Human probiotics


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Role of probiotics in Human nutrition and Healthcare

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Human probiotics

  1. 1. HUMAN PROBIOTICS AND PREBIOTICS INTRODUCTION Probiotics are live microorganisms that are similar to the beneficial microorganisms found in humans and are hence generally regarded as safe (GRAS). The concept of probiotics (which means, “for life”) was introduced in early 20th century by Elie Metschnikoff. Though they have been in existence for many years, the proper appreciation of their benefits is only a recent phenomenon. More and more research is being undertaken to validate their efficacy; so far, such activities have demonstrated their effectiveness in cases of antibiotic associated diarrhea, irritable bowel syndrome, lactose intolerance, oral health etc. The functional ingredients in the probiotic products help in balancing the intestinal microbiota resulting in enhanced over all health, well being and boosts immune system.
  2. 2. Human intestinal microbiota. 1, mouth; 2, pharynx; 3, tongue; 4, esophagus; 5, pancreas; 6, stomach; 7, liver; 8, transverse colon; 9, gallbladder; 10, descending colon; 11, duodenum; 12, jejunum; 13, ascending colon; 14, sigmoid colon; 15, ileum; 16, rectum; 17, anus. Image adapted from ei_0132.gif. The gut microbiota form a diverse and dynamic ecosystem, including bacteria, Archaea, and Eukarya that have adapted to live on the intestinal mucosal surface or within the gut lumen. Stomach and duodenum • Harbor very low numbers of microorganisms: < 10^3 bacterial cells per gram of contents • Mainly lactobacilli and streptococci • Acid, bile, and pancreatic secretions suppress most ingested microbes • Phasic propulsive motor activity impedes stable colonization of the lumen Jejunum and ileum • Numbers of bacteria progressively increase from approximately 104 cells in the jejunum to 10^7 cells per gram of contents in the distal ileum Large intestine • Heavily populated by anaerobes: 10^12 cells per gram of luminal contents
  3. 3. HISTORY The use of lactic acid bacteria as feed supplements goes back to pre-Christian times when fermented milks were consumed by humans. It was not until the beginning of this century that Metchnikoff, working at the Pasteur Institute in Paris, started to put the subject on to a scientific basis. He believed that the flora in the lower gut was having an adverse effect on the host and that these adverse effects could be ameliorated by consuming soured milk. In support of this he cited the observation that Bulgarian peasants consumed large quantities of soured milk and also lived to a great age; he was in no doubt about the causal relationship and subsequent events have, in part, confirmed his thesis. He isolated what he called the 'Bulgarian bacillus' from soured milk and used this in subsequent trials. This organism was probably what became known as Lactobacillus bulgaricus and is now called L. delbrueckii subsp bulgaricus which is one of the organisms used to ferment milk and produce yoghurt. After Metchnikoff's death in 1916 the centre of activity moved to the USA. The American workers showed that the yoghurt organisms could not colonise the gut and reasoned that, if the effect was to be manifested in the gut then a gut micro organism was more likely to produce the required effect. Knowledge available at that time suggested the use of L. acidophilus and many trials were carried out using this organism. Encouraging results were obtained especially in the relief of chronic constipation. In the late 1940's interest in the gut microflora was stimulated by two research developments. Firstly, the finding that antibiotics included in the feed of farm animals promoted their growth. A desire to discover the mechanism of this effect led to increased study of the composition of the gut microflora and the way in which it might be affecting the host animal. Secondly, the more ready availability of germ-free animals provided a technique for testing the effect that the newly discovered intestinal inhabitants were having on the host. This increased knowledge also showed that L. acidophilus was not the only lactobacillus in the intestine and a wide range of different organisms came to be studied and later used in probiotic preparations.
  4. 4. PROBIOTICS The microbial population of the digestive tract is largest in the caecum and proximal colon with fewer numbers and species in the mouth, stomach and small intestine. However, these smaller populations can be important for health outcomes. The relatively sparse flora of the healthy stomach is usually less than 103 organisms per gram of contents, comprising chiefly facultative anaerobes such as lactobacilli, staphylococci and streptococci. Rapid peristalsis, and the bactericidal actions of gastric secretions, limit colonisation of the upper small intestine to usually less than 105 cells/ml of fluid. Bacterial numbers, especially of the strict anaerobes, increase along the small intestine, attaining a density of between 106 - 108/ml in the distal ileum. In adult humans the bacterial population is very large, consisting of about 1014 viable cells, which account for about 100 grams or 50% of the wet mass of colonic contents (Cummings and Macfarlane, 1997) and of which about 15 grams of biomass is shed daily in faeces (Cummings and MacFarlane 1991; Hill, 1995). The colonic microflora is taxonomically diverse, consisting of over 50 genera and 400 species (Drasar, 1988; Salminen et al., 1995; Savage, 1986; Mitsuoka 1996) in which only a relatively few genera predominate. Probiotics are live microorganisms thought to be healthy for the host organism. The term "probiotics" was first introduced in 1953 by Kollath (Hamilton-Miller et al. 2003). Contrasting antibiotics, probiotics were defined as microbially derived factors that stimulate the growth of other microorganisms. In 1989 Roy Fuller suggested a definition of probiotics which has been widely used: "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance". Fuller's definition emphasizes the requirement of viability for probiotics and introduces the aspect of a beneficial effect on the host. According to the currently adopted definition by FAO/WHO, probiotics are: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host". Probiotics are commonly consumed as part of fermented foods with specially added active live cultures; such as in yogurt, soy yogurt, or as dietary supplements. At the start of the 20th century, probiotics were thought to beneficially affect the host by improving its intestinal microbial balance, thus inhibiting pathogens and toxin producing bacteria. Today, specific health effects are being investigated and documented including alleviation of chronic intestinal inflammatory diseases, prevention and treatment of pathogen-induced diarrhea, urogenital infections, and atopic diseases. The original observation of the positive role played by certain bacteria was first introduced by Russian scientist and Nobel laureate Eli Metchnikoff, who in the beginning of the 20th century suggested that it would be possible to modify the gut flora and to replace harmful microbes by useful microbes. Metchnikoff produced the notion that the aging process results from the activity of putrefactive (proteolytic) microbes producing toxic substances in the large bowel. Proteolytic bacteria such as clostridia, which are part of the normal gut flora, produce toxic substances including phenols, indols and ammonia from the digestion of proteins. According to Metchnikoff these compounds were responsible for what he called "intestinal autointoxication", which caused the physical changes associated with old age.
  5. 5. A probiotic contains thousands of genes which may potentially influence the clinical effects. Furthermore, interaction with the host, food components or endogenous substrates or the endogenous microbiota inside the gastrointestinal lumen may generate by-products or end-products with functional properties. There is a great hope that new (molecular combined with biostatistical) methods including metagenomics and metabolomics will help to dissect the complexicity. These methods should, for example, allow the discovery of novel genes and gene products, with novel properties which could be later used as drugs (eg, small molecules or proteins with antimicrobial activities, immunomodulating properties or interacting with intestinal functions). The majority of colonic bacteria are saccharolytic, and several bacterial groups, including Eubacterium, Bacteroides, Bifidodobacterium and Escherichia can ferment starch (Brown et al., 1998; Wang and Gibson 1993). Clostridium butyricum and species of Bifidobacterium in particular are effective starch utilisers in vitro (Brown et al., 1998). Numerous mechanisms are involved in the action of probiotics. New discoveries which may have an impact on the understanding of the clinical effects in IBS include the demonstration of the presence of anti-inflammatory microorganisms in the endogenous microbiota (especially the phylum Firmicutes), (Sokol H,Pigneur B,Watterlot L,et al. Faecalibacterium prausnitzii is an antiinflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients; Proc Natl Acad Sci U S A 2008;105:16731–6.) Bär et al reported that conditioned media from the probiotic strain Escherichia coli Nissle 1917 modulates contractility of muscle strips isolated from humans. (Bär F, Von Koschitzky H, Roblick U, et al. Cell-free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study. Neurogastroenterol Motil 2009;21:559–66) Four controlled studies in humans showed that B lactis DN-173-010 shortened the colonic transit time and two trials also showed reduction of bloating in patients IBS or functional disorders. (Agrawal A, Houghton LA, Morris J, et al. Clinical trial: the effects of a fermented milk product containing Bifidobacterium lactis DN-173-010 on abdominal distension and gastrointestinal transit in irritable bowel syndrome with constipation. Aliment Pharmacol Ther 2009;29:104–14; Guyonnet D; Schlumberger A, Mhamdi L, et al,. Fermented milk containing Bifidobacterium lactis DN-173 010 improves gastrointestinal well-being and digestive symptoms in women reporting minor digestive symptoms: a randomised, double-blind, parallel, controlled study. Br J Nutr 2009;102:1654–62) Previous studies showed that L farciminis CIP 103136 suppresses stress-induced hypersensitivity in response to colorectal distension and it was recently reported that pretreatment with this probiotic was associated with a decreased expression of Fos protein (a marker of neuronal activation) in the spinal cord of stressed female rats. (Ait-Belgnaoui A, Eutamene H, Houdeau E, et al. Lactobacillus farciminis treatment attenuates stress-induced overexpression of Fos protein in spinal and supraspinal sites after colorectal distension in rats. Neurogastroenterol Motil 2009;21:477–80) Conditioned media from L paracasei NCC2461 reduced visceral hypersensitivity associated with antibiotic treatment in mice and normalised substance P expression in the myenteric and submucosal plexuses.
  6. 6. (Verdú EF, , Bercik P, Verma-Gandhu M, et al. Specific probiotic therapy attenuates antibiotic induced visceral hypersensitivity in mice. Gut 2006;55:182–90) L acidophilus NCFM has also been reported to increase the visceral threshold in rats (as effectively as morphine), and to induce opioid or cannabinoid receptors on HT29 cells (a property which was not shared by a variety of other bacterial strains). (Rousseaux C, Thuru X, Gelot A, et al. Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors. Nat Med 2007;13:35–7 Role of Probiotics • • • • • • • • • • • • • • • • To combat diseases like as Irritable Bowel Syndrome (IBS), dyspepsia, chronic diarrhea or constipation. To curdle milk To detoxify To eliminate pathogens To exist symbiotically To fight against certain cancers. To help in Lactic intolerance To help in producing short chain fatty acids (SCFAs) To help liver and kidneys in discharging their functions To help regulate the immune response To improve mineral absorption To improve Total digestible Nutrients (TDN) of the food intake. To inhibit LDL accumulation To maintain optimum micro flora To reduce Triglycerides To reduce the stress owing to high levels of Antibiotics Activities of Probiotic Bacteria • • • • • • • • • • • • Anti colon cancer effects Anti Milk allergy Anti-diarrheal effects Cholesterol lowering Correction of Hypertension Immune system modulation Improved tolerance to milk Intestinal health maintenance Reduction of Lactose intolerance Suppression of harmful intestinal microbe activities Suppression of pathogen translocation Vaginal/urinary tract health maintenance QUALITY PARAMETERS OF THE PROBIOTICS • Adhere to gut epithelial tissue • Be able to persist, albeit for short periods, in the gastrointestinal tract • Demonstrate non-pathogenic behavior • Exhibit resistance to technology processes (i.e., viability and activity in delivery vehicles) • Have the ability to influence metabolic activities
  7. 7. • Modulate immune responses • Prove resistant to gastric acid and bile acid. Lactic acid-producing bacteria are common components of probiotics. They are popular choices because of the historical belief that these bacteria are desirable members of the intestinal microflora, arising from the fact that lactic acid bacteria have long been used in the manufacture of dairy foods and are thus "generally regarded as safe", and because the consequent large-scale-culture and preservation methods for lactic acid bacteria in a viable state have already been developed by the dairy industry. LIST OF SOME OF THE COMMONLY USED HUMAN PROBIOTICS WHICH ARE CONSIDERED AS GRAS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Bacillius mesentericus Bacillus brevis Migula Bacillus circulans Jordon emend Ford. Bifidobacterium animalis Bifidobacterium bifidum Bifidobacterium breve Bifidobacterium infantis Bifidobacterium lactis Bifidobacterium longum Clostridium butyricum Escherichia coli strain Nissle 1917 Faecalibacterium prausnitzii Lactic acid Bacillus Lactobacillus acidophilus Lactobacillus animalis Lactobacillus brevis Lactobacillus bulgaricus Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus. Lactobacillus gasseri Lactobacillus johnsonii. Lactobacillus paracasei Lactobacillus plantarum Lactobacillus reutri Lactobacillus rhamnosus, Rhodospirillum rubrum Saccharomyces boulardii Streptococcus faecium Streptococcus faecalis. Streptococcus lactis Streptococcus salivarius Streptococcus salivarius subsp. Thermophilus Few Examples of probiotic strains in products Strain (alternative designations) Bifidobacterium animalis DN 173 010 Bifidobacterium animalis subsp. lactis Bb12 Bifidobacterium breve Yakult Bifidobacterium infantis 35624 Bifidobacterium lactis HN019 (DR10) Brand name Activia Chr. Hansen Producer Danone/Dannon Bifiene Align Howaru Bifido Yakult Procter & Gamble Danisco
  8. 8. Bifidobacterium longum BB536 Morinaga Milk Industry Enterococcus LAB SF 68 Escherichia coli Nissle 1917 Lactobacillus acidophilus LA-5 Lactobacillus acidophilus NCFM Lactobacillus casei DN-114 001 Bioflorin Mutaflor Chr. Hansen Danisco Actimel, DanActive Lactobacillus casei CRL431 Lactobacillus casei F19 Chr. Hansen Cultura Cerbios-Pharma Ardeypharm Danone/Dannon Arla Foods Information on few global suppliers of probiotics and prebiotics Company BioGaia Description Lactobacillus reuteri culture comes in three different, producer-friendly forms: freeze-dried powder, freeze- dried DVS (Direct Vat Set) granules, and frozen pellets Producer and seller of probiotic mix including L. acidophilus and L. casei The “nu-trish” brand probiotic culture range consists of Probio-Tec, Yo-Fast, and other nutrish culture blends with a well-defined viscosity profile that ferment quickly Producer of Enterococcus LAB SF 68 The company’s cultures division produces, develops, and markets starter cultures, media, coagulants, and enzymes for cheese, fresh dairy, and other food products, and also supplies probiotic cultures for foods and supplements, as well as natural food protectants URL Producer of several brands of fermented dairy products containing probiotics The Lafti line of probiotics is formulated for stability, survivability, and concentration, and includes L. acidophilus (Lafti L10), L. casei (Lafti L26), and Bifidobacterium (Lafti B94) GTC Nutrition NutraFlora short-chain fructo-oligosaccharides (scFOS) are a cane sugar or beet sugar–derived natural prebiotic fiber m Lallemand This Canadian supplier delivers probiotics and biosupplements to the nutraceuticals, functional-foods, and pharmaceuticals industries The Hi-Maize brand corn-based resistant starch has multiple benefits, including acting as a prebiotic for digestive health BeneoSynergy1 is the unique, patented oligofructose-enriched inulin prebiotic used in the landmark SynCan project on synbiotics and colon cancer Bio K + Chr. Hansen Cerbios-Pharma Danisco Danone DSM National Starch Orafti
  9. 9. Probi This biotech company develops and patents probiotic strains, including L. plantarum 299v and L. rhamnosus 271. L. plantarum 299 has not yet been commercialized, but it is in the outlicensing phase Proctor & Gamble “Align” is a probiotic supplement produced by P&G. Align capsules contain Bifidobacterium infantis 35624 Producer of Bacillus clausii strains O/C, NR, SIN, and T, marketed in Europe, Asia, and South America as Enterogermina Frutafit inulin and Frutalose fructooligosaccharides (FOS) are soluble dietary fibers with bifidogenic/prebiotic properties, suitable for a variety of food systems to enrich fiber, reduce calories, and replace sugars and fats Solvay Producer of lactulose (Duphalac) for treatment of constipation and hepatic encephalopathy Valio The Lactobacillus rhamnosus GG probiotic is the most researched in the world and was recently licensed to Dannon for the U.S. yogurt market. The Gefilus family containing LGG is marketed worldwide VSL#3 is a mixture of eight strains with 450 billion live bacteria per packet The company sells mixtures of probiotic strains for different indications Sanofi-Aventis Sensus VSL Pharmaceuticals Winclove Evidence-based indications for probiotics and prebiotics in gastroenterology Disorder, action Product Recommended dose Treatment of acute infectious diarrhea in children L. rhamnosus GG 1010–1011 cfu, twice daily L. reuteri ATTC 55730 1010–1011 cfu, twice daily L. acidophilus + B. infantis (Infloran strains) 109 cfu each, three times daily S. cerevisiae (boulardii) lyo 200 mg, three times daily Treatment of acute infectious diarrhea in adults Enterococcus faecium LAB SF68 108 cfu, three times daily Prevention of antibioticassociated diarrhea in children S. cerevisiae (boulardii) lyo 250 mg, twice daily L. rhamnosus GG 1010 cfu, once or twice daily B. lactis Bb12 + S. thermophilus 107 + 106 cfu/g of formula Enterococcus faecium LAB SF68 108 cfu, twice daily S. cerevisiae (boulardii) lyo 1 g or 3 × 1010 cfu per day L. rhamnosus GG 1010–1011 cfu, twice daily Prevention of antibioticassociated diarrhea in adults
  10. 10. L. casei DN-114 001in fermented milk with L. bulgaricus + S. thermophilus B. clausii (Enterogermina strains) Prevention of nosocomial diarrhea in children 1010 cfu, twice daily 2 × 109 spores, three times daily 5 × 1010 cfu, once daily L. acidophilus CL1285 + L. casei Lbc80r L. rhamnosus GG 1010–1011 cfu, twice daily B. lactis BB12 + S. thermophilus 108 + 107 cfu/g of formula B. lactis BB12 109 cfu, twice daily L. reuteri ATTC 55730 109 cfu, twice daily L. casei DN-114 001 in fermented milk with L. bulgaricus + S. thermophilus 1010 cfu, twice daily L. acidophilus + B. bifidum (Cultech strains) 2 × 1010 cfu each, once daily S. cerevisiae (boulardii) lyo 2 × 1010 cfu per day Oligofructose 4 g, three times per day L. rhamnosus GG 6 × 109 cfu, twice daily B. clausii (Enterogermina strains) 2 × 109 spores, three times daily AB yogurt with unspecified lactobacilli and bifidobacteria 5 × 109 viable bac, twice daily S. cerevisiae (boulardii) lyo 1 g or 5 × 109 cfu per day L. casei DN-114 001 in fermented milk with L. bulgaricus + S. thermophilus 1010 cfu, twice daily Reduces symptoms associated with lactose maldigestion Regular yogurt with L. bulgaricus + S. thermophilus Yogurt not heat-treated after pasteurization contains suitable cultures to improve digestion of the lactose in the yogurt Alleviates some symptoms of irritable bowel syndrome B. infantis 35624 108 cfu, once daily L. rhamnosus GG 6 × 109 cfu, twice daily VSL# 3 mixture 4.5 × 1011 cfu, twice daily Prevention of C. difficile diarrhea in adults Adjuvant therapy for H. pylori eradication L. rhamnosus GG, L. rhamnosus LC705, B. breve Bb99, and Propionibacterium freudenreichii ssp. shermanii 1010 cfu, once daily B. animalis DN-173 010 in fermented milk with L. bulgaricus + S. thermophilus 1010 cfu, twice daily Maintenance of remission of ulcerative colitis 5 × 1010 viable bac, twice daily E. coli Nissle 1917
  11. 11. Prevention and maintenance of remission in pouchitis VSL# 3 mixture of 8 strains (1 S. thermophilus, 4 Lactobacillus, 3 Bifidobacterium) 4.5 × 1011 cfu, twice daily Treatment of constipation Lactulose 20–40 g per day Oligofructose > 20 g per day B. infantis, S. thermophilus, and B. bifidum 0.35 × 109 cfu each strain, once daily L. acidophilus + B. infantis (Infloran strains) 109 cfu each, twice daily Prevention of postoperative infections Synbiotic 2000: 4 bacteria strains and fibers including the prebiotic inulin 1010 cfu + 10 g fibers, twice daily Treatment of hepatic encephalopathy Lactulose 45–90 g per day Prevention of necrotizing enterocolitis in preterm infants PREBIOTICS
  12. 12. A prebiotic is a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health. (Gibson et al. 2010. Food Science and Technology Bulletin: Functional Foods 7 (1) 1–19.) Unlike probiotics, most prebiotics are used as food ingredients—in biscuits, cereals, chocolate, spreads, and dairy products, for example. Commonly known prebiotics are: 1 Oligofructose 2 Inulin 3 Galacto-oligosaccharides 4 Lactulose 5 Breast milk oligosaccharides There is a growing list of candidate prebiotics such as polydextrose, soybean oligosaccharides, isomaltooligosaccharides, gluco-oligosaccharides, xylooligosaccharides. palatinose, gentio-oligosaccharides and sugar alcohols (such as lactitol, sorbitol and maltitol). However, the evidence for these, especially in humans, is not as well advanced as it is for FOS and GOS. 6 Effects of Prebiotics • Metabolic effects: production of short-chain fatty acids, fat metabolism, absorption of ions (Ca, Fe, Mg) • Enhancing host immunity (IgA production, cytokine modulation, etc.) SPRCIFIC PRODUCTS BASED ON THE END USAGE: Functional foods and beverages - dairy products, non Dairy beverages, breakfast cereal, baked goods, fermented meat products, dry-food probiotics;
  13. 13. animal feed probiotics; dietary supplements - food supplements, nutritional supplements and specialty nutrient. Selection of microbes for probiotic-based strategies. Applications: Human application - regular consumption, probiotic therapy, prevention of diseases and probiotic application for animals.
  14. 14. Probiotic bacteria have a variety of uses in the present society. Primarily, they are used as a preventative measure against a variety of infections. In the study performed by Frick et al, the scientists tested whether strains modulating inflammatory reponses in vivo could prevent DSS induced acute colitis. Key results were that they found that the probiotic bacteria increased the resistance of mice to the infection. They also saw that the probiotic bacteria inhibited inflammatory processes in vivo. The mouse model of oral Yersiniosis showed that the administration of viable B. adolescentis (the probiotic bacteria in question) protected mice from the generalization of Y. enterocolitica infection. In fact, in mice models with the dextran sodium sulfate-induced- colitis, B. adolescentis attenuated the development of colitis, indicating anti-inflammatory properties. (Frick et al.) Other uses for probiotics involve the commensal bacteria to be used as a neonatal nutrition source. For example, research has shown that probiotic bacteria are transferred through breast milk from mother to infant (Olivares et al.) As mentioned previously, having proper intestinal flora is very important to gastrointestinal health. Thus, for infants that are not breast-fed, proper supplements must be given with the bacteria added to it, to ensure gut health. MECHANISM OF ACTION BY PREBIOTICS AND PROBIOTICS The intestinal microflora likely plays a critical role in inflammatory conditions in
  15. 15. the gut, and potentially probiotics could remediate such conditions through modulation of the microflora. Different strains of Lactobacillus have shown to have varying degrees of adherence. Researchers tested the adhesive capacity of selected enterococci to human, canine, and porcine intestinal music was investigated in order to select for potential probiotic strains with good adhesive properties for human or animal use. The key results were that a strong correlation was observed for the adhesion to human and canine intestinal mucus and also between porcine and canine or human mucus. Two surface proteins have been identified that are associated with association and may be involved in adhesion of enteroccoci: the aggregation substance and enterococcal surface proteins (Laukova et al.) Thus adhesion of these probiotics blocks other pathogenic bacteria. While it has been established that adherence is key to Lactobacillus resistance of various gastrointestinal pathogens, studies have been done to examine where exactly the lactobacillus adheres to, to provide said resistance. The key results were that there were four sites of recovery for the probiotic bacteria: the cecum, the transverse, the descending colon, the sigmoid colon (Morelli et al.). While competition is a strong mechanism of resistance, it is not the only one used by probiotics. Evidence has been given to show that probiotic bacteria Lactobacilli exert bacteriostatic or bactericidal effects against bacterial pathogens, namely, Helicobacter pylori (Cruchet et al.) Further research with this pathogen/probiotic shows that Lactobacillus not only kills it, but also prevents IL-8 release (Hamilton-Miller et al.) The authors speculate that the mechanism by which Lactobacillus blocks Helicobacter pylori is combination of the following: competition for nutrients, competition for exclusion, production of inhibitory compounds, and immunomodulatory stimuli (Hamilton-Miller et al.). Another species of bacteria that is affected by probiotics is N. gonorrhoeae. Studies show that when probiotics are given to individuals who are infected with N. gonorrhoeae, symptoms are alleviated. The mechanism by which this is achieved, is again, when the Lactobacillus administered binds to the cells and therefore preventing the adhesion of the pathogenic bacteria. ( The direct effect of probiotics on infants’ intestinal systems by measuring changes in systemic levels of cytokines and inflammatory markers has also been evaluated. The researchers found that there was a significant increase in the levels of CRP (Creactive protein) in infants treated with LGG, who had AEDS. CRP is an indication of inflammation. IL-6 induces gene activation of CRP in hepatocytes and it stimulates CRP secretion (Viljanen et al.) Post inoculation with probiotic bacteria, the protein composition of the host organism also changes. A study was done to evaluate exactly what and how changes, and the result this causes on the organism. studies were performed in-vivo by inoculation of Lactobacillus fermentum into the rabbit jejunum for 4 hours. After inoculation, the bacteria were analyzed for proteome. The scientists performing this study found changes in protein biomarkers that are beneficial for Lactobacillus and intestinal
  16. 16. epithelial cells in response to interactions. The key results were that there were undetectable values for Lactate dehydrogenase when L. fermentum is incubated inside rabbit jejunum. This indicates that lactate utilization is limited as a fuel in intestinal bacterium. There is also a decrease in dihydrolipoamide dehydrogenase, which is a component of the pyruvate dehydrogenase complex. This suggests that oxidation of pyruvate via the krebs cycle is low post incubation. And since substrate oxidation is associated with the production of oxygen free radicals, which are potentially toxic to bacteria, these changes in energy metabolism would favor the survival of L. fermentum in the intestine. Lastly, there is an increase in Glycoside hydrolase, which plays a role in mucin degradation on the surface of the intestine. The release of glycoside hydrolase from the bacterium may participate in the regulation of mucin turnover and thus integrity of the intestinal epithelium. Another important finding from this study is that the levels of some regulatory proteins were elevated in response to incubation with the probiotic, and some of these proteins would help protect against the inflammatory injury that occurs in response to bacteria. They summarized that proteins can serve as biomarkers for the metabolic changes that are beneficial for the lactobacillus and intestinal epithelial cells (Yang et al.) Administration of lactobacilli and bifidobacteria could theoretically modify the flora leading to decreased β-glucuronidase and carcinogen levels (Hosada et al., 1996). Furthermore, there is some evidence that cancer recurrences at other sites, such as the urinary bladder can be reduced by intestinal instillation of probiotics including L. casei Shirota (Aso et al., 1995). In vitro studies with L. rhamnosus GG and bifidobacteria and an in vivo study using L. rhamnosus strains GG and LC-705 as well as Propionibacterium sp. showed a decrease in availability of carcinogenic aflatoxin in the lumen (El-Nezami et al., 2000; Oatley et al., 2000). ( Intravenous, intraperitoneal and intrapleural injection of L. casei Shirota into mice significantly increased NK activity of mesenteric node cells but not of Peyer's patch cells or of spleen cells (Matsuzaki and Chin, 2000), supporting the concept that some probiotic strains can enhance the innate immune response. (Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria, October 2001) In a series of randomized, double blind, placebo controlled clinical trials, it was demonstrated that dietary consumption of B.lactis HN019 and L. rhamnosus HN001 resulted in measurable enhancement of immune parameters in the elderly (Arunachalam et al., 2000; Gill et al., 2001; Sheih et al., 2001). Some probiotic strains were shown to inhibit the growth of enteropathogens, such as Salmonella enteritidis, enterotoxigenic Escherichia coli, and Serratia marcesens, in vitro and in this respect may offer considerable therapeutic potential. This finding, together with more recent evidence showing that Lactobacillus GG exerts antagonist activity against Salmonella typhimurium C5 infection both in vitro and in vivo (50), provides a basis for the clinical use of probiotics in suppression of pathogens. ( Potential for probiotics microorganisms to modulate the immune response and prevent onset of allergic diseases has been demonstrated.
  17. 17. Ability of lactobacilli to reverse increased intestinal permeability, enhance gut-specific IgA responses, promote gut barrier function through restoration of normal microbes, and enhance transforming growth factor beta and interleukin 10 production as well as cytokines that promote production of IgE antibodies (Kalliomaki et al., 2001; Isolauri, 2001). Certain microorganisms can contribute to the generation of counter-regulatory Thelper cell immune responses, indicating that use of specific probiotic microorganisms could redirect the polarized immunological memory to a healthy one (McCracken and Lorenz, 2001). There is preliminary evidence that use of probiotic lactobacilli and metabolic byproducts potentially confer benefits to the heart, including prevention and therapy of various ischemic heart syndromes (Oxman et al., 2001) and lowering serum cholesterol (De Roos and Katan, 2000). There is some clinical evidence to suggest that oral and vaginal administration of lactobacilli can eradicate asymptomatic (Reid et al., 2001a; 2001b) and symptomatic Bacterial vaginosis (Hilton et al., 1995; Sieber and Dietz, 1998). One study of day care centres in Finland showed that probiotic use reduced the incidence of respiratory infections and days absent due to ill health (Hatakka et al., 2001). Probiotic bacteria containing β-galactosidase can be added to food to improve lactose maldigestion (Kim and Gilliland, 1983). Studies have also shown that some infections are more prevalent in organisms and people without a certain baseline level of probiotic bacteria. In addition to this, research has demonstrated that having the probiotic bacteria in one’s system before being infected leads to better recovery. For example, Helicobacter pylori infections are seen more heavily in people that we know already have a lower baseline level of Lactobacillus johnsonii La1. The clinical studies that were conducted show that the Lactobacillus family of bacteria has positive effects on patients with Helicobacter pylori infections. Results show that when the mouse models that were being tested were fed L. salivarius prior to the challenge with H. pylori, no colonization was achieved. In another study, the scientists involved studied in vivo the effects of probiotics in infants with atopic eczema dermatitis syndrome (AEDS) and those infants with cow’s milk allergy (CMA). They had a total of 230 in vivo subjects and found that in atopic children, there were more coliforms and clostridia and fewer bifidobacteria and lactobacillus bacteria than nonatopic children (Viljanen et al.). Studies were conducted on the specific effect of probiotic bacteria on the specific effect of probiotic bacteria on the post treatment inflammation marker, C-reactive protein (CRP). The data presented in this particular study shows that there is a significant increase in the CRP levels in the Lactobacillustreated infants with AEDS, than those infants in the control group. This is beneficial since the production of Human CRP leads to inhibition of production of different inflammatory cytokines and chemokines. Human CRP also enhances the production
  18. 18. of IL-10, which displays potent abilities to suppress the antigen presentation capacity of antigen presenting cells. It is also stimulatory towards certain T cells, mast cells and B cells (Viljanen et al.). Studies also show that certain probiotic bacteria can protect against various fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum. These are of special importance to humans because these are common pathogens that infect fish used for human consumption. It has been deduced that L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for safe treatment in aquaculture (Nikoskelainen et al.) Adhesion, as will be discussed later, is one of the mechanisms by which probiotics are thought to benefit the host organism. In a particular study conducted by Nikoskelainen and his colleagues, it was deduced that L. rhamnosus and L. bulgaricus were the most adhesive to the gastrointestinal tract. This ability is thought to be the way by which probiotics suppress pathogen growth.
  19. 19. PROOF OF ACTIVITY Scientists have come up with techniques to follow and track the progress of the probiotic bacteria through the gastrointestinal tract. In a study mentioned previously, a tests were performed in vivo and the scientists stained the bacteria with cFDA-SE and under observation, realized that the bacteria was localized in the rectum and also in the jejunum and ileum. This is important since they know that the probiotic bacteria can localize and adhere to the GI tract, and thus provide competition to the pathogenic bacteria (Bouzaine et al.) Another method of tracking the progress of the probiotic through the GI tract is through GFP staining. Scientists realized that bacteria can be difficult to follow through the gastrointestinal tract, so they decided to label it with a plasmid containing GFP. They used rats for the in-vivo models, and then took samples from the stomach, jejunum, ileum, caecum, colon and rectum to test for presence of EcN. They found that the EcN-GFP was detectable in high numbers in the stomach and in lower numbers in the caecum but not in the rectum at the beginning of the experiment. They also found out that the organisms were localized close to the epithelial surface of the stomach, cecum, and rectum and also in the lumen of the intestine. The transformation of EcN to obtain EcN-GFP in this study had no detectable influence on the probiotic microorganism regarding adhesion on and induction of IL-8 secretion of HT-29 cells and allowed the detection in mixed microbial environments (Schultz et al.)
  20. 20. SAFETY Probiotics as treatment are still a novel concept in the medical world, and leads to questions and doubts in the minds of many. However, one can rest assured that studies are heavily underway to test for the safety and reliability of probiotics. Research has been done to determine any toxicity present through the usage of probiotic bacteria, Bacillus (subtilis and indicus). Researchers tested guinea pigs and rabbits since they are considered the most sensitive lab animals. No noticeable differences could be seen in the animals tested, thus they can assume that the probiotic bacteria are harmless (Hong et al.).
  21. 21. MICROENCAPSULATION_ A NOVELTY In order to provide the consumers with the health benefits, probiotic food must contain probiotic bacteria in sufficient number and with high viability when reaching the human intestine, where they deliver the beneficial effect. The problem lies in the sensibility of these bacteria to stress and extreme physiological conditions, thus the survival of probiotics in current food is low and results in less than the recommended effective daily intake as well as insufficient number of bacteria reaching consumers intestine. It is, therefore, necessary to protect probiotic bacteria added in food products until they reach the human large intestine. Providing probiotic living cells with a physical barrier against adverse environmental conditions (via microencapsulation, for instance) receives currently considerable interest, and quite a few research works have been conducted on the topic. Based on a novel technology, it is aimed at devising a innovative microencapsulation process to protect bacteria in food systems achieving at the same time a particle size less than 30 μm (under the consumer sensory perception). The microcapsules formulations were designed to effectively protect the bacteria by an adequate polymer coating thus enabling them to reach the human intestine with the necessary viability level.
  22. 22. MARKET SURVEY Beneficial microorganisms can be used in food, feed, drinking water; over pond water mediums; in the environment. Beneficial microorganisms can be used in controlling • Bacterial, fungal and viral infections • Cancer • Cholesterol • External parasites • Insects • Internal parasites • Obesity • Pests Beneficial microorganisms can be used as alternate antibiotics, pesticides, insecticides, growth promoters. Beneficial microorganisms can be used as • Anifungals • Antibiotic s • Antivirals • Enzyme producers • FCR Improvers • Growth promoters • Gut acidifiers • Immuno modulators • Insecticides • Meat tenderizers • Oxygen liberators • Parasiticides • Pesticides • Pollutant degraders • Protozoacides • Toxin binders • Zoothamnicides Role of Probiotics • To combat diseases like as Irritable Bowel Syndrome (IBS), dyspepsia, chronic diarrhea or constipation. • To curdle milk • To detoxify • To eliminate pathogens • To exist symbiotically • To fight against certain cancers. • To help in Lactic intolerance • To help in producing short chain fatty acids (SCFAs) • To help liver and kidneys in discharging their functions
  23. 23. • • • • • • • To help regulate the immune response To improve mineral absorption To improve Total digestible Nutrients (TDN) of the food intake. To inhibit LDL accumulation To maintain optimum micro flora To reduce Triglycerides To reduce the stress owing to high levels of Antibiotics Activities of Probiotic Bacteria • Anti colon cancer effects • Anti Milk allergy • Anti-diarrheal effects • Cholesterol lowering • Correction of Hypertension • Immune system modulation • Improved tolerance to milk • Intestinal health maintenance • Reduction of Lactose intolerance • Suppression of harmful intestinal microbe activities • Suppression of pathogen translocation • Vaginal/urinary tract health maintenance FACTORS THAT MAKE USAGE OF PROBIOTICS AS THE ONLY BEST ALTERNATIVE • • • • • Abuse of Antibiotics Abuse of Growth promoting antibiotics and chemicals Abuse of Hormones Abuse of Pesticides, insecticides Abuse of dewormers, parasiticides, disinfectants, sanitizers etc Probiotics, belonging to the functional group of gut flora stabilisers within the category of zootechnical feed additives (according to the Regulation EC No 1831/2003) is a fast growing market The probiotics market has been one of the prime beneficiaries of the recent fad over functional foods. Rising levels of health consciousness and the ageing baby boomer population are a few of the drivers helping in the growth of the market. The other major market factor driving the overall probiotics market in the present and expected to continue to do so in the future is the influence exerted by the women buyer segment. It is generally observed throughout the globe that traditionally women are responsible for the buying decisions of the foods and beverages (F&B) category in families. Since they tend to be more aware about the new products and their health benefits, they try and incorporate more beneficial foods in the families' diet. Hence it is no surprise that women have more knowledge about probiotic F&B and consequently not only drive their consumption, but also act as the opinion makers. This gathers more importance considering the fact that the probiotic Foods & Beverages segment is expected to command over 75% of the overall probiotics market in 2009.
  24. 24. Probiotic dairy products are expected to command the highest market share among all the probiotic foodstuffs, accounting for almost 70% in the year 2009 and reaching a market size of almost $24 billion by the end of 2014. The biggest markets for these products are Europe and Asia; the U.S. market has slowly but surely opened up to these products in the recent past and is expected to grow at a CAGR of 17% from 2009 to 2014, the biggest contributor being probiotic cultured drinks followed by probiotic yogurts. Though the market base of probiotic products is comparatively lesser in the U.S., the market is expected to grow at an astounding rate of almost 14% in the same period driven by the large scale acceptance of - the probiotic yogurts in spoonable single serve packs, probiotic cultured drinks in single shot packaging form and probiotic dietary supplements. Products that fall under the niche category presently, such as probiotic chocolates, probiotic ice creams and probiotic baked products are expected to enjoy a much larger market share due to the belief that consumers are ready to pay an extra amount for fortified products if confident of 'extra' benefits. However, probiotic cheese, probiotic butter etc are fated to the status of ultra-niche products due to their conventional image as unhealthy dietary products. The global market estimate of functional foods has been up to 73 Billion € and an annular growth rate of 8-16%. In a recent study undertaken by Leatherhead Food RA, the market for functional foods in the United Kingdom, France, Germany, Spain, Belgium, Netherlands, Denmark, Finland, and Sweden was reviewed. The results of the study showed that the probiotic yogurt market in these 9 countries totalled >250 million kg in 1997, with France representing the largest market, having sales of 90 million kg, valued at US$219 million. The German market for probiotic yogurts is growing rapidly; for example, during 1996–1997, it increased by 150%, whereas the UK market grew by a more modest 26% during the same period. On average, probiotic yogurts accounted for 10% of all yogurts sold in the 9 countries studied, with Denmark having the highest proportion (20%) of probiotic yogurts, followed by Germany and the United Kingdom (both at 13%) and then France (11%). On the lower end of the scale were the Netherlands and Belgium (both at 6%) and then Finland and Sweden (both at 5%). Seen as crucial to market expansion in Europe is further clarity on the use of health claims. The market for functional foods in Europe could ultimately account for 5% of total food expenditure in Europe, which, based on current prices, would equate to US$30 billion. In 2004, the global market value of probiotics was €32 million, with a forecasted annual growth of approximately 3%. However, due to the ban of antimicrobial feed additives, the probiotic market in Western Europe showed an annual growth of more than 7%. In 2006, Western Europe produced around 296 tons of probiotics, with a value of €15.5 million. With 1012 CFU (equivalent to about 100g) usually added to a ton of mixed feed, approximately 3 million tons of feed containing probiotics was produced last year. ( In Japan a standard was developed by the Fermented Milks and Lactic Acid Bacteria Beverages Association stipulating that a product contain 1 x 107 viable bifidobacteria/g or mL product to be considered a probiotic food.
  25. 25. Probiotic Foods & Beverages segment is expected to command over 75% of the overall probiotics market in 2009. Probiotic dairy products are accounting for almost 70% in the year 2009 and reaching a market size of almost $24 billion by the end of 2014. Probiotic dairy products market in USA is expected to grow at a CAGR of 17% from 2009 to 2014. Probiotic chocolates, probiotic ice creams and probiotic baked products are expected to enjoy a much larger market share. According to a new market research report, 'Probiotics Market (2009-2014)' ( hnologies-and-global-market-69.html), published by Markets and Markets (, the global probiotics market is expected to be worth US$ 32.6 billion by 2014, with the Europe and Asia accounting for nearly 42% and 30% of the total revenues respectively. The global market is expected to record a CAGR of 12.6% from 2009 to 2014. Europe forms the largest market for probiotics with an estimated $13.5 billion by 2014. Its 12.2% CAGR from 2009 to 2014 is driven by consumer demand for healthenhancing probiotic products, such as probiotic yogurts, other probiotic dairy products and probiotic dietary supplements. Asia is the second largest segment, growing at with an estimated CAGR of 11.2% to reach $9.0 billion by 2014. India is also fast emerging as a potential market for probiotics in food. Probiotic product industry in India was estimated to be around Rs 20.6 million with a projected annual growth rate of 22.6% until 2015.
  26. 26. STATUTORY REGULATIONS: One major attribute of probiotics is the lack of adverse events associated with their use. (Gregor Reid; Canadian Research and Development Centre for Probiotics, Lawson Health Research Institute, and Departments of Microbiology & Immunology, and Surgery, University of Western Ontario, London, Canada; Current Pharmaceutical Design, 2005, 11, 11-16) International guidelines on probiotics in food broadly specify the kind of tests that may be required to determine the safety and to assess the health claim of a probiotic product in food. Such tests are based on the current understanding of the subject. The regulatory mechanism for probiotics differs from country to country and also even within a country. In India there are no regulatory guidelines for probiotic foods upto 2011. However a Task Force was constituted by ICMR in 2011 and the guidelines proclaimed by it are given below. 2. GUIDELINES AND REQUIREMENTS FOR PROBIOTIC PRODUCTS 2.1. Scope: The guidelines deal with the use of probiotics in food and provide requirements for assessment of safety and efficacy of the probiotic strain and health claims and labeling of products with probiotics. Note: These guidelines are not meant for probiotics which by definition would come under drugs, beneficial microorganisms not used in foods or genetically modified microorganisms (GMOs). 2.2 Definition of Probiotics: Probiotics are ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ (FAO/WHO, 2002). 2.3 Genus, species and strain identification: Effects of probiotics are strain specific. Strain identity is important to link a strain to a specific health effect as well as to enable accurate surveillance and epidemiological studies. Both phenotypic and genotypic tests should be done using validated standard methodology. Nomenclature of the bacteria must conform to the current, scientifically recognized names as per the International Committee on Systematics of Prokaryotes (ICPS) (available at The current molecular techniques used for identification include PCR based techniques, 16S rRNA sequencing and DNA finger printing techniques like Ribotyping and Pulsed Field Gel Electrophoresis (PFGE). It is recommended that probiotic strains in use in India should be deposited in an internationally recognized culture collection/repositories. 2.4 In vitro tests to screen potential probiotic strains: The following in vitro tests* with standard methodology are recommended for screening putative probiotic strains: 2.4.1 Resistance to gastric acidity. 2.4.2 Bile acid resistance.
  27. 27. 2.4.3 Antimicrobial activity against potentially pathogenic bacteria (acid and bacteriocin production). 2.4.4 Ability to reduce pathogen adhesion to surfaces 2.4.5 Bile salt hydrolase activity These tests are based on the hostile gut environment which they mimic under in vitro conditions. The cultures evaluated as probiotics based on these tests should be subjected to preclinical validation in appropriate animal models before clinical trials are conducted in human subjects. 2.5 In vivo safety studies in animal models: Assessment of the acute, subacute and chronic toxicity of ingestion of extremely large amounts of probiotics should be carried out for all potential strains. Such assessment may not be necessary for strains with established documented use. 2.6 In vivo efficacy studies in animal models: To substantiate in vitro effects, appropriate, validated animal models must be used first, prior to human trials. 2.7 Evaluation of safety of probiotics for human use: In recognition of the importance of assuring safety, even among group of bacteria that are Generally Recognized as Safe (GRAS)**, probiotics strains needs to be characterized at a minimum with the following tests: 2.7.1 Determination of antibiotic resistance patterns. It should be ascertained that any given probiotic strain is not at significant risk with regard to transferable antibiotic resistance. 2.7.2 Assessment of undesirable side-effects. 2.7.3 If the strain under evaluation belongs to a species that is a known mammalian toxin producer or of hemolytic potential, it must be tested for toxin production and hemolytic activity respectively. Assessment of lack of infectivity by a probiotics strain in immunocompromised individuals would be an added measure. 2.8 Evaluation of efficacy studies in humans: The principal outcome of efficacy studies on probiotics should be proven with similar benefits in human trials, such as statistically and clinically significant improvement in condition, symptoms, signs, wellbeing or quality of life, reduced risk of disease or longer time to next occurrence or faster recovery from illness. Each of the parameter should have proven correlation with the probiotics tested. Probiotics delivered in food may not be tested in Phase 3 studies (effectiveness), unless the product makes a specific health claim wherein it becomes imperative to generate the required evidence necessitating carrying out Phase 3 studies. If a probiotic food has a record of documented long and safe use outside the country, the data regarding this could be reviewed and deemed as sufficient to allow its marketing within the country. However, labeling of health benefits may require evaluation in a different manner. While taking into account studies done abroad, efficacy studies of probiotics (which are of proven benefit in ‘other’ populations) should also be conducted on Indian subjects. It is recommended that such ‘bridging’ human trials should comply with the principles laid down by the Drug Regulatory Authority. Adverse effects, if any, should be monitored and incidents reported to the appropriate authority. 2.9 Effective dosage of probiotic strain / strains: The minimal effective dose or the level of viable cells of the probiotic strain in terms of cfu/ml/day in the carrier food
  28. 28. that demonstrates general health promoting functions or well being or specific health claims in target population should be clearly indicated. 2.10 Labeling Requirements: In addition to the general labeling requirements under the food laws, the following information should also be mentioned on the label (23,39): Genus, species and strain designation following the standard international • nomenclature. The minimum viable numbers of each probiotic strain should be specified at the level at • which efficacy is claimed and at the end of shelf- life. Evidence-based health claim(s) should be clearly stated. • The suggested serving size to deliver the minimum effective quantity of the probiotic • related to the health claim. Proper storage conditions to be mentioned. 2.11 Manufacturing and handling procedures: Adequate quality assurance programmes should be in place. Good Manufacturing Practices should be followed in the manufacture of probiotic foods. The Codex General Principles of Food Hygiene and Guidelines for Application of Hazard Analysis and Critical Control Point (HACCP) (40) should be followed. Guidelines for evaluation of candidate probiotic strains Strain Identification by Phenotypic and Genotypic Methods • • Genus, Species and Strain Deposit strain in an Internationally Recognized collection ↓ Screening of Potential Probiotic Strains • In vitroTests ↓ In vivo studies in validated animal models for: •Safety •Efficacy ↓ In vivo studies in humans for clinical evaluations • Phase1 (safety) • Phase 2 (efficacy) • Phase 3 (effectiveness)* ↓ PROBIOTIC FOODS ↓ Labeling Requirements • Genus, Species, Strain •Minimum viable numbers of probiotics at the level at which efficacy is claimed and at the end of shelf- life. • Health claim(s) • Serving size for efficacy • Storage conditions * Only required if a specific health claim is made
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