Covalent Modification of Emulsified beta-Casein Resulting from Lipid Peroxidation

Competitive displacement of adsorbed protein from emulsion droplets by the surfactant Tween 20 has been used to determine the influence of the oil phase and aging on the behavior of beta-casein, the displaced protein being analyzed by reverse-phase high performance liquid chromatography HPLC. Unlike soluble beta-casein or the protein displaced from tetradecane droplets where aging had no effect on the appearance of the HPLC profile, the protein displaced from a soya oil emulsion interface was observed to change. As the soya oil emulsion aged, the retention time of the protein decreased. Mass spectrometry of the modified protein showed that the molecular weight increased, indicating that some form of covalent modification was occurring. Gas chromatography-mass spectrometry of steam distillates of the samples showed the presence of a variety of aldehydes in microfluidized soya oil samples that were not present in either the original oil or the tetradecane emulsions. Aldehydes, particularly alpha,beta-unsaturated aldehydes (enals), which are the major components formed in these soya oil emulsions, are known to react with nucleophilic amino acid side chains such as lysine. This is the probable cause of the observed modification of the emulsified protein. These aldehydes, whose concentration increased with storage time, are formed by peroxidation of the unsaturated fatty acyl chains present in the soya oil as a result of the microfluidization process used in the preparation of the emulsions. The tryptic peptide pattern also changed with age due to modification of the primary structure of the protein. Potential consequences of these chemical changes arising as a result of microfluidization are discussed. Copyright 1999 Academic Press.