Abstract

The co-existence of proteins, lipids and riboflavin (RF) in milk together with the harsh conditions encountered during processing (e.g. high temperatures, light exposure) results in oxidative damage. Proteins represent -30 % of the dry mass of milk, with caseins accounting for -80 % (28 g L-1). Due to their high abundance and amphiphilic nature, caseins are targets for both hydrophilic and lipophilic oxidants. Although caseins are key milk components, and highly abundant, most previous work has employed non-biological dilute solutions. In this work we have investigated oxidative modification of alpha s-, beta- and kappa-caseins elicited by AAPH-derived oxygenated radicals, or RF-mediated photo-oxidation, at both low and high protein concentrations, to determine whether and how oxidative damage and resulting structural modifications are modulated by the protein concentration. The data obtained demonstrate that the pathways leading to casein modification are dependent on both the protein concentration and the oxidant employed. AAPH-mediated oxidation was more efficient than RF-induced photo-oxidation, in respect to the number of moles of amino acid side-chains consumed per mole of oxidant generated, the extent of damage detected by SDS-PAGE, and immunoblot detection of oxidation products. Quantification of amino acid consumption and product generation, using UPLC and LC/MS, demonstrates the occurrence of short chain reactions, with the chain-length dependent on the protein concentration. LC/MS peptide mass mapping analyses provide data on the sites of modification. Molecular crowding, arising from high casein concentrations and casein-casein interactions, therefore favors the occurrence of radical chain events that enhance the extent of protein oxidative damage.