濒危药用植物杜仲产活性成分内生真菌的研究
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摘要
选用珍稀药用植物杜仲,采用常规的真菌分离方法,从中分离出了44株内生真菌,并以此为对象进行了以下研究工作。
根据黄酮类和环烯醚萜类化合物所具有的颜色反应,初步筛选出可能生产黄酮类化合物的菌株3株——DZ2、DZY5、DZ11。进一步通过紫外-可见光光谱扫描,并检测其产物稳定性,最终确定菌株DZ11可能产黄酮类化合物——槲皮素,并且产量相对稳定,故作为下一步的实验菌株。
绘制DZ11菌株的生长曲线和槲皮素产量的变化曲线,通过紫外分光光度法和TLC法测定槲皮素的最高产量,分别为0.418mg/L和0.340mg/L。利用HPLC法分析发现DZ11菌株所产槲皮素在发酵液和菌丝体内都存在,但菌丝体内含量相对较少。通过常规湿法装柱,对DZ11菌株发酵液进行柱层析分离,得到6种不同Rf值的成分,其中第5部分有着与槲皮素标准品相同的紫外-可见光光谱。经再次纯化,通过质谱检测其[M+H]~+的m/z为303.38,与槲皮素的分子量302.23正好相符。再对照样品和槲皮素标准品的红外吸收图谱,发现它们的红外光谱图几乎相同,也就是说明它们含有相同的结构基团或化学键。由此确定DZ11菌株所产的活性物质中含有槲皮素。
通过对不同固体培养基中菌落的生长速率和长势分析,发现在豆芽汁培养基上不但菌丝生长较快而且菌丝较为浓密。通过对不同液体培养基中DZ11的生物量和槲皮素产生量的对比,筛选出适合DZ11菌丝生长及产物槲皮素合成的豆芽汁液体培养基。在基本液体培养基的基础上,通过单因素分析实验,确定实验室发酵DZ11菌株的最佳碳源、氮源、PH值和温度分别是蔗糖、脲、7.0和28℃。通过正交试验,得出适合菌丝生长的培养基为:把豆芽汁液体培养基中的蔗糖量改为25g.L~(-1),添加脲2g.L~(-1),装量为120ml/300ml;适合槲皮素积累的培养基的为:把豆芽汁液体培养基的蔗糖量改为30g.L~(-1),添加脲2.5g.L~(-1),装量为100ml/300ml。
根据DZ11菌株的显微结构特征,对DZ11菌株进行了分类,初步确定为:真菌门,半知菌类,腔孢纲,球壳孢目,球壳孢科中的刺茎点菌属。
Forty-four endophytic fungi strains were isolated from the stems,roots and leaves of a rare medicinal plant Eucommia Ulmoides Oliv.The further researches were as follows.
According to the characteristic color reactions of flavonoids and iridoids compounds,three strains,DZ2、DZY5、DZ11,which could produce flavonoids compounds were screened out in the preliminary selection.By UV-visible spectroscopy scanning and stability detection of its quercetin production,DZ11 was selected.
Through drawing growth curve of DZ11 strain and yield curve of quercetin,the maximum yields of quercetin which were 0.418mg/L and 0.340mg/L were got by UV spectrophotometry and TLC method,respectively.After HPLC analyzing,it could be found that quercetin exists in both inside and outside of hyphae,but the former had a lower content.Through column chromatography,6 components with different Rf-value were obtained from DZ11 fermentation broth,the fifth ingredient among which had the same UV-visible spectrogram as standard quercetin.After further purification to the fifth part and mass spectrography,the mass over charge ratio(m/z)of the[M+H]~+ was 303.38,which was the same as the molecular weight of quercetin.Each functional group and chemical bond absorption band of quercetin was found in infrared spectrum.These results proved that one of secondary metabolite produced by DZ11 strain was quercetin.
According to the growth rates and growth potential of the colonies,bean sprouts solid medium was chosen.The bean sprouts liquid medium was also suitable for the mycelial growth of the strain DZ11 and the output of quercetin.Based on this liquid medium,by changing different carbon source,nitrogen source,pH-value and culture temperature,the culture conditions were optimized.The optimized conditions of both mycelia growth and quercetin-production were sucrose,urine,pH7.0 and the temperature 28℃.The fermentation conditions of DZ11 were optimized further by the orthogonal experiment of L16(4~3).The results showed that the suitable conditions for its mycelial growth were an improved bean sprouts liquid medium with 25g.L~(-1)as sucrose, 2g.L~(-1)urine as nitrogen and 120ml/300ml as the liquid medium quantity;the suitable conditions for querctetin production were an improved bean sprouts liquid medium with 30g.L~(-1)sucrose as carbon source,2.5 g.L~(-1)urine as nitrogen and 120ml/300ml as the liquid medium quantity.
The strain DZ11 was classified mainly according to its microscopic morphological characters.DZ11 should be a strain belonging to the Fungi Imperfecfi, Coelelomyctes,Sphaeropsidales,Sphaeropsidaceae,Chaetophoma Cke.
根据黄酮类和环烯醚萜类化合物所具有的颜色反应,初步筛选出可能生产黄酮类化合物的菌株3株——DZ2、DZY5、DZ11。进一步通过紫外-可见光光谱扫描,并检测其产物稳定性,最终确定菌株DZ11可能产黄酮类化合物——槲皮素,并且产量相对稳定,故作为下一步的实验菌株。
绘制DZ11菌株的生长曲线和槲皮素产量的变化曲线,通过紫外分光光度法和TLC法测定槲皮素的最高产量,分别为0.418mg/L和0.340mg/L。利用HPLC法分析发现DZ11菌株所产槲皮素在发酵液和菌丝体内都存在,但菌丝体内含量相对较少。通过常规湿法装柱,对DZ11菌株发酵液进行柱层析分离,得到6种不同Rf值的成分,其中第5部分有着与槲皮素标准品相同的紫外-可见光光谱。经再次纯化,通过质谱检测其[M+H]~+的m/z为303.38,与槲皮素的分子量302.23正好相符。再对照样品和槲皮素标准品的红外吸收图谱,发现它们的红外光谱图几乎相同,也就是说明它们含有相同的结构基团或化学键。由此确定DZ11菌株所产的活性物质中含有槲皮素。
通过对不同固体培养基中菌落的生长速率和长势分析,发现在豆芽汁培养基上不但菌丝生长较快而且菌丝较为浓密。通过对不同液体培养基中DZ11的生物量和槲皮素产生量的对比,筛选出适合DZ11菌丝生长及产物槲皮素合成的豆芽汁液体培养基。在基本液体培养基的基础上,通过单因素分析实验,确定实验室发酵DZ11菌株的最佳碳源、氮源、PH值和温度分别是蔗糖、脲、7.0和28℃。通过正交试验,得出适合菌丝生长的培养基为:把豆芽汁液体培养基中的蔗糖量改为25g.L~(-1),添加脲2g.L~(-1),装量为120ml/300ml;适合槲皮素积累的培养基的为:把豆芽汁液体培养基的蔗糖量改为30g.L~(-1),添加脲2.5g.L~(-1),装量为100ml/300ml。
根据DZ11菌株的显微结构特征,对DZ11菌株进行了分类,初步确定为:真菌门,半知菌类,腔孢纲,球壳孢目,球壳孢科中的刺茎点菌属。
Forty-four endophytic fungi strains were isolated from the stems,roots and leaves of a rare medicinal plant Eucommia Ulmoides Oliv.The further researches were as follows.
According to the characteristic color reactions of flavonoids and iridoids compounds,three strains,DZ2、DZY5、DZ11,which could produce flavonoids compounds were screened out in the preliminary selection.By UV-visible spectroscopy scanning and stability detection of its quercetin production,DZ11 was selected.
Through drawing growth curve of DZ11 strain and yield curve of quercetin,the maximum yields of quercetin which were 0.418mg/L and 0.340mg/L were got by UV spectrophotometry and TLC method,respectively.After HPLC analyzing,it could be found that quercetin exists in both inside and outside of hyphae,but the former had a lower content.Through column chromatography,6 components with different Rf-value were obtained from DZ11 fermentation broth,the fifth ingredient among which had the same UV-visible spectrogram as standard quercetin.After further purification to the fifth part and mass spectrography,the mass over charge ratio(m/z)of the[M+H]~+ was 303.38,which was the same as the molecular weight of quercetin.Each functional group and chemical bond absorption band of quercetin was found in infrared spectrum.These results proved that one of secondary metabolite produced by DZ11 strain was quercetin.
According to the growth rates and growth potential of the colonies,bean sprouts solid medium was chosen.The bean sprouts liquid medium was also suitable for the mycelial growth of the strain DZ11 and the output of quercetin.Based on this liquid medium,by changing different carbon source,nitrogen source,pH-value and culture temperature,the culture conditions were optimized.The optimized conditions of both mycelia growth and quercetin-production were sucrose,urine,pH7.0 and the temperature 28℃.The fermentation conditions of DZ11 were optimized further by the orthogonal experiment of L16(4~3).The results showed that the suitable conditions for its mycelial growth were an improved bean sprouts liquid medium with 25g.L~(-1)as sucrose, 2g.L~(-1)urine as nitrogen and 120ml/300ml as the liquid medium quantity;the suitable conditions for querctetin production were an improved bean sprouts liquid medium with 30g.L~(-1)sucrose as carbon source,2.5 g.L~(-1)urine as nitrogen and 120ml/300ml as the liquid medium quantity.
The strain DZ11 was classified mainly according to its microscopic morphological characters.DZ11 should be a strain belonging to the Fungi Imperfecfi, Coelelomyctes,Sphaeropsidales,Sphaeropsidaceae,Chaetophoma Cke.
引文
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