Molecular characterisation of interactions between β-lactoglobulin and hexanal – An off flavour compound

Using molecular docking and multispectral analytical techniques, the present work investigates the binding characteristics between hexanal and β-lactoglobulin at near neutral pH and ambient temperature. Fluorescence quenching demonstrates that binding between β-lactoglobulin and hexanal is of low to medium strength, having a KA ranging from 9.6 × 102 to 4.0 × 104 M−1, with UV–vis and MALDI-TOF analysis suggesting that interactions are non-covalent in nature. Analysis of secondary structure using both FTIR and CD, shows that the complexation of the ligand with the protein induces an increase in β-sheet structure and a corresponding decrease in α-helical components. The method of continuous variation reveals a 1:1.5 (2:3) binding ratio under the present experimental conditions, suggesting that each β-lactoglobulin dimer may bind 3 hexanal molecules. Molecular docking simulations were consistent with these findings, showing three potential binding locations with distinct binding energies for the dimer. One binding site is located within the hydrophobic calyx of each monomer, while the third site is located at the interface between the two monomers. Knowledge achieved presently may advance our understanding of the fate of flavour compounds in dairy based beverage formulations.