Microbial stability and safety of traditional Greek Graviera cheese: characterization of the lactic acid bacterial flora and culture-independent detection of bacteriocin genes in the ripened cheeses and their microbial consortia.

The microflora of four batches of traditional Greek Graviera cheese was studied at 5 weeks of ripening, and 200 lactic acid bacteria (LAB) isolates were phenotypically characterized and screened for antilisterial bacteriocins. The cheeses were also analyzed for organic acids by high-performance liquid chromatography and for the potential presence of 25 known LAB bacteriocin genes directly in cheese and their microbial consortia by PCR. All batches were safe according to the European Union regulatory criteria for Listeria monocytogenes, Salmonella, enterobacteria, and coagulase-positive staphylococci. The cheese flora was dominated by nonstarter Lactobacillus casei/paracasei (67.5%) and Lactobacillus plantarum (16.3%) strains, whereas few Streptococcus thermophilus (3.8%), Lactococcus lactis subsp. lactis (0.6%), and Leuconostoc (1.9%) organisms were present. Enterococcus faecium (9.4%) and Enterococcus durans (0.6%) were isolated among the dominant LAB from two batches; however, enterococci were present in all batches at 10- to 100-fold lower populations than mesophilic lactobacilli. Sixteen E. faecium isolates produced antilisterial enterocins. In accordance, enterocin B gene was detectable in all cheeses and enterocin P gene was present in one cheese, whereas the consortia of all cheeses contained at least two of the enterocin A, B, P, 31, L50A, and L50B genes. Plantaricin A gene was also amplified from all cheeses. Mean concentrations of lactic, acetic, citric, and propionic acids in the ripened cheeses exceeded 1.5% in total, of which approximately 0.9% was lactate. Thus, organic acid contents constitute an important hurdle factor for inhibiting growth of pathogens in traditional Graviera cheese products, with LAB bacteriocins, mainly enterocins, potentially contributing to increased cheese safety.