枯草芽孢杆菌微囊泡的高效分离及其抗炎活性研究

Study on the Efficient Isolation and Anti-bacterial Activity of Bacillus Subtilis Micro-vesicles

建立枯草芽孢杆菌(Bacillus subtilis)微囊泡(Microvesicles,MV_(S))的高效分离方法,评估枯草芽孢杆菌微囊泡(BS-MV_(S))的抗菌和抗炎活性。通过筛选B.subtilis 2-1和B.subtilis 579菌株培养基和培养条件提高BS-MV_(S)的产量;使用超速离心和密度梯度离心对BS-MV_(S)进行纯化,采用滤纸片扩散法检测BS-MV_(S)的体外抑菌活性,筛选抑菌活性较好的BS-MV_(S),通过建立DSS诱导的溃疡性结肠炎模型,进行BS-MV_(S)的抗炎活性研究。确定了高产BS-MV_(S)的培养基为添加0.5%葡萄糖的LB培养基,培养时间为24 h,提高了BS-MV_(S)分离效率。抗菌活性研究结果表明1×10^(10)颗粒数/mL的BS_(2-1)-MV_(S)和BS_(579)-MV_(S)对白色念珠菌的抑菌效率分别为135%、99.2%,对金黄色葡萄球菌的抑菌效率分别为90%、62.5%。基于体外抗菌实验结果,对BS_(2-1)-MV_(S)进行体内抗炎活性研究,结果表明BS_(2-1)-MV_(S)可以有效改善DSS诱导的溃疡性结肠炎的病症症状,包括小鼠体重、疾病活动指数、结肠长度和组织病变程度等,有较强的抗炎活性。结论:通过优化培养基和培养条件提高MV_(S)的产量,BS_(2-1)-MV_(S)对白色念珠菌、金黄色葡萄球菌的抑菌效率均高于BS_(579)-MV_(S),且BS_(2-1)-MV_(S)具有较强的抗炎活性,为今后新型高效抗炎药物的开发奠定基础。

To establish an efficient isolation method of Bacillus subtilis Microvesicles(MV_(S))and evaluate the antibacterial and anti-inflammatory activities of B.subtilis microvesicles(BS-MV_(S)).Methods:The yield of BS-MV_(S) was increased by screening B.subtilis 2-1 and B.subtilis 579 strains in medium and culture conditions.BS-MV_(S) was purified by ultra-fast centrifugation and density gradient centrifugation,and the in vitro antibacterial activity of BS-MV_(S) was detected by filter paper diffusion method,and better antibacterial activity was screened.The anti-inflammatory activity of BS-MV_(S) was studied by establishing a DSS induced ulcerative colitis model.Results:LB medium with 0.5%glucose was selected as the medium for high yield of BS-MV_(S).The culture time was 24 h,and the separation efficiency of BS-MV_(S) was increased.The antibacterial activity of BS_(2-1)-MV_(S) and BS_(579)-MV_(S) with 1×10^(10) particle number/mL were 135%and 99.2%against Candida albicans,and 90%and 62.5%against Staphylococcus aureus,respectively.Based on the results of antibacterial experiments in vitro,the anti-inflammatory activity of BS_(2-1)-MV_(S) in vivo was studied.The results showed that BS_(2-1)-MV_(S) can effectively improve the symptoms of DSS induced ulcerative colitis in mice,including body weight,disease activity index,colon length and tissue lesion degree,and has strong anti-inflammatory activity.Conclusion:By optimizing the medium and culture conditions,the yield of MV_(S) was increased.The antibacterial efficiency of BS_(2-1)-MV_(S) against C.albicans and S.aureus was higher than that of BS_(579)-MV_(S),and BS_(2-1)-MV_(S) had strong anti-inflammatory activity,which laid the foundation for the development of new high-efficiency anti-inflammatory drugs in the future.