动物营养学报2023，Vol.35Issue(12)：8024-8035,12.DOI:10.12418/CJAN2023.727
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
摘要
Abstract
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 As-pergillusflavus 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 concen-tration(MIC)of Bl0-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 per-meability,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 mo-lecular 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 0.96 μg/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,Bl0-Glu and B10-LCI can exert their excellent anti Aspergillusflavus effect by inhibiting the growth and reproduction of Aspergillusflavus and destroying its cell structure.[Chinese Journal of Animal Nutrition,2023,35(12):8024-8035]关键词
解淀粉芽孢杆菌/黄曲霉/生长抑制/葡聚糖水解酶/抗菌肽LCIKey words
Bacillus amyloliquefaciens/Aspergillusflavus/growth inhibition/glucan hydrolase/antibacterial peptide LCI分类
农业科技引用本文复制引用
李天溪,龙淼..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[J].动物营养学报,2023,35(12):8024-8035,12.基金项目
国家自然科学基金(32273074,31972746,31872538,31772809) （32273074,31972746,31872538,31772809）
辽宁省教育厅重点项目(LJKZ0632) （LJKZ0632）
