Spatial Distribution of the Metabolically Active Microbiota within Italian PDO Ewes' Milk Cheeses

Italian PDO (Protected Designation of Origin) Fiore Sardo (FS), Pecorino Siciliano (PS) and Pecorino Toscano (PT) ewes’ milk cheeses were chosen as hard cheese model systems to investigate the spatial distribution of the metabolically active microbiota and the related effects on proteolysis and synthesis of volatile components (VOC). Cheese slices were divided in nine sub-blocks, each one separately subjected to analysis and compared to whole cheese slice (control). Gradients for moisture, and concentrations of salt, fat and protein distinguished sub-blocks, while the cell density of the main microbial groups did not differ. Secondary proteolysis differed between sub-blocks of each cheese, especially when the number and area of hydrophilic and hydrophobic peptide peaks were assessed. The concentration of free amino acids (FAA) agreed with these data. As determined through Purge and Trap (PT) coupled with Gas Chromatography-Mass Spectrometry (PT-GC/MS), and regardless of the cheese variety, the profile with the lowest level of VOC was restricted to the region identified by the letter E defined as core. As shown through pyrosequencing of the 16S rRNA targeting RNA, the spatial distribution of the metabolically active microbiota agreed with the VOC distribution. Differences were highlighted between core and the rest of the cheese. Top and bottom under rind sub-blocks of all three cheeses harbored the widest biodiversity. The cheese sub-block analysis revealed the presence of a microbiota statistically correlated with secondary proteolysis events and/or synthesis of VOC.