姬松茸多糖的提取、纯化和结构分析
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摘要
本论文对姬松茸子实体多糖进行了系统的提取、纯化研究，并对所得一个大分子量单组分进行了一些理化性质研究和结构分析。
1、提取时，先进行了一些单因素试验，然后对确定出的料水比、浸提温度、浸提时间三个因素进行了正交试验，得出了水提时的最佳工艺条件，而经生产经济核算，最终确定料水比：1:30；浸提温度：120℃；浸提时间：3h，该条件下，多糖得率为8.12％。
另外实验发现微波预处理8～12分钟可使提取率从8.12％提高到约9.15％；提取次数以两次较经济，两次累计提取率可达93.10％；醇沉工艺优化条件为3倍体积95％乙醇沉淀6h。
按上述最佳提取工艺条件进行提取，实际多糖得率为9.12％。
2、纯化时，系统地进行了脱蛋白研究。采用了Sevag法、酶法结合Sevag法、三氯乙酸法、1万分子截留量膜超滤、阴阳离子交换树脂法脱蛋白。
用弱碱性阴离子和弱酸性阳离子交换树脂一次串联进行粗多糖脱蛋白，效果十分理想。该法条件温和、有部分脱色效果、易工业化，蛋白脱除率达92.36％，多糖回收率为82.85％，经1万截留分子量透析膜透析，冷冻干燥后所得姬松茸多糖精粉为淡黄色粉末，其多糖含量达89.27％，蛋白质含量为4.55％。
3、对所得姬松茸多糖精粉进行了多种凝胶柱层析，先用Sephadex G-200葡聚糖凝胶层析分离成大分子量多糖部分和小分子量多糖部分，然后对大分子量多糖部分进行Sephacryl S-400凝胶层析，分离得到一个分子量超过200万且含有少量蛋白(1.5％)的多糖组分，经凝胶HPLC纯度鉴定为均一性物质，命名组分A.B.M.F-VHMW，该组分含量占多糖精粉的28.96％；对小分子量多糖部分进行Sephadex G-100葡聚糖凝胶柱层析，结果表明主要有三个组分，经凝胶HPLC标准分子量对照分析，三个组分分子量分别为7.2万、4.8万和1.3万；相对含量比例为：38.2％:16.1％:45.7％。
4、对所得单组分A.B.M.F-VHMW进行了一些理化性质和结构分析。单组分A.B.M.F-VHMW为白色粉末状固体，可溶于水，不溶于乙醇、乙醚、丙酮等有机溶剂，其比旋光度为[α]_D~(25)=+62.5°。凝胶HPLC(HPGPC)分析其分子量
大于200万。经2.omol几HZSO;完全水解后HPLC分析，确定由单一葡萄糖组成。
红外光谱(IR)、‘H和’3c核磁共振谱(NMR)分析确定其糖链结构主要为a-
(1~6)毗喃型D一葡聚糖，是含有1.5%蛋白的蛋白聚糖。
本研究的特色和创新之处主要表现在以下几点:
1、独创性地将微波处理与加压水提工艺结合起来，大大提高了多糖提取得
率。
2、在纯化时，首次采用弱碱性阴离子和弱酸性阳离子交换树脂串联进行脱
蛋白处理，各方面效果均较理想。
3、对所得姬松茸多糖精粉进行了S叩hadexo一200、s即hacryls一400和
SePhadexG一100凝胶柱层析，对其中一个大于200万分子量组分A.B.M.F一vHMw
进行了收集和冻干，并进行了一些理化性质和结构分析，结果表明组分
A.B.M一VHMW的糖链结构主要为a一(l~6)毗喃型D一葡聚糖，并含有1 .5%
蛋白的蛋白聚糖，参考国内外文献均未见报道。
In this dissertation, the extraction and purification of polysaccharides from Agaricus Blazei Murill fruiting body were systematic studied. A high molecular weight polysaccharide mono-fraction was purified, and some physicochemical characters and structural properties of this fraction were researched.
The results are mainly as following:
1. Some mono-factors in extracting the fruit bodies of Agaricus Blazei Murill with water were firstly studied, then through orthogonal test, the most rational technologic factors were obtained, with manufacture considerated, the finally rational technology condition were: 100 grams Agaricus Blazei Murill powder was soaked with 3000ml water (weight of material :weight of water=l:30), firstly treated with microwave for 10 minutes ,then extracted at temperature of 120C for 3h; Vacuum concentrated to proper volume, then it was precipitated with 3 times volume of 95% ethanol for 6h at room temperature, and the yield of polysaccharide was up to 9.12%, which was higher than that mentioned in literature.
2. When in period of purification for above crude polysaccharides, Sevag method, enzyme decomposition with Sevag method, trichloroacetic acid(TCA) sedimentation, ultrafiltration , anion and cation exchange resin chromatography were studied to remove the protein in the polysaccharides.
Based on experimental results and general consideration, it was suitable to adopt using anion and cation exchange resin in series chromatography once. After that, 92.36% protein of crude polysaccharides could be removed, the recovery rate of polysaccharide was 82.85%, and the ratio value of polysaccharides content to protein content was up to 19.6.Then after dialysis and freeze dryness, light yellow powder was produced, we called it as fine Agaricus Blazei Murill polysaccharides. In this powder, polysaccharide and protein content was 89.27% and 4.55%.
Several gel column chromatography were used to separate fine polysaccharide. Firstly, higher molecular and lower weight sections were gained by using Sephadex G-200 gel chromatograph. Then a polysaccharide fraction containing 1.5% protein was gained by Sephacryl S-400 chromatographing to the higher molecular. The purity of this fraction was proved by HPGPC and its molecular was more than 2 X 106 dalton
and was named A.B.M.F-VHMW. The portion of A.B.M.F-VHMW was 28.96% of fine polysaccharides. And the low molecular weight was chromatographed by Sephadex G-100 gel filtration, and three fraction were gained , the molecular weight of them was 7.2 X 104(38.2%), 4.8 X104 (16.1%), 1.3 X 104(45.7%) separately.
3.Some physicochemical characters and structural properties of A.B.M.F-VHMW were researched. The optical rotation degree of A.B.M.F-VHMW was [a]D25- +62.5?, and it was soluble in water, insoluble in ethanol, aether, acetone. After completely hydrolyzation by 2.0mol/L H2SO4 and analysed by HPLC it was confirmed that A.B.M.F-VHMW was made up of glucose only. Then through IR, HC NMR, the results showed that the main polysaccharide chain was a - (1 6 ) D-Glc. and 1.5% combined protein was contained.
The creative points are as folio wings:
1 .Micro-wave treated and high pressure were Combined originally and the yield was greatly improved when in extraction,.
2.For the first time, anion and cation exchange in series chromatography resin were adopted to remove the protein, and the ideal results were gained.
3.An A.B.M.F-VHMW polysaccharide fraction which molecular was more than was purified by several gel filtration, and some physicochemical characters and structural properties of it were researched, the results showed that the main polysaccharide chain was a - (1 ?) D-Glc contained with 1.5% combined protein, and it was not reported in domestic and overseas literature.
1、提取时，先进行了一些单因素试验，然后对确定出的料水比、浸提温度、浸提时间三个因素进行了正交试验，得出了水提时的最佳工艺条件，而经生产经济核算，最终确定料水比：1:30；浸提温度：120℃；浸提时间：3h，该条件下，多糖得率为8.12％。
另外实验发现微波预处理8～12分钟可使提取率从8.12％提高到约9.15％；提取次数以两次较经济，两次累计提取率可达93.10％；醇沉工艺优化条件为3倍体积95％乙醇沉淀6h。
按上述最佳提取工艺条件进行提取，实际多糖得率为9.12％。
2、纯化时，系统地进行了脱蛋白研究。采用了Sevag法、酶法结合Sevag法、三氯乙酸法、1万分子截留量膜超滤、阴阳离子交换树脂法脱蛋白。
用弱碱性阴离子和弱酸性阳离子交换树脂一次串联进行粗多糖脱蛋白，效果十分理想。该法条件温和、有部分脱色效果、易工业化，蛋白脱除率达92.36％，多糖回收率为82.85％，经1万截留分子量透析膜透析，冷冻干燥后所得姬松茸多糖精粉为淡黄色粉末，其多糖含量达89.27％，蛋白质含量为4.55％。
3、对所得姬松茸多糖精粉进行了多种凝胶柱层析，先用Sephadex G-200葡聚糖凝胶层析分离成大分子量多糖部分和小分子量多糖部分，然后对大分子量多糖部分进行Sephacryl S-400凝胶层析，分离得到一个分子量超过200万且含有少量蛋白(1.5％)的多糖组分，经凝胶HPLC纯度鉴定为均一性物质，命名组分A.B.M.F-VHMW，该组分含量占多糖精粉的28.96％；对小分子量多糖部分进行Sephadex G-100葡聚糖凝胶柱层析，结果表明主要有三个组分，经凝胶HPLC标准分子量对照分析，三个组分分子量分别为7.2万、4.8万和1.3万；相对含量比例为：38.2％:16.1％:45.7％。
4、对所得单组分A.B.M.F-VHMW进行了一些理化性质和结构分析。单组分A.B.M.F-VHMW为白色粉末状固体，可溶于水，不溶于乙醇、乙醚、丙酮等有机溶剂，其比旋光度为[α]_D~(25)=+62.5°。凝胶HPLC(HPGPC)分析其分子量
大于200万。经2.omol几HZSO;完全水解后HPLC分析，确定由单一葡萄糖组成。
红外光谱(IR)、‘H和’3c核磁共振谱(NMR)分析确定其糖链结构主要为a-
(1~6)毗喃型D一葡聚糖，是含有1.5%蛋白的蛋白聚糖。
本研究的特色和创新之处主要表现在以下几点:
1、独创性地将微波处理与加压水提工艺结合起来，大大提高了多糖提取得
率。
2、在纯化时，首次采用弱碱性阴离子和弱酸性阳离子交换树脂串联进行脱
蛋白处理，各方面效果均较理想。
3、对所得姬松茸多糖精粉进行了S叩hadexo一200、s即hacryls一400和
SePhadexG一100凝胶柱层析，对其中一个大于200万分子量组分A.B.M.F一vHMw
进行了收集和冻干，并进行了一些理化性质和结构分析，结果表明组分
A.B.M一VHMW的糖链结构主要为a一(l~6)毗喃型D一葡聚糖，并含有1 .5%
蛋白的蛋白聚糖，参考国内外文献均未见报道。
In this dissertation, the extraction and purification of polysaccharides from Agaricus Blazei Murill fruiting body were systematic studied. A high molecular weight polysaccharide mono-fraction was purified, and some physicochemical characters and structural properties of this fraction were researched.
The results are mainly as following:
1. Some mono-factors in extracting the fruit bodies of Agaricus Blazei Murill with water were firstly studied, then through orthogonal test, the most rational technologic factors were obtained, with manufacture considerated, the finally rational technology condition were: 100 grams Agaricus Blazei Murill powder was soaked with 3000ml water (weight of material :weight of water=l:30), firstly treated with microwave for 10 minutes ,then extracted at temperature of 120C for 3h; Vacuum concentrated to proper volume, then it was precipitated with 3 times volume of 95% ethanol for 6h at room temperature, and the yield of polysaccharide was up to 9.12%, which was higher than that mentioned in literature.
2. When in period of purification for above crude polysaccharides, Sevag method, enzyme decomposition with Sevag method, trichloroacetic acid(TCA) sedimentation, ultrafiltration , anion and cation exchange resin chromatography were studied to remove the protein in the polysaccharides.
Based on experimental results and general consideration, it was suitable to adopt using anion and cation exchange resin in series chromatography once. After that, 92.36% protein of crude polysaccharides could be removed, the recovery rate of polysaccharide was 82.85%, and the ratio value of polysaccharides content to protein content was up to 19.6.Then after dialysis and freeze dryness, light yellow powder was produced, we called it as fine Agaricus Blazei Murill polysaccharides. In this powder, polysaccharide and protein content was 89.27% and 4.55%.
Several gel column chromatography were used to separate fine polysaccharide. Firstly, higher molecular and lower weight sections were gained by using Sephadex G-200 gel chromatograph. Then a polysaccharide fraction containing 1.5% protein was gained by Sephacryl S-400 chromatographing to the higher molecular. The purity of this fraction was proved by HPGPC and its molecular was more than 2 X 106 dalton
and was named A.B.M.F-VHMW. The portion of A.B.M.F-VHMW was 28.96% of fine polysaccharides. And the low molecular weight was chromatographed by Sephadex G-100 gel filtration, and three fraction were gained , the molecular weight of them was 7.2 X 104(38.2%), 4.8 X104 (16.1%), 1.3 X 104(45.7%) separately.
3.Some physicochemical characters and structural properties of A.B.M.F-VHMW were researched. The optical rotation degree of A.B.M.F-VHMW was [a]D25- +62.5?, and it was soluble in water, insoluble in ethanol, aether, acetone. After completely hydrolyzation by 2.0mol/L H2SO4 and analysed by HPLC it was confirmed that A.B.M.F-VHMW was made up of glucose only. Then through IR, HC NMR, the results showed that the main polysaccharide chain was a - (1 6 ) D-Glc. and 1.5% combined protein was contained.
The creative points are as folio wings:
1 .Micro-wave treated and high pressure were Combined originally and the yield was greatly improved when in extraction,.
2.For the first time, anion and cation exchange in series chromatography resin were adopted to remove the protein, and the ideal results were gained.
3.An A.B.M.F-VHMW polysaccharide fraction which molecular was more than was purified by several gel filtration, and some physicochemical characters and structural properties of it were researched, the results showed that the main polysaccharide chain was a - (1 ?) D-Glc contained with 1.5% combined protein, and it was not reported in domestic and overseas literature.
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