Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese

Abstract

A b s t r a c tDuring 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). 280and Tetragenococcus halophilus) have previously been detected in Cotija cheese (Morales et al., 2011) without identification of those producing bacteriocins.The present work, aimed to identify the halotolerant lactic acid bacteria isolated from Cotija cheese and its antimicrobial activity. The bacteriocin produced by the isolate were also characterized. Experimental Materials and MethodsScreening for bacteriocin-producing lactic acid bacteria. Cotija cheese samples were collected from the mountain range of Michoacán, México as 20 g of cheese were added to 180 ml of peptone water. Then, the mixture was serially diluted with peptone water and plated on MRS and M-17 agar (Oxoid, Basingstoke, Hampshire, England) and supplemented with cycloheximide at 1 µg/ml to prevent growth of yeast and molds. The plates were incubated at 28°C for two to three days. The isolates were evaluated by cell morphology and Gram's stain reaction. The isolates were screened for bacteriocin production using the spot on the lawn assay using MRS plates (Moraes et al., 2010). Five µl of each isolated strain was spotted on MRS plates and incubated at 28°C for 24 h. After incubation, the plates were overlaid with 10 ml of BHI soft agar and inoculated with 10 5 CFU/ml of every tested pathogenic indicator strain. Then, the inhibition zone around the spots revealed potency of antibacterial activity.Biochemical identification of isolated bacterio cinproducers. Phenotypic identification was performed using Gram´s staining and an H 2 O 2 production test. Fermentation of carbohydrates was done by API 50 CHL system (BioMérieux, Inc,.Hazelwood Missouri, USA).Identification of the isolate by 16S rDNA sequencing and phylogenetic analysis. Bacteriocin-like substance producers were identified by 16S rDNA sequencing to confirm the results obtained from the biochemical identification. DNA was extracted using the modified method described by Wilson (2001)...

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“…cholerae O1 Ogawa . 50 The inhibitory activity of Pediococcus spp. (isolated from cheese) against Listeria species was reported by Cavicchioli et al 51 They indicated that bacteriocins produced by E .…”

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes

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“…Ped. acidilactici QC38 which produces the bacteriocin Pediocin PA-1 was isolated from Cotija cheese, a ripened artisanal product of raw milk, produced in the state of Michoacán [ 48 ]. Ent.…”

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