Unravelling the effect of autochthonous scotta-derived vs. commercial starter cultures in pilot-scale hard sheep milk cheese production: A multi-omics and sensory combined approach.

Autochthonous whey-based (scotta-innesto) starters are increasingly recognized as a valuable resource to preserve microbial biodiversity and sensory identity in hard sheep milk cheeses produced under PDO-type specifications. In this study, two indigenous scotta-innesto cultures collected in Sardinia (Italy) in the 1960s were compared with a widely used commercial starter in pilot-scale hard sheep milk cheese manufacture produced according to Pecorino Romano PDO specifications. To this end, an integrated multidisciplinary approach was employed by combining starter metagenomics, culture-dependent microbiology, LC-HRMS-based untargeted metabolomics, targeted aroma volatile analysis, and descriptive sensory analysis. The autochthonous consortia were dominated by Lactobacillus delbrueckii and Streptococcus thermophilus and showed a higher abundance of protease and peptidase genes as well as pathways linked to acetyl-CoA metabolism and alcohol formation. Conversely, the commercial starter comprised higher proportions of Lactobacillus helveticus and Lactococcus spp., together with the enrichment of the acetoin and diacetyl pathways. Moreover, by focusing on ripening, cheeses produced with the autochthonous starters showed a lower accumulation of purine catabolites, such as hypoxanthine, and higher levels of 1-methyladenosine, methionine and dimethylglycine, suggesting a potential enhancement of purine salvage and biosynthetic activity. Also, the starter culture influenced the synthesis and the accumulation of selected key aroma compounds, with higher 2-hexanol and 1-butanol in cheeses inoculated with autochthonous starters, while acetoin and ketones were found as key aroma markers in commercial-starter cheeses. Finally, saltiness and pungency, hardness, and crystal perception emerged as the most discriminant sensory attributes, with autochthonous-starter cheeses showing higher intensity scores.