Functional Food

Pre- and pro-biotics: the intestinal soothers

M.K. Rana

THERE is a substantial increasing public interest in foods and food components to promote health and lower the risk of non-infectious death causing diseases like cardiovascular heart disease, diabetes, obesity, constipation and certain cancers, especially colo-rectal that are largely associated with diet and human lifestyle. With the augmenting cost of health care people are looking for alternate approach to the prevention and treatment of ailments. It is well establish fact that holistic approach to health prevention has always been better than cure. In advanced countries the people have developed their interest in the potentials of probiotics as therapies and preventive agents since probiotics could be of great use in such conditions either through direct competition with pathogens or the synthesis of specific antibacterial or antiviral compounds. Oral ingestion of probiotics has been documented in several reports to reduce the duration of infectious diseases and the severity of dental caries.

The potentials of probiotics to provide such health benefits can be coupled with the intake of particular live organisms in the form of fermented foods, like curd and yogurt; the health promoting features of these exogenous live organisms, mostly species of lactic acid bacteria (LAB), are just contrasted with the putrefactive actions of intestinal flora. Intake of fermented-milk products enriched with specific strains of Lactobacillus acidophilus or Bifidobacterium bifidum by healthy people led to enhance phagocytosis of Escherichia coli. Probiotics in gastrointestinal tract keep the pathogenic agents under control.

The mechanism for health benefits such as lowering of plasma cholesterol and reduction of cardiovascular heart disease risk is still not so clear but it is believed that only a relatively small proportion of ingested organisms survives passage through the stomach and small intestine and colonizes the large intestine. The consumption of probiotics if discontinues, the organism is washed out of the gastrointestinal tract due to a capacity of the host’s microflora to remain stable in the face of exogenous organisms, therefore, to exert sustained effect incessant ingestion of probiotics appears to be obligatory. Enhanced colonization seems to be a viable option, which means promoting the survival of ingested probiotics to ensure that greater numbers of live organisms reach the large bowel and/or enhancing their residence time in that viscous environment. The caecum and colon are the major sites of bacterial colonization in the human gastrointestinal tract. Relatively few bacterial species and numbers are found in the stomach or small intestine. An ingested probiotic must survive passage through these internal organs and take up active residence in the large bowel. This process can be facilitated by prebiotics, which when combined with probiotics, are synbiotics.

Historical Concepts of Probiotics

As soon as the man is born, he becomes habitat of intense microflora, and later the gastrointestinal tract, the mucous membranes and the skin become the reservoir of microflora, however, all these microorganisms in healthy individuals are inoffensive and sometimes even very useful, thus, probiotic bacteria are generally said to be of human origin and considered non-pathogenic. The origin of cultured dairy products is thought to be as old as the sunrise of civilization and is found mentioned in the sacred books of Hindus and the Bible. Numerous traditional soured milks or cultured dairy products such as kefir, koumiss, leben, yogurt, and curd were developed and therapeutically used before the existence of bacteria was documented. By the 1950s, curd or yogurt was often used to re-establish the balance of gut flora generally upset by regular use of antibiotics and to alleviate or prevent conditions such as constipation, diarrhoea, dyspepsia, cystitis, chronic ulcerative colitis, mucous colitis, and dermatitis.

In the beginning of 20th Century, specific bacteria were presumed to be beneficial to human health. In a book “The Prolongation of Life, published in 1907, the author hypothesized that the bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus)involved in the fermentation of yogurt suppress the putrefactive fermentation of intestinal flora, and per se ingestion of yogurts played a significant role in maintaining good health. Bifidobacteria in breast-fed infants also played a key role in setting up a concept that specific bacteria played a role in maintaining good health, as bifidobacteria displaced the pathogenic bacteria in the gut. The fermented milk products containing L. acidophilus bacteria have therapeutic effects, especially a settling effect on digestion, but their colonization and growth in human gut were essential for effectiveness. A successful Lactobacillus therapy depends on definite principles: it is necessary to use only that organism(L. acidophilus), which is found to be a natural inhabitant of the intestine; the organism should be non-pathogenic and capable of establishing itself in the gut, and should retain viability during processing and transit through the gastrointestinal tract, as a large number of viable cells (107-109) are the prerequisite for swift establishment of this useful flora, and a further important factor for successful therapy is the intake of a high milk-based diet. The live microbiotic strains currently being used predominantly as probiotics in food ingredients are given below in Table 1.

The Probiotics

The term probiotic has been derived from the Greek word meaning ‘for life’, and over the time scientists of various working group evolved the definition of probiotics. In 1960s, reaction against the side effects of antibiotics led to augmented interest of scientists in the use of potential live bacteria as food supplements. In 1965, Lilly and Stillwell first time employed the word probiotic to describe the substances secreted by one organism to stimulate the growth of another, and in 1989, Fuller defined the probiotic as a live microbial food supplement, which beneficially influences the host by improving its intestinal microbial balance. Huis in’t Veld and Havenaar defined probiotics as a mono- or cocktail of cultures of live microbiota, which when given orally to man affects beneficially by escalating the effectiveness of native microflora, and this definition developed the concept of probiotics in a number of ways. It didn’t restrict probiotic actions to only the gut microflora but included the possibility of application to microbial communities at other sites, like respiratory tract, digestive tract, urogenital tract, and skin. A short time ago an EC-supported group of European scientists suggested that probiotics for use in human diet are best defined as live microbial food ingredients that are beneficial to human health.

The term probiotics, by the commonly accepted definition, is applied to live nonpathogenic microbial food supplements that favourably influence the intestinal microbial balance and contribute to the health of the host. An optimum gut microflora balance is considered to be one in which beneficial bacteria such as the Gram-positive lactobacilli and bifidobacteria predominate over potentially injurious bacteria. The probiotics that are usually given orally to help the host to fight against illness and ailments are very beneficial for human, however, for these beneficial effects, the probiotic bacteria must have the ability to colonize in human gut, adhere to intestinal cells, resist to acidity and bile toxicity, antagonize towards pathogens, yield antibiotic substances, offer proven health-benefits and a history of safe use in human beings. Probiotic bacteria that satisfy all these criteria are numerous, i.e., Lactobacilli, Bifidobacterium, and Streptococcus thermophilus; some of these organisms also known as friendly-bacteria or designer bugs are commercially available in the form of pure and mixed cultures.

One the most commonly known probiotic, Lactobacillus acidophilus, is naturally occurring bacterium in foods such as curd, and grains and meat products. Lactobacilli play an important role in the control of undesirable microbiota in the gastrointestinal and urogenital tracts. Besides indigenous lactobacilli, which naturally reside in the human gastrointestinal tract, distinct lactobacillus strains from fermented food products have shown beneficial effects on gut health. These probiotic bacteria are live microbial food supplements with health benefits to the host by increasing the population of intestinal microorganisms.

The Prebiotics

Prebiotics are the non-digestible part of the foods that the probiotics need to stimulate the metabolism of foods. They feed the beneficial bacteria and modify the composition of intestinal microflora as a result the probiotics can predominate. The latest concept of prebiotics envisages the use of non-digestible carbohydrates, predominantly oligosaccharides and growth factors, which are specific to a limited number of bacterial species from the colonic microflora. They are designed to favour the proliferation and activity of useful bacteria and to inhibit the activity of harmful colonic bacteria. Prebiotics are the complex carbohydrates-oligosaccharides such as inulin and short chain sugars, which are being extensively used. They pass undigested from the lower intestine to the colon where beneficial bacteria consume them, releasing vitamins and other nutrients. They may also curb the growth of food pathogenic bacteria such as Salmonella and Campylobacter by changing the colonic environment.

Prebiotics as Supportive to Probiotics

Prebiotics are the growth substrates for beneficial colonic bacteria, and being non-digestible food ingredient these affect the host beneficially by stimulating the growth and activity of a number of colonic bacteria, and per se improve the health of the host. A prebiotic acts to stimulate the growth of microflora in the large bowel, however, this concept fails to notice the fact that synbiotics have the health potential, and that increasing the numbers and the activity of ingested organisms within the colon by enhancing their survival as they pass through the hostile environment of the upper gastrointestinal tract will have the same net effect as stimulating growth of related organisms in the large bowel. A capsule containing probiotics could be a prebiotic if it increases the population of favourable organisms in the large bowel.

Prebiotics are different from colonic foods in that the latter serve as general fuel for endogenous colonic microflora, thus, providing the host with energy, metabolic substrates and essential micronutrients. Prebiotics, i.e., oligosaccharides,principally the fructo-oligosaccharides and galacto-oligosaccharides are usually indigestible by human digestive enzymes and have well-documented effects on the large-bowel microflora. Although they are classified as colonic foods but they also satisfy the criteria for prebiotics. however, other food ingredients and components regarded formerly as colonic foods, e.g., partially hydrolyzed guar gum, appear to promote the populations of probiotics. One dietary component showing great potential as a prebiotic and colonic food is resistant starch, which is emerging as a major factor in the bacterial ecology of the gut.

Starch, which offers more than 50% of the daily energy intake largely from grains, is a sizeable part of human diets. however, in affluent societies, where interest in prebiotics and probiotics is on the increase, its average consumption is much lower, possibly as little as 25% of the daily energy intake. Starch is the only naturally occurring food polysaccharide, which can be digested by the intrinsic digestive enzymes. Earlier it was thought that starch digestion in small intestine got completed since a very little of starch was noticed in faeces, but later the extensive studies demonstrated that an ample proportion of the starch escapes in the large bowel. This escaped starch was named as resistant starch and was defined as the sum of starch and products of starch degradation not absorbed by the small intestine of even healthy individuals. Starch may be digested slowly in the small intestine but if the rate of passage of the food is sufficiently slow, digestion may complete and no resistant starch, which includes oligosaccharides and other products of small intestinal starch hydrolysis, will be left undigested.

In foodstuffs, the resistant starch exists because of a variety of reasons. Raw starches in unripe banana and other starchy fruits are digested poorly, however, their digestibility can be enhanced by cooking, especially in water, which gelatinizes the raw starch giving greater access to a-amylase enzyme. Disruption of food structure by either milling or chewing also enhances digestion by activating the enzymes involved in digestion of food. Though cooking increases starch digestibility but subsequent cooling of cooked food leads to the formation of crystallites that are usually resistant to digestion. This process is termed retro-gradation and the resistant starch content of foods appears to increase when they are subject to increasing number of heating and cooling cycles. Chemical structure is an important factor in starch digestibility, especially amylose (amylopectin). Most food starches largely have about 70% amylopectin, and after digestion the remainder is amylose. The greater the content of amylose, the more difficult the starch is to gelatinize and the more susceptible to retro-gradation. Ungelatinized high-amylose starches (60-70% of the total starch) are resistant to amylolysis and are used commercially as an ingredient to raise the resistant starch content of processed foods. The factors such as chewing may also have impact on the amount of resistant starch in a food, as chewing dictates the particle size of an ingested food, with highly masticated foods having a smaller particle size. Large particles travel more rapidly through the gut than smaller ones so greater mastication would be expected to increase digestibility. Studies have shown that the digestibility of finely ground rice is greater than that of coarse rice, however, all species of resistant starch do not have the similar effects, especially as prebiotics on microflora in the large-bowel.

The fermentation of complex carbohydrates by the microflora present in large-bowel is important for good human health. The metabolic products, especially short-chain fatty acids (SCFA) have emerged as important metabolic fuels for colonocytes as well as having specific actions that promote normal colonic function. It is assumed that NSP (major components of dietary fiber) are the principal fermentative substrates. The studies show that majority of population is generally consuming <20g NSP per capita per day, which is too below than the normally required quantity (60-80g substrate per day per capita) to sustain 1013-1014 organisms population needed in large bowel of healthy human, and resistant starch is the substance that most likely fills this carbohydrate gap. Indeed, it appears plausible that some populations at low risk of large bowel disease including cancer consume relatively little NSP but their diets are high in starch. It could be suggested that eating foods containing resistant starch can lower the frequency of probiotics consumption.

Most probably resistant starch has health-promoting actions on colonic microflora above and beyond the prebiotic effect. Studies show that ingestion of resistant starch plus the usual hydration therapy reduces fluid loss in children with cholera-induced diarrhoea and also reduces the recovery time to half. Both resistant starch (as green banana) and NSP facilitate recovery in children from infectious diarrhoea. Ingesting cooked rice, which is good source of resistant starch, lowers the occurrence and severity of the disease. This benefit is thought to be due to increased fluid absorption through greater SCFA production, as these acids stimulate the uptake of water and cations (Na+, K+ and Ca2+). Short chain fatty acids also appear to modulate the muscular activity of the large bowel and to promote the flow of blood through the viscera; both these actions could assist in lowering the severity of diarrhoea. It is possible that resistant starch could limit the viability of cholera organism in the gut. The bacteria are supposed to adhere to starch granules very much as the bifidobacteria, and per se are removed from the site of infection. Both, the high-amylose starch and the rice product also lower the gut pH, which is a further means of biocontrol for infection causing latent pathogens. In this way given such probiotics orally to the patients can speed up the recovery from diarrhoea, and the maximum effectiveness can be achieved if the prebiotic is used as a mixture of oligosaccharide and resistant starch, as these agents in combination seem to have additive effects.

Inclusion of Prebiotics in Infant Formulas

The foremost idea of adding prebiotics to infant formulas is to modulate the intestinal microflora, and the general aim is to promote long-term effects on the health of infants. The galacto-oligosaccharides obtained from lactose by an enzymatic reaction are incorporated in infant formulas. The infant formula containing metabolites obtained from the fermentation of milk with Bifidobacterium breve and Streptococcus thermophilus appears to have active components to stimulate the growth of bifidobacteria, which are considered as traditional prebiotics. In adults, the ingestion of fructo-oligosaccharides and galacto-oligosaccharides stimulate the population of bifidobacteria and lactobacilli in faecal but do not show similar actions in infants.
In infants receiving prebiotics supplemented formulas, there was a dose-dependent softening of stool, a decrease in stool pH and an increase in defecation frequency. The use of prebiotics has been proposed in the treatment of constipation and bowel discomfort. Fructo-oligosaccharides consumption has been found to be associated with a reduction in febrile events, vomiting, antibiotic use and day-care absenteeism.

Mechanisms of Probiotics to be Defensive

The production of secondary metabolites, i.e., lactate and specific compounds such as bacteriocins, inhibits the growth of disease causing pathogens in large bowel, and this inhibitory effect could be easily enhanced through the immune stimulatory effects of probiotics. The dietary complex carbohydrates help in emptying large bowel where they provide preventive and therapeutic actions and consequently influence the health of large bowel either through physical bulking or through fermentation of resistant starch and oligosaccharides. The foods containing high insoluble fiber (cereals bran) are particularly effective in promoting bowel emptying. Human studies suggest that some forms of resistant starch increase butyrate production, whereby raising butyrate levels in large bowel could be of positive health benefit and particularly important in the distal colon, which is the major site for most of the large bowel diseases. Short-chain fatty acids production and availability predominate in the proximal large bowel where fermentation is greatest, reflecting substrate supply. Resistant starch can modify large bowel and faecal short-chain fatty acids favorably. Lactic acid bacteria (lactobacilli and bifidobacteria) are found most abundantly in gastrointestinal tract of adult and milk-fed infants where bifidobacteria fermentation appears to predominate. Yogurt does not contain natural free butyrate at high levels but the currently available probiotics stimulate butyrate production in the human colon.

Mucosal epithelial surfaces such as urogenital tract, respiratory system and gastrointestinal tract are the sites at which the host encounters a numerous species of microorganisms derived from the surrounding atmosphere, and through which the pathogens invade the host or colonize to initiate infections. It is well known that the gastrointestinal tract harbours a rich microflora, which entertains a symbiotic interaction with its host, though such extensive mucosal surfaces are well protected. In addition to host-derived innate immunity, colonized mucosal organs are also protected by the native microflora, which creates a barrier effect against harming pathogens, and body systems have the different mechanisms such as competition for metabolic substrates, occupation of available niches at mucosal level, and the production of regulatory short-chain fatty acids and bacteriocins. Increasing beneficial components of gastro-intestinal microflora, health benefits can be rendered to the host at the non-sterile mucosal surfaces, thus, in diet live bacteria (probiotics), which interact with and improve the functions of intestinal microflora, should regularly be inoculated, and a bacterial substrate, i.e., oligosaccharides (prebiotics) that promote the growth of beneficial microbiota such as bifidobacteria should also be administered in the diet.

The stomach is such an organ that is almost sterile because of very low pH (1-2); this constitutes a very efficient non-specific mechanism of defense against infections because it constrains the survival of pathogenic bacteria, thus, gastric acidity is the rust barrier effect usually occurs in the gastrointestinal tract, however, probiotic live bacteria that are administered to healthy people also have to face the stress of high acidity, hence, very few probiotic microbiota, i.e., Lactobacillus reuteri (various strains), Lactobacillus gasseri strain ADH, Lactobacillus johnsonii strain La1 (formerly L. acidophilus), Lactobacillus caseilrhamnosus strain GG, Lactobacillus casei (various strains), Lactobacillus plantarum 299, Bifidobacterium spp., and Saccharomyces boulardiiare, are actually able to survive in high numbers passage through the stomach and the intestine.

Probiotics protect the host against pathogens by two mechanisms (1) the barrier effect or colonization resistance, and (2) modulation of the host’s own defense mechanism, thus, in order to antagonize small infection causing agents such as Salmonella spp., Listeria monocytogenes, Escherichia coli, Shigella flexneri, Botrytis, Yersinia spp. and certain viruses such as reovirus and poliovirus, the most beneficial strains of probiotic organisms have to be present in an environment where no truly stable microflora exists. The barrier effect of probiotics in small intestines needs the presence of the protective strains at cellular target of the pathogen. Studies have demonstrated that adhesive strains of lactic acid bacteria produce a better mucosal barrier effect against disease causing pathogens in small bowel than non-adhesive ones. Few strains of Lactobacilli are able to secrete bacteriocins (anti-bacterial substances) that are actively against Salmonella typhimurium. Probiotics can modulate the host immune system, both at a local mucosal immune level and systemically. In using probiotics to promote immune function and thereby afford the host better protection the foremost aim is to enhance non-specific immunity without triggering a pro-inflammatory response that could potentially be harmful. Human studies show that both L. johnsonii La1 and L. salivarius UCC 118 can modulate the immune system in healthy humans, boosting innate immune defense mechanisms, and these strains do not induce inflammatory responses.

Therapeutic Competence of Probiotics

Probiotics have the potential to offer major benefits to human health. Increasingly probiotics are being used as prophylactic with promising results among potentially vulnerable groups, and being evaluated as adjuncts in the treatment of many ailments such as inflammatory bowel disease, food allergy, oral rehydration therapy, Helicobacter Pylori infection, bladder cancer and urogenital infections. Probiotics are energy salvage, modulation of cell growth and differentiation, antagonism against pathogens, innate immunity against infection, immune stimulation, reduction of blood lipids and serum cholesterol, overcoming lactose intolerance, correcting gastrointestinal disorders, inhibiting Helicobacter pylori responsible for peptic, gastric and duodenal ulcers, anticarcinogenicity, diminishing the recurrence of superficial bladder tumours, reduction of harmful intestinal microbial enzyme activity, decreasing faecal mutagenicity, reduction of the duration of rotavirus diarrhoea and production of vitamins. They stimulate the antibody and cell-mediated immune responses. In healthy gut, the immune system is able to create a balance between protective mucosal immunity and tolerance to dietary antigens, but in food allergies this balance is impaired. Probiotic intervention may help to alleviate the symptoms of food allergy by modulating the immune response through beneficial modification of the intestinal flora.

In patients suffering from hyperacidity, the regular use of antacids or H1 blockers (Cimetidine, Ranitidine, Lansprazole, Omeprazole, Acidoprazole, Rabeprazole, Esomeprazole etc.) that suppress acid secretion leads to reduced gastric acidity (increased stomach pH) and subsequent colonization by potential pathogenic bacteria such as Helicobacter pylori,the causative organism in many peptic ulcers and strongly associated with gastric cancer, becomes more prevalent with age and the consequences are generally mal-absorption and higher risk of infectious problems due to Gram (-ve) bacteria. Numerous reports indicate that probiotics may contribute a lot to the control of H. pylori infections. The recent epidemiological reports reveal that the consumption of whey-based medium fermented by Lactobacillus johnsonii La1 strain is noticed associated with lower incidence of ulcers and bladder cancer to some extent in specific populations. Some strains of lactic acid bacteria have been shown to inhibit.

In hospitalized patients, during treatment diarrhoea associated with antibiotic is a major complication as a result of frequent use of antibiotics, which disrupt the steady state of the colonic microflora, and consequently overgrowth of opportunistic disease causing pathogens in small intestine. Consumption of foods such as fermented milks containing probiotic strains and prebiotic factors have been advised to improve physical condition of general population. Probiotics are actively useful in avoidance of intestinal infections. Furthermore, it seems increasingly important to find alternatives to standard antibiotic treatments for gastrointestinal infections. The regular consumption of probiotics and prebiotics in a preventive fashion could diminish the requirements for antibiotic treatments in subjects that are at risk of infections. Probiotics could prevent medical complications such as antibiotic-associated diarrhoea or septic conditions. Administration of probiotic bacteria can be beneficial in reducing the incidence and duration of acute diarrhoea, though it is imperative that safe probiotic strains are used, and probiotic species should be adapted to different microenvironments of gastrointestinal tract to accomplish their protective function. The probiotics such as Saccharomyces boulardii, Lactobacillus casei GG 4 and Enterococcus faecium have been suggested for use either to prevent or to treat the diarrhoea associated with antibiotics. A mixture of Lactobacillus acidophilus and Lactobacillus bulgaricus has showed high efficacy in this respect. Clostridium difficile can also be associated with recurrent colitis, and the use of probiotics has been shown to be more beneficial in this situation. The probiotic yeast S. boulardii has been proven to have some beneficial effects in diarrhoea associated with HIV.

The term inflammatory bowel disease (IBD) is implied for a variety of incurable immune mediated diseases with unknown etiology that results in chronic relapsing inflammation of the gut. The two major clinical forms of IBD that significantly impair the quality of life of patients and substantially increase the risk of intestinal cancer are Crohn’s disease and ulcerative colitis. Besides genetic predisposition, environmental factors including the composition and activity of intestinal microbiota also play a role in the development of these diseases. However, probiotics may be useful in controlling IBD by modulating the useful intestinal microbiota. The reduction in inflammation is also translated to a reduction in cancer incidence. A reduction in Enterococci and Clostridia having strong association with colon cancer has also been noticed in healthy humans upon consumption of probiotic L. salivarius UCC 118, which establishes beneficial modifications to the intestinal microbiota, resulting in reduced inflammation, and therefore, a decrease in cancer incidence. In most of the cases, the probiotics improve the quality of life, and thus, patients are requested to continue probiotic therapy. It is safe to use the probiotic strain L. salivarius UCCl18, which does not exacerbate Crohn’s disease. Probiotics may bestow prophylactic benefits against infection, especially in infants borne at premature stage and in HIV positive subjects.

Market dynamics

The modulation of composition of microflora might be more easily obtained by ingesting prebiotics that favour the production of antibacterial metabolites such as short-chain fatty acids or bacteriocins that are implicated in the control of newcomers into a steady existing microflora. There are validations that the currently used probiotic microbes are safe in their use. Commercialization of this probiotic concept has played a key role in growing awareness of the role of gut flora in health and in publicizing information concerning potential benefits of probiotic therapy. In last few years the sale of probiotic yogurts containing Lactobacillus acidophilus and Biofidobacterium cultures has increased in Australia and USA too. In Asian and European countries too, the consumers are now buying prebiotic formulated food products to boost the populations of Biofidobacterium bacteria in the gut. Presently, the five-bifidobacteria species that have captivated the attention in dairy industry for manufacturing probiotic milk products are- Bifidobacterium adolescentis, B. bifidum, B. breve, B. infants and B. longum.

Safety of Probiotics

The nutraceutical and pharmaceutical industries are widely using the probiotic microorganisms without causing any health problems to the consumers. It has been demonstrated by the working group scientists that probiotic strains in certain cases can significantly ameliorate measurable clinical end-points of disease, and prominently can make the life easeful. In most of the cases, probiotics have been shown to be safe and considerably promise, however, some pathologies and side effects have been described for Saccharomyces boulardii and lactobacilli. Risk factors have been identified for lactobacilli that are involved in the production of some proteinases and glycosidases, platelet aggregation. One should take care of that the probiotic microorganisms should not boost up gut permeability, which is the prime cause of allergy or inflammation. o

Table 1: Live microbiotic strains currently being used as probiotics in food ingredients

Lactobacilli Bifidobacteria Non-Lactic acid bacteria
Lactobacillus acidophilus Bifidobacterium adolescentis Bifidobacterium cereus
Lactobacillus casei Bifidobacterium animalis Clostridium butyricum
Lactobacillus crispatus Bifidobacterium bifidum Escherichia coli
Lactobacillus johnsonii La Bifidobacterium breve Saccharomyces boulardii
Lactobacillus paracasei F19 Bifidobacterium infantis  
Lactobacillus plantarum UCC 35624  
Lactobacillus reuteri Bifidobacterium lactis Bb12  
Lactobacillus rhamnosus Bifidobacterium longum  
Lactobacillus salvarius    




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