Sub-denaturing thermal pretreatment modulates lactoferrin-quercetin interactions: Structural dynamics, binding mechanisms, and functional synergy

Heat treatment, as one of the most common processing methods in protein modification, has unclear mechanisms affecting protein-polyphenol interactions. This study investigated the effects of sub-denaturing temperature pretreatment on protein structure-function relationships and their interactions with polyphenols using the quercetin-lactoferrin (Que-LF, QLF) system as a model. Results demonstrated that among 25-55 °C gradient heat treatments, LF pretreated at 40 °C (LF40) formed metastable aggregates through moderate structural unfolding, with significantly exposed hydrophobic cavities that enhanced binding affinity to Que ( K a =7.11×10 5 L/mol). Preheating optimized the conformational flexibility and spatial compatibility of LF binding pockets, enabling Que to embed into hydrophobic cavities via hydrophobic interactions ( ΔH = 23.82 kJ·mol -1 , ΔS = 188.27 J·mol -1 ·K -1 ) and multiple hydrogen bonding networks. The QLF40 complex exhibited optimal binding free energy (-85.1 kJ/mol) and conformational stability. Furthermore, Que binding synergistically improved thermal stability (2 °C increase in denaturation temperature) and antioxidant activity (64.7% DPPH and 98.4% ABTS scavenging rates) of the complex through masking hydrophobic sites, strengthening hydrogen bond networks, and inducing local structural rigidity. This study provides theoretical foundation for designing efficient active ingredient delivery systems based on controlled protein structural modification.