【作者】 魏明；

【导师】 姜绍通；

【作者基本信息】 合肥工业大学 ， 农产品加工及贮藏工程， 2007， 博士

【摘要】 霍山石斛是我国珍贵的药用植物，产于安徽霍山及其邻近地区，石斛多糖具有抗肿瘤、增强人体免疫等功效。由于霍山石斛在自然环境下生长缓慢，加上人工过度采集，其自然资源已濒临灭绝。目前对霍山石斛的研究主要集中在组织培养方面，但试管苗的脱瓶移栽问题尚未解决。霍山石斛类原球茎是霍山石斛的体细胞胚胎，具有和植株同样的物质代谢和发育潜能。利用霍山石斛类原球茎悬浮培养生产活性多糖是解决霍山石斛资源短缺问题的有效途径之一。本文对霍山石斛类原球茎悬浮培养过程进行了系统的动力学分析，并构建了动力学模型；考察了植酸和多胺对霍山石斛类原球茎细胞生长和多糖合成的影响；采用二步培养法对霍山石斛类原球茎合成多糖进行了研究；在10 L气升式反应器中进行了初步扩大培养。不同培养条件下，霍山石斛类原球茎悬浮培养细胞生长曲线呈现较为典型的S型，低浓度时，蔗糖是细胞生长的限制性因素，提高蔗糖浓度可以明显促进细胞生长，蔗糖浓度在30g／L时，类原球茎生物量达到最大；但蔗糖浓度超过30 g／L后对细胞生长具有抑制作用。硝酸盐浓度为30 mmol／L时，有利于细胞生长，高浓度时对细胞生长有抑制作用。磷酸盐是细胞生长的限制性因素，提高磷酸盐浓度可以促进细胞生长，缩短培养周期，当磷酸盐浓度为2.5mmol／L时，生物量达最大，为28.7g DW／L。不同培养过程中多糖总产量变化规律相似。在一定范围内，增加起始蔗糖浓度有利于多糖积累，但在过高蔗糖浓度下，多糖产量反而下降。硝酸盐浓度为30mmol／L时，有利于多糖积累。初始磷酸盐浓度对多糖的积累影响显著，当磷酸盐浓度为0.312 mmol／L时，多糖积累量最大为2.46g／L。多糖合成不仅与类原球茎细胞的生长密切相关，而且与细胞内还原糖浓度有关。霍山石斛类原球茎对培养基中蔗糖的利用采用先水解后吸收的方式，可以用带底物抑制的酶反应动力学方程来描述该过程。类原球茎对葡萄糖和果糖的吸收没有明显差别，细胞内还原糖的积累与培养基的组成有密切关系，各种培养条件下类原球茎对蔗糖的得率系数也因此不同。类原球茎对磷酸盐的吸收比较快，细胞内磷酸盐的积累水平与培养基初始磷酸盐浓度有关，细胞内磷酸盐的积累水平对类原球茎细胞的生长和多糖积累有重要影响。构建了霍山石斛类原球茎悬浮培养过程的桔构化动力学模型。模型中生物相被分为四个部分：细胞内还原糖、中间代谢产物，代谢产物多糖、细胞的呼吸损失。非生物相指细胞外部培养基，包括蔗糖和还原糖两部分。模型的模拟结果与实验测定值基本相符，能用于霍山石斛类原球茎的悬浮培养过程预测。在培养基中添加2.5g／L的植酸可以抑制过氧化物酶和多酚氧化酶的活性，提高细胞活力，促进细胞生长和多糖的合成，最终生物量为29.4g DW／L，多糖产量为2.06g／L。添加0.6mmol／L的腐胺和精氨能提高内源多胺的含量，促进霍山石斛类原球茎细胞的生长和多糖的合成，最终生物量为32.6g DW／L，多糖产量为2.20g／L。磷是霍山石斛类原球茎细胞生长和多糖积累的有效调控因素。可以在适合类原球茎增殖的蔗糖浓度下，调节磷酸盐浓度，使碳源流向多糖合成。利用二步法培养，第一步培养磷酸盐浓度为2.5 mmol／L，第二步培养磷酸盐浓度为0.312 mmol／L，结果多糖产量和多糖含量分别为5.22g／L和11.9％，明显高出其它方法。建立的二步法培养模型基本反映了在二步培养中霍山石斛类原球茎增殖和多糖积累的变化规律。进行了霍山石斛类原球茎悬浮培养的初步放大试验，通气量在0.5 L／min较适宜。发酵罐中获得的最大生物量是摇瓶的86.5％，多糖产量是摇瓶的145％。在反应器中，类原球茎细胞生长和多糖积累的动力学特性和摇瓶中相似，但发酵罐培养中各种基质消耗同摇瓶相比出现滞后现象，且利用率偏低。通过流加补料培养，生物量提高到44.7g DW／L，多糖产量提高到8.15g／L。更多还原

【Abstract】 Dendrobium huoshanense, a precious wild medicinal plant in China, distributes in Huoshan County, Anhui Province. Polysaccharides isolated from Dendrobium can inhibit the growth of tumor cells and possess immunological activity. Because of a deficiency in the wild source of Dendrobium huoshanense and excessive collection, it has been on the verge of extinction. Tissue culture techniques have been used for the in vitro mass multiplication of Dendrobium huoshanense, but the culture system has not been shown to be more efficient. Protocorm-like bodies (PLBs)are actually somatic embryos that can be induced from the explants of Dendrobium and produce some active substances. A new approach has been considered as a feasible alternative for the production of active polysaccharides using protocorm-like body cultures of Dendrobium huoshanense. A structural kinetic model was developed to describe the cultivation process. Furthermore, the impact of phytic acid and polyamines on cell growth and polysaccharide synthesis, the regulation of cell growth and polysaccharide synthesis by two-stage cultivation and primary scale-up of PLB suspension culture in 10 L air-lift bioreactor were investigated.The kinetics of cell growth, polysaccharide synthesis and consumption of nutrients were analyzed in suspension cultures of PLBs. The suspension culture process of PLBs could be divided into three phase: lag phase, rapidly growth phase, and stationary phase under different conditions. Sucrose was a growth limiting nutrient on low concentration condition. Increase in sucrose concentration stimulated cell growth and increased final PLB harvest. Sucrose at 30 g/L gave the highest biomass of PLBs. Substrate inhibition was observed when the concentration was above 30 g/L. Nitrate at 30 mmol/L was beneficial for cell growth. Phosphate was a limiting factor for cell growth. Increase in phosphate concentration increased cell growth rate and shortened the culture period. A total of 2.5 mmol/L medium phosphate gave the highest biomass(28.7g DW/L).The trends of polysaccharide production variation were the same under different conditions. Increase in sucrose concentration could improve polysaccharide production to some extent, while higher sucrose concentration inhibited polysaccharide production. Nitrate at 30 mmol/L was beneficial to accumulation of polysaccharide. Initial phosphate concentration was the most effective on polysaccharide accumulation. The maximum polysaccharide production(2.46g/L) could be obtained at 0.312 mmol/L phosphate concentration. The accumulation of polysaccharides not only correlates with cell growth but also with intracellular reducing sugar level.PLBs rapidly hydrolyzed extracellular sucrose before absorbing it. The hydrolyzing process could be described using substrate saturation kinetic equation. Glucose and fructose were absorbed at the same time. Intracellular accumulation of reducing sugar was closely related to medium composition. Biomass yield coefficients of sucrose under different culture conditions were different. Intracellular phosphate accumulation level related to the initial phosphate concentration in the medium. Intracellular phosphate level affected the cell growth and accumulation of polysaccharides.A structural model was developed. The whole culture system was separated into biotic phase and abiotic phase. The former was divided into 4 compartments: reducing sugar, middle metabolites, product, and respiration loss. The later included sucrose and reducing sugar. Calculated result coincided well with experimental data. The model could be used for description of cultivation process.The medium supplemented with phytic acid 2.5g/L was beneficial for cell growth and polysaccharide synthesis. The cell dry weight and production of polysaccharides were 29.4g DW/L and 2.06g/L, respectively. The phytic acid could inhibit the activities of peroxidase and polyphenol. Putrescine and spermine at 0.6mmol/L improved intracellular polyamine contents and stimulated cell growth and polysaccharide production. The production of biomass and polysaccharides was 32.6g DW/L and 2.20g/L, respectively.Phosphate is an effective factor in the regulation of cell growth and the accumulation of polysaccharides. Two-stage cultivation can be used to produce polysaccharides. In the first step, the PLBs were grown in a medium with 2.5 mmol/l phosphate and in the second step, they were grown in a medium with 0.312 mmol/1 phosphate. By two-stage cultivation, polysaccharide production and content reached 5.22 g/l and 11.9 %, respectively, which were higher than that of other methods. The model could describe the two-stage cultivation process.Primary scale-up of PLB suspension cultures in 10 L air-lift bioreactor was investigated. Aeration rate at 0.5 L/min was beneficial for cell growth and polysaccharide synthesis. The maximum biomass and total polysaccharide production in bioreactor were 86.5 % and 145 %, respectively, compared to that in flask culture. The kinetics of cell growth and accumulation of polysaccharides in the bioreactor were the same as in flask culture. Consumption of nutrients was much more slowly and had lower utilization efficiency than that in flask culture. The biomass (44.7 g DW/L) and polysaccharide production (8.15 g/L) were obtained by feeding culture.更多还原