【作者】 王亮；

【导师】 龚国利；

【作者基本信息】 陕西科技大学 ， 中药学， 2020， 硕士

【摘要】 三七(Panaxnotoginseng(Burkill)F.H.Chen ex C.H.),属于伞形目五茄科人参属多年生的宿根直立草本植物,享有“参中之王”、“南国神草”、“金不换”等美誉,其药用具有悠久的历史。黑斑病是三七栽培生产中常见的一大病害,常年发病率在10%～20%,严重时高达80%以上,已成为三七的毁灭性病害之一。三七的黑斑病由人参链格孢(Alternariapanax Whetzel)浸染引起,病原隶属于半知菌亚门,丝孢纲,丛梗孢目,暗色孢科,链格孢属。多抗霉素(Polyoxin)又名多氧霉素、多效霉素或多抗菌素,是一种用现代生物工程技术生产的肽嘧啶核苷类广谱农用抗生素,近年来被应用到三七黑斑病的生物防治上取得良好的效果。本论文研究多抗霉素的工业化发酵生产技术,首先基于陕西麦可罗生物科技公司的多抗霉素生产菌株,选育对三七黑斑病病原菌人参链格孢具有高效抑制作用的菌株,进一步研究发酵工艺和产物分离提取工艺,提高多抗霉素生产效率,降低生产成本。本论文主要研究结果如下:1、采用琼脂扩散法,进行高效抑制三七黑斑病病原菌的多抗霉素产生菌株的选育,建立了一套快速和高效的选育方法。以三七黑斑病病原菌为检验菌,运用自制具盖玻璃大盘双层培养基抑菌圈法、摇瓶筛选法和生物管碟法测定生物效价技术,选育出高效抑制三七黑斑病病原菌的多抗霉素产生菌金色产色链霉菌(Streptomyces aureochromogenes)—18#。2、采用单因子及正交实验对防治三七黑斑病的多抗霉素产生菌金色产色链霉菌—18#的摇瓶发酵工艺进行优化,获得了最佳的发酵培养基配方和发酵条件。发酵培养基配方:玉米粉7%、冷榨黄豆饼粉3%、酵母粉0.8%、鱼粉 0.75%、KH2PO4 0.02%、NaCl 0.2%、CaCO3 0.5%、pH6.5。发酵条件:300 mL广口三角瓶装液量50 mL、发酵温度28℃、转速200 r/min,发酵培养周期为120h。在此工艺条件下,选育出的金色产色链霉菌—18#多抗霉素平均发酵效价4374.6 μg/mL,相对增长率38.7%。3、应用 5 L—15 L—50 L三平行九联Distributed Control System控制系统发酵罐,将选育出的金色产色链霉菌—18#用优化发酵培养基和发酵条件,进行了三个罐批的发酵工艺实验。通过发酵过程检测调控生化指标生物效价、菌体浓度、总糖、还原糖、氨氮、磷含量,溶氧、pH值等参数,优化该生产菌株的发酵工艺。4、采用截留相对分子质量为300～1000 Da的卷式纳滤膜分离系统进行多抗霉素分离提取,分离提取后的提取液多抗霉素含量大于4%,pH 3.50～5.00。选育了高效抑制三七黑斑病病原菌的多抗霉素产生菌金色产色链霉菌—18#高产菌株,优化了该菌的摇瓶和小试发酵工艺,并建立了纳滤膜分离提取多抗霉素分离提取工艺,本研究为防治三七黑斑病的多抗霉素产业化奠定了基础。更多还原

【Abstract】 Panax notoginseng,is a perennial erect herb of the genus Panax in the umbelliferae family,enjoying the reputation of "King of Ginseng","Nanguo Shencao”and "Jinbuhuan”etc.It’s medicinal use has a long history.Black Spot Disease is a common disease in the cultivation and production of Panax notoginseng,with an annual incidence of 10%to 20%,and in severe cases as high as 80%or more,has become one of the destructive diseases of Panax notoginseng.The Black Spot Disease of Panax notoginseng is caused by the contamination of Alternaria panax Whetzel.The pathogen belonging to the Deuteromycotina,Hyphomycetes,Moniliales,Dematiaceae,Alternaria.Polyoxin,a nucleosides of peptide pyrimidines broad-spectrum agricultural antibiotic produced by modern bioengineering technology,has been applied to the biological control of Panax notoginseng Black Spot Disease in recent years with good effect.This thesis research industrial fermentation production technology of Polyoxin,first of all,based on the Shaanxi Microbe Biotechology CO.,LTD production strains of Polyoxin,selection of Panax notoginseng Black Spot Disease pathogen Alternaria panax Whetzel efficiency of the strains of inhibition,further study on fermentation process and product separation extraction process,improve production efficiency,reduce production costs.The main results were as follows:1.The agar diffusion method was used for the selection and selection of Polyoxin-resistant strains that effectively inhibited the pathogen of Panax notoginseng and established a set of rapid and efficient selection methods.Taking Panax notoginseng pathogenic bacteria as the test bacteria,the self-made glass-plated double-layered culture medium antibacterial circle method,shake flask screening method and biological tube dish method were used to determine the biological titer technology Streptomyces aureochromogenes—18#.2.The single-factor and orthogonal test were used to optimize the shaking flask fermentation conditions of the Polyoxin-producing bacteria Streptomyces aureochromogenes—18#for the control of Panax notoginseng Black Spot Disease,and the best fermentation medium was obtained recipe and fermentation conditions.Fermentation medium formula:corn flour 7%,cold-pressed soybean cake flour 3%,yeast flour 0.8%,fish meal 0.75%,KH2PO4 0.02%,NaCl 0.2%,CaCO3 0.5%,pH 6.5.Fermentation conditions:300 mL wide mouth triangular bottle with a volume of 50 mL,fermentation temperature of 28℃,rotation speed of 200 r/min,and fermentation culture period of 120 h.Under the conditions of this process,the average fermentation titer of Streptomyces aureochromogenes—18#Polyoxin was 4374.6 μg/mL,and the relative growth rate was 38.7%.3.Using 5 L—15 L—50 L triple parallel nine-link Distributed Control System control system fermentation tank,the selected Streptomyces aureochromogenes-18#was optimized fermentation medium and fermentation conditions,three batch fermentation experiments were carried out.Detecting by a fermentation process biological potencytotal,cell density of biochemical indexes,sugar regulation,reducing sugar,ammonia,phosphorus content,dissolved oxygen,pH other parameters of the fermentation process to optimize production strains.4.The use of a roll-type nanofiltration membrane separation system with a relative molecular mass of 300～1000 Da for separation and extraction of Polyoxin,after separation and extraction,the Polyoxin content of the purified solution was greater than 4%with a pH of 3.50-5.00.A high-yielding strain of Streptomyces aureochromogenes—18#was selected to inhibit the pathogenic bacteria of Panax notoginseng Black Spot Disease,and the shaking flask and small-test fermentation processes were optimized.The separation and extraction process of Panax notoginseng Black Spot Disease by nanofiltration membrane was established.This study laid a foundation for the industrialization of Polyoxin against Black Spot Disease of Panax notoginseng.更多还原