Identification et caractérisation de bactériocines produites par deux bactéries lactiques (Enterococcus faecium CWBI-B1430 et Enterococcus mundtii CWBI-B1431) isolées à partir de fromages frais artisanaux péruviens

Doctoral thesis (2011)

A screening of lactic acid bacteria (LAB) with bacteriocin-like inhibitory activity against Listeria monocytogenes was performed. The screening was conducted in 27 traditional artisanal-produced cheeses ... [more ▼]

A screening of lactic acid bacteria (LAB) with bacteriocin-like inhibitory activity against Listeria monocytogenes was performed. The screening was conducted in 27 traditional artisanal-produced cheeses obtained from different Peruvian regions. Two thousand two hundred and seventy seven strains (2277) were isolated, of which 20 exhibited bacteriocin-like inhibitory activity, two strains stand out for having higher activity against listeria, as well as stability at pH value of 2-11 and a thermal stability. The two strains were identified using 16 S ribosomal DNA method, and were registered in the GenBank database of NCBI (National Center for Biotechnology Information). These were Enterococcus faecium CWBI-B1430 (accession number EF591817) and Enterococcus mundtii CWBI-B1431 (accession number EF591816). Two of the major factors concerned in a potential health risk associated with the genus Enterococcus were evaluated, and it was demonstrated that they were sensitive to vancomycin and showed no hemolytic activity. Genetic studies showed the presence of four putative bacteriocin genes in both strains, these were enterocin A, enterocin B, enterocin P, and mundticin KS. The reverse transcription-PCR analysis was realized, the expression of the bacteriocin genes enterocin A, enterocin B, and mundticin KS in CWBI-B1430 was showed, while bacteriocin genes enterocin P and mundticin KS was demonstrated in CWBI-B1431. These results are the first report of the expression of mundticin KS in E. faecium and enterocin P in E. mundtii. Also, the expression was studied at different temperatures, unlike the enterocin P, where only a positive signal was obtained at 12 and 37°C. All other enterocin showed positive signal at 12, 30, 37 and 42°C. The production of bacteriocins on a small scale, in both strains was studied. It was investigate the influence of initial pH (4.5, 5.0, 5.5, 6.0, 6.5 and 7.0), temperature (25, 30 and 37°C) and oxygen level (anaerobic, microaerobic and aerobic). The conditions for maximizing the production of bacteriocins are : initial pH of 7.0, at 37 °C, and microaerobic conditions for CWBI-B1430 strain; initial pH of 6.5, at 37 °C, and an aerobic condition for CWBI-B1431. The influence of the source of carbon and nitrogen, as well as their concentration in the bacteriocin production, was studied. The study of the production of short-chain fatty acids by these strains of multiproducing bacteriocins, showed the propionic acid as the most important. The purification study confirmed that the two bacteria in question are multi-producing of bacteriocins, as shown by the obtained chromatograms, showing different molecular weights, different charge and hydrophobicity level. The results of in situ application of the two selected strains or their supernatant does not show the same efficiency as those obtained in vitro. Factors such as temperature and food composition may be the origin of these results. Therefore, we propose the use of both strains in the food industry, for being multi-producing bacteriocins, have broad spectrum of inhibition and because the substances that produce have functional properties that are useful in the food industry. [less ▲]