Detection, Identification, and Diffusion of Yeasts Responsible for Structural Defects in Provolone Valpadana PDO Cheese Using Multiple Research Techniques

The aim of this work was to identify the microbial agent(s) responsible for a structural defect in Provolone Valpadana, Protected Designation of Origin (PDO) cheese, and to establish their spread along the production line. Repeated sampling of defective cheeses and analyses of processing intermediates following two inspections at the cheese factory identified yeasts as the main causative agents. Microbiological analysis highlighted an almost constant presence of yeasts, which dominate over the other microbial groups. Forty yeast isolates from defective cheeses were identified by sequencing the D1/D2 region of the 26S rRNA gene. Saccharomyces cerevisiae, Kluyveromyces marxianus, and Debaryomyces hansenii dominated in all sampled cheeses, followed by D. tyrocola, Pichia kudriavzevii, and Torulaspora delbrueckii. Yeast and bacterial metabarcoding on three cheeses with a yeast count > log 4.0 CFU/mL indicated D. hansenii as the dominant yeast taxon and confirmed the absence of gas-producing bacterial taxa. RAPD-PCR analysis suggested the presence of yeast biofilms in the dairy environment or along the production line, as confirmed by the repeated isolation of specific genotypes of S. cerevisiae, K. marxianus, and D. hansenii in different defective cheeses sampled between April and August 2023, as well as in samples taken following two inspections at the production site, during cheese processing and ripening.