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得到了迅速发展。实际上，这些系统已应用于有用化合物如食品添加剂和药物的工业生产。然而，关于将固定化植物细胞用于生产有用的天然化合物的报道很少。因此，本课题首先研究了固定化植物细胞的方法，然后研究了固定化植物细胞在皂苷、黄酮类化合物等反应中的应用。固定化微生物细胞的基质有多种，但海藻酸钙因其操作条件温和、细胞活力保持率高等优点，特别适合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藻酸钙珠中。当它们在补充有生长培养基的烧瓶中在旋转振荡器中培养时，固定化细胞在珠中略有增加。使用这些固定化的活植物细胞，该项目是为了解决接下来的三个问题而进行的。(1)在生产有用化合物中的应用。(2)通过透化作用连续生产细胞内化合物。(3)生物反应器连续生产系统的开发。因此，我们首次进行了固定化小粒咖啡细胞生产咖啡因和可可碱的研究。接下来，人参皂苷和类黄酮的刺根突变体细胞分泌的DMSO（二甲基亚砜）处理，从培养的根和固定化细胞的细胞活力没有影响，分别。最后，我们成功地利用黄连培养根为改良的生物反应器，连续生产小檗碱及其相关化合物。

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.