Thermally induced conformational transition and binding affinity of β-lactoglobulin and capsaicin: A multi-spectroscopic and molecular dynamics simulation study.

This study investigates the effect of heat treatment (25-95 °C) on the interaction between β-lactoglobulin (β-LG) and capsaicin (CAP) through multi-spectroscopic, physicochemical, and molecular dynamics (MD) simulation approaches. We reveal a non-monotonic, temperature-dependent binding profile governed by β-LG's multistage conformational transitions. Binding affinity increases from 25 to 65 °C, driven by a molten globule-like transition that exposes hydrophobic binding sites. A sharp decline at 75 °C, due to vigorous aggregation and site burial, marks a critical temperature threshold. Notably, partial binding recovery occurs at 85-95 °C, resulting from aggregate rearrangement. Beyond its role as a ligand, CAP actively modulates β-LG aggregation by occupying hydrophobic patches, as evidenced by consistently smaller particle sizes and lower turbidity in CAP-supplemented groups. Molecular docking and MD simulations, including MM-PBSA free energy calculations and free energy landscape analysis, confirm stable binding within β-LG's hydrophobic cavity and reveal that temperature-driven reshaping of conformational substates directly governs binding thermodynamics, with optimal affinity at 65 °C. These findings establish 65 °C as the optimal processing temperature for CAP retention, while suggesting that prolonged heating at 75 °C should be avoided. The strong correlations between simple physicochemical indicators (turbidity, solubility) and binding affinity offer a practical pathway for low-cost, real-time quality monitoring in industrial dairy processing.