Improving Antioxidant Activity of β-Lactoglobulin by Nature-Inspired Conjugation with Gentisic Acid

Dietary phenolic compounds display strong antioxidant capabilities but face limited practical applications as a result of their poor biocompatibility (high immune resistance). Some food proteins possess mild antioxidant capabilities but are often not sufficient to maintain a reactive oxidative species balance. In this study, we overcome these barriers by covalently conjugating a natural phenolic antioxidant, gentisic acid (GA), onto an antioxidant protein, β-lactoglobulin (βLG). Upon optimization of conjugation conditions, we confirm the formation of βLG-GA conjugates with mass spectrometry, Fourier transform infrared spectroscopy, and ultraviolet-visible absorption. Surface charge analysis revealed a saturation molar ratio of 150:1 (GA/βLG), while far-ultraviolet circular dichroism revealed substantial changes in the protein secondary structure upon conjugation. The antioxidant capability of resultant conjugates was probed by monitoring the decay of 1,1-diphenyl-2-picrylhydrazyl radical content via time-resolved electron paramagnetic resonance spectroscopy, which suggested two possible pathways to scavenge radicals, i.e., the antioxidant GA on the protein surface and the protein conformational change that exposes more antioxidant amino acids. To our best knowledge, this work is the first report on the fabrication of a dual-effect antioxidant biopolymer using a nature-inspired template via covalent linking with the antioxidant mechanism probed. Our findings are essential for opening a new route to design functional materials with enhanced antioxidant activity and biocompatibility.