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Promising perspectives for ruminal protection of polyunsaturated fatty acids through polyphenol-oxidase-mediated crosslinking of interfacial protein in emulsions

  • N. De Neve (a1), B. Vlaeminck (a1), F. Gadeyne (a1), E. Claeys (a1), P. Van der Meeren (a2) and V. Fievez (a1)...


Previously, polyunsaturated fatty acids (PUFA) from linseed oil were effectively protected (>80%) against biohydrogenation through polyphenol-oxidase-mediated protein crosslinking of an emulsion, prepared with polyphenol oxidase (PPO) extract from potato tuber peelings. However, until now, emulsions of only 2 wt% oil have been successfully protected, which implies serious limitations both from a research perspective (e.g. in vivo trials) as well as for further upscaling toward practical applications. Therefore, the aim of this study was to increase the oil/PPO ratio. In the original protocol, the PPO extract served both an emulsifying function as well as a crosslinking function. Here, it was first evaluated whether alternative protein sources could replace the emulsifying function of the PPO extract, with addition of PPO extract and 4-methylcatechol (4MC) to induce crosslinking after emulsion preparation. This approach was then further used to evaluate protection of emulsions with higher oil content. Five candidate emulsifiers (soy glycinin, gelatin, whey protein isolate (WPI), bovine serum albumin and sodium caseinate) were used to prepare 10 wt% oil emulsions, which were diluted five times (w/w) with PPO extract (experiment 1). As a positive control, 2 wt% oil emulsions were prepared directly with PPO extract according to the original protocol. Further, emulsions of 2, 4, 6, 8 and 10 wt% oil were prepared, with 80 wt% PPO extract (experiment 2), or with 90, 80, 70, 60 and 50 wt% PPO extract, respectively (experiment 3) starting from WPI-stabilized emulsions. Enzymatic crosslinking was induced by 24-h incubation with 4MC. Ruminal protection efficiency was evaluated by 24-h in vitro batch simulation of the rumen metabolism. In experiment 1, protection efficiencies were equal or higher than the control (85.5% to 92.5% v. 81.3%). In both experiments 2 and 3, high protection efficiencies (>80%) were achieved, except for emulsions containing 10 wt% oil emulsions (<50% protection), which showed oiling-off after enzymatic crosslinking. This study demonstrated that alternative emulsifier proteins can be used in combination with PPO extract to protect emulsified PUFA-rich oils against ruminal biohydrogenation. By applying the new protocol, 6.5 times less PPO extract was required.


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Present address: Research Group Marine Biology, Department of Biology, Faculty of Sciences, Ghent University, Krijgslaan 281-S8, B-9000 Gent, Belgium.



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Ackman, RG and Sipos, JC 1964. Application of specific response factors in the gas chromatographic analysis of methyl esters of fatty acids with flame ionization detectors. Journal of the American Oil Chemists Society 41, 377378.
Ambrose, DJ, Kastelic, JP, Corbett, R, Pitney, PA, Petit, HV, Small, JA and Zalkovic, P 2006. Lower pregnancy losses in lactating dairy cows fed a diet enriched in α-linolenic acid. Journal of Dairy Science 89, 30663074.
Baumann, E, Chouinard, PY, Lebeuf, Y, Rico, DE and Gervais, R 2016. Effect of lipid supplementation on milk odd- and branched-chain fatty acids in dairy cows. Journal of Dairy Science 99, 63116323.
Dickinson, E 1999. Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology. Colloids and Surfaces B: Biointerfaces 15, 161176.
Elis, S, Freret, S, Desmarchais, A, Maillard, V, Cognié, J, Briant, E, Touzé, J-L, Dupont, M, Faverdin, P, Chajès, V, Uzbekova, S, Monget, P and Dupont, J 2016. Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science 164, 121132.
El-Sherbiny, M, Cieślak, A, Szczechowiak, J, Kołodziejski, P, Szulc, P and Szumacher-Strabel, M 2016. Effect of nanoemulsified oils addition on rumen fermentation and fatty acid proportion in a rumen simulation technique. Journal of Animal and Feed Sciences 25, 116124.
Ercili Cura, D, Lille, M, Partanen, R, Kruus, K, Buchert, J and Lantto, R 2010. Effect of Trichoderma reesei tyrosinase on rheology and microstructure of acidified milk gels. International Dairy Journal 20, 830837.
Fievez, V, Vlaeminck, B, Jenkins, T, Enjalbert, F and Doreau, M 2007. Assessing rumen biohydrogenation and its manipulation in vivo, in vitro and in situ . European Journal of Lipid Science and Technology 109, 740756.
Friedman, M 1997. Chemistry, biochemistry, and dietary role of potato polyphenols. A review. Journal of Agricultural and Food Chemistry 45, 15231540.
Gadeyne, F, De Neve, N, Vlaeminck, B, Claeys, E, Van der Meeren, P and Fievez, V 2016a. Polyphenol oxidase containing sidestreams as emulsifiers of rumen bypass linseed oil emulsions: interfacial characterization and efficacy of protection against in vitro ruminal biohydrogenation. Journal of Agricultural and Food Chemistry 64, 37493759.
Gadeyne, F, De Neve, N, Vlaeminck, B, Van Der Meeren, P and Fievez, V 2016b. Transfer to the milk of rumen bypass CLA emulsions created by potato tuber peel polyphenol oxidase. Proceedings of the 14th Euro Fed Lipid Congress, 18 to 21 September 2016, Ghent, Belgium, Abstracts, pp. 165–165.
Gadeyne, F, De Neve, N, Vlaeminck, B, Van der Meeren, P and Fievez, V 2017. In vitro post-ruminal digestion of rumen bypass emulsions encapsulated by interfacial crosslinking using polyphenol oxidase from potato tuber peels. Journal of Dairy Science 100 (suppl. 2), 95.
Gadeyne, F, De Ruyck, K, Van Ranst, G, De Neve, N, Vlaeminck, B and Fievez, V 2016c. Effect of changes in lipid classes during wilting and ensiling of red clover using two silage additives on in vitro ruminal biohydrogenation. The Journal of Agricultural Science 154, 553566.
Gadeyne, F, Van Ranst, G, Vlaeminck, B, Vossen, E, Van der Meeren, P and Fievez, V 2015. Protection of polyunsaturated oils against ruminal biohydrogenation and oxidation during storage using a polyphenol oxidase containing extract from red clover. Food Chemistry 171, 241250.
Hassim, HA, Lourenço, M, Goel, G, Vlaeminck, B, Goh, YM and Fievez, V 2010. Effect of different inclusion levels of oil palm fronds on in vitro rumen fermentation pattern, fatty acid metabolism and apparent biohydrogenation of linoleic and linolenic acid. Animal Feed Science and Technology 162, 155158.
Isaschar-Ovdat, S, Davidovich-Pinhas, M and Fishman, A 2016. Modulating the gel properties of soy glycinin by crosslinking with tyrosinase. Food Research International 87, 4249.
Isaschar-Ovdat, S, Rosenberg, M, Lesmes, U and Fishman, A 2015. Characterization of oil-in-water emulsions stabilized by tyrosinase-crosslinked soy glycinin. Food Hydrocolloids 43, 493500.
Lanier, JS and Corl, BA 2015. Challenges in enriching milk fat with polyunsaturated fatty acids. Journal of Animal Science and Biotechnology 6, 2634.
Le Roes-Hill, M, Palmer, Z, Rohland, J, Kirby, BM and Burton, SG 2015. Partial purification and characterisation of two actinomycete tyrosinases and their application in cross-linking reactions. Journal of Molecular Catalysis B: Enzymatic 122, 353364.
Livingstone, KM, Lovegrove, JA and Givens, DI 2012. The impact of substituting SFA in dairy products with MUFA or PUFA on CVD risk: evidence from human intervention studies. Nutrition Research Reviews 25, 193206.
Lock, AL and Bauman, DE 2004. Modifying milk fat composition of dairy cows to enhance fatty acids beneficial to human health. Lipids 39, 11971206.
Ma, H, Forssell, P, Partanen, R, Buchert, J and Boer, H 2011. Improving laccase catalyzed cross-linking of whey protein isolate and their application as emulsifiers. Journal of Agricultural and Food Chemistry 59, 14061414.
Macierzanka, A, Bordron, F, Rigby, NM, Mills, ENC, Lille, M, Poutanen, K and Mackie, AR 2011. Transglutaminase cross-linking kinetics of sodium caseinate is changed after emulsification. Food Hydrocolloids 25, 843850.
Maier, C, Ensenberger, S, Irmscher, SB and Weiss, J 2016. Glutaraldehyde induced cross-linking of oppositely charged oil-in-water emulsions. Food Hydrocolloids 57, 221228.
Maier, C, Oechsle, AM and Weiss, J 2015. Cross-linking oppositely charged oil-in-water emulsions to enhance heteroaggregate stability. Colloids and Surfaces B: Biointerfaces 135, 525532.
Marengo, M, Miriani, M, Ferranti, P, Bonomi, F, Iametti, S and Barbiroli, A 2016. Structural changes in emulsion-bound bovine beta-lactoglobulin affect its proteolysis and immunoreactivity. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1864, 805813.
Mayer, AM 2006. Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry 67, 23182331.
Osborne, VR, Radhakrishnan, S, Odongo, NE, Hill, AR and McBride, BW 2008. Effects of supplementing fish oil in the drinking water of dairy cows on production performance and milk fatty acid composition1. Journal of Animal Science 86, 720729.
Palmquist, DL 2009. Omega-3 fatty acids in metabolism, health, and nutrition and for modified animal product foods. The Professional Animal Scientist 25, 207249.
Ribeiro, ES 2018. Lipids as regulators of conceptus development: implications for metabolic regulation of reproduction in dairy cattle. Journal of Dairy Science,, Published online by American Dairy Science Association 22 November 2017.
Sato, ACK, Perrechil, FA, Costa, AAS, Santana, RC and Cunha, RL 2015. Cross-linking proteins by laccase: effects on the droplet size and rheology of emulsions stabilized by sodium caseinate. Food Research International 75, 244251.
Shingfield, KJ, Bonnet, M and Scollan, ND 2013. Recent developments in altering the fatty acid composition of ruminant-derived foods. Animal 7, 132162.
Thalmann, C and Lötzbeyer, T 2002. Enzymatic cross-linking of proteins with tyrosinase. European Food Research and Technology 214, 276281.
Winters, AL and Minchin, FR 2005. Modification of the Lowry assay to measure proteins and phenols in covalently bound complexes. Analytical Biochemistry 346, 4348.
Wolf, WJ 1993. Sulfhydryl content of glycinin: effect of reducing agents. Journal of Agricultural and Food Chemistry 41, 168176.
Wolff, RL, Bayard, CC and Fabien, RJ 1995. Evaluation of sequential methods for the determination of butterfat fatty acid composition with emphasis on trans-18:1 acids. Application to the study of seasonal variations in french butters. Journal of the American Oil Chemists’ Society 72, 14711483.
Xu, R, Teng, Z and Wang, Q 2016. Development of tyrosinase-aided crosslinking procedure for stabilizing protein nanoparticles. Food Hydrocolloids 60, 324334.
Yang, M, Liu, F and Tang, C-H 2013. Properties and microstructure of transglutaminase-set soy protein-stabilized emulsion gels. Food Research International 52, 409418.
Zeeb, B, Beicht, J, Eisele, T, Gibis, M, Fischer, L and Weiss, J 2013. Transglutaminase-induced crosslinking of sodium caseinate stabilized oil droplets in oil-in-water emulsions. Food Research International 54, 17121721.
Zeeb, B, Fischer, L and Weiss, J 2014. Stabilization of food dispersions by enzymes. Food & Function 5, 198213.


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