【作者】 李卫旗；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2004， 博士

【摘要】 热凝胶多糖（Curdlan，又名可德兰）是由细菌发酵生产的胞外直链无分枝β-（1，3）-D-葡聚糖，因其特定的热凝胶性和独特的分子结构而在食品、轻工、医药、保健等领域有广泛的应用开发价值。本文从诱变筛选高性能生产菌株着手，研究了Curdlan的发酵特性，优化了发酵培养基的组成和发酵培养条件，分析了Curdlan间歇发酵过程中发酵液的流变学特性及气液传质状态，并提出了Curdlan的分批发酵动力学模型，探讨了Curdlan的提取、分离和纯化工艺，对Curdlan样品的分子结构及其改性进行了探索研究。主要的研究结果如下： 1．利用⁶⁰Co γ射线辐照对出发菌——粪产碱杆菌（Alcaligenes faecalis）变种GM-24的菌体细胞和原生质体进行同步处理诱变处理。结果表明，适当剂量的⁶⁰Co γ辐射对产Curdlan菌株GM-24的菌体细胞和原生质体均有较好的正突变诱变作用，而对原生质体的诱变效果尤为明显，原生质体经⁶⁰Co γ辐射后，其正突变率、最优突变株的多糖产量提高幅度、突变株的遗传稳定性都高于菌体细胞，尚未发现国内外有相关的文献报导。 与出发菌株GM-24相比，最终选得的原生质体突变株A38的生产性能有了显著改善，其糖转化率从38.7％提高至58.2％，Curdlan产量从15.35 g．L^-1上升到22.3 g．L^-1，提高了50.4％，发酵周期时间缩短了18％。A38的遗传性能稳定，连续翻接5代后发酵性能维持良好，。 2．对菌株A38的底物组分单因素实验表明，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，酵母粉为最佳氮源，适量的碳酸钙不仅可以促进Curdlan的发酵生产，而且能够维持发酵液的pH的稳定。 正交实验表明氮源对Curdlan产物得率的影响最显著，碳源影响显著，MgSO₄．7H₂O有较显著影响，KH₂PO₄的影响较不显著。作为辅助因素，尿嘧啶对Curdlan发酵的影响比柠檬酸钾明显。 利用响应面分析法（RSM）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，KH₂PO₄0.22，MgSO₄．7H₂O0.31，CaCO₃ 2.0，柠檬酸钾0.2。菌株A38在优化的培养基中发酵培养72h，所得Curdlan最高产量为26.32g．L^-1，比培养基优化前提高了18％。 发酵结果表明菌株A38菌体生长与产Curdlan为部分偶联型，在菌体对数生长阶段没有Curdlan产出，Curdlan在氮源基本消耗完后菌体进入平衡期后开始生产。 3．发酵条件的单因素实验表明，发酵种子液的适宜种龄为10h接种量为10％（V／V），摇瓶装液量为500ml瓶装100～125ml，摇床转速200rpm，助溶氧剂PPE和一!菜油有助于改善发酵液的溶氧水平，适宜的发酵温度为30一32℃，培养基初始pH为7.0，产胶阶段pH为5.2一6。，发酵周期为72h左右。 应用响应面设计（RSM）优化的C盯dlan发酵的培养条件为:种龄9.9h，接种量9.8%，摇床转速208rpm，发酵培养基初始pH7.0，产胶阶段pH为5.2，发酵温度为30℃，发酵周期70.911，在优化的培养条件下生产Curdlan，得到的发酵多糖产量为29.88g.L’‘，比培养条件优化之前提高了13.6%。 3.7L自动发酵罐实验表明以优化的培养基和培养条件进行分批发酵，考察罐发酵过程中Curdlan浓度与发酵液粘度的变化趋势，首次得到用发酵液粘度预测C盯dlan产量浓度的模型方程。 4.通过对C盯dlan发酵液在不同阶段流变特性的分析，发现C盯dlan发酵液在平衡期阶段由牛顿型流体转为假塑性流体，流体流变特性趋于复杂，发酵过程的溶氧传质机制好坏，主要取决于反应器搅拌转速的影响。具体反映在: （1）搅拌转速在600r/min以内时，搅拌转速越高，菌体的生长速率越快，最终细胞千重越大，细胞产率也较高，但发酵周期却基本相同。 （2）当转速从35Or/min提高到60Or/min时，多糖的终产量由13.459/L上升到29.49/L。但继续提高转速达75Or/min后，多糖产量迅速下降，多糖的得率系数变化也呈相同的变化趋势。 （3）Curdtan的最终分子量随着搅拌转速的提高不断下降，在35Or/min时是63500 Da，到900r/min时则只有2650ODa。 （4）适当的高搅拌转速（6O0r/min）下，发酵体系处于良好的搅拌状态，前期有效体积为100%，到产糖最高时（6Oh）也有86%。而低转速搅拌时，有效体积随着发酵时间的推进不断下降，Curdlan合成的产量也明显降低。 Curdlan传氧系数kLa是生物反应系统中的一个限制性因素，搅拌转速和kLa之间几乎成正比关系，C盯dlan浓度的增高也明显降低了气体在溶液中的k，a值。 5.用Logistie方程和Luedeking一piret方程分别描述A3s菌体生长、产物合成动力学规律，并据发酵过程中的底物消耗主要用于菌体生长与产物合成及相关的代谢规律，首次对分批发酵动力学进行了初步探索，得到其发酵动力学方程为: （1）菌体生长动力学方程0 .2e0·155‘0 .9723+0.0277eol55‘（2）月包外多糖生成动力学方程S=50.040 .04e0155‘0 .9723+o.0277eo4155‘+8 .O183ln（0.9723+O.O277e。”，‘）（3）底物消耗动力学方程P=10.96161n(0.9723+0.0277eo·”，‘0 .52更多还原

【Abstract】 Curdlan is an exopolysaccharide composed entirely of β- (1,3)- D-glucosidic linkages which is produced by bacteria. The unique features of Curdlan, thermic gels and its special molecular structure has attracted the extensive attention of the food industry, light industry, medicine and healthcare. In this study, the new mutant strain for Curdlan was induced and its characters for Curdlan fermentation were studied, the ideal culture medium and fermentation conditions were investigated. The rheological property and oxygen mass transfer were both studied in Curdlan broth, and the batch process and dynamics model were determined , the progresses of isolation, purification and separation of Curdlan from fermentation broth were studied. The structure of the purified Curdlan which was separated and purified from fermentation broth was analyzed, and the process for its structure modification was also studied. The main results were as following:1. The cells and protoplasts of initial strain, whose name was Alcaligenes Species var. GM-24 , were treated by 60Co γ radiation respectively for the first time, while both of the mutants from protoplasts and cells had improved their productivity of Curdlan , the productivity and genetic stability of former were much greater than those of the latter.In contrast with the strain GM-24, the final mutant A38, which was induced by 60Co γ radiation from protoplast of the GM-24, had greatly improved its productivity properties. Its rate of sugar transmission was increased to 58.2% from 38.7% , the productivity of Curdlan was increased to 15.35 g.L-1 from 22.3 g.L-1, meanwhile , the fermentation term was reduced 18%.The genetic properties of strain A38 were very stable, so it was choosed as fermentation strain in follow-up study.2. The single factor experiments for the composition in culture medium indicated that glucose was the optimal carbon resource, while sucrose, molasses and solubility starch could be also used to produce Curdlan , and that yeast extract was the optimal nitrogen resource . Furthermore, reasonable calcium carbonate could not only promote the Curdlan yield but maintain the pH of fermentation broth.The result of orthogonal test indicated that the effect of four principal factors on Curdlan fermentation was: glucose > yeast extract > MgSO4 7H2O > KH2PO4 . As an assistant factor in culture medium, uracil had greaterinfluence on Curdlan production than citrate kalium.The composition of fermentation culture medium optimized with respond surface methodology(RSM )was (g.L-1): glucose 49.5, yeast extract 7.64, KH2PO4 0.22, MgSO4 7H2O 0.31, uracil 0.32, citrate kalium 0.2, CaCO3 2.0. The Curdlan yield was 26.32gL-1 when the strain A38 was cultured in optimized medium, it was 18% higher than that in culture medium before being optimized .The result of fermentation indicated that Curdlan produced by strain A38 was associated partially with cell growth , The Curdlan had not to be yielded from the cells until the nitrogen resource was almost consumed up and the cells entered stationary phase.3.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 , 30-32 shaker rotation speed, 200 rpm; initial pH, 7.0 ;and pH for Curdlna, yielding 5.2-6.0; fermentation time was around 72 hours . PPE and rap oil could help oxygen to dissolve in the fermentation broth and promote Curdlan production.The optimal conditions for Curdlan fermentation with RSM was: inoculum age , 9.9h; inoculum size , 9.8%(v/v); shaker rotation speed , 208rpm; initial pH 7.0; pH for Curdlna yielding , 5.2; fermentation temperature, 30 fermentation term,70.9 hours. Curdlan production reached the maximal point 29.88g.L-1 under the optimal culture conditions, this was 13.6% higher than that before the culture conditions were optimized .The op更多还原