Investigation of free radical grafting modification of whey protein isolate by polyphenols with different hydroxyl...

This study selected three structurally similar polyphenols with varying numbers of hydroxyl groups (p-Coumaric acid [pCA], Caffeic acid [CA], and Chlorogenic acid [CGA]) for free radical grafting modification with whey protein isolate (WPI), to investigate the influence of hydroxyl numbers in polyphenols on grafting efficacy with WPI and to explore their reaction mechanisms. Ultraviolet-visible spectroscopy, proton nuclear magnetic resonance spectroscopy and LC-MS/MS results indicate that WPI and polyphenols form WPI-polyphenols conjugates through covalent bonding, with the binding extent being WPI-CGA (58.08 mg/g) > WPI-CA (35.31 mg/g) > WPI-pCA (32.64 mg/g). Meanwhile, compared to WPI-pCA and WPI-CA, WPI-CGA exhibits higher flexibility, antioxidant capacity, and emulsifying activity, along with more pronounced structural changes. Notably, WPI-CGA demonstrated lower surface hydrophobicity than native WPI, whereas WPI-pCA and WPI-CA showed opposite trends. Based on these findings, it is indicating that polyphenols with different numbers of hydroxyl groups exhibit distinct covalent bonding sites on WPI, with more hydroxyl groups resulting in higher reactivity and binding affinity. These findings provide new insights into the radical grafting mechanism of WPI with polyphenols and the controlled preparation of WPI-polyphenol conjugates.