Protection of polyunsaturated oils against ruminal biohydrogenation and oxidation during storage using a polyphenol oxidase containing extract from red clover.

Polyunsaturated fatty acid (PUFA) are to a large extent subject to biohydrogenation in a ruminal environment, which results to the healthy value of these PUFA being lost upon dietary addition to ruminants. PUFA are also prone to lipid oxidation upon storage. Therefore, it was tested whether emulsions could be protected against in vitro ruminal biohydrogenation and oxidation during storage by using protein extracts rich in polyphenol oxidase, an enzyme responsible for browning of plant tissues. PUFA rich emulsions were made with a protein extract from red clover (Trifolium pratense L.) before adding a synthetic diphenol (4-methylcatechol) to induce protection. Results after in vitro incubation confirmed the hypothesis and indicated the potential to prevent PUFA in linseed or fish oil from ruminal biohydrogenation and oxidation during storage through addition of 4-methylcatechol to the emulsions. Protection depended on the amount of oil present and protein concentrations in the emulsions. Protection efficiency increased with increasing the amounts of diphenol present in the emulsion per unit interfacial surface area. It is suggested that protection is caused by an effective encapsulation by cross-linking of the protein layer at the emulsion interface. For the first time, a method is described to protect PUFA using an enzyme abundantly available in nature, polyphenol oxidase, in combination with 4-methylcatechol.

Lipid metabolism in mixtures of red clover (Trifolium repens) and perennial ryegrass (Lolium perenne) in lab scale silages and in vitro rumen incubations.

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.

Red clover polyphenol oxidase and lipid metabolism.

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.

Monitoring of vitamin E status of dry, early and mid-late lactating organic dairy cows fed conserved roughages during the indoor period and factors influencing forage vitamin E levels.

Analysis of blood plasma of 60 cows selected on six Flemish organic dairy farms revealed that on average 12% of all samples and on one farm up to 28% of the organic cows showed plasma vitamin E concentrations less than 3.0 µg/ml, which is considered the minimum level to avoid health risks due to vitamin E shortages. Furthermore, this study showed early lactating and dry cows to be more at risk in relation to animals in mid-late lactation. In European organic farming, vitamin supplements are only allowed if granted by the local authority to satisfy daily requirements. Therefore, the vitamin E content of the feedstuffs used on the farms was determined. Grass clover silage (GCS) and mixed silage had significantly more vitamin E than hay, maize or grain (p < 0.05) [mean (SD): 52 (35), 29 (20), 4.5 (1.7), 4.9 (4.4) and 7.1 (3.8) mg/kg DM, respectively]. Apparently, variation in the vitamin E content in the silage samples was huge. Hence, the vitamin E content of ryegrass, white and red clover was determined in a second lab scale experiment and the effects of wilting, DM content and supplementation of ensiling additives were investigated. Fresh ryegrass had a higher vitamin E content than white and red clover (p < 0.05) [156 (11.3), 49.3 (0.67) and 74.3 (5.73) µg/g DM, respectively]. These differences remained after the wilting or ensiling. Supplementation of formic acid or lactic acid bacteria at ensiling had no significant effect on the vitamin E content. Overall, it can be concluded that GCS is the most important source of vitamin E in organic dairy farming. A legal possibility for case-related supplementation should be retained in organic dairy farming as approximately 18% of all dry and early lactating cows were at risk of vitamin E shortage.

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

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.

In vitro study of red clover polyphenol oxidase activity, activation, and effect on measured lipase activity and lipolysis.

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).

Effect of grazing pastures with different botanical composition by lambs on rumen fatty acid metabolism and fatty acid pattern of longissimus muscle and subcutaneous fat.

In order to study the effect of grazing pastures with a different botanical composition on rumen and intramuscular fatty acid metabolism, 21 male lambs were assigned to three botanically different pastures: botanically diverse (BD) (consisting for 65% of a variety of grass species); Leguminosa rich (L) (consisting for 61% of Leguminosae) and intensive English ryegrass (IR) (with 69% Lolium perenne). Pastures were sampled weekly for 12 weeks for analysis of their fatty acid content and composition and on nine occasions to determine the botanical composition. Ruminal and abomasal contents were sampled at slaughter and muscle and subcutaneous fat 24 h after slaughter. All samples were prepared and analysed for fatty acid composition. The L pasture showed a higher fatty acid content (29.8 mg/g dry matter (DM) v. 18.5 and 25.5 mg/g DM, for BD and IR pastures, respectively), but the sum of the proportions of the major polyunsaturated fatty acids, C18:2 n-6 and C18:3 n-3, were similar for the three pastures (69.9, 69.4 and 71.1% of fatty acids methyl esters (FAME) for BD, L and IR pastures, respectively). The BD pasture was richer in C18:2 n-6 (18.2% of FAME), while IR pasture had a higher C18:3 n-3 content (57.2% of FAME). Rumen data showed that animals grazing the BD pasture presented higher proportions of biohydrogenation intermediates, mainly C18:1 t11, C18:2 t11c15 and CLA c9t11, suggesting an inhibition of biohydrogenation. These changes were associated with shifts in the rumen microbial population as indicated by differences in the rumen pattern of volatile fatty acids, microbial odd- and branched-chain fatty acids. In L pasture animals, the content of C18:2 n-6 and C18:3 n-3 in the abomasum and subcutaneous fat was higher. Finally, higher proportions of C20:4 n-6, C20:5 n-3 and C22:5 n-3 and higher indices for elongation and desaturation activity in the intramuscular fat of BD grazing animals suggest some stimulation of elongation and desaturation of long-chain fatty acids, although this also might have been provoked partially by reduced fat deposition (due to a lower growth rate of the animals).