engleski

Authenticity of Fresh Sheep Cheese Karakačanski Skakutanac

AIMS: (i) to obtain an initial insight into the biodiversity of the cheese microbiota during production season and shelf life period by culture-independent molecular approach ; (ii) to assess the structure and dynamic of indigenous viable microbial community associated with the cheese by culture-dependent molecular approach and to preserve autochthonous microbial consortia ; (iii) to determine the size distributions of casein micelles in bulk raw sheep milk, casein particles in curd and the cheese by Dynamic Light Scattering (DLS) during lactation period. EXPERIMENTS: Eleven cheeses were obtained from a dairy farm at 14 day intervals during lactation period of east Friesian sheep in 2007. Bacterial bulk DNA was isolated by Maxwell®16 DNA instrument (Tissue DNA Purification Kit) from 99 microbial consortia harvested from three culture media (M17, Rogosa, CATC) on the 1st, 2nd and 3rd day of the cheese shelf life and directly from 33 cheeses, respectively. The extracted bulk DNA samples (n = 132) were used as the templates 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 reach the number of E+07 to E+08 CFU/g, while Leuconostoc pseudomesenteroides, Enterococcus faecalis, and Lactobacillus versmoldensis were identified only at lower dilutions (-2, -3). Dual culture-independent molecular approach also unequivocally confirmed predominance of indigenous lactococci in the cheese. The samples for DLS analysis (bulk milk, n = 11 ; curd, n = 10 ; cheese, n = 10) were collected in two weeks intervals during lactation of east Friesian sheep. Casein micelles and particles were isolated from the samples and suspended in buffer with pH value adjusted to the pH value of corresponding sample. A significant variation of casein micelles sizes (mean sizes by number, dh, varied from 87 - 213 nm) was observed in milk during lactation. No such variation was observed for particles in cheese (dh = 11.77 – 14.99 ) and curd samples (dh = 11.54 – 16.45 nm). CONCLUSION AND IMPACT OF THE STUDY: The indigenous microbiota of the cheese demonstrated very homogenous structure and stability during production season indicating specific microbial ecosystem. The isolated and preserved autochthonous microbial consortia of the vanishing cheese might be used as a valuable pool of strains for further genetic and functional characterizations and development of indigenous starter culture. The DLS method demonstrated to be a promising tool for evaluation of the cheese nanostructure. :::: PAPERS: (1)Pogačić, T.*, D’Andrea, M., Kagkli, D-M., Corich, V., Giacomini, A., Baldan, E., Čanžek Majhenič, A., Obermajer, T., Rogelj, I., Samaržija, D. (2011): Biodiversity of microbial consortia isolated from traditional fresh sheep cheese Karakačanski skakutanac. Mljekarstvo / Dairy 61 (3) 208-219. IF = 0. (2)Pogačić, T.*, Samaržija, D., Corich, V., D’Andrea, M., Kagkli, D-M., Giacomini, A., Čanžek Majhenič, A., Rogelj, I. (2010): Microbiota of Karakačanski skakutanac, an artisanal fresh sheep cheese studied by culture-independent PCR-ARDRA and PCR-DGGE. Dairy Science & Technology, 90 (4) 461–468. The original publication is available at www.dairy-journal.org, IF = 1.154. (3)Pogačić, T., et al. (manuscript): Size distribution of casein particles in raw sheep milk, cheese curd and fresh sheep cheese studied by Dynamic Light Scattering during lactation period.

traditional fresh sheep cheese Karakačanski skakutanac; indigenous microbiota; microbial consortia; bulk DNA; 16 S rRNA gene; PCR-ARDRA; PCR-DGGE; Lactococcus lactis; casein micelle; DLS; casein particles

Istraživanja su provedena tijekom 2007 i 2008 godine u: Zavodu za mljekarstvo Agronomskog fakulteta Sveučilišta u Zagrebu ; Zavodu za fizičku kemiju Instituta Ruđer Boškoviću Zagrebu ; Katedri za mljekarstvo Biotehničkog fakulteta Sveučilišta u Ljubljani ; Laboratoriju za mikrobiologiju, Conegliano Campus, Sveučilište u Padovi ; Zavodu za poljoprivrednu biotehnologiju Agronomskog fakulteta Sveučilišta u Padovi ; BMR Genomics, Sveučilište u Padovi. U Zavodu za mljekarstvo su pripremljeni uzorci za DLS analizu koja je provedena u Zavodu za fizičku kemiju tijekom 2007. Kultiviranje mikrobnih konzorcija, izolacija DNA iz konzorcija i izolacija DNA direktno iz sira je provedena na Katedri za mljekarstvo od 17.3.2008 do 8.4 2008. Molekularne analize (PCR-ARDRA, PCR-DGGE, sekvenciranje 16S rRNA gena) na 132 DNA izolata su provedene u navedenim istraživačkim institucijama Sveučilišta u Padovi od 5.5.2008 do 31.7.2008. Eksperimentalni rad u institucijama u Hrvatskoj i Sloveniji je financiran iz projekta Funkcionalni mliječni proizvodi od ovčjeg mlijeka (178-1782128-2124 ; MZOS RH). Eksperimentalni rad u institucijama u Italiji je financiran samo iz projekta LATSIER (R