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Cotija cheese is an artisanal matured Mexican cheese from unpasteurized milk. This work determined the microbiological characteristics and behavior of Staphylococcus aureus, Salmonella spp., Listeria monocytogenes and staphylococcal toxin during cheese elaboration and ripening. Sixteen 20-kg cheeses were used, eight inoculated with 6 log CFU/mL of each pathogen, and eight uninoculated, and samples were taken at each stage of the process. In the uninoculated samples, the survival of S. aureus and L. monocytogenes decreased after 30 days of ripening. The average counts of S. aureus in milk, curd, and serum were 7 log MPN /mL, and 8.7 log MPN /g in cheese, decreasing from day 15. Salmonella spp. counts (initial inoculum: 7.2 log MPN /mL) decreased after 24 h, and L. monocytogenes counts (8.7 log MPN/g at 24 h) decreased from day 15 in the cheese. Salmonella spp. and L. monocytogenes were not detected in any sample after 60 days of ripening, unlike S. aureus, which was detected at the end of the study. Lactic acid bacteria counts were 9 log CFU/mL in milk and whey and 7.2 log CFU/g in cheese. Pathogens behavior during the ripening process reduces the health risks by consuming products made with unpasteurized milk when subjected to ripening.

During a screening of lactic acid bacteria producing bacteriocin from Cotija cheese, the strain QC38 was isolated. Based on the 16S rRNA gene nucleotide sequencing (516 pb accession no KJ210322) and phylogenetic analysis, the isolate was identified as Pediococcus acidilactici. Neutralized cell-free supernatant was tested for antimicrobial activity against 17 Gram-negative and Gram-positive pathogens. Growth inhibition was achieved against Listeria monocytogenes (supplier or indication or source), Staphylococcus aureus, Vibrio vulnificus, Vibrio cholerae O1 Ogawa, Vibrio cholerae NO 01 and Salmonella enterica subsp. Enterica serovar Typhimurium. Bacteriocin-like substance, after heating at 121°C for 15 min it remained stable and its antimicrobial activity was observed at pH ranging from 1.0 to 10.0 but inactivated by α-chymotrypsin and proteinase K. Strain QC38 was able to grow in 1–9% NaCl concentration. The plate overlay assay showed an approximate size of bacteriocin-like substance between 3.4 and 6.5 kDa. P. acidilactici QC38 harboured a plasmid that contains a gene for a pediocin (PA-1).