Culture-dependent and -independent Approaches

17 Oscypek is a traditional Polish, scalded-smoked cheese, with a protected designation of origin 18 (PDO) status manufactured from raw sheep's milk without starter cultures in the Tatra Mountains 19 region of Poland. This study was undertaken in order to get some insight on the microbiota which 20 develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use 21 of both culturing and the culture-independent methods of PCR-DGGE and pyrosequencing of 16S 22 rRNA gene amplicons. Culturing and culture-independent techniques are known to give 23 complementary results on the diversity and dynamics of microbial populations, thus providing a better 24 description of the cheese ecosystem of traditional Oscypek. Culture-dependent technique and PCR-25 DGGE fingerprinting detected the predominant microorganisms in the traditional Oscypek whereas the 26 next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. 27 Besides members of the most abundant bacterial genera in dairy products, e.g. Lactococcus, 28 Lactobacillus, Leuconostoc, Streptococcus and Enterococcus, identified by all three methods, other 29 subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly 30 Enhydrobacter) and various minor bacteria were identified by pyrosequencing. In addition to bacteria, 31 a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing 32 methods enabled the determination of a number of viable microorganisms from different microbial 33 groups and their isolation for potential future applications in specific cheese starter cultures. 34 35 INTRODUCTION