LAUSR

Dietary phenolic compounds display strong antioxidant capabilities but face limited practical applications due to their poor biocompatibility (high immune-resistance). Some food proteins possess mild antioxidant capabilities but are often not sufficient to maintain ROS 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 (MS), fourier transform infrared (FTIR) spectroscopy, and ultraviolet-visible (UV-Vis) absorption. Surface charge analysis revealed a saturation molar ratio of 150:1 (GA: βLG), while far-ultraviolet circular dichroism (CD) revealed substantial changes in protein secondary structure upon conjugation. The antioxidant capability of resultant conjugates was probed by monitoring the decay of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical content via time-resolved electron paramagnetic resonance (EPR) spectroscopy, which suggested two possible pathways to scavenge radicals, i.e. the antioxidant GA on protein surface and the protein conformational change that expose more antioxidant amino acids. To our best knowledge, this work is the first report on the fabrication of dual effect antioxidant biopolymer using a nature inspired template via covalent linking with antioxidant mechanism probed. Our findings are essential for opening a new route to design functional materials with enhanced antioxidant activity and biocompatibility.