Physical Modification of Whey Protein by Interacting with Methyl Hesperidin: Impacts on Antioxidant Activity and Underlying Mechanism

Simple SummaryWhey protein is a widely utilized food ingredient known for its favorable nutritional profile and functional properties. Nevertheless, its inherent antioxidant capacity is relatively limited, which restricts its application as a natural antioxidant in the food industry. This study aims to investigate an environmentally friendly method to enhance the antioxidant activity of whey protein. In this process, a natural flavonoid derivative, methyl hesperidin, was introduced into a whey protein solution. Through physical mixing, it was observed that the two components could spontaneously bind to form a non-covalent complex. Furthermore, the microenvironment of amino acid residues in whey protein was altered with changed conformation, leading to enhancement in its antioxidant capacity. Additionally, molecular docking studies revealed that methyl hesperidin inserted itself into the hydrophobic cavity of the whey protein. These findings not only propose a novel approach for developing healthier antioxidants but also contribute to a deeper understanding of the interaction mechanisms between proteins and flavonoids.In this work, the possibility of enhancing the antioxidant capacity of whey protein (WP) through non-covalent interaction with methyl hesperidin (MH, a hesperidin derivative) was assessed. The underlying mechanism was analyzed in terms of multi-spectroscopy methods, thermodynamic analysis, and molecular docking simulation. The data indicated that MH could spontaneously bind to WP and form a non-fluorescent complex when physically mixed together. The presence of MH statically quenched the intrinsic fluorescence of WP, changed the microenvironment of amino acid residue, and altered the secondary and tertiary structure of WP, which in turn enhanced the antioxidant capacity of WP. The underlying mechanism may be assigned to hydrophobic interactions, which promoted MH inserting itself into the hydrophobic cavity in WP. The methoxy group on the B ring of MH may form hydrogen bonds with amino acids, which enhances the freedom of the phenyl hydroxyl group, resulting in higher antioxidant capacity than other hesperidin structural analogs. This research would enrich the theoretical basis about the interaction between protein and hesperidin-based derivatives, and it may supply valuable information for its application in the food and medicine fields.