In situ covalent modification of soy protein isolate during alkaline extraction: effects of polyphenol structure and concentration on protein yield, conformation, functional properties, and emulsifying performance

This study first investigated in situ polyphenol covalent modification during alkaline extraction to form SPI-polyphenol conjugates. The effects of polyphenol type (gallic acid, GA; epigallocatechin gallate, EGCG; tannic acid, TA) and concentration (0.5–5 %, w /w) on yield, structure, functionality, and emulsifying properties were evaluated. The results demonstrated that polyphenol structure significantly influenced the covalent binding efficiency and protein yield, with EGCG at 5 % showing the highest binding (89.78 mg/g) and yield (43.35 %). SDS-PAGE, spectroscopy, and amino acid analysis confirmed covalent bonding and conformational changes, with secondary structure shifting from β-sheet to α-helix, decreased fluorescence, and increased UV absorption. Conjugates exhibited improved antioxidant activity, with EGCG at 5 % reaching 99.5 % ABTS scavenging and highest reducing power. Nanoemulsions stabilized by conjugates (0.4 % w /w, oil-to-water ratio 5:95  v /v) showed 40-day stability, inhibited oil-phase peroxide formation, and enhanced lutein protection. This provides a green SPI modification strategy and insights into polyphenol-protein conjugation design.