An exploratory dual-platform metabolomics reveals region- and milk source-associated signatures in commercial cheeses

Commercial cheeses exhibit distinct compositional profiles shaped by milk substrate, production practices, and geographical terroir. However, the relative contribution of geographic origin versus milk source to cheese metabolomes remains poorly quantified, particularly in unstandardized commercial products. This exploratory study investigated region-associated metabolic signatures in commercially available cheeses, with a focus on comparing the relative contributions of geographic origin and milk source to metabolomic variance. We applied complementary volatile (HS-SPME-GC-MS) and non-volatile (UHPLC-MS/MS) metabolomic profiling to 32 commercial cheeses from Hungary (n = 11) and 3 Australian regions (Queensland, New South Wales, Victoria; n = 21), representing cow (n = 24) and goat/sheep (n = 8) milk sources. Multivariate ANOVA and random forest analyses revealed pronounced metabolic separation by geographic origin, which accounted for a substantially larger proportion of variance than milk source. Hungarian cheeses were enriched in citrate-derived flavor compounds (e.g., 2,3-butanedione, 2,3-butanediol), while Australian samples exhibited elevated lipid oxidation and branched-chain amino acid catabolites. Non-volatile profiles further highlighted regional differences in fatty acid composition and dipeptide abundance, with phenylalanine metabolism emerging as a consistent discriminant pathway. Correlation-based association networks revealed interconnected volatile and non-volatile metabolic modules. Since cheese variety, ripening protocols, and starter cultures were uncontrolled, observed patterns reflect region-associated metabolic signatures rather than causal terroir effects. These findings provide a preliminary metabolomic landscape for authenticity tracing and hypothesis-driven fermentation research. Future controlled trials integrating metagenomics and standardized production parameters are needed to isolate the specific contributions of geography, milk substrate, and processing technology.