In Vitro Study of Red Clover Polyphenol Oxidase Activity, Activation, and Effect on Measured Lipase Activity and Lipolysis

Abstract

The goal of this paper was, first, to study the effect of red clover polyphenol oxidase (PPO) activity on protein-bound phenols and measured lipase activity in vitro and, second, to study the effect of PPO activation, measured as an increase in protein-bound phenols, as a result of degrees of damaging (not damaged, crushed, and freeze/thawed) of red clover before wilting on measured enzyme activity and in vitro lipid metabolism when incubated in a phosphate buffer. There was a positive relation between PPO activity and the occurrence of protein-bound phenols with a concomitant decrease in measured lipase activity, indicating a possibility to a direct inhibition of enzymes as a result of protein-bound phenols. Furthermore, damaging can activate PPO in red clover, measured as an increase in protein-bound phenols during wilting [0.7-20.6 nmol of tyrosine equiv (mg of protein)(-1)], again with a concomitant decrease in measured lipase activity [41.3-20.3 mumol of p-nitrophenyl butyrate (PNPB) min(-1) (mg of protein)(-1)]. Lipid metabolism during incubation of these forages in a phosphate buffer with ascorbic acid was only influenced by damaging when wilted for 24 h, with a lower lipolysis in crushed and freeze/thawed (52.9 and 32.6%, respectively, after 8 h of incubation) material compared to all other treatments (on average 60.4% after 8 h of incubation).

Red clover polyphenol oxidase and lipid metabolism

“…A second indication was a higher lipase activity in a protein extract of red clover after incubation for 24 h when phenols were scavenged (using polyvinylpyrrolidone) compared with the incubation in which phenols were still present (between 56.7 6 4.24 and 19.8 6 1.03 nmol methylumbelliferyl oleate (MBF)/min per mg DM, respectively; Van Ranst et al, 2006b). In a more elaborate study (Van Ranst et al, 2009c), in which a synthetic diphenol was added to a purified protein extract of red clover and incubated for 120 s, there was a clear correlation between measured lipase activity and the amount of protein-bound phenols (Figure 1). Furthermore, the increase in proteinbound phenols during these incubations was lower when PPO was partially inhibited using NaCN and no increase was found when PPO was almost entirely inhibited at the highest NaCN concentration.…”

“…Applying this theory to the results of the FT and wilted red clover in the study by Van Ranst et al (2009c) would suggest an inhibition of plant lipases due to the multiple binding of quinones to proteins, which leads to lower in silo lipolysis. However, lipids were not encapsulated due to the lack of Van Ranst, Lee and Fievez major cross-linking in and between proteins, explaining the lack of difference in biohydrogenation between fresh and wilted red clover.…”

“…In vitro lipolysis of forage mixed in a phosphate buffer demonstrated that a high and/or activated PPO activity results in a lower plant lipasemediated lipolysis (Lee et al, 2004;Van Ranst et al, 2006a;Lee et al, 2007;Van Ranst et al, 2009c). In all studies a control (non-activated, low or inhibited PPO activity) was compared with treatments in which PPO activity was promoted (activation) or was high (compared with the control).…”

“…Van Ranst, Lee and Fievez lipolysis during incubation in the presence of ascorbic acid over 24 h in which the wilted red clover was prepared under three damage regimes: not damaged (A), crushed (B) and (FT; C) (Van Ranst et al, 2009c). Owing to the presence of ascorbic acid, no PPO activity was possible during incubation.…”

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

“…PPO is a diphenol oxidizing enzyme, which is particularly active in red clover (Lee et al, 2004). It is stress activated as it is normally present in a latent form and thus activation due to damaging could be necessary to ensure sufficient activity and provoke sufficient amounts of protein-bound phenols for a protective effect toward lipolysis (Lee et al, 2009;Van Ranst et al, 2009b). Wilting could be of importance because PPO needs oxygen to oxidize diphenols to quinones; presumably, not enough oxygen is present after ensiling in well-sealed silages.…”