Limited changes in physical and rheological properties of peroxidase-cross-linked apo-α-lactalbumin after heat treatment

Abstract The current study aims at elucidating the effect of heating on the physical properties of peroxidase-cross-linked (apo) α -lactalbumin ( α -LA) aggregates with hydrodynamic radii ( R H ) of 25 nm – 100 nm. We consider the effects of heating at the single protein level, and at the particle level for both dilute and dense dispersions of the cross-linked α -LA. From far-UV circular dichroism (CD) spectroscopy, we find that changes in the secondary structure of the cross-linked α -LA are limited upon heating but thermoreversible. Cross-linking already leads to a complete disappearance of all tertiary structure, and heating therefore leads to no further changes of tertiary structure as detected by near-UV CD. The surface hydrophobicity of the cross-linked α -LA, as probed using a fluorometric assay (ANSA), remains unchanged upon heating, whereas heating leads to a significant increase of surface hydrophobicity for uncross-linked α -LA. Prolonged heating leads to a moderate decrease of the particle size of α -LA aggregates, while heating does not affect its electrophoretic mobility. As a consequence of the insensitivity of the physical properties of the cross-linked α -LA to heating, we find that dilute dispersions of the cross-linked α -LA are stable against heat-induced aggregation over a wide range of pH values and salt concentrations. Furthermore, for dense dispersions of α -LA aggregates, that form physical hydrogels, we find that heating has a very limited impact on the rheology of the hydrogels. Our results may be of importance in designing heat-insensitive protein ingredients for novel food formulations.