微酸性电解水对蜡样芽孢杆菌芽孢的杀灭特性与机理研究

The presence of spores in food with germination ability could affect the shelf life of products and food safety. Slightly acidic electrolyzed water (SAEW) with broadly effective bactericidal activity and friendly environmental advantages has been found could effectively inactivate spores, but its mechanism of action is not yet clear. Bacillus cereus which easily cause food poisoning and hence will be taken as the treatment object in this study to evaluate the characteristic of SAEW against spores under different conditions. On this basis, we plan to study the inactivation mechanism of SAEW by determining variations of spores’ structure and germination ability. Scanning electron microscope, transmission electron microscope and flow cytometer will be used to detect the effect of SAEW on changes of spores’ morphology and structure. We will adopt the technologies of differential interference contrast microimaging combined with optical tweezers Raman spectra, Western blot and RT-PCR to determine the variations of spore germination-related proteins and gene expression, dynamic germination procedure in group and in individual, and dynamic procedure of DPA release, respectively. From this study, we would like to verify and confirm the pathway and the target to inactivation spores by SAEW and finally clarify the inactivation mechanism of SAEW on B. cereus spores. This study will provide a new approach for spores killing and lay a theoretical basis for SAEW application in the area of food safety.

食品中残存具有萌发能力的芽孢是影响产品货架期和食品安全性的重要因素。申请人前期研究发现具有广谱高效、安全环保特点的微酸性电解水（Slightly acidic electrolyzed water,SAEW）能够有效杀灭芽孢，但其作用机理尚不明确。本项目以易引起食物中毒的蜡样芽孢杆菌芽孢为对象，研究不同条件下SAEW对芽孢的杀灭特性。在此基础上，从破坏芽孢结构与影响芽孢萌发两方面探究SAEW杀灭芽孢的机理。采用扫描电镜、透射电镜、流式细胞仪等测定SAEW对芽孢形态和各部分结构的损伤；运用微分干涉差显微成像联合光镊拉曼光谱技术、Western blot及RT-PCR等方法测定芽孢群体与个体萌发动态、DPA释放动态、萌发相关蛋白和基因表达的变化，确定SAEW的作用途径与作用位点，阐明SAEW对芽孢的杀灭机理。本研究将为芽孢的杀灭提供新途径，为SAEW在食品安全中的应用奠定理论基础。

食品中残存具有萌发能力的芽孢是影响产品货架期和威胁其安全水平的重要因素。本研究以蜡样芽孢杆菌芽孢为研究对象，探究不同处理条件下微酸性电解水（SAEW）对芽孢的杀灭特性。在此基础上，采用扫描电镜、透射电镜等观测SAEW对芽孢形态和结构的损伤情况，并进一步探究了SAEW处理对芽孢生长和萌发的影响，揭示SAEW对芽孢萌发的影响规律，最终确定SAEW杀灭芽孢的作用途径和作用位点，阐明其杀灭机理。主要研究内容及结果如下：.（1）SAEW杀灭芽孢的基本特性.采用有效氯浓度为40-120mg/L的SAEW分别处理芽孢纯悬液1-50min，并结合温和热处理（60℃），考察SAEW对芽孢的杀灭效果以及对芽孢热抗性损失、2,6-吡啶二羧酸（DPA）释放和芽孢悬液粒径分布的影响，并以NaClO作为对照。结果表明，SAEW对芽孢的杀灭效果高于NaClO，且灭活效果与处理时间和有效氯浓度呈正相关，温和热处理有助于提高其灭活效果。芽孢热抗性损失和DPA释放量随着SAEW处理时间和有效氯浓度的增加而增加。此外，SAEW处理使得芽孢悬液粒径增加，有芽孢聚集体形成。.（2）SAEW对芽孢形态与结构的破坏作用.采用场发射扫描电镜和透射电镜观测SAEW对芽孢形态和结构的影响。结果表明，SAEW处理后的芽孢表面出现凹陷褶皱、破损破裂等形态变化，以及芽孢衣、皮层、芽孢内膜和芽孢核等结构破坏。SAEW处理破坏了芽孢衣，降低了芽孢抗性，但未破坏芽孢DNA。溶菌酶能够提高处理后芽孢的活性。SAEW处理后芽孢对高渗培养基抗性显著降低，在亚致死温度（85℃）下芽孢DPA释放量显著增加，萌发后易被PI染色，表明SAEW后芽孢内膜受损，透性增加。上述研究表明，SAEW处理破坏了芽孢内部结构，其中芽孢内膜、芽孢衣等结构的破坏可能是杀灭芽孢的主要原因。.（3）SAEW对芽孢萌发的影响.SAEW处理后的芽孢生长速度明显减缓，其减缓程度与有效氯浓度和处理时间呈正相关。在营养萌发剂L-缬氨酸和非营养萌发剂十二烷胺诱导下，SAEW处理均能够抑制芽孢萌发的进行，可能是SAEW处理破坏了芽孢内膜上相关萌发受体，芽孢萌发受阻从而无法正常生长。