Solvent-free pH-driven encapsulation of hesperidin by ovalbumin: binding, antioxidant activity, and gel network properties

Abstract Hesperidin (HES) is a citrus flavanone glycoside with promising bioactivities but limited aqueous solubility, which restricts its utilization in food systems. Here, a solvent-free pH-driven strategy was developed to encapsulate HES using ovalbumin (OVA) as a protein carrier, and the structural responses and antioxidant performance of the resulting OVA-HES systems were systematically evaluated at the alkaline driving stage (pH 12) and after neutralization (pH 7). Encapsulation efficiency (EE) increased monotonically at pH 12 and reached 74.75% at 0.8 mg/mL HES, whereas EE exhibited a bell-shaped dependence after neutralization and peaked at 51.64% at 0.6 mg/mL. Fluorescence quenching, Stern-Volmer analysis and docking collectively supported complex formation dominated by static quenching and multiple hydrogen-bond interactions, with stronger binding at pH 12 (K = 49.3 × 1010 L/mol) than at pH 7 (K = 3.45 × 1010 L/mol). Moderate HES loading enhanced free sulfhydryl exposure (maximum at 0.6 mg/mL) and improved 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical scavenging ability, with a peak value of 64.51% at pH 7. Fourier transform infrared spectroscopy, water-holding capacity, low-field nuclear magnetic resonance, and scanning electron microscope analyse further indicated that intermediate HES levels (0.4–0.6 mg/mL) promoted a denser and more continuous gel network, whereas excessive HES induced heterogeneous aggregation and reduced water retention. Overall, this work demonstrates a pH- and dosage-dependent route for incorporating HES into OVA matrices and provides mechanistic insights for designing antioxidant protein gels via solvent-free encapsulation.