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PROBIOTICS, PREBIOTICSAND THE GUT MICROBIOTAbyNino Binns

2 Concise Monograph SeriesINTRODUCTIONMicrobes, or micro-organisms, include bacteria, fungi,yeasts and algae. They can be found everywhere onEarth, including hostile environments like volcanoes,the ocean bed and deserts. They are incredibly diverseand have adapted over millions of years to occupy theirown particular niches. As far as humans are concerned,microbes are best known for their role in causingdisease, but their power has also been harnessed formillennia to the benefit of humankind. They are usedin the production of fermented foods including dairyproducts, breads, vegetables and, of course, wines andbeers to name but a few. Owing to their potential forvery selective action, microbes are also crucial to thedevelopment and production of pharmaceuticals suchas antibiotics and to the production of food ingredientssuch as vitamins and citric acid. Microbes are alsoinvolved in the production of many other chemicals andenzymes and are used in waste processing.Most of the 10 14 bacteria in the gut are found in the largeintestine (colon) and, over the past 30 years or more,interest in the gut microbial population – the microbiota– and its environment has intensified. Numerousresearch studies have shown that, far from being passiveinhabitants of the gastrointestinal (GI) tract, the habitualresidents of the gut (commensal micro-organisms)interact with their host in a very intricate manner. Theymay modulate the effect of potentially harmful bacteria,impact the host’s GI tract, digestion, metabolism andimmune system, and might even influence functionsbeyond the gut.The concept that food-borne bacteria can be beneficialto health emerged at the turn of the twentieth centuryand is usually attributed to Nobel Prize-winningRussian scientist Ilya Metchnikoff. He hypothesised thatconsuming large amounts of fermented milk productsthat contained Lactobacillus bacteria (“soured milk”)could prolong and improve the quality of life becausethese bacteria entered the colon and limited the activitiesof undesirable microbes. Metchnikoff therefore saw theintestinal tract as an organ that could be manipulatedto improve health by adding exogenous bacteria tothe gut. As a result, commercial yogurts and fermentedmilks gained some popularity after the First World War,but it was not until the 1980s that the sales of productscontaining probiotics began to grow rapidly - first inJapan and then extending to Europe during the 1990s.Probiotic bacteria may be defined as ‘live microorganismswhich, when administered in adequateamounts, confer a health benefit on the host’ (FAO/WHO2001). They can interact with commensal bacteria andcan also have a direct impact on the host. Disentanglingthese interactions is one of the key challenges for futureresearch. Other key challenges are to understand theirmechanisms of action, to elucidate more specificallywhich probiotic strains can offer which health benefitsand to define the intake levels needed to achieve thoseeffects.The prebiotic concept developed more recently. TheJapanese were the first to recognise the value of nondigestibleoligosaccharides, initially in animal feedwhere their addition to the feed of piglets helped relieveand prevent scouring (diarrhoea). Japanese researchersalso recognised the value of oligosaccharides in humanmilk and later demonstrated that consumption of fructooligosaccharidesand galacto-oligosaccharides led to anincrease in intestinal bifidobacteria and stimulated theirgrowth in the human gut. However, it was not until 1995that the scientific concept for human gut microbiotamodulation by “prebiotics” was introduced. Since then,a wealth of research information has accumulated. Aprebiotic may be defined as “a selectively fermented

6 Concise Monograph SeriesRecent research also suggests that the normal microbiotais not simply a collection of micro-organisms, but reflects aninter-relationship between different groups that may worktogether to the benefit of the host. In addition, the currentthinking is that harbouring a wide diversity of organisms inthe GI tract is beneficial to the host.Bacterial fermentation andmetabolismAs living organisms, all microbes require a source ofenergy in order to grow and reproduce. Many microbesferment carbohydrates (saccharolytic fermentation), anactivity that is harnessed by humans in the production ofvarious food products. For example, in wine production,yeast ferments the sugars in grape juice to yield alcohol.In yogurt production, bacteria such as lactobacilli andstreptococci ferment milk sugar (lactose) to lactic acidto develop the characteristic tart flavour. In sauerkrautproduction, the bacteria naturally present in cabbageferment sugars to lactic acid in the absence of oxygenand the presence of 2-3% salt.In like manner, microbes in the first part of the colon meettheir energy needs by fermenting dietary and endogenousresidues that have escaped digestion and absorption inthe upper GI tract (Table 2 and Figure 2). Many microbesTABLE 2.Bacteria, their mode of action on substrates and the products of fermentation (adapted from Salminen, 1998)Bacteria Mode of action on substrates Fermentation productsBacteroides Saccharolytic, peptolytic, aa-fermenting Ac, Pr, Su, AmEubacteria Saccharolytic, some aa-fermenting species Ac, Bu, La, Am, SulBifidobacteria Saccharolytic Ac, La, f, EtOHRuminococci Saccharolytic AcPeptostreptococci Saccharolytic, some aa-fermenting species Ac, La, AmPeptococci aa-fermentation Ac, Bu, La, AmClostridia Saccharolytic, some aa-fermenting species Ac, Pr, Bu, La, EtOH, Am, SulLactobacilli Saccharolytic LaPropionibacteria Saccaharolytic, lactate fermentation Ac, Pr, AmActinomyces Saccharolytic Ac, PrStreptococci Carbohydrate and aa-fermentation La, Ac, Am, SulMethanobrevibacter Chemolithotrophic CH4Escherichia Carbohydrate and aa-fermentation Mixed acids, AmDesulfovibrio Various Ac, SulFusobacteria aa-fermentation, assimilation of carbohydrates Bu, Ac, La, Am, Sulaa, amino acid; Ac, acetate; Am, amines; Bu, butyrate; EtOH, ethanol; f, formate; La, lactate; Pr, propionate; Su, succinate; Sul, sulphides

Probiotics, Prebiotics and the Gut Microbiota 7FIGURE 2.Diagram showing the principle metabolic activity in the colonSource: Prof. R. Rastall, University of Reading, UKmetabolise carbohydrates and dietary fibre 1 , includingpolysaccharides (such as pectins, hemicelluloses, gums,inulin and resistant starches), oligosaccharides (such asraffinose, stachyose, fructo-oligosaccharides, galactooligosaccharidesand resistant dextrins), sugars (lactulose,non-absorbed lactose and non-absorbed fructose) andpolyols (such as mannitol, lactitol, maltitol and isomalt).The main species in the colonic microbiota that fermentcarbohydrates belong to the genera Bacteroides,Bifidobacterium, Ruminococcus, Eubacterium andLactobacillus. This microbial action results in the productionof the short chain fatty acids (SCFA) acetic, propionic andbutyric acids and of lactic acid (which is mostly convertedto acetic and propionic acid by gut microbes). The natureof the fermentation products depends partly on thesubstrate fermented and the type of bacteria (Table 2) andalso on other individual host factors. SCFA are absorbed,enhancing the uptake of water and salts, and are used as asource of energy by the host. Butyric acid is also the majorsource of energy of the epithelial cells lining the colon andcan impact cell growth and differentiation.The gases hydrogen, methane and carbon dioxide are alsoproduced and may contribute to the equilibrium of themicrobiota. In addition, these gases can cause flatulenceand distension, which can lead to intestinal discomfort ifthe dietary intake of fermentable substrates is suddenlyincreased.1. Note that the legal definition of dietary fibre differs around the world. The term dietary fibre is used here only in a general sense to refer to thedietary components listed.

8 Concise Monograph SeriesBacteria also metabolise other components found intheir environment (Figure 2). In addition to foodstuffsconsumed by the host and not fully digested, substratesfor bacterial growth include degraded bacterial cells andhost-derived mucins, enzymes and sloughed-off intestinalcells. Peptococci and clostridia species metabolise proteinsas a source of nitrogen for growth and yield branchedchain fatty acids such as isobutyrate and isovalerate aswell as a range of nitrogenous and sulphur-containingcompounds, some of which may be harmful. For example,ammonia, amines and phenolic compounds can, undercertain conditions, lead to the formation of carcinogens,particularly in the left, descending colon where putrefactiveconditions can prevail. Phytochemicals such as isoflavonesand polyphenols may also be metabolised, yielding smallercomponents like equol and small phenolic molecules thatare more readily absorbed. The impact of this microbialactivity on human health is still under investigation.As bacteria grow in numbers, they contribute to the bulkof the stools that form in the rectum. High stool bulk isrelated to a shorter gut transit time and also to a lowerrisk of constipation and bowel cancer. Although nonfermentabledietary fibre sources such as wheat bran fibreare the most important contributors to stool bulk, bacterialmass resulting from the fermentation of more solubledietary fibres and carbohydrate residues also contributesto the bulk.The GI epithelial barrier and immunesystemThe GI tract is sometimes described as the body’s largestimmune organ. It represents the host’s greatest areaof mucosal contact with the environment and containsas many as 80% of all antibody-producing cells. Theintestinal microbiota is also a vital part of the body’sdefence system.At birth, the GI tract is essentially sterile and, in addition, thenewborn’s immune system is not fully mature. The immunesystem only becomes functionally mature as a result ofexposure to the myriad of foreign substances encounteredby the naive intestinal tract. Studies on animals raised ingerm-free conditions have shown that the immune systemis poorly developed in such animals and that they havelower levels of immunoglobulins and fewer specialisedimmune cells in their intestinal mucosa. Germ-free animalsare thus much more susceptible to disease than are thosethat are conventionally reared. It is also known from thesestudies that microbial antigens, derived from the intestinalmicrobiota as well as the environment, play a crucial role inthe maturation of gut-associated lymphoid tissue (GALT)and normal resistance to disease.The GALT is organised into different compartments suchas lymph nodes, lymph follicles and Peyer’s patches(Figure 3). The GALT limits the passage of bacteriaand food antigens from the GI lumen through theintestinal mucosa. It does, however, allow the passageof antigens (minute samples of viable or dead bacteriaand protein and peptide fragments) using specialisedcells such as the M cells that cover the Peyer’s patchesand the dendritic cells that act as sentinels along themucosa. These so-called antigen-presenting cells (APCs)process and present the antigens to lymphocytes, atype of immune cell. The APCs are thus very importantin stimulating a balanced immune response and, as isincreasingly documented, having an impact beyond thegut (see the section “Cross-talk with the host”). It hasbeen hypothesised that reduced exposure to exogenousmicrobes in developed and industrialised countries hasled to increased incidence of chronic immune dysfunction,leading to atopic (allergic) and auto-immune disorders orinflammatory bowel disease, because of changes in theway the immune system has matured. This is known asthe “hygiene hypothesis”.

Probiotics, Prebiotics and the Gut Microbiota 9FIGURE 3.Schematic overview of the lymphoid elements of the gut-associated lymphatic systemPeyer’s patches (PP) and mesenteric lymph nodes (MLN) are organised intestinal lymphoid follicles. (A–C) Pathways of intestinal antigen uptake:luminal antigen can be taken up by (A) intestinal epithelial cells, (B) interdigitating lamina propria dendritic cells, and (C) M cells. The lymphaticdrainage of PP and villus lamina propria goes to the MLNs (direction of lymph flow is indicated by arrows). Reproduced from Spahn and Kucharzik(2004) with permission from BMJ Publishing Group Ltd.The integrity of the epithelial lining of the GI tract is crucialto health and the disruption of this intestinal barrier mayincrease the risk of certain intestinal disorders or diseases.Epithelial cells have become specialised and adopt anumber of strategies for defence against pathogens.Goblet cells secrete mucins (high molecular weightglycoproteins), which act as a layer that helps protectthe underlying epithelial cells from mechanical damageand the direct action of chemical compounds that areingested or derived endogenously from gut secretions.The amount and composition of mucus produced by thegut varies by site. The small intestine has a thick, quitemobile layer of mucus whereas the colon has two layers:a mobile layer similar to that of the small intestine anda second thinner layer that is much more viscous andimpermeable than the mobile mucous layer. Althoughmicrobes reside predominantly in the lumen of the GItract, they are also associated with the mucous layerand may adhere to the cells lining certain areas of thesmall intestine if the mucous layer is compromised. Here,beneficial microbes may compete with pathogens.Both the mucous layer and the epithelial cells are designedto allow selected nutrients and other dietary componentsto penetrate and, in some cases, pass through them.

10 Concise Monograph SeriesIn addition, some components pass through the intercellularspaces. Proteins known as occludins and claudinshelp police the small intercellular space (tight junction)between cells to control access by foreign molecules andparticles.Specialised Paneth cells, located in the crypts of thesmall intestine, produce antibacterial peptides known asdefensins as well as defensive enzymes (such as lysozyme)and cytokines that help protect the host from pathogenicmicro-organisms.Techniques for exploring the GImicrobiotaIn the past, microbes taken from their initial source(whether food, blood, tissue or excreta) werecharacterised after culturing them in a laboratory. Thecultured micro-organisms could then be counted andidentified by microscopy, biochemical observation andother taxonomical (identification) tests (see section“Characterisation and taxonomy” for information ontaxonomy).Faecal sampling has always been the mainstay of analysisof the human gut microbiota, especially given the limitedaccessibility of other GI sites. An inherent limitation ofthis approach is that the micro-organisms expelled in thefaeces and cultured in the laboratory do not necessarilyaccurately reflect what can be found in different segmentsof the gut, particularly the upper gut. Even colonic biopsysamples may not accurately reflect the in vivo situationbecause, prior to their excision, the colon is cleared withlaxatives, which disturbs the endogenous microbiota.Another challenge in understanding the composition ofthe gut microbiota is that numerous microbes have not yetbeen successfully cultivated under laboratory conditions.In the early 1990s, research scientists developed atechnique called fluorescence in situ hybridisation (FISH).By using fluorescent probes directed to highly variableregions of the 16S ribosomal ribonucleic acid (rRNA)within the bacterial cells, different species and even subspeciesof bacteria could be identified and quantified.From the mid-1990s, sequence analysis of 16S ribosomalDNA, often obtained by polymerase chain reaction(PCR), was possible, which enabled microbiologists todetect and identify micro-organisms without the needto culture them. Simultaneously, sequence analysisrevealed a far greater diversity than previously detectedby culturing. These techniques have allowed moreaccurate detection and identification of specific species,especially ones that were previously unknown or difficultto culture, from faecal or intestinal samples. Cultureindependentanalysis of faecal samples has thus led toan increased understanding of the complexity of theintestinal microbiota. Modern techniques also allow veryhigh numbers of samples to be analysed in parallel andthus have increased the knowledge of inter-individualvariation and stability of the microbiota within individuals.The co-development of high-throughput DNAsequencing technology and information technology (bioinformatics)has enabled clustering and analysis of largeamounts of data such that researchers have embarkedupon major new projects to study the human microbiome– a term that refers to the collective genomes of allmicro-organisms present in an ecosystem, in this case,the human body. The Human Microbiome Project (USAled)and the MetaHIT project (Europe-led) compriselarge research consortia that have started to study andcharacterise the complete microbial population of thehuman intestine and other parts of the body, with theaim of associating the composition and function ofthe microbiome with health and disease. A great deal

Probiotics, Prebiotics and the Gut Microbiota 11of current research on probiotics and prebiotics alsointerfaces with these ongoing research programmeson commensal bacteria. All these projects will help toshed light on the role of microbes, both commensal andingested, in human health.Analysis of the intestinal microbiota has made tremendousprogress, in particular in the past two decades with themainstream use of various molecular techniques. Thesetechniques have made it possible to both investigatethe unknown members of the microbiota and theirfunctionality as well as follow specific strains. A numberof challenges remain, however. Analysis primarily remainsrestricted to faecal samples that may not be representativeof the microbiota higher up the GI tract or the microbiotaassociated with the mucosal surfaces. On the analyticalside, new techniques allow the accurate and quantitativeanalysis of the microbiota and, although the detectionlimits may currently still be too high to capture all the minorcomponents of the intestinal microbiota, it is reasonableto assume this will improve in the future. More powerfulcomputers and new statistical algorithms will also berequired to deal with the ever-increasing amount of data.THE PROBIOTIC CONCEPTDefinition and historyThe word “probiotic” (origins: Latin pro meaning “for”and Greek bios meaning “life”) was first used in 1954to indicate substances that were required for a healthylife. Out of a number of definitions, the most widely usedand accepted definition is that proposed by a joint FAO/WHO panel (FAO/WHO, 2001): “Live micro-organismswhich, when administered in adequate amounts, confera health benefit on the host”.As mentioned, the original proposal that certain bacteriacould benefit human health is usually attributed to IlyaMetchnikoff, who worked at the Pasteur Institute at thebeginning of the twentieth century. His insights still haveresonance today:“The dependence of the intestinal microbes on thefood makes it possible to adopt measures to modify theflora in our bodies and to replace the harmful microbesby useful microbes” and “systematic investigationsshould be made on the relation of gut microbes toprecocious old age, and on the influence of diets whichprevent intestinal putrefaction in prolonging life andmaintaining the forces of the body.”A French paediatrician, Henry Tissier, also publishedinformation at around the same time about his work onyoung children with diarrhoea. He found that their stoolscontained fewer unusual Y-shaped (“bifid”) bacteriathan did stools from their healthy peers and suggestedthat patients with diarrhoea could be treated with these“bifid” bacteria to help restore a healthy gut microbiota.Until recently, high quality scientific research supportingthe purported benefits of probiotics was limited, partly

14 Concise Monograph Seriesor if any safety concerns can be defined and excluded,the organism may be granted QPS status. Thus, forany strain of micro-organism that can be unequivocallydemonstrated to be from a qualified QPS group (suchas Lactobacillus or Bifidobacterium), further safetyassessment is limited to tests for antibiotic resistance. Ifa microbe is not covered by QPS, then a comprehensiveassessment of safety is likely to be required before it canbe used in the food supply.Application of probiotics in foodProbiotic organisms are used in a variety of foods, themain category being dairy products, but they are alsopresent as food supplements in capsule or tablet form.Since viability is an essential property of a probiotic, thefinal product must contain an adequate amount of livingprobiotic(s) until the end of its shelf life. A health claim forthe addition of probiotics to foods or food supplementsshould only be made if there are documented benefitsbased on good quality human trials conducted with therelevant food product containing the specific strain thatis the subject of the claim and using relevant endpoints.These studies should also be able to demonstrate thesafe, effective dose of the probiotic organism in food.Like legislation on food safety, regulation of health claimsfor foods varies by country or region and any claims oncommercial products containing probiotics must adhereto requirements, which in some cases include pre-marketapproval of the claim by the regulatory authorities.THE PREBIOTIC CONCEPTDefinition and historyAs mentioned, the Japanese were the first to recognise thevalue of fermentable oligosaccharides, initially in feedingpiglets and later, during the 1980s, with the identificationof human milk oligosaccharides. However, it was notuntil 1995 that the prebiotic concept for modulation ofgut microbiota was introduced. Although a number ofdefinitions have been proposed, there is as yet no fullagreement on a single definition of a prebiotic. The mostrecent was agreed at the 2010 Meeting of the InternationalScientific Association of Probiotics and Prebiotics (ISAPP)(Gibson et al., 2011):“A dietary prebiotic is a selectively fermented ingredientthat results in specific changes, in the compositionand/or activity of the gastrointestinal microbiota, thusconferring benefit(s) upon host health.”Characterisation of prebioticingredientsAlthough not stipulated as a requirement in the definitionof a prebiotic, to date only carbohydrate compoundshave been studied with regard to prebiotic activity.Most research has been carried out on fructans (i.e. thepolysaccharide inulin or fructo-oligosaccharides (FOS)derived from various crops or from sucrose) and galactooligosaccharides(GOS). For these ingredients, selectivefermentation and a shift in the microbiota have beenconfirmed in human studies and they have been linkedto potential health benefits. Candidate or emergingprebiotics require additional evidence in humans beforethey can be fully established as prebiotic. Such candidate

Probiotics, Prebiotics and the Gut Microbiota 15prebiotics include the disaccharide lactulose, furtheroligosaccharides and resistant dextrins, polysaccharidessuch as polydextrose, arabinoxylans and resistant starchesas well as some polyols such as lactitol and isomalt.Some prebiotics occur naturally in foods such as chicory,cereals, agave and milk. However, most foods containonly trace levels, so the approach of refining the activeingredients from these foods crops or of producingthem by synthesis (e.g. enzymatic, chemical or thermalprocesses) has been undertaken in order to attain levels infoods whereby a prebiotic effect may occur.Many prebiotics and candidate prebiotics today fall intothe nutritional and regulatory definition of dietary fibre andare labelled as nutrients of that category. They share withdietary fibre the properties of resistance to digestion and(for some fibres) fermentability, but established prebioticsare distinguished from dietary fibre by the selectivity oftheir fermentation. Note that mono- and disaccharides aretypically not considered as dietary fibre according to EUand CODEX definitions.Criteria for prebiotic selectionPrebiotics have an action complementary to, but distinctfrom, probiotics. Probiotics are exogenous micro-organismsthat are ingested to promote a specific health effect. Incontrast, the prebiotic concept is based on the selectivestimulation of the host’s own beneficial microbiota, theprebiotic being the substrate that is (selectively) fermented,stimulating the growth and activity of the particular microorganismor group of micro-organisms of interest and thusleading to the desired health effect.It is essential to measure the effect of the candidateprebiotic on bacterial growth; it is not enough simplyto know that fermentation has taken place. Although invitro tests can be used to screen potential candidates, theincrease in target microbes must be quantified in humantrials after a short feeding period at acceptable levels ofintake in order to establish prebiotic status. Furthermore,human feeding trials are essential in order to demonstratea health benefit.The main site of action for prebiotics is the colon. Thus,a prebiotic should resist the effects of gastric acidity anddigestive enzymes in order to reach the colon intact. Oncethere, prebiotics confer their purported benefits throughthe stimulation of the growth and/or the metabolicactivities of the bacteria that ferment them. The foremosttarget genera for prebiotic action are bifidobacteria andlactobacilli, although this may change as knowledge ofthe microbial diversity and functionality expands. It can,however, not be excluded that prebiotics have a directeffect on health e.g. through the immune system or animpact on binding of microbes to receptors.Prebiotics and probiotics may be combined into“synbiotics”. In this case, the effects of the two componentsshould be synergistic. The probiotic may be stimulated togrow in the gut by fermenting the prebiotic and/or theprebiotic may support a more favourable gut environmentin which the probiotic may better compete.Application of prebiotics in foodAs noted above some prebiotics or candidate prebioticsare naturally occurring and widely consumed at low levelsin the normal diet. The commercial prebiotic ingredientsGOS and fructans are used in infant foods when their safetyand efficacy has been demonstrated; in some countriesthis may require premarket approval. In foods for generalconsumption, the target level of intake of prebiotic rangesfrom 2 to 20 g per day, depending on the ingredientand the desired effect. These amounts can be readilyincorporated into a variety of foods such as cereals, bread,confectionery, biscuits, yoghurts, table spreads, saucesand drinks. Similarly to the case of probiotics, the healthbenefits of candidate prebiotics need to be demonstratedin clinical trials.

16 Concise Monograph SeriesHEALTH EFFECTSOF PREBIOTICS ANDPROBIOTICSResearch challengesIn order to demonstrate that probiotics and prebioticshave beneficial effects on human health, evidence shouldbe provided by nutritional intervention studies in humansubjects. Supportive evidence may be gathered fromanimal feeding studies (in vivo studies) as well as fromlaboratory studies that examine blood or tissue samplestaken from humans or animals (ex vivo studies), or byexamining isolated cells that are grown in culture in thelaboratory and subject to various experimental conditions(in vitro studies). These non-human studies can provideinsights into mechanisms of action, but are not suitableper se to substantiate a human health benefit.One of the factors that has hampered progress in researchinto the health impact of functional foods, includingprobiotics and prebiotics, is the lack of generally acceptedbiomarkers of GI health and immune function. In thiscontext, biomarkers are surrogate markers of healthendpoints just as blood cholesterol level is a well-acceptedrisk factor of disease. Accepted markers of GI functioninclude stool bulk and the transit time through the GItract, and these can be used to demonstrate the benefitof prebiotics and probiotics. There are numerous markersused in relation to the immune system but knowledge islacking about the predictive value of single markers offunction, such as immune cell function, cytokine levels,or antibody production, in overall immune health. Therelevance of these immune markers remains to beestablished, even when more than one marker is used. Theabsence of agreed markers means that clinical endpointssuch as reduced susceptibility to an infection, enhancedresponse to a vaccine or reduction in the duration ofvalidated symptoms are still more widely accepted asevidence of benefit than are changes in a biomarker.Another challenge that is common to all research in humansis inter-individual variation, i.e. the variability in resultsobserved for a specific endpoint in different subjects. Interindividualvariability depends on a wide range of factorsincluding host genetics, diet, microbiota, age, nutritionalstatus and other lifestyle factors. Researchers try to controlfor these differences but must include sufficient subjectnumbers to allow for variation. In addition, the effects ofan intervention may be more evident in people at high riskof, or diagnosed with, a disease than they are in healthysubjects. This often poses a question as to whether theeffect would be observed in healthy people.In all cases, it is clear that prebiotics and probiotics mustbe consumed regularly in order to confer a benefit.Impact on the GI tract of prebioticsand probioticsGut microbiotaAn increased proportion of bifidobacteria and lactobacilli isthought to represent a “healthier” microbial composition.This is partly based on evidence from infants, which isdiscussed later in this section as well as in the section onmechanisms. These bacteria are more likely to fermentcarbohydrates and produce acids, and they generally lackpotential toxicity.There is ample evidence in human subjects, includinginfants, as well as in animal and in vitro studies thatestablished prebiotics increase the proportion ofbifidobacteria and sometimes lactobacilli present in thegut microbiota while having no measurable effects onother groups of bacteria.

Probiotics, Prebiotics and the Gut Microbiota 17In the case of probiotics, the consumption of adequatedoses of Lactobacillus strains often results in a measurableincrease in the lactobacilli in the faeces, and in somecases there may be a decrease in unfavourable organismssuch as staphylococci. For pre-term infants, who usuallyharbour reduced numbers of bifidobacteria, there isgood evidence that the ingestion of bifidobacteria notonly increases their numbers but may also reduce thenumbers of clostridia. In practice, the effect of prebioticsand probiotics on the microbiota is somewhat variablebut also difficult to measure because of the factorsdiscussed in the section “Techniques for exploring theGI microbiota”.In addition to considering an increase in the number orproportion of certain microbes, it is also important toconsider their functional capacity, which may be changedby prebiotic or probiotic consumption in the absence ofan alteration in number or proportion. Recent humandata on probiotics using new techniques have enabledmeasures of components that reflect the genes thatare being actively expressed at any given time. The linkbetween gene expression and health outcomes will nodoubt be the subject of future research.Transit time and stool bulkingThere is strong evidence that prebiotics and probioticscan influence gut function. This effect for prebiotics isthought to be due to their fermentation in the colon,resulting in increased bacterial mass and osmotic waterbindingcapacity that contribute to increased stoolweight, increased stool frequency and softer stools.There is also some evidence that SCFA, especiallybutyrate, have a positive effect on the endotheliumand on peristalsis, which improves transit. Becausethere is an inverse link between stool mass and transittime, prebiotics may also decrease transit time. In somestudies, prebiotics are reported to reduce symptoms ofintestinal discomfort, such as bloating, abdominal painand flatulence. Studies on certain strains of probioticbacteria have demonstrated an impact on gut function,as revealed by normalisation of transit time and reductionof self-reported minor digestive discomfort symptoms.An improved transit time may reduce putrefactive activityin the left colon, as indicated by some studies that havefound reduced levels of polyamines and metabolitessuch as cresol and indoles.These stool-regulating effects are considered to bebeneficial to gut health by decreasing the risk ofconstipation. An improvement of stool function is likely tobe important with respect to the general population sincedietary fibre intakes in developed countries are almostuniversally lower than recommended and the number ofpeople reporting digestive problems is extremely high(more than 80% in some surveys of women). As withall dietary fibre, too-high an intake of prebiotics mayneed to be avoided by certain individuals because overconsumptioncould lead to bloating and, in severe cases,to watery stools. However, this subsides if consumptionis reduced or stopped.Chronic inflammatory gut conditionsThe inflammatory bowel diseases (IBD) are seriousconditions with an as-yet unknown cause. They includeCrohn’s disease (CD), which can affect the whole gutthough mainly affects the small intestine, and ulcerativecolitis (UC), which is usually restricted to the large bowel.IBD is associated with a breakdown of the normal barrierfunction provided by the gut epithelial lining and itsassociated mucus. Whether the inflammation causes thebreakdown of the barrier or if a breakdown of the barrierallows inflammation to develop is not clear. It is knownfrom studies on germ-free animals compared to normalanimals that germ-free animals are less susceptible toexperimental IBD and that the presence of commensal

18 Concise Monograph Seriesbacteria can initiate and/or exacerbate inflammatorybowel conditions. CD and UC may thus result from aninappropriate mucosal immune response to the GImicrobiota in genetically susceptible individuals. There isalso some evidence from clinical studies that the balanceof different groups of commensal bacteria might bealtered in IBD patients.Numerous studies of both probiotics and prebioticsin animal models have shown a positive impact on theprevention or treatment of IBD. Clinical studies in CDsubjects have not been effective in prolonging remissionof CD, but there are promising data indicating thatsome probiotics are useful in maintaining remission inUC. In another inflammatory bowel condition known aspouchitis, which can occur after surgery to treat UC, onemixture of probiotic strains appeared to be effective inhelping maintain remission. The potential for prebioticsand synbiotics to help the management of IBD has beenshown in several small studies with fructans, mainly in thereduction of inflammatory markers, but as yet the datado not allow a final conclusion. Although there are stillinsufficient data to draw firm conclusions on the effect ofpre- or probiotics on IBD, importantly, none of the trialsconducted thus far have raised concerns regarding theirsafety in patients with IBD at the levels of intake tested.Irritable bowel syndromeIrritable bowel syndrome (IBS) is a distressing conditionthat is characterised by an array of symptoms such asabdominal pain, bloating and altered bowel habits thatmay often alternate between constipation and diarrhoea.As similar symptoms can be observed from time to time inthe general population, a specific set of criteria (known asthe Rome criteria) was developed to improve consistencyof diagnosis of IBS. In industrialised countries, IBS affectsbetween about 5 and 20% of the adult population, withrates higher in women and older people. Recently, therehas been interest in the role of inflammatory processes asa potential cause of IBS. In addition, in a certain subset ofsubjects, it appears that previous gut infections play a rolein onset of IBS (post-infectious IBS). Furthermore, in somestudies, lower levels of bifidobacteria have been observedin subjects with IBS compared with healthy subjects.Because of the lack of good therapy for IBS and theidentification of abnormal microbiota in IBS subjects, bothprobiotics and prebiotics have been investigated for theirability to help subjects manage this condition. A coupleof probiotic preparations have been shown to providereduction in a global symptom score (the sum of a numberof different symptom scores) and in reducing abdominalpain; however, no change in diarrhoea, constipation orbloating was confirmed. In other studies, some strainshad no effect or resulted in worsening of symptoms. Forsome prebiotics, studies showed that low doses led toan improvement in the condition, whereas a larger loadled to an enhancement of the perceived symptoms.Thus, additional research will be required to determine ifconsistent benefits can be observed by those experiencingIBS if they use prebiotics and probiotics.Impact on the GI tract specific toprebioticsColon cancerColon cancer has been linked to diets low in dietary fibreand thus the potential for prebiotics to reduce coloncancer risk has also been investigated, mainly usingin vitro techniques and animal models. Results fromanimal studies with endpoints such as DNA damage,aberrant crypt foci as well as tumours in the colonsuggest that prebiotics may reduce the risk of coloncancer. This is supported by ample in vitro evidence.Synbiotics were investigated in a few animal studies andwere found to be more effective than either prebioticsor probiotics alone. One synbiotic study (SYNCAN

Probiotics, Prebiotics and the Gut Microbiota 19project) in humans found a reduction in DNA damageand a reduction in cell proliferation in colon biopsies.Potential mechanisms for a prebiotic effect on coloncancer risk have been identified in animal studies andinclude changes in gut bacterial enzyme activities, whichmodify the fermentation products, and up-regulation ofapoptosis (programmed cell death – in this case of thepre-cancerous cells). However, definitive evidence thatcertain prebiotics might reduce the risk of colon cancerin human subjects is lacking and requires more robust,multi-centre, prospective human trials.Prebiotics in early life nutritionOligosaccharides with fucosyl, galactosyl and sialylstructures are found in human breast milk and arethought to promote healthy microbiota. Interventionstudies show that infant formula supplemented with GOSand fructans, alone or in combination, help stimulate thebifidobacteria that are characteristic of breast-fed infantsin a dose-dependent manner. Further, infants fed formulawith oligosaccharides have microbiota, a stool pH and aSCFA pattern similar to those of breast-fed infants. Thestool consistency and stool frequency of prebiotic-fedinfants (softer and more frequent) is also closer to thatseen for breast-fed infants than for those fed standardformula. The use of specific GOS and fructan prebioticsin infant formula is widespread practice and acceptedas safe. The range of the resulting benefits of theseprebiotics as well as of other prebiotic candidates is stillan active area of research by experts in the field.Mineral absorptionOne specific, well-established effect of prebiotics is onmineral absorption. There is a wealth of data showingthat prebiotics increase calcium absorption and increasegrowth and skeletal mass in rats. In addition, thereare some studies showing enhanced absorption ofmagnesium and iron. Further evidence for an improvedmineral absorption is available from pigs, which areconsidered a better model for the human than are rodents.Numerous human intervention studies consistently showan increase in calcium absorption. So far, there is onelong-term human intervention study that assessed theeffects of prebiotics on bone health. The study was inadolescents and used a combination of FOS and longchaininulin (50/50). After one year, bone mineral densityand bone mineral content were significantly higher atcertain bone sites in the supplemented group. Whetherthis effect is common to all prebiotics or unique to theparticular formulation requires further substance-specificresearch.Gut hormones and food intakeNumerous studies in rodents, mainly with fructans,show a consistent effect of feeding prebiotic fibres inreducing food intake and decreasing fat mass, thoughnot necessarily body weight. Additional data from thesestudies suggests that the mechanism for this is likelyto be SCFA-stimulated secretion of gut peptides suchas glucagon-like peptide (GLP)-1, peptide YY (PYY)and oxytomodulin and reduced secretion of ghrelin,all of which are secreted by endocrine-type cells inthe mucosa. These peptides are known to affect foodintake in animals and humans. Overall, evidence froman increasing number of studies in human subjects,mainly with fructans, supports an effect of daily prebioticconsumption in reducing appetite, lowering body weightor fat mass, altering gut peptide levels in blood andimproving glucose tolerance. Some, but not all, of thesestudies examined the composition of the gut microbiota;where examined, shifts in the microbiota were confirmed.The impact of SCFA on glucose and lipid metabolismmay also be important.

20 Concise Monograph SeriesImpact on the GI tract specific toprobioticsLactose malabsorptionAs discussed in the section on “Bacterial fermentationand metabolism”, many micro-organisms fermentlactose, the sugar found in milk and products made frommilk. Although infants rely on lactose, which contributesas much as 10% of the energy in breast milk, manypopulations around the world have a high proportion ofadults who are unable to digest this sugar. In humans, andin fact in all mammals, expression of the enzyme lactaseis down-regulated in adulthood with the exception ofsome population groups, particularly those of Europeanorigin. Lactose intolerance is a condition in which thecolonic fermentation of undigested lactose results ingastrointestinal effects such as abdominal pain, bloating,borborygmi or laxation. There is evidence that the livebacteria of yogurt are able to compensate for the lackof endogenous lactase in the human gut by digestinglactose. The typical measure of improved lactosedigestion is a reduction in breath hydrogen excretion(breath hydrogen is usually raised when undigestedcarbohydrate reaches the colon and is fermented). Thisimproved digestibility reduces the symptoms related tolactose intolerance in some lactose malabsorbers.Impact on the immune responsesGerm-free animals have, as mentioned, anunderdeveloped immune system and GI epithelium,resulting in reduced resistance to infection comparedwith conventional animals. It is thus accepted thatcommensal organisms are vital for the maturation ofthe immune system. The potential for probiotics andprebiotics to impact immune responses and to reducethe risk of infections has been the subject of a number ofhuman studies (discussed below). Such results, combinedwith evidence from mechanistic studies showing changesin certain immune parameters, support the notion thatthe effect of probiotics and prebiotics on the immunesystem can translate into measurable health benefits, butdefinitive evidence is lacking.Gastrointestinal infectionThe small intestine is the main target of many GIinfections such as rotavirus, S. typhimurium and someE. coli types. As early as 1916, it was reported thatS. typhimurium was cleared from the GI tract of healthycarriers of the organism when members of the normalgut microbiota were introduced. Probiotics have longbeen associated with a purported ability to counteractpathogenic bacteria and so recently several potentiallybeneficial strains have been tested in controlled studies.The first-line of treatment for the symptoms of diarrhoeais oral rehydration – and no other dietary treatmentshould be substituted for this, especially in infants.However, in established conditions, some probioticscan be used as an adjunct under medical supervisionwhere appropriate. Certain probiotics seem to be mosteffective in improving symptoms when the diarrhoea isthe result of a viral (rather than bacterial) infection, if theyare used early in the course of the infection and are givenin sufficient amounts. In terms of reduced susceptibilityto infection, some studies have found decreases in therisk of infection in infants (mainly in developing countries)and in institutionalised or hospitalised elderly. Efficacyis clearly strain related, i.e. some strains are effectiveand others not. In addition, there is some evidence thatspecific probiotic strains, and some prebiotics, mayreduce the risk of traveller’s diarrhoea.Some antibiotics can significantly disrupt commensalbacteria, resulting in side effects such as antibioticassociateddiarrhoea (AAD). The estimated incidenceof AAD is as high as 25% for some antibiotics and thiscan lead to patients failing to complete the course of

Probiotics, Prebiotics and the Gut Microbiota 21treatment. There is evidence that specific probiotics canreduce the risk of AAD and, indeed, several meta-analysesconclude that there may be as much as a halving of therisk of AAD in adults or the elderly, although the effect isless consistent in children. The observed effects relate toa limited number of specific probiotic strains. In the caseof prebiotics, it has been shown that FOS administrationfollowing an antibiotic treatment reduced the reoccurrenceof AAD from more than 30% in the controlgroup to less than 10% in the prebiotic group. As this wasnot associated with a decrease in subjects testing positivefor C. difficile, this could suggest that the prebiotic had astabilising effect on the microbiota, supporting a return ofeubiosis.Clostridium difficile infection is a frequent cause ofdiarrhoea in institutionalised populations, for examplein hospitals and in long-term care homes. It is oftenassociated with antibiotic use but it can occur as a resultof other risk factors such as age greater than 65 yearsor a compromised immune system owing to illness,medication or GI surgery. So far, the results of researchto investigate whether probiotics can reduce the risk ofC. difficile infection or reduce the severity or duration ofsymptoms in adults are promising but further confirmatorystudies are needed.A bacterium known as Helicobacter pylori is present inthe stomach of a small proportion of young adults butin as many as 50% of those aged 60 years and over. Itcolonises the mucous layer next to the gastric epitheliumand in some people can cause acute gastritis (i.e. pain,bloating, nausea and vomiting) and can lead to chronicgastritis and peptic ulcers. Treatment involves longtermadministration of strong antibiotics and althoughprobiotics do not speed the eradication of H. pylori, somehave been shown in several studies to reduce the sideeffects of treatment and may result in less disturbance ofthe microbiota.The microbiota in pre-term infants is restricted anddiffers in composition from those in healthy, full-terminfants. In particular, potentially beneficial bifidobacteriaare not well established in the pre-term neonatal gut.The microbiota is further challenged by environmentalbacteria from the hospital milieu and the common useof antibiotics in pre-term infants, putting this populationat increased risk of necrotising enterocolitis (NEC).Although the use of probiotics is not yet established inclinical practice, several trials have shown that specificprobiotic strains can reduce the risk of NEC. Additionalstudies are needed to clarify the preferred strain and doserecommendations. Furthermore, the use of live microbesin such a susceptible population makes confirmation ofsafety for this use a prime objective.Other infectionsThere has been a number of studies on various age groupsto investigate the potential for probiotics to impact thesusceptibility to upper respiratory tract infection (URTI)and its duration and symptoms. Studies were conductedwith a range of different strains; some strains reducedincidence, some reduced duration and most had effectson symptoms. The evidence is promising, but the rangeof strains and the variation in age groups and studydesign prevent any firm conclusions. Evidence for aneffect of prebiotics is limited to a recent, large, long-termstudy in which infants consuming formula supplementedwith a specific GOS/long-chain FOS combination wereless prone to upper URTI and associated fever than werethose infants fed formula without a prebiotic.There has also been interest in the use of probiotics inurogenital medicine. Certain probiotic strains have beenshown to improve recovery from bacterial vaginosisduring antibiotic treatment. Potential mechanisms for theeffect include anti-microbial antagonism, restoration ofbalanced microbiota or an enhanced immune response.

22 Concise Monograph SeriesVaccinationsAnimal studies have convincingly demonstratedthat certain probiotic strains can both enhance theimmune response to a vaccine and reduce the risk ofsubsequent infection. Human studies are much fewer,but an increasing number of well-controlled trials arebeing conducted. Preliminary evidence indicates thatthe response to vaccines against influenza, choleraor childhood diseases can be enhanced by selectedprobiotics, as measured by the number of subjects whorespond to the vaccine or an increase in the level of serumimmunoglobulins. The effects are strain-specific in termsof efficacy of the probiotics and, in the case of influenza,specific to the pathogen strains. In one study, there waslimited evidence that subsequent risk of infection withthe influenza virus was reduced. In the case of prebiotics,although evidence in animals seemed promising, clinicalstudies have not yet been supportive of an effect.Allergic conditionsAllergy can be defined in simple terms as an inappropriateimmune reaction or over-reaction to an otherwiseharmless foreign antigen (mostly proteins or peptides).In medical terms, it is described as a hypersensitivityreaction mediated by specific antibodies (IgE) or cellbasedmechanisms. Common allergies include reactionsto certain food proteins (e.g. milk, eggs, peanuts, treenuts, soy, wheat gluten, fish, shellfish and shrimp) or toenvironmental allergens such as pollen (hay fever), housedust mites and pet hair. Food allergies are more commonin infants and children than in adults. The most seriousform of allergy resulting in anaphylaxis (which can be fatalwhen the throat and respiratory tract swell and restrictbreathing) is rare, albeit a lifelong concern. Less severesymptoms of allergies are more common (prevalence isabout 2% for food allergies and up to 30% for respiratoryallergies) and can substantially reduce the quality of lifefor allergic subjects.As noted, the prevalence of allergy has increased inwesternised societies. There is growing evidence thatthe nature of microbiota acquired by the infant in thepostnatal period has an important bearing on maturationof the immune system. There is some indication thatatopic children tend to have a degree of dysbiosis, withmore clostridia and fewer bifidobacteria than non-atopicindividuals. In addition, data suggests that breast-fedinfants are less prone to allergic conditions. It has thusbeen suggested that prebiotics may help reduce the riskof developing atopy or reduce the associated symptomsof atopic eczema or allergic rhinitis. There is promisingevidence, based on a follow-up of one intervention,that not only can prebiotic-supplemented infant formulareduce susceptibility to atopy but that the benefits persistup to 2 years of age. Furthermore, studies in infants at highrisk of allergy who were fed supplemented formulas for 6months had reduced levels of IgE and some IgG types.There have been several studies on the impact ofprobiotics on the development of allergic symptoms ininfants at high risk of developing atopic disease. In mostof these studies, the mother consumed the probiotic priorto birth and the infant was administered the probioticafter birth. Results showed a decreased risk of eczema at2 years of age and beyond. Overall, results of the studiespoint towards strain-specificity and also hint towardstwo separate windows of opportunity: first, the maternalconsumption of probiotics during the perinatal periodand, second, the use of probiotics during weaning. Pastand ongoing studies have also targeted the managementor reduction of allergic symptoms such as those linked toatopic eczema or allergic rhinitis; results are promisingbut not yet conclusive. This probably reflects thecomplexity of the allergic diseases spectrum and the factthat a range of different clinical designs was used.

Probiotics, Prebiotics and the Gut Microbiota 23PROBIOTICS ANDPREBIOTICS:MECHANISMS OF ACTIONOverall mechanismBoth probiotics and prebiotics are thought to work largelythrough direct or indirect effects on the gut microbiotaand environment and/or on host function. In the caseof probiotics, a live micro-organism is consumed, in arange of dosages, spanning from ~10 8 to 10 12 cells/day,depending on the product. This large number of microbeshas the potential for a greater impact in the upper GI tractwhere lower densities of micro-organisms are found, butis also thought to impact the colon. Prebiotics enhancethe growth of the endogenous microbiota or possiblystimulate the growth of probiotics when providedconcurrently. Thus, probiotics and prebiotics share manycommon mechanisms of action mediated through animpact of microbes on the host and these are discussedbelow. In the case of health effects that relate only toprebiotics or only to probiotics, the mechanisms are lesswell known and have been alluded to in the section onhealth effects.Probiotics and prebiotics (via their stimulation ofcommensal organisms) act on and interact with the hostby two main modes of action, or a combination of actions(Figure 6 – see page 24):• Impact of micro-organisms or their metabolites/enzymes on the host’s GI tract and its microbiota• Interaction with the host’s cells and immune systemGI tract and its microbiotaAs noted, bifidobacteria and lactobacilli in the colonpreferentially ferment carbohydrates that escapedigestion in the upper GI tract, resulting in a reduced pHof the colon. Bifidobacteria can ferment fructans becausethey have an enzyme, β-fructofuranosidase, that otherbacteria either lack or have present at a lower activity,thus giving them a competitive advantage when exposedto fructans in the human gut. Similarly, the presenceof β-galactosidase in lactobacilli or streptococci exertsa competitive advantage in GOS fermentation. Themetabolism of prebiotic fructans by bifidobacteria yieldsmainly the acidic compounds acetate and lactate. Crossfeedingof these fermentation products to other speciesgives rise to butyrate and propionate. Butyrate andpropionate are also formed from the direct fermentationof other dietary carbohydrates.The benefits of a lower pH in the colon are that itencourages the multiplication and survival of commensalorganisms that prefer acidic conditions and generallyinhibits the ability of some pathogens to adhere, grow,translocate across the epithelium or colonise the GI tract.Furthermore, butyrate has long been known, from invitro studies on fermentable dietary fibres, to enhancemucosal cell differentiation and this may also promotethe barrier function of the epithelium.Saccharolytic fermentation concomitantly reduces thepotentially adverse effects of protein fermentation andother processes, which give rise to nitrogen and sulphurcontainingcompounds such as ammonia, N-nitroso- andazo- compounds as well as sulphides.Many bacteria produce bacteriocins, which are peptidesor proteins that are intended to reduce the survival ofcompeting organisms. Bacteriocins produced by probioticbacteria have been observed in in vitro studies to decreasethe ability of pathogens such as E. coli O157:H7 toadhere to and invade cultured intestinal cells. Bacteriocin

Probiotics, Prebiotics and the Gut Microbiota 25studies suggests that an increase in the productionof mucins may result from an enhancement of geneexpression in the mucus-producing Goblet cells that linethe GI tract. Increasing the mucous layer helps protectthe epithelial cells from potential pathogen translocationand may enhance the clearance of pathogens from theGI tract.Probiotics may also enhance the ability of specialisedPaneth cells in the small intestine to produce theantibacterial peptides known as defensins. Thishypothesis is supported by in vitro studies, using intestinalepithelial (e.g. Caco-2) cells grown in tissue culture, thathave shown that certain probiotics can stimulate humanβ-defensin mRNA expression and peptide secretion.In vitro studies suggest that probiotics and prebioticsmay affect the barrier function of the epithelium itself byenhancing the resistance of tight junctions, possibly viaan effect on tight junction proteins (e.g. occludins andclaudins). Increased expression of genes encoding tightjunction proteins has recently been shown in a studyconducted in human volunteers administered a specificLactobacillus strain.Animal and in vitro studies have found that specificprobiotics can compete with pathogens for receptorsites on epithelial cells or in the mucous layer, therebypreventing pathogens from adhering or translocating.In contrast, other probiotics may directly bind to thepathogen, thus reducing its ability to colonise theintestine. There is good evidence from studies on micethat feeding on certain probiotic strains can greatlyreduce the ability of pathogens such as S. typhimuriumand pathogenic E. coli to translocate and invade the liverand spleen. In vitro studies showed that the same strainscompete with the ability of pathogens to adhere to cells.Influence on pathogen translocation in infected animalmodels has also been shown for some prebiotics.Cross-talk with the hostThe most complex of the postulated mechanisms by whichprobiotics and stimulated endogenous microbes may actis the interaction with the GI immune cells and lymphoidtissue to modulate the immune and inflammatoryresponses of the host, which might lead to the potentialfor an impact beyond the gut (Figure 7 – see page 26).The mammalian immune system is generally consideredto consist of two major arms: the innate (or non-specificimmediate) immune response and the acquired (orspecific adaptive) immune response. Both parts of theimmune system are extremely complex and involvecells (cellular immunity) and other components secretedinto the blood (e.g. antibodies and cytokines). The twoarms work together to protect the host from pathogens(bacteria, viruses, fungi), other foreign materials (antigens)and also from tumour cells arising in the host. For moreinformation, see the ILSI Europe Concise Monograph onNutrition and Immunity in Man (ILSI, 2011).Through so-called bacterial–epithelial cell “cross-talk”,it seems that ingested and endogenous microbes canimpact both the innate and the adaptive responses of thehost immune system. The interaction between microbialcells (commensal, probiotic or pathogen) and host cellsis mediated by the interaction with specific receptorssuch as Toll-like receptors (TLR) that are associated withcells lining the mammalian GI tract. The activation ofthese receptors initiates a cascade of concerted immunesignals leading to different responses. For example,the response can ensure a balanced maturation ofT cells (Th1 versus Th2) and T-regulatory cells, whichallows an appropriate response to potential pathogensand food antigens. An inappropriate T cell response isthought to be one of the features of allergic conditions,as mentioned previously. Further, activation of theimmune pathways can also result in B cell differentiationand production of protective antibodies, such as IgA,

Probiotics, Prebiotics and the Gut Microbiota 27thus opening up the possibility of alternative mechanismsimpacting the immune system.Although studies in humans have found changes inbiomarkers such as cytokine levels and changes in thenumber and activity of immune cells, it is nevertheless ofprime importance to have studies in human subjects thatalso measure clinical outcomes. Clinical measures, suchas a reduced incidence of infection or enhanced immuneresponse to a vaccine, can then be linked to measuresof humoral or cellular immune biomarkers. Eventhough results from animal studies cannot necessarilybe extrapolated to humans, in vivo studies in animalmodels represent a valuable means of understandingthe complex signalling cascade underlying a protectiveimmune response.OVERALL CONCLUSIONSThe science around the concept of probiotics andprebiotics continues to expand. Current global researchefforts have greatly contributed to the understanding ofthe role of GI commensal organisms in their extraordinarysymbiotic relationship with humans. Continued researchinto the microbiota will no doubt help lead to an improvedinsight into the impact of probiotics and prebiotics onhuman health.Probiotics are designed to provide added functions thatcan compensate for, substitute for, or add to the gutmicrobiota, and therefore impact the host directly orindirectly through “cross-talk” with the gut microbiotaand/or the host. In addition, the effects may be local in theGI tract or systemic. Prebiotics are designed to improvethe intrinsic microbiota by selectively stimulating thosegroups that are thought important for eubiosis.Research over past decades has demonstrated potentialhealth benefits of dietary probiotics and prebiotics andcontributed to our understanding of the mechanismsby which these effects are brought about. The mostcommonly reported impact of probiotics and prebioticsis on intestinal function, including transit time, AAD andinfectious diarrhoea. Evidence continues to emergethat probiotics and prebiotics have an influence on theimmune system and thereby may enhance resistance toinfections, particularly those of the GI or respiratory tract,and help to mitigate allergies, particularly in infants andyoung children. Evidence is gradually developing for thepotential for probiotics and prebiotics to impact otherconditions of the GI tract, such as IBD, IBS and coloncancer. In the case of prebiotics, a well-established rolein enhancing calcium absorption remains to be a provenbenefit for bone health.

28 Concise Monograph SeriesAn emerging role for prebiotics and probiotics inappetite control and weight management could bevery important. An expanding area of interest for bothprebiotics and probiotics is the investigation of theirpotential for an anti-inflammatory role in conditionsbeyond the gut such as cardiovascular disease, obesityand metabolic syndrome.One critically important fact to bear in mind is thatreported benefits of probiotics should be consideredstrain-specific unless otherwise demonstrated. Prebioticsare also likely to have substance-specific effects. Thus, forboth probiotics and prebiotics it is vital that future humanstudies take this into account. Such studies, apart fromestablishing the effects of each ingredient, should alsoaim to improve our understanding of the mechanismsof action and, if possible, lead to validated biologicalmarkers.It must be remembered when considering studieson prebiotics that only a few prebiotics are currentlyestablished. Similarly, only a limited number of microbeshave been documented as probiotic. In all cases, it isclear that prebiotics and probiotics must be consumedregularly in order to confer a benefit.This monograph attempts to summarise the scienceand principles valid for prebiotics and probiotics today.It is noteworthy that these ingredients can be readilyincorporated into a balanced diet and that there is agrowing body of evidence for their potential healthbenefits.

Probiotics, Prebiotics and the Gut Microbiota 29ABBREVIATIONSAADCDCFUDPFOSGALTGIGOSIBSIBDILNECQPSSCFATLRUCURTIAntibiotic-associated diarrhoeaCrohn’s diseaseColony-forming unitsDegree of polymerisation, i.e. the number of monomers in a moleculeFructo-oligosaccharides – typically applied to mixtures of DP3–DP9Gut-associated lymphoid tissueGastro-intestinalGalacto-oligosaccharides – typically applied to mixtures of DP3–DP9Irritable bowel syndromeInflammatory bowel diseaseInterleukinNecrotising enterocolitisQualified presumption of safetyShort chain fatty acidsToll-like receptorsUlcerative colitisUpper respiratory tract infection

30 Concise Monograph SeriesGLOSSARYAntibody: A specific protein produced in the blood ortissues as part of the immune response to a foreignantigen such as a bacterium, toxin or food protein.The antibody interacts with the antigen, therebyinactivating it and thus forming the basis of immunity.Antigen: A substance that the body recognises as foreignand that can evoke an immune response. Most often,an antigen is a peptide or protein (e.g. bacterialantigen, food antigen or toxin).Atopy: A genetic susceptibility to exhibit hypersensitivityreactions (eexaggerated immune responses) tocommon antigens e.g. atopic eczema in response to acommon foodstuff.Commensal: From the Latin for “common table”. Itmeans two organisms living together in a way that iseither beneficial to both and or that, at least, is notharmful to either. Hence, commensal bacteria live inthe human gut and may be neutral or beneficial.Cytokines: Low molecular weight proteins (other thanantibodies) produced by various cell types andinvolved in cell-to-cell communication and control ofthe inflammatory and immune response. Cytokinesinclude interferons, interleukins and lymphokines.Dysbiosis: The condition of the microbiota of the gut inwhich one or a few potentially harmful micro-organismsare present in high numbers, thus creating a diseasepronesituation or resulting in otherwise noticeabledisturbances of the microbiota such as liquid stools,gastrointestinal infections or inflammations.Eubiosis: Formally referred to as “normobiosis”, thischaracterises the composition of a stable or balancedgut microbiota in a healthy individual. There isincomplete understanding of what constituteseubiosis and, hence, no general definition in terms ofbacterial composition or function.Fermentation: The anaerobic oxidation of organiccompounds to generate metabolic energy in theabsence of oxygen as an electron sink. Reductionequivalents are released as hydrogen, ammonia,hydrogen sulphide, methane, organic acids oralcohols. For example, the oxidation of carbohydratesto short chain fatty acids (SCFA), ethanol, lactic acidand/or gases to produce energy in the form of ATP.Microbe/micro-organism: Small, often single-cellorganisms including bacteria, archaea, yeast,mould, fungi, algae and plankton (fungi may alsobe multicellular). Although definitions vary, we havetaken the view that microbes do not include viruses.Microbiota: All the microbes that are found in a particularregion or habitat; hence, gut microbiota describesthe whole microbial population found in the gut orgastrointestinal tract. The term “microflora” is nolonger used.Oligosaccharide: A carbohydrate that consists of 3–9monosaccharide units joined by glycosidic linkages.Some are prebiotics.Polysaccharide: A carbohydrate comprising ten or moremonosaccharide units. Some are prebiotics.Taxonomy: The science of identifying species andarranging them into a classification.

32 Concise Monograph SeriesSalminen et al., (1998) Functional food science andgastrointestinal physiology and function. British Journalof Nutrition 80(Suppl. 1):S147–S171.Sanders, M.E., et al. (2007). Probiotics: their potential toimpact human health. Council for Agricultural Scienceand Technology (CAST), Issue Paper 36. CAST, Ames,Iowa.Sanders, M.E. (2009). How do we know when somethingcalled “probiotic” is really a probiotic? A guideline forconsumers and health care professionals. FunctionalFood Reviews, 1:3–12.Sanders, M.E. (2011). Impact of probiotics on colonizingmicrobiota of the gut. Journal of Clinical Gastroenterology,45(Suppl. 3): S115-S119.Spahn, T.W. and Kucharzik, T. (2004) Modulating theintestinal immune system: the role of lymphotoxin andGALT organs. Gut, 53:456-465.World Gastroenterology Organisation (2011) Practiceguideline: probiotics and prebiotics, October 2011.http://www.worldgastroenterology.org/probioticsprebiotics

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