Preparation and characterization of O/W Nanoemulsions stabilized by covalent complex of ball-milled soy protein isolate and Chlorogenic acid

Abstract

Ball-milled soy protein isolate (BMSPI) and chlorogenic acid (CA) covalent complexes were prepared via alkaline conjugation to investigate how mechanical pretreatment modulates protein-polyphenol structure-function relationships. At 0.5 mM CA, binding was optimized, yielding reduced particle size/turbidity, increased zeta potential (32.85 ± 2.32 mV), smoother surfaces, decreased free sulfhydryl content, and enhanced α-helix (16.85%) with reduced random coil (35%). Unlike direct SPI-CA conjugation, BMSPI tolerated higher CA (0.8 mM) via mechanically induced unfolding and disulfide cross-linking, which redirected cysteine reactivity. BMSPI-CA conjugates exhibited superior emulsifying properties, and their nanoemulsions demonstrated excellent thermal, pH, and salt-ion stability over 14 days, offering a mechanistic basis for engineering interfacial performance via physical-chemical synergy