Bacillomycin D发酵与纯化工艺优化及其抑菌活性初步研究

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4×106 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。

Fermentation and purification optimizations of bacillomycin D, and preliminary exploration for its antibiotic activity

Scale-up production technology of highly purified bacillomycin D was established to support the exploration for its agriculturally antibiotic activity. After isolation, purification and molecular identification, the bioactive compounds extracted from Bacillus amyloliquefaciens TYg 3-2 were identified as antibiotic lipopeptides bacillomycin D. The refined nature media and chemically defined available carbon and nitrogen sources, including yeast extract, peptone, glucose, and (NH₄)₂SO₄, were combined and optimized using response surface method for high-yield complex medium of bacillomycin D. Using the optimized medium, the yield of bacillomycin D in 5 L bioreactor reached 990 mg/L. The productivity reached 20.62 mg/(L·h), which was much higher than the previously reported productivity of 10.23 mg/(L·h). Biomass-supernatant separation, acid precipitation, methanol extraction, Et₂O precipitation, macroporous absorption resin column chromatography and silica gel column chromatography were conducted for efficient purification of bacillomycin D. And 94.72% purity was obtained when high-quality raw material was used. Preliminary exploration for antibiotic activity was conducted using the highly purified bacillomycin D sample. Inhibition tests in vitro demonstrated that bacillomycin D was the main effective substance against Drechslera turcica, Curvularia lunata, Ustilaginoidea virens and Alternaria longipes in vitro. And the result of pot test in vivo revealed that bacillomycin D was the promotor on biocontrol efficacy of homologous B. amyloliquefaciens TYg 3-2 biomass. Because the mixture of 500 mg/L bacillomycin D and 4×10⁶ CFU/mL B. amyloliquefaciens TYg 3-2 biomass exhibited 95.3% biocontrol efficacy against Colletotrichum orbiculare in vivo, which was higher than those of bacillomycin D alone (78.4%) and Bacillus biomass alone (0). The potential value of bacillomycin D in the field of agricultural antibiotic was preliminarily explored. And scale-up production technology of highly purified lipopeptides played an important role in this exploration. Not only could it isolate and purify target lipopeptide from fermentation broth for this agricultural antibiotic research without the loss in producing bacteria (the spore of which was the feedstock of biocontrol agent), but also can it satisfy the requirement of toxicological tests of pesticide registration in China (sample with >85% purity in grams). As a result, this research was able to support the research and development of agriculturally antibiotic value in Bacillus lipopeptides.