Phenotypic and Genotypic Characterization of Indigenous Lactobacillus Community from Traditional Istrian Ewe's Cheese
Andrea Skelin1*, Mirna Mrkonjić Fuka1, Andreja Čanžek Majhenič2, Sulejman Redžepović1, Dubravka Samaržija3and Bojana Bogovič Matijašić2
1Department of Microbiology, Faculty of Agriculture, University of Zagreb, Svetošimunska 25, HR-10000 Zagreb, Croatia
2Chair of Dairy Science, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domžale, Slovenia
3Department of Dairy Science, Faculty of Agriculture, University of Zagreb, Svetošimunska 25, HR-10000 Zagreb, Croatia
Article history:
Received October 26, 2011
Accepted February 29, 2012
Key words:
Istrian cheese, lactobacilli, PCR, PCR-DGGE, sequencing
Summary:
The objective of this study is to describe the diversity of indigenous cultivable community of the lactobacilli associated with the production of traditional Istrian cheese and to get a collection of well characterized strains. Raw milk and cheese samples were collected from three different farms in Istria during ripening. A total of 212 mesophilic and thermophilic Lactobacillus isolates as well as bulk colonies (consortia) were investigated using culture-dependent approach combining phenotyping and genotyping. Biochemical fingerprinting with PhenePlate-LB system preliminary grouped 212 isolates in 16 distinct PhP types. Only one representative isolate from each PhP cluster was further analyzed by genotyping for a reliable identification at the genus and species level by employing PCR techniques and sequencing of 16S rRNA genes. Sequence analysis of 16S rRNA revealed the presence of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus. Lactobacilli were screened for possible resistance against seven selected antibiotics: ampicillin, tetracycline, penicillin, rifampin, clindamycin, erythromycin and vancomycin. Although there was no clear pattern of antimicrobial susceptibility to most tested antibiotics, all representative isolates were resistant to vancomycin. The analysis of bulk colonies by denaturing gradient gel electrophoresis (DGGE) identified Lactobacillus plantarum and Lactobacillus brevis as predominant members of Lactobacillus population. Pediococcus pentosaceus, Pediococcus acidilactici, Streptococcus sp. and Leuconostoc mesenteroides were also detected as part of the analysed consortia. The prevalence of identified species and community members of lactobacilli agrees with other studies of raw milk cheese and represents a useful base for further selection.
*Corresponding author: 
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