源自发酵凡纳滨对虾的抗菌肽BCE3对蜡样芽孢杆菌的抑菌机制及其在米饭中的应用

The antimicrobial peptide BCE3 isolated from fermented Penaeus vannamei:inhibition mechanism on Bacillus cereus and application in rice

【目的】利用枯草芽孢杆菌(Bacillus subtilis)发酵凡纳滨对虾(Penaeus vannamei)加工废弃物,并从发酵液中鉴定出一种新型抗菌肽(命名为BCE3),探究其对蜡样芽孢杆菌(Bacillus cereus)的抑菌作用和机制。【方法】通过超高效液相色谱-质谱联用技术(ultra performance liquid chromatography tandem mass spectrometry,UPLC-MS)对发酵液中的小分子多肽序列进行鉴定,并进一步通过生物信息学筛选潜在抗菌肽;通过微量稀释法和平板涂布法分析BCE3对蜡样芽孢杆菌的最低抑菌浓度(minimum inhibitory concentration,MIC)、最低杀菌浓度(minimum bactericidal concentration,MBC)和时间-杀伤曲线(time-kill curve);通过碱性磷酸酶(alkaline phosphatase,AKP)泄漏、碘化丙啶(propidium iodide,PI)染色分析、核酸和蛋白质泄漏实验以及流式细胞术分析BCE3对细菌细胞壁和细胞膜的影响;通过DNA凝胶阻滞实验、与EB竞争性结合的荧光光谱实验以及分子对接模拟实验探究BCE3对细菌DNA的影响;通过菌落计数法探究BCE3在米饭中的抑菌作用。【结果】利用生物信息学筛选出潜在抗菌肽BCE3,其对蜡样芽孢杆菌的MIC和MBC分别为62.5μg/mL和125.0μg/mL,其可在3 h内使细菌数减少86.0%(62.5μg/mL),与乳酸链球菌素(nisin)相比具有更好的杀菌效果。BCE3破坏细菌细胞壁和细胞膜,导致细胞胞内核酸和蛋白质泄漏,并与细菌DNA结合并导致细菌死亡。另外,BCE3(125.0μg/mL)对米饭中蜡样芽孢杆菌的生长具有明显的抑制作用。【结论】BCE3能够改变蜡样芽孢杆菌细胞膜的通透性,同时与细菌的DNA结合导致细菌死亡。这些发现为BCE3应用于蜡样芽孢杆菌的防治提供了理论基础。

[Objective]To study the inhibitory effect and mechanism of a novel antimicrobial peptide(BCE3)isolated from Penaeus vannamei processing waste fermented with Bacillus subtilis against Bacillus cereus.[Methods]The small peptide sequences in the fermentation broth were identified by ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS),and the potential antimicrobial peptides were screened by bioinformatics.The minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),and time-kill curve of BCE3 against B.cereus were determined by the microdilution method and plate colony counting method.Then,the alkaline phosphatase release assay,propidium iodide(PI)staining,nucleic acid and protein leakage assays,and flow cytometry were employed to examine the effects of BCE3 on the cell wall and cell membrane of B.cereus.The effect of BCE3 on bacterial DNA was explored by the gel retardation assay,fluorescence spectroscopy,and molecular docking.Finally,the antimicrobial effects of BCE3 in rice were evaluated by the colony counting method.[Results]The potential antimicrobial peptide BCE3 screened out showed the MIC of 62.5μg/mL and MBC of 125.0μg/mL against B.cereus.The time-kill curve revealed that BCE3 reduced the bacterial count by 86.0%within 3 h(62.5μg/mL),outperforming nisin.BCE3 caused damage to the bacterial cell wall and membrane,leading to the leakage of cell contents.Moreover,it can bind with DNA to kill the bacteria.In addition,BCE3(125.0μg/mL)exerted a significant inhibitory effect on the growth of B.cereus in rice.[Conclusion]BCE3 inhibits B.cereus by altering the permeability of the cell membrane and binding to DNA,thus leading to bacterial death.These findings provide a theoretical basis for application of BCE3 in the control of B.cereus.