Isolamento e caracterização fenotípica e molecular de bactérias ácido lácticas bacteriocinogênicas em leite in natura da região oeste de Santa Catarina.

Abstract

Lactic acid bacteria (LAB) are a group of microorganisms that may be present in many foods, including raw milk, and have the capacity of produce several substances with antagonist activity, such as organic acids, hydrogen peroxide and bacteriocins, where can have the potential to be used in the conservation of foods. The aim of this study was to carry out the isolation of bacteriocinogenic LAB and perform its biochemical and molecular characterization from raw milk in the west region of Santa Catarina, Brazil. Eighteen samples of raw milk were collected from a reception tank at different days in a dairy industry. After, the isolates were inoculated in the MRS agar and M17 agar, and then incubated at 25 and 37°C, under aerobic and anaerobic conditions, being the first for 72h and the second for 48 hours. The average counting of LAB was 3.2 to 7.46 log CFU.mL-1 of milk. A total of 478 Gram-positive and catalase-negative bacteria were isolated. Using the spot-on-the-lawn method, all isolates were evaluated regarding of the antagonistic activity against Listeria monocytogenes on MRS agar, BHI agar and BHI agar with catalase (BHI + catalase). Also, the identity of the antagonism was performed through the same spot-on-thelawn method. From those, 28 isolates that showed antagonistic reaction against L. monocytogenes, and in which the antagonistic activity it was a peptide, confirmed by sensitivity test for at least one of the enzymes,pepsin, -chymotrypsin, proteinase K or trypsin, were then identified at genus and species levels by the ViteK®2 system. Twenty four isolates were identified by this device, being twenty Enterococccus faecium, three Leuconostoc pesudomesenteroides and one Enterococcus gallinarum. Among the 20 isolates identified biochemically as E. faecium, 16 were confirmed at genus level by PCR and at species level through sequencing of the gene pheS. These 16 isolates were grouped by Rep-PCR and evaluated by PCR according to the presence of enterocins genes A, B, P and L50A/B. All the isolates carried at least one of the genes of these enterocins and, despite of having generated four different molecular profiles of Rep-PCR, there was not a direct relation between the presence of enterocins genes and the molecular profiles. Moreover, these 16 isolates were evaluated by two markers of virulence: activity of the -hemolysin and sensitivity the clinical antimicrobials. None of the isolates showed activity of the -hemolysin enzyme. The sensitivity to antimicrobials was evaluated by disc diffusion method, using the antibiotics ampicillin (10μg), vancomycin (30μg), tetracycline (30μg) and penicillin G (10μg). All isolates were sensitivity to vancomycin, and had 94% to ampicillin, and 88% of sensitivity to tetracycline. The highest levels of resistance were for penicillin (44%) and tetracycline (19%). It was verified that the isolates of E. faecium from raw milk of the west region of Santa Catarina presents great potential to be used as bioconservation. However, there is a necessity to conduct further studies to verify the presence of virulence genes in these isolates and/or the feasibility of the enterocins purification for its uses in foods.