侧孢短芽孢杆菌抗菌肽Brevilacterin结构解析与抑菌机理研究

Biopreservatives have gained increasing attention in food industry due to the characteristic of high safety, while their applications are limited by the fact that only a few kinds are approved, and most of them show narrow antimicrobial spectrum. Brevilacterin, a series of antimicrobial peptides produced by Brevibacillus laterosporus, show broad antimicrobial spectrum against pathogenic and spoilage microorganisms, high thermal and pH stability, thus they show great potential to be developed as a novel promising food biopreservative. However, the structures and antibacterial mechanism remain unknown. Therefore, the aim of this project is first to elucidate the structures of Brevilacterin, then to investigate the relationship between antibacterial activity and structure, and finally to clarify the antibacterial action mode using Staphylococcus aureus and Escherichia coli as mode bacteria from cellular and molecular level. Three action targets (i.e., cell membrane, DNA, proteome) will be analyzed to gain the insight into the antibacterial mechanism of Brevilacterin: First, the effect of Brevilacterin on membrane ultrastructure, integrity, permeability, fluidity and proton motive force will be investigated by transmission electron microscopy, fluorescent spectrometry and flow cytometry, respectively; Then, the effect of Brevilacterin on DNA will be studied by florescent real-time quantitative PCR; Finally, the effect of Brevilacterin on differential expression of proteome will be investigated by iTRAQ-based quantitative proteomic analysis, and the metabolic pathways will be speculated by bioinformatics analysis. In brief, the project would elucidate the inhibitory mechanism from cellular and molecular level, so as to provide theoretical foundation for the investigation of microbial biopreservative .

生物防腐剂的高度安全性受到食品工业青睐，但现有产品种类少与抑菌谱窄等问题限制了其广泛应用。Brevilacterin为申请人前期研究的一组具有抑菌谱广、酸热稳定性高的侧孢短芽孢杆菌抗菌肽，有望被开发为新型生物防腐剂。然而，Brevilacterin的结构与抑菌机制尚未明确。因此，本项目旨在对Brevilacterin结构表征基础上，阐明其结构与抑菌活性关系；以金黄色葡萄球菌和大肠杆菌为指示菌，通过透射电镜、荧光光谱、流式细胞术等分析Brevilacterin对细胞膜渗透性、完整性、流动性、质子动势等的影响；荧光实时定量PCR等技术分析Brevilacterin对DNA的影响；iTRAQ技术分析Brevilacterin对蛋白质组表达差异，生物信息学技术分析对代谢通路影响，从细胞和分子水平阐明Brevilacterin的抑菌机理。项目实施可为研究微生物源新型生物防腐剂奠定理论基础。

天然食品防腐剂是控制微生物污染、保障食品安全的重要手段。项目组前期研究选育出抗菌肽高活力菌株侧孢短芽孢杆菌S62-9，研究发现其抗菌肽（Brevilaterin）具有高效、稳定抗菌活性，有望开发为新型的天然食品防腐剂，但其结构及抗菌机制尚未明确。因此，本项目首先采用发酵-液相色谱法成功分离五种高纯抗菌肽组分Brevilaterin A、B、C、D、E；然后，通过MALDI-TOF/TOF-MS、2D-NMR及圆二色谱等技术完成Brevilaterin结构解析，确定各组分一级结构均为含13个氨基酸残基的阳离子脂肽，二级结构主要为无规则卷曲。Brevilaterin具有广谱抗菌活性，尤其对革兰氏阳性菌具有高效抗菌活性，但组分间抗菌活性无显著差异。.在结构及活性研究基础上，项目从细胞水平、模型膜角度及分子水平层层深入，进一步揭示了Brevilaterin的抗菌作用机制：Brevilaterin通过使细胞膜发生去极化作用，增加膜渗透性，破坏细胞膜完整性，最终导致细胞死亡；采用模型膜明确了Brevilaterin与膜的结合位点为磷酸基；基于转录组、实时荧光定量PCR及蛋白组学等技术，在分子水平上深入挖掘了Brevilaterin作用下单增李斯特氏菌关键差异基因及关键蛋白的变化，发现Brevilaterin主要影响单增李斯特氏菌多条细胞膜相关的代谢途径，包括抑制肽聚糖生物合成、寡肽转运、ATP合成等过程，促进钾离子转运，同时激活阳离子抗菌肽抗性等应激途径。.综上，本项目完成了侧孢短芽孢杆菌抗菌肽Brevilaterin的结构解析工作，并从细胞及分子水平揭示其抗菌作用机制，为Brevilaterin的进一步开发应用奠定了理论基础。项目实施期间共计发表学术论文23篇，其中SCI论文12篇，EI收录7篇，核心4篇；授权发明专利2项；培养2名博士研究生、6名硕士研究生毕业，培养1名博士后出站。完成项目书约定的研究内容及考核指标。