Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
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.Svrha je ovog istraživanja bila utvrditi raznolikost autohtone mikrobne zajednice laktobacila u istarskom siru te detaljno genetički definirati sojeve. Uzorkovanje mlijeka i svježeg sira provodilo se na tri obiteljska poljoprivredna gospodarstva na području Istre. Ukupno 212 izolata mezofilnih i termofilnih laktobacila, te ukupna zajednica (konzorcij) istraženi su pomoću fenotipskih i genotipskih metoda ovisno o izolaciji na selektivnim hranjivim podlogama. Primjenom biokemijskog testa PhenePlate-LB, 212 izolata preliminarno je svrstano u 16 skupina prema načinu korištenja supstrata. Po jedan reprezentativni izolat laktobacila iz svake PhP skupine genotipski je analiziran PCR analizom i sekvencioniranjem 16S rRNA gena te je određeno kojem rodu i vrsti pripadaju. Sekvencijskom su analizom određene ove vrste: Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei i Lactobacillus rhamnosus. Dodatno je testirana osjetljivost svakog reprezentativnog izolata na sljedeće antibiotike: ampicilin, tetraciklin, penicilin, rifampicin, klindamicin, eritromicin i vankomicin. Kod svih izolata utvrđena je rezistencija na vankomicin, iako nije jasno utvrđen mehanizam osjetljivosti na ostale testirane antibiotike. PCR DGGE analizom ukupne zajednice laktobacila (konzorcija) utvrđeno je da prevladavaju vrste Lactobacillus plantarum i Lactobacillus brevis. Također su pronađene vrste Pediococcus pentosaceus, Pediococcus acidilactici, Streptococcus sp. i Leuconostoc mesenteroides. Prisutnost određenih vrsta u kompleksnoj mikrobnoj zajednici laktobacila istarskog sira potvrđuje rezultate brojnih studija o tradicijskim sirevima, što predstavlja važan autohtoni genetički potencijal za daljnju selekciju izolata
