REDUCTION OF PROTEOLYSIS DURING ENSILAGE OF PERENNIAL RYEGRASS BY PROTEASE INHIBITORS

Two experiments were carried out to assess the effectiveness of protea se inhibitors in reducing proteolysis during ensilage. Perennial ryegr ass (Lolium perenne) (279 g dry matter (DM) kg(-1), experiment 1 and 1 70 g DM kg(-), experiment 2) was ensiled in laboratory silos for 82 an d 50 days, respectively. With the exception of the application of form ic acid (1.5 g kg(-1)) plus formalin (3 g kg(-1)) in experiment 1, all silages had pH values of less than 4.0 and lactic acid concentrations of greater than 100 g kg(-1) DM. Application of the cysteine endopept idase inhibitor E-64 (1-trans-epoxysuccinyl-leucylamido(4-guanidino) b utane, 22.5 mg kg(-)) reduced proteolysis during ensilage as measured by a reduction in the quantities of soluble non-protein N in silage to 0.76 and 0.91 of control values (528 and 643 g kg(-1) total N) in exp eriments 1 and 2, respectively. In experiment 2 increasing the applica tion rate of E-64 from 22.5 to 45 mg kg(-1) further reduced the extent of proteolysis from 0.91 to 0.75 of control soluble non-protein N con centrations. Proteolysis was also reduced in experiment 2 by two other cysteine endopeptidase inhibitors, antipain (32 mg kg(-1), 0.81 of co ntrol) and cystatin (1.2 mg kg(-1); 0.89 of control). Pepstatin A (300 mg kg(-1)), an inhibitor of aspartic acid endopeptidases, did not red uce soluble non-protein N concentrations in experiment 1 but did do so in experiment 2 (0.81 of control). Treatment of grass at ensiling wit h Lactobacillus plantarum (10(6) g(-1)) reduced silage non-protein N c oncentrations to 0.83 of the control. The effects of E-64 and pepstati n A and of E-64 and L plantarum in experiment 2 were additive. Cystein e endopeptidases therefore play an important role in protein breakdown during ensilage.