药用菌液体培养和多糖活性研究
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摘要
以本实验室野外采集并纯化培养得到的赤芝(Ganoderma lucidum (leyss.:Fr.) Karst)、蝉花(Cordyceps sobolifera (Hill.) Berk. et Br)、硫磺菌(Laetiporus sulphureus (Fr.) Murrill)为实验材料,以菌丝产量为目标,探索3种药用菌的液体发酵培养基优良配方。通过对菌丝体和发酵液多糖的提取,研究不同种类药用菌多糖得率。采用Lewis肺癌实体瘤实验模型,研究不同种类多糖对肿瘤抑制率的高低;以及采用脾细胞增殖实验研究多糖对免疫细胞的增殖刺激作用,探索不同种类多糖的功效作用,为野生药用菌的开发利用提供参考数据。
主要研究结果:
1.在适宜的培养条件下,通过对3种药用菌培养基配方比较实验,筛选出适合各种药用菌生长、菌丝体产量高的液体培养基优良配方。
①赤芝液体培养基配方:葡萄糖30g,豆粉20g,(NH4)2SO4 2g ,KH2PO41g ,MgSO4 0.8g ,VB60.02g,水1000mL。
②蝉花液体培养基配方:马铃薯200g,豆粉5g,葡萄糖20g,KH2PO42g,水1000mL。
③硫磺菌液体培养基配方:葡萄糖30g,玉米粉10g,酵母提取物5g,KH2PO4 1.0g,CaCO32.0g,MgSO40.5g,水1000mL。
2.通过对3种药用菌菌丝体和发酵液的多糖提取研究,结果表明相同的提取方法,不同药用菌菌丝体、发酵液多糖提取率均有显著差异,其中赤芝菌丝体、发酵液多糖提取率均为最高,分别为18.5%和23.3%。
3.测定3种药用菌多糖中多糖含量和蛋白质含量,3种菌丝体多糖多糖含量由高到低依次是赤芝菌丝体多糖>蝉花菌丝体多糖>硫磺菌菌丝体多糖,3种发酵液多糖具多糖含量由高到低依次是赤芝发酵液多糖>硫磺菌发酵液多糖>蝉花发酵液多糖。
4.通过3种药用菌菌丝体粗多糖对Lewis肺癌的体内抑制作用的研究,表明3种真菌菌丝体多糖对肿瘤均有一定抑制效果。其中,对Lewis肺癌的最大抑制率是赤芝高剂量组为50.2%,其次是蝉花高剂量组为46.3%;硫磺菌多糖的最大抑瘤率仅为35.7%,低于其他2种多糖的最大抑瘤率。
5.脾细胞增殖实验结果表明6个样品的增强免疫力作用依次为蝉花发酵液多糖>蝉花菌丝体多糖>赤芝发酵液多糖>硫磺菌发酵液多糖>赤芝菌丝体多糖>硫磺菌菌丝体多糖;药用菌发酵液多糖对细胞增殖作用的效果优于菌丝体多糖。
To investigate the best formula of fermentation medium of three medicinal mushroom by utilizing mycelium as the target product,the Ganoderma lucidum (leyss.:Fr.) Karst,Cordyceps sobolifera (Hill.) Berk. et Br and Laetiporus sulphureus (Fr.) Murrill,which were collected from field acquisition and purification were employed as the experimental materials. Different types of extracting rate of polysaccharides were determined through extracting the polysaccharide of mycelium and fermentation liquid. Furthermore,Lewis lung cancer solid tumors were used to investigate the influencing of polysaccharides to the tumor inhibitory rate. The splenocyte proliferative experiment was used to investigate the stimulatory effects of polysaccharides to the immune cells,in order to exploring the function of different kinds polysaccharides. This can establish scientific basis for the exploring of wild medicinal fungi.
Main results are as follows:
1.In the appropriate cultural environment,the excellent formula liquidcultural medium for the fungi growth and mycelium production were selected by the comparative experiment of cultural medium formula of medicinal fungi.
①The optimum liquid medium of G.lucidum :Glucose 30g,soybean powder 20g,(NH4)2SO4 2g ,KH2PO41g ,MgSO4 0.8g ,VB0.02g,H2O 1000ml。
②The optimum liquid medium of C. sobolifera :potato 200g,soybean powder 5g,Glucose 20g,KH2PO4 2g,H2O 1000ml。
③The optimum liquid medium of L. sulphureus :Glucose 30g,corn meal 10g,yeast extract 5g ,KH2PO4 1.0g, CaCO3 2.0g , MgSO4 0.5g, H2O 1000ml。
2.The polysaccharide extraction of three kinds of mycelium and fermentation liquid were investigated. The results indicated that there were significant differences of the yield rate among distinct medical mycelium and fermentation liquid by the same extracting method. The yield rate of polysaccharide from mycelium and fermentation liquid of G. lucidum was 18.5% and 23.3% respectively and was highest.
3.The polysaccharide content and protein content of three medical mushroom were determined. The results shows that the seriatim of the mycelium polysaccharide content is G. lucidum > C. sobolifera > L. sulphureus and the seriatim of the polysaccharide content in Polysaccharide from fermentation liquid is G. lucidum > L. sulphureus > C. sobolifera .
4.The inhibition function of three types of medical mycelium to Lewis lung cancer was studied. The results indicated that all of the three types of mycelium polysaccharide can inhibit the tumor. The inhibition ratio of G. lucidum polysaccharide to Lewis lung cancer is highest, which reached to 50.2%. The inhibition ratio of the C. sobolifera polysaccharide and the L. sulphureus polysaccharide was 46.3% and 35.7% respectively.
5.The splenocyte proliferation assay showed that the seriatim of the increasing immunity effect of six samples was C. sobolifera polysaccharide of fermentation liquid>C. sobolifera polysaccharide of mycelium >G. lucidum polysaccharide of fermentation liquid >L.sulphureus polysaccharide of fermentation liquid >G. lucidum polysaccharide of mycelium >L. sulphureus polysaccharide of mycelium. The effects of medicinal mushroom polysaccharide of fermentation liquid were better than that of medicinal mushroom polysaccharide of mycelium.
主要研究结果:
1.在适宜的培养条件下,通过对3种药用菌培养基配方比较实验,筛选出适合各种药用菌生长、菌丝体产量高的液体培养基优良配方。
①赤芝液体培养基配方:葡萄糖30g,豆粉20g,(NH4)2SO4 2g ,KH2PO41g ,MgSO4 0.8g ,VB60.02g,水1000mL。
②蝉花液体培养基配方:马铃薯200g,豆粉5g,葡萄糖20g,KH2PO42g,水1000mL。
③硫磺菌液体培养基配方:葡萄糖30g,玉米粉10g,酵母提取物5g,KH2PO4 1.0g,CaCO32.0g,MgSO40.5g,水1000mL。
2.通过对3种药用菌菌丝体和发酵液的多糖提取研究,结果表明相同的提取方法,不同药用菌菌丝体、发酵液多糖提取率均有显著差异,其中赤芝菌丝体、发酵液多糖提取率均为最高,分别为18.5%和23.3%。
3.测定3种药用菌多糖中多糖含量和蛋白质含量,3种菌丝体多糖多糖含量由高到低依次是赤芝菌丝体多糖>蝉花菌丝体多糖>硫磺菌菌丝体多糖,3种发酵液多糖具多糖含量由高到低依次是赤芝发酵液多糖>硫磺菌发酵液多糖>蝉花发酵液多糖。
4.通过3种药用菌菌丝体粗多糖对Lewis肺癌的体内抑制作用的研究,表明3种真菌菌丝体多糖对肿瘤均有一定抑制效果。其中,对Lewis肺癌的最大抑制率是赤芝高剂量组为50.2%,其次是蝉花高剂量组为46.3%;硫磺菌多糖的最大抑瘤率仅为35.7%,低于其他2种多糖的最大抑瘤率。
5.脾细胞增殖实验结果表明6个样品的增强免疫力作用依次为蝉花发酵液多糖>蝉花菌丝体多糖>赤芝发酵液多糖>硫磺菌发酵液多糖>赤芝菌丝体多糖>硫磺菌菌丝体多糖;药用菌发酵液多糖对细胞增殖作用的效果优于菌丝体多糖。
To investigate the best formula of fermentation medium of three medicinal mushroom by utilizing mycelium as the target product,the Ganoderma lucidum (leyss.:Fr.) Karst,Cordyceps sobolifera (Hill.) Berk. et Br and Laetiporus sulphureus (Fr.) Murrill,which were collected from field acquisition and purification were employed as the experimental materials. Different types of extracting rate of polysaccharides were determined through extracting the polysaccharide of mycelium and fermentation liquid. Furthermore,Lewis lung cancer solid tumors were used to investigate the influencing of polysaccharides to the tumor inhibitory rate. The splenocyte proliferative experiment was used to investigate the stimulatory effects of polysaccharides to the immune cells,in order to exploring the function of different kinds polysaccharides. This can establish scientific basis for the exploring of wild medicinal fungi.
Main results are as follows:
1.In the appropriate cultural environment,the excellent formula liquidcultural medium for the fungi growth and mycelium production were selected by the comparative experiment of cultural medium formula of medicinal fungi.
①The optimum liquid medium of G.lucidum :Glucose 30g,soybean powder 20g,(NH4)2SO4 2g ,KH2PO41g ,MgSO4 0.8g ,VB0.02g,H2O 1000ml。
②The optimum liquid medium of C. sobolifera :potato 200g,soybean powder 5g,Glucose 20g,KH2PO4 2g,H2O 1000ml。
③The optimum liquid medium of L. sulphureus :Glucose 30g,corn meal 10g,yeast extract 5g ,KH2PO4 1.0g, CaCO3 2.0g , MgSO4 0.5g, H2O 1000ml。
2.The polysaccharide extraction of three kinds of mycelium and fermentation liquid were investigated. The results indicated that there were significant differences of the yield rate among distinct medical mycelium and fermentation liquid by the same extracting method. The yield rate of polysaccharide from mycelium and fermentation liquid of G. lucidum was 18.5% and 23.3% respectively and was highest.
3.The polysaccharide content and protein content of three medical mushroom were determined. The results shows that the seriatim of the mycelium polysaccharide content is G. lucidum > C. sobolifera > L. sulphureus and the seriatim of the polysaccharide content in Polysaccharide from fermentation liquid is G. lucidum > L. sulphureus > C. sobolifera .
4.The inhibition function of three types of medical mycelium to Lewis lung cancer was studied. The results indicated that all of the three types of mycelium polysaccharide can inhibit the tumor. The inhibition ratio of G. lucidum polysaccharide to Lewis lung cancer is highest, which reached to 50.2%. The inhibition ratio of the C. sobolifera polysaccharide and the L. sulphureus polysaccharide was 46.3% and 35.7% respectively.
5.The splenocyte proliferation assay showed that the seriatim of the increasing immunity effect of six samples was C. sobolifera polysaccharide of fermentation liquid>C. sobolifera polysaccharide of mycelium >G. lucidum polysaccharide of fermentation liquid >L.sulphureus polysaccharide of fermentation liquid >G. lucidum polysaccharide of mycelium >L. sulphureus polysaccharide of mycelium. The effects of medicinal mushroom polysaccharide of fermentation liquid were better than that of medicinal mushroom polysaccharide of mycelium.
引文
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