Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and -independent approaches

Abstract

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.

Fig 1

Flowchart of manufacturing and ripening stages of the traditional Polish cheese Oscypek.

Fig 2

Diversity and dynamics of bacterial, fungal, and LAB (represented by lactococci and lactobacilli) populations, as shown by analysis of their DGGE profiles. The 16S rRNA genes of bacteria and the D1 domains of the 26S rRNA genes of fungi were amplified by using universal prokaryotic (A) and eukaryotic (B) primers and specific primers for the lactococcus-enterococcus-streptococcus group (C) and the lactobacillus-leuconostoc-pediococcus group (D). The order of cheese samples is the same in all panels and corresponds to the order of samples in Table 1. Capital roman numerals represent four different producers. F, fresh; Sm, smoked; S, fresh salted; C, curd. Numbered bands were purified and identified by reamplification, sequencing, and sequence comparison. The identities of the bands are summarized in Table 3.

Fig 3

Major taxonomic groups in traditional Oscypek by the percentage of assigned reads. Only genus-level reads that represented at least 0.4% of the total pool of reads across the three samples are shown. unc., unclassified.