Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2009
Publication Date: 1/27/2010
Citation: Marita, J.M., Hatfield, R.D., Brink, G.E. 2010. In Vitro Proteolytic Inhibition, Polyphenol Oxidase Activity, and Soluble O-Diphenols in Grasses and Cereals. Journal of Agriculture and Food Chemistry. 58:959-966. Interpretive Summary: Producing high-quality forage in cool, humid regions remains a challenge due to environmental conditions. Ensiling forage is typically used to produce a high-quality feed in these regions. However, ensiling conditions must be optimal and often require silage additives in order to minimize the potentially high degree of protein degradation during ensiling. Red clover is an exception, with high protein levels at harvest that are maintained during the ensiling process. We have studied the red clover ensiling process and found that decreased protein degradation is due to an enzyme, polyphenol oxidase (PPO), and its substrates, o-diphenols. These substrates are special phenolic compounds that react with the PPO to produce highly reactive o-quinones that, in turn, react with a wide range of other molecules including proteins. This is the same type of reaction that occurs when many fruits (apples, bananas, avocadoes, etc.) are cut and exposed to air, resulting in a browning of the exposed tissue. Besides red clover, many grasses are also ensiled to produce animal feed. We were interested in knowing if any of the typical forage grasses contained PPO activity and, if they did, would protein degradation also be decreased as seen in red clover. Fifteen grasses were analyzed for PPO activity and o-diphenol substrates. Orchardgrass, meadow fescue, ryegrass, and smooth bromegrass had high levels of PPO activity and exhibited some browning reaction upon grinding and exposure to air. Analysis of phenolics in these grasses indicated the typical o-diphenols found in red clover were not present in the grasses with high PPO activity. If it is possible to treat high-PPO grasses with appropriate o-diphenols in order to decrease protein degradation, this would result in more efficient use of silage protein by ruminants such as dairy cows and decrease the nitrogen losses in animal urine. Decreasing protein degradation during ensiling decreases the amount of supplemental protein that must be added to the diet, resulting in a cost saving to the farmer. The PPO process has the potential to treat silages of all types to prevent excess nitrogen loss, ultimately increasing profitability of operations for farmers and significantly lessening nitrogen pollution to the environment.
Technical Abstract: Harvesting and storing high-quality forage in the cool humid regions of agricultural production remains a challenge due to the potentially high degree of protein degradation during ensiling. Red clover is an exception with high protein levels at harvest that are maintained during ensiling. Decreased proteolytic activity in red clover is due to polyphenol oxidase (PPO) activity and appropriate o-diphenol substrates. This project was undertaken to determine if PPO activity and appropriate o-diphenol substrates were present in a range of C3 grasses. The amount of PPO activity measured in each grass varied significantly, depending upon the individual grass species. All grasses were analyzed for levels of caffeic and chlorogenic acids, two of the most common o-diphenol substrates found in plants. Orchardgrass, meadow fescue, ryegrass, and smooth bromegrass exhibited the highest PPO activities (4-25 µmoles mg-1-min-1) with chlorogenic acid as the preferred substrate. There were differences among the most active grasses as to which o-diphenol substrates were the best utilized. This suggests potential differences among the individual PPO enzymes. Most of the grasses with PPO activities did not contain chlorogenic and caffeic acids, although some grasses such as tall fescue and timothy contained significant amounts of chlorogenic acid (10-40 µmoles g-1 fresh weight) and lacked PPO activity. Generally, the addition of caffeic acid to isolated grass extracts resulted in proteolytic inhibition in grasses with substantial PPO activity. Such results suggest that several important grass species contain PPO activity, but may lack the appropriate o-diphenol substrates to effectively inhibit proteolysis. Initial results suggest that proteolytic inhibition can be achieved with the addition of caffeic acid.