Causal Relationship between Microbial Ecology Dynamics and Proteolysis during Manufacture and Ripening of Protected Designation of Origin (PDO) Cheese Canestrato Pugliese

Pyrosequencing of the 16S rRNA gene, community-level physiological profiles determined by the use of Biolog EcoPlates, and proteolysis analyses were used to characterize Canestrato Pugliese Protected Designation of Origin (PDO) cheese. The number of presumptive mesophilic lactococci in raw ewes' milk was higher than that of presumptive mesophilic lactobacilli. The numbers of these microbial groups increased during ripening, showing temporal and numerical differences. Urea-PAGE showed limited primary proteolysis, whereas the analysis of the pH 4.6-soluble fraction of the cheese revealed that secondary proteolysis increased mainly from 45 to 75 days of ripening. This agreed with the concentration of free amino acids. Raw ewes' milk was contaminated by several bacterial phyla:Proteobacteria (68%; mainlyPseudomonas ),Firmicutes (30%; mainlyCarnobacterium andLactococcus ),Bacteroidetes (0.05%), andActinobacteria (0.02%). Almost the same microbial composition persisted in the curd after molding. From day 1 of ripening onwards, the phylumFirmicutes dominated.Lactococcus dominated throughout ripening, and most of theLactobacillus species appeared only at 7 or 15 days. At 90 days,Lactococcus (87.2%),Lactobacillus (4.8%; mainlyLactobacillus plantarum andLactobacillus sakei ), andLeuconostoc (3.9%) dominated. The relative utilization of carbon sources by the bacterial community reflected the succession. This study identified strategic phases that characterized the manufacture and ripening of Canestrato Pugliese cheese and established a causal relationship between mesophilic lactobacilli and proteolysis.