Influence of protease inhibitors on nitrogen distribution in ensiled perennial ryegrass and the utilisation of silage nitrogen for growth by rumen bacteria in vitro

Protease inhibitors were added to perennial ryegrass (Lolium perenne; dry m atter (DM), 188 g kg(-1) fresh weight (FW); water soluble carbohydrates, 17 5 g kg(-1) DM) at ensilage and their influence on silage nitrogen (N) distr ibution and the ability of soluble N fractions prepared from these silages to support the growth of Megasphaera elsdenii, Prevotella bryantii and Sele nomonas ruminantium in vitro were investigated. Control silages were well f ermented (pH 3.9), ammonia N and peptide N concentrations were 0.09 and 0.0 8 of total N, respectively, and the silage had undergone extensive proteoly sis (soluble non-protein N, (NPN) 0.68 of total N). Formic acid treatment ( 5.4 g kg(-1) FW) restricted silage fermentation, decreased soluble NPN (0.4 9 of total N) and ammonia-N concentrations and increased peptide N concentr ations to 0.21 of total N. Two metallo-protease inhibitors, 8-hydroxyquinol ine (5 g kg(-1) FW) and 1,10-phenanthroline (2.4 g kg(-1) FW) restricted si lage fermentation and increased peptide-N concentrations to values similar to those obtained with formic acid treatment. Both of these additives decre ased ammonia-N concentrations and, in addition, 1,10-phenanthroline decreas ed soluble NPN concentrations to 0.5 of total N, A third metallo-protease i nhibitor, bestatin had no effect. Inhibitors of chymotrypsin-like enzymes h ad little effect on silage fermentation but markedly different effects on N distribution: N-acetyl-L-tyrosine ethyl ester (20 mg kg(-1) FW) was withou t effect, whereas tosyl phenylalanyl chloromethyl ketone (TPCK, 42 mg kg(-1 ) FW) decreased the concentrations of soluble NPN (0.59 of total N) and amm onia N and increased peptide-N to 0.22 of total N. The cysteine-protease in hibitor,1-trans epoxysuccinyl-leucylamido-(4-guanidino) butane (22.5 mg kg( -1) FW), decreased total soluble NPN (0.45 of total N) and ammonia-N concen trations with no effect on peptide-N concentrations. Silage extracts and N fractions purified from silage extracts were supplied as sources of N and g lucose added to growth media for rumen bacteria and specific growth rates w ere determined turbidimetrically. M. elsdenii grew more rapidly in medium c ontaining silage extracts than in complete medium, whereas P. bryantii and S. ruminantium grew more slowly. None of the protease inhibitors resulted i n silage extracts which supported significantly more rapid growth of any of the bacteria compared to the control silage. However, 8-hydroxyquinoline a nd 1,10-phenanthroline were toxic to the bacteria when silage juices were a dded to the medium. The results suggest that grass protein is degraded by m etallo- and cysteine proteases during ensilage and that silage peptide N co ncentrations may be manipulated by the use of formic acid, chelators and TP CK. Rumen bacteria grew well on silage extracts and N fractions prepared fr om silage extracts, but there was no indication that increasing the concent ration of silage peptide N beyond 17 mu g peptide N ml(-1) supported higher growth rates. (C) 1998 Elsevier Science B.V.