Studies on the continuous production of natural medicines by the immobilized plant cell bioreactors.

近年来，固定化微生物细胞系统发展迅速。实际上，这些系统已经应用于食品添加剂和药物等有用化合物的工业生产。然而，关于固定化植物细胞在生产有用的天然化合物方面的应用的论文很少。因此，在本课题中，我们首先研究了固定化植物细胞的方法，然后将固定化的植物细胞应用于皂素、黄酮类和生物碱的各种反应。人们已经使用了各种基质来固定化微生物细胞，但海藻酸钙由于其操作条件温和、细胞活力保持率高等优点而特别有利于植物细胞的固定化。在无菌条件下，将完整的植物培养细胞包埋在海藻酸钙微珠中。将固定化细胞放入加有摇瓶培养液的摇瓶中培养，固定化细胞的数量略有增加。利用这些固定化的活植物细胞，本项目的开展是为了解决以下三个问题：(1)应用于有用化合物的生产。(2)通过渗透作用连续生产胞内化合物。(3)生物反应器连续生产系统的开发。因此，我们首先进行了固定化阿拉比卡咖啡细胞生产咖啡因和可可碱的研究。在此基础上，分别用二甲基亚砜(DMSO)处理培养的根细胞和固定化细胞，在不影响细胞活力的情况下，分泌人参皂苷和甘草黄酮类化合物。最后，在改进后的生物反应器中，利用黄连培养的根，成功地实现了黄连素及其相关化合物的连续生产。

The immobilized microbial cell systems have rapidly developed during recent years. Practically, these systems have been applied to the industrial production of useful compounds such as foodstuff additives and drugs. However, there are few papers concerning the application of immobilized plant cells to the production of useful natural compounds. So, in this project we investigated first the method to immobilize a plant cell and next the application of the immobilized plant cell to various reactions concerning saponin, flavonoid and alkaloids.A variety of matrices to immobilize microbial cell have been employed, but calcium alginate was especially favorable to plant cells because of several operational advantages, i.e. mild conditions and high retention of cell viability. Intact plant cultured cells were entrapped in calcium alginate beads under sterile conditions. The immobilized cells slightly increased in the beads when they were cultured in a flask supplemented with the growing medium in a rotary shaker. Using these immobilized living plant cells, this project was carried out to resolve the next three problems.(1) The application to the production of useful compounds. (2) Continuous production of intracellular compounds by permeabilization. (3) Development of continuous production system using bioreactor. As the result, we carried out first the production of caffeine and theobromine by the immobilized Coffea arabica cells. Next, saponin of Panax ginseng and flavonoids of Glycyrrhiza echinata mutant cells were excreted by DMSO(dimethylsulfoxide) treatment with no effect on the cell viability from the cultured roots and the immobilized cells, respectively. Finally, we succeeded in the continuous production of berberine and its related compounds directly using Coptis japonica cultured root for the revised bioreactor.