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This book is an outcome of 20 years of experience in probiotics and I thank my research students, collaborators, research grant agencies and mentors, who helped me in enriching knowledge with constant support. All my 12 research students and some post graduate students helped in reviewing the literature and created a strong database. My heartfelt thanks to all the Research Scholars of our Probiotic Laboratory particularly Dr Asha, Dr Shobha K Jayanna, Dr Somaraja P K, Dr Sunil Kumara S, Dr Swamy C T and Dr Vasudha M. My special thanks to Dr Rashmi B S, Research scholar, for strong commitment during her study, who contributed significantly in shaping this book. Many thanks to Dr Prashantkumar S Chakra, MS R Soundarya, Mrs Shwetha G S and Ms Pruthvi S M for contributing apt information at right time and their commitment in designing this book. Many of my MSc project students provided timely information in rewinding/improvising the Book. I thank all the authors, reviewers and scientists whose works have been embodied in this book. I would like to thank UGC, DST-DBT, DAE-BRNS (Govt of India) and VGST (RFTT) and VGST-KSTePs (CISEE) for providing funding resources for the R & D works. Most of the information cited in the book are the derivatives of our published works in diverse peer reviewed journals such as Current Science, Experimental and Therapeutic Medicine, Current Microbiology, Journal of Basic Microbiology, Journal of applied Microbiology, Probiotics and Antimicrobial Proteins, Toxicon, J Mental Health Prevention, GenomeA, Biomedical Reports, Microbial Cell Factories, The Microbe, J Functional Nutrition and others. I wish to acknowledge the blessings of my mentors Dr K M Shankar, Professor of Aquaculture, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, for instilling scientific spirit in me and Dr Indrani Karnunasagar and Dr Iddya Karunasagar, NUCCSER- KSHEMA, Deralakatte, Mangalore for always being caring and source of inspiration. My heartfelt thanks to my better half and best friend Dr Devaraja T N, Head and Chief Scientist, ICAR-TKVK, Davangere, for his unconditional motivation, support and providing scientific inputs time and again. My heartfelt thanks to our lovely daughters, Ms Abhijna Tanvi D and Ms Olavu Siri D, for their everlasting love and cooperation during the preparation of this book. My special thanks to my mother-in law and all my family members for the constant support in this enduring and memorable journey of scientific documentation.

The term probiotic means “for life” and it is currently used to refer bacteria that are associated with beneficial effects for humans and animals. The credit for first observation made on the positive role of some selected bacteria was attributed to Eli Metchnikoff, the Russian born Nobel Prize recipient, worked at the Pasteur Institute in the beginning of the last century. Metchnikoff suggested that - “the dependence of the intestinal microbes on the food makes it possible to adopt measures to modify the microflora in our bodies and to replace the harmful microbes by useful microbes”. Interim Henry Tissier, a French paediatrician, observed that children with diarrhea in their stools had a low number of bacteria which are characterized by a peculiar Y shaped morphology. On the contrary, these “bifid” bacteria were abundant in healthy children. Based on these observations, Tissier suggested that “bifid” bacteria could be administered to patients with diarrhoea to help restore a healthy gut microflora. These two scientists were the first to give scientific intimation for the use of probiotics when even the term probiotics was not coined. Later in 1965, the term probiotics is coined by Lilly and Stillwell to describe substances produced by one microorganism, that stimulate the growth of another. Subsequently, Parker defined probiotics as organisms and substances that contribute to intestinal balance. Further, Fuller (1989) improved the Parker’s definition of probiotics as “live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance”. Havenaar and Huis in’t Veld (1992) extended the probiotic definition with the description: “A viable mono or mixed culture of microorganisms which, applied to animal or man, beneficially affects the host by improving the properties of the indigenous microflora”. At the end of the millennium, Salminen and his coworkers (1999) proposed the following definition: “probiotics are microbial cell preparations or components of microbial cells that have a beneficial effect on the health & well- being of host”. Whereas FAO/WHO (2001) defined probiotics as “living bacteria that, when administered in adequate amounts, confer a health benefit on the host”. This definition for probiotics is widely accepted.

Introduction Probiotics, prebiotics and synbiotics are interrelated terms. Probiotic is discussed in detail in previous chapter. In this chapter, prebiotics and its effect on gut along with probiotics are emphasized. Prebiotics can be defined as non digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial to health. They were f irst identified and named by Marcel Roberfroid in 1995. He defined prebiotic as a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health. Further, FAO defined prebiotic as “a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota.”Whereas the term synbiotics refers to a product that contains both probiotics and prebiotics in the form of synergism. A synbiotic has been defined as “a mixture of prebiotics and probiotics that beneficially affect the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract, by selectively stimulating the growth and activating the metabolism of one or a limited number of health promoting bacteria, and thus improving host welfare”. As described above, the combined use of prebiotics and probiotics is often described as synbiotic, but the United Nations Food & Agriculture Organization (FAO) recommends that the term “synbiotic” be used only if the net health benefit is synergistic.

Introduction From earlier chapters, it is evident that Lactobacilli and Bifidobacteria are the two important genera that have potent applications in food microbiology and human nutrition due to their role in food and feed production, preservation and also due to probiotic potentialities. Most of the bacterial species are still unknown indicating that the identification and classification of bacteria is a hard task since the beginning of microbiology and consequently our knowledge about bacterial ecology is very low. Mean while the expectations about novel bacterial products are high. Therefore, bacterial identification has become a growing field of interest in microbiology. Since, Lactobacilli and Bifidobacteria genera include a large number of species and strains exhibiting important properties in an applied context, chiefly in the area of probiotics as food and therapeutics, an updated list of species belonging to these two genera, their phylogenetic relationships and other relevant taxonomic information are of great significance. The classification and identification of a probiotic isolate at strain level point out its typical habitat and origin. Moreover, the species, or genus name, may also indicates the strain’s safety and technical applicability for its use in probiotic products. Taxonomy helps to place a newly identified organism in a relevant group and enables the scientific communication. Further it facilitates the construction of databases that can be used for rapid identification of organisms at laboratory level easily. Thus, taxonomy of Lactobacilli and Bifidobacteria helps to study these organisms scientifically and allows characterizing and arranging them in an orderly manner which promote the scientific community to explore the benefits from those organisms for the betterment of mankind.

Introduction Preceding chapters discuss the essentials of probiotics, their role in maintaining gut health in combination with prebiotics and significance of systematic classification of probiotic isolates. Further, to draw an idea how probiotics function in the gut, it is essential to know the interaction of probiotics with other microorganisms in the gastrointestinal milieu - the site of their probiotic action. Knowledge in the ecology of probiotic microorganisms in host environment such as gut gives an ideal picture of their functioning manner and the factors (both host and microbial) responsible for it. Study of microbial ecology in the gastrointestinal tract is very difficult since gut harbors highly diversified microflora that varies not only at species level but also at individual level. The diversity of gut microflora in two healthy individuals of same species may exhibit variations in their composition and it may be influenced by geodemographic factors also. In this regard, the present chapter deals with the study of ecology of Lactobacillus- most commonly used probiotic bacteria in the gastrointestinal tract which in turn helps in framing selection criteria for screening of potential probiotics. Recent studies on interaction between human and gut microflora discussed that co-evolution between microorganisms and their host have contributed for the development of a very complex microbial diversity in the gut. Highly diversified microflora in the human gut is a consequence of natural selection at two levels, top-down and bottom-up selection pressure. Hierarchy theory expounds that higher levels compel lower levels for possible organizational solutions i. e. organisms at the higher levels govern the structure or population dynamics of an ecosystem while on contrary lower levels control the structure of ecology when bottom-up level is the selection pressure. In case of interaction between human and gut microflora, being host human is at higher level in the hierarchy for selection and as mentioned above, top down selection pressure is driven by higher levels; host selects the beneficial microorganisms for functional redundancy leading to the development of a microbial community with divergent microbial divisions. Chances for lateral gene transfer also thought to influence the selection process and there by structure of the microbial community. On the other hand, competition among the members of host selected microbiota exert bottom up selection pressure resulting in the development of highly specialized genomes with functionally prominent set of genes. Natural selection at host level obviously favors functionally constant microbial communities which benefits the host with functional redundancy encoded in their genomes. Mean while bottom-up cell-level selection pressure aspire to favor niche specialization to avoid competition between the members of microbial community. In order to be a part of specialized niche, to become more potent microorganisms adapt a particular set of abiotic and biotic factors within the habitat by acquiring new sets of genes essential for their survival and thus escape the competition from decrepit microbial species.

Introduction Cell surface components such as adhesins, polysaccharides, and proteins play major roles in the adherence of probiotic bacteria to the intestinal epithelium, interactions that might lead to pathogen exclusion and immunomodulation of host cells. Probiotic bacteria have the ability to produce exocellular polymers called Exopolysachcharides (EPSs). These exocellular polymers present in the surface of many lactic acid bacteria, including Lactobacilli.The adhesive properties of lactobacilli are directly linked to their surface properties which are influenced by the structure and composition of their cell wall. The cell envelope of lactobacilli, bacteria, is composed of the bilipidic plasma membrane with embedded proteins encompassed by the cell wall. The bacterial cell wall consists of a thick multilayered sacculus made of peptidoglycan (PG) with teichoic acids (wall teichoic acids (WTA) and/or lipoteichoic acids (LTA)), exopolysaccharides (EPS), proteinaceous filaments called pili, and proteins that are anchored to the cell wall through different mechanisms (Figure 1). Some species of lactobacilli possess an additional paracrystalline layer of proteins surrounding the PG layer, referred to as the S layer. These macromolecules together may play crucial roles in determining species and strain-specific characteristics of lactobacilli by influencing host-microbe interactions and microbial adaptations to the changing host environment. Several studies implicate that cell surface components, either individually or collectively, involve in host-microbe interactions. Lactobacilli beneficially affect the host in many ways by producing abundant variety of EPSs through the activity of extracellular glycosyl transferases. Approximately 30 species of lactobacilli are described as EPS producers. Among them, the best known are L. casei, L. acidophilus, L.brevis, L. curvatus, L. delbrueckii bulgaricus, L. helveticus, L. rhamnosus, L. plantarum, L. johnsonii, etc.

Introduction It became clear from the discussions of previous chapters that probiotics perform metabolic functions such as fermenting indigestible dietary residues and endogenous mucus, production of vitamins, and absorption of minerals. They also play key role in intestinal epithelial cell proliferation and differentiation, and the homeostasis of the immune system. Further, probiotics are also known to involve in modifying gut pH, antagonizing pathogens through the production of antimicrobial compounds, competing with pathogens for recept or sites as well as for available nutrients and growth factors, stimulating immunomodulatory cells of the host, and produce a wide range of enzymes such as lactase, amylase, lipase, protease, phytase and so on. The molecular mechanisms of probiotics are largely unknown and opens avenue for research in probiotics. Moreover, current research in probiotics and health consciousness in the public lead to an increased interest towards probiotics from the public, researchers, governmental organizations (such as the WHO/FAO) and medicine and food companies. In this chapter, application of probiotics in treating various diseases and disorders are discussed.

Diversity of gut microbial community The human microbiota (the collection of microbes that live on and inside us) consists of about 100 trillion microbial cells that outnumber our ‘‘human’’ cells 10 to 1, and that provide a wide range of metabolic functions that we lack. It has been estimated that they encode 100-fold more unique genes than our own genome. Furthermore, the gut microbes contribute to energy harvest from food, and changes of gut microbiome may be associated with bowel diseases or obesity. To understand and exploit the impact of the gut microbes on human health and well-being it is necessary to decipher the content, diversity and functioning of the microbial gut community. If we consider ourselves as superior organisms encompassing these microbial symbionts, by far the majority of genes in the system are microbial. In this sense, completing the human genome also requires the characterization of the microbiome (the collection of genes in the microbiota). Methods of counting viable and non-viable bacteria in gut Before the advent of genome sequencing and genome-wide analysis method, our knowledge of molecular characteristics of diverse gut microbial population was extremely limited. To obtain an estimate of cultivability, microbial ecologists generally compare microscopic counts with total viable counts. The total viable count made on a nonselective agar based medium estimates the number of colony forming units (cfu) per gram of sample. The finding that total viable counts are typically lower than total microscopic counts was thought to be due to the number of dead cells. Indeed, dead bacteria in feces may constitute up to one third of the total bacterial community. However, recent nucleic acid based studies indicated that a majority of bacteria in a variety of ecosystems are different from those described in culture.

Introduction Let food be thy medicine and medicine be thy food- the age-old quote by Hippocrates, is certainly the tenet of today. Microbial cultures have been used for millenniums in food and alcoholic fermentations and in the past century have undergone scientific scrutiny for their ability to prevent and cure a variety of diseases. Later it became popular in improving human and farm animal nutritive values and recently in aquaculture also. The growing interest in the understanding the role of food in human health has moved from its primary role as a source of energy to the subtle action of biologically active food components on human health. Henceforth functional foods or nutraceuticals are of great demand in present time. The growing scientific evidence suggests that the food supplements containing beneficial bacteria can provide an array of benefits to the host. Now a day’s probiotic fermented foods are in great demand due to the awareness of beneficial aspects of probiotics among people. Several industries focused on the improvement of conventional fermented foods scientifically in order to fetch good market for their products. Particularly, animal feed companies and researchers have been looking for alternative products and strategies that can help to maintain animal gut health in order to prevent or reduce the prevalence of pathogens in the food chain. After understanding the mechanism and beneficial aspects of probiotics in detail at molecular level in earlier chapters, now it is relevant to discuss the applied part of probiotics. In this chapter, commercial importance of probiotics and probiotics derived products at industrial level are emphasized.

Introduction Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for production of energy and lactic acid. Lactic acid bacteria includes different major genera: Lactococcus, Enterococcus, Lactosphaera, euconostoc, Melissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Other genera are Aerococcus, Microbacterium, Propionibacterium and Bifidobacterium. Through carbohydrate fermentation along with lactic acid, various organic acids are produced due to which there is a fall in pH level and creates unfavorable environment or inhibits growth of some undesired microorganisms. The low pH makes organic acids lipo -soluble, allowing them to invade into cytoplasm of pathogens by rupturing cell membrane. LAB are generally regarded as safe (GRAS), and have a significant role in the preservation of foods and fermented products. Under controlled conditions they can be used as natural competitive microbiota or as specific starter cultures. Antagonistic substance like bacteriocins are produced from lactic acid bacteria that inhibits the activity and growth of other bacteria and these bacteriocins are active against various pathogens in minimum concentration. These are antimicrobial peptides modified by post -translational mechanisms and exported to the extracellular medium. Till date about 239 bacteriocin producing LAB genome and 789 putative bacteriocin producing genes cluster are stored in the public databases. Many gram positive and gram negative bacteria in the environment produce bacteriocins.

Introduction The gut microbiota is the complex and dynamic community of microorganisms that includes various bacteria, fungi and other microorganisms, proliferate and lead a symbiotic/mutual relationship with the host system. These microorganisms protect the host against pathogens by unique mechanisms, helps to maintain shape and strengthen the intestinal epithelium and boost immune response. Diverse bacterial metabolites can immigrate the host bloodstream and other parts of the body via various channels in humans and animals, such as gut-microbiota-brain, gut microbiota-skin, gut-vagina, gut liver, gut-bones etc. However, many aspects of modern lifestyle would alter the gut microbiota and lead to numerous diseases including inflammatory bowel disease, diabetes mellitus, obesity, and metabolic syndrome. Probiotics and other beneficial microbes exert many beneficial effects on their hosts by modulating the gut microbiota. While the term probiotic is reserved only for well-characterised strains with clinically proven health benefits, other beneficial microorganisms include, organisms responsible for the fermentation of foods viz. yogurt, kefir, kombucha, kimchi and many others that have been shown to have distinct health benefits. Interestingly, major group of these beneficial organisms belong to lactic acid bacteria.

A Absorption: 4, 20, 23, 24, 25, 55, 73, 74, 75, 77, 84, 99, 107 Acidic: 2, 10, 20, 52, 55, 75, 76, 78, 79 Acidophilus: 14, 15, 16, 35, 36, 38, 39, 40, 41, 57, 61, 65, 70, 79, 86, 100, 107, 108, 109, 115, 121, 134 Acids: 2, 4, 5, 6, 17, 23, 25, 27, 29, 30, 35, 36, 38, 39, 56, 57, 60, 62, 65, 75, 76, 84, 92, 95, 102, 106, 108, 113, 114, 119, 123, 132, 133, 134 Action: 2, 3, 6, 9, 16, 17, 18, 49, 50, 56, 57, 61, 62, 74, 77, 81, 83, 99, 102, 104, 105, 107, 108, 114, 117, 118, 124, 125, 126, 129, 132 Activation: 3, 60, 61, 78, 81, 103, 133, 135 Addition: 3, 4, 5, 6, 8, 9, 11, 12, 15, 16, 23, 24, 25, 39, 40, 42, 52, 57, 58, 59, 60, 66, 67, 69, 74, 75, 76, 79, 82, 96, 100, 102, 103, 104, 108, 109, 110, 114, 127, 133, 135