpe

(MRS, Oxoid, England) agar plates by pour plate method (Awan and Rahman,

2005). MRS agar plates were incubated at 37oC for 48 h an-aerobically.

Morphologically distinct and well isolated colonies were picked and transferred

to new MRS agar plates by streaking. Finally, pure colonies were obtained.

Hyun-jue Kim et al. (2006), obtained raw milk from the Sunhwa Dairy

Farm (Suwon, Korea) and stored at 5°C. In order to isolate LAB, raw milk was

cultured in Lactobacillus MRS broth, then spread onto Bromocrezol Purple Agar

(BCP agar). After it was incubated at 37°C for 48 h, under either aerobic or

anaerobic conditions, the colony were subcultured more than 3 times in

Lactobacillus MRS agar. And then it was dissolved in skim milk solution

containing 20% glycerol and stored at -70°C for further use. Each experiment

used a stock freezer vial for medium inoculation.

Fifty-four fermented vegetables, 9 silages and 4 grasses were used as

sources to isolate lactic acid bacteria by Kimoto et al. (2004). They were gathered

in Hokkaido and Okinawa area in Japan. MRS broth (Difco, Laboratories,

Detroit, MI) to which 1.6% (w/v) of agar and 0.8% (w/v) CaCO3 were added was

used to isolate lactic acid bacteria strains from non-dairy plant materials. One-

gram of each sample was homogenized with 9 mL of 0.85% (w/v) sterilized NaCl

solution by shaking for several minutes. From the suspensions, serial dilutions

were made in NaCl solution and plated by spreading 0.1 mL onto the surface of

MRS agar. After anaerobic incubation at 30oC for 48 h, colonies which dissolved

36

CaCO3 and formed clear zones around their own colonies on the medium plate

were isolated at random. The broths inoculated with each colony were cultivated

at 30oC and tested for catalase reaction. Briefly, the cultures were centrifuged,

and 15% (v/v) H2O2 solution was added to the pellets. Catalase-positive was

determined for gas production. Isolated strains were stocked as frozen cultures in

MRS broth with 15% (v/v) glycerol at –80°C.

2.3.1 Identification of Lactic acid bacteria.

2.3.1.1 Physiological, morphological and biochemical characterization of

Lactic Acid Bacteria.

Identification of LAB isolates was performed by examination for cell

morphology using optical microscopy, Gram staining, catalase activity and gas

production from glucose. The carbohydrate fermentation patterns were

determined by using API 50 CHL test kit (bioMerieux, Marcy l’Etoile, France).

Growth at different temperatures, pH and Salt concentrations was studied in MRS

broth. An overnight culture was inoculated at 5 % (v/v) into MRS broth. Growth

at different temperatures was observed in MRS broth after incubation at 10, 25,

30, 37 and 45°C for 3 days. Salt tolerance was determined in MRS broth

containing 3.0, 4.5 and 6.5%. (w/v) NaCl at 30°C for 3 days. Growth at pH of

3.0, 4.0, 5.0, 6.0 and 7.0 was determined in MRS broth after incubation at 30°C

for 7 days. Glucose fermentation and acid production was performed in Nutrient

agar (Merck, Darmstadt, Germany) supplemented with 0.5 % (w/v) glucose and

0.005 % (g/L) bromocresol purple as a pH indicator. Ten µL of the overnight

culture of LAB isolates were then dropped on the prepared agar. After overnight

incubation, acid production was noted by the formation of yellow color zone

around the drops of isolates due to acid production (Baradaran et al., 2012).

Identification of the Lactobacilli was performed according to their

morphological, cultural, physiological and biochemical characteristics (Kandler

and Weiss, 1986; Sharpe et al., 1979): Gram reaction, production of catalase,

carbohydrate fermentation patterns, growth at 15 and 45°C in the lactobacilli de

Man Rogosa and Sharpe (MRS) broth as described by Bergey’s Manual of syste-

matic Bacteriology, methyl red and Voges-Proskauer test in MRVP medium,

37

nitrate reduction in nitrate broth, and indole production in Tryptone broth.

Purified cultures were maintained at -20°C in MRS broth with 10% glycerol and

enriched in MRS broth by incubating at 37°C for 24 h (Kumar and Murugalatha,

2012). The isolates were characterized for gram and catalase reaction and

fermentative catabolism of various carbohydrates. The strains were also tested for

their ability to grow at lower pH values (2.0, 2.5, 3.0, 3.5, 4.0, 4.5). The

fermentative behavior was determined for various carbohydrates in Bromothymol

blue lactose broth (peptone, 0.35 g; Tryptone, 0.35 g; NaCl, 0.5 g; Bromothymol

blue, 0.004 g distilled water, up to 100; pH:7.0) supplemented with inverted

Durham’s tube and 1.5% of carbohydrate to be tested (arabinose; fructose;

galactose; glucose; lactose; maltose; mannitol; raffinose; sucrose, xylose). The

strains showing positive gram reaction and negative catalase test were selected

and the glycerol stocks were stored at -80oC (Naeem et al., 2012).

Initial typing of the representative isolates was based on colony and cell

morphology, Gram, catalase and oxidase reactions. Carbohydrate fermentation of

isolates was investigated using API 50CHL (BioMerieux, Marcy-L’Etolie,

France) galleries (Kpikpi et al., 2010). Growth of isolates was assessed in MRS

broth at 15, 37 and 45oC and at pH 4.4, 7.0, 8.6 and 9.6 by incubating at 37oC

(Patil et al., 2010). Salt tolerance was tested by incorporating 6.5, 10.0 and 15.0%

(w/v) sodium chloride in MRS broth. Lactic acid and carbon dioxide production

was tested in MRS broth containing inverted Durham’s tube in the absence of

citrate in the medium. Production of ammonia in MRS broth containing 0.3%

arginine and 0.2% sodium citrate replacing ammonium citrate was monitored

using Nessler’s reagent. The ability of LAB to ferment various sugars was

examined using HiCarbohydrateTM kit, HiMedia Laboratories Pvt. Ltd, India.

Kostinek et al. (2008) observed Cell morphology by using phase contrast

microscopy at 1000 × magnification (Leitz, Jena, Germany), isolates were Gram-

stained, and catalase activity was determined. Production of gas from glucose in

MRS broth and determination of the presence of d-meso-di-aminopimelic acid in

the cell wall (mDAP) and of the type of lactic acid enantiomer produced were

done using the methods of Schillinger und Lücke. The fermentation of specific

38

sugars was tested according to the method of Jayne-Williams. Growth of

presumptive enterococci was tested in MRS broth containing 6.5% (w/v) NaCl or

in MRS broth at pH 9.6, at 10°C or 45°C, respectively.

2.3.1.2 Molecular characterization of Lactic Acid Bacteria

The sequence analysis of 16S ribosomal RNA (16S rRNA) gene was

employed for identification of bacterial strains. Genomic DNA of selected

isolates was isolated by the protocol devised by Rodringuez & Tait, (1983) with

some amendments. ~1.5 Kb long fragment of the 16S rRNA gene was amplified

from the extracted DNA using eubacterial universal primers [Forward primers:

P8 (5’ AGAGTTTGATCCTGGCTCAG IDENTIFICATION OF LACTIC ACID

BACTERIA FROM FRUIT JUICES ‘3) and Reverse primers: PC1544 (5’

AAGG AGGTGATCCAGCCGCA 3’)] specific for 16S rRNA gene. The

amplified products were directly cloned in pTZ57R/T vector using InsTAclone

Cloning Kit (Fermentas) according to the instructions provided by the

manufacturer. The clones were confirmed for the presence of amplicons via

double digestion of plasmids using restriction enzymes Pst1 and Eco R1. The

confirmed clones were repurified using QIA spin Miniprep Kit # 27106. The

clones were sequenced using universal primers. The partial genome sequence of

approximately 1500 bp long 16S rRNA gene was obtained and sequence

homologies were analysed by comparative studies using 325 “The National

Center for Biotechnology Information (NCBI) using weblink

(http://www.ncbi.nlm.nih.gov/) and Basic Alignment Search Tool (BLAST). The

sequences were then aligned with two closest sequences via ClustalV- Multiple

Sequences Alignment using web links

(http://www.ebi.ac.uk/Tools/msa/clustalw2/). The sequences were then submitted

to Genbank databases (Naeem et al., 2012).

Genomic DNA of LAB isolates was extracted using the conventional

method described by Sambrook et al. (2001) with minor modifications. Briefly,

1.5 mL of overnight culture was harvested by centrifugation at 10,000 x g for 2

min and the pellet was washed with 1 ml of sterile distilled water. The cell was

then resuspensed in 0.2 ml of solution Ι (10 mM Tris-HCl, PH 8.0, 10 mM

39

EDTA, pH 8.0, 20 mM glucose, 50 mM Sodium chloride) containing lysozyme

(10 mg/ml) and incubated at 37oC for 45 min. After that, 20 µL of proteinase K

(20 mg/ml) and 100 µL of solution II (10 % SDS) were added and the mixture

was inverted gently and incubated for 1 hour at 60oC. Following that, a volume of

300 µl of ice-cold neutralizing solution (sodium acetate 3M pH 5.2) was added

and mixed by gentle inversion before placing on ice for 5 min to allow

precipitation. The cell debris was pelleted by centrifugation at 14,000 x g for 12

min at 4°C and the supernatant was transferred to a new micro-centrifuge tube.

Equal volume of phenol-chloroform-isoamylalcohol (PCI; 25:24:1) was added

and mixed gently. Next, the two phases were separated by centrifugation at

13000 x g for 10 min. The upper aqueous layer containing DNA was carefully

transferred to a new 1.5 mL micro-centrifuge tube. This step was repeated once

or twice. Then, the DNA was precipitated by the addition two volumes of cold

absolute ethanol, followed by gentle mixing, incubated at 4°C for 1 h, and

centrifugation at 13000 x g at 4°C for 15 min. The DNA pellet was rinsed with 70

% (v/v) cold ethanol; followed by air dried prior to dissolve in 50 µL of warm

distilled water. Subsequently, 5 µL of 10 mg/mL RNase was added and incubated

at 37°C for 20 min. Finally, the DNA sample was kept at 4°C for working use

and at -2oC for long term storage. The extracted DNAs were used directly in PCR

reactions to amplify the 16S rDNA gene from LAB. The 16S rDNA region was

amplified by using primers 27F 5'- AGAGTTTGATCCTGGCTCAG-3' (8-28)

and E. coli numbering 1542–1522 R 5'- AAGGAGGTGATCCAGCCGCA-3'.

The PCR reaction mixture (25 µl) contained 2.5 µl PCR buffer (Fermentas,

USA), 0.5 µl of 10 mM dNTPs (0.2 mM), 2.5 µl of 25 mM MgCl2 (1.5 mM), 0.5

µl of each primers; and 0.2 µl (1 unit) of Taq DNA polymerase (Fermentas,

USA). Some of the reactions were carried out with the same amount of Pfu DNA

polymerase (Fermentas) and 1 x Pfu PCR buffer with MgSO4 (Fermentas) in

replace of the Taq DNA polymerase. The volume of the mixture was adjusted to

25 µl with 18.3 µl of sterile distilled water containing 100 ng of extracted

genomic DNA. A positive control, consisting of 50 ng of Escherichia coli DNA

and a negative control (no DNA) were included in each amplification. An aliquot

(5 µl) of each amplification reaction was analyzed on 1 % (w/v) agarose gel in 1

40

× TBE buffer (pH 8). PCR was performed in a PCR master Cycler (Eppendorf,

Germany) and the reaction parameters were 30 cycles of denaturation at 94°C for

45 s, annealing at 56°C for 60 s, and extension at 72°C for 90 s (Baradaran et al.,

2012).

Each isolate was grown anaerobically on MRS agar for 48 h at 30oC. An

isolated colony was suspended in 1ml of autoclaved Milli – Q water in a

microfuge tube, centrifuged for 1 min at 12000g and the supernatant discarded.

DNA was isolated from the pellet using the InstaGene matrix (Bio-Rad, Hercules,

CA, USA) following the instructions of the manufacturer. The resulting

supernatant was used as DNA template for PCR. The amplification of the

repetitive DNA elements of isolates was carried out in 25µl of reaction mixture

containing 1.5µl of DNA template, 2.5µl PCR – buffer(x10), 4µl of dNTP

(1.25mM), 1.5µl MgCl2 25mM), 4µl of primer GTG5;(5pmol/µl) (5 � -

GTGGTGGTGGTGGTG - 3 �), 0.25µl of Formamide, 0.25µl of BSA (Bovine

Serum Albumin, 0.1mg/ml), 10.8µl of autoclaved Milli Q water an 1.5µl of Taq

polymerase. The amplification was performed with 30 PCR cycles in a

thermocycler (Trio – Thermoblock, Biometra, Germany). The cycling program

was started with an initial denaturation at 94oC for 5 min followed by 30 cycles

of denaturation at 94oC for 30s, annealing at 45oC for 30s and elongation at 65oC

for 8 min. The PCR was ended with a final extension at 65oC for 16 min and the

amplified product cooled at 4oC The DNA fragments were separated by applying

10µl of each PCR product with 2µl of loading dye to 1.5% agarose gel. A 1kb

DNA marker (GeneRuler DNA ladder, Fermentas) was included as standard for

the calculation of the fragments. The gel was run in 1x TBE buffer (108g

Trisbase/l, 55g boric acid/l and 40 ml of 0.5 M EDTA, pH 8.0) for 5 h at 120V.

The gel was then stained with ethidium bromide for 20 min, washed with distilled

water and photographed under UV illuminator using a digital camera. Cluster

analysis of gels was carried out using BioNumerics Version 2.5 software

(Applied Maths, SINT – MARTENS – LATEM, Belgium) based on the Pearson

Coefficient and the Unweighted Pair Group Method using Arithmetic averages

(UPGMA). PCR reaction was carried out by mixing 1µl of extracted DNA with a

mixture containing 5µl PCR – buffer, 8µl dNTP (1.25mM), 3µl of MgCl2

41

(25mM), 1µl of primer 0011F, 1µl of primer 1510 r, 0.5µl formamide, 0.25µl Taq

polymerase and 30.25µl of autoclaved Milli Q water. The amplification was

carried out in 30 PCR cycles, first denaturation at 94oC for 5 min then 30 cycles

at 99oC for 90s, 52oC for 30s and 72oC for 90s. The final extension was carried

out at 72oC for 7 min and the product cooled at 4oC. The PCR product was

purified using QIAquick purification Kit (Qiagen, Germany). The sequencing

was performed and a database search was carried out in GenBank database using

BLAST programme (Kpikpi et al., 2010).

Patil et al. (2010), extracted genomic DNA from all the isolates as per the

method of Delos Reyes Gavilan et al. (1992) with minor modifications. The

DNA was extracted by phenol-chloroform method and precipitated with absolute

alcohol. After air drying, the DNA pellets were dissolved in 10 mM Tris-1 mM

EDTA (TE) buffer and analyzed by electrophoresis on 1% agarose gel containing

1 µg/mL ethidium bromide with Tris- acetate EDTA (TAE) buffer at 50 mA for 1

h. The 16S rRNA gene sequence of LAB was amplified using universal primers

of E. coli 16S rRNA gene sequence from 18-27 bp as forward primer (5′ -AGA

GTT TGA TCC TGG CTC AG- 3′) and 1471-1492 bp as reverse primer (5' -TAC

GGC TAC CTT GTT ACG ACT T- 3'). The reaction was carried out in

Eppendorf Master Cycler. The PCR conditions were standardized as follows:

initial denaturation at 94oC for 5 min, 30 cycles of denaturation of 94oC for 30 s,

annealing at 54oC for 30 s and extension at 72oC for 90 s and final extension at

72oC for 5 min. The PCR products were analyzed by 1% agarose gel

electrophoresis with 1 µg/mL ethidium bromide and visualized with hand held

UV trans-illuminator. The 16S rDNA amplicon in the gel was excised and

purified from agarose using Gel extraction kit (Advanced Micro Device Pvt Ltd,

India) as per the manufacturer’s instructions. The purified DNA was sequenced

using ABI Sequencer Model 3700. The 16S rRNA gene sequences determined

(ca. 600-700) were aligned along with the sequences of the type strains obtained

from the GeneBank using the CLUSTAL_X program version 1.8211. Distance

matrices for aligned sequences were calculated by the two-parameter method12.

A phylogenetic tree was constructed by neighbor-joining method13 with the

programme PHY-LIP (version 3.64) available at.http://evolution.

42

genetics.washington.edu/phylip.ht ml. Confidence values of individual branches

in phylogenetic tree were determined by bootstrap analysis based on 1000

samplings (Felsenstein, 1985).

The total genomic DNA of all strains was isolated. Repetitive extragenic

palindromic (rep)-PCR-based typing used for presumptive identification of LAB

strains was done using the primer (GTG). Gel electrophoresis, fingerprint

analyses, and grouping of rep-PCR fingerprints was performed as described

before [6]. The almost- complete 16S rRNA gene of selected strains was

amplified by PCR as described by (Kostinek et al., 2005) and sequenced at

GATC Biotech (Konstanz, Germany). The nucleotide sequences were imported

into the Bionumerics (version 2.5) program (Applied Maths, Sint-Martens-Latem,

Belgium). These sequences were aligned using multiple alignment and similarity

was calculated by global cluster analysis, i.e., UPGMA clustering with Jukes and

Cantor correction and discarding of unknown bases. The nucleotide sequences

were deposited in the GenBank database and received the accession numbers

EU147300 for Lactobacillus fermentum BFE 8253; EU147301, EU147302,

EU147303, and EU147304 for Lactobacillus brevis strains BFE 8325, BFE 8266,

BFE 8285, and BFE 8359, respectively; EU147305, EU147306, EU147307, and

EU 147308 for Lactobacillus plantarum strains BFE 8239, BFE 8200, BFE 8202,

and BFE 8348, respectively; and EU147309, EU147310, EU147311, EU147312,

EU147313, EU147314, EU147315, and EU147316 for Pediococcus acidilactici

strains BFE 8245, BFE 8246, BFE 8387, BFE 8384, BFE 8230, BFE 8390, BFE

8260, and BFE 8262, respectively. The nucleotide accession numbers for the L.

plantarum DSM 20174T, L. fermentum ATCC 14931T, L. brevis DSM 20054T,

and P. acidilactici DSM 20284T type strains used for sequence comparisons

were D79210, M58819, M58810, and M58833, respectively (Kostinek et al.,

2008).

2.4 EFFECTS OF DIFFERENT PARAMETERS ON LAB FOR

PROBIOTIC POTENTIAL Despite the important progress made in the field of probiotics, prediction

of the precise mechanism of action is difficult, due, on one side to the complex

43

interactions that can occur between food, microbes and host cells and on the other

side, to the fact that the probiotic effect is strain dependent. Most of probiotic

products are containing lactic acid bacteria (LAB), microorganisms considered as

commensal and harmless for the host organisms.

The purpose of the present work was to characterize the probiotic effects

of Lactic Acid Bacteria viz., resistance to pH variation and taurocholic acid

sodium salt concentrations, antimicrobial activity, ability to metabolize lactose

and cholesterol, the ability to adhere to eukaryotic cells and competition with

enteropathogenic Salmonella enteritidis, Shigella flexnerii and enteropathogenic

Escherichia coli, immunomodulatory effect.

2.4.1. Resistance to acidic environment

Bacteria used as probiotic strains are joined in the food system with a

journey to the lower intestinal tract via the mouth. In this food system, probiotic

bacteria should be resistant to the enzymes like lysozyme in the oral cavity. Then

the journey will be going on in the stomach and enter the upper intestinal tract

which contain bile. In this stage strains should have the ability to resist the

digestion processes. It is reported that time at the first entrance to release from the

stomach takes three hours. Strains need to be resistant to the stressful conditions

of the stomach (pH 1.5-3.0) and upper intestine which contain bile (Chou and

Weimer 1999, Çakır 2003).

To show probiotic sufficiencies, they should reach to the lower intestinal

tract and maintain themselves overthere. Because of desirable point the first

criteria is looking for probiotic strains is being resistant to acid and bile. Bile

acids are synthesized in the liver from cholesterol and sent to the gall –bladder

and secreted into the duodenum in the conjugated form (500-700 ml/day). In the

large intestine this acids suffer some chemical modifications (deconjugation,

dehydroxylation, dehydrogenation and deglucuronidation) due to the microbial

activity. Conjugated and deconjugated bile acids show antimicrobial activity

especially on E. coli subspecies, Klebsiella spp., and Enterococcus spp. in vitro.

44

The deconjugated acid forms are more effective on gram positive bacteria

(Dunne, et al. 2001, Çakır 2003).

The tested LAB strains isolated from examined samples of probiotic dairy

products differed considerably in their resistance to acid. After 3h of exposure to

pH2, the best survival was observed with strains of L. acidophilus and B. lactis

(>2.0 log cycles reduction). While other strains (L. casi and L. plantarum)

displayed loss of viability of more than <2 log cycles (Dardir, 2012).

The ability of selected strains to survive in low pH conditions were

analyzed after subjecting the strains to a low pH shock by Naeem et al. (2012).

Most of the strains were found to survive between pH ranges of 2.0-4.5. But

some of the strains could survive at pH range of 2.0- 2.5. Pelinescu et al. (2011)

tested Lactococcus lactis strains for their sensitivity to different pH values. The

results showed that resistance to acid, respectively alkaline pH is strain

dependent. Maximum growth ratio for all strains was obtained at pH 6.0 and 7.0.

The most resistant strain to acid pH range between 1.0 and 3.0 was Lactococcus

lactis ssp lactis CMGB 32 (viability between 4.85% and 35.46%). For the pH

range 3.0 – 5.0 an increased resistance and maximum viability rates were

observed for Lactococcus lactis ssp. lactis CMGB 30 (75.24% at pH 5.0). At pH

8.0 the strain viability was strain dependent and viability values were 62.5% to

90%.

Screening of acid tolerant strains in presence of acidic phosphate buffer

(pH=2.5) led to the cultivation of 66 isolates of Lactobacillus spp. from 14

different fermented dairy products. Most of them were isolated from cheese and

yogurt; 34 and 14 respectively. All isolates survived exposure to PBS at pH 6,

with negligible loss of viability, results not shown. However, only 18 isolates

exhibited resistance to pH 2.5, indicated by survival at levels of at least 108

CFU/ml. Thirty strains were judged resistant to pH 2.5 as indicated by survival at

levels of 106-1010 CFU/ml (Ebrahimi et al., 2011).

Isolate OPA4 and AL1 have excellent adaptability to acid atmosphere,

because an increase in growth at 3 pH levels in 24-h period. At pH 2 the two

45

isolates did not grow, because the pH of 2 is a very extreme pH for growth of

microorganisms, including lactic acid bacteria which are generally well adapted

to living in habitats with a relatively low pH environment. At pH 4, 5 and 7 both

isolates are able to grow well, the exponential increase in growth occurred in the

observation at 24th h because of the incubation period is long enough from the 4th

h up to the 24th h resulting in significant cell division. Isolate OPA4 achieve the

best growth at pH 7 whereas at pH 5 isolates AL1 (Sarkono et al., 2010).

Hoque et al. (2010) observed the growth of their isolated Lactobacillus

spp. in various pH values ranges from 2.5 to 8.5. The reason for choosing this pH

range was to determine whether LAB species can grow in acidic and alkaline

conditions and also to predict the optimum pH value for good growth. From the

experimental results, it was found that the isolated Lactobacillus spp. from

yoghurts is able to survive in extreme acidic pH (pH 2.5 to 3.5) and basic pH (pH

7.5 to 8.5). Maximum growth (OD= 2.054) of isolated lactobacilli from Bogra

yoghurts was observed at pH 5.0 and for lactobacilli isolated from Khulna

yoghurt maximum growth (OD= 1.93) was observed at pH 6.5. (Sansawat and

Thirabunyanon, 2009). Kalui et al. (2009) demonstrated that all the 18 L.

plantarum strains tested were tolerable to pH 2.5 after exposure for 3h and 10%

of these strains could not at pH 2.

Iñiguez-Palomares et al. (2007) revealed that, from all isolated strains,

just 20 survived at pH 3.0 and conjugated porcine bile salts (CPBS) conditions in

45% or more. Survival at pH 3 is significant because ingestion of probiotic

bacteria with food or dairy products raises the pH in stomach to 3.0 or higher.

Resistant strains belonged to three identified species, Lactobacillus salivarius

being the most common with 15 strains. Others strains showed good survival to

low pH (more than 50%), but they were discarded, because the resistance to

CPBS was less than 0.1%. There were no statistical differences in the survival

percentage (p>0.05), between the two main 1 species isolated. Mourad and Nour-

Eddine, (2006) showed the results of acid tolerance (survival percentage of L.

plantarum strains at various pH values). All tested strains survived an incubation

periods of 2h to 6h at pH 2.0 and pH 3.0 with decrease in survival percentage

46

when the exposure time progresses for strains. Generally, L. plantarum OL12,

OL15, OL16 and OL33 strains survived acidic conditions better than the rest of

strains. At pH 2.0, L. plantarum OL15 strain showed the highest survival

percentage (65±0.3%, 53±2% and 28±1.4%) after 2, 4 and 6 h incubation period,

respectively. No growth occurred after incubation at pH 1 for 2 h.

2.4.2. Resistance to Bile Salt Bile salt tolerance is the second selection criterion for probiotics.

Resistance to bile salts generally considered as an essential property for probiotic

strains to survive the conditions in the small intestine. Bile salts are synthesized

in the liver from cholesterol and are secreted from the gall bladder into the

duodenum in the conjugated form in volumes ranging from 500 to700 ml per day.

The relevant physiological concentrations of human bile range from 0.1 to 0.3%

(Dunne et al., 2001) and 0.5% (Mathara et al., 2008). The three strains were

resistant to all tested taurocholic acid sodium salt concentrations (i.e. 0.5%, 1%,

1.5%, 2%, 3% and 4%) (Figure 2), the greatest resistance being obtained for the

strain Lactococcus lactis ssp lactis CMGB 30 and Lactococcus lactis sp. lactis

CMGB 31 (Pelinescu et al., 2011).

15 selected acid tolerant lactobacilli isolates were assayed for bile salt

tolerance. All isolate demonstrated good capacity to resist bile salts by presenting

surviving percentage greater than 50% under exposure to 0.2% bile salts after 24h

at 37°C. These isolates were further investigated for their safety properties

including sensitivity to antibiotic, haemolysis and gelatinase activity (Sieladie et

al., 2011).

Screening and selection of the 107 lactobacilli isolates under the acidic

conditions using rapid selective method resulted in four groups. Sixty-six isolates

out of the 107 tested demonstrated poor tolerances to acidic condition, 34 isolates

showed good tolerance, 1 isolate demonstrated very good tolerance and 6 isolates

presented excellent tolerance. Among the 41 lactobacilli isolates demonstrating

at least good tolerance under the acidic conditions using rapid selective method,

18 best isolates were screened for their ability to tolerate acidic condition in citric

47

acid, pH 3 after 5 h. Fifteen of these isolates demonstrated high tolerance to

acidic conditions of pH 3 after 5h of exposure in citric acid at 37°C by showing

surviving percentage greater than 50%. The highest resistance to acidic

conditions was observed in isolate 11RM with maintenance levels of 83.46 after

exposure to pH 3. Surviving percentage of one isolate (103RM) was below 50%

after 5 h of exposure in pH3. Two isolates (66RM and 82RM) lost their viability

under exposure in citric acid pH 3 after 5 h at 37°C. Additionally, the two

methods used to screen isolates based on their tolerance to acidic conditions

showed almost similar results. The fifteen isolates demonstrating surviving

percentage greater than 50% were selected for the further investigations (Sieladie

et al., 2011).

Hoque et al. (2010) isolated Lactobacillus spp. From both yoghurts

(Bogra and Khulna regions of Bangladesh) are able to tolerate up to 0.3% of bile

concentrations.

Sarkono et al. (2010) performed tests for resistance toward bile liquid

used method that was developed by Gilliand et al. (1984) in which he uses bile

concentration of 0.05%, 0.15%, and 0.30%. As a comparison, other researcher

Ljung et al. (2002) tested the resistance of isolates Lactobacillus paracasei subsp.

paracasei F19 in 20% bile and continues to show growth on incubation time of

2 h.

2.4.3 Adhesion properties of Lactic acid bacteria as a potential probiotic

strain

An important property of probiotic bacteria is to adhere on the surface of

intestinal mucosal surface. This would help in improving immune system,

competition with pathogens, maintain metabolic activity and prevent pathogens

to adhesion and colonization. So, for this adhesion probiotic bacteria would

exhibit strong autoaggregation and hydrophobic characters.

Three different solvent were used to evaluate hydrophobic/hydrophobic

cell surface properties and acidic-basic character (Nuraida et al., 2011). The

results revealed that most isolates showed negative affinity to xylene. Low

48

affinity to xylene indicates hydrophylic properties of cell surface. While

Lactobacillus A15 and R23 indicated being slightly hydrophobic as they have

positive affinity to xylene i.e. 15.24% and 9.43% respectively. Lactobacillus A15

and R23 shows higher affinity to chloroform as compared to xylene and ethyl

acetate indicating that the cell surface of A15 and R23 act as electron donor and

has basic character. Meanwhile, lactobacilli A27, A29, B10, B13, B16, R14, and

R26 shows higher affinity to ethyl acetate indicating the cell surface character of

most lactobacilli isolates being electron acceptor and acidic. The results revealed

that autoaggregation ability was relatively low, i.e. ranged between 4.13%-

39.10% (Nuraida et al., 2011). The highest autoaggregation ability shows by

Lactobacilli R23 (39.10%), followed by B13 (31.8%), B16 (29.42%), B10

(28.04%), R14 (26%), and A15 (14.96%). The lowest autoaggregation ability was

shown by A29 (4.13%) and R26 (4.93%).

The autoaggregation percentage of the tested isolates was determined

during a period of 5 h (Sansawat and Thirabunyanon, 2009). In the beginning, the

percentage of autoaggregation ranged between 10.5–14.2%, and then continually

increased every hour. In the final 5th hour, the autoaggregation registered a high

percentage of 35.7–42.2. Coaggregation of these isolates with Aeromonas

hydrophila was expressed as per cent reduction in the absorbance of a mixed

suspension after 5 h. The rates of both isolates were 11.1 and 11.6% for B.

subtilis P33 and P72 respectively. The use of n-hexadencane, xylene, and toluene

to evaluate the hydrophobic cell surface properties of the tested Bacillus isolates

showed a rather consistent result. The hydrophobicity of B. subtilis P33 and P72

strains was 25.6–30.0 % in n-hexadecane, 32.2–36.1 % in xylene, and 30.3-

31.6% in toluene. Hydrophobicity measured by xylene extraction was from 0 to

67 %. No statistically significant correlations were found between aggregation

and hydrophobicity of the tested strains. On the other hand, all B. bifidum strains

were hydrophobic with H% from 16.0 to 67.3. zero values of H% were measured

in strains belonging to the species B. adolescentis, B. longum, and B. dentium,

except for strain A3 of B. dentium (H% = 18.9). Control strains from culture

collections had either Agg or Hydrophobic abilities with the exception of B.

breve ATCC 15700 which showed H% = 45.0 (Valkova et al., 2008).

49

20 strains that survived to pH 3.0 and conjugated porcine bile salts

(CPBS) conditions were included to further characterization (Iñiguez-Palomares

et al., 2007). They showed significant differences (p<0.05) in their

autoaggregation and hydrophobicity properties. Strains 5, 6, 8, 9, 10, 11, 13, 18

and 20 showed an autoaggregation percentage superior to 40%, but strain 13 had

less than 30% for hydrophobicity, for which reason it was discarded as a potential

probiotic. For hydrophobicity, strains 7, 15 and 19 showed less than 10%.

Altogether, 8 strains showed autoaggregation and hydrophobicity percentages

superior to 40%, from these, 6 correspond to L. salivarius, 1 to L. reuteri and 1 to

L. mucosae. This indicates that these strains possess autoaggregative and

hydrophobic characteristics which are related to adhesion to epithelia.

2.4.3 Antibiotic Sensitivity

In the literature some authors have claimed that antibiotic resistance is an

essential criterion for bacteria to be probiotic while others claimed for its

sensitivity. According to world health organisation WHO, 2001 and European

Food Safety Authority-EFSA, 2008 bacteria used as probiotics for humans and

animals should not carry any transferable antimicrobial resistant genes. Thus the

susceptibility of our LAB isolates to the clinically important antimicrobials, is

beneficial as it minimizes the chances of disseminating resistance genes to

pathogens both in the food matrix/or in the gastrointestinal tract.

All 15 isolates bacterial strains were tested for their susceptibility and

resistance against 10 available antibiotics. Almost all strains were sensitive to

50% of the 10 antibiotic used in the test but maximum sensitivity was observed

for oxacillin and kanamycin (Naeem et al., 2012). Strain 43 was resistant to 7

antibiotics out of 8, strains 10, 9, 7, 47 were found to be resistant to 6-8

antibiotics, strains 22, 18, 12, 8, 6, 2, 4, 27 and 42 were resistant to 5-8 antibiotics

(Lavanya et al., 2011). Almost all the strains tested were resistant to penicillin

and 10% were susceptible to ampicillin, ($ lactum antibiotics). Around 20% of

the strains were resistant to kanamycin and streptomycin (aminoglycosides), 70%

were resistant to rifampicin, 20% to trimethoprim and only 6% were resistant to

bacitracin.

50

From the MIC values of 9 tested antibiotics it was found that,

Lactobacillus spp. isolated from Bogra yoghurt of Bangladesh sensitive to

amoxicillin (MIC = 2 µg/ml), moderately sensitive to gentamycin (MIC = 8

µg/ml), clindamycin (MIC = 8 µg/ml), azithromicin (MIC = 8 µg/ml) and

resistant to kanamycin (MIC = 256 µg/ml), nalidixic acid (MIC > 1024 µg/ml),

metronidazol (MIC > 1024 µg/ml), cefradine (MIC = 128 µg/ml) and tetracyclin

(MIC = 32 µg/ml). On the other hand, Lactobacillus spp. isolated from yoghurt of

Khulna region of Bangladesh were sensitive to gentamicin (MIC = 16 µg/ml),

clindamycin (MIC = 8 µg/ml) and resistant to amoxicillin (MIC = 256 µg/ml),

tetracycline (MIC = 128 µg/ml), kanamycin (MIC > 1024 µg/ml), nalidixic acid

(MIC > 1024), metronidazol (MIC > 1024 µg/ml), azithromicin (MIC > 1024

µg/ml) and cefradine (MIC > 1024 µg/ml). Isolated Lactobacillus spp. from

yoghurt of Khulna region has shown broad range of resistances to most of the

antibiotics including amoxicillin (Hoque et al., 2010). Mourad and Nour-Eddine,

(2006) showed that all strains were susceptible to penicillin G, ampicillin,

vancomycin, cloramphenicol, clindamycin, rifampicin and ciprofloxacin. Three

strains (OL16, OL23 and OL53) were totally susceptible to all antibiotics tested.

Most strains showed resistance to 4 of the 11 antibiotics tested, i.e. to cefoxitin (2

strains: OL12 and OL40), oxacillin (3 strains: OL12, OL40 and OL15),

tetracycline (4 strains: OL2, OL7, OL9 and OL15) or kanamycin (8 strains; OL2,

OL7, OL9, OL12, OL15 OL33, OL36 and OL40). Three strains (OL12, OL15

and OL40) have showed a multiple resistance to 3 different antibiotics (both L.

plantarum OL12 and OL40) resist to cefoxitin, oxacillin and kanamycin.

2.3 ANTIMICROBIAL PROPERTIES

Lactobacillus species can produce a variety of metabolites that are

inhibitory to compete bacteria including psychotrophic pathogen. This effect

could be due to combination of many factors such as metabolites of lactic acid

bacteria which may be inhibitory product to other pathogen and food spoilage

organism. This effect could be due to combination of many factors such as

metabolites of lactic acid bacteria which may be inhibitory to other pathogens

and food spoilage organism (Yukeskdag and Aslim, 2010).

51

Table 6. Antimicrobial peptides of Lactic acid bacteria

S. No. Produce Main target organism References

Organic acid

a) Lactic acid Putrefactive and gram –ve bacteria, some fungi.

b) Acetic acid Putrefactive bacteria, clostridia, some yeast and some fungi

1.

c) Hydrogen peroxide

Pathogens and spoilage organism especially in protein rich food

Yukeskdag and Aslim, 2010

Enzymes 2.

Lactoperoxidase system

Pathogens and spoilage causing bacteria (milk and dairy product with hydrogen peroxide)

3. Lysozyme (by recombinant DNA)

Undesired gram +ve bacteria

Low molecular weight metabolites

a) Revterin Wide spectrum of bacteria, yeast, mold

b) diacetyl Gram –ve bacteria

4.

c) Fatty aicd Different bacteria

Breidt & Fleming, 1997

Bacteriocins

I. Lantibiotics Ribosomally produced peptides that undergo extensive post-translational modification Small (<5 kDa) peptides containing lanthionine and methyl lanthionine Ia. Flexible molecules compared to Ib Ib. Globular peptides with no net charge or net negative charge

II. Nonlantibiotics Low-molecular-weight (<10 kDa), Heat stable peptides Formed exclusively by unmodified amino acids Ribosomally synthesized as inactive peptides that get activated by posttranslational cleavage of the N-terminal leader peptide IIa. Anti-listerial single peptides that contain YGNGGVXC amino acid motif near their N termini IIb. Two peptide bacteriocins IIc. Bacteriocin produced by the cell’s general sec-pathway

III. Nonlantibiotics High-molecular-weight (>30 kDa), heat labile proteins

5.

IV Others Complex bacteriocins carrying lipid or carbohydrate moieties, which appear to be required for activity Such bacteriocins are relatively hydrophobic and heat stable

Klaenhammer, 1993; Belkum and Stiles, 2000

As indicated previously, the intestinal microflora is a complex ecosystem.

Introducing new organisms into this highly competitive environment is difficult.

52

Thus organisms that can produce a product or products that will inhibit the

growth or kill existing organisms in the intestinal milieu have a distinct

advantage. The growth media filtrates and sonicates from the bacterial cells of

prospective probiotics should be tested for bactericidal and bacteriostatic activity

in well-plates against a wide variety of pathogens. The ability of probiotics to

establish in the gastrointestinal tract will be enhanced by their ability to eliminate

competitors.

2.5.1. Antagonism among bacteria

Bifidobacteria produce acetic and lactic acids in a molar ratio of 3:2. L.

acidophilus and L. casei produce lactic acid as the main end product of

fermentation. In addition to lactic and acetic acids, probiotic organisms produce

other acids, such as hippuric and citric acid. Lactic acid bacteria also produce

hydrogen peroxide, diacetyl and bacteriocin as antimicrobial substances. These

inhibitory substances create antagonistic environments for foodborne pathogens

and spoilage organisms.

The antagonistic activity of the selected three Lactobacillus isolates L2,

L4, L5 to inhibit the growth of enteropathogens was investigated. All the three

isolates inhibited the growth of E.coli. The L2 isolate has strongest inhibitory

activity against the gastro intestinal enteropathogens like E.coli, Enterococcus

faecalis, Pseudomonas fluorescence, Pseudomonas auregenosa, Staph aureus,

Salmonella typhimurium and Proteus mirabilis (Fig.1) and less antagonistic

activity was observed against Bacillus megaterium1684 and Xanthomonas

campestris. The Lactobacillus spp. L2 having probiotic properties and strong

inhibitory activity was selected for the optimization of bacteriocin production

using various sources. Furthermore the tested isolates (strains) were able to

inhibit the growth of human enteropathogens (Meera and Devi, 2012).

An agar spotting method was used to assess the antimicrobial properties

of dominant LAB strains isolated from traditional maari against a panel of Gram

negative and Gram positive microorganisms, which included food spoilage and

pathogenic bacteria by Kabore et al. (2012). The antimicrobial properties were

53

variable according to the LAB strains. The results showed that all Enterococcus

faecium strains (L9, L104, L117, L134 and L154), E. casseliflavus strains (L142

and L152) and P. acidilactici strains (L87 and L169) were able to inhibit seven

strains of Bacillus cereus, seven strains of Salmonella spp., one strain of E. coli,

three strains of Listeria monocytogenes and one strain of M. luteus. Furthermore,

it is interesting to note that E. faecium strains as well as P. acidilactici strains

were, in general, more effective against the pathogenic microorganisms than E.

casseliflavus strains as regards the inhibition zones displayed. Among the tested

LAB strains, the greatest inhibition zone (16 mm) was against B. cereus 11, and

the least (1.5 mm), was against Salmonella thompson strain and L.

monocytogenes Scott A strain. However, no inhibition was observed against

Yersinia enterocolitica strains. None of the LAB strains tested was active against

Yersinia strains. No inhibitory effect of MRS on any of the pathogenic strains

tested was observed. Large and clear inhibition zones were obtained from E.

faecium L154 and P. acidilactici isolates (L87 and L169) using the spot assay.

The culture supernatants obtained from isolated strain were tested for

antimicrobial activity against the laboratory test organisms and some fruit

spoilage bacteria like E.coli and Staphylococcus aureus and Pseudomonas.

aeruginosa and Candida albicans. The isolated strain inhibits the growth of the

E.coli, Pseudomonas. aeruginosa and Staphylococcus aureus. However, there is

no inhibitory activity on Candida albicans (Ravi et al., 2011). All isolates

inhibited the growth of all pathogenic strains when agar spot method was used.

The free-cell neutralized supernatant of 14 out of the 15 lactobacilli tested did not

inhibit the growth of the tested pathogenic indicators. It was also noticed that, the

neutralized free-cell supernatant from the culture of the isolate 29V inhibited the

growth of all pathogenic indicators (Sieladie et al., 2011).

Antibacterial activity was tested on different enteropathogenic bacteria

isolated from poultry used as indicators (Bakari et al., 2011). Agar diffusion

methods describe by Tagg and McGiveen, (1971) was used. The growth

inhibition showed a clear zone around the tested colonies. The inhibitory zone

varied from 9 mm to 26 mm. The main revelation in this study was that strains

54

were active both against Gram positive (Enterococcus avium, Enterococcus

cloacae), and Gram negative (Escherichia coli, Proteus mirabilis, and

Salmonella arizonae) bacteria. The result of bacterial growth inhibition test with

the indicator diffusion method showed that seven among ten isolates showed the

ability to inhibit the growth of bacteria, characterized by the formation of clear

zones around the wells with varied sizes (Sarkano et al., 2010). Three isolates

had the ability to inhibit the three bacterial indicators as well as the isolates

OPA3, OPA4 and AL1. Three isolates could inhibit the growth of two indicator

bacteria namely OPA5, OPA6 and OPA7. Meanwhile, only one isolate which is

only able to inhibit the growth of one indicator bacteria namely OPA1 isolates,

whereas three other isolates namely OPA1, RL1 and KA1 did not have the ability

to inhibit any bacterial indicator.

The isolated probiotics from milk of domestic animals, L. rhamnosus

(G119b), L. plantarum (G95a, C68a) were strong antagonistic (Score 17)

followed by commercial probiotic preparations Pre-Pro kid, PBiotics kid, Sporlac

powder, LactoBacil plus, Gastroline containing probiotics (Score 9-15), standard

probiotic bacterial strains L. plantarum (MTCC 2621), L. rhamnosus (MTCC

1048) (Score 11-12) against enteric pathogens. This may be due to the production

of acetic and lactic acids that lowered the pH of the medium or competition for

nutrients, or due to production of bacteriocin or antibacterial compound

(Tambekar et al., 2009; Bezkorvainy, 2001). Chuayana et al. (2003) reported that

different milk products were able to inhibit the growth of S. aureus, E. coli, Ps.

aeruginosa, S. typhi, Serratia marcescens and Candida albicans (Tambekar and

Bhutada, 2010).

The antimicrobial activity of the 10 isolates of LAB and their degree of

inhibition against the test pathogens were studied by Bhattacharya and Dass,

(2010). From total of 10 lactic acid bacteria, the culture supernatants of 7 isolates

yielded zones of inhibition when tested against the indicator strains. The

diameters of the inhibition zones ranged from 9 to 12 mm. the highest diameter

(12 mm) was recorded for the culture supernatants of C2 and F2 on S. aureus and

the smallest of 9 mm for E1 and F3 on S. aureus. No zone of inhibition was

55

observed against Pseudomonas. Ten bile salt tolerant LAB were used to study the

antimicrobial activity using non-neutralized and neutralized supernatant by agar

diffusion method. Out of 10, the non-neutralized supernatant of 6 LAB strain s

(As99-1, As100-2, As101-3, As102-4, As105-7, and As112-9) showed the

growth inhibition activity in E. coli and Salmonella sp, whereas no clear zone

were pronounced in neutralized supernatant of 10 LAB. The diameter of growth

inhibition zones varied from 0 to 11 mm and 0 to 10.5 in E. coli and Salmonella

sp., respectively in all tested LAB. No inhibition effects were found by As103-5,

As104-6, As106-8, and As113-10 strains against both E. coli and Salmonella sp.

(Bhakta et al., 2010).

8 strains that displayed superior autoaggregation and hydrophobicity

properties were included in this experiment for antagonism. Whole cultures were

used and halos of inhibition more than 20 mm against E. coli K88 were observed.

There were no significant differences (p>0.05) between strains (Iñiguez-

Palomares et al., 2007).

The extracts of eight strains of lactic acid bacteria gave zones of

inhibition onto the indicator pathogenic strains tested. The Table 1 gives the

results of inhibition (inhibition diameter), indicators strains inhibited are

Escherichia coli 105182 CIP Enterococcus faecalis 103907CIP, Staphylococcus

aureus ATCC 25293, Bacillus cereus 13569 LMG. The diameters of inhibition

are included between 8 mm and 12 mm. The biggest diameter of 12 mm

inhibition is obtained with the extract of strain S1 (Lactobacillus fermentum) on

Enterococcus faecalis, as for the smallest diameter is obtained with the extract of

strain S5 (Leuconostoc mesenteroides) on the same indicator strain Enterococcus

faecalis. The most inhibited indicators strains are the most part of gram positive

bacteria (Enterococcus faecalis 103907CIP, Staphylococcus aureus ATCC

25293, Bacillus cereus 13569 LMG), a single gram negative indicator bacteria

(Escherichia coli 105182 CIP) was inhibited by the extracts of bacteriocins.

Gram positive indicator bacteria are much more sensitive to bacteriocin of our

lactic acid bacteria strains than gram negative indicator bacteria (Savadogo et al.,

2004).

56

2.5.2 Bacteriocin production by Lactic Acid Bacteria

Many bacteriocin producing strains belonging to several genera and

species of LAB have been isolated. Their bacteriocins are bactericidal to

sensitive cells and death occurs very rapidly at a low concentration. Normally a

range of gram +ve bacteria is sensitive to bacteriocin while producer strain is

immune to its own bacteriocin and often is sensitive to other bacteriocin limited

studies indicate that although gram –ve bacteria are resistant they become

sensitive to bacteriocin once their outer membrane is destabilized by physical,

chemical or other stresses.

Musikasang et al. (2012) reported that the bacteriocins produced from

selected LAB showed strongly inhibitory activity toward many Gram-positive

bacteria. However, all of the Gram-negative indicators used were not inhibited by

any of the bacteriocins produced. Bacteriocins produced by LAB have attracted

great interest in terms of safety, but most of them only inhibit some Gram-

positive pathogenic bacteria. However, some bacteriocins are effective against

Gram-negative spoilage and pathogenic bacteria such as sakacin C2 produced by

Lactobacillus sake C2 strongly inhibited E. coli ATCC 25922. This activity

against many Gram-negative bacteria was not frequently seen in bacteriocins

from LAB (Gao et al., 2010). In addition, the combination of bacteriocin with

some natural antimicrobial compounds is able to enhance the inhibition of Gram-

negative pathogen such as interactions of nisin, lysozyme, and EDTA had an

effect on the growth of S. Typhimurium (Gill and Holley, 2000).

The bacteriocins obtained showed antimicrobial activity against E. coli, S.

aureus and B. cereus. For E. coli, there was 10 mm zone of inhibition activity of

the bacteriocins, while for B. cereus there was poor bacteriocin activity or no

activity against it (5 mm zone of inhibition) using the bacteriocin from

Lactobacillus plantarum. For Staphylococcus species, there was 10 mm zone of

inhibition activity. This was closely followed by the bacteriocins from

Streptococcus thermophilus, Lactobacillus brevis, Lactobacillus fermentum,

Pediococcus cerevisiae and Leuconostoc mesenteroides (Okereke et al., 2012).

The bacteriocin obtained from LAB6 showed the maximum zone of inhibition

57

compared to all the other strains. It showed a zone of inhibition of 7 mm against

V. parahaemolyticus, 6 mm against L. monocytogenes, Listeria spp. 5 mm against

E. coli, Salmonella spp. S. aureus and Yersinia spp and 4mm against Shigella

spp. Vibrio cholerae and Lactobacillus vulgaris, whereas the supernatant of the

same strain showed zone of inhibition of 5 mm against Vibrio paraheamolyticus,

4 mm against L. monocytogenes, S. aureus and Yersinia spp and 3 mm against E.

coli, Listeria spp, Salmonella spp, Vibrio cholerae and Lactobacillus vulgaris

(Indira et al., 2011).

Antibacterial activity of bacteriocin produced by isolated probiotics

showed that, L. rhamnosus (G119b), L. plantarum (G95a, C68a) were strong

(Score 27-31) antibacterial than bacteriocin of commercial probiotic preparations

Pre-Pro kid, Sporlac powder, LactoBacil plus, P-Biotics kid, Gastroline

containing probiotics (Score 20-24) and standard probiotic bacterial culture of L.

plantarum (MTCC 2621), L. rhamnosus (MTCC 1048) (Score 20-21) against

enteric bacterial pathogens (fig. 3). Bacteriocins of L. rhamnosus (G119b), L.

plantarum (G95a, C68a) were stable at 121oC and in acidic as well as alkaline pH

(3 to 9). Moghaddam et al, (2006) reported that bacteriocins of L. acidophilus, L.

bulgaricus were stable between pH 3 and 10 while L. helveticus was found to be

sensitive to pH 10. Bacteriocins of all the selected commercial probiotic

preparations were stable at 900C and acidic to neutral pH i.e. (3 to 7) except P-

Biotics kid was stable up to 1000C. Bacteriocins of standard probiotic strains

were stable up to 1000C and pH 3 to 7 (Tambekar and Bhutada, 2010).

Sapatnekar et al. (2010) extracted bacteriocin from Lactic acid bacteria. The

action of bacteriocin was checked on the indicator organism (E.coli).

Antimicrobial activity of the extracted and purified bacteriocin was checked on

the available indicator organism (E.coli). Clear and distinct zones of inhibition

were seen on the plates.

Maximum production of bacteriocin was obtained in MRS broth

containing at least 1-2% glucose or xylose. Also, MRS medium with 1% NaCl

found that, the antibacterial activity increased. The inhibitory activity was

maximal at the beginning of the stationary phase and remained stable long after

58

growth had ceased, even in the presence of the producer cells. Zone inhibition of

S. aureus against supernatant of lactobacilli by agar spot method, blank disk, and

agar well diffusion assay are showed by Nowroozi et al. (2004). Ahern et al.

(2003) reported that Thuricin 439 was shown to affect growth of all other tested

B. cereus, B. thuringiensis strain as well as affecting L. innocua 4202. However,

it had no effect on growth of any other gram +ve bacteria like B. subtilis, B.

coagulans several clostridium sp. Lactococcus lactis, S. aureus, Lactobacillus

sakei, gram –ve bacteria like Citrobacter frevndii, E. coli, Klebseilla pneumoniae,

Pseudomonas spp. Salmonella typhimurium, mold (Aspergillus niger),

Penicillium rogueforkii spp.

A strain can sometimes produce more than one type of bacteriocin, e.g.

Lactococcin A, B, M by Lac lactis SSP cremoris. Strains of same species

generally produce same bacteriocin e.g. Pediocin PA-1/ACH by different P.

acidilactici strains. However, stains of same species can also produce different

bacteriocin e.g. Sakacin A and Sakacin P produced by two strains of L. sake and

strains of different sp. and different genera can produce same bacteriocin, e.g.

Pediocin PA-1/ACH produced by Pediococcus acidilactici, Pediococcus

pentosaceous, Pediococcus parvidus, Lab plantarum strains. Strains from

different sub-species of same species can produce different bacteriocins, e.g.

Nisin A Lactocin 481 produced by different strains of L. lactis, SSP Lactis.

Different sp. in a genus can produce different bacteriocin, e.g. Enterococcin EF

S2 and enterocin 900 produced by strain of Enterococcus faecalis and

Enterococcus facium, respectively. Natural variants of same bacteriocin can be

produced by different strains of same species, e.g. Nisin A and Nisin Z by L.

Lactis SSP lactis strain ATCC 11454 and ATCC 7962, respectively, also by

different sp. e.g. Leucocin A and mesenterocin by Leuconostoc gelidim and

Leuconostoc mesenteroides, respectively (Ray et al., 2001). Papanthanasopoulos

et al. (1997) reported multiple bacteriocin of Leuconostoc mesenteroides. Each

bacteriocin was tested against 16 indicators. Leucocin C-TA33a was active. 9 of

16 indicator including Listeria, Enterococcus, Cornobacterium, Leuconostoc

strain while Leucocin B-TA33a only inhibited four Leuconostoc/Weissela

indicator strain. 14 out of 16 indicator strain tested were inhibited by Leucocin

59

A-TA33a, e.g. Cornobacterium mobile DSM 4848 was specifically inhibited by

Leucocin C-TA33a, W. paramesenteroides DSM 120288 by Leucocin B-TA33a,

Lactobacillus curvatus DSM 20019, Enterococcus durans CIP 55125,

Pediococcus pentosaceus LMA 596. Lc mesenteroides CIP 5417, L.

mesenteroides subsp. dextranicum J24 by Leuconin A-TA33a.

Sudirman et al. (1993) isolated semidry sausages on MRS agar. They

found that cell free supernatant of an overnight culture of SB 13 in MRS medium

was active against 27 strains of gram +ve and two gram –ve bacteria. Strains

were tested in presence of catalse by agar well diffusion assay. S. aureus, B.

cereus four thermophilic sp. of Lactobacillus were inhibited. 38 strains of

Listeria monocytogenes were inihibited (out of 41 tested). 12 strains of

lactococcus and 4 sp. of mesophilic lactobacilli, two sp. of Pediococus two gram

–ve E. coli and Pseudomonas fluresence were not inhibited. Vignolo et al. (1993)

reported that antimicrobial activity of neutrilized supernatant fluid of

Lactobacilus casei CRL 705 was active against Lact. Casei, Lac lactis,

Leuconostoc lactis, Listeria monocytogenes, S. aureus, Streptococcus pyogenes,

Bacillus subtilis. Gram –ve bacteria tested were inhibited by antibacterial

compound and included food borne pathogens E. coli, Proteus vulgaris,

Klebsiella pneumoniae, Salmonella typhimurium, Psudomonas aeroginosa.

Lewus et al. (1991) isolated ten strains of bacteriocin producing LAB

from retail cut of meat. These strains showed inhibitory activity against

psychotrophic pathogen including 4 strains of L. monocytogenes two strains of

Aeromonas hydrophila, two strains of S. aureus. Park et al. (2003) reported that

bacteriocin produced from Lactococcus lactis subsp. lactis inhibited strains of

Clostridium perifringes, C. difficle, L. monocytogenes Vanomycin resistant

enterococcus and one out of four Methicillin resistant S. aureus strain as well as

closely related lactic acid bacteria.

2.5.3. Effect of Proteolytic and amylolytic enzymes on inhibition activity

Antimicrobial action of probiotic Lactobacilli may be manifested by one

or combination of the following actions including competition for nutrients,

60

adhesion and production of different antimicrobial metabolites such as organic

acids, H2O2, bacteriocins, etc. LAB produce lactic acid and other organic acids

thus lower the pH of the environment and consequently inhibit the growth of the

bacterial pathogens. Alokomi et al. (2000), observed that the lactic acid produced

by Lactobacillus acts as a permeabilizer of the Gram-negative bacterial outer

membrane, allowing other antimicrobial substances produced by the host to

penetrate and thereby increasing the sensitivity of pathogens to these

antimicrobial molecules, Pithva et al. (2011). Production of H2O2 by

Lactobacillus spp. may be a non-specific antimicrobial defence mechanism as

hydrogen peroxide inhibits both Gram-positive and Gram-negative organisms

(Reid, 2002; Reid and Burton, 2002).

LAB’s are also known to produce antimicrobial peptides named

“Bacteriocin”. It is therefore more interesting with respect to probiotics that

individual strains may inhibit growth or adhesion of pathogenic microorganism

by extracellular synthesized products like bacteriocin and it is not merely an

effect of acidic pH. There are many evidences reporting secretory antibacterial

components produced by LAB having broad range of activity against Gram-

positive and Gram-negative organisms (Nomoto, 2005), which are independent of

lactic acid and hydrogen peroxide. However the overall antimicrobial activity of

LAB is generally due to a synergistic action of lactic acid, proteinaceous

substances and other antimicrobial substances viz. H2O2 etc.

The proteinaceous character of the bacteriocin substance was confirmed.

After treating NCFS with α-chymotrypsin, the inhibitory activity toward L. sakei

subsp. sakei JCM1157 of 12 selected LAB strains disappeared. The destruction of

the antimicrobial activity by proteases suggested that the compound in the

supernatant could be a peptide or bacteriocin (Bromberg et al., 2005). In addition,

the inhibitory activities of NCFS after being treated with trypsin were decreased

0.5 fold Musikasang et al. (2012).

Antimicrobial compounds produced by the isolates were inactivated by all

the proteolytic enzymes (pepsin and trypsin). No reduction in the zone was

61

encountered when the bacteriocins were treated with amylase catalase and lipase

Bhattacharya and Dass, (2010).

Ogunbanawo, (2003) reported that antimicrobial activity was lost or

unstable after treatment with all proteolytic enzymes whereas treatment with

Lipase, Catalase, Phospholipase C, Lysozyme, -amylase, Dextranase and UV

light did not affect activity of bacteriocin. Bacteriocin produced by L.

planatarum showed decrease in activity when treated with pronase E.

The bacteriocin of Bacillus thuringiensis was resistant to several proteases

and nucleases, as reported by Cherif et al. (2001). Similarly, some other Bacillus

sp. produced bacteriocin which was resistant to the proteolytic action of trypsin

and papain, but sensitive to proteinase K and pronase E (Bizani and Brandelli,

2002). The bacteriocins of Bacillus thermoleovorans were sensitive to protease

type XI and pepsin (Novotony and Perry, 1992).

The antimicrobial activity of Lactococcin R from Lactococcus lactis was

lost completely when treated with protease type IV, pronase E, proteinase K,

pepsin and -chymotrypsin. But it was not inactivated by treatment with

lysozyme, ribonuclease A, cellulase, lipase, catalase, peroxidase, amylase,

dextranase, glucosidase or organic solvent (Yildirim and Johnson, 1998).

Yildirim and Johnson, (1998) reported that bacteriocin Lactococcin R produced

by Lactococcus lactis subsp. cremoris R isolated from Radish was sensitive to

some proteolytic enzymes viz. proteinase K, pronase E, pepsin, chymotrypsin but

resistant to trypsin, papain, catalase, lysozyme, lipase and organic solvent.

Similarly, Suma et al. (1998) reported that plantaricin LP84 was sensitive to

trypsin and chymotrypsin.

Sudirman et al. (1993) reported that catalase had no effect because

inhibition of H2O2 was ruled out. Proteases destroyed the activity completely

chymotrypsin, Pronase E or partially trypsin. Pepsin had no effect at pH 7 but

inactivation occurred at pH 3.0. Park et al. (2003) reported that bacteriocin from

Lactococcus lactis subsp. lactis was inactivated by proteolytic enzyme but not

affected by lysozyme, lipase, catalase, B. glucosidase. Naclerio et al. (1993)

62

reported that the antimicrobial activity of cerein by Bacillus cereus was not

affected by treatment with Lysozyme, RNase, while it was completely lost after

treatment with trypsin, chymotrypsin and protease K, thus suggesting the

proteinaceous nature of bacteriocin. Motta and Brandelli (2002) reported the

bacteriocin by Brevibacterium linens was sensitive to the proteolytic action of

trypsin, papain and pronease E. Vaughan et al. (1992) observed that helveticin V-

1829 totally inactivated by treatment with proteinase K, ficin, trypsin, pronase

but papain had no effect on activity. In addition phospholipase C and Lysozyme

did not inactivate the bacteriocin. The sensitivity of helveticin V-1829 to

proteolytic enzyme indicated that it is proteinaceous in nature.

Chapter-3

MATERIALS AND METHODS

3.1 COLLECTION OF SAMPLES Isolation of lactic acid bacteria was done from different food samples viz.,

seera, sauerkraut, dough, dosa batter, cheese, jalebi batter, milk, lassi, tea leaves,

garlic pickle, home made butter, honey and chhang. Traditional fermented food

samples viz. seera, dough and chhang were collected from district Hamirpur,

Lahaul and Spiti district of Himachal Pardesh. Other fermented food items like

jalebi batter, dossa batter and lassi were procured from Solan market. Whereas,

Saurkraut was procured from Department of Food science and Technology, Dr.

Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.). All

samples were collected in clean and sterilized polythene bags or test tubes and

stored in refrigerator until further use.

3.2 ISOLATION OF LACTIC ACID BACTERIA In total 13 food samples were taken for isolation of potential lactic acid

bacteria. Out of 13 food samples, two samples were in solid form and rests of

them were liquids. The samples which were in solid form were crushed properly

in a clean sterilized pestle mortar in presence of distilled water and then

homogenized for 15 min on vortex mixture and the liquid samples were taken as

such. From these samples, stock was made by adding 1 ml of sample in 9 ml of

distilled water. All samples were serially diluted by serial dilution range of 10-1

to 10-9. The samples (0.1 ml each) from each dilution were mounted by spread

plate method on sterilized petriplates containing solidified media Man, Rogosa,

Sharpe (MRS) agar for isolation of bacterial colonies. Plates were incubated at

37oC for 48 h under anaerobic conditions. After incubation, individual colonies

were selected and purified using streak plate technique on MRS medium. The

isolates were primarily examined according to their colony morphology, catalase

reaction and gram reaction. Gram positive, catalase negative cocci and bacilli

colonies were taken to the glycerol stocks as lactic acid bacteria.

64

Composition of Man, Rogosa, Sharpe (MRS) agar

i) Peptone : 10 g

ii) Beef extracts : 10 g

iii) Yeast extract : 5 g

iv) Dextrose : 20 g

v) Ammonium citrate : 2 g

vi) Distilled water : 1000 ml

3.2.1 Gram Staining (Gram, 1984) Cultures were grown in appropriate mediums at 37°C for 24 h under

anaerobic conditions. Cells from fresh cultures were used for gram staining. After

incubation cultures were transferred aseptically into 1.5 ml eppendrof tubes and

centrifuged for 5 min at 6000 rpm. Then, supernatant was removed and cells were

resuspended in sterile water. Gram staining procedure was applied according to

Gram, 1984. Then, under light microscopy gram reaction of purified isolates was

explored.

3.2.2. Catalase Test (Aneja, 2003)

Catalase test was performed with isolates in order to observe their catalase

reaction. Overnight cultures of isolates were grown on MRS agar at suitable

conditions. After 24 h 3% hydrogen peroxide solution was dropped onto

randomly chosen colony. Also fresh liquid cultures were used for catalase test by

dropping 3% hydrogen peroxide solution onto 1 ml of overnight cultures.

Therefore, isolates which did not give gas bubbles were chosen for further study.

Since, LAB’s are catalase negative.

3.2.3. Long Term Preservation of Isolates

Gram positive and catalase negative isolates of lactic acid bacteria were

preserved in MRS broth medium containing 20% (v/v) glycerol as frozen stocks

at -80°C. The glycerol stocks of samples were prepared by mixing 0.5 ml of

active cultures and 0.5 ml MRS medium including 40% sterile glycerol.

65

3.2.4. Biochemical tests

Following biochemical tests were performed with selected isolates viz.

carbohydrate fermentation, Citrate utilization, H2S production, MRVP test and

casein hydrolysis.

3.2.4.1 Carbohydrate fermentation (Aneja, 2003)

Tubes of fermentation media (pH 7.3) were inoculated with different

isolated LAB separately followed by the incubation at 350C for 48 h. then tubes

were examined for the change in colour.

Composition of fermentation broth (pH 7.3) Peptone : 10.0 g

Glucose : 5.0 g

Sodium chloride : 15.0 g

Phenol red : 0.018 g

Distilled water : 1000 ml

pH : 7.3

3.2.4.2 Citrate utilization (Aneja, 2003)

Slants of simmon’s citrate agar (pH 6.9) were inoculated with each of the

isolates by streak inoculation. At the same time control was also run. All the

tubes were incubated at 370C for 48 h. Stabs were observed for the coloration of

the medium.

Composition of Simmon’s citrate agar medium

Ammonium dihydrogen phosphate : 1.0 g

Dipotassium phosphate : 1.0 g

Sodium chloride : 5.0 g

Sodium citrate : 2.0 g

Magnesium sulphate : 0.2 g

Agar : 15 g

Bromothymol blue : 0.8 g

66


pH : 6.9

3.4.2.3 H2S production (Aneja, 2003) Stabs of SIM agar medium (pH 7.3) were inoculated with the isolated

LAB separately followed by incubation at 370C for 48 h. Tubes were examined

for the presence or absence of black coloration along the line of stab inoculation.

Composiion of SIM agar:

Peptone : 30.0 g

Beef extract : 3.0 g

Ferrous ammonium sulphate : 0.2 g

Sodium thiosulphate : 0.025 g

Agar : 3.0 g


pH : 7.3

3.4.2.4 Methyl-Red and Voges-Proskauer (MRVP) test (Aneja, 2003) Tubes of MRVP broth (pH 6.9) were inoculated with the isolated LAB

separately followed by the incubation at 350C for 48 h. Then tubes were

examined for the change in colour of methyl red for MR test and crimson-to ruby

pink for VP test.

Composition of MRVP broth:

i) Peptone : 7.0 g

ii) Dextrose/Glucose : 5.0 g

iii) Potassium phosphate : 5.0 g

iv) Distilled water : 1000 ml

v) pH : 6.9

3.4.2.5 Casein hydrolysis (Aneja, 2003) Skimmed milk agar medium was autoclaved at 15 lb pressure for 15 min.

the medium was poured into sterile petridish and isolates were allowed to

solidify. The plates were streaked with isolated LAB’s followed by incubation at

67

370C for 24 h in an inverted position. Presence or absence of clearance around the

line of growth was examined.

3.3 PRELIMINARY SCREENING OF ISOLATED LACTIC ACID

BACTERIA BY BIT/DISC METHOD

3.3.1 Procurement of indicator bacteria

Different indicator bacteria viz., Staphylococcus aureus IGMC,

Enterococcus faecalis MTCC 2729, Listeria monocytogens MTCC 839,

Clostridium perfringens MTCC 1739, Leucononstoc mesenteroids MTCC 107,

and Bacillus cereus were used to check antagonistic activity of the isolates. All

the indicators used to check the antagonistic activity of given isolates were

maintained on nutrient agar (composition same as given in 3.4.1c) slants at 4oC.

All indicators were sub cultured periodically at 35oC.

3.3.2 Growth of indicator microorganisms

3.3.2a Growth of bacterial isolates and indicator bacteria

A loopful of the bacterial isolates as well as the indicator bacteria was

added into a test tube containing 10 ml of nutrient broth having pH 7.0. The

cultures were incubated at 35oC until they reached 1.0 OD which was checked

periodically after every 24 h.

Composition of nutrient broth:

i) Peptone : 5 g

ii) Beef extract : 3 g

iii) NaCl : 5 g


v) pH : 7.0

3.3.3 Antimicrobial activity

The antimicrobial activity of bacterial isolate against the indicator

bacterial strains was checked by bit/disc method.

68

3.3.3a Bit/Disk preparation of bacterial isolates (Barefoot and

Klaenhammer, 1983)

3.3.3b Lawn preparation of indicators

1 ml of inoculums of each indicator bacteria (1.0 OD) was swabbed

properly on pre-poured sterilized petriplates using sterilized cotton bud. The

swabbing was done in such a way that indicator culture covered the whole

surface of nutrient agar plate.

3.3.3c Bit/Disk preparation

The bacterial isolates (1.0 OD) were grown on MRS agar plate for 24 h at

37oC. Then with the help of sharp, sterilized borer bit of 10 mm diameter of

isolates was cut. The bit of bacterial isolates was kept on lawn of indicator

bacteria with the help of sterilized inoculating needle in such a way that surface

on which isolates grew faced the lawn of indicator bacteria and the activity was

noted in terms of zone of inhibition formed around the bit. The diameter of zone

formed was measured as its zone size.

3.4 PRELIMINARY SCREENING ON THE BASIS OF BILE SALT

TOLERANCE (DORA AND GLENN, 2002)

Preliminary selection of bile sat tolerant LAB isolates was done by

adjusting bile salt concentration of MRS broth to 0.3, 1 and 2% and then

sterilized by autoclaving at 121oC for 15 min. 24 h active culture was inoculated

1% v/v into adjusted bile salt concentrated broth media and incubated at 37oC for

72 h. Cell growth was monitored periodically at 620 nm. Control without bile salt

was also prepared. The percent difference between the variation of optical density

(OD) of culture without bile salts (∆OD0% BS ) and the variation of optical density

of culture containing 0.3, 1 and 2 % bile salts (∆OD0.3, 1 or 2% BS ) gives an index of

surviving isolates and expressed as follows:

Surviving (%) = (∆OD0% BS - ∆OD0.3, 1 or 2% BS/ ∆OD0% BS) × 100

69

3.5 PRELIMINARY SCREENING ON THE BASIS OF ACIDITY

TOLERANCE (PELINESCU ET AL., 2009)

Preliminary selection of acidity tolerance of lactic acid bacteria was done

by adjusting pH of MRS broth such as 3 and 4 pH by using 1 N HCL and then

sterilized by autoclave at 121oC for 15 min. Active culture 24 h were inoculated

(1% v/v) into adjusted pH broth media and incubated at 37oC for 72 h. Cell

growth monitored periodically spectrophotometrically at 540 nm. The percent

difference between the variations of optical density (OD) at pH3/4 (∆ODpH3/4)

would give an index of surviving isolates and expressed as follows:

Surviving (%) = (∆ODpH7 - ∆ODpH3/4/ ∆ODpH7) × 100

3.6 MICROBIAL PROFILE OF FOOD ITEMS OF FINALLY

SCREENED LAB HAVING HIGH PROBIOTIC POTENTIAL

For studying the microbial diversity, collected samples of each product

wasprocessed immediately on the selected media, viz. Potato dextrose agar

(PDA) for yeast and molds, Czapek Dox agar for fungi and Nutrient agar for

other bacteria. From these samples, stock was made by adding 1 ml of sample in

9 ml of distilled water. All samples were then serially diluted by serial dilution

range of 10-1 to 10-9. The samples (0.1 ml each) from each dilution were

mounted by spread plate method on sterilized petriplates containing different

media viz. PDA, Czapek dox and nutrient agar for isolation of bacterial, fungal

and yeast colonies. These plates were incubated at 37oC for 48 h/72 h under

aerobic conditions. The number of colonies of bacteria, yeast and moulds that

appeared on plates were counted and expressed as cfu/g of the sample.

3.6a Composition of Potato dextrose agar (PDA) (Aneja, 2003)

i) Potato (peeled) : 200 g

ii) Dextrose : 20 g

iii) Agar : 15 g


70

3.6b Czapek dox agar ( pH – 7.3) (Aneja, 2003)

i) Sodium nitrate : 2.0 g

ii) Dipotasium hydrogen phosphate : 1.0 g

iii) Magnesium sulphate : 0.5 g

iv) Potassium chloride : 0.5 g

v) Ferrous sulphate : 0.01 g

vi) Sucrose : 30.0 g

vii) Agar : 15.0 g

viii) Distilled water : 1000 ml

3.6c Nutrient agar (Aneja, 2003)

i) Peptone : 5 g

ii) Beef extract : 3 g

iii) NaCl : 5 g

iv) Agar powder : 20g

v) Distilled water : 1000ml

vi) pH : 7.0

3.7 MOLECULAR CHARACTERIZATION OF SIX SELECTED

ISOLATES USING 16S RRNA GENE TECHNIQUE Six best screened bacterial isolates were identified at genomic level by

using 16S rRNA gene technique as given below.

3.7a Isolation of genomic DNA

Genomic DNA of six bacterial isolates were isolated by using DNA prep

kit (Banglore genei, India make)

Reagents

i) Lysis buffer I

ii) Lysis buffer II

iii) Wash buffer I

iv) Wash Buffer II

71

v) Absolute ethanol

vi) Elution buffer

vii) RNase A

viii) Proteinase k

ix) Lysozyme

Procedure

18 h old bacterial culture was centrifuged at 10,000 rpm for 10 min.

Supernatant obtained after centrifugation was discarded and pellet was suspended

in 100µl of bacterial lysis buffer containing lysozyme at a final concentration of

20 µg/ml and incubated at 37oC for 30 min. Then 180 µl of lysis buffer I and 20

µl of Proteinase k was added followed by incubation at 55oC for 1-3 h. To the

solution 4 µl RNase A (100 mg/ml) was added followed by vortexing. This

mixture was incubated at room temperature for 5 min. 200 µl of lysis buffer II

was added followed by slow vortexing. Then incubation of 20 min was given at

70oC. 200 µl of absolute ethanol was added and mixed properly by vortexing.

Genei column was kept in a 2 ml of collection tube and mixture prepared above

was added in it which was centrifuged at 10,000 rpm for 5 min. Collection tube

with a flow through was discarded. Genei column was kept in a fresh 2 ml

collection tube in which 500 µl of wash buffer I (diluted with 3 volumes of

ethanol) was added. Column containing wash buffer was spinned at 10,000 rpm

for 1 min. Collection tube with wash sample was discarded. Then the column was

again kept on fresh collection tube, 500 µl of wash buffer II (diluted with 3

volumes of ethanol) was added in column followed by centrifugation at 10,000

rpm for 3 min. Wash fraction collected after centrifugation was discarded and

collection tube was retained for next step. Spin the empty column for 2 min at

10,000 rpm. After spinning collection tube was discarded and Genei column was

placed in a new 1.5 ml vial and incubated for 2 min at 70oC at dry bath. 200 µl of

elution buffer was added in a column which was incubated for 5 min. at room

temperature. Finally DNA was eluted by spinning column at 10,000 rpm for 1-2

min. Eluted DNA was stored at -20oC.

72

3.7b PCR amplification of 16S rRNA region PCR amplification was done to confirm the identity of the bacterial strain

and the small sub unit 16S rRNA genes were amplified from the genomic

DNA with 16SU (5’AGAGTTTGATCMTGGCTCAG3’) and 16SD

(5’ACCTTGTTACGACTT3’) universal primers to get an amplicon size of 700

bp. Amplification were carried out in 50 µl reaction volume consisting of 10 x

buffer, 5.0 µl; 2mM dNTPs, 5.0 µl; 3 U/µl Taq DNA polymerase, 0.33 µl;

100ng/µl of each primer, 2 µl; 50 – 100 ng template DNA, 1µl and H2O 34.67 µl

in a Astech thermocycler using the PCR conditions 95oC for 2 min

(denaturation), 58oC for 1 min (annealing) and 72oC for 1 min (extention). The

total numbers of cycles were 40, with the final extension of 72oC for 10 min. The

amplified products (50µl) were size separated on 0.8% agarose gel prepared in

1% TAE buffer containing 0.5 µgml-1 ethidium bromide and photographed with

the gel documentation system (Alpha Imager 2200). A 100 bp ladder was used as

molecular weight size markers.

3.7c Purification of the PCR product The PCR product was purified from contaminating products by electro

elution of the gel slice containing the excised desired fragment with Qiaquick gel

extraction kit (Sigma). The elution was carried out in 30 µl of nuclease free

water.

3.7d Nucleotide sequencing

Sequencing Preparation-The PCR amplicons obtained by amplifying PCR

products was diluted in Tris buffer (10 mM, pH 8.5), dilutions used was 1:1000.

In order to obtain the DNA concentration required for sequencing (30 ng/µl), the

sequencing reaction required 8 µl DNA. The primer used in all sequencing

reactions was 16 SU at a concentration of 3 µM. Sequencing was then performed

using an automated sequencer (ABI PRISM 310, Applied Biosystem, USA).

3.7e BLAST analysis

Translated nucleotide sequence was then analyzed for similarities by

using BLASTN tool (www.ncbi.nlm.nih.gov:80/BLAST/)

73

3.7f Inference

Lactic acid bacterial isolate F3 was identified as L. fermentum, F8 as

Lactobacillus sp., F11 as L. crustorum, F14 as L. acidophilus, F18 as L. delbreuckii

subsp. bulgaricus and F22 as L. plantarum.

3.8 SECONDARY SCREENING OF DIFFERENT PARAMETERS ON

SIX SCREENED LACTIC ACID BACTERIA FOR ASSESSMENT

OF PROBIOTIC POTENTIAL 3.8.1 Autoaggregation properties (Kos et al., 2003)

The culture of each of screened six isolates was grown in MRS Broth for

18 h at 37oC. The pellet was washed twice in phosphate-buffered saline (PBS)

and re-suspended in similar solution to reach number of cells of 10-8 cfu/ml.

Autoaggregation was determined by measuring their absorbance at 0 h (A0) and

after 5 h (At) incubated at room temperature. Measurement of absorbance was

done by taking 0.1 ml of upper aqueous phase and diluted with 3.9 ml PBS. The

absorbance was measured at 600 nm. Percentage of autoaggregation was

calculated using the following formula:

Autoaggregation (%) = [1- (At/A0)] x 100.

3.8.2 Cell surface hydrophobicity (Mishra and Prasad, 2005)

Hydrophobicity of cell surface was assessed based on MATS (Microbial

Adhesion to Solvent). Lactic acid bacteria were harvested after growth for 16-18

h at 37oC by centrifugation for 15 min at 5000 rpm, and then washed twice in

PUM buffer.

And finally suspended in the same buffer at the level of 10-8 cfu/ml. The

absorbance of the suspension was measured at 600 nm (A). Five ml of cell

suspension in PUM buffer was taken into clean and dry round bottom test tubes.

Then, 1 ml of different hydrocarbon viz. xylene, toluene, ethyl acetate and

chloroform was added and mixed by vortexing at high speed for 1 min. The tubes

were left undisturbed for 1 h at 37oC to allow the phase separation. The lower

aqueous phase was carefully removed with a sterile Pasteur pipette and final

74

absorbance (A0) was recorded at 600 nm. The decreased absorbance in aqueous

phase was taken as measure of cell surface hydrophobicity (H %), calculated

using following equation:

Hydrophobicity % = [(A-A0)/A] x 100

Where A and A0, were the absorbance before and after hydrocarbons

(xylene, toluene, ethyl acetate and chloroform) extraction respectively.

Composition of PUM buffer:

i) K2HPO4.3H2O : 22.2 g

ii) KH2PO4 : 7.26 g

iii) Urea : 1.8 g

iv) MgSO4.7H2O : 0.2 g

v) pH : 7.1

3.8.3 Acid tolerance (Gotcheva et al., 2002)

Then pH tolerance of screened LAB isolates was performed. Each

bacterial isolate grown on MRS broth and incubated at 37oC overnight then

subcultured into fresh MRS broth and incubated for 24 h at 37oC. The bacterial

culture then centrifuged at 5000 rpm for 10 min at 4oC and pellets were washed

twice in sterile phosphate buffer saline PBS, (0.1 M PO4 buffer + 0.8% NaCl,

pH-7.2) and resuspended in PBS. Each strain diluted 1/100 in PBS at pH 1, 2 and

3 and incubated for 1, 2 and 3 h. Counts of surviving bacterial colonies were

determined after plating the bacterial isolates on MRS agar with appropriate pH

and incubating them aerobically at 37oC for 48 h. Control samples without

acidification were also prepared. Percent survivability of the isolates was

calculated using the formula given below:

% Survivability = (log cfu 3rd h / log cfu 0th h) × 100

3.8.4 Antibiotic sensitivity test Antibiotic sensitivity of the isolated bacterial cultures was determined

using discs of different antibiotics. The interaction to antibiotics of LAB strains

75

was tested through HiMedia® Antimicrobial susceptibility tests discs. Each of

Six LAB strains were tested against 10 available antibiotics with following

concentrations.

Antibiotic Concentration (mcg)

i) Ampicillin (AMP) : 10

ii) Gentamycin (GEN) : 10

iii) Chloromphenicol (C) : 30

iv) Ofloxacin (OF) : 10

v) Tetracycline (TE) : 25

vi) Co-trimoxazol (COT) : 30

vii) Methicillin (MET) : 30

viii) Vancomycin (VA) : 30

ix) Cefotaxime (CTX) : 30

x) Cephalothin (CEP) : 30

The freshly grown, well isolated bacterial colonies were spreaded over the

pre-dried MRS agar plates to form a growth lawn. Antibiotic discs were placed

on spreaded plates at appropriate distances and incubated for 24 h at 37oC. The

results were noted down in terms of zones of clearance formed around

corresponding discs.

3.9 TESTING OF EXTENDED INHIBITORY SPECTRUM OF

LACTIC ACID BACTERIA AGAINST SPOILAGE

CAUSING/FOOD BORNE PATHOGENS

3.9.1 Bit/Disc method (Barefoot and Klaenhammer, 1983)

3.9.1.1 Procurement of indicator bacteria

Finally screened LAB isolates were further tested for their broad range

inhibitory spectrum by bit/disc and well diffusion method against in addition to

six test indicators already mentioned in section 3.5.1 additional four test

indicators viz. Pseudomonas syringe, Pectobacterium carotovorum, Escherichia

coli and Streptococcus mutans were used. All the indicators used to check the

antagonistic activity of given isolates were maintained on nutrient agar

76

(composition same as given in 3.4.1c) slants at 4oC. All indicators were sub

cultured periodically at 35oC.

3.9.1a Lawn preparation Lawn was prepared the same as mentioned in section 3.5.3b

3.9.1b Bit/Disc preparation

Bit/Disc was prepared the same as mentioned in section 3.5.3c

3.9.2 Well diffusion method (Kimura et al., 1998)

The antibacterial activity of screened six lactic acid bacteria was done by

well diffusion method.

3.8.2a Lawn preparation

Lawn was prepared the same as mentioned in section 3.5.3b

3.9.2b Preparation of cell free supernatant of bacterial isolates

Bacterial isolates were grown up to 1.0 OD in MRS broth. The isolates

with 1.0 OD were centrifuged at 12,000 rpm at 4oC for 15 min. The supernatant

of each isolate was collected in sterilized test tubes and the pellet was discarded.

In the well diffusion method, well of 7 mm diameter and 5 mm depth were cut on

the lawn laid in the nutrient agar plates with the help of sharp borer. 200 µl of

culture supernatant of each of bacterial isolate was poured into the wells. The

plates were then incubated at 35oC for 24 h and the zones of inhibition formed

around the wells were measured.

3.10 BACTERIOCIN PRODUCTION DURING GROWTH CYCLE

3.10.1 Inoculum preparation of bacterial isolate

100 ml of MRS broth (pH 6.5) was seeded with 10 ml of bacterial isolate

of 2.0 OD. Bacterial isolates were incubated for 96 h at 37oC at 150 rpm on

rotatory shaker. OD of each isolate was noted down periodically after every 24 h

at 540 nm.

77

To detect maximum production of metabolites, the culture of six isolates

were centrifuged at 10,000 x g at 4oC for 30 min after every 24 h and then well

diffusion method was repeated with these isolates against their respective

indicators. Both neutralized and unneutralized supernatants were poured into the

wells of molten nutrient agar. The neutralized supernatant was prepared by

neutralizing the supernatant of the bacterial culture with 1 N NaOH. The plates

were kept for incubation at 37oC for 24 h and result were observed as clear halos

of inhibition formed around the wells.

4.0 EFFECT OF PROTEOLYTIC ENZYMES – PEPSIN, TRYPSIN

AND PROTEINASE K ON THE ACTIVITY OF SIX SCREENED

LABS’ SUPERNATANT

To check the bacteriocinogenic nature of six selected isolates, effect of

different proteolytic enzymes was determined on supernatant of different cultures

as given below:

4.0.1 Lawn preparation of indicator bacteria Lawn of S. aureus was prepared in petriplates as described in section 3.5.3b

4.0.2 Enzyme activity

I. Control I (C) : Supernatant

II. Enzyme reaction 1 (ER1) : 0.25 mg of enzyme trypsin (Sigma chemicals)

was dissolved in 1 ml of 0.5 M phosphate buffer and then added to

supernatant of six LAB isolates in the ratio of 1:1 in the wells cut on the

lawn of indicator.

III. Enzyme reaction 2 (ER2) : 0.25 mg of enzyme pepsin (Sigma chemicals)

was dissolved in 1 ml of 0.5 M phosphate buffer and then added to

supernatant of six LAB isolates in the ratio of 1:1 in the wells cut on the

lawn of indicator.

IV. Enzyme reaction 3 (ER3) : 0.25 mg of enzyme proteinase K (Sigma

chemicals) was dissolved in 1 ml of 0.5 M phosphate buffer and then

78

added to supernatant of six LAB isolates in the ratio of 1:1 in the wells cut

on the lawn of indicator.

The preparations C, ER1, ER2, ER3 and ER4 were incubated for 1 h at 37oC.

The enzyme reaction and both the enzyme control were assayed by well diffusion

method as given in 3.7.2, against their respective indicators.

4.1 EFFECT OF AMYLOLYTIC ENZYME – AMYLASE ON THE

ACTIVITY OF SIX SCREENED LAB SUPERNATANT

I. Control I (C) : Supernatant

II. Enzyme reaction 4 (ECR4) : 0.25 mg of enzyme amylase (Sigma

chemicals) was dissolved in 1 ml of 0.5 M phosphate buffer and then added to

supernatant of six LAB isolates in the ratio of 1:1.

Rest of the process was same as given in section 4.4.2

4.2 CUMULATIVE PROBIOTIC POTENTIAL (TAMBEKAR AND

BHUTADA, 2010)

The cumulative probiotic potential of six screened lactic acid bacteria was

calculated using standard score card as given in Appendix II.

4.3 COMPATIBILITY TEST OF ISOLATED LACTIC ACID

BACTERIA

Compatibility of different isolates was checked by using Cross streak

method.

4.3.1 Cross Streak method (Barefoot and Klaenhammer, 1983)

In this method, on the prepoured properly sterilized MRS agar plate’s two

bacterial isolates were cross streaked against each other. Then these plates were

incubated at 37oC for 24 h and their growth patterns were noticed. The strain

showing best compatibility was chosen for probiotic consortia formulations.

4.4 STATISTICAL DESIGN The experimental data was analyzed by completely randomized design

(CRD).

Chapter-4

RESULTS AND DISCUSSION

4.1 ISOLATION OF LACTIC ACID BACTERIA

4.1.1 Collection of samples: Lactic acid bacteria from different food sources

were isolated and exhibited in Table 1. Different food sources used in the present

study were Seera, Saurkraut, Dough, Dosa Batter, Cheese, Jalebi Batter, Human

Milk, Lassi, Tea Leaves, Garlic Pickle, Homemade Butter Honey and Chhang.

These food samples were either in the solid form or in the liquid form. Food

items in solid form were Seera, Dough, Cheese, Saurkraut, Homemade Butter,

Tea leaves, Garlic pickle while, Dosa Batter, Jalebi Batter and Honey were semi

solid, Lassi and Human Milk were in liquid form. Among the food samples

Seera, Dough, Chhang are common local fermented food/beverage of Himachal

Pradesh. These were collected from district Hamirpur, Lahaul and Spiti and

Kullu of Himachal pradesh. Jalebi Batter and Dosa batter were procured from

the Solan market while the foods like Lassi, Cheese, Butter, Garlic pickle were

either homemade while Saurkraut was taken from the Department of Food

Science and Technology of Dr. Y.S. Parmar University of Horticulture and

Forestry, Solan. The selected food items were collected either in sterilized

polythene bags or sterilized test tubes for solids and liquids respectively.

4.1.2 Isolation: In total, 22 bacteria were isolated anaerobically on MRS agar

medium at pH 6.5. The morphological characters i.e. color, form, elevation and

margins of bacterial isolate were noted down and presented in Table 1 and Fig

(1a, 1b and 1c). The color of colonies varied from transparent white, white, cream

and yellow. Majority of isolates (55%) were cream in color, 32 % were white in

color, 9% were transparent white while only 4% were yellow in color. All

isolates exhibit form mainly in two types i.e., circular and punctiform. Out of 22

isolates, isolate F1, F2, F11, F13, F18 and F22 (27%) were punctiform and rest F3, F4,

F5, F6, F7, F8, F9, F10, F12, F14, F15, F16, F17, F19, F20 and F21 were circular with

overall percent of 73%. All isolates had entire margin and had two different

80

elevations (flat and raised). Out of total, 50 % Isolates had flat elevation and 50

% had raised elevation i.e., F1, F2, F3, F4, F5, F6, F8, F11, F12, F16, and F22 had flat

elevation while F7, F9, F10, F13, F14, F15, F17, F18, F19, F20 and F21 had raised

elevation.

As Himachal Pradesh is a rich repository of indigenous fermented foods.

Therefore in the present study fermented foods of Himachal Pradesh along with

other food sources were included to isolate lactic acid bacteria from them. Since,

traditional fermented foods are least studied and these are supposed to be treasure

hunts of rare microorganisms, therefore isolation of lactic acid bacteria was

carried out with selected food items as mentioned in Table 1 with a main

aimexploring a high probability of new/unique LAB having high probiotic

potential. Similar studies have been performed in literature cited below:

Hoque et al., (2010) isolated lactobacilli from yoghurts and differentiated

them as Isolate-1 (1.0 mm, white, rough, irregular, round and white colony),

Isolate-2 ( small 0.1-0.5 mm, rough, dull and brownish colony), Isolate-3 (white,

shiny, smooth and 1.0mm diameter) and Isolate-4 (2.0-2.5 mm, white, shiny,

smooth and disc like colony). Bacteriocin producing isolate A75 was already

isolated from ‘Marcha’-a traditional fermented food of North East in the

Microbiology Research Lab, Basic Science, UHF, Nauni. Given isolate was

found to have creamish, smooth and round colony (2mm), was rod shaped i.e.

bacilli, gram positive and endospore former, Gupta (2010).

Kapoor (2007) isolated 102 lactic acid bacteria on MRS medium (pH-

5.5). Different food sources including traditional fermented foods like Dal vari,

Vari kandal, Soya nuggets (Nutri), Saurkraut, Nashasta, Butter, Whey, Curd,

Dosa batter, Idli batter, Dough, Milk cake, Powdered milk etc were taken from

different parts of Himachal Pradesh for the isolation of LAB’s. Isolates were

characterized on the basis of their morphological, cultural, physiological and

biochemical properties. These bacterial isolates had a great variations in their

morphological characters i.e. colour, texture and shape. The colour of colonies

varied from transparent, white, and cream to off white. The texture of bacterial

81

colonies had a variation ranging from smooth, wavy, rough, shinning, wrinkled,

and elevated to globulated while colony size of 24 h old culture varied from 0.5

to 4 mm.

Table 1. Isolation of Lactic Acid bacteria (LAB) from different food sources

showing their morphological characteristics

Sr.

No. Isolate Source Form Elevation Margins Colour

1. F1 Seera Punctiform Flat Entire White

2. F2 Saurkraut Punctiform Flat Entire White

3. F3 Dough Circular Flat Entire Cream

4. F4 Dosa Batter Circular Flat Entire Cream

5. F5 Cheese Circular Flat Entire Yellow

6. F6 Cheese Circular Flat Entire Transparent White

7. F7 Jalebi Batter Circular Raised Entire White

8. F8 Jalebi Batter Circular Flat Entire Cream

9. F9 Human Milk Circular Raised Entire Cream

10. F10 Human Milk Circular Raised Entire White

11. F11 Human Milk Punctiform Flat Entire Cream

12. F12 Lassi Circular Flat Entire White

13. F13 Lassi Punctiform Raised Entire Cream

14. F14 Lassi Circular Raised Entire Cream

15. F15 Tea Leaves Circular Raised Entire Cream

16. F16 Garlic Pickle Circular Flat Entire Cream

17. F17 Garlic Pickle Circular Raised Entire Cream

18. F18 Homemade Butter

Punctiform Raised Entire White

19. F19 Honey Circular Raised Entire Cream

20. F20 Honey Circular Raised Entire White

21. F21 Chhang Circular Raised Entire Cream

22. F22 Chhang Punctiform Flat Entire Transparent white

Incubation time : 48 h Incubation temperature : 37oC Growth media : MRS Agar

4.2 SCREENING AND IDENTIFICATION OF LACTIC ACID

BACTERIA SHOWING HIGH PROBIOTIC POTENTIAL

4.2.1 Physiological and Biochemical characterization

Physiological and Biochemical characterization of 22 isolated lactic acid

bacteria has been done and their characteristics have been noted down in Table 2

82

and Fig (2a, 2b, 2c and 2d). Gram’s staining has been performed to check the

gram’s reaction and shape of the bacteria. The gram reaction of the isolates was

determined by light microscopy after gram staining. Since, LAB’s are known to

be gram positive. Hence, they appeared blue-purple color by gram staining. The

results showed that all were gram positive (100%) and maximum (91%) were

found to be rod shaped bacteria except F1 and F2, which were cocci with overall

percent of 9% shown in Fig 2(b). Further Catalase test of the isolates was

performed and all isolates were found to be catalase positive shown as in Fig

2(c). Catalase is an enzyme produced by many microorganisms that breaks down

the hydrogen peroxide into water and oxygen and causes gas bubbles. The

formation of gas bubbles indicates the presence of catalase enzyme.

2H2O2 → 2 H2O + O2

As generally Lactic acid bacteria are gram-positive and catalase-negative

in nature. Therefore, 22 isolates shown in Table 2 and were tentatively identified

as Lactobacilli and Lactococci. Mode of growth of isolated anaerobic lactic acid

bacteria was ascertained on the basis of their sensitivity to oxygen. The growth

conditions revealed that 64% isolates were strictly anaerobes and 36% isolates

were facultatively anaerobic in nature as shown in Fig 2d. These results were

compared with the previous studies given below:

Hoque et al., (2010) reported that bacteria isolated from different

yoghurts were identified as Lactobacillus spp. by observing their colony

morphology, physiological as well as some biochemical characteristics.

Microscopically they were gram-positive, rod shaped, non-motile, catalase

negative and absence of endospores. Naeem et al., (2012) isolated 15 lactic acid

bacterial strains from 20 fruit samples collected and analyzed during present

course of work. All isolates were primarily identified on the basis of colony/cell

morphology and biochemical tests. Majority of the colonies on MRS agar plates

were small whitish to off-white in colour; gram positive, catalase negative and

were able to grow under anaerobic conditions suggest the relatedness of these

species with lactic acid bacteria.

Fig. 1a.Differentiation of isolated LAB’s on the basis oftheir form

Fig. 1b. Morphology of isolated LAB’s on the basis of their elevation

Fig. 1c. Morphology of isolated LAB’s on the basis of colour

83

Table 2. Biochemical characteristics of isolated Lactic acid bacteria and their tentative identification

Gram Staining Sr. No. Isolate Source Shape Gram’s

reaction

Catalase test Carbohydrate

utilization Caesin

Hydrolysis Citrate

utilization MRVP

test H

2S

production

Growth Conditions Tentative

Identification

1. F1 Seera Coccus +ve -ve AG

- + + +VP

+ - Anaerobic Lactococcus

2. F2 Saurkraut Coccus +ve -ve AG

- + + +VP

+ - Anaerobic Lactococcus

3. F3 Dough Rods +ve -ve AG

- + + +VP

+ - Facultative anaerobic Lactobacillus

4. F4 Dosa Batter Rods +ve -ve AG

- + + +VP

+ - Anaerobic Lactobacillus

5. F5 Cheese Rods +ve -ve AG

- + + +VP


6. F6 Cheese Rods +ve -ve AG

- + + +VP


7. F7 Jalebi Batter Rods +ve -ve AG

- + + -VP

- - Anaerobic Lactobacillus

8. F8 Jalebi Batter Rods +ve -ve AG

- + + +VP


9. F9 Human Milk Rods +ve -ve AG

- + + -VP


10. F10

Human Milk Rods +ve -ve AG- + + -VP


11. F11

Human Milk Rods +ve -ve AG- + + +VP


12. F12

Lassi Rods +ve -ve AG- + + +VP


13. F13



14. F14



15. F15

Tea Leaves Rods +ve -ve AG- + + +VP


16. F16

Garlic Pickle Rods +ve -ve AG- + + +VP


17. F17

Garlic Pickle Rods +ve -ve AG- + + +VP


18. F18

Homemade Butter Rods +ve -ve AG- + + +VP


19. F19

Honey Rods +ve -ve AG- + + +VP


20. F20

Honey Rods +ve -ve AG- + + +VP


21. F21

Chhang Rods +ve -ve AG- + + +VP


22. F22

Chhang Rods +ve -ve AG- + + +VP


84

Various biochemical tests have been performed viz., carbohydrate

utilization, casein hydrolysis, citrate utilization, MRVP test and H2S production

with isolated LAB. All 22 isolates were able to ferment carbohydrate but no one

of them was able to produce gas during fermentation. All were found to

hydrolyze casein and utilize citrate while, no one was found responsible for H2S

production. Methyl Red and Voges-Proskauer (MRVP) test was performed for

high acid production during carbohydrate fermentation. F7, F9 and F10 showed

poor acid production while, rest of them were found to be high acid producers.

These were tentatively identified as Lactobacillus or Lactococcus.

Lactic acid bacteria is an important group of bacteria being placed in

group 19 with important biochemical characters that is gram’s reaction, catalase

negative, carbohydrate utilizing, casein hydrolysis as authenticated in Bergey’s

manual of Determinative Bacteriology (7th Edn).

Baradarn et al., (2012) studied carbohydrate fermentation patterns of the

LAB isolates by API 50 CHL test kit. Observation of result was carried out at 24

h and 48 h in which at both periods, similar profiles were obtained. Positive

reaction was indicated by change in color of media from violet to yellow. Kimoto

et al., (2009) reported that all isolated strains fermented glucose, trehalose,

sucrose, mannitol, fructose, and maltotriose, but did not ferment raffinose. Most

strains fermented lactose. Some strains fermented arabinose, but some strains did

not. The ability to hydrolyze esculine was determined by a BBL kit. The ability

was detected in all isolated strains, but not in reference strains including strain

ATCC 19435, type strain of Lactococcus lactis subsp. lactis. On the basis of

these results, they were tentatively identified as Lactococcus lactis.

Hoque et al., (2010) isolated lactobacillus spp. from two regional

yoghurts in Bangladesh. Seven isolates of bacteriocin producing LAB were

isolated from curd, dossa batter and idlli batter and were identified as species of

Lactobacillus (Bhattacharya and Dass, 2010).

Sieladie et al., (2011), isolated one hundred and seven colonies of

lactobacilli from thirty-two samples of raw cow milk were screened for their

Fig. 2a. Gram’s reaction of isolated LAB’s

Fig. 2b. Morphology of isolated LAB’s on the basis of their shape

Fig. 2c. Catalase test of isolated LAB’s

Fig. 2d. Mode of growth conditions of isolated LAB’s

85

probiotic use. Ebrahimi et al., (2011) isolated and identified new potential

probiotic lactobacilli from traditional Iran dairy products. The isolates were

screened for their probiotic potential activities, including acid and bile resistance,

antagonistic activity and cholesterol removal. Screening of acid and bile tolerant

strains from 14 different samples led to the identification of 20 isolates of

Lactobacillus spp.

4.2.2 Preliminary screening of Lactic acid bacteria

Preliminary screening of 22 LAB’s was done on the basis of their

antagonistic pattern, bile salt tolerance and acidity tolerance to select best isolates

out of them for further studies.

4.2.2.1 Antagonistic spectrum of LAB by Bit/Disc method

Tentatively identified Lactobacilli/Lactococci were further tested for their

antagonistic activity against selected food borne/spoilage causing bacteria viz.,

Staphylococcus aureus IGMC, Enterococcus faecalis MTCC 2729, Listeria

monocytogens MTCC 839, Clostridium perfringens MTCC 1739, Leucononstoc

mesenteroids MTCC 107, and Bacillus cereus. The data on inhibitory spectrum

of Lactobacillus/Lactococcus by bit/disc method is shown in Table 3, Plate 1(a)

and 1(b) and Fig 3. Those isolates having clearance zones less than 9 mm

diameter against their respective test strain indicated poor activity, while the

other strains which made appreciable halos greater than 12 mm shown to have

good and strong antimicrobial activity against their corresponding indicators.

Antagonistic pattern of different Lactobacillus/Lactococcus varied against test

pathogens i.e., some showed antagonism against maximum number of test

indicators viz. isolates F1, F3, F4, F5, F6, F8, F11, F14, F15, F18 and F22 inhibited 6

test indicators. Isolate F2, F9, F13 and F20 were found to inhibit five test indicators,

whereas, isolate F10, F12, F17, F19 and F21 found to inhibit four test indicators,

while isolates F16 and F7 found to inhibit only two and one indicator respectively.

The inhibitory action of LAB is mainly due to accumulation of main

primary metabolites such as lactic and acetic acids, ethanol, carbon dioxide; or

antimicrobial compounds such as formic, benzoic and acids, hydrogen peroxide,

86

Table 3. Preliminary Screening of isolated Lactic acid bacteria on the basis of antagonistic pattern against test indicators by bit/disc

method

Sr. No. Name of isolate Source S. aureus (mm)

E. faecalis (mm)

L. monocytogens (mm)

C. perfringens (mm)

L. mesenteroids (mm)

B. cereus (mm)

Mean Percent

Inhibition (%) 1. F

1 Seera 16.0 17.5 16.0 11.0 22.0 15.0 16.25 100

2. F2 Saurkraut 15.0 14.0 14.0 - 12.0 12.0 11.5 83.3

3. *F3 Dough 26.0 23.5 17.0 23.0 21.0 25.0 22.6 100

4. F4 Dosa Batter 14.0 16.0 14.0 13.0 15.4 16.0 14.7 100

5. F5 Cheese 16.0 20.0 16.0 14.0 16.0 20.0 17.0 100

6. F6 Cheese 14.0 20.0 16.0 13.0 15.0 16.0 15. 7 100

7. F7 Jalebi Batter - - - 12.5 - - 2.1 16.7

8. *F8 Jalebi Batter 25.0 25.5 19.0 21.0 16.0 22.0 21.4 100

9. F9 Human Milk 23.0 23.6 - 15.0 12.5 13.5 14.6 83.3

10. F10

Human Milk 17.0 20.0 - 18.0 10.0 - 10.8 66.7

11. *F11

Human Milk 25.0 25.0 8.5 16.0 15.0 20.0 18.1 100

12. F12

Lassi 13.0 13.0 22.0 - 13.0 - 10.2 66.7

13. F13

Lassi 17.0 18.0 22.0 13.5 19.0 - 14.9 83.3

14. *F14

Lassi 25.0 25.0 23.0 19.0 21.0 20.0 22.5 100

15. F15

Tea Leaves 21.0 16.0 10.0 16.0 20.0 12.0 15.8 100

16. F16

Garlic Pickle - 13.0 - - 14.0 - 4.5 33.3

17. F17

Garlic Pickle 12.0 14.0 - 23.75 15.0 - 10.8 66.7

18. *F18

Homemade Butter 23.0 21.0 25.0 21.0 14.0 19.0 20.5 100

19. F19

Honey 20.0 14.0 - - 21.0 12.0 11.2 66.7

20. F20

Honey 22.0 16.0 20.0 15.0 17.0 - 15.0 83.3

21. F21

Chhang 20.5 16.0 - 16.0 17.0 - 11.6 66.7

22. *F22

Chhang 23.0 19.0 19.0 20.0 23.0 17.0 20.2 100

Zone size > 20 mm = strong activity Zone size > 12 mm = good activity Zone size < 9 mm = poor activity *Showing broadest/strongest antagonism

0

5

10

15

20

25

30

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22

Fig 3. Antagonistic potential of Lactic acid bacteria against test indicators

Isolates

An

tago

nis

m

87

diacetyl and acetoin (Yukeskdag and Aslim, 2010). In addition, LAB has shown

to possess inhibitory activities due to the bactericidal effect of protease sensitive

bacteriocins (Jack et al., 1995). By producing these antimicrobial compounds,

probiotic microorganisms gain an edge over other microorganisms to survive in

the adverse conditions of gastrointestinal tract (El-Naggar, 2004).

The studies pertaining to explore the antimicrobial activity of different

bacteria against food borne pathogens by bit/disc method have been well

documented in literature. The inhibitory substances produced by lactic acid

bacterial strain act differently on different indicator strains. Hamid et al. (2012)

when tested the bacteriocidal effect of the few isolated LAB bacteria by disc

diffusion assay, demonstrated a clear zones against Gram negative pathogens

Salmonella typhimurium and Escherichia coli. Among all isolates S1#1, S2#8

and S2#25 showed inhibitory zones of at least 10 mm diameter on S.

typhimurium while, isolates S1#9, S1#10, and S1 #18, S1 #19 and S1 #20 showed

inhibition of at least 10 mm size on E. coli indicator bacteria. Further test using

the supernatant solutions, isolates S1#8, S1#9, S1#10, S1#18, S1#19 and S1#20

gave inhibition of variable in size (between 5.6 to 10.6 mm) on S. typhimurium.

The supernatant from isolates S1#8, S1# 9, S1# 10 and S1#19 gave inhibition of

sizes (between 8.0 to 9.3 mm) on E. coli. Similarly, Bhatacharya and Dass (2010)

studied antimicrobial activity of the 10 isolates of LAB and their degree of

inhibition against test indicator. From a total of 10 lactic acid bacteria, the culture

supernatants of 7 isolates yielded zones of inhibition when tested against the

indicator strains. The diameters of the inhibition zones ranged from 9 to 12 mm.

The highest diameter (12 mm) was recorded for the culture supernatants of C2

and F2 on the Staphylococcus aureus and the smallest of 9 mm for E1 and F3 on

Staphylococcus aureus. No zone of inhibition was observed against

Pseudomonas sp.

Thus, Antagonistic pattern on the basis of percent inhibition and the mean

of zone of inhibition of Lactic acid bacteria was one of the important factors for

preliminary screening. Out of 22 isolates, six isolates viz., F3, F8, F11, F14, F18 and

88

F22 (Fig 6) showed as the best strains on the basis of broadest and strongest

antagonism.

4.2.2.2 Bile Salt Tolerance

For the preliminary screening of 22 isolates were studied for their bile

tolerance at different concentrations viz., 0.3, 1.0 and 2 %. The growth was

monitored spectrophotometricaly at 600 nm after 24 h and percent survival was

calculated. According to the results, a great variation in the percent survival was

observed as given in Table 4 and Fig 4. Most of the isolates showed maximum

percent survival in 0.3% bile concentration, while gradual decrease in the percent

survival was observed when same isolates were grown in 1 and 2% bile salt

concentration. Isolate F3, F8, F11, F14, F18 and F22 showed maximum percent

survival in these three bile concentrations. Isolate F3 showed 89% survival in

0.3% bile concentration, 21.8% survival in 1% and survival of 12.1% in 2% bile

concentration. Same is in the case of isolate F8 where 45.3; 19.5and 16.6%

survival was observed at 0.3, 1 and 2% bile salt respectively. Isolate F11 showed

16.0 % survival at 0.3%, 13.8% survival at 1% and 11.1% survival at 2% bile salt

concentrations. Whereas, isolate F14 showed 88, 54 and 39.1% survivability in

these three bile salt concentrations. Isolate F18 also showed good survivability

with 81, 22 and 20% at the rate 0.3, 1 and 2%, while isolate F22 showed 12, 11,

and 9% survival at these three bile salt concentrations. These six isolates were

selected for next experiment as they showed in overall highest bile salt tolerance.

Bile salt tolerance is the second selection criterion for probiotics.

Resistance to bile salts is generally considered as an essential property for

probiotic strains to survive the conditions in the small intestine. Bile salts are

synthesized in the liver from cholesterol and are secreted from the gall bladder

into the duodenum in the conjugated form in volumes ranging from 500 to 700

ml per day. The relevant physiological concentrations of human bile range from

0.1 to 0.3% (Dunne et al., 2001) and 0.5% (Mathara et al., 2008). Thus, it is

necessary that efficient probiotic bacteria should be able to grow in bile salt with

concentration ranging from 0.15 - 0.30% (w/v) (Šuškovi et al., 2000).

0

10

20

30

40

50

60

70


Per

cen

t su

rviv

al

(%)

Isolates

Fig 4. Percent survival of Lactic acid bacteria in the presence of bile salt

89

Table 4: Preliminary screening of isolated Lactic acid bacteria on the basis of percent survival in the presence of bile salt

Sr. No. Isolate Source 0.3% 1% 2% Mean

1. F1 Seera 15.2 0.03 0.02 5.08

2. F2 Saurkraut 12.0 0.06 0.02 4.02

3. *F3 Dough 89.0 21.8 12.1 40.97

4. F4 Dosa Batter 13.0 11.0 9.0 11.0

5. F5 Cheese 15.6 12.0 0.06 9.22

6. F6 Cheese 1.5 1.0 1.0 1.17

7. F7 Jalebi Batter 8.0 7.0 5.0 6.67

8. *F8 Jalebi Batter 45.3 19.5 16.6 27.13

9. F9 Human Milk 14.0 9.0 7.0 10.00

10. F10

Human Milk 15.0 6.0 1.0 7.33

11. *F11

Human Milk 26.0 13.8 11.1 17.0

12. F12

Lassi 7.0 5.0 2.0 4.67

13. F13

Lassi 16.0 5.0 4.0 8.33

14. *F14

Lassi 88.0 54 39.1 60.37

15. F15

Tea Leaves 12.0 9.0 7.0 9.33

16. F16

Garlic Pickle 10.0 10.1 3.0 7.7

17. F17

Garlic Pickle 5.6 10.6 9.0 8.4

18. *F18

Homemade Butter 81.0 22.0 20.0 41.00

19. F19

Honey 14.0 7.0 6.0 9.0

20. F20

Honey 11.0 9.0 9.0 9.7

21. F21

Chhang 11.7 6.0 6.0 7.90

22. *F22

Chhang 58.1 11.0 9.0 26.0

Mean 25.86 11.4 8.05 CD 0.31 0.40 0.30

Surviving (%) = (∆OD0% BS - ∆OD0.3, 1 or 2% BS/ ∆OD0% BS) × 100 *Showing highest bile salt tolerance

90

Sieladie et al. (2011) reported that their all isolates demonstrated good

capacity to resist bile salts by presenting surviving percentage greater than 50%

under exposure to 0.2% bile salts after 24h at 37°C. Jamaly et al. (2011) showed

that only ten strains were observed resistant to 0.3% of Ox-bile (percentage of

resistance ≥ 50%), corresponding to survival percentages ranging from 54.05 ±

2.45 to 89.07 ± 1.26 % after 24 h incubation. The other strains such as LPL3,

LPAR4 and LPAR5, were inhibited dramatically in presence of bile. And,

between bile resistant strains, LPL2, LPAR1, LPAR2, LPAR9, LPAR11 and

LBR bacteria were found having capability to grow also at high Ox-bile

concentrations (0.5% and 1%). Kalui et al. (2009) reported that 18 of the 19

Lactobacillus plantarum tested were able to grow in broth supplemented with

0.3% bile salts following exposure to pH 2.5.

4.2.2.3 Acidity Tolerance

Preliminary screening of acidity tolerance LAB strains was done on the

basis of their percent survival in the presence of low pH i.e., 3 and 4. The growth

was monitored at 600 nm after 24 h and percent survival was calculated.

According to the results, 22 isolates have shown less survival and low growth at

low pH (Table 5 and Fig 5). Isolate F3, F8, F11, F14, F18 and F22 showed higher

survival percentage as compared to other isolates. Isolate F3 showed 11.2 and

29.3% survival in the presence of 3 and 4 pH whereas, isolate F8 showed 6.3 and

18.8% survival. Isolate F11 exhibited 13.2 and 25.3% survival and isolate F14

showed 15.3 and 25.6% of survival. F18 and F22 were capable of expressing 12.8,

28.4, 19.6 and 29.9% survival at pH 3 and 4 respectively.

In vitro survival of bacterial strains in low pH is a more accurate

indication of the ability of strains to survive passage through the stomach. The

organisms taken orally have to face stresses from the host which begin in the

stomach, with pH between 1.5 and 3.0 (Corzo and Gilliland, 1999). In the

stomach, there is formation of gastric acid - a digestive fluid which has a pH of

1.5 to 3.5 and is composed of hydrochloric acid (HCl) (around 0.5%, or 5000

ppm, and large quantities of potassium chloride (KCl) and sodium chloride

(NaCl). The acids play a key role in digestion of proteins, by activating digestive

91

Table 5: Preliminary screening of isolated Lactic acid bacteria on the basis of percent survival in acidic pH

S.No. Isolate Source 3 pH 4 pH Mean

1. F1 Seera 1.3 12.6 6.95

2. F2 Saurkraut 1.5 4.7 3.10

3. *F3 Dough 11.2 29.3 20.25

4. F4 Dosa Batter 4.0 8.2 6.10

5. F5 Cheese 3.5 12.2 7.85

6. F6 Cheese 0.6 9.0 4.80

7. F7 Jalebi Batter 6.0 8.4 7.20

8. *F8 Jalebi Batter 6.3 18.8 12.55

9. F9 Human Milk 2.4 16.2 9.30

10. F10

Human Milk 2.1 19.9 11.00

11. *F11

Milk 13.2 25.3 19.25

12. F12

Lassi 6.1 12.6 9.35

13. F13

Lassi 2.7 6.12 4.41

14. *F14

Lassi 15.3 25.6 20.45

15. F15

Tea leaves 0.1 0.7 0.40

16. F16

Garlic Pickle 4.9 5.7 5.30

17. F17

Garlic Pickle 1.5 4.6 3.05

18. *F18

Homemade Butter 12.8 28.4 20.60

19. F19

Honey 5.8 14.6 10.20

20. F20

Honey 7.9 13.7 10.80

21. F21

Chhang 5.0 14.9 9.95

22. *F22

Chhang 19.6 29.9 24.75

Mean 6.10 14.61 CD 0.28 0.25

Surviving (%) = (∆ODpH7 - ∆ODpH3/4/ ∆ODpH7) × 100 *Depicting highest survival

92

enzymes, and making ingested proteins unravel so that digestive enzymes can

break down the long chains of amino acids.

Every microorganism has a minimal, a maximal and an optimum pH for

growth and metabolism. Microbial cells are significantly affected by the pH of

their immediate environment because they apparently have no mechanism for

adjusting their internal pH. In general, microorganisms are able to grow over a

wide pH range from 1.0 to 11.0, Padan et al. (1981). Despite this remarkable

tolerance, most bacteria maintain a neutral cytoplasm. Even acidophilus, for

which the optimal pH for growth may be as low as 2.0, have cytoplasmic pH near

7.0 (Marshall and Law, 1984). Lactic acid bacteria generally grow and remain

viable within a medium pH range of 4.5 to 7.0 (Kashket, 1987). During growth

and fermentation, pH of the medium decreases because of the accumulation of

organic acids, primarily lactic acid. However, the pH within the cytoplasm of

fermenting lactic acid bacteria remains more alkaline than the medium

surrounding the cells (Kashket, 1987), largely because the cells rapidly excrete

protonated lactic acid, via a carrier-mediated process, into the extracellular

medium (Gatje et al. 1991 and Konings et al, 1989). In addition, the membrane is

relatively impermeable to extracellular protons (and lactate molecules) that are

produced during fermentation. Accordingly, a pH difference between the

cytoplasm and the medium, a pH gradient (ApH) is formed. The formation and

maintenance of ApH is important not only for pH homeostasis but also as a

component of the proton motive force (Mitchell, 1973).

Similar to our study, Iñiguez-Palomares et al. (2007) observed that from

all isolated strains, just 20 survived at pH 3.0 and conjugated porcine bile salts

(CPBS) conditions in 45% or more. Survival at pH 3 is significant because

ingestion of probiotic bacteria with food or dairy products raises the pH in

stomach to 3.0 or higher. Resistant strains belonged to three identified species,

Lactobacillus salivarius being the most common with 15 strains. Others strains

showed good survival to low pH (more than 50%), but they were discarded,

because the resistance to CPBS was less than 0.1%. In this work, all of three

species were resistant to pH 3.0 and CPBS. Maxwell and Stewart (1995) found

that L. acidophilus, Lactobacillus fermentum and Lactobacillus lactis were

0

5

10

15

20

25

30


Per

cen

t su

rviv

al

(%)

Isolates

Fig 5. Percent survival of Lactic acid bacteria in the presence of acidic pH

93

resistant to these adverse conditions in adult pigs. From those species, 20 strains

survived the gastrointestinal transit more than 45%. Gómez-Zavaglia et al. (1998)

and Kociubinski et al. (1999) obtained resistant strains to gastrointestinal transit

over 23%. Those species were Bacillus pseudolongum, Bacillus infantis, Bacillus

animalis and Bacillus breve. Aside, Ibrahim and Bezkorovainy (1993) worked

with strains of Bacillus bifidum, Bacillus breve, Bacillus infantis and Bacillus

longum, which were resistant to the adverse conditions of digestive tract. In

general, variable results have been documented in respect the resistance of low

pH and bile salts of the Lactobacillus and Bifidobacterium strains (Clark and

Martin, 1994; Chung et al., 1999; Mishra and Prasad, 2005).

Therefore, on the basis of preliminary screening, six isolates viz., F3, F8,

F11, F14, F18 and F22 based on broadest/strongest antagonism; highest bile salt

tolerance and acidity tolerance thus finally were screened for further

characterization and probiotic evaluation related experiments.

Table 5(a). Final screening of six Lactic acid bacteria with high probiotic

potential

Isolate Name Food source F3 Dough

F8 Jalebi Batter

F11 Human Milk

F14 Lassi

F18 Homemade Butter

F22 Chhang

4.2.2.4 Microbial profile of the food sources of finally screened LAB strains

The microbiological analysis of dough, jalebi batter, human milk, lassi,

homemade butter and chhang revealed that it had consortia of microorganisms

consisting mainly of lactic acid bacteria, bacillus spp. and yeasts. Colony forming

unit (cfu) count of all the samples has been evaluated on different media viz.

nutrient agar, Czapeck dox and potato dextrose agar (PDA) which ranged

between 11 × 106 to 108 × 108cfu/g/ml as given in Table 5(b). Microbial isolates

found in these food samples were identified on the basis of morphological and

94

biochemical characteristics. The microflora of these fermented food items mainly

dominated yeast - Saccharomyces cerevisiae, Lactic acid bacteria (Lactobacillus

fermentum, L. crustorum, L. acidophilus, L. plantarum etc.) and Bacillus spp. The

above results are corroborated by earlier findings which also encountered similar

genera in similar food items (Muyanjaet al., 2003; Hesseltine, 1983; Adegoke

and Oguntimein, 1995). Saccharomyces cerevisiae has been reported from

various fermented foods and beverages such as bhalle, beer, burukutu, bourbon

whiskey, coffee beans, cidar, Merissa, fufu, tape, ogi, puto, dosa, idli, papdam,

kecap, laochao, warri, scotch whiskey, etc. (Padmaja and George 1999; Batra and

Millner 1974, 1976; Soni and Sandhu 1990).

Table 5(b). Total microbial profile of the food sources of finally screened

Lactic Acid Bacteria

cfu/g*/ml** Sr.

No. Food item

Nutrient

Agar CzapekDox

Agar Potato

Dextrose

Agar

Predominant

microflora

1. Dough 47 × 106* 22 × 104* 20 × 106* Saccharomyces

cerevisisae, Bacillus

spp., Lactobacillus

plantarum, kocuria spp.

2. Jalebi batter 20 × 106* 35 × 108* 27× 106* Lactobacillus fermentum,

Lactobacillus plantarum,

Bacillus spp.,

Sacccharomyces

cerevisisae

3. Human milk 57 × 106** -Nil- 14 × 106** Lactobacillus crustorun,

Lactobacillus nantensis,

Bacillus spp.

4. Lassi 34 × 106** 17 × 106** -nil- Lactobacillus fermentum,

Lactobacillus

acidophilus,

Saccharomyces

cerevisisae, Bacillus spp.

5. Homemade butter

108 × 108* 32 × 104* 96 × 106* Lactobacillus spp.,

Saccharomyces


6. Chhang 11 × 106* 25 × 106* 34 × 108* Lactobacillus plantarum,

Saccharomyces


*cfu/g **cfu/ml

Lactobacillus fermentum F3

Lactobacillus fermentum strain

ULAG10JN944697.1


ULAG16 JN944678.1

Lactobacillus fermentum

FR873956.1


FJ861097.1


KLD51.0642 FJ861095.1


EF113958.1


AB690181.1


IMAU:60180 AB626052.1

Fig 6 . Phylogenetic tree of Lactobacillus fermentum F3

95

4.2.2.5 Genotypic Characterization

The best selected six lactic acid bacteria were identified at genomic level

by using 16S rRNA gene technique. Genomic DNA of six best selected isolates

was isolated using DNA purification kit (Bangalore Genei, make). The isolated

DNA was used in PCR to amplify small subunit of 16S rRNA using universal

primer having expected product size of 1500 bp. The PCR product so obtained

after amplification was visualized using ethidium bromide on 2% agarose gel

(Plate 3, 5, 7, 9, 11, 13). Amplified PCR products were purified and got

sequenced by the services provided by Xceleris, India. Pvt. Ltd to confirm the

results.

Nucleotide Sequencing

Following sequences of best screened six isolates were obtained after

sequence analysis.

Sequence of isolate F3 GACCCTCCCCGCTGAGCCCCCGCGTGGCCGGCTCCTAGAGGTTACCCAACCGACTTTGGATGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCCGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTATCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTTGCGGTCGTACTCCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGCGGAAACCCTCCAACACCTATCACTCATCGTTTACTGTCATGGACTACAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCACCGTCAGTTGCAGACCAGGTAGCCGCCTTCACCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCCTGCACTCAAGTTATCCAGTTTCCATGCACCTCTCCGGTTTAACACGAAGGCTTTCACATCAAACTTAGAAACCGCCTGCACTCTCTTTACGCCAATAAATCCAGGATAACGGTTGTCACCTACTATTACTGGTGGCTGCTGGCCCGTTATTCACCTGTGACTTTCCGTTTTCCAGCTCCCGCTCAGCTGCAGTCGACGCGTTAGGCCCAATTGATTAGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTATGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCAA

96

AACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTAACAAGGCAATGATGCATACCCGAGTTGAGAGACTGATCGGCCACGATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGTTCGTAAAGCTCTGTTGTTAAAGAACAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCCGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATC

Sequence similarity search for the F3 (BLAST, NCBI) showed 98%

homology with the available sequence of Lactobacillus fermentum accession

number JN703792.1. Phylogenetic tree of Lactobacillus fermentum F3 with

respect to other lactic acid bacteria as inferred by neighbour joining method has

been presented in Fig 6.

Table 6. Genotyping of finally screened lactic acid bacterial isolate F3

Name of

isolate

Source

Closest homologue

(organism)

Identity

(%)

16S rRNA

Identification

F3 Dough Lactobacillus fermentum JN703792.1

98% Lactobacillus

fermentum

Sequence of isolate F8

GGGTTCCCGGTAGCGCTATACGCGTCCCTGTGGCTCTGCGGCGCCCCGCGCTATATGTATATTTGTGGGGACCGTGAAGGCGGAGGGGTGTCGGCTATTTTTTGTTTCAGCTCCCCCCCGCTCGCGGTGCTAATTACTTCCCGCTCGGCAAGAGCTATCGTATAGCCGGAATTATTGTAAATTAAGAGAGATGGATGGTGAGCTGTCATACTTCGTGCATGAACCGAATACCAAAATTTTCCCTCATCTGCCACTTTCGCCGACCAACGTTGTTCTCATAAGTCTCTTATAAAACCTGGCGTCTCCCTTTCACTTCTGGATGGACCTGCCGTGCATTACCTTGTTGGTGGGGTCCGACCTTA


homology with the available sequence of Lactobacillus sp. Lu7 accession number

JX188048.1. Phylogenetic tree of Lactobacillus sp. F8 with respect to other lactic

acid bacteria as inferred by neighbour joining method has been presented in

Fig 7.

Lactobacillus sp. F8

Lactobacillus sp. Lu2

DQ471800.1


JX188046.1


GU145273.1


JX094502.1

Lactobacillus casei strian SM-G

JX003592.1


JX188047.1


JX188048.1

Fig 7. Phylogenetic tree of Lactobacillus sp. F8

Lactobacillus sp. S4-3

GU125569.1

Lactobacillus mindensis

IMAU:10199 GU125536.1

Lactobacillus crustorum F11

Lactobacillus crustorum strain

IMAU60028 FJ749757.1

Lactobacillus nantensis strain

LP33 NR_043114.1

Lactobacillus alimentarius strain

FMA224 AB626076.1

Lactobacillus crustorum

AB626073.1

Lactobacillus nantensis

AB626067.1


IMAU:10213 AB626056.1

Fig 8. Phylogenetic tree of Lactobacillus crustorum F11

97


Name of

isolate

Source Closest homologue

(organism)

Identity

(%)

16S rRNA

Identification

F8 Jalebi Batter Lactobacillus sp. JX188048.1

98% Lactobacillus

sp. Sequence of isolate F11

GATTTCTCCCCCCTATAGGGCTACCACCACCGTATGATTGAATGTTACAAACTCTCATGCTGTGAAGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCTTGCTGATCCGCGATTACTAGCGATTCCAACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATCGGTTTTAAGTGATTTGCTTACCCTCGCGAGTTCCCAACACGTTGTACCGACCATTGTAACACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCGTCCGGTTTGTCACCGGTAATCTCACCAGAGTGCCCAACTGAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAAAGACTAATCTCTTAGCTTTTCATGGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCGACCGGGGCCAACTCCGAAATCAACCACCCTGAAGAGCTATTATCGCTTCACGATTTAAACTTGGTGAGTGCATCACGGGTGAGTAACACATGGTCAACCTGCCCACAATCATGGGAAAACATCTGGAATCCGGTGTTAATACCGCATAACCACTACTTTCCCCGATCTTTGCTTGAAAGATGGCTCTGCTATCGCTTTTGGATGGACCCCCGGCGTATTAGCTAGTTGGTGAGGTAATATCTCACCGAGGCTTCGATACGTTTGCAACCTTAAAGGGTGCCCCCCCCCTTTGTCACTGACCCAGGTCCAACTCTTATAC


homology with the available sequence of Lactobacillus crustorum strain: NBRC

107159 accession number AB626073.1. Phylogenetic tree of Lactobacillus

crustorum F11 with respect to other lactic acid bacteria as inferred by neighbour

joining method has been presented in Fig 8.


Name of

isolate


(organism)

Identity

(%)

16S rRNA

Identification

F11 Human Milk


AB626073.1 96% Lactobacillus

crustorum

98


CGGGTGTGTGGCCCACATAAATAGTTAGCCAACCGGTATTAAGTAGTAGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGG CAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCAAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAACTTCCGGCACTGAAGGGCAGAAACCCTCCAACAAGTAGACAGTGAGAAGAATACAAGCGAAGGCCTACTTCTAGAGACCAACTCTGTCAATTCGCGTTTCCGAGTCTAAGTAATCCTGGTGTTTGCTTCATAGTGTGGCTGTGAATGCATTTACCTGTCGGACTGGGGATGAATTTGCTTCTACGCAAGTTTCGGTGAGTAACGTTGTTTTCATGGCTGCTTTTAAATGAGCGCTTCTCGCTTTCACTTCTGGATGCACCTGCGGTGCATTAACTTGTTGGTGGGGTAACGGCCTATACCAGGGATTGATGTCTATCCGAATTAAAAGACTAATCCCAGTATGATGGGACGGAGAGCCGGGCCCATTCTAAG


homology with the available sequence of Lactobacillus acidophilus strain LA1

accession number HE793099.1. Phylogenetic tree of Lactobacillus acidophilus

F14 with respect to other lactic acid bacteria as inferred by neighbour joining

method has been presented in Fig 9.


Name of

isolate


(organism)

Identity

(%)

16S rRNA

Identification

F14 Lassi Lactobacillus acidophilus

HE793099.1 99% Lactobacillus

acidophilus

Lactobacillus fermentun strain 7.2

JX272057.1

Lactobacillus fermentun strain 6.1

JX272056.1

Lactobacillus acidophilus F14

Lactobacillus fermentun strain Lb11

JX202610.1

Lactobacillus fermentun JQ844571.1

Lactobacillus delbrueckii subsp.

bulgaricus LB2 HE793100.1

Lactobacillus fermentum strain 40

Mosuero JQ973612.1

Lactobacillus acidophillus LA1

HE793099.1

Lactobacillus fermentum LF4

HE650147.1

Fig 9. Phylogenetic tree of Lactobacillus acidophilus F14


bulgaricus F18


bulgaricus JN944701.1


JN944700.1


ULAG33 JN9444697.1


PL9006 AF477499.1

Lactobacillus fermentum strain:

JCM8592 AB690181.1

Lactobacillus fermentum isolate: 1.5.2

FR873956.1

Lactobacillus fermentum strain : JCM 8593

AB690182.1

Lactobacillus fermentum strain : JCM 8587

AB690176.1

Fig 10. Phylogenetic tree of Lactobacillus delbreuckii subsp. bulgaricus F18

99


ATATTGGGGGTCCCTCATTAAGGTAGCAAAAACGGTATGATGTATATTACAACTCTCATGGCGCGTCGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTCGTCACCGGCGGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCAACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGGACTACCATGGTATCTAATCCTGTTCGCTACCCATGCTTTCAGTCTCAGCGTCTGTTGCAGACCAGGTACCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGGCCCTCAAGTTATCTTGTTTCCCGATGCACTTCTCCGGTTAAGCCGAATGTTTTACATCAAAACTAAAAACCGCCGCACTCCTCTTTACCCCAAATAAATCCGGAAATACCTTAGCGTAGGCTACAGAATCCCGTTTACCGAGCTGGAGAAAAAGCTATAGGTATTGCCCTGGTTGATTTTGGTAATATTGAGTAACGGAGTGTGAACTGACGTATGTAACGTGCTAGACCTGGGGAGAAATTTGGAATCATCCGCCAGTATCGAAGAATAACGATGATCTCAGGAACAACTTATAACCGAGCTTTCTCGCTTTCCCTTCTGGATGCACCTGCCGTGCATTAGCTTGTTGGTGGGGTTACCGCCTTACCCGGCCGCGAAGCATACCCGAATTTAAAGACTGATTAGTTTATATACAGGGACGCACGGGTTAT

Table 10. Genotyping of finally screened lactic acid bacterial isolate F18 Name of

isolate


(organism)

Identity

(%)

16S rRNA

Identification

F18 Homemade Butter

Lactobacillus

delbrueckii subsp. bulgaricus

HE793100.1

97% Lactobacillus

delbrueckii subsp. bulgaricus


homology with the available sequence of Lactobacillus delbrueckii subsp.

bulgaricus strain LB2 accession number HE793100.1. Phylogenetic tree of L.

100

delbrueckii subsp. bulgaricus F18 with respect to other lactic acid bacteria as

inferred by neighbour joining method has been presented in Fig 10.


AGGGCACCGGTCGGGGGCTCGCGTCTCCGTCGGCTCTTAGGTTGCAAAGCGGTATGATGGTATATTCAACTATCTTGGGGTTTTCTAGGTGTGTGGCAAGGGCAGGGAACGTATTCCCCCCCCCCTTTTGACCCCCAAATCTTTCGATTCCCACTTCCCGTATGCCACTTGCTTTCTACCATCCTATCTTAGAATGGCTTTCCCCCTTCGCTTACCCTCGCGACTTCCCCCTCGTTCTTCGTGGGCTCGCCTCCGCCTACAGGTGCACATCGACCCAACTCTCGATATTGATTTATAGCTTGCTCACGATTTACATTTGACTGAGAGGTCAACTGGTGATTAACACGTGCGAATCCTGCCCTCAAACCATGCGGTCCACGTGGTTATGCATGCTAATACCCCGTTTCCACTTGTACCCCCCGGCCCCAGTTTGAGTTACCCATTTGGCTATCACTTTTTGACCGCTCCCTCGCATATTAATCATGATGCAGGCACCAATCAATACCAGAGTTCGTTCCACTTGCATGTATTAGGCACGCCCCCCGCGTTCGTCCTGAACCAGGATCAAACTCTTAAG


homology with the available sequence of Lactobacillus plantarum strain X14

accession number EF426256.1. Phylogenetic tree of Lactobacillus plantarum F22

with respect to other lactic acid bacteria as inferred by neighbour joining method

has been presented in Fig 11.

Baradaran et al. (2012), extracted the genomic DNA of Pediococcus

pentosaceus K1 using the conventional method. Amplification of 16S rDNA gene

was carried out by PCR. The resultant 1.5 kb PCR product was analysed, run on

the agarose gel and purified using Wizard SV Gel and PCR Clean up Kit

(Promega, Madison, Wi, USA) according to the manufacturer’s instructions

(Figure 2). The 16s rDNA sequences were aligned using BLAST algorithm

(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), and compared with the published

sequences of 16S rDNA gene of different LAB strains deposited in NCBI

databases.

Uncultured bacterium clone S69 JX133400.1

Uncultured bacterium clone MID42_3820 JX109392.1

Uncultured bacterium clone MID44_10296

JX107792.1

Lactobacillus plantarum F22

Lactobacillus strain X14

EF426256.1

Uncultured bacterium clone L12_aae48e11

EU468374.1

Uncultured bacterium clone

L12_aae49e06 EU468403.1

Uncultured bacterium clone

armae072 EU467609.1

Lactobacillus plantarum isolate:L4-1

AB550299.1

Fig 11. Phylogenetic tree of Lactobacillus plantarum F22

101

Table 11. Genotyping of finally screened lactic acid bacterial isolate F22 Name of

isolate


(organism)

Identity

(%)

16S rRNA

Identification

F22 Chhang Lactobacillus plantarum

EF426256.1 96% Lactobacillus

plantarum

The sequences of isolates F3, F8, F11, F14, F18 and F22 have been submitted

to NCBI for accession numbers.

4.3 FINAL ASSESSMENT OF PROBIOTIC ATTRIBUTES OF

SCREENED LACTIC ACID BACTERIA

4.3.1 Autoaggregation on the basis of sedimentation rate:

The autoaggregation percentage of the six screened lactic acid bacteria

was determined during a period of 5 h. autoaggregation capacity of bacterial

isolates was measured by comparing the initial absorbance at 600 nm with

absorbance of 1st, 2nd, 3rd, 4th and 5th h respectively. The autoaggregation

percentage was measured by A0- (At/A0) × 100.

The autoaggregation of screened LAB’s was exhibited in Table 12

(a,b,c,d,e and f) and Fig 12. Table 12(a) depicted the autoaggregation of L.

fermentum F3 on the basis of sedimentation rate for 5 h. Initially, the percentage

of autoaggregation was 15.8 that increased continuously and finally registering a

high percentage of 89.2. For Lactobacillus sp. F8 the autoaggregation percentage

was 3.42 in the 1st h and in the final 5th h, it was found to be 70.3% as given in

Table 12(b). Similarly, L. crustorum F11 and L. acidophilus F14, the

autoaggregation percentage was 25.1 and 15.3% respectively in 1st h with a

gradual increase in the percentage afterwards reaching 79.1 and 60% at 5 h

respectively as shown in Table 12(c) and Table 12(d). For L. delbreuckii subsp.

bulgaricus F18 and L. plantarum F22, it was noticed that, in the 1st h,

autoaggregation % was 1.0 and 31.4 and in the final 5th h, the autoaggregation

registered a high percentage of 68 and 79.5% respectively as given in Table 12(e)

and Table 12(f). Above mentioned results showed that all the six strains exhibited

strong autoaggregating phenotype as all the strains had autoaggregation more

than 40%. The minimum required autoaggregation percentage for good probiotic

strain has been recommending more than 40% (Del Re et al. 2000 and Pérez et

al. 1998).

102

Table 12: Estimation of *autoaggregation of screened LAB’s

a) Lactobacillus fermentum F3

Time OD600♦ 0h 1.640

**Autoaggregation (%)

1h 1.380 15.8 2h 1.060 35.3 3h 0.849 48.2 4h 0.513 68.7 5h 0.178 89.2

*Autoaggregation in terms of sedimentation rate ♦OD600 = Mean of the results from three separate experiments **Autoaggregation % = 1-(At/A0) ×100 b) Lactobacillus sp. F8

Time OD600♦ 0h 1.750


1h 1.690 3.42 2h 1.490 14.8 3h 1.360 22.2 4h 1.170 33.1 5h 0.519 70.3

* ♦ Same as in Table 12(a) **

c) Lactobacillus crustorum F11

Time OD600♦ 0h 1.350


1h 1.010 25.1 2h 0.700 42.1 3h 0.500 62.9 4h 0.446 66.9 5h 0.282 79.1


d) Lactobacillus acidophilus F14

Time OD600♦

0h 1.850


1h 1.567 15.3

2h 1.352 26.9

3h 1.209 34.6

4h 0.978 47.1

5h 0.740 60.0

* ♦ Same as in Table 12(a)

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5

L. fermentum F3 Lactobacillus sp. F8

L. crustorum F11 L. acidophilus F14

L. delbrueckii F18 L. plantarum F22

Time (h)

Au

toa

gg

reg

ati

on

(%

)

Fig 12. Comparison of the autoaggregation ability of screened LAB cells resuspended in buffer after growing in MRS broth

103

e) Lactobacillus delbrueckiisubsp. bulgaricus F18

Time OD600♦ 0h 1.540


1h 1.524 1.0 2h 0.962 37.5 3h 0.686 55.4 4h 0.677 56.0 5h 0.493 68.0


f) Lactobacillus plantarum F22

Time OD600♦ 0h 1.650


1h 1.131 31.4 2h 0.909 44.9 3h 0.861 47.8 4h 0.772 53.2 5h 0.338 79.5


The concept of aggregation ability includes autoaggregation,

characterized by clumping of cells of the same strain. According to Rickard et al.

(2003), bacterial aggregation is an integral process of biofilm formation which

proceeds in the form of adhesion and multiplication events. The cell surface

properties of bacteria are thought to play an important role in autoaggregation. It

has been suggested that (lipo) teichoic acids, proteins, and carbohydrates on the

bacterial surface, soluble proteins, or pheromones are involved in the aggregation

ability of bacteria (Ocaňa and Nader-Macias, 2002). Glucan production has

shown to play an important role in cell aggregation in Gram-positive bacteria

(Lynch et al., 2007). Autoaggregation of probiotic strains appears to be necessary

for adhesion to epithelial cells, with coaggregation resulting in a barrier that

prevents colonization by pathogenic microorganisms (Reid et al., 1990; Boris et

al., 1997; Del Re et al., 2000).

Sansawat and Thirabunyanon, (2009), determined the autoaggregation

percentage during a period of 5 h and showed that, in the beginning, the

104

percentage of autoaggregation ranged between 10.5-14.2%, and then

continuously increased every hour. In the final 5th h, the autoaggregation

registered a high percentage of 35.7-42.2. Nuraida et al. (2011), measured

sedimentation rate over a period of 5 h and the results revealed that

autoaggregation ability was relatively low, i.e. ranged between 4.13% - 39.10%

where, the highest autoaggregation ability showed by lalctobacilli R23 (39.10%),

followed by B13 (31.8%), B16 (29.42%), B10 (28.04%), R14 (26%), and A15

(14.96%) upto by A29 (4.13%) and R26 (4.93%). Morelli dan Callegari (2006)

stated that strains with high autoaggregation properties generally show

hydrophobic properties of cell surface.

Kos et al. (2003), measured sedimentation rate over a period of 5 h and

showed that the strain L. acidophilus M92 exhibited a strong autoaggregating

phenotype and also reported that autoaggregation could be related to cell surface

component, because it was not lost after washing and suspending of the cells in

phosphate buffer saline.

4.3.2 Bacterial hydrophobicity

The adherence to gut is an important criterion to select probiotic bacteria.

Indeed, the probiotic ability to adhere to the intestinal epithelium is regarded as a

prerequisite to colonize the human GIT for exerting beneficial effects, such as the

exclusion of enteropathogenic bacteria (Collado et al., 2007; Juntunen et al.,

2001). The ability of probiotics to adhere to epithelia is studied in vitro by

evaluating the cells surface hydrophobicity toward xylene, toluene, chloroform

and ethyl acetate. The results are noted down in Table 13 and Fig 13 of percent

hydrophobicity for the screened LAB’s. Table 13(a) was depicting percent

hydrophobicity for L. fermentum F3. L. fermentum F3 showed 20.4% adhesion

towards xylene, 13.3% adhesion towards toluene, 50.5% adhesion towards

chloroform and 11% towards ethyl acetate. While, Lactobacillus sp. F8 showed

11, 50.1, 28.6 and 20.8% adhesion towards xylene, toluene, chloroform and ethyl

acetate respectively as given in Table 13(b). L. crustorum F11 and L. acidophilus

F14 exhibits maximum adhesion i.e, 42.3 and 42.8% adhesion for xylene and 54.5

and 44.8% adhesion for toluene respectively as depicted in Table 13(c) and 13(d).

105

Whereas they both showed minimum adhesion i.e., 11.5 and 13.3% for

chloroform and 7 and 12% for ethyl acetate respectively. Table 13(e) and 13(f)

indicated that L. delbreuckii subsp. bulgaricus F18 showed 21.8% adhesion

towards xylene, 33.8% towards toluene and 42.3% towards chloroform and 22%

towards ethyl acetate, whereas L. plantarum F22 showed 28.7, 36.1, 50.1 and

20.4% adhesion towards xylene, toluene, chloroform and ethyl acetate

respectively.

Table 13. Expression of adhesion by screened LAB’s to different

hydrocarbons

a) Lactobacillus fermentumF3:

S.

No. Name of

hydrocarbon OD600♦ % Hydrophobicity** Indication ◘

1. Xylene 0.582 20.4 2. Toluene 0.945 13.3 3. Chloroform 0.540 50.5 4. Ethyl acetate 0.634 11.0

Moderate

♦OD: Mean of results from three different experiments **Hydrophobicity % = [(A-A0)/A] x 100 ◘ Indication: Strong = Hydrophobicity (> 40% for Xylene/Toluene) Moderate = Hydrophobicity (> 20% for Xylene/Toluene) Low= Hydrophobicity (< 20% for Xylene/Toluene)

b) Lactobacillus sp. F8:

S.

No. Name of



Strong

♦ ** Same as in Table 13(a)

◘

c) Lactobacillus crustorumF11:

S.

No. Name of



Strong


◘

106

d) Lactobacillus acidophilus F14:

S.

No. Name of



Strong


◘

e) Lactobacillus delbrueckiisubsp. bulgaricusF18:

S.

No. Name of



Moderate

♦ ** Same as in Table 13(a) ◘

f) Lactobacillus plantarumF22:

S. No. Name of



Moderate

♦ ** Same as in Table 13(a) ◘

The strains which showed % hydrophobicity greater than 40% for xylene,

toluene and chloroform were taken as strong hydrophobic and the strains which

showed % hydrophobicity greater than 20% for xylene, toluene and chloroform

were taken as moderate hydrophobic whereas % hydrophobicity less than 20%

for xylene, toluene and chloroform were low hydrophobic. All the six LAB

0

10

20

30

40

50

60

L. fermentum F3 Lactobacillus sp.

F8

L. crustorum F11 L. acidophilus

F14

L. delbrueckii

F18

L. plantarum F22

Xylene Toluene Chloroform Ethyl acetate

Hyd

rop

hob

icit

y

(%)

Screened LAB’s

Fig 13. Comparison of the hydrophobicity of screened LAB cells resuspended in buffer after growing in MRS broth

Autoaggregation (%)

Fig 14. Relationship between auto-aggregation (%) ability and hydrophobicity (%) of screened six isolates – L. fermentum F3, ∆∆∆∆– Lactobacillus sp. F8, O –L. crustorum F11, – L. acidophillus F14, – L. delbrueckii subsp. Bulgaricus

F18, – L. plantarum F22

Hyd

rop

ho

bic

ity (

%)

107

isolates showed strong hydrophobicity greater than 40% for non-polar solvents

like xylene, toluene and chloroform and showed low hydrophilic character as all

showed less affinity for ethyl acetate.

Bacterial adhesion towards xylene, toluene, chloroform and ethyl acetate

was tested to assess hydrophobicity of the bacterial cell wall. The four different

solvents were studied, out of this xylene, toluene and chloroform, which are a

non-polar solvents, thus demonstrated hydrophobic cell surface which is highly

desirable probiotic attributes, while ethyl acetate is a polar solvent, thus

demonstrated hydrophilic property of a cell surface.

Strong hydrophobicity and high autoaggregation are considered desirable

traits for conferring probiotic status to six LAB isolates as shown in Fig 14.

The Gram-positive cell wall of lactic acid bacteria consists mainly of

peptidoglycans, (lipo) teichoic acids, proteins and polysaccharides (Delcour et

al., 1999). The inner layer of the cell wall consists of a peptidoglycan network,

the sacculus, which is made up of linear polysaccharide chains which are

themselves made up of alternating n-acetylglucosamine and n-acetyl-muramic

acid units extensively crosslinked by two short peptides (Streyer, 1981; Delcour

et al., 1999). The peptidoglycan layer of the cell wall of lactic acid bacteria is

covered by a variety of substances. The most important of these substances are

(lipo) teichoic acids, neutral and acidic polysaccharides, and (surface) proteins

(Delcour et al., 1999). Teichoic acids form a diverse class of substances whose

basic structure is a linear polymer of a polyol (such as glycerol or various

monosaccharides) linked by phosphodiester bridges (Streyer, 1981; Delcour et

al., 1999). Lipoteichoic acids are anchored into the cytoplasmic membrane by

their lipidic tail whereas teichoic acids are covalently attached to the sacculus. As

its phosphate groups are strong acids, (lipo) teichoic acids display a pronounced

polyelectrolyte character. The polysaccharides associated with the bacterial cell

wall and the extracellular polysaccharides of lactic acid bacteria are either neutral

or acidic (Delcour et al., 1999; Ricciardi and Clementi, 2000). Because of their

abundance and their presence at the outer surface of the cell wall, extracellular

108

and cell-wall associated polysaccharides are expected to determine to a large

extent the surface properties of microorganisms. The most abundant surface

proteins in many Lactobacillus species are the S-layer proteins (Mozes and

Lortal, 1995; Delcour et al., 1999; Smit et al., 2001). Up to now, S-layers have

been found in strains of the species L. brevis, L. acidophilus, L. crispatus, L.

helveticus, L. amylovorus, and L. gallinarum (Delcour et al., 1999; Smit et al.,

2001; Ventura et al., 2002) but not in species like L. johnsonii and L. gasseri

(Ventura et al., 2002). S-layer proteins are usually small proteins of 40–60 kDa

with generally highly stable tertiary structures (Engelhardt and Peters, 1998). S-

layer proteins are noncovalently bound to the cell wall and assemble into surface

layers with high degrees of positional order often completely covering the cell

wall (Lortal et al., 1992; Engelhardt and Peters, 1998; Sleytr et al., 2000). In

contrast to most bacterial species, the S-layer proteins in lactobacilli are highly

basic, with an isoelectric point above pH = 9 (Smit et al., 2001; Ventura et al.,

2002; unpublished data). Because it fully covers the cell wall and because of the

high isoelectric point of the S-layer protein, the S-layer may be expected to have

appreciable effects on the properties of the cell wall of many Lactobacillus

strains although its precise functionality is not known (Delcour et al., 1999; Smit

et al., 2001).

The presence of surface proteins in lactobacilli can be deducted from the

elevated isoelectric point and the high hydrophobicity of the surface. (Lipo)

teichoic acids render the surface strongly negatively charged and hydrophobic at

the same time. Surfaces rich in polysaccharides are generally weakly charged and

are hydrophilic. Hydrophobic compounds like xylene, toluene and chloroform

can adsorb on sites on or within the cell wall. If the absorbing moieties are at the

outer surface, this will render the bacterial surface very hydrophobic. Thus our

results revealed that, the cell surface of the LAB isolates was rich in lipo

(teichoic) acids and are strongly negatively charged so they showed strong

affinity to non-polar solvents. Therefore, they are highly hydrophobic (Schär-

Zammaretti and Ubbink, 2003).

109

In one of the study Nuraida et al. (2011), studied three different solvents

to evaluate hydrophobic/hydrophobic cell surface properties. The results revealed

that most isolates showed negative affinity to xylene. Low affinity to xylene

indicates hydrophilic properties of cell surface. While Lactobacillus A15 and R23

indicated being slightly hydrophobic as they have positive affinity to xylene i.e.

15.24% and 9.43% respectively. Similarly, Sansawat and Thirabunyanon (2009),

studied n-hexadencane, xylene, and toluene to evaluate the hydrophobic cell

surface properties of the tested Bacillus isolates showed a rather consistent result.

The hydrophobicity of B. subtilis P33 and P72 strains was 25.6–30.0 % in n-

hexadecane, 32.2–36.1 % in xylene, and 30.3-31.6% in toluene. Surface

hydrophobicity was determined in order to test for possible correlation between

this physico-chemical property and the ability to adhere to the intestinal mucus as

suggested by Wadstrom et al. (1987).

In order to complete probiotic criteria, the hydrophobicity and adherence

properties of selected bacteria strains were performed by Jamaly et al. (2011).

The calculated value for the hydrophobicity ranged from 37.80 to 85.67, 21.06 to

88.00 and 76.33 %, respectively, for Lactobacillus paracasei, Lactobacillus

plantarum, and Lactobacillus brevis

The desirable property of probiotic bacteria is their colonization in

intestinal wall. This colonization is necessary in order to exert its beneficial

effects (Tuomola et al., 2001). In probiosis, it is important to evaluate surface

properties, like autoaggregation and hydrophobicity, because they are used as a

measurement directly related to adhesion ability to enterocytic cellular lines

(Pérez et al., 1998; Del Re et al., 2000). Autoaggregation besides also determines

the capacity of the bacterial strain to interact with itself, in a nonspecific way.

Aside, when that hydrophobicity is high (more than 40%), it indicates the

presence of hydrophobic molecules in the bacterial surface, like surface array

proteins; wall intercalated proteins, cytoplasmic membrane protein and lipids.

(Ofek and Doyle, 1994; Pérez et al., 1998; Bibiloni et al., 1999; Bibiloni et al.,

2001).

110

4.3.3 Potential of screened LAB’s for acidity tolerance

To resist acidic pH of gastric juices is an important characteristic of

probiotic lactic acid bacteria. Acid tolerance of the screened LAB’s was studied

by suspending bacterial cells in phosphate buffer saline of different pH 1.0, 2.0

and 3.0 following incubation for 1, 2 and 3h. It was observed in this experiment

that cells of both the isolates could tolerate an incubation of 1 to 3h at pH 1.0 to

3.0. Table 24 was depicting % survival of six screened LAB’s at different pH for

different time interval. L. fermentum F3 showed survival of 79.3% after 60 min at

pH 1.0. Whereas, at pH 2.0 and 3.0 it showed survival of 88.1 and 99% at pH 2.0

and 3.0 after 180 min of incubation period as shown in Table 14(a) and Fig 15.

Table 14(b) and Fig 16 were depicting % survival of Lactobacillus sp. F8 at pH

1.0, 2.0 and 3.0 for different time intervals. It showed 71.6% survival at pH 1.0

after an incubation of 120 min and showed 91 and 98.5% survival at pH 2.0 and

3.0 after 180 min of incubation. While, L. crustorum F11 survived 83% at pH 1.0

after 120 h of incubation and showed a survival of 79.8 and 94.2% after 180 min

at pH 2.0 and 3.0 as shown in Table 14(c) and Fig 17. Similar results depicted in

Table 14(d) and Fig 18 for L. acidophilus F14. It showed 69% survival after 120

min at pH 1.0 and 83.6 and 85.3% survival at pH 2.0 and 3.0 after 180 min. For

L. delbreuckii subsp. bulgaricus F18 and L. plantarum F22 highest % survival was

98.9 (60 min), 90 (120 min), 85.3 (180 min) and 98.7% (180 min) respectively

(Table 14(e), (f) and Fig 19, 20).

In order to gain health benefit, probiotic bacteria need to survive passage

through the gastrointestinal tract. This means that they must tolerate hydrochloric

acid in the stomach and bile salts in the small intestine. In vitro survival of

bacterial strains in low pH is a more accurate indication of the ability of strains to

survive passage through the stomach. The organisms taken orally have to face

stresses from the host which begin in the stomach, with pH between 1.5 and 3.0

(Corzo and Gilliland, 1999). LAB isolates were able to tolerate hydrochloric acid

(strong acid) more than lactic acid (weak acid). At low pH, undissociated

lipophilic acid molecules of weak acids inhibit microorganisms by entering the

cells and dissociating to hydrogen ion within the cells. This causes a decrease of

the internal pH (Adam and Moss, 1995; Girgis et al., 2003). The ability of LAB

111

isolates to grow at high acid conditions may be due to their acid tolerant response

(ATR). The ATR had been observed in Leuconostoc mesenteroides (McDonald et

al., 1990), Lactobacillus plantarum (McDonald et al., 1990), Enterococcus hirae

(Belli and Marquis, 1991) and Lactoccoccus lactis (Hartke et al., 1996). In

addition, resistance of LAB isolates may be due to the function of ATPase to

maintain intracellular pH and protect the cells during exposure to acids by

transferring protons out of the cell membrane (Girgis et al., 2003).

Table 14. Potential of screened LAB’s for acidity tolerance

a) Lactobacillus fermentum F3 Cell survival (Log cfu/ml)* **% Cell Survival

Incubation time (min) Incubation time (min)

pH

0 60 120 180 Mean 60 120 180 Mean

1.0 6.000 4.760 0.000 0.000 2.690 79.3 0.0 0.0 26.4 2.0 6.000 5.481 5.362 5.286 5.532 91.3 89.4 88.1 89.6 3.0 6.034 6.009 5.982 5.974 6.000 99.6 99.1 99.0 99.2

Control 6.041 6.042 6.040 6.043 6.068 100 100 100 100 Mean 6.045 5.573 4.346 4.326 CD 0.08

*Log cfu/ml: Mean of results from three separate experiments **% Suvivability = (log cfu 3rd h / log cfu 0th h) × 100

b) Lactobacillus sp. F8

Cell survival (Log cfu/ml)* **% Cell Survival


pH

0 60 120 180 Mean 60 120 180 Mean

1.0 5.982 5.006 4.286 0.000 3.818 83.7 71.6 0.0 51.8 2.0 6.017 5.665 5.496 5.464 5.660 94.1 91.3 91.0 92.1 3.0 6.034 6.017 5.892 5.946 5.972 99.7 98.0 98.5 98.7

Control 6.059 6.058 6.061 6.060 6.059 100 100 100 100 Mean 6.023 5.686 5.434 4.367 CD 0.007

* **} Same as Table 14(a)

c) Lactobacillus crustorum F11



pH

0 60 120 180 Mean 60 120 180 Mean

1.0 5.965 5.242 4.951 0.000 4.040 87.9 83.0 0.0 56.9 2.0 5.928 5.889 5.611 4.758 5.546 99.3 94.6 79.8 91.2 3.0 5.973 5.946 5.908 5.612 5.860 99.5 98.9 94.2 97.5

Control 5.982 5.980 5.981 5.982 5.981 100 100 100 100 Mean 5.962 5.764 5.614 4.088 CD 0.012


112

d) Lactobacillus acidophilus F14



pH

0 60 120 180 Mean 60 120 180 Mean

1.0 6.026 5.156 4.156 0.000 3.835 85.6 69.0 0.0 51.5 2.0 6.032 5.665 5.464 4.103 5.316 93.9 90.5 68.0 84.1 3.0 6.050 6.009 5.946 5.927 5.983 99.3 98.2 97.9 98.5

Control 6.098 6.098 6.097 6.099 6.098 100 100 100 100 Mean 6.051 5.732 5.415 4.032 CD 0.007


e) Lactobacillus delbrueckii subsp. bulgaricus F18



pH

0 60 120 180 Mean 60 120 180 Mean

1.0 6.017 5.464 4.758 0.000 4.060 90.8 79.1 0.0 56.6 2.0 6.051 5.738 5.663 5.059 5.628 94.8 93.1 83.6 90.5 3.0 6.066 6.000 5.946 5.918 5.982 98.9 90.0 85.3 91.4

Control 6.070 6.072 6.071 6.070 6.071 100 100 100 100 Mean 6.051 5.818 5.609 4.262 CD 0.006


f) Lactobacillus plantarum F22



pH

0 60 120 180 Mean 60 120 180 Mean

1.0 6.008 5.888 5.612 4.794 5.575 98.0 93.4 79.7 90.4 2.0 6.042 6.034 5.982 5.611 6.001 99.9 99.0 92.8 97.2 3.0 6.059 6.066 6.034 5.982 6.035 100 99.6 98.7 99.4

Control 6.082 6.081 6.083 6.081 6.082 100 100 100 100 Mean 6.048 6.017 6.011 5.617 CD 0.24


Kaboré et al. (2012), studied the effects of low pH (2.5) on the viability of

the LAB strains. Considerable variations among the strains were observed

Enterococcus faecium L104 was the most acid sensitive of all the strains tested,

losing its viability (<1 CFU/ml of viable cells) in less than 2 h at pH 2.5. The

other sensitive strains which lost viability in acidic conditions were Enterococcus

faecium strains L117, L134, L9 and Enterococcus casseliflavus strains L142 and

pH

Lo

g c

fu/m

l

% s

urv

iva

l

Fig 15. Acidity tolerance range of Lactobacillus fermentum F3

0

1

2

3

4

5

6

7

Control

(7.0)

3.0 2.0 1.0

0

20

40

60

80

100

120Incubation time

Percent survival

pH

Lo

g c

fu/m

l

% s

urv

iva

l

Fig 16. Acidity tolerance range of Lactobacillus sp. F8

0

1

2

3

4

5

6

7

Control

(7.0)

3.0 2.0 1.0

0

20

40

60

80

100

120Incubation time

Percent survival

pH

Lo

g c

fu/m

l

% s

urv

ival

Fig 17. Acidity tolerance range of Lactobacillus crustorum F11

5.3

5.4

5.5

5.6

5.7

5.8

5.9

6

6.1

6.2

Control

(7.0)

3.0 2.0 1.0

84

86

88

90

92

94

96

98

100

102Incubation time

Percent survival

pH

Lo

g c

fu/m

l

% s

urv

ival

Fig 18. Acidity tolerance range of Lactobacillus acidophilus F14

0

1

2

3

4

5

6

7

Control

(7.0)

3.0 2.0 1.0

0

20

40

60

80

100

120Incubation time

Percent survival

pH

Lo

g c

fu/m

l

% s

urv

iva

l

Fig 19. Acidity tolerance range of Lactobacillus delbrueckii subsp. bulgaricus F18

0

1

2

3

4

5

6

7

Control

(7.0)

3.0 2.0 1.0

0

20

40

60

80

100

120Incubation time

Percent survival

pH

Lo

g c

fu/m

l

% s

urv

iva

l

Fig 20. Acidity tolerance range of Lactobacillus plantarum F22

0

1

2

3

4

5

6

7

Control

(7.0)

3.0 2.0 1.0

0

20

40

60

80

100

120Incubation time

Percent survival

113

L152. From all the tested strains, only P. acidilactici L87 survived over a period

of 4 h at pH 2.5. A slight decrease in cell viability (96.2%) at pH 2.5 was

observed with P. acidilactici L169. For E. faecium L154, the results showed a

decrease in cell viability of around 28% within 4 h. MRS broth (Oxoid CM0359)

pH 7 was used as positive control, a good growth was observed for all the LAB

strains tested in MRS broth pH 7 (Oxoid CM0359). From the beginning (0 h) to

the end of the experiment (4 h), the pH varied from 2.43 to 2.59. Similarly, Bacha

et al. (2009), observed that, ninety two (92.9 %) of the total tested isolates did not

survive exposure to pH 2.5 for 3 h. The survival rates of 79 isolates (79.8%) were

below 10% after 3 h of incubation in the same pH environment. Only 5 (13.2%)

isolates of the pediococci, mainly Pediococcus pentosaceus 1, and two isolates of

lactococci, both belonging to Lactococcus lactis spp. lactis, managed to survive

at pH 2.5 for 3 h with 100% survival rate. None of the lactobacilli was recovered

from pH 2.5 after 3 h incubation. At pH 2.0, almost all strains did not survive the

stringent acidity of the simulated gastric medium.

Bhakta et al. (2010), employed LAB strains for their abilities to grow at

pH 2.5 for 2 h to select the acid tolerant strains, 55 isolates were selected as acid

tolerant strains from 103 As-resistant isolates.

So it could be said that all the screened six isolates could be possible

probiotic candidate bearing acid tolerance properties.

4.3.4 Detection of Antibiotic sensitivity for screened LAB’s

Selected LAB’s were tested for antibiotic sensitivity/resistance with a

range of antibiotic discs (Hi-media Make). Different antibiotic discs used were

Ampicillin (10 mcg), Gentamycin (10 mcg), Chloromphenicol (30mcg),

Ofloxacin (10 mcg), Tetracycline (25 mcg), Co-trimoxazol (30 mcg), Methicilin

(30 mcg), Vancomycin (30 mcg), Cefotaxime (30 mcg) and Cephalothin (30

mcg). The entire six screened LAB’s showed sensitivity against most of the

tested antibiotics as shown in Table 15. Lactobacillus sp. F8, L. crustorum F11 and

L. plantarum F22 showed 80% sensitivity towards the antibiotics whereas, L.

fermentum F3, L. acidophilus F14 and L. delbreuckii subsp. bulgaricus F18

114

Table 15: Detection of Antibiotic sensitivity for screened LAB’s Sr.

No. Isolate Methicilin

(MET) Vancomycin

(VA) Cephalothin

(CEP) Ampicillin

(AMP) Gentamycin

(GEN) Tetracycline

(TE) Co-

trimoxazol (COT)

Chloramphenicol (C)

Cefotaxime

(CTX) Ofloxacin

(OF) %

Sensitivity

1. L. fermentum F3 S R S S S S S S S S 90

2. Lactobacillus sp. F8 S S S S S S S R S R 80

3. L. crustorum F11

S S R S S S S S R S 80

4. L. acidophilus F14

S S S S S S S R S S 90

5. L. delbreuckii subsp. bulgaricus F

18

S S S S S S R S S S 90

6. L. plantarum F22

S S S R S S R S S S 80

R: Resistant; S: Sensitive

115

exhibited 90% of sensitivity towards these ten test antibiotics. Lactobacilli are

usually sensitive to inhibitors of protein synthesis such as chloramphenicol,

erythromycin, clindamycin and tetracycline and resistant to glycopeptides

(neomycin, kanamycin, streptomycin and gentamycin) (Charteris et al., 1998;

Coppola et al., 2005; Zhou et al., 2005) but in our study, all screened six LAB’s

were found to be sensitive to protein inhibitors viz. ampicillin, tetracycline and

chloramphenicol as well as to glycopeptides i.e. vancomycin. It is well known

that vancomycin is an antibiotic belongs to glycopeptide antibiotics inhibits the

peptidoglycan synthesis which is an important structural component of the

bacterial cell wall. Therefore, Gram-positive bacteria, including lactic acid

bacteria are especially vulnerable to vancomycin treatment (Reynolds, 1989). The

antibiotic susceptibility of all the six screened LAB’s makes it crucial for the

safety point of view to their use as potential probiotics because probiotic bacteria

may act as potential reservoir of antimicrobial resistance genes. Therefore, when

these probiotic strains enter the gut, they interact with the native microbiota and

gene transfer can occur. Probiotics might contribute to the transfer of antibiotic

resistance genes to other commensal bacteria or pathogens present in the GIT.

The occurrence of large numbers of transferable resistance genes within the

intestinal microbiota is undesirable due to the potential risk of acquisition by

pathogens present in the GIT and subsequent antibiotic treatment failure (Licht &

Wilcks, 2005).

According to world health organisation WHO, 2001 and European Food

Safety Authority-EFSA, 2008 bacteria used as probiotics for humans and animals

should not carry any transferable antimicrobial resistant genes. Thus the

susceptibility of our LAB isolates to the clinically important antimicrobials, is

beneficial as it minimizes the chances of disseminating resistance genes to

pathogens both in the food matrix/or in the gastrointestinal tract. It could thus be

concluded that our isolates L. fermentum F3, Lactobacilllus sp. F8, L. crustorum

F11, L. acidophilus F14, L. delbreuckii subsp. bulgaricus F18 and L. plantarum F22

are not reservoir of transferable resistance genes and can be used as probiotics.

Similar studies have been reported by several authors. Maurad and Nour-

Eddine (2006), showed the results obtained for antibiotic susceptibility of 11

116

strains. All strains were susceptible to penicillin G, ampicillin, vancomycin,

cloramphenicol, clindamycin, rifampicin and ciprofloxacin. Three strains (OL16,

OL23 and OL53) were totally susceptible to all antibiotics tested. Most strains

showed resistance to 4 of the 11 antibiotics tested, i.e. to cefoxitin (2 strains:

OL12 and OL40), oxacillin (3 strains: OL12, OL40 and OL15), tetracycline (4

strains: OL2, OL7, OL9 and OL15) or kanamycin (8 strains; OL2, OL7, OL9,

OL12, OL15 OL33, OL36 and OL40). Three strains (OL12, OL15 and OL40)

have showed a multiple resistance to 3 different antibiotics (both L. plantarum

OL12 and OL40) resist to cefoxitin, oxacillin and kanamycin. Sieladie et al.

(2011), studied fifteen potentially probiotic lactobacilli isolates for antibiotic

susceptibility using the agar diffusion method. All of them were sensitive to

penicillin, ampicillin, amoxicillin, erythromycin, tetracycline, chloramphenicol,

and doxycycline. Three isolates (20RM, 48RM, 53RM) demonstrated

intermediate resistance to cotrimoxazole. Notable observation is the resistance

towards ciprofloxacin expressed by all isolates.

Naeem et al. (2012), tested susceptibility and resistance of 15 isolates

against 10 available antibiotics. Almost all strains were sensitive to 50% of the 10

antibiotics used in the test but maximum sensitivity was observed for oxacillin

and kanamycin. Ahire et al. (2011) found susceptibility of Lactobacilli ST2 to

most of the antibiotics and recommended antibiotic sensitivity of Lactobacilli as

positive trait to be used as probiotic culture.

4.3.5 Extended broad range inhibitory spectrum of screened LAB’s

Finally screened LAB isolates were further tested for their broad range

inhibitory spectrum by bit/disc and well diffusion method against additional four

test pathogens i.e., Pseudomonas syringe, Pectobacterium carotovorum,

Escherichia coli and Streptococcus mutans besides six already explored

indicators (section 4.2.2.1) with an aim to find out the broad antagonistic

potential of the isolates. The data on inhibitory spectrum of finally screened LAB

isolates by bit/disc and well diffusion method is shown in Table 16 and 17, Plate

14(a) and 14(b). The entire screened LAB isolates exerted good/strong activity

117

Table 16. Extended inhibitory spectrum of screened LAB’s by Bit method Sr.

No. Isolate Source S. aureus

(mm) E. faecalis

(mm) L.

monocytogens (mm)

C.

perfringens (mm)

L.

mesenteroids (mm)

B. cereus (mm)

P.

syringe (mm)

P.

caratovorum (mm)

E. coli (mm)

S. mutans (mm)

1. L. fermentum F3 Dough 25.0 23.0 18.0 23.5 21.0 25.0 25.0 18.0 21.0 28.5

2. Lactobacillus sp. F8 Jalebi batter 24.0 24.5 20.0 21.5 15.0 22.0 24.0 15.0 16.0 25.0

3. L. crustorum F11

Human Milk 24.0 13.5 15.0 15.0 24.5 20.0 22.0 12.0 15.0 17.0


Lassi 26.0 22.0 22.5 20.0 21.0 20.0 22.0 14.0 14.0 25.0

5. L. delbeuckiiubsp. bulgaricus F

18

Homemade butter

23.0 24.0 24.0 21.5 14.5 19.0 27.5 18.0 20.0 27.5

6. L. plantarum F22

Chhang 23.5 19.0 20.0 21.5 22.5 17.0 25.5 16.0 19.0 26.0

CD 1.59 1.07 0.95 1.12 1.07 1.22 1.08 1.49 0.72 1.07

* Zone size < 10 mm = Poor activity Zone size > 15 mm = Good activity Zone size > 20 mm = Strong activity

118

against all the test indicators. The zone of inhibition for these sensitivity strains

ranged between 10 to 30 mm.

Antimicrobial action of probiotic Lactobacilli may be manifested by one

or combination of the following actions including competition for nutrients,

adhesion and production of different antimicrobial metabolites such as organic

acids, H2O2, bacteriocins, etc. LAB produce lactic acid and other organic acids

thus lower the pH of the environment and consequently inhibit the growth of the

bacterial pathogens. Alokomi et al. (2000), observed that the lactic acid produced

by Lactobacillus acts as a permeabilizer of the Gram-negative bacterial outer

membrane, allowing other antimicrobial substances produced by the host to

penetrate and thereby increasing the sensitivity of pathogens to these

antimicrobial molecules, Pithva et al. (2011). Production of H2O2 by

Lactobacillus spp. may be a non-specific antimicrobial defence mechanism as

hydrogen peroxide inhibits both Gram-positive and Gram-negative organisms

(Reid, 2002; Reid and Burton, 2002).

LAB’s are also known to produce antimicrobial peptides named

“Bacteriocin”. It is therefore more interesting with respect to probiotics that

individual strains may inhibit growth or adhesion of pathogenic microorganism

by extracellular synthesized products like bacteriocin and it is not merely an

effect of acidic pH. There are many evidences reporting secretory antibacterial

components produced by LAB having broad range of activity against Gram-

positive and Gram-negative organisms (Nomoto, 2005), which are independent of

lactic acid and hydrogen peroxide. However the overall antimicrobial activity of

LAB is generally due to a synergistic action of lactic acid, proteinaceous

substances and other antimicrobial substances viz. H2O2 etc.

Our results showed that, all the six screened LAB isolates were having a

broad range of inhibitory spectrum towards both gram positive and gram negative

pathogenic bacteria which is a desirable character to suppress the growth of

various pathogens. Similar to our study, a broad range inhibitory spectrum was

studied by Forestier et al. (2001), they observed that the cell free L. casei subsp.

rhamnosus Lcr35 supernatant inhibited the growth of human pathogenic bacteria:

119

Table 17. Extended inhibitory spectrum of screened LAB’s by Well Diffusion method Sr.

No. Isolate Source S. aureus

(mm) E. faecalis

(mm) L.

monocytogens (mm)

C.

perfringens (mm)

L. mesenteroids (mm)

B. cereus (mm)

P. syringe (mm)

P.

caratovorum (mm)

E. coli (mm)

S. mutans (mm)

1. L. fermentum F3 Dough 27.0 20.0 24.0 25.0 20.0 25.0 26.0 16.2 18.0 22.0

2. Lactobacillus sp. F

8

Jalebi batter 26.5 20.6 24.0 22.5 14.0 22.0 24.0 18.0 16.0 24.0

3. L. crustorum F11

Human Milk 29.5 20.0 25.0 24.0 20.0 20.0 24.0 15.0 15.0 20.0


Lassi 20.0 24.0 20.0 22.0 15.0 20.0 26.0 20.0 18.0 24.0

5. L. delbeuckii subsp. bulgaricus F

18

Homemade butter

20.0 22.2 24.0 22.0 15.0 19.0 14.0 22.0 20.0 29.0

6. L. plantarum F22

Chhang 22.0 30.0 28.0 23.5 20.0 17.0 26.0 20.6 19.0 25.0

CD 1.85 0.84 1.14 0.93 0.90 0.92 0.99 1.16 2.24 0.55

*Same as in Table 18

120

Enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli,

Klebsiella pneumoniae, Shigella flexneri, Salmonella typhimurium, Pseudomonas

aeruginosa, Enterococus faecalis, and Clostridium difficile.

4.3.6 Inhibitory spectrum of screened LAB’s during their growth phase

The growth curve of the isolates followed a sigmoid curve pattern based

on measuring bacterial turbidity level OD540 nm. The bacterial cultures were

incubated at 37oC in MRS broth (6.5 pH) for different interval of time (6 to 90 h).

Optical density and inhibition zones were measured after 6 h of interval at 540

nm. The growth was initiated at 0 h with optical density of 0.080 in L. fermentum

F3, 0.066 in Lactobacillus sp. F8, 0.071 in L. crustorum F11, 0.093 in L.

acidophilus F14, 0.098 in L. delbreuckii subsp. bulgaricus F18 and 0.065 in L.

plantarum F22. The log phase has been extended between 24 to 42 h and

stationary phase preveiled between 42 to 78 h. For all the six isolates, the

maximum inhibition against 3 test pathogens taken in the present study (E. coli,

S. aureus and L. monocytogens) was noticed in the late log phase and in

beginning of the stationary phase. This indicated peak period of inhibition was in

between 42 to 60 h (OD 1.87 onwards) for L fermentum F3 followed by 36 to 54 h

for Lactobacillus sp. F8, 42 to 60 h for L. crustorum F11 and 48 to 60 h for L.

acidophilus F14 with OD ranging from 1.45 onwards for different spp. Similarly,

strongest suppression for test pathogens was detected by L. delbreuckii subsp.

bulgaricus F18 and L. plantarum F22 in 42 to 54 h and 36 to 42 h with OD range of

1.58 and 1.74 respectively as shown in Fig. 21, 22, 23, 24, 25 and 26.

The inhibitory action of LAB is mainly due to accumulation of main

primary metabolites such as lactic and acetic acids, ethanol, carbon dioxide; or


diacetyl and acetoin and a proteinaceous compound called bacteriocin

(Yukeskdag and Aslim, 2010).

The metabolites of selected LAB have shown antagonistic activities

against all organisms used in this experiment during their growth phase. All

bacteria undergo constant change or growth. In general the bacterial growth curve

0

0.5

1

1.5

2

2.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

0

4

8

12

16

O D 5 4 0

E . c o l i

S . a u r e u s

L . mo n o c y t o g e n s

Time interval (h)

OD

540

Zo

ne s

ize

(m

m)

Fig 21. Inhibitory spectrum of Lactobacillus fermentum F3 during its growth phase against three different test indicators

0

0.5

1

1.5

2

2.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

0

4

8

12

16

O D 5 4 0

E . c o l i

S . a u r e u s


Time interval (h)

OD

540

Zo

ne

siz

e (

mm

)

Fig 22. Inhibitory spectrum of Lactobacillus sp. F8 during its growth phase against three different test indicators

0

0.5

1

1.5

2

2.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

0

4

8

12

16

O D 5 4 0

E . c o l i

S . a u r e u s


Time interval (h)

OD

540

Zo

ne

siz

e (

mm

)

Fig 23. Inhibitory spectrum of Lactobacillus crustorum F11 during its growth phase against three different test indicators

0

0.5

1

1.5

2

2.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

0

4

8

12

16

O D 5 4 0

E . c o l i

S . a u r e u s


Time interval (h)

OD

540

Zo

ne s

ize

(m

m)

Fig 24. Inhibitory spectrum of Lactobacillus acidophilus F14 during its growth phase against three different test indicators

0

0.5

1

1.5

2

2.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

0

4

8

12

16

O D 5 4 0

E . c o l i

S . a u r e u s


Time interval (h)

OD

540

Zo

ne s

ize (

mm

)

Fig 25. Inhibitory spectrum of Lactobacillus delbreuckii subsp. bulgaricus F18 during its growth phase against three different test

indicators

0

0.5

1

1.5

2

2.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

0

4

8

12

16

O D 5 4 0

E . c o l i

S . a u r e u s


Time interval (h)

OD

540

Zo

ne

siz

e (

mm

)

Fig 26. Inhibitory spectrum of Lactobacillus plantarum F22 during its growth phase against three different test indicators

121

consists of four phases i.e., lag phase, exponential phase, stationary phase, and

death phase predict different changes in the metabolic activities of bacteria.

According to it, in the late exponential and the beginning of the stationary

phase of LAB isolates there was maximum number of cells in the growth medium

due to constant binary fission of the cells and the cell number is high, thus in turn

the metabolites secreted by them are found maximum at that time. The increase

in the population of lactic acid bacteria in the growth medium leads to maximum

production of lactic acid, H2O2 and other metabolites which attributed to lowered

pH of the medium and maximum inhibition of pathogenic bacteria. The

antimicrobial effect of lactic acid might be due to undissociated form of acid

which penetrate the membrane and liberate hydrogen ion in the neutral cytoplasm

thus leading to inhibition of vital cell functions of the bacterial pathogens. The

inhibitory effect of hydrogen peroxide produced by LAB has also been reported.

Saranya and Hemashenpagam, (2011), obsereved that inhibition of

S.aureus and Pseudomonas is due to hydrogen peroxide produced by certain

LAB strains which contribute to their inhibitory activity against other

microorganism.

4.3.7 Effect of different enzymes on the activity of screened LAB’s

supernatant against test indicator Table 18 and Plate 15 were depicting the effect of amylolytic and

proteolytic enzymes on the six screened LAB’s supernatant. The supernatant of L.

fermentum F3, Lactobacillus sp. F8, L. crustorum F11, L. acidophilus F14, L.

delbreuckii subsp. bulgaricus F18 and L. plantarum F22 was treated with

proteolytic enzymes i.e., pepsin (ER1), proteinase k (ER2) and trypsin (ER3) and

amylolytic enzyme-amylase (ER4) @ 0.25 mg/ml in the ratio 1:1

(Supernatant:Enzyme) and welled into the lawn of indicator strain S. aureus. It

was observed that when supernatant of these six screened LAB’s was treated

separately with the given enzymes, then a decrease was observed in the zone size

after the treatment. The results revealed that in all the finally screened LAB

122

Table 18. Effect of different enzymes on the activity of supernatant of LAB’s against test indicator

Zone size (mm)

Treatments Isolates Control

(C*) Trypsin (E

R1*)

Decrease in

zone size

(%)

Pepsin (E

R2**)

Decrease in

zone size

(%)

Proteinase k (E

R3***)

Decrease in

zone size

(%)

Amylase (E

R4****)

Decrease in

zone size

(%)

L. fermentum F3 30.0 20.0 33.3 22.0 26.7 20.0 33.3 22.0 26.7

Lactobacillus sp. F8 28.0 18.0 35.7 16.0 42.9 16.0 42.9 22.0 21.4

L. crustorum F11

30.5 10.0 67.2 12.0 60.7 12.0 60.7 14.0 54.0

L. acidophilus F14

28.5 12.0 58.0 18.0 36.8 14.0 50.9 18.0 33.3

L. delbreuckiisubsp. Bulgaricus F

18

26.5 10.0 62.3 22.0 13.2 12.0 54.7 22.0 13.2

L. plantarum F22

30.0 16.0 46.7 16.0 46.7 16.0 46.7 22.0 26.7

*C : Control (Supernatant) ** E

R1: Enzyme reaction - Trypsin

*** ER2

: Enzyme reaction - Pepsin

**** ER3

: Enzyme reaction - Proteinase K

***** ER4

: Enzyme reaction - Amylase

123

isolates, there was 13.2 to 67.2% decrease in the zone size with proteolytic

enzymes and 13.2 to 54.0% decrease was observed with amylase.

According to our results the percent decrease in the inhibitory activity of

the screened LAB’s revealed that the inhibitory action is not only due to the

primary metabolites (lactic and acetic acids, ethanol, carbon dioxide; or


diacetyl and acetoin) but there are certain proteinaceous compound like

bacteriocins or carbohydrate moieties which contribute significantly to inhibit the

growth of pathogenic microorganism. According to the classification of

bacteriocins of LAB’s given by (Klaenhammer, 1993; Belkum and Stiles, 2000),

bacteriocins are broadly classified into four classes. 1st class is Lantibiotics,

which are ribosomally produced peptides that undergo extensive post-transational

modifications, 2nd class is nonlantibiotics, which are low-molecular weight (<10

kDa), heat stable proteins, 3rd class is nonlantibiotics, which are high molecular

weight (>30 kDa), heat labile proteins, while 4th class is a complex bacteriocins

carrying lipid or carbohydrate moieties, which appear to be required for activity.

Such bacteriocins are relatively hydrophobic and heat stable and exert strong

antagonistic effect against a broad range of challenging human pathogens, food

borne pathogens and spoilage causing microorganisms.

Gautam (2006), studied the activity of bacteriocin isolated from L. brevis

and B. mycoids. The bacteriocins produced by L. brevis and B. mycoides showed

resistance to the spoilage causing microorganisms in milk, cheese and apple

juice. The purified bacteriocin of L. brevis and B. mycoides was found sensitive

to trypsin thus, proving their proteinaceous nature. The use of bacteriocin as food

preservative has been found quite satisfactory and comparable to the chemical

preservative. Attri (2007), studied the effect of bacteriocin lenticin isolated from

B. lentus against food borne and spoilage causing microorganisms. Gupta (2009),

studied the antagonistic effect of purified bacteriocin of Bacillus sp. A75. It

showed strong antagonistic activity against food borne pathogens.

124

Bhattacharya and Dass, (2010), observed that antimicrobial compounds

produced by the isolates were inactivated by all the proteolytic enzymes (pepsin

and trypsin). No reduction in the zone was encountered when the bacteriocins

were treated with amylase catalase and lipase. Aween et al. (2012) studied the

effect of enzymes RNase and Proteinase K on L. acidophilus 1 supernatants

showed variable inhibitory activity against S. typhimurium, E. coli and E.

aerogenes. Supernatants H008-E and H008-D (obtained from pure honey from

Cameron Highlands, Malaysia) were slightly sensitive to both enzymes

Proteinase K and RNase II when tested against E. coli and S. typhimurium,

indicating the protein-like compound produced by these L. acidophilus strain.

Similarly, Maurad and Meriem (2008) reported that antibacterial activity of L.

plantarum isolated from butter made from camel milk against indicator strain of

Lactococcus lactis B8 was lost when treated with α-chymotrypsin and proteinase K.

4.3.8 Compatibility of screened LAB’s for probiotic formulations

In order to formulate probiotic consortia, compatibility of screened six

LAB’s was determined by cross streak method. In this method all the six

screened LAB’s were cross streaked against each other on prepoured MRS agar

plates followed by incubation at 350C for 48h under anaerobic conditions. It was

observed that compatibility between the six stains varied for each other. Highest

compatibility of 80% has noticed for Lactobacillus sp. F8/L. acidophilus F14 with

rest of four isolates except L. delbreuckii subsp. bulgaricus F18 and L. crustorum

F11 respectively followed by L. plantarum F22 and L. fermentum F3 showing 60%

compatibility, L. delbreuckii subsp. bulgaricus F18 showed 40% compatibility

whereas, L. crustorum F11 showed only 20% compatibility to its other

counterparts as shown in Table 19.

Probiotics can be bacteria, moulds, yeast. But most probiotics are

bacteria. Among bacteria, lactic acid bacteria are more popular. Lactobacillus

acidophilus, L. casei, L. lactis, L. helviticus, L. salivarius, L. plantarum, L.

bulgaricus, L. rhamnosus, L. johnsonii, L. reuteri, L. fermentum, L. delbrueckii,

125

Table 19. Compatibility of screened LAB isolates

Compatibility*

Isolates L. fermentum

F3

Lactobacillus

sp. F8

L. crustorum

F11

L. acidophilus

F14

L. delbreuckii

subsp.

bulgaricus F18

L. plantarum

F22

Percent

Compatibility

(%)

L. fermentum F3 R + - + - - 40

Lactobacillus sp. F8 + R + + - + 80

L. crustorum F11 - + R - - - 20

L. acidophilus F14 + + - R + + 80

L. delbreuckii subsp.

bulgaricus F18

- - - + R + 40

L. plantarum F22 - + - + + R 60

*Compatibility = + or – R= Reference strain

126

Streptococcus thermophilus, Enterococcus faecium, E. faecalis, Bifidobacterium

bifidum, B. breve, B. longum and Saccharomyces boulardii are commonly used

bacterial probiotics. A probiotic may be made out of a single bacterial strain or it

may be a consortium as well (Gilliland and Speak, 1977). The most commonly

utilized probiotic preparations include specific strains of — either alone or in

combination — Lactobacilli, Streptococci and Bifidobacteria as these three

genera are important components of the gastrointestinal flora, are considered to

be harmless, and might be capable of preventing the overgrowth of pathogenic

organisms (Wadher, 2010).

Regular intake of probiotics (i.e., a fermented milk drink containing a

mixture of L. rhamnosus GG, Bifidobacterium, L. acidophilus, and S.

thermophilus ) has been demonstrated to reduce potentially pathogenic bacteria in

the upper respiratory tract of humans (Wang, 2004). Some in vitro and

experimental animal studies, indicates that probiotics may have the potential to

reduce colon cancer risk in experimental animals, intake of yogurt and specific

probiotic cultures has been shown to reduce the development of precancerous lesions

(aberrant crypts) and chemicallyinduced tumors, although the findings appear to be

both species- and strain-dependent (Wollowksi et al., 2001).

4.3.9 Cumulative probiotic score of screened LAB isolates

The probiotic potential of the bacterial strains is based upon assigned

cumulative probiotic score. Cumulative probiotic potential is the sum of score of

bile tolerance, acid tolerance, autoaggregation capacity, hydrophobic capacity,

antibiotic sensitivity and antimicrobial activity. The probiotic potential for

screened LAB isolates was calculated by following formula:

Observed score

**Probiotic potential = Maximum score X 100

In the present investigation probiotic potential for L. fermentum F3,

Lactobacillus sp. F8, L. crustorum F11, L. acidophilus F14, L. delbreuckii subsp.

bulgaricus F18 and L. plantarum F22 was adjudged 91.7, 100, 95.8, 100, 91.7 and

127

Table 20. Cumulative probiotic effect of screened LAB isolates

Score Probiotic characters Indication

L. fermentum

F3

Lactobacillus

sp. F8

L. crustorum

F11

L. acidophilus

F14

L. delbreuckii subsp.

bulgaricus F18

L. pantarum

F22

Acidity tolerance Resistant = 1

Sensitive = 0

1 1 1 1 1 1

Bile salt tolerance Resistant = 1

Sensitive = 0

1 1 1 1 1 1

Autoaggregation capacity Positive = 1

Negative = 0

1 1 1 1 1 1

Hydrophobic Capacity (Xylene/Toluene) >40% Strong = 1

(Xylene/Toluene) >20% Moderate = 0.5

(Xylene/Toluene) <20% Low = 0

0.5 1 1 1 0.5 0.5

Antagonistic activity 5-10 = 0.25

10-20 = 0.50

15-20 = 0.75

>20 = 1

1 1 0.75 1 1 1

Antibiotic sensitivity Antibiotic sensitive =1

Antibiotic resistant =0

1 1 1 1 1 1

Total 5.5/6.0 6.0/6.0 5.75/6.0 6.0/6.0 5.5/6.0 5.5/6.0

**Probiotic Potential (%) 91.7 100 95.8 100 91.7 91.7

Observed score

**Probiotic potential = Maximum score

X 100

128

91.7% respectively as shown in Table 19. Thus, all the screened six LAB’s in the

present study had highly qualified the score and are being recommended for their

use as commercial probiotics. Generally, commercially available probiotic

preparations have probiotic score in the range of 75 to 85%. The present study

revealed that these six LAB’s follows the criteria of FAO/WHO (2002) for

identifying the status of good probiotics.

Similar study to evaluate probiotic potential of lactic acid bacteria has

been done by other authors Tambekar and Bhutada (2010); Obadina et al. (2006).

Authors compared the probiotic potential of lactic acid bacteria with

commercially available probiotic preparations viz. pre-pro kid, P-Biotics kid,

Sporlac powder, Lactobacill plus, Gastroline and Standard probiotic bacterial

strains viz. L. plantarum (G95 a), L. rhamnosus (G119b) and reported that

isolated strains have equal potential as that of commercially available probiotic

preparations and probiotic bacterial strains.

Hence, this study affirms the use of six isolated LAB’s identified as L.

fermentum F3, Lactobacillus sp. F8, L. crustorum F11, L. acidophilus F14, L.

delbreuckii subsp. bulgaricus F18 and L. plantarum F22 bearing notably high

probiotic potential in the development of new pharmaceutical and functional

foods to the impart to betterment of the health of public and thus fulfils the main

objectives.

Chapter-5

SUMMARY

In the present investigation entitled “Isolation of lactic acid bacteria and

to study their potential as probiotics” an attempt has been made to isolate lactic

acid bacteria from different food sources, their screening, characterization on

biochemical as well as molecular level and furthermore to explore their probiotic

potential. The major findings of the work include:

In total, 22 lactic acid bacteria were isolated from different food sources

including indigenous foods like seera, sauerkraut, dough, dosa batter, cheese,

jalebi batter, human milk, lassi, tea leaves, garlic pickle, homemade butter, honey

and chhang. The morphological and biochemical characteristics of all the isolates

were studied. All were found to be gram positive and catalase negative and were

tentatively identified as lactobacilli/lactococci. Further, these 22 isolates were

preliminary screened on the basis of their antagonistic activity, bile salt tolerance

and acidity tolerance. The test indicator used in the present study were,

Staphylococcus aureus IGMC, Enterococcus faecalis MTCC 2729, Listeria

monocytogens MTCC 839, Clostridium perfringens MTCC 1739, Leucononstoc

mesenteroids MTCC 107, and Bacillus cereus. Among 22 isolates, six lactic acid

bacterial isolates viz. F3, F8, F11, F14, F18 and F22 showed broadest and strongest

antagonism with zone size greater than 15 mm, showed highest bile salt tolerance

(0.3-2%) with survival upto 60% and acidity tolerance (3 & 4pH) with survival

range of 24.7%. On the basis of broadest and strongest antagonism, highest bile

salt tolerance and highest acidity tolerance, these six lactic acid bacteria were

finally screened as best isolates for further characterization and probiotic

potential assessment.

The six screened lactic acid bacteria, i.e., F3, F8, F11, F14, F18 and F22 were

isolated from dough, jalebi batter, human milk, lassi, homemade butter and

chhang respectively were further characterized for their molecular identification

by 16S rRNA gene technique. Isolate F3 was identified as L. fermentum F8 as

130

Lactobacillus sp. , F11 as L. crustorum, F14 as L. acidophilus, F18 as L. delbreuckii

subsp. bulgaricus and F22 as L. plantarum.

These six finally screened LAB isolates were tested for their probiotic

potential by evaluating autoaggregation capacity, hydrophobic capacity, acidity

tolerance, antibiotic susceptibility and cumulative probiotic potential. In

probiosis, it is important to evaluate surface properties, like autoaggregation and

hydrophobicity, because they are used as a measurement directly related to

adhesion ability to enterocytic cellular lines. All the screened lactic acid bacteria

showed good autoaggregation capacity. L. fermentum F3 showed 89%

autoaggregation, Lactobacillus sp. F8 showed 70.3%, L. crustorum F11, L.

acidophilus F14, L. delbreuckii subsp. bulgaricus F18 and L. plantarum F22

showed 79.1, 60.0, 68, 79.5% autoaggregation respectively. The autoaggregation

percentage exhibited by all the screened six lactic acid bacteria was very high as

40% had been considered minimum level for adhesion abilities. All the strains

showed moderate to strong hydrophobicity. Lactobacillus sp. F8, L.crustorum F11

and L. acidophilus F14 showed strong hydrophobicity whereas, L. fermentum F3,

L. delbreuckii subsp. bulgaricus F18 and L. plantarum F22 showed moderate

hydrophobicity as measured for xylene and toluene was found to be greater than

20%. All the six screened lactic acid bacteria were found to be high acidity

tolerant strains. L. fermentum F3, Lactobacillus sp. F8, L. crustorum F11, L.

acidophilus F14, L. delbreuckii subsp. bulgaricus F18 and L. plantarum F22

showed survival of 26.4, 51.8, 56.9, 51.5, 56.6 and 90.4% respectively at pH 1.0

after 3 h of incubation. All six screened LAB’s strains were found to be sensitive

for maximum antibiotics used in the present study. L. fermentum F3, L.

acidophilus F14 and L. delbreuckii subsp. bulgaricus F18 showed 90% sensitivity.

Whereas, Lactobacillus sp. F8, L. crustorum F11 and L. plantarum F22 exhibited

80% sensitivity towards antibiotics respectively. Thus, the susceptibility of

screened LAB isolates showed that they are not reservoir of transferable

resistance genes and are safe to be used as probiotics.

All the six screened LAB isolates were tested for their broad range

inhibitory spectrum against a panel of different test strains i.e., Pseudomonas

131

syringe, Pectobacterium carotovorum, Escherichia coli and Streptococcus

mutans besides six already explored indicators. All isolates showed a broad and

strong inhibitory spectrum against both gram-positive and gram-negative

pathogenic microorganisms which is a desirable character to suppress the growth

of various pathogens. The overall antimicrobial activity of LAB isolates is

generally due to the synergistic action of lactic acid, bacteriocin and other

antimicrobial substances viz. H2O2 etc. Inhibitory spectrum during growth phase

of screened LAB isolates showed that the maximum production of these

inhibitory metabolites were found to be in between the late exponential phase and

in the beginning of the stationary phase. The effect of proteolytic and amylolytic

enzyme on LAB’s supernatant was also tested. A decrease noticed in the zone

size after enzymatic treatment, proved that the inhibitory action of the LAB

isolates is not only due acidic effect, but there are some proteinaceous and

carbohydrate moieties which also contribute significantly to inhibit the growth of

pathogenic bacteria.

The cumulative probiotic score to the screened LAB isolates was

conffered on the basis of the sum of score attained for bile tolerance, acid

tolerance, autoaggregation capacity, hydrophobicity, antibiotic sensitivity and

antimicrobial activity. The assigned probiotic potential was as high as 100% for

Lactobacillus sp. F8 and L. acidophilus F14, 95.8% for L. crustorum F11 and 91.7,

91.7 and 91.7% for L. fermentum F3, L. delbreuckii subsp. bulgaricus F18 and L.

plantarum F22 respectively. Thus, all the screened LAB isolates in the present

study had qualified the high score to be potential probiotics and based upon the

present findings for their use as commercial probiotics.

Hence, this study affirms the use of L. fermentum F3, Lactobacillus sp. F8,

L. crustorum F11, L. acidophilus F14, L. delbreuckii subsp. bulgaricus F18 and L.

plantarum F22 in the development of new pharmaceutical and functional food to

impart the betterment of the heath of public as these six strains isolated in the

present study have been proven safe as well as highly effective probiotics.

Chapter-6

REFERENCES

Adam M R and Moss M O. 1995. Food Microbiology.The Royal Society of Chemistry, Cambridge, UK.

Adams M R. 1990. Topical aspects of fermented foods. Trends in Food Science

& Technology 1: 141-144.

Adegoke GO and Oguntimein G B. 1995.Microbiological and biochemical changes during the production of Sekete- a fermented beverage made from maize. Journal of Food Science Technology 32(6): 516-518.

Aguirre M & Collins M D. 1993. Lactic acid bacteria and human clinical infection. Journal of Applied Bacteriology 75: 95-107.

Ahern M, Verschueren S and Sinderen D V. 2003. Isolation and characterization of a novel bacteriocin produced by Bacillus thuringiensis strain B439. FEMS

Microbiology Letter 220: 127-131.

Ahire J J, Patil K P, Chaudhary B L and Chincholkar S B. 2011. A potential probiotic culture ST2 produces siderophore 2, 3-dihydroxybenzoylserine under intestinal condition. Food Chemistry 127: 387-393.

Aiba Y, Suzuki N, Kabir A M, Takagi A and Koga Y. 1998.Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus

salivarius as a probiotic in a gnotobiotic murine model. American Journal of

Gastroenterology 93: 2097–2101.

Alokomi H L, Skytta E, Saarela M, Mattila-Sandholm T, Latva-Kala K, and Helander I M. 2000. Lactic acid permeabilizes gram negative bacteria by disrupting the outer membrane. Applied and Environmental Microbiology 66: 2001–2005.

Aneja K R. 2003. In: Experiments in microbiology, Plant pathology and Biotechnology. Biochemical activities of microorganisms, 4th edition, New age International Publishers, New Delhi.245-275 pp.

Aween M M, Hassan Z, Muhialdin B J, Noor H M and Eljamel Y A. 2012. Evaluation on antimicrobial activity of lactobacillus acidophilus strains isolated from honey. American Journal of Applied Sciences 9(6): 807-817.

Axelsson, L T. 1993. Lactic acid bacteria: Classification and Physiology: Lactic Acid Bacteria. S. Salminen and A. V. Wright (ed.). Marcel Dekker, INC., New York: 1-52.

Bacha K, Mehari T, Ashenafi M. 2009. In-Vitro probiotic potential of lactic acid bacteria isolated from from ‘Wakalim’, A traditional Ethiopian fermented beef. Ethiopia Journal of Health Sciences 19(1): 21-27.

134

Bakari D, Tatasadjieu N L, Mbawala A and Mbofung C M. 2011. Assessment of physiological properties of some Lactic acid bacteria isolated from the intestine of chickens use as probiotics and antimicrobial agents against enteropathogenic bacteria. Innovative Romanian Food Biotechnology 8: 33-40

Baldwin C, Millette M, Oth D, Ruiz M T, Luquet F M and Lacroix M. 2010. Probiotic Lactobacillus acidophilus and L. caseimix sensitize colorectal tumoral cells to 5- fluorouracil-induced apoptosis. Nutrition Cancer 623: 371-378.

Baradaran A, Foo H, Sieo C CandRahim R A. 2012. Isolation, identification and characterization of lactic acid bacteria from Polygonumminus. Romanian

Biotechnological Letters 17(3): 7245-7252.

Barefoot S F and Klanhammer T R. 1983. Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Applied and

Environmental Microbiology 45(6): 1808-1815.

Batra L R and Millner P D. 1976.Asian fermented foods and beverages. DevIndMicrobiol 17:117–128.

Batra L R and Millner P D. 1974. Some Asian fermented foods and beverages

and associated fungi. Mycology 66: 942–950.

Belkum V M J and Stiles M E. 2000.Non-lantibiotic antibacterial peptides from lactic acid bacteria Natural Product Report 17: 323–335.

Belli W A and Marquis R E. 1991. Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture. Applied and

Environmental Microbiology 57: 1134-1138.

Bengmark S. 2000. Colonic food: pre- and probiotics. American Journal of

Gastroenterology 95: S5-S7.

Bettache G, Fatma A, Miloud H and Mebrouk K. 2012.Isolation and identification of lactic acid bacteria from dhan, a traditional butter and their major technological traits. World Applied Sciences Journal 17(4): 480-488.

Bezkorvainy A. 2001. Probiotics: Determinants of survival and growth in the gut. American Journal Clinical Nutrition 73: 399-405.

Bhakta J N, Ohnishi K, Munekage Y and Iwasaki K. 2010.Isolation and probiotic characterization of arsenic-resistant Lactic acid bacteria for uptakingarsenic.International Journal of Chemical and Biological

Engineering 3(4): 167-174.

Bhatacharya S and Dass A. 2010. Study of physical and cultural parameters on the bacteriocins produced by Lactic acid bacteria isolated from traditional Indian fermented foods. American Journal of Food Technology 5(2): 111-120.

Bhatia S J, Kochar N, Abraham P, Nair N G and Mehta A P. 1989. Lactobacillus

acidophilus inhibits growth of Campylobacter pylori in vitro. Journal of

Clinical Microbiology 27: 2328–2330.

135

Bibiloni R., Pérez P F, Garrote L G, Disalvo E A and Antoni G L D. 2001. Surface characterization and adhesive properties of Bifidobacteria. Methods

Enzymol 336: 411-427.

Bibiloni R., Perez P F, and Antoni G L D. 1999. Factors envolved in adhesion of bifidobacterial strains to epithelial cells in culture. Anaerobe 5: 483-485.

Bizani D and Brandelli A. 2002. Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8A. Journal of Applied Microbiology 93: 512-519.

Boris S, Suárez J E, and Barbés C. 1997.Characterization of the aggregation promoting factor from Lactobacillus gasseri vaginal isolate.Journalof

Applied Microbiology 83:413-420.

Boutron-Ruault M C. 2007.Probiotiqueset cancer colorectal.Nutrition and

Clinical Méthods 21: 85-88.

Brady L J, Gallaher D D, Busta F F. 2000. The role of probiotic cultures in the prevention of colon cancer. The Journal of Nutrition 130: 410S-414S.

Breed R S, Murry E G D and N R Smith (eds). 1957. Bergey's Manual of Determinative Bacteriology (7th edn). Williams and Wilkinns. Baltimore, USA

Breidt F and Fleming H P. 1997. Using lactic acid bacteria to improve the safety of minimally processed fruits and vegetables. Food Technology 51: 44-46, 48, 51.

Çakır İ. 2003. Determination of some probiotic properties on Lactobacilli and Bifidobacteria. Ankara University Thesis of Ph.D.

Campbell-Platt G. 1987. Fermented foods of the world - a dictionary and guide. London, Butterworths. ISBN: 0-407-00313-4

Caplan M S and Jilling T. 2000. Neonatal necrotizing enterocolitis: possible role of probiotic supplementation. Journal of Paediatrics and Gastroenterology

Nutrition 30: S18–S22.

Charteris W P, Kelly P M, Morelli L, Collins J K. 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species. Journal of Food Protection 61: 1636-1643.

Cherif A, Quzari H, Daffonchio D, Cherif H, Bem S K, Hassen A, Jaaua S and Boudabous A. 2001. Thuricin 7: a novel bacteriocin produced by Bacillus

thuringiensis BMG 1.7, a new strain isolated from soil. Letters in Applied

Microbiology 32: 243-247.

Chou L S and Weimer B. 1999.Isolation and characterization of acid and bile tolerant isolates from strains of Lactobacillus acidophilus.Journal of Dairy

Science 82: 23-31.

136

Chuayana E L, Ponce C V, Rosanna M and Cabrera E C. 2003.Antimicrobial activity of probiotics from milk products. Philippine Journal of Microbiology

and Infectious diseases 32(2): 71-74.

Chung H, Kim Y B, Chun S L and Ji G E. 1999.Screening and selection of acid and bile resistant bifidobacteria. International Journal of Food Microbiology

47: 25-32.

Clark P A and Martin J H . 1994. Selection of bifidobacteriafor use as dietary adjunts in cultured dairy foods: III – Tolerance to simulated bile concentrations of human small intestine. Culture Dairy Products Journal 29: 18-21.

Coconnier M, Lievin V, Bernet-Camard M F, Hudault S H and Servin A L. 1997. Antibacterial effect of the adhering human Lactobacillus Acidophilus

strain LB. Antimicrobial Agents Chemoth 41: 1046-1052.

Collado M C Meriluoto J and Salminen S. 2007a. Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus. Letters in

Applied Microbiology 45: 454-460.

Collado M, Meriluoto J and Salminen S. 2007.In vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus. Food

Research International 40: 629-639.

Collins J K, Thornton G and Sullivan G O. 1998. Selection of probiotic strains for human applications. International Journal of Systematic Bacteriology 8: 487-490.

Collins M D, Farrow J A E, Phillips B A, Ferusu S and Jones D. 1987. Classification of Lactobacillus divergens, Lactobacillus pisciolaand some catalase-negative, asporogenous, rod-shaped bacteria from poultry in a new genus, Carnobacterium, International Journal of Systematic Bacteriology 37: 310-316.

Coppola R, Succi M, Tremonte P, Reale A , Salzano G, Sorrentino E. 2005. Antibiotic susceptibility of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. Lait 85: 193-204.

Corzo G and Gilliland S E. 1999.Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts. Journal of Dairy Science 82: 466-71.

Crittenden R G, Martinez N R and Playne M J. 2003.Synthesis and utilization of folate by yoghurt starter cultures and probiotic bacteria. International Journal

of Food Microbiology 80: 217-222.

Dardir H A. 2012. In vitro evaluation of probiotic activities of Lactic acid bacteria strains isolated from novel probiotic dairy products. Global

Veterinarias 8(2): 190-196.

Davis C D and Milner J A. 2009. Gastrointestinal microflora, food components and coloncancer prevention. Journal of Nutritional Biochemistry 20: 743–752.

137

De Man J, Rogosa M and Sharpe M. 1960. A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23: 130-135.

Del Re B, Sgorbati B, Miglioli M and PalenzonaD. 2000. Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Letters in Applied Microbiology 31: 438–442.

Delcour J, Ferain T, Deghorain M, Palumbo E, and Hols P. 1999. The biosynthesis and functionality of the cell-wall of lactic acid bacteria. Antonie

van Leeuwenhoek 76: 159-184.

Delos Reyes-Gavilan C G, Limsowtin G K Y, Tailliez P, Seachaud L and Accolas J P. 1992. Lactobacillus helveticusspecific DNA probe detects restriction fragment polymorphism in this species. Applied Environmental


Dora I A P and Glenn R G. 2002. Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut. Applied Environmental


Dugas B, Mercenier A, Lenoir-Wijnkoop I, Arnaud C, Dugas N and Postaire E. 1999. Immunity and probiotics. Trends Immunology Today 20(9): 387-390.

Dunne C, O’Mahony L, Murphy L, Thornton G, Morrissey D, O’Halloran S, Feeney M, Flynn S, Fitzgerald G, Daly C, Kiely B, O’Sullivan G, Shanahan F and Collins J K. 2001. In vitro selection criteria for probiotic bacteria of human origin: Correlation with in vivo findings. American Journal Clinical

Nutrition 73: 386S-392S.

Dylewski J, Psaradellis F and Sampalis J. 2010. Efficacy of a probiotic formula in the reduction of antibiotic-associated diarrhea, A placebo controlled double-blind randomized, multicenter study. Archives of Medical Science 61: 56-64.

Ebrahimi M T, Ouwehand A C, Hejazi M A and Jafari P. 2011. Traditional Iranian dairy products: A source of potential probiotic lactobacilli. African

Journal of Microbiology Research 5(1): 20-27.

El-Nagger M Y M. 2004.Comparative study of probiotic culture to control the growth of Escherichia coli and Salmonella Typhimurium. Biotechnol 32: 173-180.

Engelhardt H, and Peters J. 1998. Structural research on surface layers: a focus on stability, surface layer homology domains, and surface layer cell wall interactions. Journal of Structural Biology 124: 276-302.

European Food Safety Authority. 2008. Update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. EFSA Journal 732: 1-15.

FAO/WHO. 2001. Food and Agricultural Organization of the United Nations and World Health Organization: Guidelines for the evaluation of probiotics in food. WWW.fao.org/es/esn/food/foddanfood_probio.

138

Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.

Fernandes C F, Shahani K M and Amer M A. 1987. 'Therapeutic role of dietarylactobacilli and lactobacillicfermented dairy products'. FEMS

Microbiology review 466: 343-356.

Fleck Ž C , Savić V, Kozačins L, Njari B, Zdolec N, and Filipović I. 2012. Identification of lactic acid bacteria isolated from dry fermented sausages. Veterinarski Arhiv 82(3): 265-272.

Fooks L J, Fuller R, Gibson G R. 1999. Prebiotics, probiotics and human gut microbiology. International Dairy Journal 9: 53-61.

Forestier, C, Champs CD, Vatoux C, and Joly B. 2001. Probiotic activities of Lactobacillus casei rhamnosus: in vitro adherence to intestinal cells and antimicrobial properties. Research in Microbiology 152: 167–173.

Fuller R. 1989. Probiotics in man and animals: A review. Journal of Applied

Bacteriology 66: 365-378.

Fuller R. 1991.Probiotics in human medicine.Gut; 32: 439-442.

Gao Y, Jia S, Gao Q, Tan Z. 2010. A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage. Food Control 21: 76–81.

GatjeG, Muller V, and Gottschalk G. 1991. Lactic acid excretion via canier-mediated facilitated diffusion in Lacrobucillus helveticus. Applied

Microbiology and Biotechnology 34: 778.

Ghafoor A, Naseem S, Younus M and Nazir J. 2005. Immunomodulatory Effects of Multistrain Probiotics Protexin™ on Broiler Chicken Vaccinated Against Avian Influenza Virus H9. International Journal of Poultry Sciences 410: 777-780.

Gibson G R and Robertfroid M B. 1995. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. American Journal

Clinical Nutrition 1401-1412.

Gill A O, Holley R A. 2000. Surface application of lysozyme, nisin, and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna. Journal of Food

Protection 63: 1338–1346.

Gilliland S E, Speck M L. 1977. Deconjugaton of bile acids by intestinal lactobacilli. Applied Environmental Microbiology 33: 15–18

Gilliland S E, Stanley T E and Bush L J. 1984. Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. Journal of Dairy Science

67: 3045-3051.

Girgis H S, Smith J, Luchansky J B and Klaenhammer T R. 2003. Stress adaptation of lactic acid bacteria. In Microbial Stress Adaption and Food Safety, A.E. Yousef and V.K. Juneja, editors. CRC Press, Boca Raton, U.S.A., pp. 159-211.

139

Gismondo M R, Drago L and Lombardi A. 1999. Review of probiotics available to modify gastrointestinal flora. International Journal of Antimicrobial Agents

12: 287-292.

Gómez-Zavaglia A, Kociubinski G, Pérez P and De Antoni,G. 1998. Isolation and characterization of Bifidobacterium strains for probiotic formulation. Journal of Food Protection 61: 865-873.

Gorbach S L, Chang T W and Goldin B. 1987.Successful treatment of relapsing Clostridium difficile colitis with Lactobacillus GG. Lancet 2: 1519.

Gotcheva V, Hristozova E, Hristozova T, Guo M, Roshkova Z and Angelov A. 2002. Assessment of potential probiotic properties of lactic acid bacteria and yeast strains. Food Biotechnology 16: 211-225.

Gram H C. 1884. Uber die isoliertefarbung der Schizomuceten in Schnitt- und Trockenpraparaten (In German). Fortschritte der Medizin 2:185-189.

Grosu-Tudor S S and Zamfir M. 2012. Probiotic potential of some potential lactic acid bacteria isolated from Romanian fermented vegetables. Annals of RSCB

XVII(1): 234-239.

Guarner F, Perdigón G, Corthier G, Salminen S, Koletzko B and Morelli L. 2005. Should yoghurt cultures be considered probiotic? British Journal of Nutrition

93: 783–786.

Gupta A. 2010. PCR detection of bacteria isolate no. A75 and application of bacteriocin produced from it as food biopreservative. UHF MSc. Thesis.

Hamid M and Abulfazal B. 2012. Isolation and molecular study of potentially Lactobacilli in Traditional white cheese of Tibriz in Iran. Annals of Biological

Research 3(5): 2213-2216.

Hamid T H T B, Khan A J, Jalil M F and Azhar N S. 2012.Isolation and screening of Lactic acid bacteria, Lactococcus lactis from Clariasgariepinus (African catfish) with potential use as probiotic in aquaculture. African

Journal of Biotechnology 11(29): 7494-7499.

Hartke A, Bouché S, Giard J C, Benachour A, Boutibonnes P and Auffray Y. 1996. The lactic acid stress response of Lactococcus lactis subsp. lactis. Current Microbiology 33: 194-199.

Helland M H, Wicklund T and Narvhus J A. 2004.Growth and metabolism of selected strains of probiotic bacteria in maize porridge with added malted barley. International Journal of Food Microbiology 91: 305-313.

Hesseltine C W. 1983.Microbiology of oriental fermented foods.Annual Review

of Microbiology 37: 575-601.

Hirayama K, and Rafter J. 2000. The role of probiotic bacteria in cancer prevention. Microbes and Infection 2: 681-686.

140

Holzapfel W H, Haberer P, Geisen R, Björkroth J and Schillinger U. 2001. Taxonomy and important features of probiotic microorganisms in food nutrition. American Journal Clinical Nutrition 73: 365S-373S.

Hoque M Z, Akter F, Hossain K M, Rahman M S N, Bilah M M and Islam K M D. 2010. Isolation, identification and analysis of probiotic properties of lactobacillus spp. from selective regional yoghurts. World Journal of Dairy

and Food Sciences 5(1): 39-46.

Ibrahim S and Bezkorovainy A. 1993. Survival of bifidobacteria in the presence of bile salt. Journal of Animal Sciences 62: 351-354.

ICMR-DBT. 2011. Guidelines for Evaluation of Probiotics in Food. Indian

Journal of Medical Research 134: 22-25.

Indira K, Jayalakshami S, Gopalakrishnan A and Sirinivasan M. 2011. Biopreservative potential of marine lactobacillus spp. African Journal of

Microbiology Research 5(16): 2287-2296.

Iñiguez-Palomares C, Pérez-Morales R and Acedo-Félix E. 2007. Evaluation of probiotic properties in Lactobacillus isolated from small intestine of piglets. Microbiologia 49(3-4): 46-54.

Jack R W, Tagg J R and Ray B. 1995. Bacteriocins of gram positive bacteria. Microbiology Review 59: 171-200.

Jamaly N, Benjouad A and Bouksaim M. 2011. Probiotic potential of lactobacillus strains isolated from known popular traditional Moroccan dairy products. British Microbiology Research Journal 1(4): 79-94.

Juntunen M, Kirjavainen P V, Ouwehand A C, Salminen S J, and Isolauri E. 2001. Adherence of probiotic bacteria to human intestinal mucus in healthy infants and during rotavirus infection. Clinical and Diagnostic Laboratory

Immunology 8: 293-296.

Kaboré D, Sawadogo-Lingani H, Dicko M H, Diawara B and Jakobsen M. 2012. Acid resistance, bile tolerance and antimicrobial properties of dominant lactic acid bacteria isolated from traditional “marri” baobab seeds fermented condiment. African Journal of Biotechnology 11(5): 1197-1206.

Kalui C M, Mathara J M, Kutima P M, Kiiyukia C and Wongo L E. 2009. Functional characteristics of Lactobacillus plantarum and Lactobacillus

rhamnosus from ikii, a Kenyan traditional fermented maize porridge. African

Journal of Biotechnology 8: 4363-4373.

Kandler O, Weiss N, Sneath P H A, Mair N S, Sharpe M E and Holt J G (eds). Regular, non-sporing, gram-positive rods.Bergey’s manual of systematic

bacteriology. Williams and Wilkins, London, MD. 2: 1208-1234.

Kapoor R. 2007. PCR detection of bacteria isolate no. A75 and application of bacteriocin produced from it as food biopreservative. UHF MSc. Thesis.

Kashket E R, Blanchard A G and Metzger W C. 1980. Proton motive force during growth of Srreptococcus lactis cells. Journal of Bacteriology 143: 128.

141

Kaur I P, Chopra K and Saini A. 2002. Probiotics: Potential pharmaceutical applications. European Journal of Pharmaceutical Sciences 15: 1-9.

Khetarpaul N &Chauhan B M. 1990. Fermentation of pearl millet flour with yeasts and lactobacilli: in vitro digestibility and utilisation of fermented flour for weaning mixtures. Plant Foods for Human Nutrition 40: 167-173.

Kim H, Shin H, Ha W, Yang H and Lee S. 2006. Characterization of lactic bacterial strains isolated from raw milk. Asian-Australian Journal of Animal

Sciences 19 (1): 131-136.

Kimoto H, Nomura M, Kobayashi M, Okamoto T and Ohmomo S. 2004. Identification and probiotic characteristics of Lactococcus strains from plant materials. Japan Agricultural Research Quarterly 38(2): 111-117.

Kimura H, Sashihara T, Matsusaki H, Sonomoto K and Ishizaki A. 1998. Novel bacteriocin of Pediococcus sp. ISK-1 isolated from well – aged bed of fermented rice bran. Annals of New York Academy of Science 864: 345-348.

Klaenhammer T R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiology Reviews 12: 39–86.

Kociubinski G, Perez P F, Añon M C & De Antoni G L. 1999. A method for the screening of lactic acid bacteria with high inhibitory power. Journal of Food

Protection 59: 739.

Konings W N, Poolman B and Driessen A J M. 1989. Bioenergetics and solute transport in lactococci. CRC Critical Review of Microbiology 16: 419.

Kos B, Sŭškovic´ J, Vukovic´ S, Sˇ impraga M, Frece J and Matošic´ S. 2003. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus

M92. Journal of Applied Microbiology 94: 981–987.

Kostinek M, Ban-Koffi L, Ottah-Atikpo M, Teniola D, Schillinger U, Holzapfel W H and Franz C M A P. 2008. Diversity of predominant Lactic acid bacteria associated with cocoa fermentation in Nigeria. Current Microbiology, pp 1-15

Kostinek M, Specht I, Edward VA, Schillinger U, Hertel C, Holzapfel WH, Franz CMAP. 2005. Diversity and technological properties of predominant lactic acid bacteria from fermented cassava used for the preparation of Gari, a traditional African food. Systematic Applied Microbiology 28: 527–540.

Kpikpi N E, Glover R L K, Dzogbefia V P, Nielsen D S and Jakobsen M. 2010.Isolation of lactic acid bacteria from kantong, a condiment produced from the fermentation of kapok (Ceibapentandra) seeds and cassava (Manihotes culentum) flour. Report and Opinion 2(8): 1-7.

Kumar A M and Murugalatha N. 2012.Isolation of Lactobacillus plantarumfrom cow milk and screening for the presence of sugar alcohol producing gene. Journal of Microbiology and Antimicrobials 4(1): 16-22.

Lavanya V, Sowmiya S, Balaji S and Muthivelan B. 2011.Screening and characterization of Lactic Acid Bacteria from fermented milk.British Journal

of Dairy Sciences 2(1): 5-10.

142

Lewus C B, Kaiser A and Montiville T J. 1991. Inhibition of food – borne pathogens by bacteriocins from lactic acid bacteria isolated from meat. Applied and Environmental Microbiology 57(6): 1683-1688.

Licht T R and Wilcks A. 2005.Conjugative gene transfer in the gastrointestinal environment. Advances in Applied Microbiology 58C:77-95.

Lin M, Savaiano D and Harlender S. 1991. Influence of nonfermented dairy products containing bacterial starter cultures on lactose maldigestion in humans. Journal of Dairy Science 74: 87-95.

Ljung A, Lan J, Yanagisawa N. 2002. Isolation, selection and characteristics of Lactobacillus paracasei subsp. Paracasei F19. Microbial Ecology Health

Disease 14 (3): 4-6.

Lortal S, Van Heijenoort J, Gruber K, and Sleytr S. 1992. S-layer of Lactobacillus helveticus ATCC12046: isolation, chemical characterization and re-formation after extraction with lithium chloride. Journal of Genetics

and Microbiology 138: 611-618.

Lynch D J, Fountain T L, Mazurkiewicz J E and Banas J A. 2007. Glucan-binding proteins are essential for shaping Streptococcus mutants biofilm architecture. FEMS Microbiology Letters 268: 158-165.

Mahrous H, Shaalan U F and Ibrahim A M. 2011.The role of some probiotic Lactic acid bacteria in the reduction of cholesterol on mice. International

Research Journal of Microbiology 2(7): 242-248.

Marshall V M and Law B A. 1984. The physiology and growth of dairy lactic-acid bacteria. In: Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk. F. L. Davies and B. A. Law, ed. Elsevier Applied

Science Publications New York, NY.

Marteau P, de Vrese M, Cellier C J and Schrezenmeir J. 2000. Protection from gastrointestinal diseases with the use of probiotics. American Journal Clinical

Nutrition 73: 430S–436S.

Mathara J M, Schillinger U, Guigas C, Franz C, Kutima P M, Mbugua S K, Shin H.-K, Holzapfel W H. 2008. Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya. International

Journal of Food Microbiology 126: 57–64.

Matthes H, Krummenerl T, Giensch M, Wolff C, Schulze J. 2010. Clinical trial, probiotic treatment of acute distal ulcerative colitis with rectally administered Escherichia coli Nissle 1917 EcN. Complementary and Alternative Medicine

10: 13.

Maurad K and Meriem K H. 2008. Probiotic characteristics of Lactobacillus

plantarum strains from traditional butter made from camel milk in arid regions (Sahara) of Algeria. Grasas Aceites 59: 218-224.

Maxwell F J and Stewart C S. 1995. The Microbiology of the Gut and the Role of Probiotics, pp. 155-177. In Varley M.A (ed). The neonatal Pig Development and Survival.Ed. Cab International. Leeds, UK.

143

McDonald L C, Fleming H P and Hassan H M. 1990. Acid tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum. Applied and

Environmental Microbiology 56: 2120-2124.

Meera N S and Devi M C. 2012. Partial characterization and optimization of parameters for bacteriocin production by probiotic Lactic acid bacteria. Journal of Microbiology and Biotechnology Research 2(2): 357-365.

Metchnikoff E. 1908. The prolongation of life: Optimistic studies. G P Putnam's sons, New York.

Mishra V and Prasad D. 2005. Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. International Journal of

Food Microbiology 103: 109-115.

Mitchell P. 1973. Performance and conservation of osmotic workby proton-coupled solute porter systems. Journal of Bioenergy 463.

Mombelli B, Gismondo M R. 2000. The use of probiotics in medical practice. International Journal of Antimicrobial Agents 16: 531-536.

Morelli L and Callegari M L. 2006.Taxononomydan biology of probiotics. In: Goktepe I, Juneja V K, Ahmedna M, editors. Probiotics in Food Safety and

Human Health. Boca Raton: Taylor and Francis.

Mourad K and Nour-Eddine K. 2006. In vitro preselection criteria for probiotic Lactobacillus Plantarum strains of fermented olives origin. International

Journal of Probiotics and Prebiotics 1(1): 27-32.

Mozes N and Lortal S. 1995. X-ray photoelectron spectroscopy and biochemical analysis of the surface of Lactobacillus helveticus ATCC12046. Microbiology

141: 11-19.

Mundt J O. 1986. Enterococci: Bergey's Manual of Systematic Bactriology. Sneath P H A, Mair N S, Sharpe M E and Holt J E (ed.). Williams and Wilkins, Baltimore. 2: 1063-1065.

Musikasang H, Sohsomboon N, Tani A and Maneerat S. 2012. Bacteriocin-producing Lactic Acid Bacteria as a probiotic potential from Thai indigenous chickens. Czech Journal of Animal Science 57(3): 137-149

Muyanja BK, Naruhus J A and Langsrud T. 2003.Isolation, characterization and identification of lactic acid bacteria from Bushera: A Ugandan traditional fermented beverage. International Journal of Food Microbiology 80(3): 201-210.

Naclerio G, Ricca E, Sacco M and DeFelice M. 1993. Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus. Applied and Environmental

Microbiology 59(12): 4313-4316.

Naeem M, Ilyas M, Haider S, Baig S and Saleem M. 2012.Isolation characterisation and identification of lactic acid bacteria from fruit juices and their efficacy against antibiotics. Pakistan Journal of Botany 44: 323-328.

144

Nanasombat S, Phunpruch S, and Jaichalad T. 2012. Screening and identification of lactic acid bacteria from raw sea foods and Thai fermented seafood products for their potential use as starter cultures. Songklanakarin Journal of

Science and Technology 34(3): 255-262.

Neha. 2006. Production and characterization of bacteriocin from lactic acid bacteria and to study its biopreservative effect. UHF MSc. Thesis.

Nobaek S, Johansson M L and Molin G. 2000. Alteration of intestinal microflora is associated with reduction in abdominal bloating and pain in patients with irritable bowel syndrome. American Journal of Gastroenterology 95: 1231–1238.

Nomoto K. 2005. Prevention of infections by Probiotics. Journal of Bioscience

and Bioengineering 100: 583-592.

Novotony J F and Perry J J. 1992. Characterization of bacteriocins from two strains of Bacillus thermoleovorans, a thermophilic hydrocarbon – utilizing species. Applied and Environmental Microbiology 58(8): 2393-2396.

Nowroozi J, Mirzaii M and Norouzi M. 2004. Study of lactobacillus as probiotic bacteria. Iranian Journal Public Health 33(2): 1-7.

Nuraida L, Anggraeni D and Dewanti-Hariyadi R. 2011. Adherence properties of Lactic acid bacteria isolated from breast milk. The 12th Asean Food Conference 16-18.

Obadina A O, Oyewole O B, Sanni L O and Tomlins K I. 2006. Bio-preservative activities of Lactobacillus plantarum strains in fermenting Casssava ‘fufu’. African Journal of Biotechnology 5(8): 620-623.

Obizoba I C &Atii J V. 1991.Effect of soaking, sprouting, fermentation and cooking on nutrient composition and some anti-nutritional factors of sorghum (Guinesia) seeds. Plant Foods for Human Nutrition 41: 203-212.

Ocaňa V S and Nader-Macías M E. 2002. Vaginal lactobacilli: self- and coaggregation ability. British Journal of Biomedical Sciences 59:183-190.

Ofek I and Doyle R J. 1994. Bacterial Adhesion to Cells and Tissues, pp. 80-84. Chapman & Hall. New York.

Ogunbanwo S T, Sanni A I and oniludeAA. 2003. Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1. African Journal of Biotechnology 2(8): 219-227.

Okereke H C, Achi O K, Ekwenye U N and Orji F A. 2012.Antimicrobial properties of probiotic bacteria from various sources. African Journal of

Biotechnology 11(39): 9416-9421.

Orla-Jensen S. 1919. The lactic acid bacteria, Koeniglicher Hof Boghandel. Copenhagen

O'Sullivan, M.G. 1996. Metabolism of bifidogenic factors by gut flora- an overview. IDF Bul. 313: 23-30.

145

Ouwehand A C, Salminen S and Isolauri E. 2002. Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek 82: 279–289.

Padan E, ZelbersteinDandSchuldiner S. 1981.pH homeostasis in bacteria. Biochemistry and Biophysics Acta 650: 151.

Padmaja G and George M. 1999.Oriental fermented foods; biotechnological approaches. In: Marwaha S S and Arora J K (eds) Food processing: biotechnological applications. Asiatech Publishers Inc, New Delhi, pp 143–189.

Papathanasopoulos M A, Krier F, Junelles A M R, Lefebue G, Lecaer J P, Von Holy A and Hastings J W. 1997. Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissela strains. Current Microbiology 35: 331-335.

Paredes-López O & Harry G I. 1988. Food biotechnology review: traditional solid-state fermentations of plant raw materials - application, nutritional significance and future prospects. Critical Reviews in Food Science and

Nutrition 27: 159-187.

Park Y H, Jong Gun K, Young Won S, HyungSoo K, Young-Jun K, Taehoon C, Hun K S and Kwang Wang Youn W. 2008. Effects of Lactobacillus

acidophilus 43121 and a mixture of Bifidobacterium longum on the serum cholesterol level and fecal sterol excretion in hipercholesterolemia induced pigs. Biosciences and Biotechnology Biochemistry 72: 70581-70588.

Parker R B. 1974.Probiotics, the other half of the antibiotic story. Animal

Nutrition and Health 29: 4-8.

Parvez S, Malik K A, Kang S A and Kim H Y. 2006. Probiotics and their fermented food products are beneficial for health. Journal of Applied

Microbiology ISSN: 1364-5072.

Patil M M, Pal A, Anand T and Ramana K V. 2010. Isolation and characterization of lactic acid bacteria from curd and cucumber. Indian

Journal of Biotechnology 9: 166-172.

Pelinescu D R, Sasarman E, Chifiriuc M C, Stoica I, Nohita A M, Avram I, Serbancea F, Dimov T V. 2009. Isolation and identification of some Lactobacillus and Enterococcus strains by a polyphasic taxonomical approach.RomanianBiotechnological Letters 14: 4225-4233.

Pelinescu D, Chifiriuc M C, Ditu L M, Sarbu I, Bleotu C, Vassu T, Stoica I, Lazar V, Corcionivoschi N and Sasarman E. 2011. Selection and characterization of the probiotic potential of some Lactic acid bacteria isolated from infant feces. Romanian Biotechnological Letters 16(3): 6179-6189.

Pelletier C, Bouley C, Cayuela C, Bouttier S, Bourlioux P and Bellon-Fontaine M N. 1997. Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains. Applied and Environmental Microbiology 63: 1725–1731.

146

Pérez P F, Minnaard Y, Disalvo A and Antoni G L D. 1998. Surface properties of bifidobacterial strains of human origin. Applied Environmental


Pithva S, Ambalam P, Dave J M and Vyas B R M. 2011.Antimicrobial peptides of probiotic lactobacillus strains. In: Science against microbial pathogens: communicating current research and technological advances. A. Méndez-Vilas (Ed.). pp. 987-991.

Pochapin M. 2000. The effect of probiotics on Clostridium difficile diarrhea. American Journal Gastroenterology 95: S11–S13.

Prakash S and Jones M L. 2005. Artificial cell therapy: New strategies for the therapeutic delivery of live bacteria. Journal of Biomedicine and

Biotechnology 1: 44-56.

Quewand A C and Salminen S J. 1998. The health effects of cultured milk products with viable and non-viable bacteria. International Dairy Journal 8: 749-758.

Rafter J. 2003. Probiotics and colon cancer. Best Practice and Research Clinical

Gastroenterology 17(5): 849-859.

Ravi V, Prabhu M and Subramanyam D. 2011.Isolation of bacteriocin producing bacteria from mango pulp and its antimicrobial activity. Journal of

Microbiology and Biotechnology Research 1(2): 54-63.

Ray B, Miller K W and Jain M K. 2001. Bacteriocins of lactic and bacteria: Current perspectives. Indian Journal of Microbiology 41: 1-21.

Reid G and Burton J. 2002. Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes and Infection 4: 319–324.

Reid G, McGroarty J A, Domingue P A, Chow A W, Bruce A W, Eisen A, andCosterton J W. 1990. Coaggregation of urogenital bacteria in vitro and in vivo. Current Microbiology 20:47-52.

Reid G. 2002. The potential role of probiotics in pediatric urology. Journal of

Urology 168: 1512–1517.

Reynolds P E. 1989.Structure, biochemistry and mechanism of action of glycopeptides antibiotics. Journal of Clinical and Microbiology Infection

Diseases 8:943–950.

Ricciardi A and Clementi F. 2000. Exopolysaccharides from lactic acid bacteria: structure, production and technological applications. Italian Journal of Food

Science 12: 23–45.

Rickard A H, Gilbert P, High N J, Kolenbrander P E and Handley P S. 2003. Bacterial coaagregation: an integral process in the developments of multi-species biofilms. Trends Microbial 11: 94-100.

Rodriguez R L andTait R T. 1983.Recombinant DNA techniques.An introduction.AdissonWesely, London. xviii-236 pp.

147

Rolfe, R.D. 2000. The Role of probiotic cultures in the control of gastrointestinal health. Journal of Nutrition 13: 396-402.

Sahlin P. 1999.Fermentation as a method of food processing: production of organic acids, pH-development and microbial growth in fermenting cereals. Ph. D Thesis.pp 56

Salminen S, Gorbach S, Lee Y K and Benno Y. 2004. Human studies on probiotics: What is scientifically proven today? Lactic Acid Bacteria

Microbiological and Functional Aspects, New York: Marcel Dekker Inc.

Salminen S, von Wright A, Morelli L, Marteau P, Brassart D, de Vos W M, Fonden R, Saxelin M, Collins K, Mogensen G, Birkeland S and Mattila-Sandholm T. 1998a. Demonstration safety of probiotics-A review. International Journal of Food Microbiology 44(1): 93-106.

Salminen, S. 1999. Probiotics: scientific support for use. Food Technology

53(11): 66.

Sambrook J and Russell D. 2001. Molecular cloning: a laboratory manual: CSHL press.

Sanders M E and Klaenhammer T R. 2001.Invited review: the scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic. Journal of

Dairy Science 84: 319–331.

Sansawat A and Thirabunyanon M. 2009. Anti-Aeromonashydrophila activity and characterization of novel probiotic strain of Bacillus Subtilis isolated from the gastrointestinal tract of giant freshwater prawns. Maejo International

Journal of Science and Technology 3(01): 77-87.

Saranya S and Hemashenpagam N. 2011.Antagonistic activity and antibiotic sensitivity of Lactic acid bacteria from fermented dairy products. Advances in

Applied Science Research 2(4): 528-534.

Sarkano, Faturrahman and Sofyan Y. 2010.Isolation and identification of Lactic acid bacteria from abalone (Haliotis asinine) as a potential candidate of probiotic. Bioscience 2(1) 38-42.

Savadogo A, Ouattara C A T, Bhassole I H N and Traore A S. 2004. Antimicrobial activities of Lactic acid bacteria strains isolated from Burkina faso fermented milk. Pakistan Journal of Nutrition 3(3): 174-179.

Schär-Zammaretti P and Ubbink J. 2003. The cell wall of lactic acid bacteria: Surface constituents and macromolecular conformations. Biophysical Journal

85: 4076-4092.

Scheinbach S. 1998. Probiotics: Functionality and commercial status. Biotechnology Advances 16(3): 581-608.

Schrezenmeir J and Vrese M. 2001.Probiotics, prebiotics, and synbiotics – approaching a definition. American Journal Clinic and Nutrition 73: 361S-364S.

148

Schultz M and Sartor R B. 2000. Probiotics and inflammatory bowel diseases. American Journal Gastroenterology 95: 19S–21S.

Sharpe M E. 1979. Identification of the lactic acid bacteria: Identification Methods for Microbiologists, 2nd ed. Soc. Appl. Bacteriol. Technical series, No. 14. F. A. Skinner and D. W. Lovelock (ed.). Academic press, London,246-255.

Shornikova A V, Casas I A and Isolauri E. 1997b. Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children. Journal of Paediatrics

Gastroenterology Nutrition 24: 399–404.

Shruthy V V, Pavithra M, Gawri S and Ghosh A R. 2011. Probiotic potential among Lactic Acid Bacteria isolated from curd. International Journal of

Research in Ayurveda & Pharmacy 2(2): 602-609.

Sieladie D V, Zambou N F, Kaktcham P M, Cresci A and Fonteh F. 2011. Probiotic properties of lactobacilli strains isolated from raw cow milk on the western highlands of Cameroon. Innovative Romanian Food Biotechnology 9: 12-28.

Sleytr U B, Sara M, and Pum D. 2000. Crystalline bacterial cell surface layers (S-layers): a versatile self-assembly system. In Supramolecular Polymers. A. Ciferri, editor. New York. 177-213.

Smit E, Oling F, Demel R, Martinez B, and Pouwels P H. 2001. The S-layer protein of Lactobacillus acidophilus ATCC4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding. Journal of Molecular Biology 305: 245-257.

Soni S K and Sandhu D K. 1990. Indian fermented foods: microbiological and biochemical aspects. Indian Journal of Microbiology 30:135–157.

Streyer L. 1981. Biochemistry, 2nd Ed. Freeman, San Francisco.

Sudirman I, Mathieu F, Michel M and Lefebvre G. 1993. Detection and properties of curvaticin 13, a bacteriocin like substance produced by Lactobacillus curvatus SB13. Current Microbiology27: 35-40.

Suma K, Misra M C and Varadaraj M C. 1998. Plantaricin LP84, a broad

spectrum heat stable bacteriocin of Lactobacillus plantarum NCIM 2084 produced in a simple glucose broth medium. International Journal of Food

Microbiology 1(40): 17-25.

Šuškovi B K, Matošic S, Besendorfer V. 2000. World Journal of Microbiology

and Biotechnology 16: 673–678.

Suvarna V C and Boby U V. 2005. Probiotics in human health: A current assessment. Current Science 88: 1744-1748.

Tagg J R, McGiven A R. 1971. Assay system for bacteriocins. Applied and

Environmental Microbiology 21: 934.

149

Tamang J P. 2010. Diversity of fermented foods, In: fermented foods and beverages of the world, edited by Tamang J P and Kailasapathy K, (CRC Press, Taylor and Francis group, New York), 41-84.

Tambekar D H and Bhutada S A. 2010. An evaluation of probiotic potential of Lactobacillus sp. from milk of domestic animals and commercial available probiotic preparations in prevention of enteric bacterial infections. Recent

Research in Science and Technology 2(10): 82-88.

Tambekar D H, Bhutada S A, Choudhary S D and Khond M D. 2009. Assessment of potential probiotic bacteria isolated from milk of domestic animals. Journal

of Applied Biosciences 15: 815-819.

Tannock G W, Crichton C, Welling G W, Koopman J P and Midtvedt T. 2005. Ecological behaviour of Lactobacillus reuteri is affected by mutant of the luxS gene. Applied Environmental Microbiology 71: 8419-8425.

Tomioka H, Tomioka K, Sato K and Saito H. 1992.The protective activity of immunostimulants against Listeria monocytogenes infection in mice. Journal

of Medical Microbiology 36: 112–116.

Tuomola E, Crittenden R, Playne M, Solauri E I and Salminen S. 2001. Quality assurance criteria for probiotic bacteria. American Journal Clinical Nutrition

73(Suppl): 393S-398S.

Tynkkynen S, Singh K V, Varmanen P. 1998. Vancomycin resistance factor of Lactobacillus rhamnosusGG in relation to enterococcal vancomycin resistance (van) genes. International Journal of Food Microbiology 41: 195-20.

Valkova E, Rada V, Smehilova M and Killer J. 2008. Auto-Aggregation and Co-Aggregation ability in bifidobacteria and clostridia. Folia Microbiology

53(3): 263-269.

Vanderhoof J A. 2000. Probiotics and intestinal inflammatory disorders in infants and children. Journal of Paediatrics and Gastroenterology Nutrition 30: S34–S38

Vaughan E E, Daly C and Fitzgerald G F. 1992. Identification, characterization of helviticin V-1829, a bacteriocin produced by Lactobacillus helveticus1829. Journal of Applied Bacteriology 73: 299-308.

Ventura M, Jankovic I, Walker D C, Pridmore R D, and Zink R. 2002. Identification and characterization of novel surface proteins in Lactobacillus

johnsonii and Lacobacillus gasseri. Applied Environmental Microbiology 68: 6172-6181.

Vignolo G M, Suriani F, Holgado A, Pescade R and Oliver G. 1993. Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages. Journal of Applied Bacteriology 75: 344-349.

Wadher K J,Mahore J G and Umekar M J. 2010. Probiotics: living medicines in health maintenance and disease prevention. International Journal of Pharma

and Bio Sciences 1(3): 1-9.

150

Wadstrom T, Andersson K, Sydow M, Axelsson L, Lindgren S, and Gullmar B. 1987. Surface properties of lactobacilli isolated from the small intestine of pigs. Journal of Bacteriology 62: 513-520.

Walker W A. 2000. Role of nutrients and bacterial colonization in the development of intestinal host defense. Journal of Paediatrics and

Gastroenterology Nutrition 30: S2–S7.

Wang K Y, Li S N, Liu C S, Perng D S, Su Y C and Wu D C. 2004. Effects of ingesting Lactobacillus –and Bifidobacterium -containing yogurt in subjects with colonized Helicobacter pylori. American Journal of Clinical Nutrition

80: 737.

Woese C R. 1987. Bacterial evolution.Microbiolology Review 51: 221-271.

Wollowski I, Rechkemmer G and Pool-Zobel B L. 2001.Protective role of probiotics and prebiotics in colon cancer. American Journal of Clinical

Nutrition 73(suppl): 451S-455S.

Yavuzdurm H. 2007. Isolation, characterization, determination of probiotic properties of lactic acid bacteria from human milk. Izmir Institute of Technology, Msc. Thesis pp. 80

Yildirim Z and Johnson M G. 1998. Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R. isolated from radish. Letters in Applied Microbiology 26(4): 297-304.

Yuksekdag Z N and Aslim B. 2010. Assessment of potential probiotic and starter properties of Pediococcus spp. isolated from Turkish-Type fermented sausages (Sucuk). Journal of Microbiology and Biotechnology 20(1): 161-168.

Zhou J S, Pillidge C J, Gopal P K, Gill H S. 2005. Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. International Journal of Food Microbiology 98: 211-217.

151

Department of Basic Sciences

Dr. Y.S. Parmar University of Horticulture & Forestry, Nauni, Solan–173 230, H.P.

Title of thesis : “Isolation of lactic acid bacteria and to

study their potential as

probiotics” Name of student : Shweta Handa Admission No. : F-2010-29-M Name of Major Advisor : Dr. (Mrs.) Nivedita Sharma

Major field : Microbiology Minor field(s) : i) Biochemistry Degree awarded : M.Sc. Year of award of degree : 2012 No. of pages in thesis : 151 + III No. of words in abstract : 380

ABSTRACT

The present investigation was carried out to isolate lactic acid bacteria from different food sources including indigenous fermented foods, their screening, characterization on biochemical as well as molecular level and further more to explore their probiotic potential. Total 22 lactic acid bacterial isolates were isolated from different food sources. All isolates were found to be gram positive, catalase negative and were preliminary screened on the basis of antagonism, bile salt tolerance and acidity tolerance. Among all, 6 isolates viz. F3, F8, F11, F14, F18 and F22 were finally screened and were identified as Lactobacillus

fermentum, Lactobacillus sp., Lactobacillus crustorum, Lactobacillus acidophilus, Lactobacillus delbreuckii

subsp. bulgaricus and Lactobacillus plantarum, respectively by 16S rRNA gene technique. These screened LAB’s were further evaluated for their probiotic potential viz., autoaggregation capacity, hydrophobicity, acidity tolerance, antibiotic susceptibility and cumulative probiotic potential. All the six LAB isolates showed good autoaggregation capacity i.e., greater that 40% after 5h and showed moderate to strong hydrophobicity towards xylene/toluene with hydrophobicity greater than 20%. These six screened LAB’s were found to be highly acidity tolerant strains as they showed survival of 26.4 to 90.4% at pH 1.0 for 3h. All the six isolates were found to be highly sensitive towards all the antibiotics tested, proving them safe for use. These screened LAB’s showed broad and strong inhibitory spectrum against both gram-positive and gram-negative pathogenic microorganisms and their growth phase depicted maximum production of inhibitory metabolites in between the late exponential phase and in the beginning of the stationary phase. Screened LAB’s supernatant was found to be sensitive to both proteolytic and amylolytic enzymes as decrease in the zone of inhibition was found. Thus, proving that the supernatant must contain proteins or carbohydrate moieties which help in the inhibitory action of these screened LAB’s. The entire screened LAB isolates were highly qualified the cumulative probiotic score and are being recommended for their use as commercial probiotics. Hence, this study affirms the use of L. fermentum F3, Lactobacillus sp. F8, L.

crustorum F11, L. acidophilus F14, L. delbreuckii subsp. bulgaricus F18 and L. plantarum F22 in the development of new pharmaceutical and functional foods to impart to betterment of the health of public as these six strains isolated in the present study have been proved safe as well as highly effective probiotics. Signature of Major Advisor Signature of student

Countersigned

Professor and Head,

Department of Basic Science,

Dr. Y.S. Parmar University of Horticulture and Forestry,

Nauni, Solan – 173 230 (HP)

Appendix -I

Anova for Table 4

1)

Source DF SS MS F

T (A) 21 47766.6 2274.1 29.93.7

A x B 44 3.440 0.07

Total 65 47770.1

2)

Source DF SS MS F

T (A) 21 8015.7 381.7 3030.9

A x B 44 5.51 0.126

Total 65 8021.2

3)

Source DF SS MS F

T (A) 21 4799.8 228.5 3222.1

A x B 44 3.1 0.07

Total 65 4802.9

Anova for Table 5

1)

Source DF SS MS F

T (A) 21 1734.8 82.6 1363.9

A x B 44 2.7 0.06

Total 65 1737.4

2)

Source DF SS MS F

T (A) 21 4776.5 227.5 4548.6

A x B 44 2.2 0.05

Total 65 4778.7

Anova for Table 16(a)

Source DF SS MS F

T (A) 3 92.8 30.9 15762.9 I (B) 3 27.4 9.1 4648.4 A x B 9 62.7 6.9 3549.52 A x B x C 32 0.06 0.001 Total 47 183.2

Anova for Table 16(b)

Source DF SS MS F


Anova for Table 16(c)

Source DF SS MS F


Anova for Table 16(d)

Source DF SS MS F

T (A) 3 39.01 13.0 664739.41 I (B) 3 28.5 9.5 484951.6 A x B 9 42.01 4.7 238659.4 A x B x C 32 0.001 0.00001 Total 47 109.5 Anova for Table 16(e)

Source DF SS MS F


Anova for Table 16(f)

Source DF SS MS F

T (A) 3 1.97 6.6 31.9 I (B) 3 1.51 5.03 24.4 A x B 9 1.95 2.2 10.54 A x B x C 32 0.7 0.02 Total 47 6.1 Appendix -II

Cumulative probiotic potential (Score Card)

Probiotic characters Indication Score

Acidity Tolerance Resistant = 1 Sensitive = 0

Bile salt tolerance Resistant = 1 Sensitive = 0

Autoaggregation capacity Positive = 1 Negative = 0

Hydrophobic Capacity (Xylene/Toluene) >40% Strong = 1

(Xylene/Toluene) >20% Moderate = 0.5

(Xylene/Toluene) <20% Low = 0

Antagonistic activity 5-10 = 0.25 10-20 = 0.50 15-20 = 0.75

>20 = 1

Antibiotic sensitivity Antibiotic sensitive =1 Antibiotic resistant = 0

Total

Probiotic Potential (%)

Appendix –III

Biochemical analysis of fermented food samples of LAB having high

probiotic potential

Food Item pH Moisture

(%) TSS

(oB)

Protein

(mg/ml) Carbohydrates

(mg/ml) Reducing

sugars

(mg/ml)

Dough 4.17 53.0 10.0 2.77 1.34 3.64

jalebi batter

3.59 72.0 8.0 2.41 1.39 2.99

Human Milk

5.7 95.0 3.0 2.80 1.25 2.45

Lassi 3.50 79.3 9.0 1.85 1.03 2.50

Homemade Butter

4.50 80.5 9.5 1.65 1.06 2.35

Chhang 3.25 75.0 12.0 1.83 1.30 1.09

CURRICULUM VITAE

Name : Ms Shweta Handa

Father’s Name : Shri. Ramesh Chand Handa

Date of Birth : 03.06.1989

Sex : Female

Marital Status : Unmarried

Nationality : Indian

Educational Qualifications :

Certificate/ Degree

Class/Grade Board/ University Year

Matriculation First CBSE, Board 2005

10+2 First CBSE, Board 2007

B.Sc. First PTU, Jalandhar 2010

Whether sponsored by some state : No

Scholarship/Stipend/ Fellowship,

any other financial assistance

received during the study period

:

No

(Shweta Handa)

pdfs.semanticscholar.org · 80 dr nivedita sharma professor department of basic sciences...

Documents