Heat-Induced Aggregation of β-Lactoglobulin:  Role of the Free Thiol Group and Disulfide Bonds

The heat-induced aggregation of bovine β-lactoglobulin, dispersed in water at neutral pH and in different concentrations (10, 30, or 50 g of dry matter/L), was studied at 65 °C, and the results are related to a kinetic model. Native PAGE and SDS−PAGE analysis under nonreducing and reducing conditions showed that on heating disulfide-linked aggregates were formed and that the average size of these aggregates increased with increasing initial β-lactoglobulin concentration. In the presence of the thiol-blocking agent N -ethylmaleimide (NEM), at a molar ratio of NEM/β-lactoglobulin monomer of 1, all thiol groups were blocked and no disulfide-linked aggregates were formed, although with native PAGE high molecular mass noncovalently linked aggregates were observed. The formation of these aggregates accelerated with increasing NEM concentration until a molar ratio of NEM/β-lactoglobulin monomer of 1 was reached. In separate experiments we studied the effect of pH (in the range pH 6.0−8.0) on the aggregation of β-lactoglobulin and related this to the pH dependent reactivity of the thiol group.