Red clover polyphenol oxidase and lipid metabolism

Abstract

Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid encapsulation in protein-phenol complexes would lead to an effective protection of lipids against ruminal biohydrogenation. From several studies it has also become apparent that the degree of PPO activation could influence the mode and degree of protection. In conclusion, this review demonstrates that protein-bound phenols and encapsulation in protein-phenol complexes, induced by PPO-mediated diphenol oxidation, could be of interest when aiming to protect lipids against pre-ruminal and ruminal degradation.Keywords: red clover, polyphenol oxidase, lipid metabolism, rumen, silage ImplicationsPolyphenol oxidase (PPO) is an enzyme that has been found to be active in red clover. There is a whole range of recent studies exploring possibilities of using this enzyme to protect membrane lipids against degradation in the forage during storage and in the rumen of ruminants. Protection of lipids against degradation is of importance as it could help to improve the fatty acid profile of ruminant products, with respect to consumer health. This review aims to summarize...

The effect of N-fertilisation rate or inclusion of red clover to timothy leys on fatty acid composition in milk of dairy cows fed a commercial silage : concentrate ratio

“…The binding of proteins to phenols, which results from PPO activity, lead to inhibition of lipolysis in the silage and as lipolysis is a prerequisite for biohydrogenation of PUFAs in the rumen, the extent of biohydrogenation is decreased. However, Van Ranst et al (2011) concluded that the importance of this inhibition of lipolysis in the silage is questionable. The PPO-mediated inhibition of lipolysis failed to protect lipids against microbial lipases, which become important during later stages of ensiling and in the rumen.…”

“…The PPO-mediated inhibition of lipolysis failed to protect lipids against microbial lipases, which become important during later stages of ensiling and in the rumen. Instead, Van Ranst et al (2011) postulated that PUFA could be protected against metabolism through encapsulation in protein-phenol complexes. In the current paper, there were, however, no significant effects on apparent recoveries of PUFA from RC-G compared with the grass silages.…”

“…Further, red clover has been shown to have lower lipolytic activities, or to protect the FA against lipolysis through encapsulation in protein-phenol complexes (Van Ranst et al, 2011). Hence, significantly higher concentrations of 18:2n-6 and 18:3n-3 have been found in milk from cows fed red clover silage than in milk from cows fed ryegrass silage (Dewhurst et al, 2003a;Al-Mabruk et al, 2004).…”

The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers1

“…Although other differences between grasses must be acknowledged, the results are not indicative of a PPO protective response in grass. There is little doubt that PPO in both red clover and grasses can deactivate lipase activity (Lee et al, 2006b;Van Ranst et al, 2011). However, for PPO to protect lipid in the ruminal environment, there appears to be a requirement for the lipid to be complexed within PBP, which would limit microbial lipase access to the PUFA-glycerol ester bond ).…”

“…A further benefit of red clover silage is a reduction in C18 PUFA biohydrogenation compared to grass silage (Lee et al, 2003). Polyphenol oxidase reduces plant mediated lipolysis (Lee et al, 2004) by inhibiting plant lipases (Van Ranst et al, 2011).…”