Efficacy of various naturally occurring caffeic acid derivatives in preventing post‐harvest protein losses in forages

Abstract

In red clover, oxidation of endogenous o-diphenols by polyphenol oxidase (PPO) inhibits post-harvest proteolyis. This system is transferable to alfalfa by providing PPO (via a transgene) and o-diphenol PPO substrates (via exogenous application). To exploit the PPO system for protein protection, it would be advantageous to produce PPO substrates in alfalfa, which lacks them. We assessed the extent of PPO-mediated proteolytic inhibition by phenolic compounds, especially those whose biosynthesis could be engineered into alfalfa. Tested compounds included o-diphenols (caffeic acid, phaselic acid, chlorogenic acid, clovamide) and monophenols (p-coumaric acid, p-coumaroyl-malic acid). In the presence of PPO, 2 mmol o-diphenol g⁻¹ protein reduced 24 h proteolysis 68-87% (P < 0.001) and as little as 0.25 mmol g⁻¹ protein still decreased 24 h proteolysis 43-60% (P < 0.001). At high concentrations, clovamide inhibited 24 h proteolysis 50% (P < 0.001) in the absence of PPO, likely due to non-PPO oxidation. Monophenol p-coumaric acid did not inhibit 24 h proteolyis, although high levels of its malate ester did exhibit PPO- and oxygen-independent inhibition (37%, P < 0.001). For PPO-mediated proteolytic inhibition, pathways for both phaselic acid and chlorogenic acid may be good targets for engineering into alfalfa. Clovamide may be useful for inhibiting proteolysis without PPO.