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Appraisal of Probiotics and Prebiotics in Gastrointestinal Infections

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Appraisal of Probiotics and Prebiotics inGastrointestinal Infections
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  • Article ID: WMC003796 ISSN 2046-1690

    Appraisal of Probiotics and Prebiotics inGastrointestinal InfectionsCorresponding Author:Dr. Asit R Ghosh,Senior Professor & Assistant Director, Centre for Infectious Diseases & Control, School of Biosciences andTechnology, VIT University, , Centre for Infectious Diseases & Control, School of Biosciences and Technology,VIT University, Vellore, INDIA, 632014 - India

    Submitting Author:Dr. Asit R Ghosh,Senior Professor & Assistant Director, Centre for Infectious Diseases & Control, School of Biosciences andTechnology, VIT University,, Centre for Infectious Diseases & Control, School of Biosciences and Technology,VIT University, Vellore, INDIA, 632014 - India

    Article ID: WMC003796Article Type: Review articlesSubmitted on:27-Oct-2012, 12:53:50 PM GMT Published on: 29-Oct-2012, 07:51:37 PM GMTArticle URL: http://www.webmedcentral.com/article_view/3796Subject Categories:GASTROENTEROLOGYKeywords:Probiotics, Prebiotics, Synbiotics, Gastrointestinal infection, Curd, Diarrhea

    How to cite the article:Ghosh AR. Appraisal of Probiotics and Prebiotics in Gastrointestinal Infections .WebmedCentral GASTROENTEROLOGY 2012;3(10):WMC003796Copyright: This is an open-access article distributed under the terms of the Creative Commons AttributionLicense(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

    Source(s) of Funding:The project was carried out by the intramural funding which was supported for the promotion of science in theVIT University, Vellore, India

    Competing Interests:There is no competing intersts

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    Appraisal of Probiotics and Prebiotics inGastrointestinal InfectionsAuthor(s): Ghosh AR

    Abstract

    A probiotic is a viable microbial dietary supplementthat beneficially affects the host through its effects inthe gastrointestinal tract (GIT). Probiotics are widelyused to prepare fermented dairy and non-dairyproducts. Several health-related effects associatedwith the intake of probiotics, including alleviation oflactose intolerance, immune enhancement, control ofdifferent kinds of diarrhea and colon cancer etc. havebeen reported in human studies. Prebiotics arenondigestible food ingredients that benefit the host byselectively stimulating the growth or activity of one or alimited number of microorganisms in the colon. Workwith prebiotics has been limited, only severaloligosaccharides and inulin-type fructans havegenerated some research-based effective data.However, the common understanding about the utilityof prebiotics is that they benefit our intestinalecosystem, improve immunity and overall health. Atpresent, claims about reduction of disease risk areonly tentative and further research is needed. Thecombination of probiotics and prebiotics in a synbiotichas not also been studied well. This combination mightimprove the survival of the bacteria crossing the upperpart of GIT, thereby enhancing their effect in the largebowel. In addition, their effects might be additive oreven synergistic. Several research groups are evolvedin India to implement the age-old system into modernpractice. Fundamental research with clinical trials isthere with the growth of Probiotic market in India.Present trends of research hopefully will ensure theimplementation of its potentials towards the benefit oflife extensively.

    Review

    General Concept:

    About a century ago, India won her first Noble prize bythe work of Viswakavi Rabindranath Tagore in theyear 1913. In his several novels, he mentioned aboutthe curd or dahi or specially misti dahi and sketched acharacter of a curd peddler, in: Dakghar (Post office).Almost during the same period, Elie Metchnikoffreceived Noble prize for his discovery of phagocytosis

    in 1908, and devoted last decade of his lifeinvestigating means of increasing human longivity andadvocating the consumption of lactic acid producingbacteria (Shruthy et al., 2011). Again our VedicSanskriti had recognized the role of diet in health andnutrition and now after several thousand of years, weare proving and improving the linkages among health,nutrition and diet with science based investigation. Theusage of panchamrit in Hindu rituals is to beconsidered as the oldest functional food or synbioticfood in the history of mankind. The utility of curd hasbeen described in Rig-Veda also. The Dadhi made outof cows milk has been described as a likeable foodnot only for the humans but of gods as well. Thefollowing mantra from Rig-Veda (10-179-3) aptlydescribes the nutritional qualities of Dadhi or curd(made from cows milk):

    Shratam manya oodh nishrat magnowsushraatam manye tadyate naviyah madhyen dinasaya sevanasya dadhana pivendervajrinpuru krijjushanah.

    However, lack of written documents and scientificevidence kept western world unaware and unnoticeduntil the publication of book, Prolongation of life in1907 by Elie Metchnikoff.

    Dahi or Dadhi or Doi or Tahri or Perugu or Mosaru orThayir are the acronyms for curd or yogurt in India.The Indian economy had been agriculture-based sincethe Vedic civilization. Cow and land were the sourcesof major economy. Almost all the oldest documentsand mythological inscriptions were cow-centric. Cowmilk and milk products are thus obviously andintimately involved with Indian society. Right from theday of Lord Krishna to the modern age, milk, andthousands of milk-products like curd, cream, butter,lassi, paneer, sandesh, rasogolla, ksheer; sreekhandetc. are in common use regularly. Specially, curd is avery essential and regular food item in India withparticular reference to south India. The curd isprepared in India by natural contamination andfermentation. Besides milk, other indigenousfermented foods have been prepared and consumedfor thousands of years and are strongly linked toculture and tradition. In the Indian subcontinent,

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    making use of fermented food and beverages usinglocal food crops and other biological resources arevery common (Roy et al., 2004). But the nature ofproducts and the base material vary from region toregion. One can contemplate that people are usingmicroorganisms, unknowingly for varied purposes.Further, such kind of undocumented knowledge oftraditional systems is in the danger of extinction due tovarious causes. The contaminants may be yeastor/and bacteria. The fermented food and beveragescontain microorganisms those may or may not bebeneficial to health, but are generally regarded as safe(GRAS). Again, the beneficial microbes are calledprobiotics with special characteristics. The metabolicingredients which support the growth of probiotics aretermed as prebiotics. Together probiotics andprebiotics constitute the functional food, calledsynbiotics (Bagad et al., 2012).The scientific understanding of probiotics and theirpotential for preventing and treating health conditionsis moving ahead. The effectiveness of probiotics isstrain-specific, and each strain may contribute to hosthealth through different mechanisms. Pathogeninhibition by probiotics may provide significant humanhealth benefits. Many reports indicate that probioticscan affect innate immunity and also protect againstpathogens by decreasing pathogenic adherence or byproduction of acids or bacteriocins (Kumari et al.,2011). The most usual application of probioticmicroorganisms is for the prevention or therapy ofgastro intestinal disorders such as irritable bowelsyndrome (Cui and Hu, 2012; Murakami et al., 2012),colitis (Rodrigues et al., 2012), colon cancer (Denipoteet al., 2010; Wollowski et al., 2001) and mostimportantly diarrhoea (Boyle et al., 2006; Cremoniniand Videlock, 2012). Several clinical trials haveevaluated their effective use in the prevention andtreatment of infectious gastro-intestinal (GI) diseasesor disturbances happened in the normal micro-flora.The ideal probiotic is one that remains viable insufficient number by adhering to the intestinalepithelium to confer a significant health benefit(Kumari et al., 2011). In the last three decades,scientists have gathered plenty of evidences of healthand nutritional benefits of probiotics. As a result manyglobal companies including India participated in themanufacturing of several different nutraceuticals andpharmaceuticals to offer health benefits to society.2. Introduction to probiotics, prebiotics, synbioticsLet food be thy medicine and medicine thy food, theage old quote by Hippocrates is certainly the tenet oftoday. The growing interest in understanding the roleof food in human health has moved from the primary

    role of food as a source of energy to the more subtleaction of biologically active food components onhuman health. Probiotics has been defined by anexpert committee as living organisms, which uponingestion exerts health benefits beyond inherentgeneral nutrition. FAO has defined it as livemicroorganisms which when administered in adequateamounts confer a health benefit on the host.Probiotics commonly stem from the category of Lacticacid bacteria (LAB). LAB are Gram positive, non-sporeforming, catalase negative, acid tolerant and strictlyfermentative with lactic acid as the major end productd u r i n g s u g a r f e r m e n t a t i o n ( h o m o - o rhetero-fermentative). LAB with probiotic potentials areknown to exert positive influence on host health andphysiology. The physiological effects include theproduction of antibacterial substances, maintenance ofhealthy microflora, reduction of cholesterol level in theblood (Starovoitova et al., 2012), stimulation ofimmune functions and the removal of carcinogens(Boyle et al., 2006; Rupa and Mine, 2012). At presentprobiotic bacteria are widely used in human andanimal nutrition because they beneficially influence thebalance of the intestinal flora of the host (Kumari et al.,2011). Implementation of probiotics is intended toreduce the use of antibiotics, with a proven low risk ofinducing or being associated with the etiology ofdisease (Szajewska, et al., 2006).Mahatma Gandhi advocated the virtues of fermentedmilk and milk products in his book The Diet Reform.The therapeutic property of curd is due to the probioticbacteria present in them. Promising probioticmicroorganisms come under the genera Lactobacillusand Bifidobacterium. In general, LAB are food-gradeorganisms witnessing a long history of safe use andare categorised as GRAS for human consumption. Butnot all curd bacteria may have the probiotic potentialsand may not be as good as it is labeled with yogurt. Toaddress the fact that how far curds we consume areprobiotic, we performed a study for two years whichrevealed that 10 of 20 samples (50%) had lactic acidbacteria and only two of LAB positive curds (10%)had the probiotic strains of Lactobacillus spp. Isolatedstrains showed several characteristics of being a goodprobiotic. Strains were acid resistant, bile salt tolerant,antibacterial, and antagonistic by slow acidificationand antimicrobial production, and adherent (Shruthy etal., 2011).Dahi or curd is a regular food component of SouthIndia and has been documented well for its beneficialeffects. It is the semisolid product obtained frompasteurized or boiled cow/buffalo milk by souringnatural or otherwise by a harmless lactic acid or other

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    bacterial culture. Unlike yoghurt which involves theuse of specific symbiotic/mixed culture of Lactobacillusbulgaricus and Streptococcus thermophilus, curdcontains a mixture various strains of LAB or yeast orcombination of both. Thus the quality of dahi may varywith the type of starter culture used. Otherwise themode of preparation is almost similar to yoghurt. Poorquality milk, unhygienic practices associated with thepreparation and the use of wild type of starter culturegive rise to poor grade dahi having less shelf-life. Theymay not have the probiotic potentials and may not beas good as yoghurt.2.1. Probiotics

    Probiotics can be bacteria, moulds or yeast. But mostprobiotics are lactic acid bacteria consist of a numberof heterogenous bacterial genera within the phylumFirmicutes. The genera Carnobacter ium,Enterococcus, Lactobacil lus, Lactococcus,Lactosphaera, Leuconostoc, Melissococcus,Oenococcus , Pediococcus, Streptococcus,Tetragenococcus, Vagococcus and Weissella arerecognized as LAB. Depending on the organism,metabolic pathways differ when glucose is the maincarbon source: homofermentative bacteria such asLactococcus and Streptococcus yield two lactatesf r o m o n e g l u c o s e m o l e c u l e(Embden-Meyerhof-Parnas pathway), whereas theheterofermentative (ie. Leuconostoc and Weissella)transform a glucose molecule into lactate, ethanol andcarbon dioxide (pentose phosphate pathway)(Sonomoto and Yokota, 2011). In addition, LABproduces small organic compounds that give thearoma and flavor to the fermented product. Allprobiotic bacteria are LAB but all LAB are not probiotic.We observed also the same phenomenon in our study(Shruthy et al., 2011). LAB has been cited to be partof human and animal microbiota. LAB constitutes anintegral part of the healthy gastrointestinal (GI)microecology and is involved in the host metabolism.LAB and gut microbiota ferment various substrates likelactose, biogenic amines (Stadnik and Dolatowski,2012) and allergenic compounds and convert intoshort chain fatty acids (SCFA), organic acids, gasesand also synthesise enzymes, vitamins (Rossi et al.,2011), antioxidants (Gowri and Ghosh, 2011;Achuthan et al., 2012) and bacteriocins (Lasta et al.,2012). With these properties, intestinal LABconstitutes an important mechanism for themetabolism and detoxification of foreign substancesentering the body. Table 1 shows the use of severalmicroorganisms with probiotic potentials.Table 1

    Commonly used microorganisms as probiotics

    See Illustration 12.1.1. Characterisation of lactic acid bacteriaPhenotypic characterisation includes identification ofcolony morphology, gram staining, fermentationpatterns or a combination of these methods. With thedeveloping technology, molecular typing is gettingmore reliable to identify and differentiate bacterialstrains. Many of these techniques are based on thepolymerase cha in react ion (PCR) us ingoligonucleotide primers to amplify targeted DNAfragments, to different taxonomical levels, fromgenus-specific differentiation, to the species-specificlevel, to sub-species level and also further to thestrain-level. In a separate study, we isolated probioticLAB from fermented non-milk food and characterizedboth phenotypically and genotypically and identifiedusing 16S rDNA where we reported a strain,Pediococcus sp. (GS4), Pom4 (Gowri and Ghosh,2010a). In addition, PCR based Pulse-Field GelElectrophoresis (PFGE) (De Baets et al., 2009; Struset al., 2012) techniques, Denaturing gradient gelelectrophoresis (DGGE) (Dalmacio et al., 2011)andtemperature gradient gel electrophoresis (TGGE)(McCartney, 2002)analysis of faecal 16S rDNA (Gowriand Ghosh, 2010; Kumaret al, 2012) gene and itsrRNA amplicons have shown to be powerfulapproaches in determining and monitoring theprobiotic community.2.1.2. Selection criteria for probioticsThere is considerable interest in probiotics for a varietyof medical conditions, and millions of people aroundthe world consume probiotics daily for perceivedhealth benefits. The probiotic must be able to surviveextremely harsh condition found in the digestive tractof the host. This involves the ability to survive the highacidity of the stomach as well as the concentrated bilefound in the small proximal of the intestine. It shouldpersist, albeit for short periods in the gastro-intestinaltract influencing metabolic activities like cholesterolassimilation, lactase activity and vitamin production.They need to avoid the effects of peristalsis, whichtend to flush out bacteria with food. The survival ofprobiotic organisms in the gut depends on thecolonization factors they possess, organelles whichenable them to resist the antibacterial mechanismsthat operate in the gut. In addition to these biologicalcriteria, probiotic intended for human use must also becommercially feasible and technologically compatiblewith the food manufacturing process. Thus they mustbe mass producible, remain viable on storage andmaintain the characteristic sensory attributes of thetraditional food. General biological criteria forprobiotics include: acid tolerance; bile salt tolerance;

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    adherence to the gut mucosa; ?-galactosidase activity;antibiotic resistance; and antimicrobial potential.2.1.2.1. Acid tolerance

    Bacteria used as probiotic adjuncts are commonlydelivered in a food system and therefore begin theirjourney to the lower intestinal tract via mouth. It wasreported that the duration of acidic stress fromentrance to release from the stomach to be 90minwhich can cause cellular damage at Low pH of 1.5.However, many of the Lactobacilli isolated from theileal samples performed well and could successfullytransit the stomach and capable of reaching theintestinal environment and function effectively. Themechanism involved in acid tolerance is not known,however studies in mice models have inferred thatgenes induced upon exposure to acid was identified toplay a role in survival of Lactobacillus strain underacidic conditions.

    2.1.2.2. Bile tolerance

    Bile acids are synthesised in the liver from cholesteroland are secreted from the gall bladder into theduodenum in the conjugated form (500700 mL/d).Microbial activity in the colon brings about extensivechemica l modi f ica t ions (decon jugat ion,dehydroxy la t ion , dehydrogenat ion , anddeglucuronidation) to the secreted bile acids (He et al.,2012). In vitro studies have shown that bothconjugated and deconjugated bile acids exhibitantibacterial activity. Several studies have shown theinvolvement of gene activation due to membranestress or oxidative damage upon exposure to bileacids. Research has shown that the permeability of L.acidophilus cells was increased in the presence of bile.Increased permeability allows more substrate to entercells so that the ?-galactosidase activity of the wholecell is increased.

    2.1.2.3. Adhesion to the gut mucosaAttachment is considered as a necessary first step inthe colonisation of the host mucosal surfaces. HT-29and Caco-2 cells are human intestinal cell linesexpressing morphologic and physio logiccharacteristics of normal human enterocytes that havebeen exploited to elucidate the mechanisms mediatingadhesion. And it explains that mechanism of adhesionis not unique for all Lactobacilli. Most Lactobacilli havebeen shown to adhere via binding to collagen type Iand a few strains to other proteins such as fibronectin,fibrinogen and lactoferrin.2.1.2.4. ?-galactosidase activity?-Galactosidase reduces lactose intolerance and is animportant therapeutic property of probiotic bacteria.

    The prevalence of lactose intolerance variesdepending on ethnic origin and is caused by thedeficiency in ? Galactosidase, resulting in the inabilityto digest the disaccharide lactose. On reaching thelarge bowel, the undigested lactose is degraded bybacterial enzymes, leading to osmotic diarrhoea.Lactose intolerant individuals develop diarrhoea,flatulence and abdominal pain after consumption ofmilk. LAB with probiotic potential contain the ?Galactosidase enzyme which aids in lactose digestionin the small intestine after being released frombacterial cells upon exposure to bile acids (Zrate andChaia, 2011).2.1.2.5. Antibiotic resistance

    One of the most important safety aspects of LAB istheir resistance to antimicrobial drugs that might betransferred to other possible, pathogenic bacteria.Moreover, the resistance profile of LAB to antibioticswill enable to clarify their potential in minimizing thenegative effects of antibiotic therapy on the hostbacterial ecosystem. The Lactobacillus species havebeen found susceptible to many cell wall synthesisinhibitors like penicillin and ampicillin, howeverresistance to vancomycin has been demonstratedbeing intrinsic.2.1.2.6. Antimicrobial potentialProbiotics have the ability to produce antimicrobialcompounds against pathogenic and carcinogenicbacteria and also to adhere to and colonise theintestinal gut mucosa. LAB is known to producebioactive molecules such as organic acids (Lactic,acetic and propionic acid), Carbon dioxide, H2O2, lowmolecular weight substances as well as protein-likeantimicrobials called Bacteriocins (Gomes et al.,2012). Inhibition of the growth of pathogenic bacteriabecause of antimicrobials is biologically significant asit is thought to be that of amensalism, a means of onebacterium gaining advantage over another competingbacterium. In our study, several of our probiotic isolatehave shown to produce bacteriocin like Pediocin fromPediococcus spp. GS4 with molecular weight of 9.9kDa that inhibits the growth of Staphylococcus aureus(ATCC 25923), Listeria monocytogens (ATCC 15313)and diarrhoeagenic bacteria including Shigelladysenteriae type1, S. sonnei, Salmonella typhimurium,Vibrio cholerae O1 and O139 (Gowri and Ghosh,2010; Gowri and Ghosh, 2010a; Shruthy et al., 2011).2.1.2.7. Organic acidsLAB is characterised by the accumulation of organicacids and the accompanying reduction in pH. It hasbeen proposed that the low external pH causesacidification of the cell cytoplasm, while the

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    undissociated acid, being lipophilic, can diffusepassively across the membrane (Schellenberg et al.,2012). The undissociated acid acts by collapsing theelectrochemical proton gradient, or by altering the cellmembrane permeability which results in disruption ofsubstrate transport systems. One among the organicacids is the lactic acid, a major metabolite of LABfermentation. It is in equilibrium with its undissociatedand dissociated forms, and the extent of thedissociation depends on pH. Different microorganismsvary considerably in their sensitivity to lactic acid.Heterofermentative LAB strains are known to produceacetic and propionic acids in addition to lactic acidthrough metabolic pathways. These acids interact withthe cell membrane and cause intracellular acidificationand protein denaturation.2.1.2.8. Hydrogen peroxideH2O2 is produced as a result of the action offlavoprotein oxidases or nicotinamide adeninedihydroxy nucleotide (NADH) peroxidase in thepresence of oxygen. It has been reported that theproduction of H2O2 by Lactobacillus and Lactococcusstrains inhibi ted Staphylococcus aureus ,Pseudomonas spp. and various psychotrophicmicroorganisms in foods. The antimicrobial activity ofH2O2 may result from the oxidation of sulphydrylgroups thereby causing denaturation of a number ofenzymes, as well as the increased membranepermeability as a result of peroxidation of membranelipids (Schellenberg et al., 2012). This may cause DNAdamage through the production of bactericidal freeradicals such as superoxide (O2-) and hydroxyl (OH.)radicals with H2O2 serving as a precursor for the freeradical cascade. In raw milk, H2O2 activates thelactoperoxidase system, producing hypothiocyanate(OSCN-), higher oxyacids (O2SCN- and O3SCN-) andintermediate oxidation products that are inhibitory to awide spectrum of Gram-positive and Gram-negativebacteria.

    2.1.2.9. Carbon dioxideCarbondioxide is also one of the end products ofheterofermentative metabolism. CO2 has effectivelyinhibited the growth of many food spoilagemicroorganisms, especially Gram-negativepsychrotrophic bacteria. With CO2 at 10% lowering thetotal bacterial counts by 50% and at 20-50% exhibitedstrong antifungal activity. CO2 could play a role increating an anaerobic environment which inhibitsenzymatic decarboxylation reaction and theaccumulation of CO2 in the membrane lipid bilayermay cause a malfunction in permeability.2.1.2.10. Aroma compounds

    Diacetyl is produced by strains within all genera ofLAB by citrate fermentation (Gallardo-Escamilla et al.,2005). The antimicrobial effect of diacetyl has beenknown since the 1930s. It inhibits the growth ofGram-negative bacteria by reacting with thearginine-binding protein, thus affecting the arginineutilisation. Acetaldehyde is another aroma compoundproduced by L. delbrueckii subspecies bulgaricus bythe action of a threonine aldolase, which cleavesthreonine into acetaldehyde and glycine. It canaccumulate in the product at a concentration of about25 ppm. I t was shown experimental ly thatStaphylococcus aureus, Salmonella typhimurium andE. coli in dairy products are completely inhibited byacetaldehyde at a concentration of 10-100 ppm.2.1.2.11. Fatty acidsSome Lactobacilli and Lactococci possessing lipolyticactivities produce significant amounts of fatty acidswith antimicrobial potential under specific conditions.The unsaturated fatty acids are active againstGram-positive bacteria, and the antifungal activity offatty acids is dependent on chain length, concentration,and pH of the medium. Reuterin is produced duringthe anaerobic growth of Lb. reuteri by the action ofglycerol dehydratase, a heterofermentative speciesinhabiting the gastrointestinal tract of humans andanimals which has been chemically identified to be3-hydroxypropanal (?-hydroxypropionaldehyde), ahighly soluble pH-neutral compound which is inequilibrium with its hydrated monomeric and cyclicdimeric forms. Reuterin exhibits a broad spectrum ofantimicrobial activity against certain pathogenicGram-positive and Gram-negative bacteria, yeast,fungi and protozoa (Schaefer et al., 2010).2.1.3. Bacteriocins as antimicrobials

    Some LAB strains ribosomally synthesise antimicrobialpeptides, or bacteriocins, targeted to inhibit otherGram-positive bacteria (Gomes et al., 2012). Bacteriocins permeate the outer membrane ofGram-negative bacteria and induced the inactivation inconjunction with other enhancing antimicrobialenvironmental factors, such as low temperature,organic acid and detergents. Bacteriocins produced byLAB are classified into three main groups, lantibioticsbeing the most documented and industrially exploited.The groups are lantibiotics (Class I), non lantibiotics,small heat-stable peptides (Class II) and largeheat-labile protein (Class III) The lantibiotic nisinnaturally produced by Lactococcus lactis ssp. lactis iscommercially available as food additive E234. Besidesthe production of bacteriocins, some LAB is able tosynthesise other antimicrobial peptides includinglow-molecular-mass metabolites, and/or cyclic

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    dipeptides.2.1.4. Role of probiotics in human healthAttempt has been made to unify the hypothesis forhealth benefits achieved using probiotics (Heinemanet al., 2012). Several results include lowering thefrequency and duration of antibiotics-associatedclostridial diarrhea, rotaviral infection, chemotherapyand Travellers diarrhea; stimulation of immunity anddecrease in unfavourable metabolites like ammoniaand procarcinogenic enzymes in the colon. Somemore benefits have been experienced over the recenttimes which specify the i) reduction of Helicobacterpylori infection (Baryshnikova, 2012), ii) reduction ofallergic symptoms (Matsuda et al., 2012), iii) relieffrom constipation (Liu, 2011; Malaguarnera et al.,2012), iv) relief from irritable bowel syndrome(Thomson et al., 2012), v) beneficial effects on mineralmetabolism (Lamberti et al., 2011; Rossi et al., 2011),vi) cancer prevention (Escamilla et al.,2012; Maroof etal., 2012), and vii) reduction of cholesterol andtriacylglycerol plasma concentrations (Wang et al.,2012).The role of probiotic organisms as alternative orcomplementary therapy in combating a large numberof disorders, especially gastrointestinal and their abilityto enhance immune response has attracted globalattention. A brief outline has been drawn here tointroduce the some health benefits of probiotics.2.1.4.1. Lactose intolerance

    Most of the worlds population (60-90% ofnon-caucasians and 6-12% of Caucasians) becomelactose intolerant after weaning, this stems from a90-95% decline in the production of lactase. Thepresence of lactose alters the osmotic balance in thecolonic lumen and develops symptoms includingabdominal bloating, cramping, flatulence and diarrhea.The contribution of lactase by the bacterial culturesused to manufacture the probiotic curd (yoghurt) isthought to mediate enhanced lactose digestion. Ingeneral, results have indicated that probioticseffectively improve the digestion of lactose and thiseffect seems to be more cell-density dependent thanstrain specific.2.1.4.2. Cholesterol reductionThe WHO has predicted recently that by 2030,cardiovascular diseases will remain leading causes ofdeath, affecting approximately 23.6 million peoplearound the World. The risk of heart attack is threetimes higher in those with hypercholesterolemia,compared to those who have normal blood lipidprofiles. The cholesterol-lowering activity of certainfermented milk and dairy products with some species

    of Lactobacillus, Bifidobacterium, Enterococcus, andStreptococcus has been demonstrated in rats,hamsters, and pigs. A human strain with similarcholesterol assimilating properties has beendiscovered. L. acidophilus designated strain NCFMhad an appreciable ability to assimilate cholesterol.The in vitro studies have evaluated a number ofmechanisms proposed for the cholesterol-loweringeffects of probiotics and prebiotics. One of themechanisms includes enzymatic deconjugation of bileacids by bile-salt hydrolase of probiotics. Oncedeconjugated, bile acids are less soluble andabsorbed by the intestines, converted to coprostanol,leading to their elimination in the faeces. 2.1.4.3. Irritable bowel syndromeIrritable Bowel Syndrome (IBS) is the most commonfunctional gastrointestinal disorder with a reportedprevalence in the general population between12%22%. IBS is characterised by a collection offunctional gastrointestinal symptoms such asabdominal pain, defecatory frequency and/orconstipation. The etiology of IBS is still not clear andnumerous factors are involved in the damage to themucosa, including microorganisms, psychologicalfactors and nutritional habits. In addition, thegut-associated immune system is up regulated asevidenced by increased inflammatory cytokines suchas interleukin 1, 6, and 10. The upregulatedgastrointestinal (GI) associated immune tissue isknown to stimulate discharge of enterochromaffin cellsand other cells, which release serotonin and/orhistamine resulting in GI symptoms. The type ofcolonising microflora may play an important role inregulating immunity. IBS patients host an intestinalmicroflora containing few Lactobacilli and a decreasein Bifidobacteria faecal concentration. In normalconditions, an immunologic tolerance is maintainedtoward the commensal enteric bacteria which preventintestinal inflammation. This controlled homeostaticresponse is lost in susceptible individuals that developchronic aggressive cellular immune response at theintestinal level. Oral administration of L. rhamnosusGG (LGG) in patients with Crohns disease resulted inthe promotion of the intestinal IgA immune response,reduction in the pain and severity scores in patients.Positive role of probiotics in IBS are convincing but atrend to the benef ic ial ef fect of bacter ialsupplementation as an adjunct to treatment isemerging.2.1.4.4. Colon cancerColorectal cancer is one of the leading causes ofcancer morbidity and mortality in many countries and itis thought to be caused by an interaction between

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    dietary factors and genetic predisposition. Diet isestimated to be responsible for 30 to 60 percent of allcancers. Genotoxic carcinogens including heterocyclicaromatic amines, which are formed during the cookingof meat, increase the potential risk factor of coloncancer in high-meat consumers. Epidemiologicalstudies have shown that consumption of fermentedmilk products, especially those containing probioticbacteria, may help reduce the risk of cancer at anumber of sites. Lactobacil lus acidophilus,Lactobacillus casei Shirota strain and LGG have beenshown to have inhibitory properties on chemicallyinduced tumours in animals. There is some evidencethat probiotics can interfere at various stages of thecancer process, such as prevention of DNA damage inthe colon by l ive bacteria, suppression ofpre-neoplastic changes in the colon and suppressionof colon tumours in animals. Preliminary studies on theeffect of probiotic consumption on cancer appear to bepromising. However further studies are needed beforethe beneficial effects of probiotics in the prevention ofhuman colon cancer can be confirmed.

    2.1.4.5. Probiotics and dental health

    Probiotics may be beneficial for the oral microflora bypreventing the growth of harmful bacteria such asStreptococcus mutans. A few studies are available onthe dental effects of LAB and fermented products.Russian Scientists have reported that probioticBifidobacteriuim spp. can reduce gingival andperiodontal inflammation. Lactobacillus GG (ATCC53103) has been studied for the colonising propertiesin the oral cavity. In healthy volunteers, it wasobserved to colonise oral mucosa for up to two weeksafter completion of the oral intake. Some of thehypothetical mechanisms of probiotics action in theoral cavity include binding of oral micro-organisms toproteins, action on plaque formation and on itscomplex ecosystem by competing and intervening withbacterial attachments and involvement in metabolismof substrate, production of chemicals that inhibit oralbacteria like antioxidants (Gowri and Ghosh, 2011).2.1.5. Features of probiotics to develop afunctional product An effective probiotic is required to function undervarious environmental conditions and has to survive inmany different forms. The candidate probiotic shouldpossess some characteristics:1. Acid tolerance and tolerance to human gastric juice.2. Bile tolerance (an important property for survival inthe small intestine)3. Adherence to epithelial surfaces and persistence inhuman GI tract.

    4. Immuno-stimulation,

    5. Antagonistic activity against pathogens such as,Helicobacter pylori, Salmonella typhimurium, Listeriamonocytogens and Clostridium difficle.6. Antimutagenic and anticarcinogenic properties.7. Should maintain viability and activity in the carrierfood for long periods under storage and fieldconditions (industrial scale) before consumption.2.2. Prebiotics

    Prebiotic is a non-digestable food ingredient thatconfers benefits on the host by selectively stimulatingthe growth and/or the activity of one bacterium or agroup of bacteria in the colon and thus improves hosthealth (Gibson and Roberfroid, 1995; Ooi and Liong,2010; Gourineni et al., 2011; Salvini et al., 2011; Yeoet al., 2009) and promotes the growth of certainprobiotics. Thus, the prebiotic approach advocates theadministration of non-viable entities. Dietarycarbohydrates, such as fibres, are candidate prebiotics,but most promise has been real ized wi thnon-digestible oligosaccharides, because of theirselective metabolism. As prebiotics exploit non-viablefood ingredients, their applicability in diets is wideranging. Prebiotics could also be termed as Colonicfoods, i.e., food that enter the large intestine (colon)and which serve as substrates for endogenous andautochthonous colonic bacteria, thereby indirectlyproviding the host with energy, metabolic substratesand essential nutrients. Resistant starch andnon-starch polysaccharides (NPS) are classified ascolonic foods but not as Prebiotics, as they are notmetabolized by a limited number of beneficial bacteria.However, some non-digestible oligo-saccharides areclassified as both colonic foods as well as Prebiotics.Fructo-oligosaccharides (FOS), transgalactosylatedd isacchar ides (TOS/GOS) and soybeanoligosaccharides (SO) are the only productsrecognized widely and are used as food ingredientsthat meet all the requisites of probiotic classification.Besides these, xylo-oligosaccharides (XOS), lactuloseand lactitol are also considered as Prebiotics. Theprebiotic activity of FOS has been confirmed in bothlaboratory and human trials. This is because thesecarbohydrates have a specific colonic fermentationdirected towards bifidobacteria. Bifidobacteria are ableto break down and utilize fructo-oligosaccharides dueto their possession of a ?-fructofuranosidase enzyme,providing a competitive advantage in a mixed cultureenvironment like the human gut. XOS are used asfood ingredients owing to their technological propertiesand healthy effects. Xylobiose (X2) (degree ofpolymerization; DP=2) is considered a member of

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    XOS with beneficial effect in GI tract. Results obtainedin vivo using rats proved the administration of XOSresulted in increased amounts of Bifidobacterium spp.with the increase of SCFA. It has been observed thatingested X2 is not excreted in feces or in urine in the24h following oral administration. Since X2 cannot behydrolysed either by saliva, pancreatin, gastric juice,or intestinal mucosa homogenate, suggest X2 isutilized by intestinal microflora. GOS are another classof prebiotics that are manufactured and marketed inEurope and Japan. These consist of a lactose corewith one or more galactosyl residues linked via ?1?3,?1?4 and ?1?6 linkages. They have found applicationin infant formula foods. Table 2 shows theclassification and sources of Prebiotics.

    Table 2

    Classification and sources of PrebioticsSee Illustration 2Prebiotics are with following characteristics: i) they areparts of edible plants; ii) they are carbohydrates thatare composed of mixture of oligosaccharides/ andpolysaccharides (degree of polymerization differs); iii)resist hydrolysis by human digestive enzymes; iv) donot appear to be significantly absorbed in the smallintestine (exception remains with very short chainoligosaccharides); v) they are not metabolized by thehost; and vi) they are excreted as such in the urine.However, the dietary intake of OS is difficult toestimate, but it may reach 3-13 g/day per person.Certain studies indicate that daily intake of 5-20 g ofInulin and FOS selectively promotes the growth ofBifidobacteria. Such studies are not available in India.A large number of beautiful and excellent reviewarticles are available to gain interesting informationabout pro- and pre-biotics. However, the commonunderstanding about the utility of Prebiotics is that theybenefit our intestinal ecosystem, improve immunityand overall health. Probiotics along with Prebioticshave been significantly used for the clinical benefit inthe prevention and management of gastrointestinaland non-gastrointestinal conditions(Sazawal et al.,2010; O Donnell et al., 2011) like necrotizingenterocolitis, Travellers diarrhea, allergic colitis,inflammatory bowel diseases and in pediatricsgastrointestinal disorders, by increasing biomass ofprobiotics and stool bulking.2.3. SynbioticsA further approach is synbiotics, where probiotics andprebiotics are combined. Synbiotics are preparationsthat contain probiotics as well as prebiotics. Synbioticsfurther improve the efficacy of the probiotic andprebiotic when used individually. This occurs because

    the prebiotic boosts the growth of the survivingprobiotic once it reaches the area of the intestinewhere it would multiply. The symbiotic conceptcombines efficacious probiotic strains with specificprobiotic compounds in a single product (Zhang et al.,2010; Fotiadis et al., 2008; Bengmark, 2012; Martin etal., 2012). Synbiotic is defined as a mixture ofprobiotics and prebiotics that beneficially affects thehost by improving the survival and implementation oflive microbial dietary supplement in the GI tract.Synbiotics are mostly prepared by adding prebioticingredients / dietary fibre or value added ingredientsalong with probiotics having some intention of healthadvantage. More than 350 products have beenapproved under Food for specific health use (FOSHU)as on 1st April,2011 with a claim to control GI functions(helps to maintain good GI condition and to improvebowel movement), which include oligosaccharides,Lactobacillus, Bifidobacteria, dietary fibres/non-digestible oligosaccharides. Several synbioticformulations have been reported in literature. Amongall, preparation with cassava starch or inulin, milk andthree different LAB; lactulose, inulin with L. acidophilusLA-5 and B. bifidum BB-12; inulin, FOS and honeywith L. acidophilus; sugar with inulin and probiotcs;raftiline, raftilose with honey, milk and LAB are worthmentioning. In our study, we prepared severalformulations (capsule) for delivery of probiotics (ourstrains Pediococcus sp.GS4, L. lactis and L. gasseri)for health benefits. Our preparations contain severaldifferent Prebiotics as excipients which include XOS,FOS, inulin, skim milk, dulcitol, xylitol, lactitol, honey,ascorbic acid in permutation and combination. It isalso reported that Prebiotics products are available inthe form of tablet or powder and mint wafers, whichcontains inulin and calcium (500 mg), manufactured bythe company, Naturally Vitamines under brand nameof InufloraTM and Inu-Lean respectively. StonyfieldCompany, sells synbiotic products in blendedflavoured yoghurt form containing six live activeculture and inulin. A large number of scientificliteratures are there to claim its use in different clinicalgastrointestinal (GI) conditions as displayed in Table3.

    Table 3

    Clinical applications of Synbiotics in several GIconditions

    See Illustration 33. Mechanisms of action of synbiotics onmucosal surface of GITGastrointestinal tract (GIT) represents a complexecosystem in which a delicate balance exists between

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    the intestinal microflora and host. It comprised of 95%obligate anaerobes including Bifidobacterium,Clostridium , Eubacterium, Fusobacterium ,Peptococcus, Peptostreptococcus, Bacteriodes andothers. About 1-10% of intestinal population iscomposed of facultative anaerobes includingLactobacil lus, Escherichia coli, Klebsiella,Streptococcus , Staphylococcus . Aerobicmicroorganisms are not present in the GIT. Thedistribution of these hosekeeping bacteria differ fromoral cavity to anus. The load of microbiota vary from102- 1012 colony forming unit (cfu) per gram of aspirateor stool and vary also with age, gastric acid secretion,diet, life style and time feeding. In general, we theHomo sapiens sapiens possess 10 times moremicrobiota than our cells content (1013). In the smallintestine, jejunum and ileum contain no more than 104-105, whereas the terminal ileal content increases by10-100 folds. On opening into the gut, the microbialload increases by million fold and reaches to 1011 to1012cfu/gram of stool. Each individual has a rather uniquecolonic flora that remains constant over long periods oftime, with minor variations conditioned by frequency ofbowel movement and diet. Uniquely, the morphologyof gut is governed by the microbiota and maintains thehomeostasis of the host (Marik, 2012).The therapeutic applications with probiotics, prebiotics,and synbiotics are moving fast in the recent decade.The effectiveness of probiotics is strain-specific, andeach strain may contribute to host health throughdifferent mechanisms. Many reports indicate thatprobiotics can affect innate immunity and also protectagainst pathogens by decreasing pathogenicadherence or by production of acids, H2O2,bacteriocins or several surface active molecules whichare inhibitory to pathogens in gastro-intestinal tract. Probiotics have been found to control intestinaldisorders, partially due to immunomodulating theinnate pattern-recognition receptors such as TLRs,NLRs and CLRs and because of serum antibodies IgG,and secretory IgA and IgM enhancing immuneresponse. Certain strains can intermittently translocateacross the intestinal mucosa without causing infection,thus influencing systemic immune events. Evidencehas been presented that some Lactobacilli can directlystimulate the immune system on the gut mucosalsurface via localised GI tract lymphoid cell foci. In arecent study, potential of twenty seven lactobacilli andsixteen bifidobacteria strains were compared tostimulate bone-marrow derived dendritic cells. Most ofthem induced strong production of IL-12 and TNF-?(DSouza et al., 2012).A number of mechanisms work to prevent harmful

    bacteria from growing on and attaching to theintestinal epithelium: production and secretion ofantimicrobial agents such as bacteriocins and organicacids, adherence via competition for the binding sitesand steric hindrance and barriers interfering withpathogens and hence promote the elimination ofharmful bacteria. The probiotic exert multiple potentialto confer health benefit to the host following severalmechanisms which include (1) competition for dietaryingredients as growth substrates, (2) bioconversion of,for example, sugars into fermentation products withinhibitory properties, (3) production of growthsubstrates, like exopolysaccharides or vitamins, forother bacteria, (4) direct antagonism by bacteriocins,(5) competitive exclusion for binding sites, (6)improved barrier function, (7) reduction ofinflammation, thus altering intestinal properties forcolonisation and persistence within, and (8) stimulationof innate immune response. A summary has beendrawn to demonstrate the possible mechanisms ofactions offered by probiotics-prebiotics or synbiotics toenhance the health benefit to humans with specialreference to GI conditions as in Table 4.Table 4

    Summary of mechanisms of action of probioticsand the impact on health including GI Tract.See Illustration 4

    4. Mechanisms of beneficial effects

    The origin of cultured dairy products dates back to thedawn of civilization and mention of them is found in theBible and the sacred books of Hinduism. However, theassociation between these products and human healthwas not realized until 1900s. During the same periodof Metchnikoff, Cohendy observed that humansubjects administered with sour milk containg L.bulgaricus were with reduced putrefaction, while HenryTissier observed that children with diarrhea had intheir stools a low number of Bifidobacteria, on thecontrary, abundant in healthy children and suggestedthat these bacteria could be administered to patientswith diarrhea to help restore a healthy gut flora in 1906.In the early 1920s, Rettger and Cheplin (1922)documented that L. acidophilus milk had therapeuticeffects, in particular, a settling effect on digestion.They believed that colonisation and growth in the gutwere essential for efficacy, and therefore, advocatedthe use of intestinal isolates. This kindled furtherinterest in understanding the survival and colonisationof Lactobacillus spp. Since then yoghurts were incommon use and were found efficacious inmaintaining the balance of the gut flora or to prevent

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    conditions, such as, diarrhoea, constipation, dyspepsia,cystitis, mucous colitis, chronic ulcerative colitis anddermatitis. Shirota focused his research on selectingstrains of intestinal bacteria that could survive passagethrough the gut and on the use of such strains L.acidophilus Shirota (subsequently named L. caseiShirota) ( Dong et al., 2010; Hori et al., 2002) was thebasis for the establishment of the Yakult Company.Gordon and colleagues noted that the success oftherapy using Lactobacillus was depended on certaincriteria: it was essential to use a non-pathogenicorganism which is a normal inhabitant of the intestineand capable of establishing itself in the gut and that alarge number of viable cells (107-109) were essentialfor the establishment of a beneficial flora. Theco-administration of a diet with high milk or lactosecontent or the growth factors required by Lactobacilliwas also considered as a key factor for successfulbacterial therapy. The scientific basis of probiotics andthe health benefits related to the consumption of foodcontaining viable LAB has branched from this historyand opened many new avenues for exploiting optimalbenefits from these microbes. The probiotics has thebest potential to be the best therapeutics for all sortsof diseases and disorders, the Panacea in near future( Martin et al., 2012).Lactobacillus and Bifidobacterium species originatingfrom human microflora have been a matter of interest.Hence Probiotics are sometimes called colonic foods.Lactobacilli have a long history of safe use in foodsand dairy products. Lactobacilli are normal inhabitantsof the intestinal ecosystem and vagina but variableamounts are found among people (ranging from 0 to106 cfu/g faeces). The therapeutic roles of someLactobacillus spp. have been demonstrated.Lactobacillus rhamnosus GG (LGG) was the firstprobiotic which received most clinical attention to date(Vandenplas et al., 2007). The Lactobacillus strainused traditionally for fermentation by the dairy industrywas unable to implant to the gut so, LGG wasdiscovered in 1985 by developing a list of idealqualities for probiotics, as mentioned earlier. LGG hasproven beneficial effects on the intestinal immunity. Itinc reases the number o f IgA and o therimmunoglobulin secreting cell in the intestinal mucosa,stimulates the local release of interferons, facilitatesantigen transport to underlying lymphoid cells whichsevers to increase antigen uptake in the Peyerspatches.Bifidobacteria are also part of the human and animalmicroflora, but species differ according to age:newborns are readily colonised by Bifidobacteriumbreve and B. infantis and colonisation is favored in

    breast-fed compared to bottle-fed infants, whereasadults more often host B. adolescentis, B. bifidum andB. longum. Presently there are about 30 speciesincluded in the genus Bifidobacterium, 10 of which arefrom human sources and 17 from intestinal tracts ofanimal or rumen. Of these, six species from humanorigin, B. adolescentis, B. breve, B. bifidum, B. lactis,B. infantis and B. longum have been used in dairyproducts. It has now been conclusively demonstratedthat some Bifidobacterium strains can surviveintestinal transit and persist transiently within the colon.The therapeutic roles of Bifidobacteria in animal modeland in human are due to four major mechanismsincluding modulation of the host immune system,resistance to infectious diseases such as againstrotavirus diarrhoea and enteropathogens , control ofinflammatory bowel disease such as Crohns disease,ulcerative colitis and pouchitis and prevention ofcancer. Persistance of ingested Bifidobacterium in thedigestive tract is dependent on the contribution of hostgenotype to the dominant microbial diversitysuggesting specific interactions between microbes andhumans. On the other hand, microflora establishmentand maintenance is highly dependent on the foodintake and style of diet. Among the other probiotics,Enterococcus faecium SF68 strain has been used inthe management of d iarrhoeal i l lnesses.Non-pathogenic probiotic yeast, Saccharomycesboulardii has also shown beneficial effect in thetreatment of diarrhea associated with antibiotic use.

    5. Clinical studies of probiotics in diarrhealdisease and other GI infectionsIn India, about 59% of prescriptions for the treatmentof diarrhoea were fixed dose combinations of anantibacterial and an antiprotozoal. Such combinationhas risks of adverse drug reactions and also increasesthe chances of drug resistance. Physicians in India areinfluenced by socio economic factors and so a cheapway for the management of diarrhoea is necessary(Reid et al., 2003; Preidis et al., 2011). Probiotics maybe seen as a natural alternative to traditional drugs,and thus patients may regard probiotics as safer, moreeffective, and less likely to pose significant health risks.Probiotic therapy is preferable to traditional drugsbecause it involve a more holistic approach to themanagement of disease, are readily available and donot require the additional costs of a physician visit.Diarrhoea is the second fatal disease in India and acommon problem of other developing countries. It hasbeen estimated that there may be 4 billion cases ofacute diarrhoea each year worldwide. All kindsinfectious diarrhea including Cholera, shigellosis andother infections lead to stunt and slow growth in

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    children below five years. Intake of curd has beenfound beneficial in a study in India (Saran, 2002). Thebalanced normal microbiota may become aberrant oninfection and synbiotics repair the physiological andimmunological processes to maintain the homeostasis.Diarrhoea is caused by infections, antibiotic therapy,and tube feeding. Infectious diarrhea is caused bymore than 50 different pathogens including virus,bacteria, fungus and protozoa and it affects allage-groups. It is the major cause of high mortality andmorbidity, as seen especially in India. In developingcountries, bacterial enteropathogens, particularlyenterotoxigenic Escherichia coli (ETEC) cause justunder half of the cases of endemic paediatricdiarrhoea and majority of all cases of Travellersdiarrhea (Ghosh et al., 1996). Probiotics have beenbest researched as therapeutics for the managementof acute infanti le diarrhoea. Rotavirus andenteropathogenic E. coli (EPEC) are leading cause ofthis condition worldwide. At least three systematicreviews have shown that use of probiotics achievesoverall reductions in the duration of diarrhoea rangingfrom 17 to 30 hours. Many mechanisms of actionexplaining how lactobacilli reduce the duration ofrotavirus-induced diarrhoea have been proposed (Reidet al., 2003; Reid et al., 2008; Preidis et al., 2011).These include competitive blockage of receptor siteswhen lactobacilli bind to receptors; signal(s) fromlactobacilli regulating secretory and motility defencesdesigned to remove perceived noxious substances;enhancement of the immune response; and productionby lactobacilli of substances that inactivate the viralparticles. However, it is not clear whether the routineuse of probiotics in acute diarrhoeal illnesses isjustified, as most acute diarrhoeal illnesses are selflimited.

    There is not much information indicating whetherprobiotics reduce the serious complications ofdiarrhoeal i l lnesses (Yan and Polk, 2006).Furthermore, the data do not provide a clearunderstanding of the type, dose, or duration ofprobiotic treatment required for achieving clinicalbenefit. Limited data suggest that the minimal effectivedose in children is 10 billion colony-forming units givenwithin the first 48 hours. Another study evaluated theuse of LGG as a prophylactic agent against diarrhoeain children. The regular administration of a daily doseof LGG, 6 days a week for 15 months, achieved a lowrate of diarrhoea, but only in nonbreast-fed infants.

    Persistent diarrhoea is defined as a diarrhoeal episodethat starts acutely but lasts for 14 days or more, and itis an important cause of morbidity and mortality inchildren under five years old in developing countries

    throughout the world. The cause of persistentdiarrhoea is not completely understood but is likely tobe complex; this in turn makes management of thecondition difficult. In a separate study, four clinicaltrials involving children with persistent diarrhea werereviewed in 2010 (Sazawal et al., 2010). Two studieswith a combined total of 324 showed that probioticsshorten the duration of diarrhoea and reduce the stoolfrequency on day 5. One study with 235 hospitalisedchildren suggested that probiotics reduced the hospitalstay. Three out of four trials reported that no adverseevents occurred. The report was satisfactory but notoverwhelming as every study is limited by few trialswith small number of participants, and therefore maynot represent a reliable estimate of probiotics' effect.Diarrhoea associated with antibiotic use and causedby Clostridium difficile is a complication of treatmentwith antimicrobial agents and occurs in about 5-25% ofpatients. C difficile is responsible for around 15-25% ofall cases of diarrhoea associated with antibiotic use,most occurring in older patients, usually in the two tothree weeks after cessation of antibiotic treatment.Lactobacilli, Bifidobacteria, and Streptococcus specieshave all been evaluated for the prevention ortreatment of diarrhoea associated with antibiotic useand found to be safe. In another study conducted in2007 showed that consumption of a readily availableprobiotic drink containing Lactobacillus casei, Lbulgaricus, and Streptococcus thermophilus, twice aday during a course of antibiotics and for one weekafterwards, reduced the incidence of diarrhoeaassociated with antibiotic use and C. difficile.Two recent hospital-based studies in 2011, reportedreduction in diarrhea duration when S. boulardii wasgiven to 186 children between 6-48 months, within 72hours after the onset of acute diarrhea. The resultsuggests a complementary treatment of acute diarrheain infants with daily oral doses of S. boulardii. In aseparate randomized clinical trial, a total of 111diarrhoeic children with median age of 40 months weretreated with synbiotics containing S. thermophilus, L.rhamnosus, L.acidophilus, Bifido lactis with FOS. Theduration of diarrhoeal episodes were one day shorterthan placebo group.The use of probiotics as prophylaxis against diarrhoeais an interesting concept that remains unproven(Culligan et al., 2009; Gratz et al., 2010). The data onthe use of probiotics to manage adults with infectivediarrhoea are limited and mixed. Twenty threeresearch papers were on the treatment of infectiousdiarrhoea with probiotics and concluded that theyappear to be a useful adjunct to rehydration therapywhen managing both adults and children (Allen et al.,

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    2004).6. Products marketed by Indian firmsA large number of research results on probiotics,prebiotics and synbiotics have been translated intoindustrial products. Due to its wide-acclaimedacceptability and simple formulation with dietary utility,probiotic market has brighter future to safeguardhuman lives. The market of functional food or designerfood like synbiotics is governed by several factorsspecially the cultural base, geographical location,demography, economy, level of education in thesociety, government interests and others. Properlyformulated probiotic-containing foods having thepotential to promote health benefits offer consumers alow risk and low cost dietary component. Although theworldwide market for probiotics or/ and synbiotics isgrowing fast and Japan, USA, Europe took theleadership, however, in India, the market has juststarted to conceive with leading companies like Amul,Nestle, Mother Dairy and Aavin making the first move.In India, these companies have come up with theirprobiotic products, which are very popular. Some ofthese are presented in Table 5.Table 5

    Indian company in the Probiotics marketSee Illustration 5

    Yakult-DANONE India, is the first Indian company with50:50 joint venture of Japanese global probioticsleader, Yakult, with the French food major, GroupDANONE, has entered the Indian probiotics marketwith launching of their product Yakult, a probiotic curdin December 2008. At least 39 probiotic drug brands,mainly in the area of gastroenterology, from 30 majorIndian companies have already created a probioticsdrug market in India worth Rs 80 crore. Major playersin the probiotics drug market in India includecompanies like Ranbaxy (Binifit), Dr. Reddy'sLaboratories, which has four probiotic brands, ZydusCadila, Unichem, JB Chem, and Glaxo SmithKline.Many Indian research institutes are working on theprobiotic technologies (Probiotic curd - PunjabAgricultural University, Ludhiana, Punjab; Probiotic icecream - National Dairy Research Institute (NDRI),Karnal, Haryana; Cereal based probiotic foods Haryana Agricultural University, Haryana, etc.).National Research Development Corporation, a Govt.of India enterprise under Ministry of Science andTechnology, has also taken act ive role incommercialization of the probiotic technologiesdeveloped in various Indian research institutes and

    laboratories for popularization of this new concept.India has a long tradition in using foods for theirhealth-promoting or functional properties, influencedby Ayurvedic medicine. These functional foods caninclude herbal extracts, spices, fruits and otheringredients for fortification and enhanced healthbenefit. Increasing health-awarness of synbiotics isfurther raising demand for diet foods and fortifiednatural foods. Nine out of ten urban Indian consumershave been reported to generally choose foods basedon health and wellness benefits.

    Concluding Remarks

    The concept of synbiotics is based on the knowledgeof providing therapeutic benefit to human beingsreducing or elimination the risk of gastrointestinaldisorders. Increasing evidences in scientific literaturepoint out that the consumption of synbiotics(Bio-yoghurt), particularly containing L. acidophilusand Bifdobacterium species offers considerable healthbenefits through different mechanisms to consumers.In addition to the nutritional favour there is a need for asustained effect to combine the skills of themicrobiology, food technologist and clinician.Undoubtedly, gut flora can protect the host againstseveral intestinal diseases. Once the prophylaxis ofprobiotics are well established with respect to theclaimed health benefits, probiotics are sure to gain firmfooting in the health food sector of the market and wellcertainly be the major portion of functional food sector.The probiotic dairy foods such as probiotic ice cream,kulfi, yoghurt drink and dietic yoghurt could becommercialized through the organized dairy for thebenefit of common people.The role of probiotic organisms as alternative orcomplementary therapy in combating a large numberof disorders, especially gastrointestinal and their abilityto enhance immune response has attracted globalattention. A number of health related effects havebeen suggested and partially established but somecan be considered reasonably well established andclinically well documented. It is hoped that the simplestformulation will be discovered soon like oralrehydration solution (ORS) to support the global healthand economy. The potentials with synbiotics ensure tobe a solution to those people where doctors never visitfor the cause of health. It will be very friendly toameliorate the public health in near future. Theincrease in metabolic activity of probiotics isfundamental to many of the currently proposedmechanisms of health promotion by prebiotics.Synergistic approach in synbiotics will add more

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    values to the mitigation, reduction or elimination ofmany existing diseases and many more diseases yetto be enlisted in the era of globalization. Truly,synbiotics has potentials to be panacea of all earthlydiseases.

    Acknowledgements

    The Author would like to thank the management of VITUniversity, Vellore, India for supporting and performingthe scientific exercise.

    References

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    21. Gourineni, V. P., Verghese, M., Boateng, J.,Shackelford, L., Bhat, N. K., Walker, L. T., 2011.Combinational effects of prebiotics and soybeanagainst azoxymethane-induced colon cancer in vivo.Journal of Nutr i t ion and Metabolism, doi:10.1155/2011/868197.22. Gowri, S., Ghosh, A.R., 2010. Study of theprobiotic potential of lactic acid bacteria isolated froma variety of Indian fermented food. Journal ofPharmacy Research 3, 2254-2257.23. Gowri, S., Ghosh, A.R., 2010a. Pediococcus spp. a potential probiotic isolated from Khadi (an Indianfermented food) and identified by 16S rDNA sequenceanalysis. African Journal of Food Sciences 4, 597-602.24. Gowri, S., Ghosh, A.R., 2011. Antioxidativepotential of probiotic bacteria from Indian fermentedfood. International Journal of Research in Ayurvedaand Pharmacy 2, 983-86.25. Gratz, S.W., Mykkanen, H., El-Nezami, H.S.Probiotics and gut health: A special focus on liverdiseases. 2010. World Journal of Gastroenterology 16,403-410.26. He, X., Zou, Y., Cho, Y., Ahn, J., 2012. Effects ofbile salt deconjugation by probiotic strains on thesurvival of antibiotic-resistant foodborne pathogensunder simulated gastric conditions. Journal of FoodProt 75, 1090-1098.27. Heineman, J., Bubenik, S., McClave, S.,Martindale, R., 2012. Fighting fire with fire: is it time touse probiotics to manage pathogenic bacterialdiseases? Current Gastroenterology Reports 14,343-348.28. Hori, T., Kiyoshima, J., Shida, K., Yasui, H., 2012.Augmentation of cellular immunity and reduction ofinfluenza virus titer in aged mice fed Lactobacilluscasei strain Shirota. Clinical and Diagnostic LaboratoryImmunology 9, 105108.29. Kumar, M., Ghosh, M., Ganguli, A., 2012.Mitogenic response and probiotic characteristics oflactic acid bacteria isolated from indigenously pickledvegetables and fermented beverages. World Journalof Microbiology and Biotechnology 28, 703-711.30. Kumari, A., Catanzaro, R., Marotta, F., 2011.Clinical importance of lactic acid bacteria: a shortreview. Acta Biomedica 82, 177-180.31. Lamberti, C., Mangiapane, E., Pessione, A.,Mazzoli, R., Giunta, C., Pessione, E., 2011. Proteomiccharacterization of a selenium-metabolizing probioticLactobacillus reuteri Lb2 BM for nutraceuticalapplications. Proteomics 11, 2212-21.32. Lasta, S., Ouzari, H., Andreotti, N., Fajloun, Z.,Mansuelle, P., Boudabous, A., Sampieri, F., Sabatier,J.M., 2012. Lacticin LC14, a new bacteriocin producedby Lactococcus lactis BMG6.14: isolation, purification

    and partial characterization. Infectious Disord DrugTargets 12, 316-325.33. Liu, L.W., 2011. Chronic constipation: currentt reatment opt ions. Canadian Journa l o fGastroenterology 25 Suppl B, 22-28.34. Malaguarnera, G., Leggio, F., Vacante, M., Motta,M., Giordano, M., Bondi, A., Basile, F., Mastrojeni, S.,Mistretta, A., Malaguarnera, M., Toscano, M.A.,Salmeri, M., 2012. Probiotics in the gastrointestinaldiseases of the elderly. Journal of Nutrition Health andAging 16, 402-410.35. Marik, P.E., 2012. Colonic flora, probiotics, obesityand diabetes. Frontiers in Endocrinology (Lausanne) 3,87. doi: 10.3389/fendo.2012.00087.36. Maroof, H., Hassan, Z.M., Mobarez, A.M.,Mohamadabadi, M.A., 2012. Lactobacillus acidophiluscould modulate the immune response against breastcancer in murine model. Journal of ClinicalImmunology. PMID:22711009.37. Martin, Franois-Pierre J., Collino, S., Rezzi, S.,Kochhar, S. 2012. Metabolomic applications todecipher gut microbial metabolic influence in healthand disease. Frontiers in Physiology 3, 113. doi:10.3389/fphys.2012.0011338. Matsuda, A., Tanaka, A., Pan, W., Okamoto, N.,Oida, K., Kingyo, N., Amagai, Y., Xia, Y., Jang, H.,Nishikawa, S., Kajiwara, N., Ahn, G., Ohmori, K.,Matsuda, H., 2012. Supplementation of the fermentedsoy product ImmuBalance effectively reducesitching behavior of atopic NC/Tnd mice. Journal ofDermatological Science 67, 130-139.39. McCartney, A.L., 2002. Application of molecularbiological methods for studying probiotics and the gutflora. British Journal of Nutrition 88 Suppl 1, S29-37.40. Murakami, K., Habukawa, C., Nobuta, Y.,Moriguchi, N., Takemura, T., 2012. The effect ofLactobacillus brevis KB290 against irritable bowelsyndrome: a placebo-controlled double-blindcrossover trial. Biopsychosocial Medicine 6, 16.41. O Donnell, M.M., Forde, B.M., Neville, B., Ross,P.R., O Toole, P.W., 2011. Carbohydrate catabolicflexibility in the mammalian intestinal commensalLactobacillus ruminis revealed by fermentation studiesaligned to genome annotations. Microbial CellF a c t o r i e s 1 0 ( S u p p l 1 ) , S 1 2 . d o i :10.1186/1475-2859-10-S1-S1242. Ooi, Lay-Gaik., Liong, Min-Tze., 2010.Cholesterol-lowering effects of probiotics andprebiotics: a review of in vivo and in vitro findings.International Journal of Molecular Sciences 11,24992522. 43. Preidis, G.A., Hill, C., Guerrant, R.L., Ramakrishna,B.S., Tannock, G.W., Versalovic, J. 2010. Probiotics,enteric and diarrheal diseases, and global health.

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    Gastroenterology 140, 814.44. Reid, G., Anukam, K., Koyama, T., 2008. Probioticproducts in Canada with clinical evidence: What cangastroenterologists recommend? Canadian Journal ofGastroenterology 22, 169-175.45. Reid, G., Jass, J., Sebulsky, M.T., McCormick,J.K., 2003. Potential uses of probiotics in clinicalpractice. Clinical Microbiology Review 16, 658-672. 46. Rettger, L.F., Cheplin, H.A., 1922. Bacillusacidophilus and its therapeutic application. Archives ofInternal Medicine 29, 357-367.47. Rodrigues, D.M., Sousa, A.J., Johnson-Henry,K.C., Sherman, P.M., Gareau, M.G., 2012. Probioticsare effective for the prevention and treatment ofCitrobacter rodentium-induced colitis in mice. Journalof Infectious Diseases 206, 99-109.48. Rossi, M., Amaretti, A., Raimondi, S., 2011. Folateproduction by probiotic bacteria. Nutrients 3, 118-34.49. Roy, B., Kala, C.P., Farooquee, N.A., Majila, B.S.,2004. Indigenous fermented food and beverages; apotential for economic development of high altitudesocieties of Uttranchal, Journal of Human Ecolology15, 45-49.50. Rupa, P., Mine, Y., 2012. Recent advances in therole of probiotics in human inflammation and guthealth. Journal of Agricultural and Food Chemistry, PMID:2289774551. Salvini, F., Riva, E., Salvatici, E., Boehm, G.,Jelinek, J., Banderali, G., Giovannini, M., 2011. Aspecific prebiotic mixture added to starting infantformula has long-lasting bifidogenic effects. Journal ofNutrition 141, 1335-1339.52. Saran, S., 2002. Use of fermented foods to combatstunting and failure to thrive. Nutrition 18, 393-396.

    53. Sazawal, S., Dhingra, U., Hiremath, G., Sarkar, A.,Dhingra, P., Dutta, A., Verma, P., Menon, V.P., Black,R.E., 2010. Prebiotic and probiotic fortified milk inprevention of morbidit ies among chi ldren:community-based, randomized, double-blind,controlled trial. PLoS One 5, e12164. 54. Schaefer, L., Auchtung, T.A., Hermans, K.E.,Whitehead, D., Borhan, B., Britton, R.A., 2010. Thea n t i m i c r o b i a l c o m p o u n d r e u t e r i n(3-hydroxypropionaldehyde) induces oxidative stressvia interaction with thiol groups. Microbiology 156(Pt6), 1589-1599.55. Schellenberg, J.J., Dumonceaux, T.J., Hill, J.E.,Kimani, J., Jaoko, W., Wachihi, C., Mungai, J.N., Lane,M., Fowke, K.R., Ball, T.B., Plummer, F.A., 2012.Selection, phenotyping and identification of acid andhydrogen peroxide producing bacteria from vaginalsamples of canadian and east african women. PLoSOne 7, e41217.

    56. Shruthy, V.V. , Pavithra, M., Gowri, S. GhoshA.R., 2011. Probiotic potentials among lactic acidbacteria isolated from curd. International Journal ofResearch in Ayurveda and Pharmacy 2, 602-609.57. Sonomoto, K., Yokota, A., (editor) 2011. Lacticacid bacteria and bifidobacteria: current progress inadvanced research. Caister Academic Press. ISBN978-1-904455-82-0.58. Stadnik, J., Dolatowski, Z.J., 2012. Biogenicamines content during extended ageing of dry-curedpork loins inoculated with probiotics. Meat Science 91,374-377.59. Starovoitova, S.A., Babenko, L.P., Timoshok, N.A.,Shynkarenko, L.N., Lazarenko, L.N., Spivak, N.Y.,2012. Cholesterol-lowering activity of lactic acidbacteria probiotic strains in vivo. MikrobiolhichnyiZhurnal 74, 78-785.60. Strus, M., Chmielarczyk, A., Kochan, P., Adamski,P., Che?micki, Z., Che?micki, A., Pa?ucha, A., Heczko,P.B., 2012. Studies on the effects of probioticLactobacillus mixture given orally on vaginal and rectalcolonization and on parameters of vaginal health inwomen with intermediate vaginal flora. EuropeanJournal of Obstetrics and Gynecology andReproductive Biology 163, 210-215.61. Szajewska, H., Ruszczy?ski, M., Radzikowski, A.,2006 . P rob io t i c s i n t he p reven t i on o fantibiotic-associated diarrhea in children: Ameta-analysis of randomized controlled trials. TheJournal of Pediatrics 149, 367-372.62. Thomson, A.B., Chopra, A., Clandinin, M.T.,Freeman, H., 2012. Recent advances in small boweldiseases: Part?I. World Journal of Gastroenterol. 18,3336-3352.63. Vandenplas, Y., Salvatore, S., Viera, M., Devreker,T. Hauser, B., 2007. Probiotics in infectious diarrhoeain children: are they indicated? European Journal ofPediatrics 166, 1211-1218.64. Wang, J., Zhang, H., Chen, X., Chen, Y.,Menghebilige, Bao, Q., 2012. Selection of potentialprobiotic lactobacilli for cholesterol-lowering propertiesand their effect on cholesterol metabolism in rats fed ahigh-lipid diet. Journal of Dairy Science 95, 1645-1654.65. Wollowski, I., Rechkemmer, G., Pool-Zobel, B.L.,2001. Protective role of probiotics and prebiotics incoloncancer. Protective role of probiotics andprebiotics in coloncancer. American Journal of ClinicalNutrition 73(2 Suppl), 451S-455S.66. Yan, F., Polk, D.B., 2006. Probiotics as functionalfood in the treatment of diarrhea. Current Opinion inClinical Nutrition and Metabolic Care 9, 717-721.67. Yeo, Siok-K., Ooi, Lay-G., Lim, Ting-J., Liong,Min-T., 2009. Antihypertensive Properties ofPlant-Based Prebiotics. International Journal of

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    Molecular Science 10, 35173530.68. Zrate, G., Chaia, A.P., 2011. Influence of lactoseand lactate on growth and ?-galactosidase activity ofpotential probiotic Propionibacterium acidipropionici.Anaerobe 18, 25-30.69. Zhang, Ming-M., Cheng, Jing-Q., Lu, Yan-R., Yi,Zhi-H., Yang, P., Wu, Xiao-T., 2010. Use of pre-, pro-and synbiotics in patients with acute pancreatitis: Ameta-analysis. World Journal of Gastroenterology 16,39703978.

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    Lactobacillus species

    L. acidophilus

    L. salivarius

    L. lactis

    L. rhamnosus

    L. gasseri

    L. reuteri

    L. paracasei

    L. casei

    L. fermentum

    L. brevis

    L. planetarum

    L. sporogens

    Bifidobacterium species

    B. bifidum

    B. longum

    B. thermophilum

    B. infantis

    B. lactis

    B. animalis

    Strptococcus species

    S. lactis

    S. cremoris

    S. alivarius

    S. intermedius

    S. thermophilus

    Other bacterial species

    Escherichia coli

    Enterococcus faecalis

    Enterococcus faecium

    Leuconostoc spp.

    Pediococcus spp.

    Bacillus spp.

    VLS#3(Four strains of Lactobacilli, three strains ofBifidobacterium, one strain of Streptococcus salivariusspp thermophilus)

    Yeast and Moulds

    Saccharomyces boulardii

    Saccharomyces cerevisiae

    Aspergillus niger

    Acanthosis oryzue

    Candida pintolopesii

    IllustrationsIllustration 1

    Commonly used microorganisms as probiotics

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    Classification Sources Manufacturing procedure

    Polyols

    Sorbitol Sugar alcohol Fermentation

    Xylitol Sugar alcohol Fermentation

    Mannitol Sugar alcohol Fermentation

    Disaccharides

    Lactulose Lactose Synthetic

    Lactitol Lactose Synthetic

    Oligosaccharides

    Fructo-oligosaccharides (FOS) Legumes, vegetables, cereals Extraction/ hydrolysis

    Soybean oligosaccharides (SO) Soybean Extraction/ hydrolysis

    Xylo-oligosaccharides (XOS) Plant sources Extraction/ hydrolysis

    (Trans) Galacto-oligosaccharides (TOS)/ (GOS) Lactose Synthetic

    Illustration 2

    Classification and sources of Prebiotics

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    Raffinose Fermentation

    Palatinose

    Iso-malto-oligosaccharides

    Lactosucrose

    Polysaccharides

    Inulin Legumes, vegetables,cereals

    Extraction/ hydrolysis

    Resistant starches Legumes, vegetables,cereals

    Extraction/ hydrolysis

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    1. Rota virus diarrhea 11. Inflammatory bowel disease

    1. Antibiotic-associated diarrhoea 12. Pancreatis

    1. Travellers diarrhea 13. Pouchitis

    1. Clostridial diarrhea 14. Colon cancer

    1. Diarrhiea of various origin 15.Food allergies and lactose intolerance

    1. Nosocomial diarrhea 16.Small bowel bacterial outgrowth

    1. AIDS diarrhea 17. Dental caries

    1. Gastroenterites 18. Cystic fibrosis

    1. H. pylori infection 19. Enhancement of oral vaccine

    1. Irritable bowel syndrome 20. Liver-gut-brain syndrome

    Illustration 3

    Clinical applications of Synbiotics in several GI conditions

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    Health Benefit Mechanisms of actions

    1. Resistance to enteric infections

    2. Secretary immune effect

    -Alteration of intestinal conditions to be lessfavourable of pathogenicity (pH, short chainfatty acids, bacteriocins)

    -Alteration of toxin binding sites toenterocytes.

    -Influence on gut flora populations.

    -Adherence to intestinal mucosa, interferingwith pathogen adherence.

    -Upregulation of intestinal mucinproduction

    -immunomodulation of pro-inflammatory(IFN, TNF-) and anti-inflammtory(IL-10) cytokines.

    1. Small Bowel decreasing bacterialinfections

    -Influence on activity of overgrown flora,toxic metabolite production

    1. Urogenital infctions -Adhesion to urinary tract and vaginal tractcells colonization resistance

    Illustration 4

    Summary of mechanisms of action of probiotics and the impact on health including GI Tract.

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    1. Infection caused by Helicobacterpylori

    -Inhibitor production (H2O2, Lactic acid,bacteriocins)

    1. Promotion of lactose digestion -production of bacterial lactase to hydrolyselactose

    1. Immune system modulation -strengthening of non-specific defenseagainst infection and tumours by productionof IL-12 and TNF-.

    -adjuvant effect in antigen-specific immuneresponses

    -Enhancement of secretary IgA production

    -conjugated leinoleic acid (CLA) of theprobiotics upregulate the PPAR to reduceinflammation and colon carcinogenesis

    1. Hepatic encephalopathy -Inhibition of urease-producing gut flora

    1. Anti-colon cancer effect -Mutagen binding carcinogen deactivation

    1. Inhibition of carcinogen producingamines and enzymes of colonicmicrobes

    -Antagonism

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    1. Effect of alergic states -Improves immune response

    -Influence on secondary bile saltconcentration

    -Prevention of antigen translocation intoblood stream

    1. Effect on blood lipids and heartdisease

    -Assimilation of cholesterol within bacterialcell

    -Increased excretion of bile salts due todeconjugation by bile salt hydrolase

    1. Antihypertensive -Antioxidative effect

    -Peptidase action on milk protein yieldstripeptides which inhibi angiotensin-1converting enzyme.

    -Cell wall components act as ACEinhibitors

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    Probiotics Products Strains used Company Name

    Dairy products

    Probiotic curd - Heritage Foods(India)ltd

    b-Activ Probiotic Dahi L.acidophillus and B.lactis strain BB12 Mother Dairy

    Nesvita Probiotic yoghurt L.acidophillus Nestle (India)

    Probiotic Ice-cream Amul Prolife - Amul Dairy (India)

    Yakult L.casei strain shirota Yakult Danone India Private Ltd.

    Pharmaceutical products

    ACTIGUT L.acidophillus, L.rhamnosus,B.bifidus

    B.longam, S.boulardii,Fructooligosaccharides

    Alembic

    BINIFIT S.faecalis, C.butyricum, Bacillusmesentericus, L.sporogenes.

    Ranbaxy

    BECELAC L.acidophillus,Vit-B6, Vit-C,Folic acid. Dr.Reddys Laboratories

    Illustration 5

    Indian company in the Probiotics market

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    BFILIN L.acidophillus, L.rhamnosus, B.bifidium,B.longum, S.thermophillus,Fructooligosaccharides

    Cipla

    EQUIPRO L.acidophillus,S.Faecalis

    Clostridiumbutyicum,

    Bacillus mesentericus

    Cadila Pharma

    ECONOVA L.Reuteri RC-14

    L.Rhamnosus GR-1

    Glenmark

    GUTRITE L.acidophillus, L.rhamnosus, B.longum,B.bifidium, S.boulardii, S.thermophillus,Fructooligosaccharides

    FDC

    GUTPROCAPS S.faecalisClostridium butyricumB.mesentericus,L.sporogenes

    Jb Chem

    PROBIZA D L.sporogenes,Fructooligosaccharides

    Zydus Cadila

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    IntroductionArticleIllustrationsIllustration 1Illustration 2Illustration 3Illustration 4Illustration 5

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