Polyphenol oxidase activity in grass and its effect on plant-mediated lipolysis and proteolysis of Dactylis glomerata (Cocksfoot) in a simulated rumen environment

Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g-1 fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant-mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic-containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 °C over five time points. At each time point (0, 1, 2, 6 and 24 h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g-1 FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24 h of incubation, namely 65.1 and 29.6 mg bound phenol g-1 protein (P <0.001) for cocksfoot and cocksfoot + ascorbate respectively. Proteolysis, measured as free amino acids released into the incubation buffer, was significantly reduced (P <0.001) with increasing PPO activity, with values after the 24 h incubation of 0.03 and 0.07 mmol L-1 g-1 FW for cocksfoot and cocksfoot + ascorbate respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (P <0.001) with increasing PPO activity, with values after the 24 h incubation of 0.43 and 0.65 for cocksfoot and cocksfoot + ascorbate respectively. Changes that occurred in protein and the lipid fractions (polar fraction, monoacylglycerol + diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid losses in silo and potentially in the rumen