In this study, we examined the ability of nisin A and a rationally assembled bank of 36 nisin derivative producing Lactococcus lactis strains to inhibit Listeria. A broth-based bioluminescence assay for screening single and combinations of bioengineered nisin derivatives using cell-free supernatants (CFS) from nisin derivative producing strains was developed. In this way, we screened 630 combinations of nisin derivative producing strains, identifying two (CFS from M17Q + N20P and M17Q + S29E) which exhibited enhanced anti-listerial activity when used together compared to when used alone, or to the nisin A producing strain. Minimal inhibitory concentration assays performed with purified peptides revealed than when used singly, the specific activities of M17Q, N20P and S29E (3.75–7.5 μM) against L. innocua were equal to, or less than that of nisin A (MIC of 3.75 μM). Broth-based growth curve assays using purified peptides demonstrated that use of the double peptide combinations and a triple peptide combination (M17Q + N20P + S29E) resulted in an extended lag phase of L. innocua, while kill curve assays confirmed the enhanced bactericidal activity of the combinations in comparison to the single derivative peptides or nisin A. Furthermore, the enhanced activity of the M17Q + N20P combination was maintained in a model food system (frankfurter homogenate) at both chill (4 °C) and abusive (20 °C) temperature conditions, with final cell numbers significantly less (1–2 log₁₀ CFU/ml) than those observed with the derivative peptides alone, or nisin A. To our knowledge, this study is the first investigation that combines bioengineered bacteriocins with the aim of discovering a combination with enhanced antimicrobial activity.

在这项研究中，我们检查了乳酸链球菌肽 A 和合理组装的 36 种乳酸链球菌肽衍生物生产乳酸乳球菌菌株抑制李斯特菌的能力。开发了一种基于肉汤的生物发光测定，用于使用来自乳链菌肽衍生物生产菌株的无细胞上清液 (CFS) 筛选生物工程化乳链菌肽衍生物的单一和组合。通过这种方式，我们筛选了 630 种乳链菌肽衍生物产生菌株的组合，确定了两种（来自 M17Q + N20P 和 M17Q + S29E 的 CFS）与单独使用相比，或与乳链菌肽 A 产生菌株相比，它们在一起使用时表现出增强的抗李斯特菌活性. 使用纯化肽进行的最小抑制浓度测定显示，与单独使用相比，M17Q、N20P 和 S29E（3.75–7.5 μM）对无害李斯特菌的比活性等于或小于乳链菌肽 A（MIC 为 3.75 μM）。使用纯化肽的基于肉汤的生长曲线分析表明，使用双肽组合和三肽组合 (M17Q + N20P + S29E) 导致L. innocua 的滞后期延长，而杀灭曲线分析证实了增强的杀菌活性与单一衍生肽或乳酸链球菌肽 A 相比，组合的增强的活性在模型食品系统（法兰克福香肠匀浆）中在冷藏 (4 °C) 和滥用 (20 °C) 下均保持不变温度条件下，最终细胞数显着减少（1–2 log ₁₀ CFU/ml) 比单独使用衍生肽或乳链菌肽 A 观察到的结果高。据我们所知，这项研究是首次将生物工程细菌素结合起来进行研究，目的是发现具有增强抗菌活性的组合。