ISOLATION OF LACTIC ACID BACTERIA AND TO STUDY THEIR POTENTIAL AS PROBIOTICS Thesis Thesis Thesis Thesis by SHWETA HANDA Submitted in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE MASTER OF SCIENCE MASTER OF SCIENCE MASTER OF SCIENCE MICROBIOLOGY COLLEGE OF FORESTRY Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan - 173 230 (H.P.), INDIA 2012
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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
7. Genotyping of finally screened lactic acid bacterial isolate F8
97
8. Genotyping of finally screened lactic acid bacterial isolate F11
97
9. Genotyping of finally screened lactic acid bacterial isolate F14
98
86
Table Title Page(s)
10. Genotyping of finally screened lactic acid bacterial isolate F18
99
11. Genotyping of finally screened lactic acid bacterial isolate F22
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
4. Morphology of isolate F8 96-97
5. Genomic DNA and PCR product of isolate F8 96-97
6. Morphology of isolate F11 96-97
7. Genomic DNA and PCR product of isolate F11 96-97
8. Morphology of isolate F14 98-99
9. Genomic DNA and PCR product of isolate F14 98-99
10. Morphology of isolate F18 98-99
11. Genomic DNA and PCR product of isolate F18 98-99
12. Morphology of isolate F22 100-101
13. Genomic DNA and PCR product of isolate F22 100-101
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
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
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)
Marteau et al. (2001)
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
Marteau et al. (2001)
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
Marteau et al. (2001)
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)
Marteau et al. (2001)
Marteau et al. (2001)
Marteau et al. (2001)
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
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
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
*Autoaggregation in terms of sedimentation rate ♦OD600 = Mean of the results from three separate experiments **Autoaggregation % = 1-(At/A0) ×100 b) Lactobacillus sp. F8
♦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)
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
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
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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
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
T (A) 3 39.9 13.32 798250.4 I (B) 3 18.4 6.1 365962.3 A x B 9 44.9 4.9 298655.1 A x B x C 32 0.001 0.00002 Total 47 103.2
Anova for Table 16(c)
Source DF SS MS F
T (A) 3 28.9 9.6 235064.8 I (B) 3 26.5 8.8 215223.7 A x B 9 43.3 4.8 117340.9 A x B x C 32 0.001 0.00004 Total 47 98.8
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
T (A) 3 31.6 10.5 612631.1 I (B) 3 23.2 7.7 449976.7 A x B 9 46.7 5.2 301899.3 A x B x C 32 0.001 0.00002 Total 47 101.5
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