Modifications of Interfacial Proteins in Oil-in-Water Emulsions Prior to and During Lipid Oxidation Claire Berton, † Marie-He ́ le ̀ ne Ropers, Dominique Guibert, Ve ́ ronique Sole ́ , and Claude Genot* INRA, UR1268 Biopolyme ̀ res Interactions Assemblages, F-44316 Nantes, France * S Supporting Information ABSTRACT: Lipid oxidation is a major cause for the degradation of biological systems and foods, but the intricate relationship between lipid oxidation and protein modifications in these complex multiphase systems remains unclear. The objective of this work was to have a spatial and temporal insight of the modifications undergone by the interfacial or the unadsorbed proteins in oil-in-water emulsions during lipid oxidation. Tryptophan fluorescence and oxygen uptake were monitored simultaneously during incubation in different conditions of protein-stabilized oil-in-water emulsions. Kinetic parameters demonstrated that protein modifications, highlighted by decrease of protein fluorescence, occurred as an early event in the sequence of the reactions. They concerned more specifically the proteins adsorbed at the oil/water interface. The reactions led in a latter stage to protein aggregation, carbonylation, and loss of protein solubility. KEYWORDS: lipid oxidation, emulsion, interface, protein, front-surface fluorescence, protein carbonylation, aggregation ■ INTRODUCTION Oxidation of polyunsaturated fatty acids (PUFA) is a chemical reaction which decreases the nutritional and sensory properties of food products. 1 It also contributes to the oxidative stress in vivo. 2-5 In these complex and multicomponent systems, lipid oxidation generally occurs simultaneously with “co-oxidation” phenomena, which affect other molecules such as proteins. 6-8 Protein oxidation is involved in various human diseases and aging 9,10 but also in the degradation of the sensory properties of food products such as texture 8,11,12 and in the loss of protein digestibility. 12-14 Since the 1970s, it is known that the proteins present in foods and food emulsions are susceptible to be attacked by the free radicals, hydroperoxides, and secondary lipid oxidation products as aldehydes, leading to the formation of various reaction products. 6,7 For the last 10 years, the question of the oxidative modifications of proteins in biological systems, food products, and related food models has been identified as an emerging subject. It addresses the nutritional, toxicological, and possibly sensory consequences of protein modifications in vivo and in the food products. 8 Indeed, the intricate radical and non radical mechanisms involving both lipid oxidation and protein modifications have not been fully elucidated yet. Time dependence between lipid and protein oxidations was observed in multiphase systems. The two phenomena were described as “correlated”, 15 “concomitant”, 16 or “simultane- ous”. 17 Lund et al. also reported a timely coincidence between lipid and protein oxidation in muscle foods. 12 It seems therefore obvious that lipid and protein oxidation are linked, but it is difficult to figure out which of the phenomena first starts. On the one hand, proteins present in food emulsions are susceptible to be attacked by the free radicals, hydroperoxides, and secondary products as aldehydes resulting from lipid oxidation, leading to the formation of various reaction products. 6,7,15,18,19 On the other hand, fatty acid oxidation can be induced by bovine serum albumin (BSA) radicals 20 and amino acid residues of β-lactoglobulin (BLG) were oxidized prior to the propagation of lipid oxidation in O/W emulsion. 21 The location of proteins and their respective concentrations in one or in the other phase have also to be taken in account when oxidation in multiphase systems is considered. In O/W emulsions, proteins are either adsorbed at the interface surrounding the oil droplets or, once the emulsifiers have covered the interface, unadsorbed in the aqueous phase. Rampon et al. revealed by front-surface fluorescence measure- ments that during oxidation of BSA-stabilized O/W emulsions unadsorbed BSA was by far less modified than BSA adsorbed onto the oil droplets. 22 It was also repeatedly found that when the concentration of unadsorbed proteins increased, lipid oxidation slowed down. 23-26 However, these studies explain only partially the links between the protein location within multiphase systems, lipid oxidation, and protein modifications. To have a better insight on the relationship between protein and lipid oxidation in protein-stabilized emulsions, we have determined the extent and kinetics of modifications of interfacial and unadsorbed proteins in oxidizing oil-in-water emulsions in relation to lipid oxidation. Lipid oxidation and protein modifications undergone by the interfacial and the unadsorbed proteins were assessed in O/W emulsions stabilized by either BLG, β-casein (BCN), or BSA with limited amounts of proteins remaining in the aqueous phases. The oxidizing conditions were the same as applied in our previous work. 23,27 ■ MATERIALS AND METHODS Materials. Rapeseed oil was purchased in a local supermarket. It was stripped by means of alumina (MP Alumina N-Super I, MP Received: February 4, 2012 Revised: June 24, 2012 Accepted: August 14, 2012 Published: August 14, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 8659 dx.doi.org/10.1021/jf300490w | J. Agric. Food Chem. 2012, 60, 8659-8671