Abstract: Objective: To evaluate the effect of preparation methods on the structural and functional properties of whey protein isolate (WPI)-chlorogenic acid (CA) conjugates. Methods: The methods tested were an alkali method, a free radical method and an enzymatic method. The reactive group content, CA amount of covalent binding, molecular structure, surface hydrophobicity, thermal stability, antioxidant activity and emulsion properties of WPI-CA conjugates were analyzed. Results: Free amino groups, tryptophan and sulfhydryl groups all participated in the grafting reaction with free amino group being the major reactive group. The CA amounts of covalent binding of the conjugates prepared by the alkali, free radical and enzymatic methods were (52.70 ± 1.81), (42.57 ± 1.85), and (63.75 ± 2.50) μmol/g, respectively. The conjugates showed less α-helix relative content, higher random coil relative content and lower fluorescence intensity than WPI. Covalent bonding of CA induced by the alkali and enzymatic methods decreased the surface hydrophobicity and increased the thermal stability of WPI, while the free radical method led to the opposite results. The emulsion properties and antioxidant activity of the conjugates were higher than that of WPI, and the antioxidant activity was positively proportional to the amount of bonded CA. Conclusion: The highest grafting efficiency was obtained by the enzymatic method, and the prepared conjugates showed best thermal stability and antioxidant activity. This study can provide a reference for the preparation and application of protein-polyphenol conjugates as antioxidant carrier materials.

Key words: whey protein isolate; chlorogenic acid; covalent complex; preparation methods; structure; functional properties