DEVELOPMENT OF A MULTIWELL ANTAGONISTIC ACTIVITY ASSAY FOR THE DETECTION OF BACTERIOCIN PRODUCTION BY LACTIC ACID BACTERIA

摘要

A total of 300 strains of lactic acid bacteria (LAB) were screened for the production of bacteriocins active against Listeria innocua or Escherichia coli by using two different techniques: the conventional well-diffusion assay and a newly developed one based on turbidity measurement of the growth of an indicator bacterium in the neutralized cell-free supernatant of the putative bacteriocin-producing strain. The latter technique, designated as the multiwell antagonistic activity assay (MW3A), offers advantages over the previously known methods. Notably, it allows testing simultaneously a large number of LAB for the production of bacteriocins against more than one indicator strain, while including appropriate controls for confirmation of the bacteriocinogenic nature of the inhibitory substances. Furthermore, five enterococcal strains were shown to produce bacteriocins active against the gram-positive and gram-negative indicator strains used in this study, suggesting that these enterococci or their bacteriocins have good potential to enhance food safety and keeping quality. The present study describes a rapid and semiautomatic nephelometry method to screen for bacteriocin-producing lactic acid bacteria (LAB). The method uses a microtiter plate where the inhibition of the growth of indicator strains in the presence of a bacteriocin is revealed by a decrease in OD as function of time. Concomitantly, this method determines the proteinaceous nature of the antimicrobial substance and its mode of action (i.e., bactericidal or bacteriostatic) while excluding the effect of interfering antimicrobials produced by LAB (e.g., hydrogen peroxide and organic acids). Furthermore, the newly described method differs from previously described ones by its ability to test for a relatively large number of putative bacteriocin-producing strains using different controls and indicator strains, and allow early detection of bacteriocin-producing strains (within 24 h). Another feature of this method is its ability to detect with confidence bacteriocins active against gram-negative bacteria. These performances have potential to be used for rapid detection of LAB having strong capacity to inhibit pathogenic or spoilage bacteria by means of bacteriocins for possible applications in food preservation strategies.