Heat-induced covalent conjugation between β-lactoglobulin and theaflavin: Structural characterization, antioxidant properties, and in vitro digestibility

Tea with milk is a widely consumed beverage in which tea polyphenols usually interact with milk proteins through non-covalent interactions. Although researchers have reported heat-induced covalent conjugation between green tea polyphenols and β-lactoglobulin, the nature of these interactions and their effects on protein properties remain poorly understood. Here, we investigated thermal processing–induced covalent conjugation between β-lactoglobulin (β-LG) and theaflavin (TF), a characteristic of black tea polyphenol, and evaluated its functional consequences. Heat treatment promoted β-LG unfolding and TF incorporation, resulting in significantly enhanced radical scavenging activity compared with free TF, with the strongest effect observed at 90 °C ( p < 0.05). Simulated gastrointestinal digestion showed that covalent conjugation accelerated gastric proteolysis and improved intestinal digestibility of β-LG, reaching approximately 74% after 120 min. These results demonstrate that heat-driven β-LG–TF conjugation modulates protein structure, antioxidant performance, and digestive behavior, providing mechanistic insight and a potential strategy for designing dairy-based matrices with improved phenolic stability and tailored digestion profiles.