Factors Affecting the Separation and Bioactivity of Red Clover (Trifoliumpratense) Extracts Assayed against Clostridium sticklandii, a Ruminal Hyper Ammonia-producing Bacterium

Red clover ( Trifolium pratense) is rich in phenolic compounds. Both the crude phenolic extract and biochanin A, an isoflavonoid component of the extract, suppress growth of Clostridium sticklandii, a bovine, Gram-positive, ruminal hyper-ammonia producing bacterium (HAB). The purpose of this study was to determine if other components of red clover extract contributed to its anti-HAB activity. Extracts of the Kenland cultivar of red clover, collected 0 h and 24 h after cutting, were separated by normal-phase thin-layer chromatography (TLC) in either ethyl acetate-hexanes (9:1, v/v) or ethyl acetate-methanol (4:1, v/v). Bands on TLC plates were assayed by either overlaying the plates with agar seeded with C. sticklandii, or setting the excised bands face-down onto plates of bacteria-seeded agar. Biochanin A inhibited C. sticklandii growth on TLC plates when as little as 8 nmol was present in the extract. An antimicrobial band, seen in a previous bioassay, was not found, suggesting that this second compound may be more labile than biochanin A.

Purification and Properties of Ornithine Racemase from Clostridium sticklandii

ABSTRACT Ornithine racemase has been purified to homogeneity fromClostridium sticklandii, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first racemase known to be highly specific to ornithine. This PLP-dependent enzyme has an M r of 92,000, with aKm for l-ornithine of 0.77 ± 0.05 mM and a kcat of 980 ± 20 s−1.

Influence of 1-[(E)-2-(2-Methyl-4-Nitrophenyl)Diaz-1-enyl]Pyrrolidine-2-Carboxylic Acid and Diphenyliodonium Chloride on Ruminal Protein Metabolism and Ruminal Microorganisms

ABSTRACT The effects 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 determined in a mixed rumen microbial population taken 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.