云芝Cov-1菌株原生质体诱变育种、发酵工艺条件优化及其复方制剂
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摘要
云芝[Corilus versicolor(L.er. Fr. )Qu’ei.]是一种世界性分布的药用真菌。云芝多糖肽具有明显的抗肿瘤、改善免疫功能、降血压、抗炎等功效。中医认为云芝具有扶正固本、补益精气的作用。本论文对云芝Cov-1菌株的选育、发酵条件的优化等方面进行了系统研究,同时对云芝糖肽的复方制剂——芝参正肝胶囊的提取工艺进行了研究。
本课题研究了酶种类、酶浓度、酶解温度和时间、渗透压稳定剂、pH值及菌龄等因素对云芝原生质体制备以及不同再生培养基对云芝原生质体再生的影响。结果表明,菌龄3 d的云芝菌丝体用3%溶壁酶,以pH 5.5、0.4 mol/L的KCl为稳定剂,30℃酶解5 h获得的原生质体产量最高;用RM1再生培养基培养,原生质体再生率最高。
采用云芝原生质体紫外诱变方法,从出发菌株筛选出一株遗传上相对稳定的适应液体培养的云芝诱变菌株——YBUV113,该菌株在综合PDA斜面上生长速度较快,长势旺盛。在液体培养基中发酵生长迅速,菌丝湿重、干重和多糖产量均与出发菌株相当。
碳、氮源是影响液体培养云芝菌丝体生长的重要因素。其中,玉米粉是其适合碳源,黄豆饼粉为其适合氮源。实验确定了云芝深层培养的较适合的培养基组成为:玉米粉2%,黄豆饼粉2%,KH2PO40.3%,K2HP040.3%。最佳摇瓶培养条件为:培养基起始pH5.5,接种量20%,摇床转速180r/min,培养3天。在上述条件下,每升发酵液可获得干菌丝体34.5g/L。糖含量53.74%,蛋白含量18.36%。
芝参正肝是云芝糖肽的复方制剂,由云芝糖肽、丹参、三七、赤芍、葛根、银杏叶6味中药组成的现代中药复方制剂。本文对芝参正肝提取纯化工艺进行了研究,通过提取方法的比较和正交试验确定了芝参正肝提取方法及其优化工艺条件。结果,渗漉提取最佳工艺条件为:药材粉碎成粗粉,用14倍药材重量的70%乙醇渗漉,渗漉速度为1mL/min;大孔树脂纯化最佳工艺条件为:上柱提取液浓度为200mg/mL,上柱流速3Bv/h,洗脱时采用60%乙醇,体积为3Bv,洗脱流速为3Bv/h。由最佳工艺提取的芝参正肝原料中有效成分丹酚酸B、芍药苷、人参皂苷Rg1和银杏总黄酮之和为45%。
Coriolus versicolor is a world wide distributing medicinal fungi which is sweet and cool, can clear away heat and eliminate inflammation, and whose indications of tracheitis, hepatitis and tumor. Its neoteric research of medicinal value is based on folk experience. With unceasingly further study, ceaseless improvement of research level and extension of application range researcher put forward that it has supplement orientation of the effect of supporting the healthy energy , invigorating energy and replenishing qi. In order to develop submerged fermentation technique and to reach high level of industrical production of Coriolus versicolor, the following aspects, including screen good strain and submerged culture process.
The conditions of protoplast formation and regeneration from the mycelia of Coriolus versicolor were studied. Experimental results indicated that the highest production rate of protoplast can be obtained by using mycelial age of 3d, 0.4mol/L KCL, 3% lywallzyme digesting for 5 hours at the condition of 30℃and pH 5.5. The highest regeneration rate was obtained on RM1 medium layers.
By means of ultraviolet mutation, a mutant strain YBUV113 was screened. The strain was well grown in PDA solid-state medium. It produces almost the same amount of mycelia and PSP(polysaccharopeptide of Coriolus versicolor) as the original strain did when grown in liquid state medium.
Carbon and nitrogen sources are important factors that effected the growen of mycelium and yield of polysaccharopeptide of Coriolus versicolor. The optumum carbon source is maize powder. The optumum nitrogen source is soybean oil meal. The suitable shaking culture conditions are as follows: initial pH of medium 5.5, inoculation quantity 20%, shaking frequency 160r/min, incubation time 3 days. Under this condition, the maximum mycelia dry weight is about 34.5g/L. Intracellular polysaccharide of 53.74% and protain of 18.36% were obtained.
The Prescription of Ziseng Zhenggan is composed of polysaccharidopeptide, salvia, panax notoginseng, red peony root, kudzu root, ginkgo biloba leaves and so on. This prescription is a self-drafted prescription based on the principle“strengthening body resistance and driving away evil, promoting blood flow and removing stasis”and consulted on some experiences of clinical medication. This prescription is also a modern Chinese traditional medicine compound preparation (including six Chinese traditional medicine drugs) with independent innovation. The study on pharmacodynamics has proved that this prescription can strengthening body resistance, promoting blood flow and remove stasis, decrease serum lipids, prevent fatty liver disease, prevent liver fibrosis, counteract hepatitis B virus and so on. Ziseng Zhenggan can be also applied to treat fatty liver disease and liver fibrosis. We choose the pattern number of macroporous resin By the method of static-state adsorption and desorption experiments, And by different extracting methods to compare every experiment, the best extracting method is confirmed. Research the dynamic adsorption of macroporous resin. By desorption experiment, the maximum adsorptive capacity of the macroporous resin and the best elution concentration can be confirmed. By using orthogonal experiment method, the optimal parameters of extracting technology and purified technology are confirmed. As a result, based on 100g as a standard, the best condition of extracting technology includes concentration of ethanol 70%, dosage of ethanol 1400mL, speed of seeping 1mL/min. The best condition of purified technology includes the flow rate of sample 3Bv/h, the volume of ethanol 3Bv, concentration of sample 200mg/mL, the flow rate of elution 3Bv/h. Active ingredients of polysaccharide-peptide,salvia,panax notginseng,red peony root, kudzu root,ginkgo biloba leaves by optimum extratction is 45%.
本课题研究了酶种类、酶浓度、酶解温度和时间、渗透压稳定剂、pH值及菌龄等因素对云芝原生质体制备以及不同再生培养基对云芝原生质体再生的影响。结果表明,菌龄3 d的云芝菌丝体用3%溶壁酶,以pH 5.5、0.4 mol/L的KCl为稳定剂,30℃酶解5 h获得的原生质体产量最高;用RM1再生培养基培养,原生质体再生率最高。
采用云芝原生质体紫外诱变方法,从出发菌株筛选出一株遗传上相对稳定的适应液体培养的云芝诱变菌株——YBUV113,该菌株在综合PDA斜面上生长速度较快,长势旺盛。在液体培养基中发酵生长迅速,菌丝湿重、干重和多糖产量均与出发菌株相当。
碳、氮源是影响液体培养云芝菌丝体生长的重要因素。其中,玉米粉是其适合碳源,黄豆饼粉为其适合氮源。实验确定了云芝深层培养的较适合的培养基组成为:玉米粉2%,黄豆饼粉2%,KH2PO40.3%,K2HP040.3%。最佳摇瓶培养条件为:培养基起始pH5.5,接种量20%,摇床转速180r/min,培养3天。在上述条件下,每升发酵液可获得干菌丝体34.5g/L。糖含量53.74%,蛋白含量18.36%。
芝参正肝是云芝糖肽的复方制剂,由云芝糖肽、丹参、三七、赤芍、葛根、银杏叶6味中药组成的现代中药复方制剂。本文对芝参正肝提取纯化工艺进行了研究,通过提取方法的比较和正交试验确定了芝参正肝提取方法及其优化工艺条件。结果,渗漉提取最佳工艺条件为:药材粉碎成粗粉,用14倍药材重量的70%乙醇渗漉,渗漉速度为1mL/min;大孔树脂纯化最佳工艺条件为:上柱提取液浓度为200mg/mL,上柱流速3Bv/h,洗脱时采用60%乙醇,体积为3Bv,洗脱流速为3Bv/h。由最佳工艺提取的芝参正肝原料中有效成分丹酚酸B、芍药苷、人参皂苷Rg1和银杏总黄酮之和为45%。
Coriolus versicolor is a world wide distributing medicinal fungi which is sweet and cool, can clear away heat and eliminate inflammation, and whose indications of tracheitis, hepatitis and tumor. Its neoteric research of medicinal value is based on folk experience. With unceasingly further study, ceaseless improvement of research level and extension of application range researcher put forward that it has supplement orientation of the effect of supporting the healthy energy , invigorating energy and replenishing qi. In order to develop submerged fermentation technique and to reach high level of industrical production of Coriolus versicolor, the following aspects, including screen good strain and submerged culture process.
The conditions of protoplast formation and regeneration from the mycelia of Coriolus versicolor were studied. Experimental results indicated that the highest production rate of protoplast can be obtained by using mycelial age of 3d, 0.4mol/L KCL, 3% lywallzyme digesting for 5 hours at the condition of 30℃and pH 5.5. The highest regeneration rate was obtained on RM1 medium layers.
By means of ultraviolet mutation, a mutant strain YBUV113 was screened. The strain was well grown in PDA solid-state medium. It produces almost the same amount of mycelia and PSP(polysaccharopeptide of Coriolus versicolor) as the original strain did when grown in liquid state medium.
Carbon and nitrogen sources are important factors that effected the growen of mycelium and yield of polysaccharopeptide of Coriolus versicolor. The optumum carbon source is maize powder. The optumum nitrogen source is soybean oil meal. The suitable shaking culture conditions are as follows: initial pH of medium 5.5, inoculation quantity 20%, shaking frequency 160r/min, incubation time 3 days. Under this condition, the maximum mycelia dry weight is about 34.5g/L. Intracellular polysaccharide of 53.74% and protain of 18.36% were obtained.
The Prescription of Ziseng Zhenggan is composed of polysaccharidopeptide, salvia, panax notoginseng, red peony root, kudzu root, ginkgo biloba leaves and so on. This prescription is a self-drafted prescription based on the principle“strengthening body resistance and driving away evil, promoting blood flow and removing stasis”and consulted on some experiences of clinical medication. This prescription is also a modern Chinese traditional medicine compound preparation (including six Chinese traditional medicine drugs) with independent innovation. The study on pharmacodynamics has proved that this prescription can strengthening body resistance, promoting blood flow and remove stasis, decrease serum lipids, prevent fatty liver disease, prevent liver fibrosis, counteract hepatitis B virus and so on. Ziseng Zhenggan can be also applied to treat fatty liver disease and liver fibrosis. We choose the pattern number of macroporous resin By the method of static-state adsorption and desorption experiments, And by different extracting methods to compare every experiment, the best extracting method is confirmed. Research the dynamic adsorption of macroporous resin. By desorption experiment, the maximum adsorptive capacity of the macroporous resin and the best elution concentration can be confirmed. By using orthogonal experiment method, the optimal parameters of extracting technology and purified technology are confirmed. As a result, based on 100g as a standard, the best condition of extracting technology includes concentration of ethanol 70%, dosage of ethanol 1400mL, speed of seeping 1mL/min. The best condition of purified technology includes the flow rate of sample 3Bv/h, the volume of ethanol 3Bv, concentration of sample 200mg/mL, the flow rate of elution 3Bv/h. Active ingredients of polysaccharide-peptide,salvia,panax notginseng,red peony root, kudzu root,ginkgo biloba leaves by optimum extratction is 45%.
引文
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2 陆佩洪等, 几种食药用真菌多糖的初步研究. 江苏食用菌, 1995, 16(5):3-4.
3 王慧杰. 药用真菌的种类与开发. 郑州牧业工程高等专科学校学报. 2005, 25(1):87-89.
4 康洁, 陈若芸. 近 10 年药用真菌成分及活性研究进展. 中国药学杂志. 2005, 40(2):88-89
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