28. Progress in the Science of Probiotics - Dr. Sisca SP.gk

7/27/2019 28. Progress in the Science of Probiotics - Dr. Sisca SP.gk

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W. Allan WalkerOlivier GouletLorenzo MorelliJean-Michel Antoine

Progress in the science of probiotics:

from cellular microbiology and applied

immunology to clinical nutrition

j Summary Probiotic research isprogressing rapidly with strongscientific-based observations. New

molecular biologic techniques forthe more accurate identification ofintestinal microflora and seminalstudies that have helped define thefunction of commensal bacteria inthe gut have been reported re-cently. In functional terms, newtechniques are operational tostudy the affect of microbialhostcrosstalk between both bacteriaand the host. Probiotics have beenshown to initiate the activation ofspecific genes localized to these

cells. Both the bacterial and hostaspects of microbiotahost cros-stalk can now be studied, inparticular thanks to simplified invivo gnotobiotic mouse models.Their functional genomic studiesenable the screening for probioticpotential and for investigating themodulated expression of genesinvolved in a broad range ofintestinal functions including reg-ulation of nutrient uptake andmetabolism, mucosal barrier andepithelial cell function, xenobioticmetabolism, and strengthening ofthe innate immune system. Animportant function of probiotics isits effect on the gut immunesystem. The latter may work byenhancing mucosal barrier func-tion, preventing apoptosis of epi-

thelial cells and ultimately,decreasing antigen uptake, espe-cially in the small bowel. Clini-

cally, there is strong evidence thatsome probiotics improve thedigestibility of lactose and othersprevent the recurrence of pouchi-tis after inflammatory bowel dis-ease (IBD) surgery. There isreasonably strong evidence for theefficacy of probiotics in childhoodinfectious gastroenteritis andantibiotic-associated diarrhea. Re-cent data suggest the potentialefficacy of probiotic strains inatopic eczema, IBD, Helicobacter

pylori gastritis, neonatal necrotiz-ing enterocolitis and as a substi-tute for inadequate initial neonatalcolonization.

j Key words acquired immu-nity cellular microbiology microbial-epithelial crosstalk gastrointestinal tract glycosylhydrolases gut-associated lymphoid tissue

host defense innate immu-nity intestinal epithelium lactic acid bacteria microbiota molecular microbi-ology mucins mucosal bar-rier probiotics toll-like receptors

W.A. WalkerPaediatrics Dept., Division of NutritionHarvard Medical SchoolBoston (MA), USA

O. GouletPaediatric Gastroenterology Dept.Necker Enfants Malades HospitalParis, France

L. MorelliMicrobiology Dept.Universita Cattolica Del Sacro CuorePiacenza, Italy

J.-M. Antoine (&)NutrivaleurDANONE VITAPOLEResearch Center D. CarassoRoute Departementale 12891767 Palaiseau Cedex, FranceTel.: +33-16/935-7000Fax: +33-16/935-7689E-Mail: [email protected]

Abbreviations: Btheta: Bacteroides the-

taioitamicron; FISH: fluorescence in situ

hybridization; HMA: human microbiota-

associated; IBD: inflammatory bowel dis-

ease; IBS: irritable bowel syndrome; NEC:

necrotizing enterocolitis; RCT: randomized

controlled trial; RIVET: recombinase-based

in vivo expression technology; TLR: toll-

like receptor

ORIGINAL CONTRIBUTIONEur J Nutr (2006) 45 [Supplement 1]:I/1I/18DOI 10.1007/s00394-006-1101-1

EJN

1101


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Introduction

This is an important year for the study of probioticsin terms of research, clinical use, and cultural andregulator constraints imposed by various societies.Probiotic use is no longer based on antidotal experi-ence but has evidenced-based scientific and clinical

studies to support its value in health and disease [ 1].The 2004 Danone International Probiotics Conven-tion constituted a documented review of these strik-ing advances. The Convention focused on thefollowing topics: recent international probioticguidelines and definition, new methods of evaluatingthe survival and function of gut flora, hostmicrobi-ota crosstalk, probiotic interactions with the immunesystem, the latest data generated by clinical studies onprobiotic use in various age groups including infancy,and a cultural comparison of the manner in whichprobiotics are perceived in Europe and the UnitedStates.

From probiotic research to guidelines

As evidenced by the substantial increase in thenumber of papers published in both scientific jour-nals and the lay press, interest in probiotics isbooming not only in the scientific community butalso in the market place among consumers. Given theextensive interest, guidelines were considered neces-sary. In response, FAO/WHO convened two compre-hensive meetings: (1) a joint FAO/WHO expertconsultants group to evaluate the health and (2)

nutritional properties of probiotics in food includingpowdered milk with live lactic acid bacteria. Twodocuments were generated as a result [2, 3]. The firstevaluated the latest information relating to probioticsbased on working papers devoted to microbiology(L. Morelli, Piacenza, Italy), the regulatory and clini-cal aspects of dairy probiotics (G. Reid, London,Ontario, Canada) and technological and commercialaspects (S. Gilliland, Stillwater, Oklahoma, USA) [2].The second document outlined general guidelines forthe assessment of probiotics [3].

FAO/WHO define probiotics as live microorgan-isms which, when consumed in adequate amounts as

part of food, confer a health benefit on the host. It isnoteworthy that no specific action of intestinal mi-croflora is emphasized since the collective beneficialeffects are comprehensive and long standing. Inaddition, both the FAO/WHO documents link probi-otics to food and to food only, thus excluding anyreference to the term biotherapeutic agents. Thesereports underscore the need for a multidisciplinaryapproach: (a) In terms of taxonomy, the consultants

recommended that probiotics be named in accor-dance with the International Code of Nomenclatureand strongly urged that, for the sake of full disclosure,probiotic strains be filed with an internationally rec-ognized culture registry. This is already required forpatent applications and, in Europe, for probiotic usein animal feeds, but not human food. (b) With regard

to health benefit, definition and measurement, theconsultants recommended that each product shouldbe labeled with the minimum daily amount requiredin order to confer specific health benefit(s). Themethods of demonstrating health benefit(s) are to bevia a randomized double-blind, well-controlled designand the studies are to be conducted with numbers ofhuman subjects sufficient to enable statistical signifi-cance to be demonstrated. (c) With respect to pro-biotic evaluation for food use, the strain has to beidentified and functionally characterized on the basisofin vitro and animal studies. Safety is to be assessedfor new strains on the basis of in vitro and/or animal

studies and phase 1 human studies and at least onedouble-blind placebo-controlled phase 2 clinical trial,preferably confirmed by a second trial. Phase 3 effi-cacy trials comparing probiotics with the standardtreatment modality for a specific condition are to bedesigned to demonstrate their biotherapeutic effect.This approach is not necessary, however, to charac-terize a probiotic food. (d) With regard to labelingand regulatory issues, the consumer is to be providedwith correct and relevant information including thegenus, species and strain, the minimum number ofbacteria to be used, the viable concentration of eachprobiotic present at the end of the shelf life, the

storage conditions, etc.The FAO/WHO expert panel agreed that the sci-

entific evidence is sufficient to indicate that healthbenefits may be derived from consuming food pro-biotics, but more research is needed for many pro-biotics in order to confirm their actual health benefitin man. The research is to be conducted using asystematic approach and in accordance withrecommended assessment guidelines.

Probiotics and functional food

Functional foods represent a new area of interest inthe field of alternative/complementary medicine. Thefirst randomized nutritional clinical trial ever re-ported in English was conducted in 1747. James Lindassessed the effects of lime (which at that time wasneither a food nor a drug but a sailors nemesis) onthe crew of a Royal Navy ship, e.g. a large studypopulation [4]. Lind randomized the crew located onthe port side of the boat to receive lime with their diet

2 European Journal of Nutrition (2006) Vol. 45, Supplement 1 Steinkopff Verlag 2006


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and the crew on the starboard side to continuereceiving the standard diet. As one would expect, theport crew fared better than the starboard crew, whodeveloped scurvy that in some cases proved fatal.However, it took the Royal Navy a 100 years toestablish the use of lime as mandatory on its ships. Ofcourse, lime contains vitamin C, but it providesadditional health active benefits other than vitamin Calone since it also contains bioactive phytochemicalswhose effects still need to be investigated. Lime mayhave been the first functional food whose benefitswere demonstrated by a nutritional clinical trial.

What is a functional food? In Europe, it is a foodand not a dietary supplement and, as such, it needs tobe palatable and balanced, e.g., in addition to itsintrinsic nutritive value it should beneficially affect a

target body function, improve health and well beingand/or reduce risk(s) of disease. The criteria for def-inition and a means of assessing health benefits forfunctional foods were suggested by a report from aEuropean commission in 2004 [5] The Quality of lifeand management of living resources program wascoordinated by the European branch of the Interna-tional Life Science Institute. On the basis of theircriteria, there is strong evidence that foods containingcertain probiotics, e.g. milk fermented with Lactoba-cillus bulgaricus and Streptococcus thermophilus,which, while tasty and balanced, has been shown tosignificantly improve lactose digestibility (Fig. 1), and

thus to fulfill the definition of a functional food.

Gut microbiota: new methods of evaluating florasurvival and functions

The structural and functional diversity of bacteria is akey characteristic of gut microbiota. While the uterusprovides a sterile environment for delivering a

newborn, the neonate is rapidly and extensively col-onized after birth in its passage through the birthcanal. Human microbiota rapidly rises to 1011 bacteriaper gram of large-intestinal content and, in adults,reaches 1014 microorganisms, ten times more organ-isms than the number of eukaryotic cells in the body[7]. The human digestive tract, in particular the colon,harbors a large portion of bacteria that are visibleunder the microscope and yet cannot be cultured. Inrecent publications [810] the fraction that can becultured has been estimated to be in the order of 30%of total microorganisms present. The proportion ofthose identifiable bacteria rises sharply throughoutlife from 0% in preterm infants to 30% in very youngchildren and 80% in adults and 87% in the elderly [ 8].

j Identification of intestinal bacteria

We need to study microbial diversity and improve ourunderstanding of the role of intestinal microbiota ingut function in order to elucidate the influence ofexogenous factors, (e.g., diet including pre- and pro-biotics, lifestyle, environment) and endogenous (e.g.,innate and acquired immunity, host physiology, ge-netic background) factors on the composition andactivity of these microbiota. In turn, those factorsstrongly influence host functions, particularly stimu-lation of active substances and gene expression. These

influences can be studied by elucidating the diversityof the gut microbiota by investigating the effects oftheir key functional activities on the host includingin situ activation or inactivation of certain substances,by identifying functional groups of bacteria and bycorrelating bacterial activity with identity.

In order to enhance our understanding of bacterialdiversity, we needed culture-independent methods ofanalysis, namely molecular tools [11, 12] that arebased on identifying unique genome sequences

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Yoghurt Milk

%o

fdigestibility

absorbedlactose

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fdigestibility

absor

bedlactose

Heat-treatment

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Yoghurt MilkMilk + addedL. bulgaricus

Milkfermented

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Fig. 1 Yogurt lactose digestibility. A. Comparison of yogurt and L. bulgaricus with or without fermentation. B. Deleterious effect of heat treatment on yogurtinduced lactose digestibility. From Martini et al. [6]

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(Fig. 2). The identification of gut microbiota requiresestablishing a comprehensive database of 16S rRNAsequences for human gut microbiota and, when thishas been done, designing 16S rRNA-targeted diag-nostic oligonucleotide probes for accurate identifica-tion.

Over the last few years, whole cell fluorescence

in situ hybridization (FISH) and dot-blot hybridiza-tion probing methods have been continuously im-proved and are now being used increasingly toidentify human gut microbiota. In-situ hybridizationcoupled with flow cytometry shows that there are onlya few, highly prevalent dominant phylogenetic groupsin the gut microbiota [1315] (Fig. 3). A pan-Euro-pean study of 91 healthy adults from five countrieshas shown that three major groups are numericallysignificant, with no significant differences between thecountries studied [16]. These include: the Bacteroidesgroup, and among Gram+ organisms, the Clostridiumleptum group [17] and the Clostridium coccoides/Eu-

bacterium rectale group [12, 17, 18]. There are severalother groups, including Bifidobacterium, Atopobium,Lactobacillus and relatives that are quantitatively lessprevalent but found in many individuals. Thus, sixdominant and prevalent phylogenetic groups havebeen identified with an increasingly large panel ofprobes. On average, however, 25% of the bacteria thatare detected with universal probes are still not iden-tified. These data do not apply to infants, in whichBifidobacteria predominate together with some Bac-

teroides if the baby is breast fed [7, 19], nor probably

to older and very elderly people [20].With regard to the dominant species, each humanfecal microbiota appears unique, e.g., specific to theindividual. Indeed, dominant species diversity is: (a)variable from one individual to the next as shown in apioneer study in 1998 [21], but appears stable overtime in a given subject on his/her usual diet [22](Fig. 3); (b) resistant, in terms of classical ecology; (c)resilient, e.g. it is able to recover its original patternwithin 3060 days of exposure to stress such as

Bifidobacteria

C. coccoides/E. rectale groupClostridium leptum group

Staphylococcus

Lactobacillus

Streptococcus

Lactococcus

Leuconostoc

Atopobium

Veillonella subgroup

Fusobacterium

Eubacterium cylindroides group

Clostridium histolyticum group

Clostridium lituseberense groupClostridium ramosum

Bacteroides/Prevotella

Bacteroides distasonisPorphyromonas

10 %

Enterobacteria

Atopobium

Fusobacterium

Fig. 2 Phylogenetic tree of thedominant fecal microflora of a healthyhuman adult derived from partialsequence data, using 16S rRNAtargeted probes. Horizontal barrepresents 10% sequence divergence

Subject 1 Subject 2

98 99 97 99

Similarity above 90%

Fig. 3 Temporal temperature gradient gel electrophoresis of 16S rDNAamplicons of fecal samples from two subjects, studied at 1- and 2-yearintervals. The dominant species diversity of human gut microbiota remainsstable for years in a given adult subject



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antibiotic treatment (de la Cochetiere and Dore,unpublished observations, 2004); and (d) not mark-edly altered by probiotics [9, 23). The lack of dis-ruption in the consortium of dominant species, mostfrequently observed in feeding trials, should be con-sidered a positive outcome.

j Functional studies of gut microbiota

Following ingestion, probiotics traverse the gut in astrain-specific, and probably a food matrix-dependentmanner, as has been demonstrated by ileal recovery ofLactobacillus casei DN114 001 [24], Bifidobacteriumsp12 [25], B. spp [26], Bifidobacterium animalis DN173 010 [27], L. plantarum NCIMB 8826 [27], and, to alesser extent, Lactococcus lactis MG [28], and by fecalrecovery ofB. sp [29] and L. plantarum NCIMB 8826[28].

The passage of probiotics through the gut is not

simply a passive act. As has been shown with humanmicrobiota-associated (HMA) mice, a derivative ofthe L. casei DN114 001 strain survives transit at highpopulation levels (>109 cfu/g content) and initiatestranscriptional activity leading to protein synthesisfor specific genes during transit. Transcriptionalactivity is initiated one and half hours post-ingestion[24].

Similar studies using intestinal fluid samples fromintubated humans are in process using L. casei DN114001 (J. Dore unpublished observations, 2004) andL. plantarum [30, 31]. In addition, a study initiated byDutch investigators using fecal bacterial genes from

healthy and diseased subjects have provided new in-sight into the metabolomics of human intestinalmicrobiota [32]. These investigators used DNA-dependent/culture-independent methods or largeDNA fragments isolated and categorized from distalintestinal mucosa in conjunction with a bank of ref-erence genes to make these observations.

The survival of ingested probiotics continues to beof importance in this context. However, since thefunctional contribution of probiotics to the ecosystemis related to its activity rather than simply its pres-ence, assessment ofin vivo adaptation of probiotics inthe gut is an important focus. This should also extend

to the overall influence of probiotics on the intestinalmicrobiota, in general, which may respond function-ally to probiotics without actual changes in compo-sition. The genome sequences of probiotic strains andgut commensals, together with recent developmentsin functional genomics of the intestinal ecosystemshould provide new perspectives in this context.

Functional studies of gut microbiota have em-ployed other methods, such as the activation orinactivation of bioactive compounds, as well as,

in situ detection of microbial activity. One example ofthe activation/inactivation of bioactive compounds isequol, a bacterial induced metabolite (produced inabout 30% of humans) of isoflavone daidzein, a soyconstituent, with greater biological activity than theinitial compound itself. Isoflavones have been sug-gested to have health-related effects in the prevention

of hormone-induced malignant degeneration, ath-erosclerosis and osteoporosis, and in the alleviation ofmenopausal symptoms [33, 34]. Since microbialmodification of food ingredients may influence theireffectiveness, the organisms catalyzing alterationwould be a good indicator of whether activity ispresent in a given subject. In situ detection ofmicrobial activity includes detection of metabolicactivities or that of mRNA. Examples are: (a) In-situdetection of catalytic activities using fluorogenicsubstrates is of considerable interest but still re-stricted to a relatively small number of enzymes(b-glucuronidase, b-galactosidase, b-glucosidase).

Using flow cytometry technology with its cell sortingcapabilities, Rigottier-Gois et al. have demonstratedthe feasibility of enrichment of metabolically activelabeled cells [15]. (b) In situ detection of mRNA,which is widely used in eukaryotic cells, requires thatthe cells be permeable to reverse transcriptase andRNA polymerase, that amplification products are notalready existent in the cells and that mRNA be presentin sufficient quantities, and its signal be detectable.This approach cannot be widely applied to bacteriasimply because they are not sufficiently permeable toenzymes and because prokaryotes has a shortenedhalf-life of mRNA and their mRNAs are much less

stable than eukaryotes.Of major concern with regard to any novel ap-

proach to the detection of relevant microbial activitiesis whether a differential expression of bacterial genesexists. Recently several seminal publications havereported that bacteria influence the expression of hostgenes in eukaryotic cells. In particular, bacteriainfluence genes involved in the regulation of nutrientuptake and metabolism, mucosal barrier function andepithelial cell activities [3537]. However, despitethese observations, very little is known about whatactually happens to the gut when probiotics are in-gested. Genomics and proteomics may be another

helpful approach. In this context there are a numberof important unanswered questions. For instance,does the consumption of certain food ingredientshave an impact on the in vivo expression of bacterialgenes? And how are the expressions of certain genesat various sites in the GI tract affected?

Two papers by Oozeer et al. have demonstrated thesurvival of and the capacity for protein synthesisinitiation of a derivative of the L. casei DN114 001strain during its transit through the GI tract of



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gnotobiotic (HMA) mice [24, 38]. The physiologicadaptation of probiotics to the GI tract environmentthrough modulation of promoter activities has alsobeen demonstrated [39]. With a view to linking ben-eficial probiotic functions to new protein synthesisand obtaining specific information on probioticadaptation, reverse transcriptase (RT-PCR) combined

with other methods appears to be promising [39].Recombinase-based in vivo expression technology(RIVET) [40, 41] is another technique which is nowbeing applied to the study of gut microbiota. Thisvery specific approach enables identification of un-ique, transiently expressed genes even after they havereverted to a basal activity state.

In addition to the development of new methods,two questions remain to be answered. (a) What is therelative contribution to the composition of gut mic-robiota of a specific host genotype compared to theinfluence of exogenous factors such as diet? Themicrobiota composition of twins show greater simi-

larity than those of unrelated subjects or maritalpartners living together [42]. This would suggest thatthe composition of the microbiota has an importantgenetic component. However, the magnitude of thatcomponent has yet to be determined. (b) Is mucosa-associated flora more important to gut function thanluminal flora, e.g., that usually obtained in fecalsamples? This question is a central issue with respectto the crosstalk studied by immunologists and mi-crobiologists. The importance of the luminal flora isillustrated by the striking spontaneous healing andprotection from postoperative recurrence of Crohnsmucosal inflammation after diversion of the fecal

stream. However, in addition to the need for a cleardefinition of what constitute mucosal bacteria (e.g. arethey adherent bacteria?), it should be noted that fecalsamples, while easily obtained, are probably not rep-resentative of the true composition of luminal mic-robiota at any site other than the rectal lumen andcertainly not representative of mucosa-associatedflora.

Hostmicrobiota crosstalk: application ofprobiotics

Communication, e.g., crosstalk, between microor-ganisms in the gut lumen and those attached to themucosal surface and the host GI tract are diverse andcomplex. They include: competition/cooperation fornutrients; intra- and interspecies communication;direct contact between components of the bacterium,e.g., lipopolysaccharide, peptidoglygans, etc., and hostcell surfaces, secretion of bacterial compounds thatcan interact with underlying intestinal epithelium and

modulins which can directly effect host cell func-tion and responses (e.g., immune response, glycosyl-ation changes, etc.) (Fig. 4). To more completelyunderstand this crosstalk, studies that identify boththe bacteria and host contribution require furtherinvestigation. For example, understanding themolecular basis for nutrient sharing among membersof the normal gut microbiota is essential if we are toappreciate how the intestines microbial community isestablished and maintained and how it may be mod-ified by probiotics to the benefit of the host. Inaddressing this issue, an in vivo gnotobiotic mousemodel, e.g. a microorganism-free state in theintestinal ecosystem has proven of particular value[43].

Using this animal model to define the specific roleof gut flora in the development of gut functions,Bacteroides thetaioitamicron (Btheta) strain VPI-5482, a dominant member of normal human distalintestinal microbiota originally isolated from the fecesof a healthy adult, has been used as a model symbiont[37, 4447] in the pioneering work of Midtvedt andGordon et al. [4749]. The organism is a readily cul-tured, Gram-negative, obligate anaerobe. It becomes aprominent member of human and murine microbiotaduring a critical phase (the suckling to weaningtransitional period) of postnatal gut development.The bacterium can be genetically modified and itscomplete genome has been sequenced [50]. The 4779-member proteome of Btheta includes diverse mole-cules that function for example as an apparatus for

1.Competition/Cooperation

for nutrients

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3. Inter - species communication

- species communication

4.Host response (e.g.immune)

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Fig. 4 Hostmicrobiota cross-talk: main subjects of the dialogue. Reprintedwith permission from M. Lecuit and JL Sonnenburg



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acquiring and hydrolyzing dietary polysaccharides, anassociated environment-sensing system consisting ofa large repertoire of extra-cytoplasmic functions andone- or two-component signal transduction systems[50]. In a recent study Gordon et al. used DNA

microarrays to identify whole genome Btheta tran-scription profiles from organisms harvested directlyfrom the mouse cecum after a ten-day colonizationperiod [44]. The authors showed that Btheta utilizesspecific gut luminal polysaccharides via a specificuptake and degradation mechanism. GC-MS analysisof the standard mouse chow diet and of the total cecalcontents harvested from microorganism-free andBtheta-associated animals has established the follow-ing: (a) The major responses in vivo occur in genesdedicated to carbohydrate transport and metabolism(glycobiome activation). (b) Expression of Bthetaglycosylhydrolases mirrors the most abundant sugarsin the environment. (c) Btheta preferentially con-sumes the subset of the monosaccharides availablethat can be metabolized with the greatest efficiency.(d) In control experiments, the repertoire of glyco-sylhydrolase genes induced in the cecum are specificto the glycan structures found in the hosts mucuswhen mice are fed a custom chow diet containingglucose and sucrose as the only fermentable carbo-hydrates (JL Sonnenburg, J. Gordon et al. unpublishedobservations, 2004). And (e) finally, Btheta is not only

able to degrade dietary plant polysaccharides [44, 50]but also host-derived polysaccharides (e.g. host mu-cus glycans) (JL Sonnenburg, J. Gordon et al.unpublished observations, 2004). Such flexible adap-tation is known to strengthen complex food webs and

is likely to promote stability in the microbial com-munity. The Btheta genome encodes more than 200glycosylhydrolases [50] (Fig. 5), many of which aresecreted to allow for extracellular processing of thosecomplex polysaccharides. Compared to Btheta, a gutcommensal and probiotic, Bifidobacterium longum,has a more modest glycan-degradation capability buta more extensive repertoire of simple transporters[47]. These features suggest that B. longum may be adirect beneficiary of the products of Bthetas poly-saccharide degradation machinery. The approachused to examine Bthetas adaptive behavior in vivocan also be extended to examine the effects of pro-biotics on members of the normal gut communitysuch as Btheta.

This same technique has been used successfully todetermine the role of microbial colonization on gutfunctions. Using gnotobiotic mice monocontaminatedwith Btheta, functional genomic studies have shownthat this symbiont modulates the expression of genesinvolved in a broad range of important intestinalfunctions including nutrient absorption, angiogene-sis, xenobiotic metabolism and strengthening of the

Fig. 5 Carbohydrate foraging by Btheta [50]. Reprinted partly, with permission from J.L Sonnenburg



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innate immune system [37, 45, 46]. In order toinvestigate the microbial determinants of beneficialversus pathogenic hostbacteria relationships, similartypes of functional genomic studies were con-ducted on microorganism-free mice colonized byListeria monocytogenes, or its non-pathogenic closerelative, L. innocua. The results revealed that the

hosts response to L. innocua was very similar to thatdocumented with Btheta. In contrast, L. monocytog-enes elicited a complex sequence of host geneexpressions that included NF-jB-dependent and IFN-responsive pathways. Of interest, a L. monocytogenesmutant for listeriolysin induced a host response thatmimics that of L. innocua (M. Lecuit, J. Gordon et al.unpublished observations, 2004). These findingssuggest that, in this case, the presence or absence of asingle gene product which enables a microorganismto access the cytoplasmic compartments of host cellscan profoundly influence the hostbacterial relation-ship.

Such studies underscore the value of the gnotobi-otic animal model in identifying the genomic andcellular factors that regulate interactions betweenbacteria and their host within the intestinal ecosys-tem. Such a powerful approach enables a step-by-stepidentification of individual organisms contributing tointestinal host responses, including that of probiotics.This approach combines sophisticated tools such aslaser capture micro-dissection to distinguish amongindividual cell types of those cells that are responsiblefor the host response and murine genetic techniques(e.g., knock-out and transgenic mice). Together withsimplified in vitro (cell culture) systems enabling

investigation of a highly controlled environment andscreening for probiotic potential, the animal modelshold the promise of providing a conceptual andexperimental framework for exploring mechanisms ofprobiotichost crosstalk in healthy and diseased hu-mans. However, the transposition of these observa-tions to humans and clinical relevance is yet a furtherstep. Thus transposition will necessitate associating,within ethical limits, clinical trials and basic mecha-nistic studies including using organ cultures of hu-man gut biopsies for microbiota exposure, repetitionof studies already performed using animal modelsystems and the use of publicly available databanks in

a bedside to bench top approach to elucidate themechanisms of crosstalk.

Probiotic interactions with the immune system

Because the mucosal immune response of the gut isso important to host defense it is likely that probi-otics will have an influence on this important gutfunction.

j Immune function as a biological marker to assesshealth benefits of probiotics

Because recent evidence indicates that probiotics (e.g.S. thermophilus and L. bulgaricus) may influence bothsystemic and gut-associated immune responses [51],systemic and intestinal immune biomarkers have

been suggested as the basis for assessing the nutrientrequirements and/or health benefits of functionalfoods including probiotics. The number and functionof circulating T-cells migrating from Peyers patchesto the intestinal mucosa (gut-homing T-cells) mayconstitute a surrogate immune marker for the effect ofprobiotics in healthy children and adults since thosecells can be identified by their expression of integrina4b7. Similarly, mucosal IgA responses can be ana-lyzed in blood and secretions, as T-cells also transitfrom organized lymphoid tissue to the mucosa via thecirculation. In vitro intestinal mucosa organ culturesof intestinal biopsies have been used as a technique to

determine the effects of exposure of normal and in-flamed gut to probiotic bacteria. Measuring diseaseparameters directly or determining surrogate markersof immune modulation in healthy subjects will notonly provide clues as to the mechanisms of probioticson immune function but should also constitute a basisfor making health claims.

j Immunological effects of probiotics

Lactobacillus species markedly inhibit TNF-a pro-duction by normal and inflamed (e.g. Crohns disease)

mucosa. The mechanism has yet to be elucidated.Compelling in vivo data generated in children show-ing that feeding pregnant women and their newbornsafter birth with probiotic bacteria reduces by one halfthe incidence of allergic eczema [52, 53]. Unfortu-nately this clinical effect is not reflected by any of thelaboratory markers associated with eczema (e.g., totalIgE, specific IgE and skin prick reactivity) but there isevidence that probiotic bacteria can inhibit Th2 re-sponses to house dust mites [54]. Further studies onatopy are ongoing in Finland. The public health valueof using probiotics may be enhanced by the observedinverse relationship with the incidence, over the last

50 years, of prototypical infectious diseases and theincidence of autoimmune and allergic disordersincluding asthma, Crohns disease, multiple sclerosisand type 1 diabetes mellitus [55]. This observationmay help explain the positive effect of probiotics onatopic eczema prevention [53]. There are also studiesto suggest the immunological effects of probiotics onthe skin (e.g., L. casei has been shown to reduceexperimental hapten-specific CD8+ T-cell-mediatedskin inflammation in the mouse) [56]. An association



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between L. casei and the usual yogurt starter bacte-riaS. thermophilus and L. bulgaricusreducesthe frequency of skin-homing CD4-CLA+ (a skin-associated antigen) cells (T. T. MacDonald et al.unpublished observations, 2004).

Some probiotics enhance while others suppressimmune responses. What mechanisms are involved?

Recent preliminary data suggest that probiotics couldact through stimulating regulatory T-cells which canactivate both of these responses. This is a major areaof investigation with respect to the immuneenhancing function of probiotics.

Most of the immunobiological effects of probioticsare likely to take place in gut-associated lymphoidtissue, including Peyers patches, in the small intes-tine. At that site, due to less difference between thenumber of probiotic and resident bacteria, probioticbacteria may compete with luminal microbiota moreeasily than in the colon, which is already heavilypopulated with indigenous bacteria. Furthermore, the

crosstalk between probiotics and the small intestinemay be different from that in the colon. In addition,some of the crosstalk effects may be age-dependent.The maturing small intestine of the newborn is ini-tially exposed to a large number of colonizing bacteriaacquired while passing through the birth canal. In theabsence of mature intestinal function (mucus pro-duction, peristalsis, etc.) large numbers of bacteriacolonize the small intestine in contrast to the presenceof large numbers of colonizing bacteria only in thedistal ileum, cecum, and colon in the mature intestine.Thus this initial early exposure of the small intestineto colonizing flora may be an important step in the

appropriate maturation of mucosal immune system.It is extremely difficult to simulate the complex

bacterialmucosal immune interaction using in vitromodels, although models of the human mucosal im-mune system have been developed using co-culturedcolon cancer lines and blood mononuclear cells. Inmany studies, probiotics have been added to immunecells in vitro. However, there does appear to be avariable response among different probiotic bacteria,both live and dead, to induce cytokine production bymacrophages and dendritic cells with large variationsin the amounts of IL-12, IL-10 and TGF-b producedby individual strains. The mechanisms of these vari-

able responses have not been elucidated and requirefurther study. It is likely, however, that the probioticchanges in regulatory T-cell activity is due to theireffect on antigen-presenting cells. Lactobacillus pro-vides a good example of a specific probiotic-immu-nological effect. As a Gram-positive bacterium,Lactobacillus express ligands for toll-like receptors(TLRs) which initiate immune responses and enablegut epithelium and immune cells to recognize bothpathogens and indigenous microbiota. With regard to

probioticTLR interaction, a recent finding is of greatinterest. It has been reported that recognition ofcommensal bacteria by TLRs is necessary for intesti-nal homeostasis, protection of epithelial cell frominjury and stimulation of repair [57]. Using in vitroepithelial cell line techniques, probiotics (e.g. VSL#3)have been shown to increase barrier function [58] via

TLR2, the toll-like cell surface receptor that recog-nizes peptidoglycans of Gram-positive bacteria [59].Thus, a probiotic signaling through the TLR2 caninhibit allergen uptake by maintaining mucosal bar-rier integrity and thereby preventing the expression ofeczema. There is also evidence that different cells inthe gut (e.g. epithelial vs. dendritic cells) expressdifferent TLRs at different stages of development. Inaddition, specific probiotics (e.g., L. rhamnosus [GG])[60] can prevent cytokine-induced apoptosis andcan inhibit NF-jB activation (e.g. L. reuteri) [61]when M-cells overlying Peyers patches allow probi-otic uptake and presentation to dendritic cells. These

observations can be confirmed in vivo by demon-strating the presence of bacteria in ileal lymphoidfollicles by endoscopic biopsy.

Thus with regard to mucosal immunology, probi-otics may increase mucosal barrier function, preventapoptosis of epithelial cells and ultimately decreaseantigen uptake, especially in the small bowel.

Clinical use of probiotics

Important clinical data with regard to probiotic use inclinical disease have recently been generated in adults,children and infants. The FAO and WHO guidelines[2, 3], albeit several years away from implementationin United Nation countries, will undoubtedly con-tribute to ensuring that reliable, clinically provenprobiotics are available in the future [62].

j Adults

The evidence for the efficacy of various probiotics isalready reasonably strong as a result of randomizedcontrolled trials (RCTs) and meta-analyses for lactosemalabsorption (significant improvement in lactose

digestibility induced by L. bulgaricus and S. thermo-philus present in yogurt) [63, 64], infectious gastro-enteritis [65] and antibiotic-associated diarrhea (e.g.Saccharomyces boulardii and Lactobacillus) [66].

Other selected clinical conditions warrant furtherstudy because of mixed clinical results. These includeinflammatory bowel disease (IBD), irritable bowelsyndrome (IBS), the effect on intestinal transit time,Helicobacter pylori gastritis and nosocomialinfections



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Inflammatory bowel disease

RCTs have been conducted with various probiotics(including VSL#3, Escherichia coli Nissle 1917,L. rhamnosus GG, S. boulardii, and other Lactobacil-lus or Bifidobacterium strains) on patients withrefractory pouchitis after ileoanal anastomosis for

ulcerative colitis, in ulcerative colitis (prevention ofrecurrence) and in Crohns disease (prevention ofspontaneous and postoperative recurrence) [67].Currently, the most convincing evidence of clinicalefficacy has been obtained with pouchitis (e.g., acutepouchitis prevention, chronic pouchitis prevention,maintenance of remission in recurrent and resistantpouchitis) which is a unique complication of IBDwhich transiently responds to antibiotics. VSL#3, aprobiotic preparation containing eight bacterialstrains has been reported to prolong the treatmentremission [6870]. In ulcerative colitis per se, E. coliNissle 1917, a probiotic not given as a food but as a

supplement, was shown to be as effective as low dosemesalamine treatment in preventing relapse [7173].However, no placebo-controlled trial is currentlyavailable. There are also studies in which probioticsare given as food (e.g. Lactobacillus rhamnosus GGand Bifidobacterium). The results, however, are not asconvincing as with those with pouchitis. Several RCTshave been conducted in the treatment of Crohnsdisease but the results are equivocal. While E. coliNissle 1917 was thought to be superior to placebo inpreventing a relapse, Lactobacillus GG, given for1 year, was of no benefit in preventing postoperativerecurrence [74]. In Crohns disease, lessons fromanimal models may be particularly useful. Forexample, a novel approach to probiotic therapy hasbeen suggested for the prevention and treatment ofcolitis in two murine models using Lactococcus lactisengineered to secrete recombinant human IL-10 [75]or trefoil factors [76]. This approach is now thesubject of pilot trials in IBD patients [67].

Irritable bowel syndrome

In IBS, the usual therapeutic interventions are gen-erally not much more effective than placebo. Theplacebo effect is marked but insufficient as a therapy

in clinical practice. Therefore, large populations ofpatients are required to assess a legitimate probioticeffect. Most published RCTs included only a smallnumber of patients for relatively short follow-upperiods. Some probiotic strains (e.g., L. Shirota) and acocktail of strains (VSL#3) appeared effective with IBSsymptoms of bloating [77, 78], while others (e.g.L. plantarum 299V) proved ineffective [79, 80]. Thetherapeutic strategy in post-infectious IBS, anincreasingly frequent entity within the IBS spectrum

in certain countries [81], may benefit from the recentfinding that L. paracasei normalizes muscle hyper-contractility in a murine model of post-infectious gutmotility dysfunction [82].

Intestinal transit time

In randomized studies, milk fermented with the Bifi-dobacterium animalis DN173 010 strain and lacticacid bacteria or yogurt bacteria significantly acceler-ated whole-gut and colonic transit time in healthyvolunteers [8385]. This effect with respect to accel-eration of both total colonic and sigmoid transit timeshas been confirmed in a double blind cross-over RCTin subjects with prolonged basal transit duration [86].These findings may prove of special interest in IBDpatients with slowed colonic transit or withconstipation.

Helicobacter pylori gastritisThrough 2004, 15 open-label or randomized clinicaltrials have addressed the effects of probiotics alone, ormore commonly as adjuncts to the standard triple-agent therapy. The most frequent shortcomings ofthese RCTs were the small study populations and theshort durations of probiotic administration and fol-low-up. Eradication of H. pylori was not observedwith probiotics alone, except in two open-label stud-ies for which 20%40% eradication was reported. Instudies combining probiotic and triple-agent therapy[(six trials and an additional three trials usingL. johnsonii La1) [87], milk fermented by L. acido-

philis La5, and B. lactis Bb12 with yogurt bacteria (S.thermophilis and L. bulgaricus)] [88] more positiveoutcomes have been noted. These included decreasedH. pylori, urease activity, as determined by 13C-ureabreath tests, and decreased gastric inflammation and/or decreased bacterial mucosal density. In no studydid addition of probiotics enhance the triple therapy-induced rate of H. pylori eradication but a signifi-cantly reduced incidence of side effects related toantibiotics was reported with L. GG [89].

Nosocomial and postoperative infections

While certain probiotic strains (S. thermophilus, L.acidophilus, L. casei DN-114 001) and a mixture ofstrains (VSL#3) may enhance intestinal barrier func-tion in animal models and in in vitro human cell lines[58, 90, 91], L. plantarum 299V and synbiotics con-taining L. acidophilus La5, B. Lactis Bb12, S. thermo-

philus and L. bulgaricus do not modify the incidenceof bacterial translocation or the postoperative septiccomplications in patients undergoing surgery [92] nordid they affect sepsis in critically ill patients in



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intensive care units [93, 94]. However, L. plantarum299V given with fiber supplements was shown to re-duce sepsis in patients with acute pancreatitis [95]and in liver transplant recipients [96].

j Infants and children

In 2001, the Committee on Nutrition of the EuropeanSociety of Pediatric Gastroenterology, Hepatology andNutrition extensively reviewed information availableon the effects of probiotics given as dietary productsto infants [97]. At the 2nd World Congress of Pedi-atric Gastroenterology, Hepatology and Nutritionheld in Paris on July 37, 2004, numerous papersregarding probiotic use in children were presented.The topics included childhood diarrheal diseases,experimental models of intestinal inflammation andbacterial infection, H. pylori gastritis, chronicconstipation, and intestinal bacterial overgrowth.

Acute diarrheal diseases

Three meta-analyses of RCTs clearly support theobservation that probiotics significantly shorten theduration of acute infectious diarrhea by a mean of18 h (8 RCTs, 773 patients) [98], 17 h (7 RCTs, 675patients) [99], and 29 h (12 RCTs, 970 patients) [100],respectively. These findings were especially true usingLactobacillus [99], in particular L. GG [98]. Two of themeta-analyses also showed that certain probioticssignificantly reduce the risk of diarrhea lasting threeor more days [98, 100]. These data confirm the con-

clusions of a previous review which suggested aclinically significant benefit of probiotic use in thetreatment of acute infectious diarrhea, particularlyrotavirus gastroenteritis in infants and children [101].Treatment of acute infectious diarrhea, especially inchildren, may prove to be a primary therapeuticapplication after probiotic therapy in pediatric prac-tice [62]. However, additional large-scale RCTs areneeded to definitively establish the clinical relevanceof this finding.

With regard to the prevention of community-ac-quired and nosocomial diarrhea, two studies estab-lished, through home visits and daycare center

evaluations respectively, that L. GG was associatedwith a reduced incidence of diarrhea in Peruvianbabies [102] but not in Finnish children aged16 years [103]. In contrast, studies in hospital-set-tings in Europe and USA demonstrated a decreasedincidence of acute infectious diarrhea. The first studywas conducted with L. GG [101], the second with B.bifidum + S. thermophilus [104] and the third, whichshowed no effect, with L. GG [105]. Effective probioticprevention of antibiotic-associated diarrhea in chil-

dren has been recently confirmed in a RCT usingS. boulardii. This study included 269 subjects aged6 months to 14 years [106].

The mechanism(s) of the probiotic effect in infec-tious diarrhea has (have) yet to be fully elucidated.Probiotics may protect the intestine by competingwith pathogens for adhesion sites, strengthening the

mucosal barrier and tight junctions between entero-cytes and/or enhancing the mucosal immune re-sponse to pathogens [107].

Experimental models of intestinal inflammation andbacterial infection

The probiotic strain E. coli Nissle 1917 activates thehuman B defensin-2 promoter in Caco-2 cells. Acti-vation does not occur following transfection of mu-tated NF-jB suggesting a possible new pathway ofprobiotic action mediated by the antimicrobial pep-tide human B defensin-2. L. plantarum 299V enemas

can reduce the induction of dextransodium sulfate-induced colitis in rats and has been shown to upre-gulate Muc2 colonic mucin gene expression. Thesefindings may prove to be clinically relevant. In amodel of colonic bacterial (Citrobacter rodentium)infection in mice, L. rhamnosus and L. acidophilusprevented colonic hyperplasia, decreased bacterialinternalization by the mucosa and reduced infection-related cell damage. A preliminary study ofL. GG in asmall number of children with active Crohns diseaseshowed therapeutic benefit and allowing for taperingof steroids [108].

Helicobacter pylori gastritis

The effects of probiotics on pediatric patients withthis condition are more disappointing. One or threeprobiotic strains (L. casei defensis, S. thermophilusand L. bulgaricus) were administered in a prospectiverandomized clinical trial (conventional triple-agenttreatment + 7 days of probiotic administration). Anincreased rate of Helicobacter pylori eradication wasonly observed when the three probiotic strains werecombined with the conventional triple-agent anti-H. pylori treatment [109].

Childhood chronic constipation

In a double blind RCT, L. GG induced no beneficialeffect to that of lactulose alone in 2- to 16-year-oldchildren with chronic constipation [110].

Intestinal bacterial overgrowth

A striking finding, to be confirmed in extensive RCTsstudies, is that killed L. acidophilus administration



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was associated with normalization of expired2H breath-test results at one month in 14 out of 16children with documented bacterial overgrowth [111].

j Neonates

A recent (19992003) randomized trial of 208 very lowor extremely low birth-weight infants given eitherBifidobacterium breve or a placebo within the first24 h after birth (Y. Yamashiro, et al. unpublishedobservations, 2004) suggests that probiotics may beuseful in the prevention of necrotizing enterocolitis(NEC). No death from infection occurred in theB. breve group as opposed to a 13.5% rate in thecontrol group. B. breve administration, which hasbeen shown to promote colonization by Bifidobacte-rium [112], may also stimulate immunologicaldevelopment in very low birth-weight infants. Onemechanism suggested is due to an increased pro-

duction, of TGF-b, a cytokine that enhances IgAproduction, immune oral tolerance, intestinal muco-sal wound healing and epithelial cell proliferation anddifferentiation. The clinical results obtained in thisJapanese study warrant further randomized, multi-center RCTs to confirm this important observation.

Cultural views on probiotics: USA vs. EUROPE

In this section cultural views are comparativelyconsidered regarding the commercial market forprobiotics with respect to distribution and sales,

sociocultural, considerations, regulatory constraints,probiotic research analysis and trends and forecasts.

j Probiotic market and sociocultural considerations

The levels of and reasons for probiotic consumptiondiffer considerably in Europe and the USA. Ninety-five percent of the consumption in Europe and lessthan 50% in the USA is accounted for by animal feeds

and dairy foods, while dietary supplements accountfor 4% in Europe and up to 56% in the USA (Fig. 6)(Frost and Sullivan Consultant Report: European andUnited States Probiotics Markets, 2003). The globalyogurt market is growing in both Europe and theUSA, but consumption is far greater in Europe (Eu-romonitor 2004). The same difference exists for

fermented dairy drinks (Fig. 7).With regard to sociocultural conditions (Frost and

Sullivan Consultant Report: European and UnitedStates Probiotics Markets, 2003), Europeans are morefamiliar with the health benefits of fermented dairyproducts, are not opposed to using such dairy prod-ucts, have some rudimentary sense of the process offermentation, need to be informed about and aresometimes skeptical about the harmlessness of bac-teria. The situation is quite different in the USA.Americans do not have a tradition of consumingfermented dairy products, which they consider tohave an unpleasant acid taste, do recognize the health

benefits of fermented dairy products but to a lesserextent than Europeans are not in general aware of thelive dimension of yogurts, associate bacteria withgerms causing disease, and have a poor under-standing of the underlying value of probiotics. How-ever, there are also some other societal issues whichcould contribute to an increase in the US probioticmarket. These include (a) an expanding interest inalternative medical practices and in the prevention ofdisease; (b) a collective attempt to seek a healthierlifestyle; and (c) unlike Europeans, a view of probi-otics as dietary supplements (e.g., nutraceuticals) andnot as food. This is illustrated by their constantly

increasing market as dietary supplements from 1990to 2000. The collective market has been estimated toreach 14 billion dollars in 2000 [113] (Fig. 8). Themarket should continue to increase and is a majorgrowth area of health-related products. Thus, Amer-icans are very interested in improving the quality oftheir health through both diet and lifestyle changes.An increasing US probiotic market has been fore-casted through 2010 (Frost and Sullivan ConsultantReport: European and United States Probiotics Mar-

67%

28%4% 1%

Europe, 2000 - 2010 US, 2003

Animal feed Dairy foodsDietary supplements Others applications

40 %4%

56%

1%

Fig. 6 Comparison of probiotic markets:Europe vs. US (from the Frost andSullivan Consultant Report: Europeanand US Probiotic Markets, 2003)



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kets, 2003). In particular, specialized nutritionalproducts for specific segments of the population (e.g.,infancy, athletes, elderly, etc.) are potential majorfuture growth areas. The US market for probioticyogurt (solid and liquid) and fermented dairy drinksis also increasing.

With regard to probiotic consumption at present,there are two proteotypic countries: (a) the Scandi-navian countries, Benelux, France, Germany, Austriaand Switzerland, in which there is a good under-standing of the probiotic concept due to a long tra-

dition of consuming fermented dairy drinks andyogurts; and (b) the USA, Ireland, the United King-dom, etc. where the concept is much less welldeveloped.

j Regulatory constraints

The regulatory constraints in Europe differ widelyfrom those in the USA and Japan, probably due to theinfluence of sociocultural traditions. In Europe there isas yet no legal definition offunctional foods and nolegislation on their use in health claims. With regard to

probiotic products, there has been no legislation onthe use of microorganisms in human food and noprohibition of functional claims with regard to pro-moting health. In contrast, important health claims forprobiotics are prohibited if they suggest diseasetreatment or disease prevention. Furthermore, the useof probiotics in animal feed is closely controlled. In theUSA, all types of health claims (for health and diseaseprevention are authorized for functional products ifthe benefits have been demonstrated. Probiotics are

generally considered dietary supplements and, assuch, are subject to the Dietary Supplement, Health

and Education Act. This act was passed by Congressin 1994 and provides a new framework for theregulation of dietary supplements by the FDA. Al-though the US restrictions seem a bit stricter thanthose in Europe, the act is still very liberal, enablingmanufacturers to sell supplements by listing labelcontents. Probiotic products require FDA approval orGRAS (Generally Recognized As Safe) status. The USregulations are thus not as stringent for functionalfoods as for the pharmaceutical products.

Market sizes - historic - retail value rsp - US$ mn - current prices

North America

Market sizes - historic - retail value rsp - US$ mn - current prices

Western Europe

US$mn

%cumulativegrowth

US$mn

%cumulativegrowth

20 000 60 -80

-60

-40

-20

0

40

20

0

-20

15 0004 000

3 000

2 000

1 000

0

5 000

10 000

5 000

01998

1999

2000

2001

2002

2003

2004

1998

1999

2000

2001

2002

2003

2004

WESTERN EUROPE USA

Fig. 7 Global fermented dairy drink market, Western Europe vs. North America: an overview up to 2004 (from Euromonitor 2004)

Sales

(dollars

x

106)

U.S. diet-supplement sales

15

10

5

01990

$3.3$3.7

$5.0

$6.5

$12

$14 est

1992 1994 1996 1998 2000

Fig. 8 Reprinted with permission from S.H. Zeisel [113]



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j Probiotic research analysis

Over the past 10 years, there have been numerouspublications on probiotics (2,632 papers) primarilyaddressing the digestive tract (1,440 papers), partic-ularly Lactobacillus than Bifidobacterium. The prin-cipal studies addressed intestinal health and disease,

but there was also considerable variety of emergingtopics. These included: the immune system, eczemaand other allergies, cholesterol levels, urinary tractinfections, colonic polyps, cancer, etc. There weremany more review articles than original publications.More publication emanated from Europe than theUSA. Only a modest number of RCTs have beenconducted. It is of interest that the largest consortiumof investigators studying the lactic acid bacterialgenome is led by the American Dairy Association andthat a number of the Lactobacilli undergoing genomesequencing are owned by that consortium. Therefore,it is likely that a considerable quantity of follow-up

results from the basic research conducted by USuniversities and corporations will emerge in thefuture.

j Trends and forecasts

Several clinically pertinent trends are likely to becomeestablished soon. For example, probiotics can be usedas surrogate flora for the initial colonization of the gutunder circumstances in which adequate colonizationdoes not occur in neonates. Based on seminal studiesby Isolauri et al. [51], probiotics may become estab-

lished as a preventative measure in allergic mothersgiving birth to allergy-prone infants. However, beforethis measure can be established as a clinical recom-mendation confirmation as to its efficacy has to beestablished by large, multi-center RCTs. The adjuvanteffects of probiotics used before standard vaccination,as was shown using L. GG with typhoid vaccination[114] may become established as standard clinicalpractice. A large multi-center RCTs of probiotics inday care centers published in 2001 has shown sig-nificant reduction in respiratory infection [103] andadditional studies suggesting protection against no-socominal diarrhea in infants using L. GG [101] may

result in new standard clinical care policies world-wide. Finally, the potential advantage of preparinggenetically engineered tailor made probiotics, suchas a study in which human IL-10, an anti-inflamma-tory cytokine, was shown to be locally secreted on theintestinal surface by a probiotic may be standardtherapy for inflammatory intestinal conditions [67] inthe future if shown through RCTs to be efficacious.

It is strongly suggested that probiotics taken asfermented foods has the added advantage of not only

providing probiotic benefits but also providing theadvantages of a healthy diet.

In summary, the potential future uses of probioticsare as follows: their use as functional foods(nutraceuticals) in dietary supplements may be asimportant as taking multi-vitamins. They could beroutinely incorporated into infant formulas and

weanling food (pediatric yogurt) to assure adequatedevelopment of mucosal immunity. With the reduc-tion of host defenses in the elderly, they could beroutinely used in nursing homes as a food source(e.g., yogurt, dairy drinks) or supplements to reducenosocominal infection. However, before these usesbecome routine additional RCTs are required toconfirm their clinical efficacy.

Conclusion

In 20012002, an expert panel from the FAO/WHO

convened to assess the use of probiotics and agreedthat adequate scientific evidence exists to indicate thatthere is potential for deriving health benefits fromconsuming food-containing probiotics. Howevermore research is needed to confirm a number of thosehealth benefits in humans by applying a systematicapproach and complying with the assessmentguidelines suggested by the FAO/WHO report.

A molecular approach to the identification of gutmicrobiota is now possible because genomic sequenceof an increasing number of bacteria are available. Inrecent years, the genomic sequences of an increasednumber of probiotics or related lactic acid bacteria

have been reported. The sequence information re-trieved may be useful to establish a better under-standing of the metabolic potential and function ofthose bacteria in the human intestinal tract. In con-

junction with classic molecular biology and culture-independent techniques, these approaches may beused to define the mechanism by which probioticsmay influence the health of the gut.

A major stumbling block exists to the clinical rec-ommended use of probiotics in humans before theycan be recommended for specific health benefits or toprevent/treat a specific illness. They include: [1] con-clusive, statistically significant effects demonstrated

by large, multi-center RCTs with a statistically reliablenumber of patients studied [2], furthermore, the spe-cific clinical effect are probiotic strain and dose spe-cific and their use can not be generalized to otherprobiotics or at other dose levels. The field of clinicalresearch on probiotics is currently expanding to in-clude studies of effects on the skin, joints, liver diseaseand obesity. In the latter condition, for example, veryrecent basic studies have shown that the conventionalgut microbiota may induce a number of changes in



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gene expression relating to body fat accumulationthrough interaction with the epithelial expression of afasting-induced adipocyte factor, e.g. angiopoietin-like protein. This new identified factor inhibits lipo-protein lipase activity and triglyceride storage inadipocytes and interferes with insulin sensitivity [36].

Much work remains to be done. However, a sci-entific motivation to consolidate basic and appliedresearch from molecular and cellular microbiologystudies to immunologic and clinical nutritionalapplication is underway.

References

1. Reid G, Guarner F, Gibson G, et al.(2004) Discussion on toll-like receptor9 signaling mediates the anti-inflam-matory effects of probiotics in murineexperimental colitis. Gastroenterology127:366377

2. Food and Agriculture Organization ofthe United Nations and World Health

Organization (FAO-WHO) (2001)Regulatory and clinical aspects of dairyprobiotics. FAO of the United Nationsand WHO working group report. On-line: http://www.fao.org/es/esn/food/foodandfood_probiocons_en.stmhttp://who.int/foodsafety/publications.fs_management/probiotics/en/

3. Food and Agriculture Organization ofthe United Nations and World HealthOrganization (FAO-WHO) (2002)Guidelines for the evaluation of pro-biotics in food. FAO of the UnitedNations and WHO working group re-port; online:FAO: http://www.fao.org/es/esn/food/foodandfood_probio-

cons_en.stm-WHO:http://who.int/foodsafety/publications.fs_manage-ment/probiotics/en/

4. Lind J (1753) Treatise on the scurvy,Millar A (ed). London

5. Conseil de lEurope. Lignes directricessur la justification scientifique desallegations sante des aliments fonc-tionnels. Document techniquewww.coe.fr/soc-sp Arch/dav 2001-4

6. Martini MC, Lerebours EC, Lin WJ,Harlander SK, Berrada NM, AntonieJM, Savaiano DA (1991) Strains andspecies of lactic acid bacteria in fer-mented milks (yogurts): effects on invivo lactose digestion. Am J Clin Nutr

54:104110467. Walker WA, Dai D (1999) Protectivenutrients for the immature gut. In:Ziegler EELA, Moro GE (eds) Nutri-tion of the very low birthweight in-fant. Lippincott Williams andWilkins, Philadelphia, pp 179197

8. Suau A, Bonnet R, Sutren M, et al.(1999) Direct analysis of genesencoding 16S rRNA from complexcommunities reveals many novelmolecular species within the humangut. Appl Environ Microbiol 65:47994807

9. Tannock GW, Munro K, Harmsen HJ,

Welling GW, Smart J, Gopal PK(2000) Analysis of the fecal microfloraof human subjects consuming a pro-biotic product containing Lactobacil-lus rhamnosus DR20. Appl EnvironMicrobiol 66:25782588

10. Hayashi H, Sakamoto M, Benno Y(2002) Phylogenetic analysis of thehuman gut microbiota using 16SrDNA clone libraries and strictlyanaerobic culture-based methods.Microbiol Immunol 46:535548

11. Namsolleck P, Thiel R, Lawson P, etal. (2004) Molecular methods for theanalysis of gut microbiota. MicrobEcol Health Dis 16:7185

12. Zoetendal EG, Collier CT, Koike S,Mackie RI, Gaskins HR (2004)Molecular ecological analysis of thegastrointestinal microbiota: a review.J Nutr 134:465472

13. Blaut M, Collins MD, Welling GW,Dore J, van Loo J, de Vos W (2002)Molecular biological methods forstudying the gut microbiota: the EUhuman gut flora project. Br J Nutr87(Suppl 2):S203-S211

14. Rigottier-Gois L, Le Bourhis AG,Gramet G, Rochet V, Dore J (2003)Fluorescent hybridisation combinedwith flow cytometry and hybridisa-tion of total RNA to analyse the

composition of microbial communi-ties in human faeces using 16S rRNAprobes. FEMS Microbiol Ecol 43:237245

15. Rigottier-Gois L, Rochet V, Garrec N,Suau A, Dore J (2003) Enumeration ofBacteroides species in human faecesby fluorescent in situ hybridisationcombined with flow cytometry using16S rRNA probes. Syst Appl Micro-biol 26:110118

16. Lay C, Rigottier-Gois L, Holmstrom K,et al. (2005) Colonic microbiota sig-natures across five northern Europecountries. Appl Environ Mibrobiol71(7):41534155

17. Sghir A, Gramet G, Suau A, Rochet V,Pochart P, Dore J (2000) Quantifica-tion of bacterial groups within human

fecal flora by oligonucleotide probehybridization. Appl Environ Micro-biol 66:22632266

18. Franks AH, Harmsen HJ, Raangs GC,Jansen GJ, Schut F, Welling GW(1998) Variations of bacterial popu-lations in human feces measured byfluorescent in situ hybridization withgroup-specific 16S rRNA-targetedoligonucleotide probes. Appl EnvironMicrobiol 64:33363345

19. Bourlioux P, Koletzko B, Guarner F,Braesco V (2003) The intestine and itsmicroflora are partners for the pro-tection of the host: report on the Da-none Symposium The Intelligent

Intestine, held in Paris, June 14,2002. Am J Clin Nutr 78:675683

20. Harmsen HJ, Wildeboer-Veloo AC,Grijpstra J, Knol J, Degener JE, Well-ing GW (2000) Development of 16SrRNA-based probes for the Corio-bacterium group and the Atopobiumcluster and their application for enu-meration of Coriobacteriaceae in hu-man feces from volunteers of differentage groups. Appl Environ Microbiol66(10):45234527

21. Zoetendal EG, Akkermans AD, DeVos WM (1998) Temperature gradi-ent gel electrophoresis analysis of 16SrRNA from human fecal samples re-

veals stable and host-specific com-munities of active bacteria. ApplEnviron Microbiol 64:38543859

22. Seksik P, Rigottier-Gois L, Gramet G,et al. (2003) Alterations of the domi-nant faecal bacterial groups in pa-tients with Crohns disease of thecolon. Gut 52:237242



16/18

23. Spanhaak S, Havenaar R, Schaafsma G(1998) The effect of consumption ofmilk fermented by Lactobacillus caseistrain Shirota on the intestinal mi-croflora and immune parameters inhumans. Eur J Clin Nutr 52:899907

24. Oozeer R, Mater DD, Goupil-FeuilleratN, Corthier G (2004) Initiation ofprotein synthesis by a labeled deriva-tive of the Lactobacillus casei DN-114001 strain during transit from thestomach to the cecum in mice har-boring human microbiota. ApplEnviron Microbiol 70:69926997

25. Pochart P, Marteau P, Bouhnik Y,Goderel I, Bourlioux P, Rambaud JC(1992) Survival of bifidobacteria in-gested via fermented milk duringtheir passage through the humansmall intestine: an in vivo study usingintestinal perfusion. Am J Clin Nutr55:7880

26. Marteau P, Pochart P, Bouhnik Y, ZidiS, Goderel I, Rambaud JC (1992)Survival of Lactobacillus acidophilusand Bifidobacterium sp. in the smallintestine following ingestion in fer-mented milk. A rational basis for theuse of probiotics in man.Gastroenterol Clin Biol 16:2528

27. Duez H, et al. (2000) A colony-immunoblotting method for quanti-tative detection of a Bifidobacteriumanimalis probiotic strain in humanfaeces. J Appl Microbiol 88:10191027

28. Vesa T, Pochart P, Marteau P (2000)Pharmacokinetics of Lactobacillus

plantarum NCIMB 8826, Lactobacillusfermentum KLD, and Lactococcuslactis MG 1363 in the human gastro-intestinal tract. Aliment Pharmacol

Ther 14:82382829. Bouhnik Y, Pochart P, Marteau P,

Arlet G, Goderel I, Rambaud JC(1992) Fecal recovery in humans ofviable Bifidobacterium sp ingested infermented milk. Gastroenterology102:875878

30. Johansson ML, Nobaek S, Berggren A,et al. (1998) Survival of Lactobacillus

plantarum DSM 9843 (299v), and ef-fect on the short-chain fatty acidcontent of faeces after ingestion of arose-hip drink with fermented oats.Int J Food Microbiol 42:2938

31. Adlerberth I, Ahrne SIV, JohanssonML, et al. (1996) A mannose-specific

adherence mechanism in Lactobacil-lus plantarum conferring binding tothe human colonic cell line HT-29.Appl Environ Microbiol 62(7):22442251

32. Gibney MJ, Walsh M, Brennan L,Roche HM, German B, Van Ommen B(2005) Metabolomics in humannutrition: opportunities and chal-lenges. Am J Clin Nutr 82(3):497503

33. Birt DF, Hendrich S, Wang W (2001)Dietary agents in cancer prevention:flavonoids and isoflavonoids. Phar-macol Ther 90:157177

34. Adlercreutz H, Mazur W (1997) Phy-to-oestrogens and Western diseases.Ann Med 29:95120

35. Rawls JF, Samuel BS, Gordon JI (2004)Gnotobiotic zebrafish reveal evolu-tionarily conserved responses to thegut microbiota. Proc Natl Acad SciUSA 101:45964601

36. Backhed F, Ding H, Wang T, et al.(2004) The gut microbiota as anenvironmental factor that regulatesfat storage. Proc Natl Acad Sci USA101:1571815723

37. Hooper LV, Wong MH, Thelin A,Hansson L, Falk PG, Gordon JI (2001)Molecular analysis of commensalhost-microbial relationships in theintestine. Science 291:881884

38. Oozeer R, Goupil-Feuillerat N, AlpertCA, et al. (2002) Lactobacillus casei isable to survive and initiate proteinsynthesis during its transit in thedigestive tract of human flora-associ-ated mice. Appl Environ Microbiol68:35703574

39. Oozeer R, Furet JP, Goupil-FeuilleratN, Anba J, Mengaud J, Corthier G(2005) Differential activities of fourLactobacillus casei promoters duringbacterial transit through the gastro-intestinal tracts of human-microbi-ota-associated mice. Appl EnvironMicrobiol 71:13561363

40. Camilli A, Mekalanos JJ (1995) Use ofrecombinase gene fusions to identifyVibrio cholerae genes induced duringinfection. Mol Microbiol 18:671683

41. Angelichio MJ, Camilli A (2002) Invivo expression technology. InfectImmun 70:65186523

42. Zoetendal EG, Akkermans AD, Ak-kermans-van Vliet WM, de VisserJAGM, de Vos WM (2001) The hostgenotype affects the bacterial com-munity in the human gastrointestinaltract. Microb Ecol Health Dis 13:129134

43. Hooper LV, Gordon JI (2001) Com-mensal host-bacterial relationships inthe gut. Science 292:11151118

44. Xu J, Gordon JI (2003) Inauguralarticle: honor thy symbionts. ProcNatl Acad Sci USA 100:1045210459

45. Hooper LV, Stappenbeck TS, HongCV, Gordon JI (2003) Angiogenins: anew class of microbicidal proteinsinvolved in innate immunity. NatImmunol 4:269273

46. Stappenbeck TS, Hooper LV, GordonJI (2002) Developmental regulation ofintestinal angiogenesis by indigenousmicrobes via Paneth cells. Proc NatlAcad Sci USA 99:1545115455

47. Hooper LV, Midtvedt T, Gordon JI(2002) How host-microbial interac-tions shape the nutrient environmentof the mammalian intestine. AnnuRev Nutr 22:283307

48. Bry L, Falk PG, Midtvedt T, Gordon JI(1996) A model of host-microbialinteractions in an open mammalianecosystem. Science 273:13801383

49. Falk PG, Hooper LV, Midtvedt T,Gordon JI (1998) Creating and main-taining the gastrointestinal ecosystem:what we know and need to know fromgnotobiology. Microbiol Mol Biol Rev62:11571170

50. Sonnenburg JL, Xu J, Leip DD, ChenCH, Westover BP, Weatherford J,Buhler JD, Gordon JI (2005) Glycanforaging in vivo by an intestine-adapted bacterial symbiont. Science307(5717):19551959

51. Meydani SN, Ha WK (2000) Immu-nologic effects of yogurt. Am J ClinNutr 71:861872

52. Kalliomaki M, Salminen S, ArvilommiH, Kero P, Koskinen P, Isolauri E(2001) Probiotics in primary preven-tion of atopic disease: a randomisedplacebo-controlled trial. Lancet357:10761079

53. Kalliomaki M, Salminen S, Poussa T,Arvilommi H, Isolauri E (2003) Pro-biotics and prevention of atopic dis-ease: 4-year follow-up of arandomised placebo-controlled trial.Lancet 361:18691871

54. Merk M, Borelli C, Korting HC (2005)Lactobacilli bacteriahost interac-tions with special regard to the uro-genital tract. Int J Med Microbiol295:918

55. Bach JF (2002) The effect of infectionson susceptibility to autoimmune andallergic diseases. N Engl J Med347:911920

56. Chapat L, Chemin K, Dubois B,Bourdet-Sicard R, Kaiserlian D (2004)Lactobacillus casei reduces CD8+ Tcell-mediated skin inflammation. EurJ Immunol 34:25202528

57. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R(2004) Recognition of commensalmicroflora by toll-like receptors isrequired for intestinal homeostasis.Cell 118:229241

58. Madsen K, Cornish A, Soper P, et al.

(2001) Probiotic bacteria enhancemurine and human intestinal epithe-lial barrier function. Gastroenterology121:580591

59. Cario E, Gerken G, Podolsky DK(2004) Toll-like receptor 2 enhancesZO-1-associated intestinal epithelialbarrier integrity via protein kinase C.Gastroenterology 127:224238



17/18

60. Yan F, Polk DB (2002) Probioticbacterium prevents cytokine-inducedapoptosis in intestinal epithelial cells.J Biol Chem 277:5095950965

61. Ma D, Forsythe P, Bienenstock J(2004) Live Lactobacillus reuteri isessential for the inhibitory effect ontumor necrosis factor alpha-inducedinterleukin-8 expression. Infect Im-mun 72:53085314

62. Reid G, Jass J, Sebulsky MT, McCor-mick JK (2003) Potential uses ofprobiotics in clinical practice. ClinMicrobiol Rev 16:658672

63. Adolfsson O, Meydani SN, Russell RM(2004) Yogurt and gut function. Am JClin Nutr 80:245256

64. Piaia M, Antoine JM, Mateos-GuardiaJA, Leplingard A, Lenoir-Wijnkoop I(2003) Assessment of the benefits oflive yogurt: methods and markers forin vivo studies of the physiologicaleffects of yogurt cultures. Microb EcolHealth Dis 15:7987

65. Brownlee IA, Havler ME, DettmarPW, Allen A, Pearson JP (2003) Co-lonic mucus: secretion and turnoverin relation to dietaryfibre intake. ProcNutr Soc 62:245249

66. DSouza AL, Rajkumar C, Cooke J,Bulpitt CJ (2002) Probiotics in pre-vention of antibiotic associated diar-rhoea: meta-analysis. BMJ 324:1361

67. Sartor RB (2004) Therapeutic manip-ulation of the enteric microflora ininflammatory bowel diseases: antibi-otics, probiotics, and prebiotics. Gas-troenterology 126:16201633

68. Gionchetti P, Rizzello F, Venturi A, etal. (2000) Oral bacteriotherapy asmaintenance treatment in patients

with chronic pouchitis: a double-blind, placebo-controlled trial. Gas-troenterology 119:305309

69. Gionchetti P, Rizzello F, Helwig U, etal. (2003) Prophylaxis of pouchitisonset with probiotic therapy: a dou-ble-blind, placebo-controlled trial.Gastroenterology 124:12021209

70. Mimura T, Rizzello F, Helwig U, et al.(2004) Once daily high dose probiotictherapy (VSL #3) for maintainingremission in recurrent or refractorypouchitis. Gut 53:108114

71. Kruis W, Schutz E, Fric P, Fixa B,Judmaier G, Stolte M (1997) Double-blind comparison of an oral Escheri-

chia coli preparation and mesalazinein maintaining remission of ulcerativecolitis. Aliment Pharmacol Ther11:853858

72. Kruis W, Fric P, Pokrotnieks J, et al.(2004) Maintaining remission ofulcerative colitis with the probioticEscherichia coli Nissle 1917 is aseffective as with standard mesalazine.Gut 53:16171623

73. Rembacken BJ, Snelling AM, HawkeyPM, Chalmers DM, Axon AT (1999)Non-pathogenic Escherichia coli ver-sus mesalazine for the treatment ofulcerative colitis: a randomised trial.Lancet 354:635639

74. Prantera C, Scribano ML, Falasco G,Andreoli A, Luzi C (2002) Ineffec-tiveness of probiotics in preventingrecurrence after curative resection forCrohns disease: a randomised con-trolled trial with Lactobacillus GG.Gut 51:405409

75. Steidler L, Hans W, Schotte L, et al.(2000) Treatment of murine colitis byLactococcus lactis secreting interleu-kin-10. Science 289:13521355

76. Vandenbroucke K, Hans W, VanHuysse J, et al. (2004) Active deliveryof trefoil factors by genetically modi-fied Lactococcus lactis prevents andheals acute colitis in mice. Gastroen-terology 127:502513

77. Koebnick C, Wagner I, Leitzmann P,Stern U, Zunft HJ (2003) Probioticbeverage containing Lactobacillus ca-sei Shirota improves gastrointestinalsymptoms in patients with chronicconstipation. Can J Gastroenterol17:655659

78. Kim HJ, Camilleri M, McKinzie S, etal. (2003) A randomized controlledtrial of a probiotic, VSL#3, on guttransit and symptoms in diarrhoea-predominant irritable bowel syn-drome. Aliment Pharmacol Ther17:895904

79. Sen S, Mullan MM, Parker TJ, Wool-ner JT, Tarry SA, Hunter JO (2002)Effect of Lactobacillus plantarum299v on colonic fermentation and

symptoms of irritable bowel syn-drome. Dig Dis Sci 47:26152620

80. Madden JA, Hunter JO (2002) A re-view of the role of the gut microflorain irritable bowel syndrome and theeffects of probiotics. Br J Nutr88(Suppl 1):S67S72

81. Spiller RC (2003) Postinfectious irri-table bowel syndrome. Gastroenter-ology 124:16621671

82. Verdu EF, Bercik P, Bergonzelli GE, etal. (2004) Lactobacillus paracaseinormalizes muscle hypercontractilityin a murine model of postinfective gutdysfunction. Gastroenterology127:826837

83. Bouvier M, Meance S, Bouley C, BertaJL, Grimaud C (2001) Effects of con-sumption of a milk fermented by theprobiotic strain Bifidobacterium ani-malis DN-173 010 on colonic transittimes in healthy humans. Biosci Mi-croflora 20:4348

84. Meance S, Cayuela C, Turchet P,Raimondi A, Lucas C, Antoine JM(2001) A fermented milk with a Bifi-dobacterium probiotic strain DN-173010 shortened oro-fecal gut transittime in elderly. Microbiol Ecol HealthDis 13:217222

85. Meance S, Cayuela C, Turchet P,Raimondi A, Lucas C, Antoine JM(2003) Recent advances in the use offunctional foods: effects of the com-mercial fermented milk with Bifido-bacterium animalis strain DN-173 010and yogurt strains on gut transit timein the elderly. Microbiol Ecol HealthDis 15:1522

86. Marteau P, Cuillerier E, Meance S, etal. (2002) Bifidobacterium animalisstrain DN-173 010 shortens the co-lonic transit time in healthy women: adouble-blind, randomized, controlledstudy. Aliment Pharmacol Ther16:587593

87. Felley CP, Corthesy-Theulaz I, RiveroJL, et al. (2001) Favourable effect of anacidified milk (LC-1) on Helicobacter

pylori gastritis in man. Eur J Gastro-enterol Hepatol 13:2529

88. Wang KY, Li SN, Liu CS, et al. (2004)Effects of ingesting Lactobacillus- andBifidobacterium-containing yogurt insubjects with colonized Helicobacter

pylori. Am J Clin Nutr 80:73774189. Armuzzi A, Cremonini F, Bartolozzi

F, et al. (2001) The effect of oraladministration ofLactobacillus GG onantibiotic-associated gastrointestinalside-effects during Helicobacter pylorieradication therapy. Aliment Phar-macol Ther 15:163169

90. Resta-Lenert S, Barrett KE (2003) Live

probiotics protect intestinal epithelialcells from the effects of infection withenteroinvasive Escherichia coli(EIEC). Gut 52:988997

91. Freitas M, Tavan E, Thoreux K,Cayuela C, Sapin C, Trugnan G (2003)Lactobacillus casei DN-114 001 andBacteroides thetaiotamicron VPI-5482inhibit rotavirus infection by modu-lating apical glycosylation pattern ofcultured human intestinal HT29-MTXcells. Gastroenterology 124(Suppl1):A-475A-476

92. McNaught CE, Woodcock NP, MacFieJ, Mitchell CJ (2002) A prospectiverandomised study of the probiotic

Lactobacillus plantarum 299V onindices of gut barrier function inelective surgical patients. Gut 51:827831

93. Anderson AD, McNaught CE, Jain PK,MacFie J (2004) Randomised clinicaltrial of synbiotic therapy in electivesurgical patients. Gut 53:241245



18/18

94. Jain PK, McNaught CE, Anderson AD,MacFie J, Mitchell CJ (2004) Influenceof synbiotic containing Lactobacillusacidophilus La5, Bifidobacterium lac-tis Bb 12, Streptococcus thermophilus,Lactobacillus bulgaricus and oligo-fructose on gut barrier function andsepsis in critically ill patients: arandomised controlled trial. Clin Nutr23:467475

95. Olah A, Belagyi T, Issekutz A, GamalME, Bengmark S (2002) Randomizedclinical trial of specific lactobacillusand fibre supplement to early enteralnutrition in patients with acute pan-creatitis. Br J Surg 89:11031107

96. Rayes N, Seehofer D, Hansen S, et al.(2002) Early enteral supply of lacto-bacillus and fiber versus selectivebowel decontamination: a controlledtrial in liver transplant recipients.Transplantation 74:123127

97. Agostoni C, Axelsson I, Braegger C, etal. (2004) Probiotic bacteria in dieteticproducts for infants: a commentaryby the ESPGHAN Committee onNutrition. J Pediatr GastroenterolNutr 38:365374

98. Szajewska H, Mrukowicz JZ (2001)Probiotics in the treatment and pre-vention of acute infectious diarrhea ininfants and children: a systematic re-view of published randomized, dou-ble-blind, placebo-controlled trials. JPediatr Gastroenterol Nutr 33(Suppl2):S17S25

99. Van Niel CW, Feudtner C, GarrisonMM, Christakis DA (2002) Lactoba-cillus therapy for acute infectiousdiarrhea in children: a meta-analysis.Pediatrics 109:678684

100. Allen SJ, Okoko B, Martinez E, Greg-orio G, Dans LF (2004) Probiotics fortreating infectious diarrhoea. Coch-rane Database Syst Rev (2):CD003048

101. Szajewska H, Kotowska M, Mruko-wicz JZ, Armanska M, Mikolajczyk W(2001) Efficacy ofLactobacillus GG inprevention of nosocomial diarrhea ininfants. J Pediatr 138:361365

102. Oberhelman RA, Gilman RH, Sheen P,et al. (1999) A placebo-controlled trialof Lactobacillus GG to prevent diar-rhea in undernourished Peruvianchildren. J Pediatr 134:1520

103. Hatakka K, Savilahti E, Ponka A, et al.(2001) Effect of long term consump-tion of probiotic milk on infections inchildren attending day care centres:double blind, randomised trial. BMJ322:1327

104. Saavedra JM, Bauman NA, Oung I,Perman JA, Yolken RH (1994) Feed-ing of Bifidobacterium bifidum andStreptococcus thermophilus to infantsin hospital for prevention of diar-rhoea and shedding of rotavirus.Lancet 344:10461049

105. Mastretta E, Longo P, Laccisaglia A, etal. (2002) Effect of Lactobacillus GGand breast-feeding in the preventionof rotavirus nosocomial infection. JPediatr Gastroenterol Nutr 35:527531

106. Kotowska M, Albrecht P, Szajewska H(2005) Saccharomyces boulardii in theprevention of antibiotic-associateddiarrhoea in children: a randomizeddouble-blind placebo-controlled trial.Aliment Pharmacol Ther 21:583590

107. Lu L, Walker WA (2001) Pathologicand physiologic interactions of bac-

teria with the gastrointestinal epithe-lium. Am J Clin Nutr 73:1124S1130S

108. Guandalini S (2002) Use of Lactoba-cillus-GG in paediatric Crohns dis-ease. Dig Liver Dis 34(Suppl 2):S63S65

109. Sykora J, Valeekova KKV, AmlerovaJJA, et al. (2004) Supplements of a oneweek triple drug therapy with specialprobiotic Lactobacillus casei immuni-tass (strain DN 114000) and Strepto-coccus thermophilus and Lactobacillusbulgaricus in the eradication of H.

pylori-colonized children:a prospec-tive randomised tr. J Pediatr Gastro-enterol Nutr 39(S1):S400

110. Banaszkiewicz A, Szajewska H (2004)Ineffectiveness of Lactobacilllus GGfor the treatment of functionalconstipation in children: a rando-mised double-blind placebo-con-trolled trial. J Pediatr GastroenterolNutr 39(S1):S67S7

111. Bargaoui K (2004) Effect of Lactoba-cilllus acidophilus LB on intestinalmicrobial pullulation in childhood. JPediatr Gastroenterol Nutr39(S1):S401

112. Li Y, Shimizu T, Hosaka A, Kaneko N,Ohtsuka Y, Yamashiro Y (2004) Ef-fects of Bifidobacterium breve sup-plementation on intestinal flora of lowbirth weight infants. Pediatr Int46:509515

113. Zeisel SH (1999) Regulation of nu-traceuticals. Science 285:18531855

114. Jung LKL (1999) Lactobacillus GGaugments the immune response totyphoid vaccination: a double-blindedplacebo-controlled study. FASEB J13:A872


28. Progress in the Science of Probiotics - Dr. Sisca SP.gk

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