Influence of disaccharide structure on prebiotic selectivity in vitro

To obtain structure-function information of a range of carbohydrates, which are available only in very small quantities, an in vitro fermentation method using 7 mg of carbohydrate, 0.7 mL of basal medium, and 1% (w/v) of fecal bacteria was validated against a pH-controlled batch culture with 150 mL of basal medium and 1.5g of test carbohydrate. This method was used to determine the influence of different glycosidic linkages and monosaccharide compositions of disaccharides on the selectivity of microbial fermentation. A prebiotic index (PI) was calculated for each disaccharide. Generally, disaccharides with linkages of 1-2, 1-4, and 1-6 generated a high PI score, with kojibiose and sophorose showing the greatest values (21.62 and 18.63, respectively). Apart from 6 alpha-mannobiose, mannose-containing disaccharicles gave a low PI due to low numbers of bifidobacteria and lactobacilli and an increase in bacteroides. The structure-function information obtained in this study may lead to a predictive understanding of how specific structures are fermented by the human gut microflora.

Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight

Gentiooligosaccharides and alternansucrase gentiobiose acceptor products were fractionated by their degree of polymerization (DP) on a Bio-Gel P2 column. Fractions were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, and incubated with human faecal bacteria under anaerobic conditions at 37 degrees C. The growth of predominant gut bacteria on the oligosaccharides was evaluated by fluorescence in situ hybridization and a prebiotic index (PI) was calculated. Lower DP gentiooligosaccharides (DP2-3) showed the highest selectivity (PI of 4.89 and 3.40, respectively), whereas DP4-5 alternansucrase gentiobiose acceptor products generated the greatest values (PI of 5.87). The production of short-chain fatty acids was also determined during the time course of the reactions. The mixture of DP6-10 alternansucrase gentiobiose acceptor products generated the highest levels of butyric acid but the lowest levels of lactic acid. Generally, for similar molecular weights, alternansucrase gentiobiose acceptor products gave higher PI values than gentiooligosaccharides.

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.

Prebiotic properties of alternansucrase maltose-acceptor oligosaccharides

alpha-(1-6) and alpha-(1-3)-linked oligosaccharides were obtained from the reaction between sucrose and maltose, catalyzed by an alternansucrase isolated from Leuconostoc mesenteroides NRRL B-21297 and separated using a Bio-Gel P2 column in six fractions. Fractions 1, 2, and 3 were mainly composed of DP3, DP4, and DP5, respectively. However, fractions 4, 5, and 6 consisted of mixtures from DP5 to IDP9, and they are identified here as DP5.7, DP6.7, and DP7.4, respectively. Potential prebiotic properties of these oligosaccharides were tested using pure and mixed cultures. Generally, in pure studies, most of the tested bacteria failed to grow or grew poorly using the DP6.7 and DP7.4 fractions and showed the greatest growth on DP3. Growth of fecal bacteria on the maltose-acceptor products was tested following an in vitro fermentation method. DP3 showed the highest prebiotic effect, followed by DP4 and DP6.7, whereas DP7.4 did not present any prebiotic activity.

Probiotics: their potential to impact human health

Probiotics—live microorganisms that when administered in adequate amounts confer a health benefit on the host—have been studied for both human and animal applications, and worldwide research on this topic has accelerated in recent years. This paper reviews the literature on probiotics, describes how probiotics work in human ecosystems, and outlines the impact of probiotics on human health and disease. The paper also addresses safety issues of probiotic use, suggests future developments in the field of probiotics, and provides research and policy recommendations. Product considerations and potential future developments regarding probiotics also are discussed. The authors conclude that controlled human studies have revealed a diverse range of health benefits from consumption of probiotics, due largely to their impact on immune function or on microbes colonizing the body. Additional, well-designed and properly controlled human and mechanistic studies with probiotics will advance the essential understanding of active principles, mechanisms of action, and degree of effects that can be realized by specific consumer groups. Recommendations include establishment of a standard of identity for the term “probiotic,” adoption of third-party verification of label claims, use of probiotics selectively in clinical conditions, and use of science-based assessment of the benefits and risks of genetically engineered probiotic microbes.

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion. which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and RI stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS RI also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10-24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS RI and with the increases in Bacteroides in cultures with both microbial EPS (RI and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids. (C) 2009 Elsevier B.V. All rights reserved.

Caseinoglycomacropeptide inhibits adhesion of pathogenic Escherichia coli strains to human cells in culture

Caseinoglycomacropeptide (CGMP) derived from kappa-casein was investigated for its ability to inhibit the adhesion of 3 strains of verotoxigenic Escherichia coli (VTEC) and 3 strains of enteropathogenic Escherichia coli (EPEC) to human HT29 tissue cell cultures. Effects on adhesion of Desulfovibrio desulfuricans, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus gasseri were also investigated. Generally, CGMP exerted effective anti-adhesive properties at a dose of 2.5 mg/mL, albeit with a high degree of strain specificity. The CGMP reduced adhesion of VTEC strains to < 50% of the control and reduced adhesion of EPEC strains to between 80 and 10% of the control. The CGMP also reduced the adhesion of L. pentosus and L. casei to 44 and 42%, respectively. A slight but significant reduction of L. acidophilus, to 81%, was observed, but no significant effects were detected with either Dsv. desulfuricans or L. gasseri. Further investigation of the dose response relationships with the E. coli strains gave IC50 values ranging between 0.12 and 1.06 mg/mL.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment wits determined in the human HT29 cell line by viable Count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coli and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected. but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 mu g ml(-1), respectively.

Inhibition of the adhesion of enteropathogenic Escherichia coli strains to HT-29 cells in culture by chito-oligosaccharides

The ability of chito-oligosaccharides (COS) to inhibit selected intestinal bacteria was investigated. COS at 2.5 mg ml(-1) had no significant effect on the adhesion of three strains of verotoxigenic Escherichia coli (VTEC), Lactobacillus pentosus, L. casei or L. gasseri to human HT29 cells in tissue culture. However, COS significantly inhibited adhesion of three strains of enteropathogenic E. coli (EPEC) to below 30% of the level of adhesion seen in the controls. Dose-response curves were constructed to further characterise the inhibition of EPEC strains to HT29 cells. (c) 2005 Elsevier Ltd. All rights reserved.

New scientific paradigms for probiotics and prebiotics

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics. Humans have evolved in symbiosis with an estimated 1014 resident microorganisms. However, as medicine has widely defined and explored the perpetrators of disease, including those of microbial origin, it has paid relatively little attention to the microbial cells that constitute the most abundant life forms associated with our body. Microbial metabolism in humans and animals constitutes an intense biochemical activity in the body, with profound repercussions for health and disease. As understanding of the human genome constantly expands, an important opportunity will arise to better determine the relationship between microbial populations within the body and host factors (including gender, genetic background, and nutrition) and the concomitant implications for health and improved quality of life. Combined human and microbial genetic studies will determine how such interactions can affect human health and longevity, which communication systems are used, and how they can be influenced to benefit the host. Probiotics are defined as live microorganisms which, when administered in adequate amounts confer a health benefit on the host.1 The probiotic concept dates back over 100 years, but only in recent times have the scientific knowledge and tools become available to properly evaluate their effects on normal health and well being, and their potential in preventing and treating disease. A similar situation exists for prebiotics, defined by this group as non-digestible substances that provide a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria. Prebiotics function complementary to, and possibly synergistically with, probiotics. Numerous studies are providing insights into the growth and metabolic influence of these microbial nutrients on health. Today, the science behind the function of probiotics and prebiotics still requires more stringent deciphering both scientifically and mechanistically. The explosion of publications and interest in probiotics and prebiotics has resulted in a body of collective research that points toward great promise. However, this research is spread among such a diversity of organisms, delivery vehicles (foods, pills, and supplements), and potential health targets such that general conclusions cannot easily be made. Nevertheless, this situation is rapidly changing on a number of important fronts. With progress over the past decade on the genetics of lactic acid bacteria and the recent, 2,3 and pending, 4 release of complete genome sequences for major probiotic species, the field is now armed with detailed information and sophisticated microbiological and bioinformatic tools. Similarly, advances in biotechnology could yield new probiotics and prebiotics designed for enhanced or expanded functionality. The incorporation of genetic tools within a multidisciplinary scientific platform is expected to reveal the contributions of commensals, probiotics, and prebiotics to general health and well being and explicitly identify the mechanisms and corresponding host responses that provide the basis for their positive roles and associated claims. In terms of human suffering, the need for effective new approaches to prevent and treat disease is paramount. The need exists not only to alleviate the significant mortality and morbidity caused by intestinal diseases worldwide (especially diarrheal diseases in children), but also for infections at non-intestinal sites. This is especially worthy of pursuit in developing nations where mortality is too often the outcome of food and water borne infection. Inasmuch as probiotics and prebiotics are able to influence the populations or activities of commensal microflora, there is evidence that they can also play a role in mitigating some diseases. 5,6 Preliminary support that probiotics and prebiotics may be useful as intervention in conditions including inflammatory bowel disease, irritable bowel syndrome, allergy, cancer (especially colorectal cancer of which 75% are associated with diet), vaginal and urinary tract infections in women, kidney stone disease, mineral absorption, and infections caused by Helicobacter pylori is emerging. Some metabolites of microbes in the gut may also impact systemic conditions ranging from coronary heart disease to cognitive function, suggesting the possibility that exogenously applied microbes in the form of probiotics, or alteration of gut microecology with prebiotics, may be useful interventions even in these apparently disparate conditions. Beyond these direct intervention targets, probiotic cultures can also serve in expanded roles as live vehicles to deliver biologic agents (vaccines, enzymes, and proteins) to targeted locations within the body. The economic impact of these disease conditions in terms of diagnosis, treatment, doctor and hospital visits, and time off work exceeds several hundred billion dollars. The quality of life impact is also of major concern. Probiotics and prebiotics offer plausible opportunities to reduce the morbidity associated with these conditions. The following addresses issues that emerged from 8 workshops (Definitions, Intestinal Flora, Extra-Intestinal Sites, Immune Function, Intestinal Disease, Cancer, Genomics, and Second Generation Prebiotics), reflecting the current scientific state of probiotics and prebiotics. This is not a comprehensive review, however the study emphasizes pivotal knowledge gaps, and recommendations are made as to the underlying scientific and multidisciplinary studies that will be required to advance our understanding of the roles and impact of prebiotics, probiotics, and the commensal microflora upon health and disease management.

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid., naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-D-hydroxyphenyl)propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols. (C) 2003 Elsevier Inc. All rights reserved.

Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications

The application of probiotics and prebiotics to the manipulation of the microbial ecology of the human colon has recently seen many scientific advances. The sequencing of probiotic genomes is providing a wealth of new information on the biology of these microorganisms. In addition, we are learning more about the interactions of probiotics with human cells and with pathogenic bacteria. An alternative means of modulating the colonic microbial community is by the use of prebiotic oligosaccharides. Increasing knowledge of the metabolism of prebiotics by probiotics is allowing us to consider specifically targeting such dietary intervention tools at specific populatiori groups and specific disease states. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Development of a fermentation system to model sessile bacterial populations in the human colon

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors. Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

Bacterial biofilms in the human gastrointestinal tract

Microbial biofilms were first described in 1936 and subsequent research has unveiled their ubiquity and physiological distinction from free-living (planktonic) microorganisms. In light of their emerging significance this review examines the bacterial biofilms within the human gastrointestinal tract. Attention is paid to the nature of these mucosally- associated populations, focusing on the protected environment afforded by the continual secretion of mucus by host epithelial cells. It also examines the attributes possessed by various bacterial species that facilitate habitation of this microenvironment. Additionally, contrasts are drawn between planktonic bacteria of the lumen and sessile (biofilm) bacteria growing in close association with host cells and food particles. In particular the different fermentation profiles exhibited by these two fractions are discussed. The potential role of these communities in host health and disease, as well as the stabilisation of the lumenal population, is also considered. Reference is made to the state of mutualism that exists between these little understood populations and the host epithelia, thus highlighting their ecological significance in terms of gastrointestinal health.