Characterisation of the yeast and mould biota in traditional white pickled cheeses by culture-dependent and independent molecular techniques.

Artisanal white pickled cheese of Western Serbia is a product of complex microbial community which detection by culture-dependent method only is hampered by its limitations. Thus, in the present study, we used a culture-independent, semi-quantitative technique based on construction of an internal transcribed spacer (ITS)-clone library from metagenomic DNA. This approach, based on direct DNA extraction followed by amplification of fungal internal transcribed regions (ITS) cloned into plasmid and restricted by endonucleases, revealed greater species richness in analysed cheeses and their by-products (17 species in total) compared to the more commonly used techniques of the culture-dependent method (8 species) and LSU-DGGE (10 species). The most frequently occurring yeast species which are commonly associated with cheeses production were Debaryomyces hansenii, Kluyveromyces lactis and Candida zeylanoides. On the other hand, Yarrowia lipolytica and Galactomyces geotrichum were detected only in one cheese sample. Moreover, some species, mainly moulds (Filobasidium globisporum, Cladosporium sp., Aspergillus sp. or Alternaria sp.) were identified only by culture-independent methods. The discrepancies between the techniques were confirmed by low correlation factor and by different indices of general biodiversity and dominance of species. The ITS-clone library approach provides the opportunity to analyse complex fungal communities associated with food products.