Heat Treatment of β-Lactoglobulin:  Structural Changes Studied by Partitioning and Fluorescence

Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of β-lactoglobulin (β-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of β-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated β-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of β-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of β-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4.0 and 9.0. The values of bimolecular quenching rate constant (k q) obtained showed that β-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated β-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of β-LG induced by heat treatment. Keywords: β-Lactoglobulin; heat treatment; aqueous two-phase systems; fluorescence quenching