Dynamic microbial and metabolic changes during Apulian Caciocavallo cheese-making and ripening produced according to a standardized protocol

The cheese microbiota plays a critical role in influencing its sensory and physicochemical properties. In this study, traditional Apulian Caciocavallo cheese coming from 4 different dairies in the same area and produced following standardized procedures have been examined, as well as the different bulk milks and natural whey starter cultures used. Moreover, considering the cheese wheels as the blocks of Caciocavallo cheeses as whole, these were characterized at different layers (i.e., core, under-rind, and rind) of the block using a multi-omics approach. In addition to physical-chemical characterization, culturomics, quantitative PCR, metagenomics, and metabolomics analysis, have been carried out post-salting and throughout ripening time (2 mo) to investigate the major shifts in the succession of the microbiota and flavor development. Culture-dependent and 16S rRNA metataxonomics results clearly clustered samples based on the microbiota biodiversity related to the production dairy plant as the result of the use of different NWS or intrinsic conditions of each production site. At the beginning of the ripening, cheeses were dominated by the Lactobacillus and, in 2 dairies (Art and SdC), Streptococcus genera associated with the NWS. The analysis allowed us to show that, although the diversity of identified genera did not change significantly between the rind, under-rind and core fractions of the same samples, there was an evolution in the relative abundance and absolute quantification, modifying and differentiating profiles during ripening. The qPCR mainly differentiated the temporal adaptation of those species originating from bulk milks and those provided by NWSs. The primary starter detected in NWS and cheese reassured the high relative concentration of 1-butanol, 2-butanol, 2-heptanol, 2-butanone, acetoin, delta-dodecalactone, hexanoic acid ethyl ester, octanoic acid ethyl ester, and VFFA during ripening, while cheeses displaying low abundances of Streptococcus and Lactococcus (dairy Del) have a lower total concentration of acetoin compared with Art and SdC. However, the sub-dominant strains and NSLAB present in cheeses are responsible for the production of secondary metabolites belonging to the chemical classes of ketones, alcohols, and organic acids, reaffirming the importance and relevance of autochthonous strains of each dairy plant although considering a delimited production area.