Structural and functional characterization of whey protein isolate-polyphenol covalent conjugates prepared via electrochemical oxidation, alkaline treatment, and free radical grafting.

Covalent conjugates of whey protein isolate (WPI) with caffeic acid (CA), chlorogenic acid (CGA), and rosmarinic acid (RA) were prepared via alkaline treatment (A), free radical grafting (F), and electrochemical oxidation (E) to investigate the effects of preparation method on their structural and functional properties. A-WPI-CGA had the highest phenolic content (61.55 ± 0.69 mg/g), A-WPI-RA showed the lowest free sulfhydryl group content (2.96 ± 0.13 μmol/g), and F-WPI-CGA showed the highest amino group consumption (316.46 ± 6.47 μmol/g). The A-WPI-polyphenol and E-WPI-polyphenol conjugates exhibited significantly lower surface hydrophobicity, smaller psizes and higher surface charges than F-WPI-polyphenol and WPI. FTIR analysis revealed blue shifts in amide A and I band for all conjugates. E-WPI-CA exhibited the best emulsifying properties (emulsifying activity index of 207.42 ± 7.33 m2/g and emulsifying stability index of 73.63 ± 2.59%), while RA-conjugates showed the highest antioxidant activity across all methods, as indicated by ABTS scavenging (71.52 ± 1.19%-95.79 ± 0.54%) and ferric reducing antioxidant power assays (92.99 ± 0.46-127.17 ± 1.66 μmol Trolox/g sample). In the conjugates prepared by the three covalent methods, cysteine, lysine, histidine, arginine, and tryptophan residues in β-lactoglobulin and α-lactalbumin, which are the main protein components of WPI, were identified as the primary sites for covalent interaction with RA. These findings elucidate the method dependent structural and functional characteristics of WPI-polyphenol covalent conjugates and provide guidance for the rational design of protein based antioxidant emulsifiers.