Importance of the agar-media in the evaluation of bacteriocin activity against the same test-microorganisms

Abstract

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms, but also on the agar-medium employed. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same test-microorganisms is an important factor to be considered. Unfortunately, no specific information about bioassays standardization to determine bacteriocin activity is available in the literature. In this work, growth inhibition by means of the agar well diffusion assays were carried out on different agar-media showing a strong dependence on the agar-medium used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The results presented in this communication show that selection of the agar-medium is crucial for the bioassay response.

Isolation of Enterococcus Species from Food Sources and Its Antibacterial Activity against Staphylococcus Aureus

“…However, this supernatant may include other medium components and/or intracellular compounds. These interfering molecules are accidently released during cell free supernatant preparation and may affect the results [11]. The molecular weight of the antibacterial substance may also affect the results of inhibition in the well diffusion method by affecting the infusion rate [12].…”

Assessing inhibitory activity of probiotic culture supernatants against Pseudomonas aeruginosa: a comparative methodology between agar diffusion, broth culture and microcalorimetry

“…However, this method is dependent on the inhibitory compound diffusing through the agar which could be affected by size, medium components and other molecules secreted by the bacteria which could possibly react (Arena et al 2016). Different sensitivities has also been demonstrated to exist between different agar medium (Azevedo et al 2018). Using IMC, inhibitory activity of neutralised CFS was detected which could not be verified with broth culture and the agar well diffusion methods due to lower sensitivities of the latter methods in recognizing subtle inhibitory effects and possibility of poor diffusion and interaction within the agar medium.…”

Recipe for Success: Suggestions and Recommendations for the Isolation and Characterisation of Bacteriocins

“…Finally, it has been observed that once removed from their habitat isolates may need to be stimulated to induce bacteriocin production. Moving away from the natural environment has previously been observed to cause a decline or inconsistencies in peptide production [59][60][61]. By inducing habitat-specific stresses through media composition and culture conditions, antimicrobial production can increase significantly [62].…”