Peptidase inhibitors as additives for ensilage : effects on silage characteristics with reference to peptide nitrogen
Novel approaches to manipulating proteolysis in ensiled perennial ryegrass (PRG) were investigated. The effects of the following on nitrogen (N) distribution in silage were investigated: E- 64, a specific cysteine-peptidase inhibitor (CPI); pepstatin A a specific aspartic-peptidase inhibitor; cystamine dihyrochloride (CYS) and N-ethylmaleimide, general CPIs, and formic acid (FA). Treatment with FA or CPIs reduced total soluble N, as a result of reduced proteolysis, and FA and CYS treatments increased peptide N concentrations (determined using fluroescamine or ninhydrin assays, and by amino acid analysis) compared to the control. Pepstatin A had little or no effect on the N constituents of silage. Characterisation of silage peptides using Sephadex G-25 suggested that they were predominantly di and tripeptides, with a small proportion of longer peptides (>7 amino acid residues). Forty additional compounds were screened for their efficacy as inhibitors of proteolysis in aqueous extracts of PRG. Five selected compounds were applied to PRG at ensilage: TPCK, a non specific CPI; chelators, 1,10-phenanthroline and 8-hydroxyquinoline (8-HQ); bestatin, a metallo-peptidase inhibitor; and N-acetyl-L-tyrosine ethyl ester (ATEE), a serine-peptidase inhibitor. When compared to the control, TPCK and 1,10- phenanthroline reduced total soluble N and increased peptide N concentrations; 8-HQ increased only peptide N concentrations. These chelators also restricted fermentation. The effects of Trypticase (peptides produced by enzymic hydrolysis of casein), silage extracts and N fractions prepared from silage extracts by cation exchange chromatography, as sources of N, on the growth of rumen bacteria, Megasphaera elsdenii, Prevotella ruminicola and Selenomonas ruminantium, supplied with glucose as an energy substrate in vitro, were investigated. No growth was observed on media containing extracts from silages produced in the presence of chelators but all bacteria grew on purified N fractions. Increasing silage peptide N therefore did not enhance microbial growth but for some treatments, silage N supported faster growth than Trypticase.