Abstract: This study employed an alkali-mediated covalent conjugation strategy to modify whey protein isolate (WPI), aiming to enhance its functional properties. The WPI-rosmarinic acid (RA) conjugate was prepared by covalently binding WPI with RA under alkaline conditions (pH 9.0). The polyphenol binding equivalent was determined using the Folin-Ciocalteu assay, and the covalent conjugation effect was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis via ultraviolet spectroscopy, fluorescence spectroscopy, and Fourier transform infrared spectroscopy (FTIR) revealed significant alterations in the secondary and tertiary structures of WPI after binding to RA, manifested by fluorescence quenching and shifts in characteristic absorption peaks. Functional evaluations demonstrated that compared with WPI, the covalent complexes had significantly enhanced 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging capacities (P < 0.05). Concurrently, the foaming capacity of the complexes was significantly improved, and the foam stability increased by approximately 40%. This study confirms that covalent modification with RA effectively modulates the structure and functional properties of WPI, providing theoretical support for developing novel functional ingredients based on protein-polyphenol complexes and holding significant value for expanding the application of whey protein in high-protein functional foods.

Key words: whey protein isolate; rosmarinic acid; covalent binding; protein functional properties