Abstract
This study develops a novel dual strategy to enhance soy protein isolate (SPI) functionality and the stability and bioavailability of hydroxytyrosol (HT) by integrating horseradish peroxidase (HRP)-mediated covalent conjugation with liposomal encapsulation. The optimal HT:HRP molar ratio for covalent complex formation was 5:1, and liposomes were prepared at a phospholipid-to-cholesterol ratio of 5:1 and 65 °C. The covalent binding efficiency of HT reached 45.53 ± 0.24%, significantly higher than that of non-covalent complexes. Spectroscopic analyses revealed strengthened UV absorption, fluorescence quenching, and conformational changes in SPI, with decreased α-helix and increased β-sheet content. The modified conjugates showed improved emulsifying properties. Liposomal encapsulation further enhanced HT retention under UV and thermal conditions. In vitro digestion demonstrated enhanced protection and higher HT availability in the intestinal phase, indicating controlled release. This strategy shows promise for developing functional food ingredients and bioactive delivery systems with improved stability and bioavailability.