Occurrence and Characterization of Non-starter Lactic Acid Bacteria Producing Pediocin PA-1/AcH-like Bacteriocin in Retail Cheddar Cheese

Lactic acid bacteria are known to produce antimicrobial compounds, including organic acids, hydrogen peroxide, and bacteriocins. However, bacteriocins have attracted strong interest as safe alternative food preservatives. In this study, bacteriocin producing non-starter lactic acid bacteria (NSLAB) were isolated from Cheddar cheese and characterized. A total of 300 NSLAB were isolated from retail Cheddar cheese. Of these, 130 isolates were found to have inhibitory activity. The isolates were grouped into five groups based on morphological and physiological characteristics and fifteen isolates were chosen as representatives for further studies. The representative isolates had antimicrobial activity against several foodborne pathogens: Listeria monocytogenes, Bacillus cereus, Salmonella typhi, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, and E. coli O157: H7. The antimicrobial activity of the supernatants was not affected by neutralization with NaOH, treatment with catalase, trypsin, lipase A and amylase. However, treatment with proteinase K resulted in partial inactivation of the supernatants suggesting that the compounds are bacteriocins. The mode of action of these bacteriocins was found to be bactericidal as revealed by Evos digital fluorescence microscopy. This was confirmed by the intra cellular detection of propidium iodide stain in most of the cells after exposure for 12 h, suggestive of a permeable membrane. Further characterization of the bacteriocins showed that they had high thermostability as activity was maintained after boiling for 6.5 h. Also autoclaving for 45 minutes had no significant effect on the activity. Similarly, storage at -20 ºC, 4 ºC, 25 ºC, 37 ºC, and 45 ºC for up to 30 days did not reduce the activity. Also, the activity was maintained at a wide pH range of one to eight. However, addition of sodium chloride 1-7% (w/w) enhanced the activity of the bacteriocins. Sonication at amplitude of 5 for 5 h (discontinuous mode) showed time dependent reduction in activity of the bacteriocins. Analysis of the fifteen representative isolates with API 50CH identified eight out of fifteen isolates to two Lactobacillus species: L. paracasei ssp. paracasei and L. brevis. The remaining isolates were identified to two genera: three Pediococcus sp. and four Lactococcus sp. 16S rRNA identified eight out of fifteen isolates to three Lactobacillus species: L. paracasei, L.=" L. paracasei, " and L. rhamnosus and the other seven isolates to Pediococcus sp. To assess species diversity, the isolates were analysed by pulse field gel electrophoresis using Not I restricted genomic DNA. Pediococccus sp. revealed only one banding pattern while Lactobacillus sp. had multiple banding patterns. However, cell wall protein analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed different banding patterns for each isolate. PCR analysis of the fifteen representative isolates using pediocin primers (PedF and PedR) identified a 322 bp fragment corresponding to pediocin in both the Lactobacillus and Pediococcus strains. Furthermore, PCR amplification with PEDRPO and PEDC1041 showed the presence of a 1044 bp DNA fragment corresponding in size to the structural gene of pediocin PA-1 in all fifteen selected isolates. Hind III digestion of pediocin PA-1 amplicons yielded two different profiles, 10 isolates showed 2 fragments while the other 5 showed one fragment suggesting possible difference in the genetic sequences of the pediocin PA-1 gene. The pediocin PA-1 structural gene seems to be located in the chromosome as no plasmid was detected under various growth conditions.