Untargeted Headspace Fingerprinting and Targeted Characterization to Reveal High-Intensity Ultrasonication Effects on the Volatilome Evolution of Milk Suspensions

This study aimed at elucidating the impact of high-intensity ultrasonication (HIU) on milk's aromatic profile and the molecular interaction behaviors between aroma compounds and β-lactoglobulin (BLG) through nontargeted headspace-gas chromatography–ion mobility spectrometry (HS-GC-IMS) and headspace solid-phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC/MS), as well as targeted multispectral characterization. GC-IMS effectively distinguished different milk dispersions with and without HIU pretreatments based on the volatile patterns, complemented by HS-SPME-GC/MS for precise classification based on its high sensitivity to detect aroma compounds, simultaneously revealing the significant effects on the evolution of volatile flavor compounds in milk suspensions after nonthermal HIU treatments. Principal component analysis (PCA) of GC-IMS data showed clear separation between all milk samples and controls with 20 min HIU. Additionally, the unbound fraction of hexanal in the headspace decreased by 12% following BLG addition. Conformational changes in BLG induced by HIU, showing more subtle structural transformation compared to thermal pretreatments, significantly altered the interaction processes between proteins and volatile flavors, thus explaining the fluctuation in the contents of headspace aldehyde-ketones following HIU pretreatments of milk suspensions. Molecular docking confirmed the binding between BLG and volatile ligands, highlighting the protein's role in flavor retention and release. The combined volatilomic and protein-structure approaches emphasized specific effects of HIU on the aroma profiling of dairy products and molecular structure of their proteins under cold sterilization conditions, providing new insights into flavor optimization and preservation.