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.

摘要 细菌素（Bacteriocins）是多种细菌，尤其是乳酸菌（lactic acid bacteria）产生的肽类物质，对腐败菌与食源性致病菌具有广谱抑菌活性。细菌素定量或定性检测技术的成功应用，不仅取决于受试微生物的灵敏度，同时也与所使用的琼脂培养基密切相关。科研人员通常采用琼脂孔穴扩散法（agar well diffusion method），以无细胞上清液（cell free supernatants）对细菌的抗菌活性进行初步筛选，但无细胞上清液在制备过程中可能意外释放其他分子（如培养基组分和/或细胞内化合物），这些物质可能干扰检测结果。针对同一受试微生物的细菌素活性检测重现性，是实验设计中需重点考量的关键因素。遗憾的是，现有学术文献中尚未见关于细菌素活性检测生物测定法（bioassays）标准化的明确规范。本研究通过琼脂孔穴扩散法开展生长抑制实验，结果显示实验结果对所用琼脂培养基存在极强依赖性，表明抑菌效果可能也与无细胞上清液中包含的渗出物的扩散行为相关。本研究结果表明，琼脂培养基的选择对生物测定反应结果至关重要。