Phenotypic, genotypic and technological characterization of wild-lactic acid bacteria strains isolated from Bitto PDO cheese.

Bitto is a Protected Designation of Origin raw milk cheese produced in a restricted Italian alpine area only during the summer transhumance. The indigenous microbial ecosystem of this artisanal cheese is considered a primary factor related to its typicality. The aim of the research was to investigate the dynamics of wild lactic acid bacteria (LAB) involved in Bitto production and to study the characteristics of LAB. A total of 210 LAB isolates from curd, whey and ripened cheese, were first molecularly analysed by means of randomly amplified polymorphic DNA (RAPD). After strain differentiation, LAB were identified at the species level using species-specific primers and 16S rRNA gene sequencing. Genotypic diversity and technological properties of major interest for cheese making (acidification ability, redox potential and caseinolytic activity) were also evaluated. The predominant species, in both curd and ripened cheese, was Enterococcus faecium, and there appeared a high degree of diversity in the genotypic and technological traits. By using 16S rRNA sequencing and RAPD-PCR as well as examining the phenotypic properties, the new isolates were shown to belong to a novel enterococcal species for which the name Enterococcus lactis has been proposed. Among the curd isolates, six bacteriocin producers were found belonging to E. faecium, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus species.