In vitro proteolytic inhibition, polyphenol oxidase activity, and soluble o-diphenols in grasses and cereals

Producing high quality forage remains a challenge due to potential protein degradation during ensiling. Ten grass species and four cereals were screened for proteolytic inhibition, polyphenol oxidase (PPO) activity, and the presence of soluble PPO substrates. In orchardgrass, ryegrass, smooth bromegrass, and meadow fescue extracts, the addition of caffeic acid resulted in 70-98% reduction in proteolysis for both greenhouse and field grown environments. These same grass extracts exhibited 10- to 1000-fold higher PPO activity compared to all other grasses and cereals, depending on the substrate supplied and environment grown. Chlorogenic acid and caffeic acid were the two abundant soluble o-diphenols. Tall fescue and timothy exhibiting low PPO activity contained the highest levels of chlorogenic acid (3-11 micromol g(-1) FW): 10- to 100-fold more than any o-diphenol in all other grasses. These results suggest that several grass species contain PPO activity, but may lack appropriate o-diphenol substrates to effectively inhibit proteolysis during ensiling while other grasses lack PPO activity but contain potential soluble substrate. Environmental factors appear to influence these parameters, creating an opportunity to exploit and enhance proteolytic inhibition during ensiling by genetic manipulation.