Deciphering the competitive binding interaction of β-lactoglobulin with benzaldehyde and vanillic acid via high-spatial-resolution multi-spectroscopic

Interaction between protein and added flavor constituents strongly affects the sensory quality and consumer acceptability of dairy products. Here, the molecular basis of how benzaldehyde and vanillic acid competitively interact with β-lactoglobulin was described by applying various complementary techniques to study the structural modification of β-lactoglobulin with benzaldehyde and vanillic acid as flavor ligands. Mass shift of β-lactoglobulin recorded by mass spectrometry spectra suggested that the covalent addition of benzaldehyde and non-covalent addition of vanillic acid both in 1:1 stoichiometric ratio. Protein limited proteolysis experiments demonstrated the benzaldehyde and vanillic acid were localized on peptide fragment of Val41-Lys60, which reduced the allergenicity of β-lactoglobulin. Molecular docking studies predicted the covalent interaction between aldehyde carbonyl group on the aromatic ring of benzaldehyde and the ε-amino group of Lys60 residue along with a series of non-covalent bonds to form adduct. The non-covalent bonds between vanillic acid and β-lactoglobulin were also predicted. In addition, fluorescence studies of the binary systems indicated that benzaldehyde/vanillic acid interacted with β-lactoglobulin through the static quenching mechanism and benzaldehyde had a higher affinity for β-lactoglobulin. The quenching constant values of the ternary systems illustrated that benzaldehyde dislodges vanillic acid from the binding site, resulting in an increased free vanillic acid concentration. This study presents detailed information about flavor interactions with dairy products and confirms the conformational changes of β-lactoglobulin due to interaction with flavor. These findings provided new insight into the interactions between β-lactoglobulin and flavors facilitating flavor regulation in dairy products.