Freda M. McIntosh

Mode of action of the yeast Saccharomyces cerevisiae as a feed additive for ruminants

Two suggested modes of action of yeast in stimulating rumen fermentation were investigated. The first, that yeast respiratory activity protects anaerobic rumen bacteria from damage by O2, was tested using different strains of yeast that had previously been shown to have differing abilities to increase the viable count of rumen bacteria. Saccharomyces cerevisiae NCYC 240, NCYC 1026, and the commercial product Yea-Sacc, added to rumen fluid in vitro at 1.3 mg/ml, increased the rate of O2 disappearance by between 46 and 89%. The same three preparations also stimulated bacterial numbers in an in vitro fermenter (Rusitec). S. cerevisiae NCYC 694 and NCYC 1088, which had no influence on the viable count in Rusitec, also had no effect on O2 uptake. Respiration-deficient (RD) mutants of S. cerevisiae NCYC 240 and NCYC 1026 were enriched by repeated culturing in the presence of ethidium bromide. S. cerevisiae NCYC 240 and NCYC 1026 stimulated the total and cellulolytic bacterial populations in Rusitec, while the corresponding RD mutants did not. Rigorous precautions to exclude air from Rusitec resulted in S. cerevisiae NCYC 240 no longer stimulating total bacterial numbers, although it still increased numbers of cellulolytic bacteria. The second hypothesis, that yeast provides malic and other dicarboxylic acids which stimulate the growth of some rumen bacteria, was examined by comparing the effects of yeast and malic acid on rumen fermentation in sheep. Three mature sheep were given 0.85 kg barley/d plus 0.55 kg chopped ryegrass hay/d either unsupplemented, or supplemented with 4 g S. cerevisiae NCYC 240/d or 100 mg L-malic acid/d either mixed with the diet or in aqueous solution infused continuously into the rumen. Yeast increased the total viable count of bacteria (P < 0.05) whereas malic acid did not, and no other effect of the treatments reached statistical significance. It was concluded, therefore, that the stimulation of rumen bacteria by S. cerevisiae is at least partly dependent on its respiratory activity, and is not mediated by malic acid.

Metabolism of Linoleic Acid by Human Gut Bacteria: Different Routes for Biosynthesis of Conjugated Linoleic Acid

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.

Rumen ciliate protozoa contain high concentrations of conjugated linoleic acids and vaccenic acid, yet do not hydrogenate linoleic acid or desaturate stearic acid

Conjugated linoleic acids (CLA) have been shown to improve human health. They are derived from the microbial conversion of dietary linoleic acid (cis-9,cis-12-18 : 2 (LA)) in the rumen. An investigation was undertaken to determine the role of ruminal ciliate protozoa v. bacteria in the formation of CLA and its precursor in animal tissues, vaccenic acid (trans-11-18 : 1 (VA)). Mixed protozoa from the sheep rumen contained at least two to three times more unsaturated fatty acids, including CLA and VA, than bacteria. Different species had different composition, with larger fibrolytic species such as Epidinium ecaudatum caudatum containing more than ten times more CLA and VA than some small species, including Entodinium nanellum. In incubations with ruminal microbial fractions (bacterial fraction (BAC), protozoal fraction (PRO)), LA metabolism was very similar in strained ruminal fluid (SRF) and in the BAC, while the PRO had LA-metabolising activity an order of magnitude lower. Using PCR-based methods, no genes homologous to fatty acid desaturase genes were found in cDNA libraries from ruminal protozoa. The absence of an alternative route of VA/CLA formation via desaturation of stearate was confirmed by incubations of SRF, BAC or PRO with [14C]stearate. Thus, although protozoa are rich in CLA and VA, they appear to lack the ability to form these two fatty acids from LA or stearate. The most likely explanation is that protozoa preferentially incorporate CLA and VA formed by bacteria. The implication of the present findings is that the flow of unsaturated fatty acids, including CLA and VA, from the rumen could depend on the flow of protozoa rather than bacteria.

Effect of adding acetogenic bacteria on methane production by mixed rumen microorganisms

Six reductive acetogenic bacteria from a variety of ruminal and non-ruminal environments were investigated for their ability to prevent the accumulation of methane when added to rumen fluid incubated in vitro. Acetitomaculum ruminis, Eubacterium limosum strains ATCC 10825 and ATCC 8486, Ruminococcus productus ATCC 35244, and two acetogenic bacteria, Ser 5 and Ser 8 isolated from 20 h old lambs, were grown in media containing glucose. The bacteria retained the ability to produce acetate from H2 when incubated in the absence of sugar, while no acetate was produced in the absence of H2. When cultures of the acetogens were added to incubations of mixed rumen microorganisms in vitro, providing between 0.05 and 0.13 mg of acetogen protein/ml, methane production decreased by about 5% after 24 h with E. limosum ATCC 8486 and Ser 5, while the other bacteria had no effect on methane production. Increasing the concentration of E. limosum ten-fold did not cause a further decrease in methane production. When E. limosum ATCC 8486 and Ser 5 were added to cultures of mixed ruminal microorganisms in the presence of 2-bromoethanesulfonic acid, which inhibited methane formation and caused H2 to accumulate, both bacteria caused substantial increases in acetate production and decreased H2 formation. It was concluded that although acetogenic bacteria can utilise H2 and CO2 to form acetate in the rumen when methanogenesis is inhibited, even large concentrations of acetogenic bacteria cannot compete for H2 with methanogenic archaea under normal circumstances. # 1999 Elsevier Science B.V. All rights reserved.

Mechanism of conjugated linoleic acid and vaccenic acid formation in human faecal suspensions and pure cultures of intestinal bacteria

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 trans-9,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 A2-183T, Roseburia inulinivorans A2-192T and Ruminococcus obeum-like strain A2-162 converted LA to VA, labelled in a manner indicating that VA was formed via C-13-labelled RA. Propionibacterium freudenreichii subsp. shermanii DSM 4902T, 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.

Differences between human subjects in the composition of the faecal bacterial community and faecal metabolism of linoleic acid

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.