深层发酵双孢菇胞外多糖的研究
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摘要
双孢菇主要以野外采集和人工栽培为主，生产周期长，占地面积大，劳动强度高，同时受季节等因素的控制，影响和制约了双孢菇的生产和充分应用。双孢菇作为近年来开发的功能成分卓越的药食两用菌之一，日益受到人们的广泛重视。但是，目前对双孢菇胞外多糖的研究较少。
本研究以双孢菇为研究对象，采用深层发酵技术，研究不同发酵条件对双孢菇胞外多糖产量的影响，并对发酵工艺进行了优化。研究并优化了胞外多糖的提取工艺；同时对双孢菇胞外多糖的分离纯化、结构以及降血糖作用进行深入研究。主要研究结果如下：
1）采用Box-Behnken响应曲面实验优化双孢菇胞外多糖发酵工艺，确定了获得最大胞外多糖（1.87g/L）时最优培养基：葡萄糖35.7g/L，KH_2PO_42.1g/L，蛋白胨3.1g/L，MgSO_4·7H_2O1g/L和玉米浆15g/L。通过摇瓶试验，确定了双孢菇菌液态深层发酵温度25oC，初始pH为7.0；通过批次发酵试验确定5L发酵罐pH控制策略：分段控制pH，用5mol/L NaOH或HCl溶液调节pH，菌体生长前期（0-40h）控制pH为7.0，40-62h控制pH6.5，62h后至发酵结束控制pH为6.0。溶氧控制策略为：搅拌转速160r/min，通风量0.9vvm。优化后的双孢菇生物量最高达到23.64g/L，胞外多糖最高达到3.71g/L，较优化前生物量15.80g/L和双孢菇胞外多糖2.78g/L分别提高了49.62%和33.45%。
2）在单因素实验的基础上，通过Box-Behnken实验设计、模型建立和响应面分析，获得了最佳提取工艺：乙醇浓度85%，pH8.0，沉淀时间22h，在此条件下双孢菇胞外多糖的提取率达到最大值2.69g/L，与预测值（2.71g/L）相一致。
3）本文选择了截留分子量为8000-14000的透析袋，通过紫外光谱分析证明，截留分子量为8000-14000的透析袋的透析效果良好。采用DEAE-纤维素层析柱和Sepharose CL-6B凝胶色谱分离纯化得到一种双孢菇胞外多糖组分ABPS1。HPGPC法测得其呈单一峰，重均分子量为5.7×10~5Da。通过单糖组成分析、红外、核磁共振等手段分析ABPS1的糖链结构，结果表明ABPS1为杂多糖，其单糖组成为葡萄糖、甘露糖和木糖，摩尔比率为6.2:1:2.5；双孢菇胞外多糖含有和β两种糖苷构型，单糖残基的构型为吡喃型，以1→3糖苷键为主链，存在1→6糖苷键的支链。
4）双孢菇胞外多糖能够清除羟基自由基、超氧阴离子和DPPH自由基，具有显著的体外抗氧化作用。同时，体外实验还表明，双孢菇胞外多糖能够抑制-葡萄糖苷酶的活性，改善血糖水平。另外，通过对胰岛β细胞的生存能力和胰岛素蛋白的分泌功能研究，表明双孢菇胞外多糖能够提高胰岛β细胞的生存能力，增加了胰岛素蛋白的分泌。
5）双孢菇胞外多糖能够改善糖尿病小鼠消瘦和毛色黯淡等症状。并且能够降低糖尿病小鼠血糖浓度，促进葡萄糖转化合成肝糖原。同时能够提高机体SOD等酶类的抗氧化酶活性，提高肝脏抗氧化能力。
Agaricus bisporus is produced mainly for wild and artificial cultivation. However, thesemethods of production have disadvantages of long production cycle, covering an area of largeand high labor intensity, affecting the production and application of Agaricus bisporus. As oneof excellent edible fungus developed in recent years, Agaricus bisporus has caused growingattention. However, there is little research on extracellular polysaccharide of Agaricusbisporus at present.
1）In this study, Agaricus bisporus was used as the research object, using submergedfermentation technology, and different fermentation conditions of extracellularpolysaccharide from Agaricus bisporus were studied and optimized. Also, the extractionprocess of extracellular polysaccharides was studied and optimized. Purification, structuralfeature and hypoglycemic effect of extracellular polysaccharide from Agaricus bisporus wasalso investigated. The main research results were as follows: A Box-Behnken factorial designwas employed to optimize the medium composition for the production of extracellularpolysaccharides from Agaricus bisporus. The optimized media was as follows: glucose35.7g/L, KH_2PO_42.1g/L, peptone3.1g/L, KH_2PO_42.1g/L, corn steep liquor15g/L. Under theseoptimum parameters, the maximum yield of extracellular polysaccharide from Agaricusbisporus was1.87g/L. After shaking flask test, fermentation temperature was set at25°C andthe initial pH was6.0. The strategy of controlled pH and dissolved oxygen were as follows:pH7.0was controlled on0-40h, pH6.5was controlled on40-62h, pH6.0was controlled on62-120h, agitation rate160r/min, aeration rate0.9vvm. Under the optimum condition, themycelial biomass reached a maximum of23.64g/L and exopolysaccharides reachedmaximum of3.71g/L respectively, which were increased by49.62%and33.45%than that ofuncontrolled fermentation.
2）Based on the single factor experiment, a Box-Behnken central composite design wasapplied to optimize the extraction process of extracellular polysaccharides from Agaricusbisporus. After response surface analysis, the optimized extraction parameters were as follows:ethanol concentration85%(V/V), pH8, sedimentation time22h. And the maximumconcentration of extracellular polysaccharides was2.69g/L under this optimized extractionparameters, which was consistent with the predicted value (2.71g/L).
3）Dialysis bag, the molecular weight cut-off of which was8000-14000, was used in thisstudy. And the excellent effect of dialysis was proved with ultraviolet spectrophotometry. Anovel Agaricus bisporus extracellular polysaccharides ABPS1purified by Sepharose CL-6B gel chromatography after DEAE-Sephadex A25chromatography. ABPS1was showed as asingle peak on high-performance gel-permeation chromatography (HPGPC) and the averagemolecular weight was5.7×10~5Da. By means of gas chromatography, infrared and NMR, thestructure of ABPS1was investigated. The analysis results showed that ABPS1was aheteroglycan which was composed of glucose, mannose and xylose with a molar ratio of6.2:1:2.5. There were two glycosidic configurations (and β) in structure of Agaricusbisporus extracellular polysaccharides. It was also revealed that the structure of ABPS1contained pyranride, with with1→3glycosidic bonds as the main chain and1→6glycosidicbonds as branched chain.
4） The extracellular polysaccharides from Agaricus bisporus have strong scavengingeffect of hydroxyl radicals, superoxide anion and DPPH radicals, and therefore it hasoutstanding performance in antioxidation. Also, in vitro model was employed to investigatethe effect of extracellular polysaccharides on alpha-glucosidase activity. It was found thatextracellular polysaccharides from Agaricus bisporus could inhibit the activity ofalpha-glucosidase. In addition, it was found that extracellular polysaccharides from Agaricusbisporus could enhance the islet β-cell viability and increase the secretion of insulin protein.
5）Agaricus bisporus extracellular polysaccharides can play an active role in reversingsymptoms of diabetes (weight loss, darker hair) in mice. And it can reduce blood glucoseconcentration in diabetic mice, promote the conversion of glucose synthesis of hepaticglycogen. At the same time, ABPS1was effective in improving the activities of SOD andother antioxidant enzyme, scavenging free radical.
本研究以双孢菇为研究对象，采用深层发酵技术，研究不同发酵条件对双孢菇胞外多糖产量的影响，并对发酵工艺进行了优化。研究并优化了胞外多糖的提取工艺；同时对双孢菇胞外多糖的分离纯化、结构以及降血糖作用进行深入研究。主要研究结果如下：
1）采用Box-Behnken响应曲面实验优化双孢菇胞外多糖发酵工艺，确定了获得最大胞外多糖（1.87g/L）时最优培养基：葡萄糖35.7g/L，KH_2PO_42.1g/L，蛋白胨3.1g/L，MgSO_4·7H_2O1g/L和玉米浆15g/L。通过摇瓶试验，确定了双孢菇菌液态深层发酵温度25oC，初始pH为7.0；通过批次发酵试验确定5L发酵罐pH控制策略：分段控制pH，用5mol/L NaOH或HCl溶液调节pH，菌体生长前期（0-40h）控制pH为7.0，40-62h控制pH6.5，62h后至发酵结束控制pH为6.0。溶氧控制策略为：搅拌转速160r/min，通风量0.9vvm。优化后的双孢菇生物量最高达到23.64g/L，胞外多糖最高达到3.71g/L，较优化前生物量15.80g/L和双孢菇胞外多糖2.78g/L分别提高了49.62%和33.45%。
2）在单因素实验的基础上，通过Box-Behnken实验设计、模型建立和响应面分析，获得了最佳提取工艺：乙醇浓度85%，pH8.0，沉淀时间22h，在此条件下双孢菇胞外多糖的提取率达到最大值2.69g/L，与预测值（2.71g/L）相一致。
3）本文选择了截留分子量为8000-14000的透析袋，通过紫外光谱分析证明，截留分子量为8000-14000的透析袋的透析效果良好。采用DEAE-纤维素层析柱和Sepharose CL-6B凝胶色谱分离纯化得到一种双孢菇胞外多糖组分ABPS1。HPGPC法测得其呈单一峰，重均分子量为5.7×10~5Da。通过单糖组成分析、红外、核磁共振等手段分析ABPS1的糖链结构，结果表明ABPS1为杂多糖，其单糖组成为葡萄糖、甘露糖和木糖，摩尔比率为6.2:1:2.5；双孢菇胞外多糖含有和β两种糖苷构型，单糖残基的构型为吡喃型，以1→3糖苷键为主链，存在1→6糖苷键的支链。
4）双孢菇胞外多糖能够清除羟基自由基、超氧阴离子和DPPH自由基，具有显著的体外抗氧化作用。同时，体外实验还表明，双孢菇胞外多糖能够抑制-葡萄糖苷酶的活性，改善血糖水平。另外，通过对胰岛β细胞的生存能力和胰岛素蛋白的分泌功能研究，表明双孢菇胞外多糖能够提高胰岛β细胞的生存能力，增加了胰岛素蛋白的分泌。
5）双孢菇胞外多糖能够改善糖尿病小鼠消瘦和毛色黯淡等症状。并且能够降低糖尿病小鼠血糖浓度，促进葡萄糖转化合成肝糖原。同时能够提高机体SOD等酶类的抗氧化酶活性，提高肝脏抗氧化能力。
Agaricus bisporus is produced mainly for wild and artificial cultivation. However, thesemethods of production have disadvantages of long production cycle, covering an area of largeand high labor intensity, affecting the production and application of Agaricus bisporus. As oneof excellent edible fungus developed in recent years, Agaricus bisporus has caused growingattention. However, there is little research on extracellular polysaccharide of Agaricusbisporus at present.
1）In this study, Agaricus bisporus was used as the research object, using submergedfermentation technology, and different fermentation conditions of extracellularpolysaccharide from Agaricus bisporus were studied and optimized. Also, the extractionprocess of extracellular polysaccharides was studied and optimized. Purification, structuralfeature and hypoglycemic effect of extracellular polysaccharide from Agaricus bisporus wasalso investigated. The main research results were as follows: A Box-Behnken factorial designwas employed to optimize the medium composition for the production of extracellularpolysaccharides from Agaricus bisporus. The optimized media was as follows: glucose35.7g/L, KH_2PO_42.1g/L, peptone3.1g/L, KH_2PO_42.1g/L, corn steep liquor15g/L. Under theseoptimum parameters, the maximum yield of extracellular polysaccharide from Agaricusbisporus was1.87g/L. After shaking flask test, fermentation temperature was set at25°C andthe initial pH was6.0. The strategy of controlled pH and dissolved oxygen were as follows:pH7.0was controlled on0-40h, pH6.5was controlled on40-62h, pH6.0was controlled on62-120h, agitation rate160r/min, aeration rate0.9vvm. Under the optimum condition, themycelial biomass reached a maximum of23.64g/L and exopolysaccharides reachedmaximum of3.71g/L respectively, which were increased by49.62%and33.45%than that ofuncontrolled fermentation.
2）Based on the single factor experiment, a Box-Behnken central composite design wasapplied to optimize the extraction process of extracellular polysaccharides from Agaricusbisporus. After response surface analysis, the optimized extraction parameters were as follows:ethanol concentration85%(V/V), pH8, sedimentation time22h. And the maximumconcentration of extracellular polysaccharides was2.69g/L under this optimized extractionparameters, which was consistent with the predicted value (2.71g/L).
3）Dialysis bag, the molecular weight cut-off of which was8000-14000, was used in thisstudy. And the excellent effect of dialysis was proved with ultraviolet spectrophotometry. Anovel Agaricus bisporus extracellular polysaccharides ABPS1purified by Sepharose CL-6B gel chromatography after DEAE-Sephadex A25chromatography. ABPS1was showed as asingle peak on high-performance gel-permeation chromatography (HPGPC) and the averagemolecular weight was5.7×10~5Da. By means of gas chromatography, infrared and NMR, thestructure of ABPS1was investigated. The analysis results showed that ABPS1was aheteroglycan which was composed of glucose, mannose and xylose with a molar ratio of6.2:1:2.5. There were two glycosidic configurations (and β) in structure of Agaricusbisporus extracellular polysaccharides. It was also revealed that the structure of ABPS1contained pyranride, with with1→3glycosidic bonds as the main chain and1→6glycosidicbonds as branched chain.
4） The extracellular polysaccharides from Agaricus bisporus have strong scavengingeffect of hydroxyl radicals, superoxide anion and DPPH radicals, and therefore it hasoutstanding performance in antioxidation. Also, in vitro model was employed to investigatethe effect of extracellular polysaccharides on alpha-glucosidase activity. It was found thatextracellular polysaccharides from Agaricus bisporus could inhibit the activity ofalpha-glucosidase. In addition, it was found that extracellular polysaccharides from Agaricusbisporus could enhance the islet β-cell viability and increase the secretion of insulin protein.
5）Agaricus bisporus extracellular polysaccharides can play an active role in reversingsymptoms of diabetes (weight loss, darker hair) in mice. And it can reduce blood glucoseconcentration in diabetic mice, promote the conversion of glucose synthesis of hepaticglycogen. At the same time, ABPS1was effective in improving the activities of SOD andother antioxidant enzyme, scavenging free radical.
引文
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