真姬菇液态发酵及菌丝体多糖的提取纯化与结构解析
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摘要
真姬菇(Hypsizigus marmoreus(Peck)Bigelow)隶属担子菌亚门、层菌纲、伞菌目、白蘑科、离褶菌族、玉蕈属，含有多种活性物质，具有很高的营养和药用价值。
本文以西北农林科技大学食品学院实验室保藏的真姬菇多糖高产菌株ZJ029为研究对象，通过考察34种营养因子对ZJ029菌株液态发酵产多糖量的影响，优化出菌株的最适发酵培养基。在此基础上，在摇瓶及5L发酵罐上研究并优化了发酵液起始pH、液体种子种龄、接种量、发酵温度、相对溶氧量等发酵工艺条件，确定出了发酵周期。在优化的营养与发酵工艺条件下发酵制得真姬菇菌丝体，对菌丝体多糖的提取、分离、纯化等工艺环节的不同技术方法进行了对比研究与分析，确定采用热碱液提取法提取多糖，运用酶解技术对粗提物脱除蛋白，以阴离子交换柱和葡聚糖凝胶柱为载体对分离得到的多糖进行纯化，同时运用纸层析、紫外光谱、红外光谱、HPLC、GC等技术方法对多糖的纯度及结构进行了综合鉴定与分析。试验取得以下主要结果：
(1)在真姬菇ZJ029菌株液态培养中，对胞外多糖产量(Y_1^)有显著影响的营养因子依次为麦芽糖(X_2)、MgSO_4(X_4)、葡萄糖(X_1)，这3个因子对胞外多糖的影响可用表达式i：Y_1^=1．21+0．093×X_1+0．14×X_2+1．11×X_4来描述。对胞内多糖产量(Y_2^)有显著影响的营养因子依次为玉米淀粉(X_3)、MgSO_4(X_4)、葡萄糖(X_1)，这3个因子对胞内多糖的影响可用表达式ii：Y_2^=-0．021+0．017×X_1+0．041＞X_3+0．23×X_4来描述。
(2)产多糖量与成本价格比相对较高的4个营养因子即玉米淀粉(X_1)、葡萄糖(X_2)、MgSO_4(X_3)、豆粕粉(X_4)与胞外多糖(Y_3^)的关系模型可用表达式iii：Y_3^=-1．27+0．86X_1+0．29X_2+1．26X_3+0．43X_4-0．025X_1~2-0．003926X_2~2-0．056X_3~2-0．031X_4~2-0．007072X_1X_2-0．093X_1X_3-0．004797X_1X_4-0．003744X_2X_3+0．00016566X_2X_4+0．002656X_3X_4来描述，该模型的校正决定系数达0．8997。与胞内多糖(Y_4^)的关系模型可用表达式iv：Y_4^=-0．1 7+0．13X_1+0．044X_2+0．20X_3+0．047X_4-0．003852X_1~2-0．0005903X_2~2-0．009031X_3~2-0．003582X_4~2-0．001116X_1X_2-0．015X_1X_3-0．003954X_1X_4-0．0008063X_2X_3+0．00007187X_2X_4+0．0016556X_3X_4来描述，该模型试验真实值与预测值之间的相关性高达97％。
(3)优化得出真姬菇ZJ029菌株深层培养的最佳营养条件为：玉米淀粉5．63g／L、葡萄糖25．11g／L、豆粕粉6．84 g／L、MgSO_43．84g／L、KH_2PO_4 3g／L、KNO_31g／L、V_(B1)10mg／L。发酵罐发酵生产的最佳工艺条件为：发酵液自然初始pH值(约为6．5)、液体种子种龄6d、接种量10％、发酵温度25℃、发酵罐相对溶氧量80％、发酵周期8d，在此营养及工艺条件下发酵，每升可获得1．98g胞内多糖和9．45g胞外多糖。
(4)真姬菇菌丝体多糖的碱提取工艺中3个关键参数即提取温度(X_1)、提取时间(X_2)、液料比(X_3)与多糖提取率(Y^)之间的关系更接近于二次多项式模型v：Y^=-339．43+6．68X_1+16．45X_2+2．56X_3-0．035X_1~2+1．66X_2~2-0．055X_3~2-0．23X_1X_2+0．007X_1X_3-0．25X_2X_3，模型的预测值与试验真实值之间的相关性达95．26％，模型能够解释84％的总变异。当提取温度在83℃～88℃、提取时间在3．4h～3．8h、料液比在1：21 g／mL～1：24 g／mL范围时，多糖得率不低于5．8％。获得最大多糖得率的工艺条件为提取温度83．95℃、提取时间3．80h、料液比1：21g／mL、提取液pH13、醇沉时乙醇用量为提取液体积的3倍，在此条件下，真姬菇菌丝体多糖碱液提取法的多糖得率为7．97％。
(5)Sevag法脱除真姬菇菌丝体多糖中蛋白质的条件是：震荡时间30min、样品：Sevag=2：1、脱除次数1次，在此条件下，蛋白质脱除率为33．8％，多糖损失率为25．1％。与木瓜蛋白酶、酸性蛋白酶、中性蛋白酶、胃蛋白酶和胰蛋白酶相比较，碱性蛋白酶更适合用来脱除真姬菇菌丝体多糖中的蛋白质。碱性蛋白酶酶解法脱蛋白的适宜条件为：酶解温度50℃、加酶量2．687 u／mg、酶解时间3．5h，此时蛋白质脱除率为63．4％，多糖损失率为8．3％。在碱提真姬菇菌丝体多糖脱蛋白过程中，碱性蛋白酶酶解法比Sevag法更有效。
(6)真姬菇菌丝体脱蛋白多糖经DEAE-Sephadex A-25柱层析后获得2种不同极性大小的多糖组分：A-25-1、A-25-3。采用纸色谱鉴定纯度表明，这2种多糖为均一组分，紫外光谱表明它们不含蛋白质和核酸；而Sephadex G-200柱层析和HPLC色谱鉴定结果表明这2种组分均不是均一多糖；进一步用Sephadex G-200层析柱对A-25-1、A-25-3组分进行纯化，可以获得TⅠ-1、TⅠ-2、TⅡ-1、TⅡ-2、TⅢ-1、TⅢ-2、NⅣ7种多糖组分，经HPLC色谱鉴定纯度，表明其中3种组分TⅡ-2、TⅢ-2、NⅣ为均一多糖，满足化学定性定量分析标准。
(7)化学分析法结合紫外光谱、红外光谱、HPLC、GC对真姬菇菌丝体纯化多糖的部分物化性质和结构研究表明，组分TⅢ-2由岩藻糖、半乳糖、葡萄糖、甘露糖4种单糖组成，其摩尔比为1．00：3．39：0．63：1．28，分子量为19136Da；该多糖主链是由β-D-半乳糖以1→3糖苷键连接而成，支链是由1→3糖苷键连接的D-葡萄糖和少量D-甘露糖组成，另外多糖链中还含有少量1→4、1→6糖苷键。组分NIV是由岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖组成，其摩尔比为1．00：0．59：1．11：2．80：2．25：1．25：0．62，分子量为522981Da；该多糖主链是由α-D-半乳糖和D-葡萄糖以1→3糖苷键连接的二糖重复单元组成，支链主要由1→4糖苷键键合的阿拉伯糖组成，另外糖链中还含有部分1→6糖苷键。
本论文的创新点在于：
(1)首次对真姬菇发酵菌丝体多糖的提取、分离、纯化技术作了系统研究，并获得了分子量分别为19136Da、522981Da的2种纯化多糖即TⅢ-2、NⅣ，明确了TⅢ-2、NⅣ的单糖组成及配比，同时对该多糖的结构进行了分析探讨。该多糖与报道的真姬菇子实体多糖的组成和结构不同，为首次获得。
(2)关于真姬菇的液态培养研究，以往的报道均以菌丝体生物量为衡量指标，摇瓶培养研究10多种营养因子对菌株生长的影响。而本文则以胞内、胞外多糖产量为指标，采用发酵罐发酵研究了34种营养因子对菌株生长的影响，并优化出了发酵工艺条件。
Hypsizigus marmoreus(Peck)Bigelow belongs to Basidiomycotina,Hymenomycetes,Agarcales,Tricholomataceae,Schizophyllum commune family,Tricholoma genera.Itcontains a variety of active substances with high nutritional and medicinal value.
This paper take Hypsizigus marmoreus ZJ029 strain,which is preserved in the laboratoryof Food Science and Engineering College of Northwest A.&F.University because ofhigh-yield polysaccharide,as the object of studies.The suitable fermentation medium wasoptimized by investigations on the effects of 34 nutritional factors on the production ofpolysaccharide from ZJ029 strain by submerged culture.On this basis,the optimizationfermentation process conditions including initial pH value of medium,liquid seed age,inoculation amount,fermentation temperature,dissolved oxygen content in fermentor andfermentation time were researched by taking shake flasks and 5L fermentors as carriers.Under the optimization fermentation medium and fermentation process conditions,abundancemycelia of Hypsizigus marmoreus were prepared as the following tests materials.Thedifferent technical methods of the extraction,separation,purification of myceliapolysaccharide were compared and analyzed.The results show that using hot alkaline solutionas extraction solution,enzymolysis isolation removal protein,ion-exchange column andsephadex column chromatography purification polysaccharide are the suitable methods.Moreover,in the last chapters,the purity and structure of polysaccharides were analyzed bymeans of paper chromatography,UV spectrum,IR spectrum,high performance liquidchromatography,gas chromatography and other technologies.In this paper,the main resultsare as follows:
(1)In the course of submerged culture of Hypsizigus marmoreus ZJ029 strain,thesignificant factors on extra-cell polysaccharide production(Y_1^) in sequence are maltose(X_2),MgSO_4(X_4),and glucose(X_1).The relationship between these three factors and extra-cellpolysaccharide production could be description with a formula i:Y_1^=1.21+0.093×X_1+0.14×X_2+1.11×X_4.The significant factors on inter-cell polysaccharideproduction(Y_2^) in sequence are cornstarch(X_3),MgSO_4(X_4),and glucose(X_1).Their relation model is a formula ii:Y_2^=-0.021+0.017×X_1+0.041×X_3+0.23×X_4.
(2)The four factors of the higher ratio of polysaccharide yield to cost price arecornstarch(X_1),glucose(X_2),MgSO_4(X_3),soybean meal(X_4) respectively.The relationshipbetween these four factors and extra-cell polysaccharide yield(Y_3^) could be description witha formula iii:Y_3^=-1.27+0.86X_1+0.29X_2+1.26X_3+0.43X_4-0.025X_1~2-0.003926X_2~2-0.056X_3~2-0.031X_4~2-0.007072X_1X_2-0.093X_1X_3-0.004797X_1X_4-0.003744X_2X_3+0.00016566X_2X_4+0.002656X_3X_4.The adjusted determination coefficient of model iii is 0.8997.The relationship between thefour factors and inter-cell polysaccharide yield(Y_4^)could be description with a formula iv:Y_4^=-0.17+0.13X_1+0.044X_2+0.20X_3+0.047X_4-0.003852X_1~2-0.0005903X_2~2-0.009031X_3~2-0.003582X_4~2-0.001116X_1X_2-0.015X_1X_3-0.003954X_1X_4-0.0008063X_2X_3+0.00007187X_2X_4+0.0016556X_3X_4.The correlation between the true value and the predictive value for the formula iv is ashigh as 97%.
(3)The optimization fermentation nutrition conditions for Hypsizigus marmoreus ZJ029are as follows:cornstarch 5.63g/L,glucose 25.11g/L,soybean meal 6.84 g/L,MgSO4 3.84g/L,KH_2PO_4 3g/L,KNO_3 1g/L,V_(B1) 10mg/L.The optimum fermentation process conditions are:natural culture medium pH value(at about 6.5),liquid seed age 6 days,inoculation amount10%,fermentation temperature 25℃,dissolved oxygen content in fermentor 80%,andfermentation time 8 days.Under the optimization nutrition conditions and fermentationprocess conditions,we can harvest inter-cell polysaccharide for 1.98g/L and extra-cellpolysaccharide for 9.45g/L.
(4)The relation model between the key factors,extration temperature(X_1),extrationtime(X_2)and extration solution-dry mycelia ratio(X_3),and the extration rate of polysaccharide(Y^) is simulated by a quadratic polynomial v:Y^=339.43+6.68X_1+16.45X_2+2.56X_3-0.035X_1~2+1.66X_2~2-0.055X_3~2-0.23X_1X_2+0.007X_1X_3-0.25X_2X_3.The correlation between the true valueand the predictive value for the formula v is as high as 95.26%.And this model couldexplain 84% of the total variation.When the extration temperature at 83～88℃,extrationsolution-dry mycelia ratio for 1:21～1:24(g/mL),extration time for 3.4～3.8h,the extrationrate of polysaccharide is not less than 5.8%.The suitable extration process conditions for thehighest extration rate of polysaccharide are as follows:extration temperature 83.95℃,extration time 3.80h,the extration solution-dry mycelia ratio 1:21(g/mL),extration solutionpH 13,and alcohol volume 3 times of extration solution.In this conditions,the extration rateof mycelia polysaccharide from fermentable Hypsizigus marmoreu ZJ029 is up to 7.97%.
(5)The appropriate conditions of removing protein from the crude myceliapolysaccharide of Hypsizigus marmoreu ZJ029 by Serag method are:shocking time 30min,the ratio of sample to Sevag reagent 2:1(V/V),shocking times 1 times.Under this conditions,the removal ratio of protein is 33.8%,and the loss ratio of polysaccharide is25.1%.Compare with papain,acid protease,neutral protease,trypsin,and pepsin,alkalineprotease is the best protease for removing protein from the crude mycelia polysaccharide ofHypsizigus marmoreu Z J029.Under the suitable conditions which are reaction temperature50℃,enzyme dosage of alkaline protease 2.687 u/mg,reaction time 3.5h,the removal ratio ofprotein is 63.4%,and the loss ratio of polysaccharide is 8.3%.The results show that thescheme of enzyme is better than the scheme of Sevag.
(6)There are 2 components(A-25-1 and A-25-3) are obtained through further isolatingploysaccharide of deproteinization with DEAE-Sephadex A-25 column chromatography.Purity identifications indicate that they are homogeneous compositions by means of paperchromatography,and no protein or nucleic acids by means of UV spectrum,but hetergeneouscompositions by means of Sephadex G-200 column chromatography and HPLCchromatography.So A-25-1 and A-25-3 are isolated by Sephadex G-200 columnchromatography again.Then 7 components,TⅠ-1,TⅠ-2,TⅡ-1,TⅡ-2,TⅢ-1,TⅢ-2,NⅣ,are obatined.Three of them,TⅡ-2,TⅢ-2,NⅣ,are identified as homogeneous compositionsby means of HPLC chromatography.
(7)By means of different methods such as chemical methods,UV spectrum,IR spectrum,HPLC chromatography,GC chromatography,the structures of polysaccharides were analyzed.The results show that sample TⅢ-2 is composed of Fuc,Gal,Glc,Man,in molar ratio of1.00:3.39:0.63:1.28.The Mw of TⅢ-2 is estimated to be 19136Da.Its main chain is made upofβ-D-1,3-Gal,and the side chains are formed withβ-D-1,3-Glc and a few ofβ-D-1,3-Man.In addition,there are a few of-1,4 and -1,6 residues in the chains.NⅣis composed of Fuc,Rha,Ara,Gal,Glc,Xyl,and Man,in molar ratio of 1.00:0.59:1.11:2.80:2.25:1.25:0.62.TheMw of NⅣis estimated to be 522981Da.The main chain of sample NⅣis made up ofα-D-1,3-Glc linkedα-D-1,3-Gal,and the side chains are formed withα-D-1,4-Ala residues.Inaddition,there are some -1,6 residues in the chains.
The new ideas and innovative points of this paper lie in the following:
(1)It's the first time to study the extraction,isolation and purification of myceliapolysaccharide from fermentable Hypsizigus marmoreus.More valuably,two polysaccharides,TⅢ-2 and NⅣ,with Mw for 19136Da and 522981Da,are obtained for the first time.Thestructures of TⅢ-2 and NⅣare analyzed,too.The structures of these two polysaccharidesare different from the polyscaaharides of Hypsizigus marmoreus fruit body.It is proved thatthey are not reported in former studies.
(2)Moreover,in previous studies,just taking mycelia biomass;as a uniqe measure indexand shake flasks as the fermentation carrier,only more than 10 nutritional factors were investigated.But in this paper,taking the production of extra-cell polysaccharide andinter-cell ploysaccharide as major measure indexes,and using fermentor as the fermentationcarrier,34 nutritional factors were investigated.
本文以西北农林科技大学食品学院实验室保藏的真姬菇多糖高产菌株ZJ029为研究对象，通过考察34种营养因子对ZJ029菌株液态发酵产多糖量的影响，优化出菌株的最适发酵培养基。在此基础上，在摇瓶及5L发酵罐上研究并优化了发酵液起始pH、液体种子种龄、接种量、发酵温度、相对溶氧量等发酵工艺条件，确定出了发酵周期。在优化的营养与发酵工艺条件下发酵制得真姬菇菌丝体，对菌丝体多糖的提取、分离、纯化等工艺环节的不同技术方法进行了对比研究与分析，确定采用热碱液提取法提取多糖，运用酶解技术对粗提物脱除蛋白，以阴离子交换柱和葡聚糖凝胶柱为载体对分离得到的多糖进行纯化，同时运用纸层析、紫外光谱、红外光谱、HPLC、GC等技术方法对多糖的纯度及结构进行了综合鉴定与分析。试验取得以下主要结果：
(1)在真姬菇ZJ029菌株液态培养中，对胞外多糖产量(Y_1^)有显著影响的营养因子依次为麦芽糖(X_2)、MgSO_4(X_4)、葡萄糖(X_1)，这3个因子对胞外多糖的影响可用表达式i：Y_1^=1．21+0．093×X_1+0．14×X_2+1．11×X_4来描述。对胞内多糖产量(Y_2^)有显著影响的营养因子依次为玉米淀粉(X_3)、MgSO_4(X_4)、葡萄糖(X_1)，这3个因子对胞内多糖的影响可用表达式ii：Y_2^=-0．021+0．017×X_1+0．041＞X_3+0．23×X_4来描述。
(2)产多糖量与成本价格比相对较高的4个营养因子即玉米淀粉(X_1)、葡萄糖(X_2)、MgSO_4(X_3)、豆粕粉(X_4)与胞外多糖(Y_3^)的关系模型可用表达式iii：Y_3^=-1．27+0．86X_1+0．29X_2+1．26X_3+0．43X_4-0．025X_1~2-0．003926X_2~2-0．056X_3~2-0．031X_4~2-0．007072X_1X_2-0．093X_1X_3-0．004797X_1X_4-0．003744X_2X_3+0．00016566X_2X_4+0．002656X_3X_4来描述，该模型的校正决定系数达0．8997。与胞内多糖(Y_4^)的关系模型可用表达式iv：Y_4^=-0．1 7+0．13X_1+0．044X_2+0．20X_3+0．047X_4-0．003852X_1~2-0．0005903X_2~2-0．009031X_3~2-0．003582X_4~2-0．001116X_1X_2-0．015X_1X_3-0．003954X_1X_4-0．0008063X_2X_3+0．00007187X_2X_4+0．0016556X_3X_4来描述，该模型试验真实值与预测值之间的相关性高达97％。
(3)优化得出真姬菇ZJ029菌株深层培养的最佳营养条件为：玉米淀粉5．63g／L、葡萄糖25．11g／L、豆粕粉6．84 g／L、MgSO_43．84g／L、KH_2PO_4 3g／L、KNO_31g／L、V_(B1)10mg／L。发酵罐发酵生产的最佳工艺条件为：发酵液自然初始pH值(约为6．5)、液体种子种龄6d、接种量10％、发酵温度25℃、发酵罐相对溶氧量80％、发酵周期8d，在此营养及工艺条件下发酵，每升可获得1．98g胞内多糖和9．45g胞外多糖。
(4)真姬菇菌丝体多糖的碱提取工艺中3个关键参数即提取温度(X_1)、提取时间(X_2)、液料比(X_3)与多糖提取率(Y^)之间的关系更接近于二次多项式模型v：Y^=-339．43+6．68X_1+16．45X_2+2．56X_3-0．035X_1~2+1．66X_2~2-0．055X_3~2-0．23X_1X_2+0．007X_1X_3-0．25X_2X_3，模型的预测值与试验真实值之间的相关性达95．26％，模型能够解释84％的总变异。当提取温度在83℃～88℃、提取时间在3．4h～3．8h、料液比在1：21 g／mL～1：24 g／mL范围时，多糖得率不低于5．8％。获得最大多糖得率的工艺条件为提取温度83．95℃、提取时间3．80h、料液比1：21g／mL、提取液pH13、醇沉时乙醇用量为提取液体积的3倍，在此条件下，真姬菇菌丝体多糖碱液提取法的多糖得率为7．97％。
(5)Sevag法脱除真姬菇菌丝体多糖中蛋白质的条件是：震荡时间30min、样品：Sevag=2：1、脱除次数1次，在此条件下，蛋白质脱除率为33．8％，多糖损失率为25．1％。与木瓜蛋白酶、酸性蛋白酶、中性蛋白酶、胃蛋白酶和胰蛋白酶相比较，碱性蛋白酶更适合用来脱除真姬菇菌丝体多糖中的蛋白质。碱性蛋白酶酶解法脱蛋白的适宜条件为：酶解温度50℃、加酶量2．687 u／mg、酶解时间3．5h，此时蛋白质脱除率为63．4％，多糖损失率为8．3％。在碱提真姬菇菌丝体多糖脱蛋白过程中，碱性蛋白酶酶解法比Sevag法更有效。
(6)真姬菇菌丝体脱蛋白多糖经DEAE-Sephadex A-25柱层析后获得2种不同极性大小的多糖组分：A-25-1、A-25-3。采用纸色谱鉴定纯度表明，这2种多糖为均一组分，紫外光谱表明它们不含蛋白质和核酸；而Sephadex G-200柱层析和HPLC色谱鉴定结果表明这2种组分均不是均一多糖；进一步用Sephadex G-200层析柱对A-25-1、A-25-3组分进行纯化，可以获得TⅠ-1、TⅠ-2、TⅡ-1、TⅡ-2、TⅢ-1、TⅢ-2、NⅣ7种多糖组分，经HPLC色谱鉴定纯度，表明其中3种组分TⅡ-2、TⅢ-2、NⅣ为均一多糖，满足化学定性定量分析标准。
(7)化学分析法结合紫外光谱、红外光谱、HPLC、GC对真姬菇菌丝体纯化多糖的部分物化性质和结构研究表明，组分TⅢ-2由岩藻糖、半乳糖、葡萄糖、甘露糖4种单糖组成，其摩尔比为1．00：3．39：0．63：1．28，分子量为19136Da；该多糖主链是由β-D-半乳糖以1→3糖苷键连接而成，支链是由1→3糖苷键连接的D-葡萄糖和少量D-甘露糖组成，另外多糖链中还含有少量1→4、1→6糖苷键。组分NIV是由岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖组成，其摩尔比为1．00：0．59：1．11：2．80：2．25：1．25：0．62，分子量为522981Da；该多糖主链是由α-D-半乳糖和D-葡萄糖以1→3糖苷键连接的二糖重复单元组成，支链主要由1→4糖苷键键合的阿拉伯糖组成，另外糖链中还含有部分1→6糖苷键。
本论文的创新点在于：
(1)首次对真姬菇发酵菌丝体多糖的提取、分离、纯化技术作了系统研究，并获得了分子量分别为19136Da、522981Da的2种纯化多糖即TⅢ-2、NⅣ，明确了TⅢ-2、NⅣ的单糖组成及配比，同时对该多糖的结构进行了分析探讨。该多糖与报道的真姬菇子实体多糖的组成和结构不同，为首次获得。
(2)关于真姬菇的液态培养研究，以往的报道均以菌丝体生物量为衡量指标，摇瓶培养研究10多种营养因子对菌株生长的影响。而本文则以胞内、胞外多糖产量为指标，采用发酵罐发酵研究了34种营养因子对菌株生长的影响，并优化出了发酵工艺条件。
Hypsizigus marmoreus(Peck)Bigelow belongs to Basidiomycotina,Hymenomycetes,Agarcales,Tricholomataceae,Schizophyllum commune family,Tricholoma genera.Itcontains a variety of active substances with high nutritional and medicinal value.
This paper take Hypsizigus marmoreus ZJ029 strain,which is preserved in the laboratoryof Food Science and Engineering College of Northwest A.&F.University because ofhigh-yield polysaccharide,as the object of studies.The suitable fermentation medium wasoptimized by investigations on the effects of 34 nutritional factors on the production ofpolysaccharide from ZJ029 strain by submerged culture.On this basis,the optimizationfermentation process conditions including initial pH value of medium,liquid seed age,inoculation amount,fermentation temperature,dissolved oxygen content in fermentor andfermentation time were researched by taking shake flasks and 5L fermentors as carriers.Under the optimization fermentation medium and fermentation process conditions,abundancemycelia of Hypsizigus marmoreus were prepared as the following tests materials.Thedifferent technical methods of the extraction,separation,purification of myceliapolysaccharide were compared and analyzed.The results show that using hot alkaline solutionas extraction solution,enzymolysis isolation removal protein,ion-exchange column andsephadex column chromatography purification polysaccharide are the suitable methods.Moreover,in the last chapters,the purity and structure of polysaccharides were analyzed bymeans of paper chromatography,UV spectrum,IR spectrum,high performance liquidchromatography,gas chromatography and other technologies.In this paper,the main resultsare as follows:
(1)In the course of submerged culture of Hypsizigus marmoreus ZJ029 strain,thesignificant factors on extra-cell polysaccharide production(Y_1^) in sequence are maltose(X_2),MgSO_4(X_4),and glucose(X_1).The relationship between these three factors and extra-cellpolysaccharide production could be description with a formula i:Y_1^=1.21+0.093×X_1+0.14×X_2+1.11×X_4.The significant factors on inter-cell polysaccharideproduction(Y_2^) in sequence are cornstarch(X_3),MgSO_4(X_4),and glucose(X_1).Their relation model is a formula ii:Y_2^=-0.021+0.017×X_1+0.041×X_3+0.23×X_4.
(2)The four factors of the higher ratio of polysaccharide yield to cost price arecornstarch(X_1),glucose(X_2),MgSO_4(X_3),soybean meal(X_4) respectively.The relationshipbetween these four factors and extra-cell polysaccharide yield(Y_3^) could be description witha formula iii:Y_3^=-1.27+0.86X_1+0.29X_2+1.26X_3+0.43X_4-0.025X_1~2-0.003926X_2~2-0.056X_3~2-0.031X_4~2-0.007072X_1X_2-0.093X_1X_3-0.004797X_1X_4-0.003744X_2X_3+0.00016566X_2X_4+0.002656X_3X_4.The adjusted determination coefficient of model iii is 0.8997.The relationship between thefour factors and inter-cell polysaccharide yield(Y_4^)could be description with a formula iv:Y_4^=-0.17+0.13X_1+0.044X_2+0.20X_3+0.047X_4-0.003852X_1~2-0.0005903X_2~2-0.009031X_3~2-0.003582X_4~2-0.001116X_1X_2-0.015X_1X_3-0.003954X_1X_4-0.0008063X_2X_3+0.00007187X_2X_4+0.0016556X_3X_4.The correlation between the true value and the predictive value for the formula iv is ashigh as 97%.
(3)The optimization fermentation nutrition conditions for Hypsizigus marmoreus ZJ029are as follows:cornstarch 5.63g/L,glucose 25.11g/L,soybean meal 6.84 g/L,MgSO4 3.84g/L,KH_2PO_4 3g/L,KNO_3 1g/L,V_(B1) 10mg/L.The optimum fermentation process conditions are:natural culture medium pH value(at about 6.5),liquid seed age 6 days,inoculation amount10%,fermentation temperature 25℃,dissolved oxygen content in fermentor 80%,andfermentation time 8 days.Under the optimization nutrition conditions and fermentationprocess conditions,we can harvest inter-cell polysaccharide for 1.98g/L and extra-cellpolysaccharide for 9.45g/L.
(4)The relation model between the key factors,extration temperature(X_1),extrationtime(X_2)and extration solution-dry mycelia ratio(X_3),and the extration rate of polysaccharide(Y^) is simulated by a quadratic polynomial v:Y^=339.43+6.68X_1+16.45X_2+2.56X_3-0.035X_1~2+1.66X_2~2-0.055X_3~2-0.23X_1X_2+0.007X_1X_3-0.25X_2X_3.The correlation between the true valueand the predictive value for the formula v is as high as 95.26%.And this model couldexplain 84% of the total variation.When the extration temperature at 83～88℃,extrationsolution-dry mycelia ratio for 1:21～1:24(g/mL),extration time for 3.4～3.8h,the extrationrate of polysaccharide is not less than 5.8%.The suitable extration process conditions for thehighest extration rate of polysaccharide are as follows:extration temperature 83.95℃,extration time 3.80h,the extration solution-dry mycelia ratio 1:21(g/mL),extration solutionpH 13,and alcohol volume 3 times of extration solution.In this conditions,the extration rateof mycelia polysaccharide from fermentable Hypsizigus marmoreu ZJ029 is up to 7.97%.
(5)The appropriate conditions of removing protein from the crude myceliapolysaccharide of Hypsizigus marmoreu ZJ029 by Serag method are:shocking time 30min,the ratio of sample to Sevag reagent 2:1(V/V),shocking times 1 times.Under this conditions,the removal ratio of protein is 33.8%,and the loss ratio of polysaccharide is25.1%.Compare with papain,acid protease,neutral protease,trypsin,and pepsin,alkalineprotease is the best protease for removing protein from the crude mycelia polysaccharide ofHypsizigus marmoreu Z J029.Under the suitable conditions which are reaction temperature50℃,enzyme dosage of alkaline protease 2.687 u/mg,reaction time 3.5h,the removal ratio ofprotein is 63.4%,and the loss ratio of polysaccharide is 8.3%.The results show that thescheme of enzyme is better than the scheme of Sevag.
(6)There are 2 components(A-25-1 and A-25-3) are obtained through further isolatingploysaccharide of deproteinization with DEAE-Sephadex A-25 column chromatography.Purity identifications indicate that they are homogeneous compositions by means of paperchromatography,and no protein or nucleic acids by means of UV spectrum,but hetergeneouscompositions by means of Sephadex G-200 column chromatography and HPLCchromatography.So A-25-1 and A-25-3 are isolated by Sephadex G-200 columnchromatography again.Then 7 components,TⅠ-1,TⅠ-2,TⅡ-1,TⅡ-2,TⅢ-1,TⅢ-2,NⅣ,are obatined.Three of them,TⅡ-2,TⅢ-2,NⅣ,are identified as homogeneous compositionsby means of HPLC chromatography.
(7)By means of different methods such as chemical methods,UV spectrum,IR spectrum,HPLC chromatography,GC chromatography,the structures of polysaccharides were analyzed.The results show that sample TⅢ-2 is composed of Fuc,Gal,Glc,Man,in molar ratio of1.00:3.39:0.63:1.28.The Mw of TⅢ-2 is estimated to be 19136Da.Its main chain is made upofβ-D-1,3-Gal,and the side chains are formed withβ-D-1,3-Glc and a few ofβ-D-1,3-Man.In addition,there are a few of-1,4 and -1,6 residues in the chains.NⅣis composed of Fuc,Rha,Ara,Gal,Glc,Xyl,and Man,in molar ratio of 1.00:0.59:1.11:2.80:2.25:1.25:0.62.TheMw of NⅣis estimated to be 522981Da.The main chain of sample NⅣis made up ofα-D-1,3-Glc linkedα-D-1,3-Gal,and the side chains are formed withα-D-1,4-Ala residues.Inaddition,there are some -1,6 residues in the chains.
The new ideas and innovative points of this paper lie in the following:
(1)It's the first time to study the extraction,isolation and purification of myceliapolysaccharide from fermentable Hypsizigus marmoreus.More valuably,two polysaccharides,TⅢ-2 and NⅣ,with Mw for 19136Da and 522981Da,are obtained for the first time.Thestructures of TⅢ-2 and NⅣare analyzed,too.The structures of these two polysaccharidesare different from the polyscaaharides of Hypsizigus marmoreus fruit body.It is proved thatthey are not reported in former studies.
(2)Moreover,in previous studies,just taking mycelia biomass;as a uniqe measure indexand shake flasks as the fermentation carrier,only more than 10 nutritional factors were investigated.But in this paper,taking the production of extra-cell polysaccharide andinter-cell ploysaccharide as major measure indexes,and using fermentor as the fermentationcarrier,34 nutritional factors were investigated.
引文
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