Polyphenol oxidase activity in grass and its effect on plant‐mediated lipolysis and proteolysis of Dactylis glomerata (cocksfoot) in a simulated rumen environment

Abstract

Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g −1 fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant-mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic-containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 • C over five time points. At each time point (0, 1, 2, 6 and 24 h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g −1 FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24 h of incubation, namely 65.1 and 29.6 mg bound phenol g −1 protein (P < 0.001) for cocksfoot and cocksfoot + ascorbate respectively. Proteolysis, measured as free amino acids released into the incubation buffer, was significantly reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.03 and 0.07 mmol L −1 g −1 FW for cocksfoot and cocksfoot + ascorbate respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.43 and 0.65 for cocksfoot and cocksfoot + ascorbate respectively. Changes that occurred in protein and the lipid fractions (polar fraction, monoacylglycerol + diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid losses in silo and potentially in the rumen. INTRODUCTIONThe enzyme polyphenol oxidase (PPO) is a copper metalloprotein which catalyses both the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones. 1 The PPO-generated o-quinones are highly reactive, electrophilic molecules which covalently modify and crosslink a variety of nucleophilic cellular constituents, such as proteins, amines and amides, leading to the formation of melanin pigments. 2 These addition products may be further oxidised to their respective quinines, and a second addition may occur resulting in the formation of crosslinked protein complexes. It has been demonstrated that such protein-phenol complexes are resistant to enzymatic digestion by proteases including trypsin, α-chymotrypsin and pepsin. 3 Lee et al. 4 have shown that the PPO enzyme can reduce the extent of both plant-mediated proteolysis and

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

“…The greater level of PPO activity in the fresh crop (not measured) resulted in greater levels of PBP in RC compared to PR and CO (Winters and Minchin, 2001). The difference between CO and PR in terms of PBP was small despite the greater reported level of PPO activity in CO compared to PR (Lee et al, 2006b;Marita et al, 2010).…”

“…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 ).…”

“…Lee et al (2006b) screened 6 grass species for PPO activity and noted activity in cocksfoot (Dactylis glomerata) comparable to red clover. They also showed in vitro that grass PPO resulted in a reduction in plantmediated proteolysis and lipolysis similar to that previously observed in red clover.…”

Red clover polyphenol oxidase and lipid metabolism

“…Often, not the same PPO activity is measured as activation and degradation of the enzyme can vary over extraction and measuring methods. Differences in procedure could, for instance, explain the lack of activity in ryegrass when measured as described by Van Ranst et al (2009a), contrary to Lee et al (2006b), who reported a significant activity.…”

“…Thus, lipolysis was reduced to a higher extent when bacterial activity was inhibited by formic acid addition Figure 3 Lipolysis during incubation expressed relative to a lipolysis in a control during incubation for 6 to 8 h, depending on the study. A, B and C represent undamaged, crushed or frozen/thawed red clover, respectively, after 24 h wilting incubated in a buffer with ascorbic acid with fresh red clover as a control; D (Van Ranst, 2009) and E (Lee et al, 2004) are red clover mixed in a buffer without ascorbic acid, with ryegrass and low polyphenol oxidase red clover as a control, respectively; F (Lee et al, 2007) and G (Lee et al, 2006b) correspond with red clover and cocksfoot without ascorbic acid compared with ascorbic acid as a control. Figure 4 Relative lipolysis during ensiling of red clover; A represents lipolysis as a mean of three treatments, which did not significantly differ red clover crushed and wilted for 4 h and crushed or undamaged and wilted for 24 h (A); red clover frozen/thawed and wilted for 4 (B) or 24 h (C), expressed relative to not damaged 4 h wilted red clover (Van Ranst et al, 2010).…”