LAUSR

In whole milk, riboflavin, lipids and proteins coexist. When exposed to light or other stressors, excited states of riboflavin (3RF) and lipid-derived peroxyl radicals are generated, which can induce oxidation and crosslinking of caseins, the major milk proteins. The nature of the crosslinks, and the involvement of di-tyrosine (diTyr) and di-tryptophan (diTrp) in these changes is unknown. In this study, we have examined changes in the structure and composition of α- and β- caseins elicited by 3RF and peroxyl radicals (ROO•) derived from thermolysis of AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride). SDS-PAGE, western blotting, fluorescence and UPLC methodologies were employed. Exposure of caseins to AAPH and 3RF resulted in extensive monomer consumption and high levels of protein cross-linking. Trp and Tyr residues were consumed, with the extent of formation of di-Tyr and diTrp dependent on the nature of the oxidant and presence of O2. With 3RF, diTyr and diTrp were primarily generated under an N2-atmosphere, with alternative pathways occurring in the presence of O2. In contrast, with ROO• low yields of diTyr and diTrp were detected, with alternative pathways likely to involve carbonyls and other oxidation products of Trp, Tyr and His mediating protein crosslinking.