Multifactorial Microvariability of the Italian Raw Milk Cheese Microbiota and Implication for Current Regulatory Scheme.

Raw milk cheese manufactory is strictly regulated in Europe by the Protected Designation of Origin (PDO) quality scheme, which protects indigenous food products based on geographical and biotechnological features. This study encompassed the collection of 128 raw milk cheese samples across Italy to investigate the resident microbiome correlated to current PDO specifications. Shotgun metagenomic approaches highlighted how the microbial communities are primarily linked to each cheesemaking site and consequently to the use of site-specific Natural Whey Cultures (NWCs), defined by a multifactorial set of local environmental factors rather than solely by cheese type or geographical origin that guide the current PDO specification. Moreover, in-depth functional characterization of Cheese Community State Types (CCSTs) and comparative genomics efforts, including metagenomically assembled genomes (MAGs) of the dominant microbial taxa, revealed NWCs-related unique enzymatic profiles impacting the organoleptic features of the produced cheeses and availability of bioactive compounds to consumers, with putative health implications. Thus, these results highlighted the need for a profound rethinking of the current PDO designation with a focus on the production site-specific microbial metabolism to understand and guarantee the organoleptic features of the final product recognized as PDO. The Protected Designation of Origin (PDO) guarantees the traceability of food production processes, and that the production takes place in a well-defined restricted geographical area. Nevertheless, the organoleptic qualities of the same dairy products, i.e., cheeses under the same PDO denomination, differ between manufacturers. The final product's flavor and qualitative aspects can be related to the resident microbial population, not considered by the PDO denomination. Here, we analyzed a complete set of different Italian cheeses produced from raw milk through shotgun sequencing in order to study the variability of the different microbial profiles resident in Italian PDO cheeses. Furthermore, an in-depth functional analysis, along with a comparative genomic analysis, was performed in order to correlate the taxonomic information with the organoleptic properties of the final product. This analysis made it possible to highlight how the PDO denomination should be revisited to understand the effect that Natural Whey Cultures (NWCs), used in the traditional production of raw milk cheese and unique to each manufacturer, impacts on the organoleptic features of the final product.