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为建立高纯度bacillomycin D(杆菌霉素D)脂肽的可放大制备工艺,促进其在农用抗生素领域应用价值的开发,经分离纯化及分子结构鉴定,确定了海洋源解淀粉芽孢杆菌Bacillus amyloliquefaciens TYg 3-2所产活性化合物为bacillomycin D脂肽类抗生素;选择精制过的天然培养基(酵母提取物和蛋白胨),与组分明确的速效碳源、氮源(葡萄糖和硫酸铵)组成复合培养基,结合响应面优化调整培养基配方,建立了bacillomycin D发酵水平高达990 mg/L的5 L罐发酵工艺,其20.62 mg/(L·h)的比生产速率远高于文献报道的10.23 mg/(L·h);在高品质原料基础上,发酵液经菌液分离、酸化沉淀、甲醇浸提、乙醚沉淀、大孔树脂吸附及硅胶柱层析工艺,可高效制备纯度达94.72%的样品。针对所制备的高纯度bacillomycin D样品进行了抑菌活性初步研究,证实其可离体拮抗德氏霉菌、玉米弯孢霉、稻绿核菌和长柄链格孢,并通过盆栽试验证实了bacillomycin D能提升同源芽孢杆菌TYg 3-2的防效,500 mg/L的bacillomycin D与4×10~6 CFU/mL的TYg 3-2菌体混合施用,对黄瓜炭疽病的防效可达95.3%,优于单独施用bacillomycin D(78.4%)和菌体(0)的防治效果。研究初步探索了bacillomycin D在农用抗生素领域的开发价值,同时其高纯度脂肽可放大制备工艺的建立至关重要,不仅能在保全生产菌(其芽孢为生防菌剂原料)的同时从其发酵液中分离和纯化目标脂肽,以促成对农用抗生素价值的深入研究,也能够满足中国农药登记相关毒理学试验对样品的要求(纯度>85%的克量级样品),其结果可为芽孢杆菌源脂肽类抗生素的研究与开发提供参考。
In order to establish a scalable process for the preparation of high-purity bacillomycin D lipopeptide, and to promote the development of its value in the field of agricultural antibiotics, the marine source Bacillus amyloliquefaciens TYg was identified through isolation, purification and molecular structure identification. The active compound produced in 3-2 is bacillomycin D lipopeptide antibiotic. The purified natural medium (yeast extract and peptone) is selected to form a composite culture with the quick-acting carbon source with definite components and nitrogen source (glucose and ammonium sulfate) Based on response surface optimization, the 5 L tank fermentation with bacillomycin D fermentation level up to 990 mg / L was established. The specific production rate of 20.62 mg / (L · h) was much higher than that reported in the literature of 10.23 mg / (L · h). On the basis of high quality raw materials, the fermentation broth can be efficiently prepared with a purity of 94.72% by bacterial liquid separation, acid precipitation, methanol leaching, ether precipitation, macroporous resin adsorption and silica gel column chromatography sample. A preliminary study on the bacteriostasis activity of the prepared bacillomycin D samples showed that the bacillomycin, the corn curdles, the green spore and the Alternaria alternata could be antagonized in vitro and the bacillomycin D could enhance the control effect of homologous Bacillus TYg 3-2. The combination of 500 mg / L bacillomycin D and TYg 3-2 strain with 4 × 10 ~ 6 CFU / mL could effectively control cucumber anthracnose 95.3%, which is better than that of bacillomycin D (78.4%) and cell (0) alone. The research initially explored the developmental value of bacillomycin D in the field of agricultural antibiotics. At the same time, the establishment of the high-purity lipopeptide scalable preparation process is of crucial importance not only in preserving the production bacteria (spores of biocontrol agent) The target lipopeptide was isolated and purified from the fermentation broth to facilitate further studies on the value of agricultural antibiotics and to meet the sample requirements (purity> 85%) of the toxicological tests related to pesticide registration in China. The results could be Bacillus lipogenic peptides derived from the research and development of reference.