Interaction mechanisms between β-lactoglobulin and food-grade molecules: Insights from multispectroscopy and molecular dynamics simulations

In dairy systems, β-LG, the predominant whey protein, is valued for its nutritional and techno-functional properties, yet it remains a major milk allergen. Modulating protein-ligand interactions presents a potential strategy to alter its characteristics. Although interactions between β-LG and polyphenols have been extensively studied, the binding mechanisms with other common food-grade molecules possessing diverse structural features remain less understood. Therefore, this study selected 3 such additives representing distinct chemical categories: galactooligosaccharides, sucrose fatty acid esters, and casein phosphopeptides, to explore how their unique functional group profiles (hydroxyl, amphiphilic, and phosphopeptide moieties, respectively) drive their interaction with β-LG. We employed a combined multispectroscopic and computational approach to build a qualitative, mechanistic portrait of these interactions. Our findings demonstrate that all 3 additives interact with β-LG, primarily via a static fluorescence quenching mechanism, which alters the protein's local microenvironment without significantly perturbing its secondary structure. These experimental observations are complemented by an in-depth computational analysis, including molecular docking, extensive molecular dynamics simulations, and binding free energy calculations. This integrated approach not only identified the specific binding modes and key interacting residues, but also quantitatively highlighted the crucial roles of hydrogen bonding and hydrophobic forces in stabilizing the complexes. Collectively, this work provides a theoretical basis for future efforts to modulate whey protein functionality and explore its potential impact on milk allergenicity.