Enumeration of amino acid fermenting bacteria in the human large intestine: effects of pH and starch on peptide metabolism and dissimilation of amino acids
Proteins and trichloroacetic acid-soluble peptides were present in high concentrations in human intestinal contents and faeces. Free amino acids were also detected in millimolar amounts in proximal and distal colon contents, with hydroxyproline, alanine, lysine and valine predominating, showing that a wide variety of organic N-containing compounds was available for fermentation by intestinal bacteria. Measurements of products of dissimilatory amino acid metabolism (ammonia, branched chain fatty acids) demonstrated that these substances occurred in all regions of the large bowel. Amino acid fermenting populations were enumerated in faeces obtained from five healthy donors by most probable number analysis. Counts ranged from 1010 to 1011 per gram dry weight faeces. Acetate, propionate and butyrate were the principal fermentation acids in the most probable number tubes. Bacteria forming branched chain fatty acids as major end products of metabolism ranged from 0.6% (isovalerate/2-methylbutyrate) to 40% (isobutyrate) of total peptide and amino fermenting populations. Plate counts also gave high values for peptide fermenting communities in the region of 1011 per gram dry weight faeces, though considerably lower numbers of organisms grew on plates containing either single amino acids or Stickland pairs. Clostridia and anaerobic Gram-positive cocci were the predominant isolates in these studies. Physiological investigations on the effects of pH and carbohydrate availability on peptide and amino acid fermentation by intestinal bacteria showed that two environmental characteristics of the proximal colon (low pH, high carbohydrate availability), reduced the rate and net ammonia production from peptides, while carbohydrate (starch) was more important in this respect in amino acid fermentation vessels. Starch reduced initial rates of production of branched chain fatty acids by approximately 35% in peptide fermentations, however, culture pH was a more significant determinant affecting formation of these metabolites. Comparisons of branched chain fatty acid formation by faecal bacteria at pH 6.8 and 5.5 showed that their production was reduced by over 60% in pH 5.5 cultures. These data demonstrate that by increasing bacterial requirements for organic N-containing compounds for use in biosynthetic reactions, and through fermentation acid production, carbohydrate availability plays a major role in regulating dissimilatory metabolism of peptides and amino acids in the human large intestine.