Potentials for developing an intake protein conserving agent for ruminants

Abstract

A series of experiments were conducted to determine effects of various protease inhibitors of synthetic, plant and microbial origin and a deaminase inhibitor on proteolytic activity of unadapted mixed rumen bacteria. Proteolytic activity was measured by the rate of release of 14 CH -lysine from 14 C-casein labelled by reductive methylation. The synthetic protease inhibitors, n-tosyl-llysine chlorometyl ketone, phenylmethylsulfonyl fluoride, p chloromercuribenzoate , iodoacetate caused signif icant (p < .01) inhibition of proteolysis. The plant protease inhibitor, soybean trypsin inhibitor type II had similar inhibitory effects. Among microbial protease inhibitors, E64 and leupeptin showed significant inhibitory effects while diprotein A and pepstatin had no inhibitory effect. These results confirmed that proteases of mixed rumen bacteria are predominantly of the cysteine type followed by serine and metalloprotease types The deaminase inhibitor diphenyliodonium chloride (DIC) did not cause inhibition of proteolysis at low concentration (0.1 mM), but showed inhibitory effects at higher concentrations (0. 4 and 0. 8 mM) . Further experiments were conducted to determine if adapted mixed rumen bacteria developed resistance to the deaminase inhibitor DIC. Various concentrations of DIC were dosed via the culture media, replacing half of the total fermentation volume daily for 37 days. Deaminase activity was measured as net transamination of [1-14C]-leucine every week. Concentration of total protein and ammonia for the control treatment continuously decreased by 15 fold during the 37 days, indicating that the system did not provide conditions adequate for microbial growth. There were no significant (p >.05) differences in total protein concentration between control and different levels of DIC during the incubation period. In the presence of 0.1 mM DIC, ammonia concentration and net transamination of leucine were significantly (p <.05) lower than the control throughout the entire incubation period. Leucine catabolic pathways, transamination and decarboxylation were equally inhibited at 0.1 mM of DIC. These results suggest that mixed rumen bacteria exposed to 0.1 mM DIC for 32 days, under the conditions of the in vitro system used in this work, did not overcome the inhibitory effect of DIC on deamination. Results of this study indicate that the microbial protease inhibitors E-64 and leupeptin and the deaminase inhibitor DIC may be potentially useful as an intake protein conserving agent for ruminants.