Bioraznolikost mikrobnih konzorcija izoliranih iz tradicionalnog svježeg ovčjeg sira Karakačanski skakutanac

The aim of this study was to assess the structure of indigenous microbial community associated with traditional fresh sheep cheese Karakačanski skakutanac and to preserve autochthonous microbial consortia. Eleven cheeses were sampled during production season (April-September) and subjected to microbiological analysis. Bacterial DNA was isolated by Maxwell®16 DNA system from 99 microbial consortia harvested from three culture media (M17, Rogosa, CATC) on the 1st, 2nd and 3rd day of the cheese shelf life. The extracted bulk DNA (n = 99) was used as a template for PCR-ARDRA and PCR-DGGE analysis. There were no dramatic shifts in the bacterial number and structure of the microbial consortia harvested on the 1st, 2nd or 3rd day of the cheese shelf life neither during period of sampling. Lactococcus lactis subsp. lactis reached the number of 107-108 CFU g-1, while Leuconostoc pseudomesenteroides, Enterococcus faecalis, and Lactobacillus versmoldensis were identified only at lower dilutions (10-2 - 10-3). This first polyphasic microbiological-molecular study of the Karakačanski skakutanac indicated the main LAB representatives associated with the cheese. Obtained autochthonous microbial consortia present a valuable pool of strains for further genetic and functional characterizations.Cilj ovog rada bio je analizirati strukturu mikrobnih konzorcija tradicionalnog svježeg ovčjeg sira Karakačanski skakutanac. Jedanaest sireva sakupljeno je tijekom sezone proizvodnje od travnja do rujna. Mikrobni konzorciji sakupljeni su sa 3 hranjive podloge (M17, Rogosa, CATC) od 11 sireva nakon prvog, drugog, i trećeg dana proizvodnje. Mikrobna DNA je izolirana iz 99 konzorcija, te korištena u PCR-ARDRA i PCR-DGGE analizi. Nije bilo promjene u strukturi mikrobnih konzorcija sakupljenih prvog, drugog i trećeg dana nakon proizvodnje, niti tijekom sezone. Utvrđena je dominantnost populacije Lactococcus lactis subsp. lactis (107-108 CFU g-1), dok su ostale vrste bakterija mliječne kiseline, Leuconostoc pseudomesenteroides, Enterococcus faecalis i Lactobacillus versmoldensis, identificirane samo na nižim razrjeđenjima (10-2-10-3). Ova prva mikrobiološko- molekularna analiza tradicionalnog sira Karakačanski skakutanac omogućila je uvid u strukturu njegove specifične mikrobne populacije. Sakupljeni mikrobni konzorciji predstavljaju značajan izvor sojeva za daljnju genetsku i funkcionalnu karakterizaciju

Microbiota of Karakačanski skakutanac, an artisanal fresh sheep cheese studied by culture-independent PCR-ARDRA and PCR-DGGE

Karakačanski skakutanac is an artisanal fresh sheep cheese produced on a small scale in a limited of eastern Croatia. It is manufactured from unrefrigirated raw sheep milk immediately after milking, without the addition of starter culture, and coagulated with industrial rennt. To date, no microbial or molecular characterization of the biodiversity of the microbiota has been performed. The objective of this study was to obtain an initial insight into the biodiversity of the microbial community associated with this cheese during the production season and shelf life period. Eleven cheeses wereobtained from a dairy farm at 14-day intervals during the lactation periodof east sheep Friesian sheep in 2007. Bacterial DNA was isolated directly from cheese on the first, second and third of the cheese shelf life, resulting in a total of 33 DNA samples. Extracted DNA was used as a template for PCR-ARDRA and PCR-DGGE analysis. the use of dual culture-independent approaches revealed similar results and indicated predominance of Lactococus lactis