Amplicon sequencing of Kefalograviera cheese after the innovative feeding strategy of dairy sheep by omega-3 fatty acids.

The microbial and metabolomic composition of sheep milk and therefore of cheese can be sig-nificantly affected by different factors across the dairy supply chain including primary production practices. Among the latter, the feeding system could drive the nutritional value and technological properties of milk and cheese. The purpose of this study was to examine the effects of two different feeding systems in a sheep flock, based on soybean (control) or flaxseed and lupin diet (experimental), on the microbiota and metabolome of Kefalograviera cheese samples produced by their milk. In particular, the microbiota present in Kefalograviera cheese samples was analyzed using 16S rRNA gene sequencing, while Ultra-High-Performance Liquid Chromatography coupled to Quadrupole Time-Off-Flight Mass Spectrometer (UHPLC-QTOF-MS) was applied to investigate the chemical profile of the cheeses, considering the different feeding systems applied. The met-agenomic profile was found to be altered by the experimental feeding system and significantly correlated to specific cheese metabolites, with Streptococcaceae and Lactobacillaceae establishing positive and negative correlations with the discriminant metabolites. Overall, more than 120 features were annotated and identified with high confidence level across the samples while most of them belonging to specific chemical classes. Characteristic analytes detected in different concen-trations at the experimental cheese samples including 5-hydroxy-L-tryptophan, N-acetyl-L-proline, itaconic, palmitate, succinic acid, 2-oxoadipic acid, hypoxanthine, and others. Therefore, taken together, our results provide an extensive foodomics approach of Kefalograviera cheese samples from different feeding regimes, investigating the metabolomic and metagenomic biomarkers that could be used to predict, optimize, and control cheese ripening outcomes, demonstrating the quality of a Kefalograviera cheese rich in omega 3 fatty acids.