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Most studies involving prebiotic oligosaccharides have been carried out using inulin and its fructo-oligosaccharide (FOS) derivatives, together with various forms of galacto-oligosaccharides (GOS). Although many intestinal bacteria are able to grow on these carbohydrates, most investigations have demonstrated that the growth of bifidobacteria, and to a lesser degree lactobacilli, is particularly favoured. Because of their safety, stability, organoleptic properties, resistance to digestion in the upper bowel and fermentability in the colon, as well as their abilities to promote the growth of beneficial bacteria in the gut, these prebiotics are being increasingly incorporated into the Western diet. Inulin-derived oligosaccharides and GOS are mildly laxative, but can result in flatulence and osmotic diarrhoea if taken in large amounts. However, their effects on large bowel habit are relatively minor. Although the literature dealing with the health significance of prebiotics is not as extensive as that concerning probiotics, considerable evidence has accrued showing that consumption of GOS and FOS can have significant health benefits, particularly in relation to their putative anti-cancer properties, influence on mineral absorption, lipid metabolism, and anti-inflammatory and other immune effects such as atopic disease. In many instances, prebiotics seem to be more effective when used as part of a synbiotic combination.

Supplementation of the human diet with prebiotic substances such as inulin and non-digestible oligosaccharides (NDO), e.g., galacto-oligosaccharides (GOS), has been associated with various health benefits. However, little information is available regarding the spatial location of their metabolism in human gut bacterial ecosystems. Therefore, the present study investigated the metabolism of inulin and GOS with respect to bacterial growth, bifidobacterial stimulatory properties and anti-mutagenicity potential, in a three-stage continuous culture model of the colon which reproduces the physicochemical characteristics of the proximal (V1) and distal (V2, V3) colons. Fermentation of both carbohydrates was rapid, and occurred primarily in V1, as evidenced by acid formation. Inulin metabolism was associated with 10-fold stimulation of lactobacillus populations, together with smaller increases in bifidobacterial cell counts in V1. However, peptostreptococci, enterococci and Clostridium perfringens also increased in this fermentation vessel. In contrast, GOS was only weakly bifidogenic in V1, although these bacteria did proliferate in V2. GOS also increased lactobacilli by an order of magnitude in V1. However, overall changes in microbial populations resulting from inulin or GOS addition were minimal in V2 and V3. Potential beneficial effects of inulin metabolism included minor reductions in beta-glucosidase and beta-glucuronidase, whereas GOS strongly suppressed these enzymes, together with arylsulphatase (AS). Growth of putatively health promoting micro-organisms was not only associated with reductions in enzymes linked to genotoxicity. For example, both carbohydrates stimulated synthesis of nitroreductase and azoreductase, throughout the fermentation system, while inulin increased AS. Colonic transit time is an important factor in bacterial metabolism in the large bowel, and these data suggest that, in some circumstances, NDO fermentation will occurprincipally in the proximal colon.

Plate counts and small subunit (SSU) rRNA abundance were used to study the effects of fructo-oligosaccharides (FOS), fructose, or galacto-oligosaccharides (GOS) on bifidobacterial populations in human faecal microbiotas. The bacteria were grown in pH-controlled anaerobic fermentation vessels. Untreated cultures and fructose-amended fermenters were used as controls. Bifidobacterium longum, B. adolescentis and B. angulatum comprised the dominant bifidobacterial populations throughout the experiment. No major differences were found in the four treatments, in terms of viable counts of the organisms or of total populations of bifidobacteria at any time point. However, large differences were observed with respect to the abundance of bifidobacterial SSU rRNA between the treatments. Greatest bifidobacterial SSU rRNA abundance was seen in FOS cultures, with the lowest in the untreated control fermentation. GOS and fructose also increased bifidobacterial SSU rRNA. Cultures supplemented with FOS and GOS were also associated with lower colony counts and SSU rRNA abundance for Escherichia coli, compared with fructose-supplemented and control fermenters. At the 24-h time point, the untreated control contained 19.8 microg of enterobacterial SSU rRNA/ml of culture fluid, compared with 11.4 microg/ml for the fructose fermentation, and 2.6 and 0.5 microg/ml for the FOS and GOS culture vessels, respectively.

Several hydrolytic and reductive bacterial enzymes (beta-glucuronidase, GN; beta-glucosidase, GS; arylsulphatase, AS; azoreductase, AR; nitroreductase, NR) involved in production of mutagenic or genotoxic metabolites were measured in human colonic contents. Cell-associated AS and extracellular GS were approximately twice as high in the distal colon compared with the proximal bowel, while AR changed little throughout the gut. Measurements of these enzymes in faeces from seven healthy donors confirmed that the majority were cell-associated, and demonstrated high levels of inter-individual variability. NR decreased four-fold between the proximal and distal colon while extracellular GN was reduced by 50%. Most probable number (MPN) analysis on faeces obtained from six healthy donors showed that counts of intestinal bacteria producing GS and AR were c. 10(10) and 10(11)/g, respectively, in all samples tested. Numbers of GN- and AS-forming organisms were between two and three orders of magnitude lower. Inter-individual carriage rates of bacterial populations synthesising NR were highly variable. Screening of 20 pure cultures of intestinal bacteria, belonging to six different genera, showed that Bacteroides ovatus, in particular, synthesised large amounts of GS, whereas B. fragilis, B. vulgatus and Bifidobacterium pseudolongum formed the highest cell-associated levels of GN. In general, bifidobacteria and Lactobacillus acidophilus did not produce significant amounts of AR. All five clostridia studied (Clostridium bifermentans, C. septicum, C. perfringens, C. sporogenes and C. butyricum) produced NR and AR, as did the bacteroides (B. fragilis, B. ovatus and B. vulgatus). Escherichia coli and C. perfringens formed large amounts of NR. Levels of AS production were invariably low and few of the organisms screened synthesised this enzyme. In-vitro studies investigating the effect of intestinal transit time on enzyme production, in a three-stage (V1-V3) continuous culture model of the colon operated at system retention times (R) of either 31.1 or 68.4 h, showed that specific activities of GS were up to four-fold higher (V3) at R = 31.1 h. Bacteriological analysis demonstrated that representative populations of colonic micro-organisms were maintained in the fermentation system, and indicated that changes in GS activity were not related to numbers of the predominant anaerobic or facultative anaerobic species within the model, but were explainable on the basis of substrate-induced modulation of bacterial metabolism.

The human large intestine contains a microbiota, the components of which are generically complex and metabolically diverse. Its primary function is to salvage energy from carbohydrate not digested in the upper gut. This is achieved through fermentation and absorption of the major products, short chain fatty acids (SCFA), which represent 40-50% of the available energy of the carbohydrate. The principal SCFA, acetate, propionate and butyrate, are metabolized by the colonic epithelium (butyrate), liver (propionate) and muscle (acetate). Intestinal bacteria also have a role in the synthesis of vitamins B and K and the metabolism of bile acids, other sterols and xenobiotics. The colonic microflora are also responsive to diet. In the presence of fermentable carbohydrate substrates such as non-starch polysaccharides, resistant starch and oligosaccharides, bacteria grow and actively synthesize protein. The amount of protein synthesis and turnover within the large intestine is difficult to determine, but around 15 g biomass is excreted in faeces each day containing 1 g bacterial-N. Whether bacterially synthesized amino acids are ever absorbed from the colon remains unclear. Finally, individual colonic micro-organisms such as sulphate-reducing bacteria, bifidobacteria and clostridia, respond selectively to specific dietary components in a way that may be important to health.

Starch supported growth of continuous cultures of Bacteroides ovatus when this carbohydrate provided the sole source of carbon and energy. Inducible amylase and alpha-glucosidase activities were inversely related to dilution rate in starch-limited and starch-excess chemostats over the dilution rate (D) range D = 0.03/h to D =0.20/h, and were partly repressed during growth under conditions of starch-excess. Preparative isoelectric focusing of B. ovatus cytoplasmic extracts indicated the existence of three distinct starch-hydrolyzing enzymes. Incubation of active fractions from the isoelectric focusing cell with maltose and a variety of low-molecular-weight oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) identified a single amylase activity, an enzyme with combined beta-amylase and glucoamylase/alpha-glucosidase properties, and also a possible pullulanase. The ability of B. ovatus to synthesize several starch-hydrolyzing enzymes with different specificities and activities may confer a significant competitive advantage to this organism in the colonic ecosystem.

The human colonic ecosystem is an extremely complex environment comprised of several hundred different strains of bacteria. Studies were undertaken to determine whether these organisms formed metabolic or genotypically distinct assemblages in the gut microbiota in relation to polysaccharide fermentation. Measurements of depolymerizing enzymes (4 polysac-charidases, 6 glycosidases) showed that specific amylase and pectinase activities were comparable in bacteria desorbed from the surfaces of food particles and in non-particulate organisms. However, xylanase, beta-xylosidase, arabinogalac-tanase, alpha-arabinofuranosidase, and beta-galacturonidase activities were always significantly greater in particulate bacteria. Short-term in vitro fermentations with both groups of bacteria showed marked differences in relative rates of starch, arabinogalactan, and mucin metabolism, while rates of fermentation product formation with pectin and xylan were broadly comparable. Significant differences were observed with respect to formation of individual fermentation products, especially when mucin or pectin were substrates, where particulate bacteria produced proportionally higher amounts of acetate. Bacteriological studies showed that communities of polymer-degrading bacteria and other groups of intestinal anaerobes growing on particulate matter were essentially similar to those occurring elsewhere in the gut lumen, at genus and species levels. In vitro colonization experiments demonstrated that a variety of polysaccharide-fermenting bifidobacteria and bacteroides--together with other cross-feeding organisms such as peptostreptococci, fusobacteria, and coliforms--rapidly attached to particulate intestinal materials.

Concentrations of phenolic compounds in human gut contents were more than fourfold higher in the distal colon (6.2 mmol kg-1) compared to the proximal bowel (1.4 mmol kg-1). Tryptophan metabolites were never found in more than trace amounts in large intestinal contents and phenol substituted fatty acids were the major products of aromatic amino acid fermentation that accumulated in the proximal colon, whereas phenol and p-cresol were more important in the distal gut, accounting for 70% of all products of dissimilatory aromatic amino acid metabolism. In vitro incubations of colonic material showed that phenol was produced most rapidly (1.0 mumol g-1 h-1), whereas indole was formed comparatively slowly (0.06 mumol g-1 h-1). Most probable number (MPN) estimations demonstrated that large populations of phenol and indole producing bacteria occur in the large intestine (range log10 9.8-11.5 (g dry wt faeces)-1, mean 10.6, N = 7). With respect to phenolic compounds, phenylacetate and phenylpropionate producers predominated, while indoleacetate-forming bacteria were the major tryptophan-utilizing organisms. Quantitation of products of dissimilatory aromatic amino acid metabolism in MPN tubes showed that phenol and phenylpropionate mainly accumulated at low sample dilutions, whereas phenylacetate, p-cresol, indoleacetate and indolepropionate were formed in greatest amounts at high sample dilutions. The significance of pH and carbohydrate availability with respect to aromatic amino acid metabolism was shown in batch culture fermentation studies, where net production of phenolic compounds by mixed populations of intestinal bacteria was reduced by approximately 33% during growth at pH 5.5 compared to pH 6.8, and by 60% in the presence of a fermentable carbohydrate. Experiments with 16 species of intestinal bacteria belonging to six different genera showed that environmental factors such as low pH and high carbohydrate availability markedly reduced dissimilatory aromatic amino acid metabolism in some organisms, but stimulated this process in others. A three-stage continuous culture model of the colon was used to investigate the effect of system retention time (27.1 or 66.7 h) on aromatic amino acid fermentation. Qualitative and quantitative increases in phenol production occurred from vessel 1 to vessel 3 in this model. Concentrations of phenolic compounds in vessel 3 were three times greater at R = 66.7 h compared to R = 27.1 h. Phenol and p-cresol were not detected in vessel 1, though formation of these metabolites increased from vessel 2 to vessel 3, in a pattern similar to that observed in the distal colon.

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H2/CO2, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.

Evidence for the occurrence of microbial breakdown of carbohydrate in the human colon has been sought by measuring short chain fatty acid (SCFA) concentrations in the contents of all regions of the large intestine and in portal, hepatic and peripheral venous blood obtained at autopsy of sudden death victims within four hours of death. Total SCFA concentration (mmol/kg) was low in the terminal ileum at 13 +/- 6 but high in all regions of the colon ranging from 131 +/- 9 in the caecum to 80 +/- 11 in the descending colon. The presence of branched chain fatty acids was also noted. A significant trend from high to low concentrations was found on passing distally from caecum to descending colon. pH also changed with region from 5.6 +/- 0.2 in the caecum to 6.6 +/- 0.1 in the descending colon. pH and SCFA concentrations were inversely related. Total SCFA (mumol/l) in blood was, portal 375 +/- 70, hepatic 148 +/- 42 and peripheral 79 +/- 22. In all samples acetate was the principal anion but molar ratios of the three principal SCFA changed on going from colonic contents to portal blood to hepatic vein indicating greater uptake of butyrate by the colonic epithelium and propionate by the liver. These data indicate that substantial carbohydrate, and possibly protein, fermentation is occurring in the human large intestine, principally in the caecum and ascending colon and that the large bowel may have a greater role to play in digestion than has previously been ascribed to it.

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.

High levels (2-565 units/g) of amylase activity were observed in human faeces. Over 92% of amylase activity in faeces obtained from healthy persons was extracellular, whereas only about 9% of activity was associated with particulate material and washed cells. Bacterial cell-bound amylases were considerably more efficient in breaking down starch, however, than were the soluble enzymes which occurred in cell-free faecal supernatant fluids. Cell population densities of anaerobic starch-hydrolysing bacteria in the stools of ten persons ranged from 1.1 X 10(10) to 3.3 X 10(12)/g of faeces. Identification of 120 starch-hydrolysing colonies isolated from the stools of six subjects showed that the predominant amylolytic bacteria belonged to the genera Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio. Mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids. Lactate was observed to be a major, though transient intermediate during starch fermentation by these cultures. Approximately 60% of starch utilized was converted to volatile fatty acids, which in the human colon would be potentially available for absorption.