Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese

Classification of 74 yeasts isolated during manufacture and ripening of Cabrales cheese was attemped by phenotypic and molecular methods. Phenotypic classification included the use of commercial kits and classical biochemical analyses. Molecular classification was performed by restriction fragment length polymorphism (RFLP) of the ITS1-5.8S-ITS2 region, sequencing and comparison of the sequences in databases. Forty-one isolates were identified by phenotypic tests, including strains of Debaryomyces hansenii (14 isolates), Kluyveromyces lactis (11 isolates) and Pichia fermentans (9 isolates). The identification was confirmed by the HaeIII and HinfI RFLP profiles and comparison of their corresponding nucleotide sequences. The phenotypic classification failed to identify many other species; 16 isolates assigned to Zygosaccharomyces were genetically related to Pichia (13 isolates) and Saccharomyces (3 isolates). Classification of a few isolates was not possible, either because yeast species are new or a lack of sequences for this region in databases.