AbstractTwo 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−1experiment 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−1DM. Application of the cysteine endopeptidase inhibitor E‐64 (1‐trans‐epoxysuccinyl‐leucylamido(4‐guanidino) butane, 22.5 mg kg−1reduced 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−1total N) in experiments 1 and 2, respectively. In experiment 2 increasing the application rate of E‐64 from 22.5 to 45 mg kg−1further 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 withLactobacillus plantarum(106g−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 andL plantarumin experiment 2 were addive. Cysteine endopeptidases therefore play an impo
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