肉苁蓉细胞和组织培养中的过程调控
文献类型：学位论文
| 作者 | 欧阳杰 |
| 学位类别 | 博士 |
| 答辩日期 | 2003 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 王玉春 |
| 关键词 | 肉从蓉 愈伤组织 细胞 苯乙醇糖贰 代谢调控 生物反应器 提取 |
| 其他题名 | Process Regulation in Cistanche deserticola Tissue and Cell Culture |
| 中文摘要 | 本文系统研究了肉从蓉愈伤组织的诱导和培养、细胞悬浮生长和次级代谢产物合成的调控、肉从蓉细胞的生物反应器培养以及有效成分提取的过程集成，为大规模培养肉从蓉细胞提供理论依据和工程基础。首先研究了从肉从蓉种子诱导愈伤组织的条件，其中将种子剥去种皮和进行热处理激活是愈伤组织形成的关键。并进行了愈伤组织在固体培养基上培养条件的优化研究，愈伤组织在添加了0.5mg 6-BA/L、10mg GA3／L、soomg cH／L和20g sucrose／L的BS培养基上，于25℃，光照强度24 p mol／mZs下培养生长最好。当接种量为1％时，在上述条件下培养30d后愈伤组织的生物量达到了111.3 g Fw/L和15．59Dw／L，苯乙醇糖贰（PeG）含量达到了10.7％，PeG产量为1.7g／L。其中愈伤组织生长的第O-12d为延滞期，第12-32d为对数生长期，第32-40d为平衡期和衰亡期。首次进行了肉从蓉细胞悬浮培养，建立了悬浮培养系。悬浮培养时大小介于0.5-1.Omm之间的细胞聚集体PeG含量最高，达11.3％，细胞种龄为25d和接种量为3-4％时细胞悬浮培养的结果最好，生物量和PeG含量均较高。对细胞悬浮培养过程中生长、PeG合成和底物消耗的动力学及其计量关系进行了研究。细胞悬浮培养的第O-8d为延滞期，第8-20d为对数生长期，第20-30d为平衡期和衰亡期。PeG的合成为生长偶联型。通过对细胞的基本元素分析，建立了细胞生长的基本计量方程式，为细胞培养的过程放大提供理论依据。通过分析PeG生物合成的代谢途径、合成中间体和关键酶，采用苯丙氨酸、酪氨酸、乙酸钠等前体以及Nd、La、Ce等稀土元素进行PeG合成的代谢调控。其中苯丙氨酸和混合稀土MR卫的作用最为显著。悬浮培养时，在培养第8d时加入O．Zmmol／L苯丙氨酸，到培养第20d时肉从蓉细胞的生物量可达5.9 g DW／L，PeG含量为18.6％，PeG产量为1.10g／L，分别是对照的95％、182％和175％。悬浮培养时，稀土元素的作用比在固体培养基里明显，当MRE在培养液中的初始浓度为0.02mmol／L时，培养20d后肉从蓉细胞的生物量、PeG含量和产量分别是对照组不添加稀土元素的126％、204％和267％。 生物反应器技术是肉从蓉组织和细胞培养工业化的关键之一。首先进行了鼓泡塔生物反应器培养肉从蓉细胞的研究，细胞在2L鼓泡式生物反应器中悬浮培养的较佳接种量为2.5％，通气量为0.010m3/h。培养20d后细胞生物量为4.6 g DW／L，增长2.4倍，PeG含量为9.0％，PeG的产量为0.42g／L。在筛网导流筒气升式生物反应器中进行了肉从蓉细胞的悬浮培养研究。该反应器可以有效降低气流给细胞带来的剪切力，并提供细胞生长可以附着的表面。肉从蓉细胞在2L筛网导流筒气升式生物反应器中悬浮培养结果明显好于鼓泡式生物反应器，较佳接种量约为5％，通气量为0．075 m3爪。此条件下，培养20d后细胞生物量为8.7 g DW／L，增长2.40倍；附网细胞PeG含量为16.3％，约为悬浮细胞的2倍（8.0％）；PeG的总产量为0.85g／L，比鼓泡式生物反应器中的PeG总产量高102％。首次研究了循环超声破碎细胞提取肉从蓉有效成分的工艺条件，以及细胞中PeG、肉从蓉多糖和甜菜碱提取的集成方法。将肉从蓉超声提取液先后过大孔吸附树脂AB-8柱、强酸型阳离子001*7交换树脂柱和Sephadex G-75凝胶柱层析，可以从1g肉从蓉中分离到52．3mg苯乙醇糖贰类化合物、125.6mg甜菜碱和24.5mg肉从蓉多糖，其回收率分别为64.4％、92.9％和53.5％。 |
| 英文摘要 | The callus induction and the cell culture of Cistanche deserticola, the metabolic regulation of secondary metabolite biosynthesis and the cell culture in bioreactors as well as an integrated technology for the extraction of effective medical substances from the cells were investigated. It is a fundamental work for the large-scale culture of C. deserticola cells. The optimum conditions for the callus induction from C. deserticola seeds were first studied. The key steps for the callus induction were that C. deserticola seeds must be treated by preheating and coat-removing, the heat condition was optimized at 50°C for lh. The optimal conditions for the callus growth and formation of phenylethanoid glycosides (PeG) were at 25 °C with light irradiation intensity of 24 u mol/m2s on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine/L, 10 mg gibberellin/L, 800 mg casein hydrolysate/L and 20 g sucrose/L. When the inoculation size was 1%, the biomass and PeG content of C. deserticola callus reached 15.5 g DW/L medium and 10.7% after 30 days culture. The cell suspension culture of C. deserticola was first established. The cell aggregates between 0.5-1.0mm had the highest PeG content of 11.3%. The biomass and PeG content were relatively high in the cell suspended culture when the cell age was 25d and inoculation size was 3-4%. The cell growth, PeG biosynthesis and kinetics of substrate consumption were also investigated. In the cell suspension culture, 0-8d was lag phase, 8-20d was exponential phase and 20-30d was stationary phase. The process of PeG biosynthesis was growth associated. The stoichiometrical relations among the cell growth, nutrient consumption and production formation were established by analysis of the elemental composition of cells. The pathway of PeG biosynthesis, precursors and the key enzymes of metabolic regulation were explored. Precursors such as L-phenylalanine, L-tyrosine, sodium acetate and rare earth elements such as Nd, La, Ce were used to regulate PeG biosynthesis. Phenylalanine and mixed rare earth elements (MRE) showed the most remarkable effects. In the suspension culture of C. deserticola cells, 0.2 mmol/L phenylalanine feeding in the 8th day of the culture gave the highest PeG production (1.10 g/L), which was 75% higher than that obtained in control culture (without precursors added). The effects of rare earth elements on the growth of C. deserticola cells and PeG biosynthesis in suspension culture were more remarkable than those on solid media. A mixture of rare earth elements (MRE, La2O3: CeO2: Pr6On: Sm2O3 = 255:175:3:1, mol/mol) had the most remarkably promoting effects. After 30d's culture, 0.02mmol/L MRE gave the highest PeG content (20.8%) and production (1.6g/L), which were 104% and 167% higher than those obtained in the control. Bioreactor technology was regarded as a key factor for the industrial production of PeG by C. deserticola cell culture. The optimal culture conditions for C. deserticola cells in 2L bubble column were airflow rate of 0.010 m3/h and inoculation size of 2.5%. The biomass was 4.6 g DW/L and PeG production reached 0.42 g/L after 30 days' culture. An internal loop airlift bioreactor with sifter riser (ILABSR) was designed. The optimal culture conditions for C. deserticola cells in a 2L ILABSR were airflow rate of 0.075 m3/h and inoculation size of 4.7%. Under these conditions, about one-fifth cells were attached to the sifter and PeG content was 16.3%, which was 104% higher than that of suspension cells. PeG production was improved to 0.85 g/L, which was 102% higher than that cultured in a 2L bubble column. The technical conditions for the extraction of effective substances from C. deserticola enhanced with ultrasonic wave and an integrated method for the extraction of PeG, betaine and polysaccharide from C. deserticola were investigated. Granular C. deserticola, lg, was treated with methanol and water under ultrasonic wave, and then the filtrate was passed through macro reticular resin (AB-8) column, strong-acidic cation exchange resin (001X 7) column and Sephadex G-75 column successively. From which 52.3mg PeG, 125.6mg betaine and 24.5mg C. deserticola polysaccharide were obtained, and the rate of recovery were 64.4%, 92.9% and 53.5% respectively. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 157 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1404]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 欧阳杰. 肉苁蓉细胞和组织培养中的过程调控[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2003. |
入库方式： OAI收割
来源：过程工程研究所
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