Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches

Abstract

The microbiota of two traditional Iranian cheeses (Lighvan and Koozeh) made of raw ewe’s milk or mixtures of ewe’s and goat’s milk without starter addition was explored by culture-independent and culture-dependent approaches. Three batches of Lighvan and one of Koozeh were subjected to culture-independent polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through manufacturing and ripening. In addition, culturing in elective media for lactic acid bacteria (M17, MRS and KAA), isolation of single colonies (n = 130), molecular identification by PCR-amplified ribosomal DNA restriction analysis and sequencing, and differentiation at the strain level by repetitive extragenic palindromic PCR was also performed. DGGE analysis showed that the dominant amplicons in all four cheese batches belonged to Lactococcus lactis and Streptococcus parauberis. In addition, Escherichia coli and Lactococcus garvieae were frequently identified in both Lighvan and Koozeh, while Streptococcus thermophilus was found occasionally. In contrast, Enterococcus faecium and Enterococcus faecalis were found to be dominant among the isolates in all batches. These species showed a high genetic diversity. The discrepancy between culturing and DGGE results suggested that dominant populations were in a nonrecoverable state in the used media. This reinforces the idea that culture-dependent and culture-independent techniques provide complementary data, ultimately affording a better description of cheese ecosystems. These data could be of help in the selection of commercial starters for industrial-scale manufacture of Lighvan and Koozeh cheeses using pasteurised milk. Alternatively, microbial analysis would allow the selection of appropriate strains for designing of specific starters for traditional cheese manufacture.