解淀粉芽孢杆菌B10抑制黄曲霉生长活性物质克隆表达及作用效果研究

Study on Bacillus amyloliquefaciens B10 Inhibiting Cloning and Expression of Growth Active Substance of Aspergillusflavus and Its Efficacy

本试验旨在从解淀粉芽孢杆菌B10中克隆表达出能够抑制黄曲霉生长的活性物质,验证其黄曲霉抑制活性,明确其抗菌作用方式。试验通过克隆表达及亲和层析纯化操作成功得到重组1,3-1,4-β-D-葡聚糖4-葡聚糖水解酶(B10-Glu)及抗菌肽LCI(B10-LCI),并对二者基本生物学信息进行分析。平板抑菌试验验证抑菌活性后,探究B10-Glu、B10-LCI最小抑菌浓度(MIC)及环境条件对二者抑菌效果的影响。通过电镜观察、细胞膜通透性及线粒体膜电位变化、活性氧(ROS)含量及过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性检测,探究B10-Glu、B10-LCI黄曲霉抑制方式。结果表明:1)B10-Glu及B10-LCI的表观分子质量分别为25及12 ku,与预测分子质量较为接近。B10-LCI最佳诱导条件为16℃诱导16 h,B10-Glu为25℃诱导12 h。2)B10-Glu及B10-LCI对黄曲霉均有清晰明显的抑制效果,抑菌圈半径分别为1.14及1.17 cm。MIC分别为0.94及0.96μg/mL。3)B10-Glu及B10-LCI均能抑制黄曲霉孢子形成;破坏其菌丝形态及细胞内部结构;提高黄曲霉细胞膜通透性,致使细胞内容物流失;破坏线粒体等细胞器,促使ROS积累并降低细胞抗氧化水平。综上所述,B10-Glu及B10-LCI能通过抑制黄曲霉生长繁殖过程、破坏其细胞结构来发挥二者的优良抗黄曲霉作用效果。

The aim of this experiment was to clone and express the active substances that could inhibit the growth of Aspergillusflavus from Bacillus amyloliquefaciens B10,verify their inhibitory activity against Aspergillusflavus and clarify their antibacterial action mode.The experiment purified recombinant 1,3-1,4-β-D-glucan 4-glucan hydrolase(B10-Glu)and antibacterial peptide LCI(B10-LCI)through cloning,expression,affinity chromatography,and other operations,and analyzed their basic biological information.After verifying their antibacterial activity through plate antibacterial tests,investigate the effects of minimum inhibitory concentration(MIC)of B10-Glu and B10-LCI,as well as environmental conditions,on their antibacterial effects.The inhibition mode of Aspergillusflavus was explored by electron microscope observation,cell membrane permeability,mitochondrial membrane potential change,reactive oxygen species(ROS)content and catalase(CAT),superoxide dismutase(SOD)activity detection.The results showed as follows:1)the apparent molecular weights of B10-Glu and B10-LCI were 25 and 12 ku,respectively,which were close to the predicted molecular weights.The optimal induction conditions for B10-LCI were 16℃for 16 h,and B10-Glu was 25℃for 12 h.2)Both B10-Glu and B10-LCI had obvious inhibitory effects on Aspergillusflavus,and the radius of inhibitory zone was 1.14 and 1.17 cm,respectively.MIC were 0.94 and 0.96μg/mL,respectively 3)Both B10-Glu and B10-LCI could inhibit the formation of Aspergillusflavus spores;destroy its mycelial morphology and cellular internal structure;improving the membrane permeability of Aspergillusflavus cells,resulting in loss of cell content logistics;destroy organelle such as mitochondria,promote ROS accumulation and reduce cell antioxidant level.In summary,B10-Glu and B10-LCI can exert their excellent anti Aspergillusflavus effect by inhibiting the growth and reproduction of Aspergillusflavus and destroying its cell structure.