Probiotics and prebiotics: can regulating the activities of intestinal bacteria benefit health?
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Enterobacteriaceae↗
Biomedical subjects
G T Macfarlane
Publications and source records attributed to G T Macfarlane.
Growth of a human intestinal Desulfovibrio desulfuricans in continuous cultures containing defined populations of saccharolytic and amino acid fermenting bacteria.
Ecological and physiological effects of the sulphate-reducing bacterium (SRB) Desulfovibrio desulfuricans on other intestinal organisms were investigated in anaerobic chemostats (dilution rate approximately 0.2 h-1). Reproducible defined bacterial communities were used in these experiments, comprising 14 different saccharolytic and amino acid fermenting species: Bifidobacterium longum, Bif. adolescentis, Bif. pseudolongum, Bif. infantis, Bacteroides thetaiotaomicron, Bact. vulgatus, Lactobacillus acidophilus, Enterococcus faecalis, Ent. faecium, Escherichia coli, Clostridium perfringens, Cl. butyricum, Cl. innocuum, Cl. bifermentans. Lactobacillus and Cl. bifermentans populations never rose above minimum detection limits (log10 2.0 and 4.0, respectively) under the experimental conditions employed in these studies. Inclusion of Des. desulfuricans in bacterial cultures (c. log10 8.4 viable cells ml-1) resulted in marked reductions (i.e. greater than 1 log) in planktonic cell population densities of several species, particularly Bif. longum, Cl. perfringens and Bif. pseudolongum. The two bacteroides species were unaffected by Des. desulfuricans, while numbers of Cl. butyricum increased. Extensive wall growth developed in the SRB culture, consisting mainly of Des. desulfuricans (log10 9.2 viable cells ml-1), Bact. thetaiotaomicron and Bact. vulgatus, with lesser numbers of facultative anaerobes, Cl. perfringens and Bif. longum. Wall growth was associated with a reduction in planktonic cell mass and increased acid production by the cultures. Chemotaxonomic study of chemostat microbiotas, on the basis of cellular fatty acid methyl ester (FAME) analyses, showed the existence of characteristic bacteroides (C15) and bifidobacterial (C18) markers, but desulfovibrio markers (i-C15:0, C16:0, i-C17:1) could be identified. The metabolic activities of saccharolytic organisms were altered in the SRB chemostat, including synthesis of a number of hydrolytic enzymes involved in carbohydrate breakdown, such as alpha-galactosidase, alpha-glucosidase and beta-galactosidase, together with several mucinolytic enzymes. High concentrations of sulphide (8.2 mmol 1-1) were detected in the SRB chemostat, suggesting that this metabolite may have been inhibitory to some species. Saccharolytic organisms growing in the SRB fermenter utilized more starch, but less galactose-containing polymers, which correlated with the observed glycosidase activities. Profound differences were also recorded with respect to fermentation product formation in the chemostats, where a major switch to acetate production occurred in the SRB culture, with concomitant reductions in propionate, butyrate and lactate, which is an important electron donor for desulfovibrios.
Amino Acids↗
Ecological and physiological studies on large intestinal bacteria in relation to production of hydrolytic and reductive enzymes involved in formation of genotoxic metabolites.
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.
Adult↗
Dissimilatory amino Acid metabolism in human colonic bacteria.
The abilities of slurries of human faecal bacteria to ferment 20 different amino acids were investigated in batch culture incubations. Ammonia, short chain fatty acids, and in some cases, amines, were the principal products of dissimilatory metabolism. The types of SCFA produced were dependent on the chemical compositions of the test substrates. Thus, acetate and butyrate were formed from the acidic amino acid glutamate, while acetate and propionate predominated in aspartate fermentations. Breakdown of the basic amino acids lysine and arginine was rapid, and yielded butyrate and acetate, and ornithine and citrulline, respectively. The major products of histidine deamination were also acetate and butyrate. However, fermentation of sulphur-containing amino acids was slow and incomplete. Acetate, propionate and butyrate were formed from cysteine, whereas the main products of methionine metabolism were propionate and butyrate. The simple aliphatic amino acids alanine and glycine were fermented to acetate, propionate and butyrate, and acetate and methylamine, respectively. Branched-chain amino acids were slowly fermented by colonic bacteria, with the main acidic products being branched-chain fatty acids one carbon atom shorter than the parent amino acid. Low concentrations of amines were also detected in these fermentations. Aliphatic-hydroxy amino acids were rapidly deaminated by large intestinal microorganisms. Serine was primarily fermented to acetate and butyrate, while threonine was mainly metabolised to propionate. Proline was poorly utilized by intestinal bacteria, but hydroxyproline was efficiently fermented to acetate and propionate. The aromatic amino acids tyrosine, phenylalanine and tryptophan were broken down to a range of phenolic and indolic compounds.
Journal Article↗
Starch utilization by Bacteroides ovatus isolated from the human large intestine.
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.
Bacteroides↗
Colonic microflora: nutrition and health.
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Animals↗
Utilization of starch and synthesis of a combined amylase/alpha-glucosidase by the human colonic anaerobe Bacteroides ovatus.
Bacteroides ovatus preferentially utilized starch and pectin when grown on a mixture of polysaccharides in batch culture, indicating that these carbohydrates are important substrates for the bacterium in the human large intestine. Further studies on starch breakdown showed that continuous cultures grew on the polysaccharide when it provided the sole carbohydrate source, to yield a single hydrolytic product at low dilution rates (D = 0.04 h-1), with an estimated molecular mass of 13 kDa. In contrast, two major types of oligomeric products were formed at higher dilution rates (D = 0.44 h-1), with approximate molecular weights of 11 and 140 kDa. Analysis of cell-associated starch-degrading enzymes produced by Bact. ovatus using ion exchange chromatography and HPLC gel-filtration showed that amylase and alpha-glucosidase activities eluted in the same fractions. The single peak containing amylase and alpha-glucosidase activities obtained by HPLC gel-filtration chromatography corresponded to a molecular mass of approximately 140 kDa, and activity staining of gels for alpha-glucosidase activity after polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate, gave an estimated molecular mass of 70 kDa, indicating this enzyme to be a dimer. After renaturation, the 70 kDa band was cut from the gels and solubilized. The extract hydrolysed gelatinized starch and p-nitrophenyl-alpha-D-glucopyranoside.
Amylases↗
Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria.
In both health and disease, the colonic microbiota plays an important role in several areas of human physiology. This complex assemblage of microorganisms endows great metabolic potential on the large intestine, primarily through its degradative abilities. Many hundreds of different types of bacteria, varying widely in physiology and biochemistry, exist in a multitude of different microhabitats in the lumen of the large gut, the mucin layer and on mucosal surfaces. Both microbiota and host obtain clear benefits from association. For example, growth substrates from diet and body tissues, together with a relatively stable environment for bacteria to proliferate are provided by the host, which in turn has evolved to use butyrate, a bacterial fermentation product, as its principal source of energy for epithelial cells in the distal bowel. The main sources of carbon and energy for intestinal bacteria are complex carbohydrates (starches, non-starch polysaccharides). Carbohydrate metabolism is of great importance in the large intestine, since generically, and in terms of absolute numbers, the vast majority of culturable microorganisms are saccharolytic. The amounts and types of fermentation products formed by colonic bacteria depend on the relative amounts of each substrate available, their chemical structures and compositions, as well as the fermentation strategies (biochemical characteristics and catabolite regulatory mechanisms) of bacteria participating in depolymerization and fermentation of the substrates. Protein breakdown and dissimilatory amino acid metabolism result in the formation of a number of putatively toxic metabolites, including phenols, indoles and amines. Production of these substances is inhibited or repressed in many intestinal microorganisms by a fermentable source of carbohydrate. Owing to the anatomy and physiology of the colon, putrefactive processes become quantitatively more important in the distal bowel, where carbohydrate is more limiting.
Adult↗
Investigations of bifidobacterial ecology and oligosaccharide metabolism in a three-stage compound continuous culture system.
BACKGROUND: Several different types of in vitro fermentation systems are currently employed to investigate pro- and prebiotic activities in the human large intestinal microbiota, ranging from simple batch cultures, with or without stirring and pH control, to more complex models involving pH controlled single and multiple-component continuous culture systems. METHODS: In this investigation, we used a three-stage continuous culture model to study the activities of colonic bacteria. This fermentation system reproduces several of the nutritional and environmental characteristics of the proximal large intestine (vessel 1) and the distal colon (vessels 2 and 3), and was validated using bacteriological, metabolic and chemical measurements made with intestinal material obtained from different regions of the large bowel. In this paper, we report studies on prospective probiotic effects of Bifidobacterium longum NCFB 2259 in relation to other bacterial populations, production of tyrosine and phenylalanine metabolites, and bacterial synthesis of enzymes involved in the formation of putatively genotoxic metabolites, including beta-glucosidase (GS), arylsulphatase (AS), beta-glucuronidase (GN), nitroreductase (NR) and azoreductase (AR). RESULTS: Bacterial activities at two different retention times were studied (31.1 and 68.4 h), which correspond to large intestinal transit times. At R = 31.4 h, significant probiotic effects were observed with respect to reductions in GS and GN, upon adding B. longum. However, despite the fact that this organism does not ferment aromatic amino acids or produce significant amounts of genotoxic enzymes, dysbiotic manifestations occurred in that both NR synthesis and dissimilatory tyrosine metabolism were stimulated. In contrast, at R = 68.4 h, GS formation increased between five and 20-fold, while GN and NR activities increased by a factor of two after adding the bifidobacterium. These data are reviewed in relation to potential health hazards that may be encountered with long-term probiotic administration. In the prebiotic experiments, the three-stage fermentation system was operated at R = 65 h. Oligofructose was added to V1 to give an initial concentration of 30 grams per litre, when the system was in steady state, to study its effects on a number of experimental parameters including bifidogenicity, bacterial growth, fermentation product formation and mutagenicity. After addition of the oligosaccharide, a multiplicity of effects were observed in V1, where synthesis of NR and AR, bifidobacterial populations and overall fermentation processes were stimulated, although these influences progressively diminished in V2 and V3. CONCLUSIONS: These studies indicate that bacterial metabolism and putative beneficial consequences associated with the breakdown of readily fermentable prebiotics in the large intestine may in some circumstances be spatially and temporally limited to the proximal bowel.
Arylsulfatases↗
Formation of a dipeptidyl arylamidase by Bacteroides splanchnicus NCTC 10825 with specificities towards glycylprolyl-x and valylalanine-x substrates.
Bacteroides splanchnicus in common with several members of the B. fragilis group constitutively produced a number of protein and peptide hydrolysing enzymes. Amongst the most active was an arylamidase, which specifically hydrolysed the dipeptidyl chromogenic substrates glycylprolyl p-nitroanilide (GPRPNA), glycylprolyl beta-naphthylamide (GP beta NA) and valylalanine p-nitroanilide (VAPNA), and had some proteolytic activity towards azocasein. No activity was detected against proline beta-naphthylamide, glycine, valanine or alanine p-nitroanilides. Physiological studies showed that the enzyme was largely cell-associated during exponential growth in batch culture, but was progressively released by the bacteria before the cells entered stationary phase. Glycylprolyl arylamidase (GPA) was completely cell-bound during growth in continuous culture, where synthesis increased concomitantly with dilution rate (specific growth rate) in both carbon- and nitrogen-limited chemostats. Gel-filtration chromatography of B. splanchnicus cell extracts yielded a single peak of GPA activity, with an apparent molecular mass of c. 160 kDa, while one peak of enzyme activity was eluted by 0.3 M NaCl during cation-exchange chromatography. Activity staining of SDS polyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded during preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possible existence of GPA isoenzymes. GPRPNA, VAPNA and azocasein were hydrolysed by the major fraction (fraction 3), while only the p-nitroanilide substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relatively broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated that while aspartic (pepstatin A), thiol (iodoacetate) and metalloprotease (EDTA, cysteine) inhibitors had little effect on hydrolysis of glycylproline p-nitroanilide, GPA was strongly inhibited (c. 80%) by 5 mM phenylmethylsulphonyl fluoride (PMSF), indicating it to be a serine enzyme.
Aminopeptidases↗
Consequences of biofilm and sessile growth in the large intestine.
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.
Bacteria, Anaerobic↗
Digestive fates of soluble polysaccharides from marine macroalgae: involvement of the colonic microflora and physiological consequences for the host.
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Agar↗
Enumeration of human colonic bacteria producing phenolic and indolic compounds: effects of pH, carbohydrate availability and retention time on dissimilatory aromatic amino acid metabolism.
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.
Amino Acids↗
Arabinogalactan utilization in continuous cultures of Bifidobacterium longum: effect of co-culture with Bacteroides thetaiotaomicron.
Studies showed that the plant cell wall polysaccharide arabinogalactan supported growth of Bifidobacterium longum in batch culture. Galactose was also utilized, but not arabinose, the other major constituent sugar of the polymer. Enzymes required for hydrolysis of arabinogalactan ('arabinogalactanase', alpha-arabinopyranosidase, beta-galactosidase) were inducible and cell-associated in B. longum, and their expression was repressed by glucose. Considerable amounts of alpha-arabinopyranosidase and beta-galactosidase were synthesized during growth on arabinogalactan, but only low levels of arabinogalactanase were detected. B. longum only grew on arabinogalactan in continuous culture under putative carbon-excess conditions. In C-limited chemostats, the bifidobacterium could not establish unless Bacteroides thetaiotaomicron was present in co-culture. The relationship between the two organisms was not simply commensal; at low specific growth rates, bacteroides cell population densities were approximately 30% lower than those recorded in axenic culture, indicating the existence of competitive interactions with the bifidobacterium. In contrast, at high specific growth rates, a mutualistic association was observed, in that Bact. thetaiotaomicron was maintained in the chemostats at high dilution rates if bifidobacteria were also present. Measurements of residual carbohydrate in spent culture fluid from C-limited chemostats indicated that a large part of the arabinogalactan molecule could not be broken down by either B. longum or Bact. thetaiotaomicron alone, or in co-culture. Formate and acetate were the major fermentation products of B. longum cultured in the presence of high concentrations of arabinogalactan, confirming that these bacteria were growing under energy-limited conditions.
Journal Article↗
Co-culture of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron in arabinogalactan-limited chemostats: effects of dilution rate and pH.
The effects of dilution rate (D = 0.04-0.38/h) and pH (5.0-6.5) on co-cultures of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron were studied in arabinogalactan-limited chemostats. B. thetaiotaomicron outcompeted B. adolescentis at all dilution rates at culture pH values between 5.0 and 6.0, although the bifidobacterium was always detected in the fermenters. At pH 6.5, however, B. adolescentis predominated in co-cultures at dilution rates above 0.24/h. Arabinogalactan degrading enzymes (beta-galactosidase, alpha-arabinofuranosidase) were strongly catabolite repressed in bacteroides at high dilution rates, but were constitutive and growth rate-associated in B. adolescentis. The increased competitiveness of B. adolescentis at high specific growth rates was not related to its ability to synthesise increased levels of depolymerising enzymes. Measurements of residual carbohydrate in pure and mixed culture chemostats showed that the bacteroides extensively digested the galactose backbone of the polymer, and to a lesser degree, the arabinose sidechains. Nevertheless, arabinose monomers and oligosaccharides (d.p. < 10) accumulated in these cultures under all growth conditions. In contrast, the bifidobacterium utilized considerably less arabinogalactan than the bacteroides, and this was reflected in the mixed culture studies. These experiments demonstrate that B. thetaiotaomicron was able to compete most successfully for this plant cell wall polysaccharide under nutritional, physiological and environmental conditions broadly similar to those encountered in the human colon, and indicate the existence of synergistic interactions between the two organisms that were growth rate dependent.
Journal Article↗
Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron.
Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.
Journal Article↗
Estimation of the fermentability of dietary fibre in vitro: a European interlaboratory study.
Five European laboratories tested a simple in vitro batch system for dietary fibre fermentation studies. The inoculum was composed of fresh human faeces mixed with a carbonate-phosphate buffer complex supplemented with trace elements and urea. Five dietary fibre sources (cellulose, sugarbeet fibre, soyabean fibre, maize bran and pectin) were used by each laboratory on three occasions to determine pH, residual non-starch polysaccharides (NSP) and short-chain fatty acid production during fermentation. Cellulose and maize bran degradabilities were very low (7.2(SE 10.8) and 6.2 (SE 9.1)% respectively after 24 h), whereas pectin and soyabean fibre were highly degraded (97.4 (SE 4.4) and 91.1 (SE 3.4)% respectively after 24 h). Sugarbeet fibre exhibited an intermediate level of degradability (59.5 (SE 14.9)%). Short-chain fatty acid production was closely related to NSP degradation (r 0.99). Although each variable was ranked similarly by all laboratories, some differences occurred with respect to absolute values. However, the adaptation of donors to the experimental substrates was not an influential factor. Interlaboratory differences could be reduced either by adding less substrate during incubations or using less-diluted inocula. In vitro fermentations with inocula made from human faeces and from rat caecal contents gave similar results. There was a close correspondence between the data obtained in the present experiment and those previously published in in vivo studies in the rat using the same fibres. The in vitro batch system tested during the present study provides a rapid means of obtaining quantitative estimates of the fermentation and the estimation of the energy content of new sources of dietary fibre.
Animals↗
Transport and metabolism of glucose and arabinose in Bifidobacterium breve.
Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-D-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using D-[U-14C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of D-[U-14C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DC had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DC by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.
Arabinose↗