【作者】 李志昂；

【导师】 李卫旗；

【作者基本信息】 浙江大学 ， 遗传学， 2003， 硕士

【摘要】 可得兰多糖（Curdlan）是微生物发酵生产的直链无分枝的β—（1-3）—葡聚糖，具有加热形成凝胶的特殊性质，在食品、轻工、医药、保健等领域有广泛的应用开发前景。本论文以诱变、筛选得到的Alcaligenes faedelis Var. C125菌株为生产菌株，研究C125菌株产Curdlan的发酵特性，应用响应面法优化了发酵培养基的组成和发酵培养条件；研究发酵后Curdlan的提取、纯化和分离工艺；分析分离纯化得到的Curdlan CD-3样品的分子结构并对其进行初步的分子结构改造研究。主要的研究内容如下： 1) 对菌株C125培养的单因素实验研究表明葡萄糖、蔗糖、糖蜜和可溶性淀粉作为发酵的碳源都可以生产Curdlan，但以葡萄糖为最佳碳源，适宜的加入量为45g.L（^-1）；酵母粉为菌株C125的最佳氮源，适宜的添加量为8.5g.L^-1；加入适量的碳酸钙(2.5g.L^-1)不仅可以促进Curdlan的发酵生产，而且能够维持发酵液的pH的稳定。 正交实验表明碳源和氮源对Curdlan发酵生产的影响显著，混合无机盐（K₂HPO₄+MgSO₄）有较显著影响，柠檬酸钾无显著影响；正交实验优化的发酵培养基组合为(g.L^-1)：葡萄糖40，酵母粉8.5，混合无机盐2.0+0.2，柠檬酸钾0.2，其Curdlan产量为28.22g．L^-1。尿嘧啶和KH₂PO₄对菌株C125发酵生产Curdlan有较大的影响，KH₂PO₄添加浓度为2g.L^-1，尿嘧啶的添加量不能低于0.3g.L^-1。 利用响应面法获得的最优培养基组合为(g.L^-1)：葡萄糖，49.5、酵母粉，7.64、尿嘧啶，0.32、KH₂PO₄，2.72和（NH₄）₂HPO₄，1.16。以优化的培养基组合发酵95小时，所得的Curdlan产量为25.03g.L^-1。菌株C125菌体生长与产Curdlan为部分偶联，发酵前期是菌体对数生长阶段，没有Curdlan产出，在氮源基本消耗完后菌体才开始产Curdlan。 2) 单因素实验表明Curdlan发酵种子液适宜的种龄和接种量分别为10h和10％；摇瓶装液量和摇床转速分别为500ml摇瓶装100～125ml培养基和200rpm；助溶氧剂PPE和菜油可以改善发酵液的溶氧水平，有助于提高Curdlan的发酵产量，而用H₂O₂作助溶氧剂则不可取；合适的发酵温度在30～32℃；发酵培养基合适的初始pH为6.5，发酵周期为95h。 应用响应面设计优化的Curdlan发酵的培养操作条件为：种龄9.9h，接种量9.8％，摇床转速208rpm，发酵培养基初始pU 6.1，发酵时间93.9h，在优化的培养条件下，发酵生产Curdlan，得到的发酵多糖产量为24.69g.L^-1。 3.7L自动发酵罐实验表明优化的培养基组成和培养条件适宜进行罐发酵，可以此为基点继续优化；考察罐发酵过程中Curdlan浓度与发酵液粘度的变化趋势和两者的关系，得到用发酵液粘度预测Curdlan产量浓度的模型方程，简化了发酵过程Curdlan浓度的检测手段及对发酵条件的控制。 3)用不同方法研究C盯dlan的提取工艺，结果表明醇析法和酸碱法是CUr以an粗多糖较合适的提取工艺。经优化的提取工艺为:醇析法添加乙醇3一5倍（v/v）、醇析次数3次;酸碱法加等体积IM的氢氧化钠溶液，使碱的终浓度在0.5M以下，ZMHCI调节PH至中性使C班dian沉淀，然后离心和醇洗。 C切dlan粗多糖用女v越笋法脱蛋白5一7次，流水逆向透析得Cur山阻半纯品CL卜2样品，cD一样品用s喇血睐G-100，S喇血 lex G.200分离和纯化得到cur山劝CD一3样品;Sepha印sex一oo和紫外光谱检测表明CD一3样品为高纯度的CUr山an;CD.3样品为白色粉末，理化实验表明具备Ourdian的特性。 4)纸层析和高碘酸钠氧化实验确定CD.3是一种以1一3葡萄糖昔键连结的单糖组份单一的多聚葡萄糖;S叩枷叨义x，200柱层析求得CD³的分子量为457以犯al;刚果红实验表明CD一3在稀碱溶液中（浓度<0 .4mo切卜1）存在三股螺旋构象。红外光谱和核磁共振图谱分析证明，CD.3具备标准Curdlan的分子结构。 采用低取代度法和SPC法制备得到C班dian硫酸化衍生物（CDRS），有效改善了C班.dian的水溶性，红外光谱显示cDRs有硫基（一0503）的吸收峰和p一D一(l一3升葡聚糖的特征吸收峰。 制备得到Ourdian梭甲基化衍生物（CM.CD）样品，能够溶解于水且保持热凝胶性能，取代度为0.5。红外图谱显示梭甲基的吸收峰和p一D一（l一3）葡聚糖的特征吸收峰;核磁共振图谱显示在c班山an的q和q位置的轻基（.OH）被梭甲基取代（一oc玩cooH）。更多还原

【Abstract】 Curdlan is a bacterial polysaccharide composed entirely of (l,3)-p-D-glucosidic linkages produced by fermentation of microorganism. One of the unique features of Curdlan is that aqueous suspensions can be thermally induced to form high-set gels, and this has attracted the extensive attention of the food industry, light industry, medicine and health protection. In this study, the Curdlan producing strain - Alcaligenes faedelis Var. C125 strain was induced and screened, and its characters for Curdlan production was studied. Culture medium and conditions of fermentation was optimized by respond surface methodology(RSM). The progress of isolation, purification and separation of Curdlan polysaccharide from fermentation broth was studied. The structure of the purified Curdlan product (CD-3) which was separated and purified from fermentation broth was analysed and its structure modification was also studied. The main results were as following:1) Glucose, sucrose, molasses and solubility starch can all be used as carbon resource to produce curdlan by single fector experiments by Alcdigenes faedelis Var. C125 strain. However, glucose was the optimal carbon source and its fitting concentration was 45gL-1; yeast extract was the optimal nitrogen source and its fitting concentration was 8.5g.L-1; Reasonable calcium carbonate(2.5g.L-1) could not only promote the Curdlan yield but also maintain the pH of fermentation broth.Carbon source and nitrogen source influenced the Curdlan fermentation significantly by orthogonal test, and mixinorganic salt (K2HPO4+MgSO4 ) influenced Curdlan fermentation significantly less than the former, but kalium citrate hadn’t significant influence. Culture medium optimized with orthogonal wasfeL-1) glucose 40; yeast extract 8.5g.L-1; mixinorganic salt 2+0.2, kalium citrate 0.2.The Curdlan yield was 28.22g.L-1 in optimized medium. Uracil and KH2PO4 could also affect Curdlan production greatly. Additonal concentration of KH2PO4 was 2g.L-1 and uracil concentration was no less than 0.3g.L-1.The composition of fermentation culture medium optimized with RSM was (g.L-1): glucose 49.5, yeast extract 7.64, uracil 0.32, KH2PO42.72, (NH4)2HPO4, 1.16; The Curdlan yield was 25.03g.L-1 at 95h in optimized culture medium, similar to that predicted by model. The Curdlan fermented by strain C125 was associated partially with cell growth. The former phase of Curdlan fermentation was logarithm phase of cell growth and did not produce Curdlan polysaccharide. The cells did not start to produce Curdln after it entered stationary phase, until nitrogen source was almost exhausted out2) The optimal culture conditions for Curdlna production selected by single factors experiments was as following: the inoculum age and inoculum size were 10h and 10%(v/v) respectively; the optimum charge quantity was 100-125ml culture medium in 500ml flask, and the optimum temperature 30癈; and shaker rotation speed, 200 rpm; initial pH, 6.5; fermentation time was 95h. PPE and rap oil used as assisting dissolved oxygen reagent could modify dissolved oxygen in the fermentation broth and promote Curdlan production. But H2O2was unfitted to be used as assisting dissolved oxygen reagent. The optimal conditions for Curdlan production with RSM was: inoculum age 9.9h; inoculum size 9.8%(v/v); shaker rotation speed 208rpm; initial pH 6.1 and culture 30℃ for 93.9h.Curdlan production reached the maximal point 24.69g.L"1similar to the result predicted by model.The optimized composition of culture medium and the optimized condition were adopted to produce Curdlna in 3.7L automatism fermentor and could continue to optimize using the former result as starting point. The connection between Curdlan concentration and viscosity of fermentation broth were studied, and obtained the model equation that used viscosity of fermentation broth to forecast Curdlan production during fennentation process of Curdlan polysaccharide. So it was simple to inspect Curdlan production and control the culture conditions of Curdlan fermentation process.3) It w更多还原