Covalent modification of whey protein isolate by rosmarinic Acid: Impacts on physicochemical and functional properties

Molecular modification has emerged as a promising strategy to enhance the functional properties of food proteins. In this study, rosmarinic acid (RA) was successfully conjugated to whey protein isolate (WPI) through a radical-mediated grafting approach, achieving a conjugation efficiency of 60.23 mg/g protein (WPI-RA 0.1). Electrophoretic analysis confirmed the formation of high-molecular-weight conjugates, while comprehensive spectroscopic characterization (FTIR, fluorescence spectroscopy, and UV absorption) revealed significant structural reorganization of WPI following RA conjugation. Notably, the α-Helix content decreased from 22.73 % (WPI-RA 0) to 17.55 % (WPI-RA 0.05), accompanied by an increase in β-turn content from 25.46 % (WPI-RA 0) to 41.82 % (WPI-RA 0.05). RA conjugation conferred multiple functional improvements to WPI. The allergenicity of WPI was significantly reduced, as evidenced by decreased IgG binding affinity (83.85 % for WPI-RA 0.1). Antioxidant capacity was markedly (P < 0.05) enhanced, with WPI-RA 0.1 demonstrating 74.37 % DPPH and 88.48 % ABTS⁺ radical scavenging activities. Additionally, the foaming and emulsifying properties of WPI-RA 0.1 were substantially improved, with foaming capacity (FC) increased to 104.39 %, foaming stability (FS) to 63.15 %, foaming half-life to 165.33 min, emulsifying activity index (EAI) to 1.11 m²/g, and emulsion stability index (ESI) to 92.37 min. Importantly, emulsions stabilized by WPI-RA exhibited superior oxidative stability during 14-day storage, with significantly reduced peroxide formation compared to native WPI. These findings demonstrate that RA conjugation effectively mitigates WPI allergenicity while enhancing its functional and antioxidant properties. This highlights its promising potential for nutritional food development, particularly in formulating hypoallergenic and high-performance protein ingredients.