How pH and temperature shape β-lactoglobulin binding to vanillin, caffeine and gallic acid

Abstract

Understanding the interaction of β-lactoglobulin (BLG) with food-derived bioactives is important for designing functional dairy formulations and protein-based delivery systems. This study evaluated the binding behavior of BLG with vanillin (VAN), caffeine (CAF), and gallic acid (GA) at pH 3 and 7 and temperatures of 298, 310, and 318 K using fluorescence spectroscopy, circular dichroism (CD), and scanning electron microscopy (SEM). All ligands caused concentration-dependent fluorescence quenching, suggesting interaction with BLG. At pH 7 and 318 K, Stern–Volmer constants (KSV) were 43.3 × 10³, 10.3 × 10³, and 10.2 × 10³ M⁻¹ for VAN, CAF, and GA, respectively, indicating stronger quenching efficiency for VAN. Binding constants (Ka) reached 65 × 10³ M⁻¹ for VAN and 83 × 10³ M⁻¹ for GA, whereas CAF showed lower affinity (4 × 10³ M⁻¹). In general, binding affinity decreased under acidic conditions, demonstrating the sensitivity of BLG–ligand interactions to environmental pH. Thermodynamic analysis indicated that VAN–BLG and GA–BLG associations were mainly driven by hydrophobic interactions, whereas CAF binding was dominated by hydrogen bonding and van der Waals forces. CD spectra revealed minor structural alterations of BLG upon ligand binding, with slightly greater spectral changes observed for GA, particularly at pH 3. SEM analysis showed distinct dried-state morphologies depending on ligand type. These findings demonstrate that ligand structure, pH, and temperature strongly regulate BLG binding behavior and highlight the potential of BLG as a tunable carrier matrix for stabilization and delivery of selected bioactive compounds in dairy-based systems.