Identification of yeasts associated with milk products using traditional and molecular techniques

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.