灵芝多糖的液体发酵、提取纯化及其硫酸化改性的研究
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摘要
本论文综述了灵芝多糖的生物学活性及其抗肿瘤、免疫调节作用的免疫学机制,讨论了影响灵芝液体深层发酵生产的主要因素,总结了有关多糖构效关系的研究并列举了常用的多糖结构修饰方法。
从11株供试灵芝菌种中筛选得到一株高菌丝体得率的灵芝菌株,为赤芝07号菌株。通过正交试验确定了适宜赤芝07号菌株液体发酵的培养基配方是：葡萄糖4%、酵母粉0.3%、KH2PO40.15%、MgSO40.1%。赤芝07号菌株摇瓶发酵的最适培养条件为：初始pH4.0,装液量40%,接种量10%,培养时间8d。经100L发酵罐中试放大试验,结果菌丝体得率为11.26g/L,胞外粗多糖得率为1.632g/L。
通过单因素试验和响应面分析法得到水提灵芝菌丝体粗多糖的最佳工艺条件为：提取温度100℃,提取时间2.45h,水料比45.72：1,提取1次。在此条件下,实际提取率2.228%与模型理论预测值的相对误差小于2%。
灵芝多糖脱蛋白的最适作用酶是木瓜蛋白酶,最佳脱蛋白方法是：粗多糖经木瓜蛋白酶酶解,再用Sevage法处理3次,其蛋白脱除率达到76.3%。紫外光谱显示在260与280 nm波长处均无显著吸收峰,说明灵芝多糖已基本去除蛋白质、核酸类杂质。
灵芝胞外多糖经超滤纯化后,高效液相色谱图显示单一的多糖峰,并测得其重均分子量为146948Da,多糖含量达到98.68%。核磁共振碳谱分析表明,灵芝胞外多糖为杂多糖,主要由3种不同的单糖组成,为α、β两种吡喃糖构型,主要由1→3和1→6糖苷键连接,其中以1→3糖苷键为主链。
采用氯磺酸-吡啶法进行灵芝胞外多糖的硫酸化改性,红外光谱表明硫酸根与灵芝多糖分子结合成酯,硫酸化产物得率为35.6%。
The biological activity of Ganoderma lucidum polysaccharide and the immunological mechanism of its anti-tumor activity, immunomodulating effect were summarized, the effects of the major factors in submerged fermentation of Ganoderma lucidum were discussed, the relationships between structure and effect of polysaccharide were studied, meanwhile, some motheds of molecular modification were listed.
A height mycelia yield strain was screened out from 11 tested strains of Ganoderma lucidum, as Ganoderma lucidum-07(GL-07). The results of orthogonal test showed that, the optimum formula of liquid fermentation medium of GL-07 was glucose 4%, yeast extract 0.3%, KH2PO4 0.15%, MgSO4 0.1%. The optimum fermentation conditions were as follow:initial pH,4.0; medium volume,40%; inoculation volume,10%; culture time,8d. The results of 100L fermentation tank pilot study showed that, mycelia yield was 11.26g/L, extracellular polysaccharide yield was 1.632g/L.
The optimal extraction condition of water-soluble polysaccharides from Ganoderma lucidum mycelia was determined by single factor method and response surface analysis, listed below:extraction temperature,100℃; extraction time,2.45h; ratio of water to raw material,45.72:1; with one-time extraction. Under these conditions, the relative error between the actual extraction rate 2.228% and the model theory predicted rate was less than 2%.
The best enzyme for deproteinization of Ganoderma lucidum polysaccharide was papain, the best way was that enzymatic hydrolyzed by papain, and then treated by Sevage method for 3 times, the removal percentage of protein reached 76.3%. The ultraviolet spectrum of deproteinized polysaccharide showed that there wasn't protein or nucleic acid because no absorption peaks at 260nm and 280nm.
Treated by ultrafiltration, extracellular polysaccharide of Ganoderma lucidum (GLP) exhibited a single polysaccharide peak on HPLC chromatogram, the weight-average molecular weight of GLP was 146948Da, the content of polysaccharide reached 98.68%. The 13C-NMR spectrum confirmed GLP was consisted of three kinds of different monosaccharide residues, as a heteropolysaccharide, and there wereα,βpyranose configuration and 1→3,1→6 glycosidic linkage.
GLP was sulfated by the means of using chlorosulfonic acid-pyridine. The infrared spectrum showd GLP was combined with sulfate, and the sulfated product yield was 35.6%.
从11株供试灵芝菌种中筛选得到一株高菌丝体得率的灵芝菌株,为赤芝07号菌株。通过正交试验确定了适宜赤芝07号菌株液体发酵的培养基配方是：葡萄糖4%、酵母粉0.3%、KH2PO40.15%、MgSO40.1%。赤芝07号菌株摇瓶发酵的最适培养条件为：初始pH4.0,装液量40%,接种量10%,培养时间8d。经100L发酵罐中试放大试验,结果菌丝体得率为11.26g/L,胞外粗多糖得率为1.632g/L。
通过单因素试验和响应面分析法得到水提灵芝菌丝体粗多糖的最佳工艺条件为：提取温度100℃,提取时间2.45h,水料比45.72：1,提取1次。在此条件下,实际提取率2.228%与模型理论预测值的相对误差小于2%。
灵芝多糖脱蛋白的最适作用酶是木瓜蛋白酶,最佳脱蛋白方法是：粗多糖经木瓜蛋白酶酶解,再用Sevage法处理3次,其蛋白脱除率达到76.3%。紫外光谱显示在260与280 nm波长处均无显著吸收峰,说明灵芝多糖已基本去除蛋白质、核酸类杂质。
灵芝胞外多糖经超滤纯化后,高效液相色谱图显示单一的多糖峰,并测得其重均分子量为146948Da,多糖含量达到98.68%。核磁共振碳谱分析表明,灵芝胞外多糖为杂多糖,主要由3种不同的单糖组成,为α、β两种吡喃糖构型,主要由1→3和1→6糖苷键连接,其中以1→3糖苷键为主链。
采用氯磺酸-吡啶法进行灵芝胞外多糖的硫酸化改性,红外光谱表明硫酸根与灵芝多糖分子结合成酯,硫酸化产物得率为35.6%。
The biological activity of Ganoderma lucidum polysaccharide and the immunological mechanism of its anti-tumor activity, immunomodulating effect were summarized, the effects of the major factors in submerged fermentation of Ganoderma lucidum were discussed, the relationships between structure and effect of polysaccharide were studied, meanwhile, some motheds of molecular modification were listed.
A height mycelia yield strain was screened out from 11 tested strains of Ganoderma lucidum, as Ganoderma lucidum-07(GL-07). The results of orthogonal test showed that, the optimum formula of liquid fermentation medium of GL-07 was glucose 4%, yeast extract 0.3%, KH2PO4 0.15%, MgSO4 0.1%. The optimum fermentation conditions were as follow:initial pH,4.0; medium volume,40%; inoculation volume,10%; culture time,8d. The results of 100L fermentation tank pilot study showed that, mycelia yield was 11.26g/L, extracellular polysaccharide yield was 1.632g/L.
The optimal extraction condition of water-soluble polysaccharides from Ganoderma lucidum mycelia was determined by single factor method and response surface analysis, listed below:extraction temperature,100℃; extraction time,2.45h; ratio of water to raw material,45.72:1; with one-time extraction. Under these conditions, the relative error between the actual extraction rate 2.228% and the model theory predicted rate was less than 2%.
The best enzyme for deproteinization of Ganoderma lucidum polysaccharide was papain, the best way was that enzymatic hydrolyzed by papain, and then treated by Sevage method for 3 times, the removal percentage of protein reached 76.3%. The ultraviolet spectrum of deproteinized polysaccharide showed that there wasn't protein or nucleic acid because no absorption peaks at 260nm and 280nm.
Treated by ultrafiltration, extracellular polysaccharide of Ganoderma lucidum (GLP) exhibited a single polysaccharide peak on HPLC chromatogram, the weight-average molecular weight of GLP was 146948Da, the content of polysaccharide reached 98.68%. The 13C-NMR spectrum confirmed GLP was consisted of three kinds of different monosaccharide residues, as a heteropolysaccharide, and there wereα,βpyranose configuration and 1→3,1→6 glycosidic linkage.
GLP was sulfated by the means of using chlorosulfonic acid-pyridine. The infrared spectrum showd GLP was combined with sulfate, and the sulfated product yield was 35.6%.
引文
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