Formation of Non-Native β-Lactoglobulin during Heat-Induced Denaturation

A mechanism describing the denaturation and aggregation behavior during heat-treatment of pure beta-lactoglobulin and beta-lactoglobulin in whey protein isolate (WPI) under selected conditions (20 to 90 gL(-1) in water at pH 7.0, 78 degrees C) is presented. A combination of reversed-phase and gel permeation chromatography was used to study the disappearance of native beta-lactoglobulin and the formation of non-native intermediates in the aggregation process. The mean reaction order for pure beta-lactoglobulin and beta-lactoglobulin in WPI were the same, 1.4. While the rate of beta-lactoglobulin denaturation was greater in WPI there was less aggregation compared to that of pure beta-lactoglobulin. More of the beta-lactoglobulin in WPI remained in a non-native monomer intermediate state after 30 min of heating. After an initial lag period, during which non-native monomers appeared, aggregates formed and rapidly reached a plateau in terms of their size. These aggregates were visualized using atomic force microscopy. There was no significant effect of protein concentration on either aggregate size or the number of exposed sulfhydryls in the heated solutions.