Genotypic characterization of non starter lactic acid bacteria involved in the ripening of artisanal Bitto PDO cheese

Bitto of Valchiavenna, an artisanal Italian cheese produced without the addition of any starter cultures, has been attributed a protected designation origin (PDO) cheese, but the strain composition of the natural microbial population colonizing this traditional dairy product is still unknown. To obtain preliminary information on the non starter lactic acid bacteria involved in its ripening, a total of 136 NSLAB isolates, randomly selected from MRS and M17 agar plates, were collected from three different cheese samples after 120 days of ripening. The new isolates were identified by combining PCR 16S–23S rDNA spacer analyses, partial 16S rRNA gene sequencing, species-specific probes and colony hybridization. Eighty-two isolates, representing 60% of the total strains selected, were homofermentative cocci: 83% of them were enterococci, with Enterococcus durans being the predominant species found. Pediococcus spp. were also isolated, together with strains of Streptococcus thermophilus. Within lactobacilli, 57% of the isolates were identified as Lactobacillus paracasei; Lact. curvatus, Lact. plantarum, Lact. fermentum, were present in a lower amount. The isolates were differentiated at strain-level by Rep-PCR analysis. This is the first effort to microbiological characterization of Valchiavenna’s Bitto; the results suggest the possibility of preserving the wild bacterial population in order to protect the typical organoleptic characteristics of this traditional raw milk cheese and to select new strains for the dairy industry.