Determination of autochthonous lactic acid bacteria from kashkaval cheese

Abstract

Autochthonous isolates of lactic acid bacteria and enterococci from Kashkaval cheese had been isolated and identified. From 6 samples of Kashkaval cheese that were traditionally produced without starter cultures, 27 isolates were isolated using selective media (MRS agar, M17 agar) and two incubation temperatures (37ºC and 43ºC). According to Gram staining, cell morophology and catalase test, it was determined that all isolates were pure culture of G (+) and catalase (-) bacteria, 12 bacilli and 15 cocci. For the purpose of diferentiation between enterococci and lactococci, growth of all cocci bacteria was examined on 6,5% NaCl, pH 9,6 and heat treatment 60ºC 20 min. Out of 15 cocci isolates, 6 isolates were able to grow at these conditions. Acidogenic activity was determinated by measuring pH values of 1% inoculated reconstituted skim milk during 24 hours. Researched isolates showed significant difference in lactic acid production. Ten isolates had good acidogenic activity (pH 4,12-4,90) and seventeen isolates were weak producers (pH 5,10 - 6,48). Identification by API CHL 50 test for lactobacilli and RAPID ID 32 Strep for cocci, showed that isolates were Lactobacillus delbrueckii ssp. delbrueckii, Lactobacillus casei, Lactobacillus plantarum Lactobacillus fermentum, Lactobacillus curvatus, Streptococcus salivarius ssp. thermophilus, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Enterococcus faecium i Enterococcus durans.

U radu su izdvojeni i okarakterisani izolati bakterija mlečne kiseline i enterokoka iz kačkavalja, proizvedenog bez dodavanja starter kulture. Iz 6 uzoraka kačkavalja različite starosti izdvojeno je 27 izolata na odgovarajućim selektivnim podlogama MRS i M17 agaru i temperaturama rasta od 37ºC i 43ºC. Postupkom bojenja po Gramu, mikroskopskim pregledom i katalaza testom je utvrđeno da svi izolati predstavljaju čiste kulture Gram-pozitivnih i katalaza-negativnih bakterija, pri čemu je utvrđeno da je 12 izolata štapićastog, a 15 okruglastog oblika.. Radi razdvajanja enterokoka od bakterija mlečne kiseline, za sve okruglaste forme je ispitana sposobnost rasta pri 6,5% NaCl, pH 9,6 i nakon termičkog tretmana na 60ºC 20 minuta utvrđeno je da od 15 izolata okruglastog oblika, 6 izolata podnosi navedene uslove. Acidogena aktivnost je određena merenjem pH vrednosti rekonstituisanog obranog mlaka sa 1 % inokuluma u toku 24 sata, pri čemu je utvrđeno da se izolati značajno razlikuju u sposobnosti stvaranja kiseline. Deset izolata je pokazalo dobru acidogenu sposonost (pH 4,12-4,90), dok je 17 izolata slabo produkovalo kiselinu (pH 5,10 - 6,48). Determinacijom dobijenih izolata primenom API sistema i to za štapićaste oblike API CHL 50 testa, a za okruglaste oblike RAPID ID 32 Strep sistema, utvrđeno je da izolati pripadaju vrstama: Lactobacillus delbrueckii ssp. delbrueckii, Lactobacillus casei Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus curvatus Streptococcus salivarius ssp. thermophilus, Lactococcus lactis ssp. lactis Lactococcus lactis ssp. cremoris, Enterococcus faecium i Enterococcus durans.