Interaction between flavonoids and β-Lactoglobulin and their antioxidant capacity under the regulation of the number of phenolic hydroxyl groups

We investigated the mechanism by which the number of phenolic hydroxyl groups in flavonoids regulates their interactions with β-lactoglobulin in food systems. Kaempferol, quercetin, and myricetin were selected as model flavonoids with increasing number of hydroxyl groups. Spectroscopy revealed that increasing the hydroxyl content enhanced protein fluorescence quenching, promoted β-sheet formation, and induced a more compact protein conformation (39.7–42.6 %). Thermodynamic results demonstrated that hydrogen bonding dominated the binding process and that the binding affinity increased with hydroxyl number (binding constant: 9.55 to 36.31 × 10 6  L·mol −1 ). Kinetic analysis showed that a higher hydroxyl content reduced the activation energy (from 40.9 to 25.4 kJ·mol −1 ), allowing flavonoid–protein binding to be completed within 5 s. Flavonoid-induced protein compaction improved the emulsion stability and antioxidant performance, depending on hydroxyl number. These findings reveal a structure–interaction–function relationship and provide guidance for designing flavonoid–protein delivery systems in food applications.