Reduction of proteolysis during ensilage of perennial ryegrass by protease inhibitors

Two experiments were carried out to assess the effectiveness of protease inhibitors in reducing proteolysis during ensilage. Perennial ryegrass (Lolium perenne) (279 g dry matter (DM) kg−1, experiment 1 and 170 gDM kg−1 experiment 2) was ensiled in laboratory silos for 82 and 50 days, respectively. With the exception of the application of formic 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 endopeptidase inhibitor E‐64 (1‐trans‐epoxysuccinyl‐leucylamido(4‐guanidino) butane, 22.5 mg kg−1 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 experiments 1 and 2, respectively. In experiment 2 increasing the application 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 concentrations. Proteolysis was also reduced in experiment 2 by two other cysteine endopeptidase inhibitors, antipain (32 mg kg−1, 0.81 of control) and cystain (1.2 mg kg−1; 0.89 of control). Pepstatin A (300 mg kg−1), an inhibitor of aspartic acid endopeptidases, did not reduce soluble non‐protein N concentrations in experiment 1 but did do so in experiment 2 (0.81 of control). Treatment of grass at ensiling with Lactobacillus plantarum (106 g−1) reduced silage non‐protein N concentrations to 0.83 of the control. The effects of E‐64 and pepstatin A and of E‐64 and L plantarum in experiment 2 were addive. Cysteine endopeptidases therefore play an important role in protein breakdown during ensilage.