Thermal examination implementing supercomputer simulations and experimental spectroscopy to identify the chemical...

To explore the thermal behaviour of flavour-protein interactions, a β-lactoglobulin monomer structure obtained from the protein databank underwent a simulated heat treatment at 353.15 K (80 °C) for 20,000 ps. A hexanal molecule acting as the ligand was docked on the host protein using the blind method approach revealing a preferential binding location situated near a specific lysine residue (LYS:60). Besides supercomputer simulations, benchtop testing in MALDI-TOF/MS and UV-vis spectroscopy were performed on the β-lactoglobulin-hexanal mixtures exposed to 80 °C for 60 min. MALDI-TOF/MS findings revealed a covalent adduct was formed with a peak shift of ∼84 Da creating a Schiff base through a condensation reaction. Secondary structure estimations indicated a significant decrease in α-helix and β-turn as well as an increase in β-sheet and unordered structures. This combined in silico and experimental approach provides a molecular-level view of how flavour-active aldehydes interact with thermally treated dairy proteins, linking processing conditions to structural changes and the fate of flavour compounds. The resulting mechanistic insights have both fundamental and industrial relevance, advancing our understanding of how thermal treatment influences flavour stability through protein-ligand interactions in dairy-based systems.