Lactic Acid Bacteria isolated from traditional and innovative alheiras as potential biocontrol agents.

ABSTRACT

From a selection of seven traditional and 14 innovative alheiras, 491 lactic acid bacteria (LAB) were isolated and tested for their antimicrobial activity against several food-borne pathogens. Among these, six strains revealed antimicrobial activity through potential bacteriocin production against 14 Listeria monocytogenes strains, Enterococcus faecalis ATCC 29212, Clostridium sporogenes ESB050, and Clostridium perfringens ESB054. Through whole genome sequencing (WGS), these strains were identified as Lactiplantibacillus plantarum (2), Leuconostoc mesenteroides (1), and Pediococcus acidilactici (3). Furthermore, several orthologues of class II bacteriocins genes were identified, including Plantaricin E, Plantaricin F, Pediocin PA, Enterocin X, Leucocin A, and Coagulin A. No virulence or antibiotic resistance genes' orthologues were detected by WGS analysis. However, the selected LAB strains showed variable phenotypic patterns related to virulence genes and antibiotic resistance when assessed through classical methodologies. None of these strains demonstrated the production of biogenic amines, gelatinase or DNase. Additionally, no hemolytic activity or lipase enzyme production was observed. However, only Lpb. plantarum 9A3 was sensitive to all tested antibiotics and was thus chosen for further examination. The bacteriocins produced by Lpb. plantarum (9A3) exhibited stability across a broad range of conditions, including temperatures from 4 to 100 °C, pH values ranging from 2 to 8, exposure to surfactants and detergents (Tween 20 and 80, SDS, EDTA 0.1, 2 and 5 mM, urea and sodium deoxycholate), and enzymes (papain and catalase). Their maximum activity (AU/mL = 12,800) against four L. monocytogenes strains was observed between 21 and 36 h of growth of Lbp. plantarum 9A3, indicating a bacteriostatic mode of action. Therefore, this strain appears to be a robust candidate for potential application as a protective strain to be used in the food industry. Not only is it safe, but it also produces stable bacteriocins (harbouring genes encoding for the production of three) effectively inhibiting significant pathogens such as L. monocytogenes and C. perfringens.