Heat-induced aggregation and covalent linkages in β-casein model systems

Conditions and mechanisms leading to covalent aggregation of beta-casein (beta CN) studied in protein solutions heat treated in the absence or presence of glucose. Under all the tested heating conditions, covalent aggregation of beta CN occurred only in the presence of glucose. Only high-MW aggregates (much greater than 100 kDa) were detectable by polyacrylamide-gel electrophoresis in the presence of SDS, whereas gel permeation chromatography in the presence of urea showed that aggregates with lower MW were formed as well. A characteristic unordered structure was observed using transmission electron microscopy for the covalent aggregates of beta CN obtained in the presence of glucose, in contrast to the mostly spherical ones due to hydrophobic interactions only. In addition, lysinoalanine (LAL), lysylpyrraline (LPA) and pentosidine (PTD), as possible crosslinking molecules in proteins, were quantified by HPLC. The highest amounts of LAL (similar to 150 mmol/mol beta CN) were found in the unglycosylated beta CN, suggesting that this molecule is mainly involved in intra-molecular reactions. Very small amounts of LPA (< 2 mu mol/mol beta CN) were found in extensively aggregated beta CN. The behaviour of PTD formation followed that of beta CN aggregation but the low values found (few mmol/mol of protein) suggest that it can only play a minor role. Although the covalent aggregation of beta CN is advanced Maillard reaction dependent, molecules other than those considered here are responsible for intermolecular crosslinking but their nature is presently unknown. (C) 1999 Elsevier Science Ltd. All rights reserved.