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Cultures of ruminal bacteria known to metabolize unsaturated fatty acids were grown in medium containing 50 [micro]g [ml.sup.-1] of geometric and positional isomers of conjugated linoleic acid (CLA) or 18 : 1 fatty acids and 37.4 % deuterium oxide to investigate the mechanisms responsible for fatty acid metabolism. Butyrivibrio fibrisolvens JW11 converted cis-9,trans-11-18 : 2 and trans-9,trans11-18 : 2 to trans-11-18 : 1 as the main product, labelled at C-9, and metabolized trans-10,cis-12-18 : 2 to trans-10-18 : 1, labelled at C-13, and smaller amounts of trans-12-18 : 1 and cis-12-18 : 1. Butyrivibrio proteoclasticus P-18 did not grow in the presence of cis-9,trans-11-18 : 2 or trans10,cis-12-18 : 2, but grew in medium containing trans-9,trans-11-18 : 2, forming 18 : 0. Propionibacterium acnes, a ruminal species that isomerizes linoleic acid to trans-10,cis-12-18 : 2, did not metabolize CLA isomers further. B. fibrisolvens metabolized small amounts of trans-10-18 : 1, trans-11-18 : 1 and cis-9-18 : 1, but the products formed were not detected. B. proteoclasticus, on the other hand, carried out substantial conversion of 18 : 1 substrates to 18 : 0. P. acnes hydrated cis-9-18 : 1 and trans-11-18 : 1 to 10-OH-18 : 0, which was further oxidized to yield 10-O-18 : 0. The deuterium enrichment in the intermediates formed during incubations with 9,11 geometric isomers of CLA was about half that of the products from trans-10,cis-12 CLA and 18 : 1 isomers, suggesting that the reduction of 9,11 geometric isomers CLA by ruminal bacteria occurs via different mechanisms compared with the metabolism of other unsaturated fatty acids. DOI 10.1099/mic.0.036442-0

Conjugated linoleic acid (CLA) is formed from linoleic acid (LA; cis-9,cis-12-18:2) by intestinal bacteria. Different CLA isomers have different implications for human health. The aim of this study was to investigate LA metabolism and the CLA isomers formed in two individuals (V1 and V2) with different faecal metabolic characteristics, and to compare fatty acid metabolism with the microbial community composition. LA incubated with faecal samples was metabolized at similar rates with both subjects, but the products were different. LA was metabolized extensively to stearic acid (SA; 18:0) in V1, with minor accumulation of CLA and more rapid accumulation of vaccenic acid (VA; trans-11-18:1). CLA accumulation at 4 h was almost tenfold higher with V2, and little SA was formed. At least 12 different isomers of CLA were produced from LA by the colonic bacteria from the two individuals. The predominant (>75 %) CLA isomer in V1 was rumenic acid (RA; cis-9,trans-11-18:2), whereas the concentrations of RA and trans-10,cis-12-18:2 were similar with V2. Propionate and butyrate proportions in short-chain fatty acids were higher in V1. A 16S rRNA clone library from V1 contained mainly Bacteroidetes (54% of clones), whereas Firmicutes (66% of clones) predominated in V2. Both samples were devoid of bacteria related to Clostridium proteoclasticum, the only gut bacterium known to metabolize VA to SA. Thus, the CLA formed in the intestine of different individuals may differ according to their resident microbiota, with possibly important implications with respect to gut health.

Faecal bacteria from four human donors and six species of human intestinal bacteria known to metabolize linoleic acid (LA) were incubated with LA in deuterium oxide-enriched medium to investigate the mechanisms of conjugated linoleic acid (CLA) and vaccenic acid (VA) formation. The main CLA products in faecal suspensions, rumenic acid (cis-9,trans-11-CLA; RA) and trans9,trans-11-CLA, were labelled at C-13, as were other 9,11 geometric isomers. Traces of trans-10,cis-12-CLA formed were labelled to a much lower extent. In pure culture, Bifidobacterium breve NCFB 2258 formed labelled RA and trans-9,trans-11-CLA, while Butyrivibrio fibrisolvens 16.4, Roseburia hominis A2-[183.sup.T], Roseburia inulinivorans A2-[192.sup.T] and Ruminococcus obeumlike strain A2-162 converted LA to VA, labelled in a manner indicating that VA was formed via C-13-labelled RA. Propionibacterium freudenreichfi subsp, shermanii DSM [4902.sup.T], a possible probiotic, formed mainly RA with smaller amounts of trans-10,cis-12-CLA and trans-9,trans-11-CLA, labelled the same as in the mixed microbiota. Ricinoleic acid (12-OH-cis-9-18:1) did not form CLA in the mixed microbiota, in contrast to CLA formation described for Lactobacillus plantarum. These results were similar to those reported for the mixed microbiota of the rumen. Thus, although the bacterial genera and species responsible for biohydrogenation in the rumen and the human intestine differ, and a second route of RA formation via a 10-OH-18 : 1 is present in the intestine, the overall labelling patterns of different CLA isomers formation are common to both gut ecosystems. A hydrogen-abstraction enzymic mechanism is proposed that may explain the role of a 10-OH-18:1 intermediate in 9,11-CLA formation in pure and mixed cultures.

Ruminant fat is rich in SFA, partly due to the biohydrogenation of dietary PUFA to SFA in the rumen. This process can be inhibited by the dietary inclusion of fish oil. The only bacteria isolated from the rumen capable of converting PUFA to SFA are closely related to Clostridium proteoclasticum. The aim of this study was to investigate if a correlation could be found in vivo between dietary fish oil inclusions and the composition of the ruminal bacterial community and specifically of C. proteoclasticum. Six Hereford x Friesian steers, prepared with ruminal and duodenal cannulae, received grass silage plus 1 of 3 concentrates resulting in total dietary fish oil contents of 0, 1, or 3% of dry matter. A dual flow marker technique was employed to estimate the relative flow of fatty acids. Steers fed the 3% fish oil diet had 100% increases in trans 18:1 flow, whereas 18:0 flow declined to 39% of steers fed the control diet. 16S ribosomal RNA-based denaturing gradient gel electrophoresis profiles obtained from ruminal digesta showed major changes in the bacterial community within steers fed the 3% fish oil diet. Quantitative PCR indicated only a weak relation between numbers of C. proteoclasticum and 18:0 flow between treatments and in individual steers (P < 0.05, but the percentage variance accounted for only 22.8) and did not provide unambiguous evidence that numbers of C. proteoclasticum in the rumen dictate the ratios of SFA:PUFA available for absorption by the animal. Understanding which microbes biohydrogenate PU FA in the rumen is key to developing novel strategies to improve the quality of ruminant products.

A survey of 30 representative strains of human gram-positive intestinal bacteria indicated that Roseburia species were among the most active in metabolizing linoleic acid (cis-9,cis-12-18:2). Different Roseburia spp. formed either vaccenic acid (trans-11-18:1) or a 10-hydroxy-18:1; these compounds are precursors of the health-promoting conjugated linoleic acid cis-9,trans-11-18:2 in human tissues and the intestine, respectively.

Flavomycin is a phosphoglycolipid antibiotic that promotes growth in ruminants. The aim of this study was to characterize the effects of flavomycin on ruminal micro-organisms and their metabolic consequences. In sheep receiving a mixed grass hay/concentrate diet, inclusion of 20 mg flavomycin [day.sup.-1] decreased ruminal ammonia and total volatile fatty acid concentrations (P< 0.001), but the acetate : propionate ratio was unchanged. Ruminal pH tended to be lower with flavomycin, and ammonia-production rates of ruminal digesta from control animals measured in vitro tended to be inhibited by flavomycin. Pure-culture studies indicated that anaerobic fungi, protozoa and most bacterial species were insensitive to flavomycin. Fusobacterium necrophorum was the most sensitive species tested, along with some hig[H.sup.-]activity ammonia-producing (HAP) species. Effects on F. necrophorum in vivo were inconsistent due to large inter-animal variation. HAP numbers appeared to be decreased. Changes in the rumen bacterial-community structure were assessed by using denaturing-gradient gel electrophoresis (DGGE) analysis of rumen digesta 16S rRNA. DGGE profiles differed from animal to animal, but remained consistent from day to day. The community structure changed when flavomycin was introduced. The roles of F. necrophorum and HAP species in ammonia formation and of F. necrophorum in the invasion of wall tissue are consistent with the observed effects of flavomycin on ruminal ammonia formation and, in other studies, on decreasing tissue-turnover rates.

Eubacterium pyruvativorans I-[6.sup.T] is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. L-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with [C.sub.2] units derived from other substrates to dispose of reducing equivalents.

Ruminal bacteria of the genus Prevotella play a crucial role in peptide breakdown in the rumen, a component of protein catabolism that leads to the inefficient use of dietary protein by ruminant animals. This is the first report of the cloning of a peptidase gene from a ruminal bacterium. Part of the dipeptidyl peptidase type IV (DPP-IV) gene from Prevotella albensis [M384.sup.T] was cloned using degenerate primers designed from conserved regions found within other known DPP-IV sequences. Flanking regions were determined by genomic walking. The DPP-IV gene was expressed in Escherichia coli. The cloned enzyme required a free N terminus and catalysed the removal of X-Pro dipeptide from proline-containing oligopeptides, where proline was the second residue from the N terminus. It was inhibited by serine protease inhibitors and the substrate analogue for mammalian DPP-IV, diprotin A. The properties of the cloned enzyme were similar to those of the native form in P. albensis and, in general, DPP-IVs from other organisms. The enzyme contained a conserved motif which is associated with the S9 class of prolyl oligopeptidases. The DPP-IV gene appeared not to be part of a contiguous operon. Regions with similarity to other putative promoters of Prevotella spp. were also identified. Construction of a phylogenetic tree demonstrated that the DPP-IV of P. albensis clusters with other DPP-IVs found in bacteria of the Cytophaga-Flexibacter-Bacteroidaceae (CFB) phylum, which are more closely related to eukaryotic DPP-IVs than the DPP-IV-like enzyme (PepX) of the lactic acid bacteria.

Some cellulolytic ruminal bacteria incorporate nitrogen from non-ammonia sources depending on the growth medium. Amino acids can also stimulate their growth on certain media. This shows that amino acids are a significant source of nitrogen for these bacteria.

Research was conducted to examine the influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, namely, Prevotella bryantii, Streptococcus bovis ES1 and Selenomonas ruminantium HD4. The bacteria were grown in media with 15 NH4Cl and additional pancreatic hydrolysates of casein or amino acids. Results suggest a wide variation in de novo synthesis between individual amino acids and individual species.

Peptides and amino acids, either present in the diet or arising from proteolysis in the rumen, are potential nutrients for the growth of ruminal microorganisms but also are liable to be degraded to ammonia and lost from the rumen. In both cases, peptides generally are metabolized by the mixed population more rapidly than amino acids. The predominant mechanism of peptide degradation is biphasic, via dipeptidyl pepti-dases, which cleave dipeptides from larger peptides followed by the action of dipeptidases. Peptides blocked at the N-terminus, or with glycine or proline as the N-terminal or penultimate N-terminal residue, are more slowly degraded than others. Dipeptidyl peptidase activity occurs only in Prevotella ruminicola among the common ruminal microbial species. In contrast, dipeptidase is present in many species, including P. ruminicola, and is particularly high in ruminal protozoa. Deamination of amino acids is carried out by a combination of numerous low activity bacteria and protozoa and a much smaller number of high activity species. Ammonia production is probably carried out mainly by the low activity species, which again include P. ruminicola, but proliferation of the high activity species may be a problem when certain diets are fed. Excessive ammonia production can be controlled by ionophores, which slow peptide metabolism, suppress growth of the high activity deaminating bacteria and cause the residual ionophore-resistant population to break down amino acids more slowly. J. Nutr. 126: 1326S-1334S, 1996. INDEXING KEY WORDS: amino acids, ammonia, peptidases, peptides, ruminal microorganisms

Abstract Background The products of protein breakdown in the human colon are considered to be detrimental to gut health. Amino acid catabolism leads to the formation of sulfides, phenolic compounds and amines, which are inflammatory and/or precursors to the formation of carcinogens, including N-nitroso compounds. The aim of this study was to investigate the kinetics of protein breakdown and the bacterial species involved. Results Casein, pancreatic casein hydrolysate (mainly short-chain peptides) or amino acids were incubated in vitro with suspensions of faecal bacteria from 3 omnivorous and 3 vegetarian human donors. Results from the two donor groups were similar. Ammonia production was highest from peptides, followed by casein and amino acids, which were similar. The amino acids metabolized most extensively were Asp, Ser, Lys and Glu. Monensin inhibited the rate of ammonia production from amino acids by 60% (P = 0.001), indicating the involvement of Gram-positive bacteria. Enrichment cultures were carried out to investigate if, by analogy with the rumen, there was a significant population of asaccharolytic, obligately amino acid-fermenting bacteria (‘hyper-ammonia-producing’ bacteria; HAP) in the colon. Numbers of bacteria capable of growth on peptides or amino acids alone averaged 3.5% of the total viable count, somewhat higher than the rumen. None of these were HAP, however. The species enriched included Clostridium spp., one of which was C. perfringens, Enterococcus, Shigella and Escherichia coli. Conclusions Protein fermentation by human faecal bacteria in the absence of sugars not only leads to the formation of hazardous metabolic products, but also to the possible proliferation of harmful bacteria. The kinetics of protein metabolism were similar to the rumen, but HAP bacteria were not found.

The consequences of 1-((E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl)pyrrolidine-2-carboxylic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were investigated in a mixed rumen microbial population obtained from sheep on a grass hay-concentrate diet. Both compounds decreased NH3 production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.