Binding temperature regulates the interaction mode and functionality of protein/peptide-polyphenol assemblies prepared under alkaline conditions

Protein/peptide-polyphenol assemblies are commonly prepared via alkaline oxidation; however, the influence of binding temperature on their emulsifying and antioxidant functionalities remains unexplored. In this study, oat protein hydrolysate (OPH) and (-)-epigallocatechin gallate (EGCG) were employed as a model system to prepare assemblies at different binding temperatures (4-60 °C, pH 9). The structural characteristics, intermolecular interactions, interfacial behavior, and emulsifying performance of the resulting assemblies were systematically investigated. At low binding temperatures (4-20 °C), hydrogen bonding served as a major contributor, rather than the expected covalent conjugation, yielding assemblies with moderate apparent molecular weights, high EGCG equivalents, and extended conformations that exposed hydrophobic residues. These structural features facilitated rapid interfacial adsorption, efficient reduction of interfacial tension, and the formation of interfacial films with high antioxidant functionality. Consequently, emulsions stabilized by these low-temperature assemblies remained stable over 30 days of storage, with lipid oxidation products being substantially reduced. In contrast, at elevated binding temperatures (particularly 60 °C), covalent bonding became significant, but excessive polymerization produced assemblies with excessively high apparent molecular weights that resulted in inadequate interfacial properties and emulsion stability. This study establishes a "temperature-formation-structure-function" relationship for protein/peptide-polyphenol assemblies, revealing that low temperature favors hydrogen-bond-dominated binding rather than the covalent cross-linking conventionally assumed for alkaline-oxidation products. These insights provide a rational basis for designing dual-functional emulsifiers tailored for oxidation-sensitive bioactive delivery.