Protection of emulsified polyunsaturated fatty acids against in vitro ruminal biohydrogenation by polyphenol oxidase: Characterization of the cross-linked emulsion

In the current study, linseed oil emulsions were cross-linked by polyphenol oxidase to study the effect of emulsion characteristics on the protection of polyunsaturated fatty acids against biohydrogenation by rumen bacteria in vitro. Polyphenol oxidase (PPO) catalyzes the oxidation of diphenolic substrate to o-quinones which are reactive towards amino- and thiol- groups on proteins, forming a complex of covalently bound protein cross-linked by protein-bound phenols (PBP). Such cross-linking of protein-stabilized emulsions was shown to yield high in vitro ruminal protection of polyunsaturated fatty acids before, by addition of PPO extract and diphenolic substrate to protein-stabilized emulsions. The current study examined the relationship between characteristics of cross-linked emulsions and in vitro ruminal protection by evaluation of two hypotheses: protection efficiency would be impaired when 1) the amount of PPO in the emulsion decreased or 2) the amount of non-adsorbed protein in the emulsion increased. Hence, in a first experiment, emulsions containing the same protein concentration and varying amounts of PPO extract (0,2-0,8 g/g) were analyzed, while in experiment 2, emulsions containing varying amounts of non-adsorbed protein and a similar amount of PPO extract (0,8 g/g) were investigated. In the latter experiment, variation in non-adsorbed protein was obtained by altering the emulsifier concentration (10 or 0 mg/g whey protein isolate) and/or removal of the majority (>90%) of the non-adsorbed protein after emulsion preparation, i.e. washing of the emulsion, before addition of PPO extract. In vitro ruminal biohydrogenation was evaluated after 24 h in a rumen-simulated environment containing rumen inoculum. Results of experiment 1 showed protection efficiency decreased for decreasing amounts of PPO extract. Additionally, in vitro ruminal protection was found to be positively correlated to mean droplet size, protein load and proportion of PBP in the free protein fraction of the emulsions after cross-linking, while proportion of PBP in the adsorbed protein layer did not differ between treatments. In experiment 2, an increase in emulsifier concentration from 10 to 50 g/g WPI lowered the amount of PBP in both protein fractions. Washing of 50 mg/g WPI emulsions, enhanced in vitro ruminal protection of 18:3n-3 and 18:2n-6 and increased the proportion of PBP in both free and adsorbed protein. Moreover, protein load and the amount of total adsorbed PBP were positively affected by washing. Comparison of emulsions with similar protein load between experiments showed in vitro ruminal protection efficiency increased for higher proportions of PBP in the adsorbed protein layer. Moreover, an excess of PBP on the possible binding positions on the adsorbed protein seemed to promote in vitro ruminal protection efficiency.