Amplicon sequence variant–level analysis reveals No stable regional microbial biomarkers in traditional Portuguese PDO cheeses

Protected Designation of Origin (PDO) cheeses are strongly linked to specific geographical areas, where local raw materials, artisanal practices, and microbial communities collectively shape product identity. Metataxonomic-based approaches have been increasingly proposed as tools for food authentication; however, the robustness of taxonomic profiling alone for defining regional biomarkers remains insufficiently tested, particularly in spontaneously fermented cheeses. Here, we conducted a two-year longitudinal analysis of bacterial and fungal communities in Portuguese PDO ewe's milk cheeses from two regions, Azeitão and Nisa, including four and two producers, respectively, using a metataxonomic analysis to 16 S and ITS.Alpha diversity analyses revealed no regional differences in bacteria or fungi richness, however some differences at the producer's level were found. Beta diversity highlighted patterns of regional variation in microbial community structures. While permutational analyses supported the idea that producer-specific effects explained a larger proportion of variance than geographical origin. The analysis of differentially abundant amplicon sequence variants (ASVs) identified 36 and 20 bacterial ASVs, and 26 and 4 fungi ASVs, capable of discriminating Azeitão and Nisa cheeses, respectively. However, further examination showed that these ASVs did not meet the criteria for true regional biomarkers, as their prevalence was uneven across producers within each region.Our findings suggest that regional identity is not primarily determined by the taxonomic composition of cheese microbiota, but rather by the functional and metabolic activities expressed during ripening. Overall, this study underscores the limitations of taxonomy-based profiling and supports the need for integrated multi-omic approaches to accurately capture PDO-specific signatures.