Multispectroscopic and molecular simulation insights into the interaction between casein and key aroma compounds in...

The molecular mechanisms governing interactions between casein (CS) and key yogurt aromas remain insufficiently elucidated. This study systematically investigated the interactions of CS with acetoin, diacetyl, and acetaldehyde through multispectroscopic techniques and integrated molecular simulations. Fluorescence quenching analysis confirmed the formation of stable ground-state complexes via static quenching (K_q ≈ 10¹¹ L·mol^-1·s^-1), with binding affinities following the order: acetoin > diacetyl > acetaldehyde (K_a ranging from 1.12 × 10³ to 2.82 × 10³ L·mol^-1 at 298-310 K). Thermodynamic evaluation (ΔG < 0, ΔH > 0, ΔS > 0) revealed a spontaneous, endothermic binding process driven predominantly by hydrophobic interactions. Notably, spectroscopic and morphological characterizations demonstrated divergent, ligand-dependent structural remodeling within the intrinsically disordered CS matrix. Ketone ligands (acetoin and diacetyl) triggered significant structural uncoiling and granular aggregation, whereas acetaldehyde promoted compact intermolecular cross-linking. Furthermore, molecular docking and 100-ns molecular dynamics simulations identified β-CS as the primary binding subunit, which utilizes its highly flexible N-terminal domain to dynamically encapsulate aroma molecules via a hydrophobic collapse mechanism. These findings offer atomic-level insights into dairy protein-flavor interactions, providing a robust theoretical foundation for targeted flavor regulation and retention in fermented dairy matrices.