Lactic acid bacteria isolated from raw milk and minas artisanal cheese with antagonistic potential against pathogens of interest in the dairy production chain

Abstract

Lactic acid bacteria (LAB) have significant potential for application in the dairy production chain due to their ability to produce bioactive molecules with antimicrobial activity, as well as other characteristics of interest to the agri-food industry and health promotion. Samples of Minas artisanal cheese (MAC) (n = 59) from five traditional regions, and raw milk samples from 65 dairy herds in the Campo das Vertentes and South of Minas Gerais regions were used to prospect LAB searching for strains with antagonistic potential against pathogens of interest in the dairy production chain. From a total of 291 LAB isolates obtained from MAC, 84 strains

were considered genetically distinct by REP-PCR and identified at the species level by MALDI- TOF. Among 352 presumptive LAB isolates from raw milk, 221 strains genetically distinct by

rep-PCR were selected for species-level identification via MALDI-TOF. The antagonistic potential of LAB was tested using the spot-on-lawn technique, and the nature of the inhibitory substance was assessed through sensitivity to proteases and amylase. Strains obtained from MAC were tested against important pathogens and contaminants in MAC: Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 5779, and Escherichia coli O157 IAL 1848, whereas LAB isolated from raw milk were evaluated against pathogens causing bovine mastitis: Staphylococcus aureus ATCC 25923, Streptococcus agalactiae ATCC 12386, E. coli O157 IAL 1848, Staphylococcus epidermidis ATCC 12228, Streptococcus uberis ATCC 700407, and E. faecalis ATCC 29212. Susceptibility to antimicrobials: ampicillin, chloramphenicol, cotrimoxazole, erythromycin, streptomycin, gentamicin, penicillin G, tetracycline, and vancomycin was assessed by disk diffusion on agar. Genes encoding antimicrobial resistance (aacA-aphD, ermA/B, tetM/O/K/L/S, blaZ, and vanA/B) were investigated via PCR. The results revealed three superior strains of the genus Lactobacillus spp. (codes 52, 177, and 272G) from MAC, susceptible to antibiotics and bioprotective against L. monocytogenes and E. coli in vitro and in coculture in milk. Among the LAB strains isolated from raw milk, the Lactococcus lactis CV151 stood out for its strong antagonistic activity against all the tested mastitis pathogens, being susceptible to all the antibiotics and showing no resistance genes; Enterococcus faecium CV167 exhibited antagonistic activity against most of the tested pathogens, with three bacteriocin-related genomic regions identified via genomic analyses; and Weissella cibaria CV04 demonstrated excellent antagonistic activity against all the tested mastitis pathogens. These results contribute to advancing the knowledge of the LAB present in milk and MAC, paving the way for applications in agribusiness and animal health.