Pulsed electric field modulates lactoferrin conformation to enhance quercetin binding and complex stability

Quercetin (QUE) is a natural flavonoid whose practical application is severely limited by poor aqueous solubility, environmental instability, and low oral bioavailability. Lactoferrin (LF), a generally recognized as safe (GRAS) glycoprotein, is a promising carrier for hydrophobic bioactives; however, its native compact globular structure causes significant steric hindrance, resulting in inefficient ligand binding. Herein, continuous-flow pulsed electric field (PEF) treatment was employed to modulate LF conformation, enhancing the binding efficiency and stability of LF-QUE complexes. The findings revealed that moderate PEF treatment (10 kV·cm^-1) induced controlled unfolding of LF, exposing internal hydrophobic cavities and increasing QUE loading capacity by 40.2% compared with untreated LF. Excessive field intensities (≥15 kV·cm^-1) triggered intermolecular disulfide cross-linking of LF, which masked specific binding sites and reduced loading performance. Multispectral analysis demonstrated that the LF10-QUE20 complex had the strongest binding affinity via a static quenching mechanism, with more binding sites than the native complex. The LF10-QUE20 complex exhibited superior colloidal dispersibility, thermal stability (denaturation temperature of 86.3 °C), and a 21.5% increase in the in vitro bioaccessibility of QUE, accompanied by a complete crystalline-to-amorphous transition of encapsulated QUE. Molecular simulations revealed that PEF treatment enhanced the binding stability of QUE to LF (binding energy: -9.0 kcal·mol^-1) mainly via hydrogen bonding, van der Waals forces, π-cation, and π-alkyl interactions. Overall, this study presents a facile, non-thermal PEF strategy to optimize protein-based delivery systems for hydrophobic bioactives in functional food applications, avoiding irreversible protein denaturation and chemical residues associated with conventional thermal or chemical modification methods.