Radical cross-linked whey protein aggregates as building blocks of non-heated cold-set gels

Abstract

In the present study whey protein aggregates were prepared by a simple method, without the need of heating, through the crosslinking of whey protein in the presence of redox initiators (Ascorbic acid + H2O2) as building blocks of non-heated cold-set gels. As an initial screening, the effects of different pH values (9.0–7.0) and redox initiators content were evaluated for gelation ability of the free radical cross-linked soluble aggregates (FRC-SAs). Then, structural alterations whey proteins in the FRC-SAs were investigated by different techniques including SDS-polyacrylamide gel electrophoresis, free sulfhydryl determination, fluorescence spectroscopy, surface hydrophobicity measurement, dynamic light scattering (DLS), circular dichroism (CD), and atomic force microscopy (AFM). Results indicated that disulfide bonds are responsible for the formation of the intermediate aggregates produced by the redox initiators, where alkaline pH led to the more extensive formation of these disulfide based mediated-aggregates. CD and fluorescence spectroscopy data suggested not only conformational alterations in the secondary structure of whey proteins are involved in the aggregation process but also changes in tertiary structure have a significant role. DLS and AFM results revealed that most of the small aggregates sticked together fo form more weighty particles, confirming free radical-triggered aggregation of proteins. Furthermore, it was observed that cross-linking at higher pH increased significantly the strength of resulting gels (P < 0.05). It is shown that electrostatic and hydrophobic interactions played a critical role in gel network formation from non-heated whey protein during acidifying step by glucono-d-lactone (GDL).