黃世佑臺灣大學：化學工程學研究所詹効松Chan, Hsiao-SungHsiao-SungChan2007-11-262018-06-282007-11-262018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/52333本研究分別對於未分化植物細胞懸浮培養系統生產二次代謝產物及具分化潛力之植物細胞，探討其細胞生長及二次代謝產物的最佳條件以及誘導體胚細胞之最佳條件。選擇人蔘細胞(Panax ginseng)，使用250 mL三角錐形瓶及7 L攪拌式生物反應器(stirred tank reactor, STR)，探討此二種培養環境中之流體剪應力對懸浮細胞生長及人蔘皂素(saponin)生產力之影響。人蔘懸浮細胞於錐形瓶培養試驗中，其細胞生質量達12 g DW/L，其值高於攪拌式生物反應器培養所得9.65 g DW/L (200 rpm)、8.83 g DW/L (300 rpm)、8.47 g DW/L (400 rpm)；然而，在人蔘皂素產率方面，於攪拌式生物反應器在300 rpm培養條件下可得最佳總皂素含量2.34 %，其值遠高於在錐形瓶培養所得之0.89 %。就兩種反應系統的動力消耗作為評估剪應力之參數，以資比較兩者之最佳培養條件。對於三角錐形瓶之動力消耗採用文獻經驗式(Büchs et al., 2000)計算；對於攪拌式反應器則使用自行組裝之小型strain gauge型扭力計測定反應器培養系統之動力消耗，以期瞭解細胞所受剪應力大小。另外，以固-液(Investigation on the optimum conditions for suspension culture of undifferentiated plant cells producing secondary metabolites (Panax ginseng producing ginseng saponin) as well as for inducing differentiated somatic embryogenic cells (Apium graveolens var. dulce, Tall Utah) were conducted. Shear stress effects on Panax ginseng suspension culture were performed using 250 mL shake flask and 7 L stirred tank reactor (STR). Biomass concentration and saponin productivity were determined at various operation conditions. The biomass concentration attained in shake flask was 12 g DW/L which was higher than that in STR (9.65, 8.83 and 8.47 g DW/L for agitation rate 200, 300 and 400 rpm, respectively). However, the productivity of total saponin in STR at 300 rpm (2.34 % DW) was much higher than that in shake flask (0.89 % DW). Taking power consumptions in the shake flask and STR as an index of shear stress intensity, its effect on the performance of cell culture in different bioreactor systems were compared. For shake flask, an empirical equation proposed by Büchs et al., (2000) was used to estimate its power consumption. As for STR, a strain gauge type torque meter was employed to estimate the power consumption. Furthermore, a solid-liquid (中文摘要………………………………………………………………………………I 英文摘要……………………………………………………………………………..III 論文架構…………….…………….………….………….…………….…..…………V 圖目錄………………………………………………………………….…………..XIV 表目錄…………………………………………………………………….………...XV 第一章 緒論 1 第二章 人蔘懸浮細胞受培養環境流體剪應力之影響………………….………….5 2.1 文獻探討…….………….………….…….……………….……….….…………..5 2.1.1 培養剪應力對於植物懸浮細胞的影響…..……………..……….….6 2.1.2植物細胞懸浮培養剪應力的定量…….……………..….……………7 2.1.3錐形瓶之動力消耗…….…………….….……………….…….….…..8 2.1.4反應器之動力消耗…….………………….……….…….….……….10 2.2 材料與方法…….…….……….…………………………………..………..…....10 2.2.1 懸浮細胞培養與反應器操作…...………………………....….….…10 2.2.2 生長狀況量測…….………………….……….…………....…..….11 2.2.3植物細胞絮聚及形態觀察………………..…….……..……..….…..11 2.2.4 皂素分析…….…………………….……………..………..…….…..13 2.2.5 動力消耗…….……………………………....……………….……...14 2.3實驗項目…….……………………………………………………….…….…….17 2.4結果與討論…….…………………………………………….……………….….21 2.4.1動力消耗及剪應力對懸浮細胞培養的影響……………….…....….21 2.4.1a 以反應器進行人蔘細胞懸浮培養之生長狀況………………....21 2.4.1b 人蔘懸浮細胞粒徑分布…….……………………………..…...21 2.4.1c 動力消耗對人蔘懸浮細胞之影響…….…………………..…….24 2.4.1d 形態觀察 …….………...……………………………………......26 2.4.1e 不同攪拌轉速對人蔘懸浮細胞生長的影響……...………….…30 2.4.1f 剪應力對人蔘皂素含量的影響…….………………………...….35 2.4.2 反應器培養後移至錐形瓶試驗，觀察比較於不同培養環境，細胞形態之變化…….…………….……..………. ….…………………….…...37 2.4.2a 反應器與錐形瓶培養動力消耗的比較…………………………37 2.4.2b動力消耗於系統中之分散狀況…….…………………...……….38 2.4.2c 利用en-US人蔘懸浮細胞西洋芹菜體胚發生逆境Ginseng Suspension CellCeleryEmbryogenesisStressthesis