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Phenotypic and genotypic characterisation of indigenous lactobacilli from traditional Istrian ewe’s cheese

INTRODUCTION: Istrian cheese is traditional Croatian dairy product made from raw ewe’s milk. Among different microbial communities involved in the fermentation process of most traditional cheeses, lactobacilli constitute a significant proportion of nonstarter lactic acid bacteria (NSLAB). The diversity and dynamics of lactobacilli population in traditional Istrian cheese were investigated using culture dependant approach combining by PCR based techniques and sequencing of 16S rRNA. METHODOLOGY: A total of 212 lactobacilli were collected from milk and cheese samples during the ripening on MRS media at 25ᵒ and 42ᵒ C. Samples were subjected to the phenotypic and genotypic characterization. Only one representative isolate from each PhP cluster was further analyzed by genotyping for a reliable identification at the genus and species level. The PCR products were sequenced for more accurate identification at the species level. Additionaly, mixtures of all colonies harvested from plate count agar (consortia) were analysed using PCR DGGE and 16 S rRNA sequencing. All representative isolates were tested for their antibiotic suspectibility using disc diffusion tests and E test method. RESULTS AND DISCUSSION: Simple biochemical fingerprinting system, PhP typing, discriminated 16 distinct clusters among 212 indigenous isolates. PCR analyses with genus-specific primers confirmed that all isolates were Lactobacillus members. Sequence analysis of 16S rRNA revealed the presence of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus casei and Lactobacillus rhamnosus. The sequence analysis of excised DGGE bands identified Lactobacillus plantarum and Lactobacillus brevis as predominant members of lactobacilli population. Pediococcus pentosaceus, Pediococcus acidilactici, Streptococcus sp. and Leuconostoc mesenteroides were also detected as a part of analysed consortia. CONCLUSIONS: The prevalence of identified species and community members of lactobacilli agrees with other studies of raw milk cheeses and represent a usefull base for further selection.

Istrian cheese; lactobacilli; PCR; PCR DGGE; sequencing

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