A metabolomics approach to fingerprint the heat-induced alterations in milk and identify potential thermal biomarkers.

The study fingerprints the heat-induced metabolic changes in milk across 15 temperature-time treatments (63-136 °C) using an untargeted metabolomics approach. A total of 2277 metabolites were detected, including organic acids, benzene, heterocyclic compounds, aldehydes, ketones, esters, amino acids, carbohydrates, alcohols, amines, fatty acids, lipids, and their derivatives, from thermally processed milk samples across 15 treatment groups. The multivariate analyses (PCA, HCA) and the KEGG pathway enrichment revealed heat-induced metabolic disruptions, particularly in the Maillard reaction products and lipid oxidation pathways. Five thousand three hundred twenty-nine differentially regulated metabolites were observed, with the most pronounced changes observed in the 135 °C vs. raw milk group. Key pathways, including those associated with amino acid and carbohydrate metabolism, were significantly enriched. Amino acid and lipid pathways were most affected by heat processing. Lactulose was consistently downregulated, while L-Proline, Adenosine, and Carnitine were upregulated, suggesting they are candidate thermal biomarkers. The findings fingerprint the thermal-induced biochemical changes in milk, and Lactulose as a potential thermal biomarker for milk quality and authenticity. Further research is required to assess the practical feasibility of the biomarker and the practices needed to optimize dairy processing techniques.