Characterization and determination of bacteriocins produced by autochthonous Lactococci

Abstract

Lactic acid bacteria (LAB), synthesized a number of different antimicrobial substances among wich bacteriocins are the most important. Bacteriocins of LAB, with regard to the GRAS (Generaly Recognized As Safe) status of LAB, can be used as food preservatives, but also in pharmaceutical industry as an alternative or supplement to antibiotic therapy. A large number of species of the genus Lactococcus sp., not only to play a key role in the production of fermented food where they contribute to specific aroma and structure of fermented products, but also they have ability to synthesize different bacteriocins. Bacteriocins are small, ribosomaly synthesized peptides or proteins with relatively narrow spectrum of antimicrobial activity which is limited to closely related species, and producer has specific mechanism of self-protection. The aim of this thesis was to select a natural isolates of lactococci which have ability to produce bacteriocins. Then, to characterize the selected bacteriocins, to determine their antimicrobial potential and analyze potential applications in the food industry. The strains Lactoccus lactis ssp. lactis BGBM50, LMG2081 and BGBU1-4, whose bacteriocins were analyzed in this paper, were determined using conventional microbiological-biochemical tests and molecular methods of determination (sequencing of gene for 16S rRNA). Testing ability of lactococci to produce bacteriocins, was performed using bacteriocin activity assay in which as sensitive strains were used lactococci, lactobacilli and pathogenic bacteria. Determinations of bacteriocins were performed using cross-resistance test between bacteriocin producer strains and by using PCR method with specific primers for the knowing genes coding for different bacteriocins. It was found that the strain L. lactis ssp. lactis BGBM50 synthesizes bacteriocin named BacBM50. Plasmid curing experiments showed that genes for bacteriocin production are located on the big plasmid of 145,5 kb...

Bakterije mlečne kiseline (BMK), sintetišu veliki broj antimikrobnih jedinjenja, među kojima značajno mesto zauzimaju bakteriocini. Bakteriocini, s obzirom na GRAS (Generalno Prepoznati Kao Sigurni) status koje imaju BMK, mogu se primenjivati kao konzervansi hrane, ali i u farmaceutskoj industriji kao alternativa ili dodatak antibiotskoj terapiji. Veliki broj vrsta roda Lactococcus sp., pored toga što zauzima ključnu ulogu u proizvodnji fermentisane hrane gde doprinose specifičnoj aromi i strukturi fermentisanih proizvoda, ima i sposobnost sinteze različitih bakteriocina. Bakteriocini su mali, ribozomalno sintetisani peptidi ili proteini koji imaju relativno uzak spektar antimikrobne aktivnosti koja je ograničena na blisko srodne vrste i prema kojoj soj proizvođač ima mehanizam specifične samozaštite. Cilj ove teze, bio je da se odaberu prirodni izolati laktokoka koji imaju sposobnost produkcije bakteriocina. Zatim, da se odabrani bakteriocini okarakterišu, da se utvrdi njihov antimikrobni potencijal i analizira potencijalna primena u prehrambenoj industriji. Sojevi L. lactis ssp. lactis BGBM50, LMG2081 i BGBU1-4, čiji su bakteriocini analizirani u ovom radu, determinisani su korišćenjem klasičnih mikrobiološko-biohemijskih metoda, kao i metoda molekularne determinacije (sekvenciranjem gena za 16S rRNK). Testiranje laktokoka na sposobnost produkcije bakteriocina, urađeno je primenom bakteriocinskih testova sa senzitivnim sojem laktokoka, laktobacila i patogenih bakterija kao indikatorima. Determinacija bakteriocina je određena testovima unakrsne inhibicije između sojeva bakteriocin proizvođača, korišćenjem PCR metoda sa specifičnim prajmerima za gene koji kodiraju različite bakteriocine i analizom sekvence genoma...