The role of reaction sequence in governing the structure and emulsification performance of ternary protein-polyphenol-polysaccharide covalent complexes

Binary and ternary covalent complexes were prepared from soy protein isolate (SPI), epigallocatechin-3-gallate (EGCG), and polydextrose (PD) by varying the reaction sequence. The covalent bonding order was a critical determinant of the complexes’ structural and functional properties. Formation of covalent linkages was confirmed by SDS-PAGE, polyphenol binding, and grafting degree. Circular dichroism spectra revealed a significant conformational transition in the secondary structure of SPI after covalent modification, with a decrease in α-helix and β-sheet content and an increase in β-turn and random coil structures. This structural shift suggests enhanced protein flexibility. In emulsion systems, the ternary complexes, particularly SPE, exhibited excellent stabilization performance, as demonstrated by a minimal droplet size (427.7 ± 6.84 nm), a high absolute zeta-potential (-33.2 ± 0.31 mV), reduced interfacial tension, and a high interfacial protein adsorption rate (82.8 ± 0.53%). These results broaden the potential application of protein, polyphenol, polysaccharide covalent conjugation technology in functional foods and establish a theoretical foundation for developing more efficient emulsion-based delivery systems. • Ternary covalent complexes were successfully prepared using two reaction sequences. • The reaction sequence critically determined their structural and emulsifying properties. • SPE exhibited the highest polysaccharide grafting degree and EGCG binding capacity. • Covalent modification enhanced structural flexibility and interfacial activity of SPI. • SPE formed emulsions with the smallest droplet size and highest storage stability.