Submitted to: Journal of the Science of Food and Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2012
Publication Date: 7/6/2012
Citation: Sullivan, M.L., Zeller, W.E. 2012. Efficacy of various naturally occurring caffeic acid derivatives in preventing post-harvest protein losses in forages. Journal of the Science of Food and Agriculture. 93:219-226.
Interpretive Summary: In an effort to help the dairy cow capture and utilize more of the protein in forages, U.S. Dairy Forage Research Center scientists have demonstrated that components found in red clover [polyphenol oxidase (PPO) and o-diphenols] prevent protein degradation when the forage is preserved by ensiling. Preventing protein degradation in preserved forages using this PPO/o-diphenol system would have significant positive economic and environmental benefits; it is estimated that it costs farmers at least $100 million annually to supplement rations with the needed true protein. Additionally, poor utilization of non-protein nitrogen by ruminants results in excretion of nitrogen waste into the environment. Unfortunately, many important forages such as alfalfa do not accumulate o-diphenols, so transferring the PPO/o-diphenol system to alfalfa may require transferring the pathways responsible for o-diphenol biosynthesis into this forage crop. This study looked at the effectiveness of several o-diphenol compounds in preventing protein degradation in conjunction with PPO. The o-diphenols tested included caffeic acid and its derivatives (phaselic acid, clovamide, and chlorogenic acid). All tested o-diphenol compounds substantially reduced post-harvest protein degradation. In the case of clovamide, a substantial reduction in protein degradation was seen even without PPO present. These results indicated that, to reduce protein degradation via the PPO/o-diphenol system, both phaselic acid and chlorogenic acid may be good targets for production in alfalfa and other forage crops. Clovamide may be useful for inhibiting protein degradation without the need for PPO. The findings of this study will be useful in adapting the PPO/o-diphenol system of protein protection to alfalfa and other forages. They will also be of interest to other researchers who are studying post-harvest physiology and to biotechnology companies interested in developing the system into a commercial product.
Technical 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 to 87% (P<0.001) and as little as 0.25 mmol g-1 protein still decreased 24-h proteolysis 43 to 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 exibit PPO- and oxygen-independent inhibition (37%, P<0.001). Thus, 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.