Bifidobacterium animalis B04所产细菌素分子结构鉴定及抑菌机理研究

Bifidobacterium animalis B04 is the first reported bacteriocin-producing Bifidobacterium strain in China. Bacteriocin B04, produced by Bifidobacterium animalis B04, showed a wide range of antimicrobial activity and better characteristics, which indicated it has significant potential as natural biopreservative in food. In this program, the molecular structure and machanism of inhibitory action of bacteriocin B04 are studied. The molecular weight, amino acid composition, amino acid sequence and three-dimensional structure of purified bacteriocin are determined by using mass spectrometry (MS), circular dichroism (CD)、fourier transform infrared spectrometer (FTIR), nuclear magnetic resonance (NMR) and X-ray. On this basis, the key structural features of bacteriocin B04 in the inhibitory process are clarified. The minimum inhibitory concentration (MIC) and mode of action of bacteriocin B04 are determined by the agar diffusion method, using the bacteriocin-sensitive Listeria monocytogens and Escherichia coli as the tested strain, respectively. The effects of bacteriocin B04 on the cellular structure integrity and membranes permeability of sensitive cells are investigated by atomic absorption spectrometry (AAS), fluophotometer with fluorescent labeling，bioluminescence detector and electronic microscopy. The effect of bacteriocin B04 on intracellular protein expression is assayed by two dimensional gel electrophoresis (2-DE) and high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). The purpose of this program is to make clear the mechanism of inhibitory action of bacteriocin B04 against Gram-positive and Gram-negative strains at the cellular and molecular level. The research results are of great scientific significances to analysis comprehensively the mechanism of inhibitory action and the relationship between structure and activity of bacteriocins from lactic acid bacteria.

Bifidobacterium animalis B04是国内首次报道的产细菌素双歧杆菌，其所产细菌素抑菌谱广、特性优良，有作为天然食品生物防腐剂的巨大潜力。本项目拟在提纯该细菌素基础上，采用质谱、圆二色谱、傅里叶红外光谱、核磁共振及X-射线衍射技术，测定其分子结构，解析在抑菌过程中起关键作用的结构特征；并分别以单增李斯特菌（G+菌）及大肠杆菌（G-菌）为测试敏感菌，通过琼脂平板扩散法确定细菌素最低抑菌浓度及作用方式，通过原子吸收光谱、生物发光测量仪、荧光探针标记、电镜观察等分析细菌素对敏感菌细胞膜通透性及细胞结构完整性的影响，通过双向凝胶电泳结合高效液相色谱-电喷雾串联质谱技术分析细胞内蛋白质的表达差异情况，旨在从细胞和分子水平探明该双歧杆菌细菌素对G+和G-细菌的不同抑菌作用机理。研究结果对于全面解析乳酸菌细菌素的抑菌机制以及结构与活性关系具有重要科学意义。

动物双歧杆菌B04是国内首次报道的产细菌素双歧杆菌，其所产细菌素bifidocin A抑菌谱广、特性优良，有作为天然食品生物防腐剂的巨大应用潜力。本项目拟在提纯该细菌素基础上，测定其分子结构，解析在抑菌过程中起关键作用的结构特征，并从细胞和分子水平探明该双歧杆菌细菌素对G+和G-细菌的不同抑菌作用机理。项目实施后所取得的重要研究结果如下：（1）确立了细菌素bifidocin A的四步高效提取纯化程序，包括收集发酵液→pH值吸附解吸提取→阳离子交换层析→葡聚糖凝胶层析→反相高效液相色谱，经此程序可获得抑菌效价达10240 AU/mL，比活力达5535 AU/mg，纯度达95%的细菌素纯品；（2）初步明确了细菌素的分子结构特征，经MALDI-TOF MS 确定其分子量为1198.7Da；氨基酸序列为Val–Phe–His–Pro–Gly–Thr–Gly–Pro–Pro–Gly–Asp(Leu)；二级结构中主要含有58%以上的无规则卷曲结构、27%左右的β折叠结构和12%左右的β转角结构，是一种非规则蛋白三级结构；（3）基本探明了细菌素对G+和G-细菌的不同抑菌作用机理，细菌素作用G+菌单增李斯特氏菌和G-菌大肠杆菌细胞后，可改变敏感菌细胞形态及破坏其内部结构，引起细胞质子驱动势耗散，形成非选择性孔洞，导致细胞死亡。同时，G-菌的各指标的变化程度明显强于G+菌。经细菌素处理前后敏感菌细胞内蛋白质表达形成差异，其中大肠杆菌共鉴定出10个明显下调及2个明显上调差异蛋白点，它们主要是参与能量代谢、细胞组成以及作为全局胁迫调节因子以适应胁迫条件与细菌素抑菌作用相关的功能蛋白；单增李斯特氏菌鉴定出4个明显上调蛋白点，它们是在维护细胞及膜孔蛋白结构整体性和细胞形态上发挥重要生理功能的蛋白。以上研究结果对于全面解析细菌素结构与活性关系以及抑菌机制具有重要的科学意义，同时为双歧杆菌细菌素作为新型生物防腐剂在食品工业中的应用也提供了理论参考。