Proteolysis of bovine β‐lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature‐modified protein and yields fragments with low immunoreactivity

Bovine β‐lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 °C at neutral pH. At these temperatures β‐lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, β‐lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High‐temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large‐sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of β‐lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of β‐lactoglobulin hydrolysis were assessed by using various β‐lactoglobulin‐specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.