蔣丙煌臺灣大學：食品科技研究所江仟琦Chiang, Chian-ChiChian-ChiChiang2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/56366本研究利用不同種類5L氣舉式發酵槽培養樟芝發酵液，並找出具有最佳抑制肝癌細胞(Hep G2 cell)與子宮頸癌細胞(HeLa cell)生長之發酵條件，並擴大生長規模，以500L氣舉式發酵槽確定生產此一發酵產品之可行性。 實驗架構可分為三個階段。第一階段主要探討適合菌絲體生長的發酵槽及通氣條件，所探討之槽體種類包括：氣泡塔發酵槽、氣舉式發酵槽以及網狀內管氣舉式發酵槽，並以攪拌式發酵槽做為對照組，且分別採用三種不同通氣量，即0.1、0.5以及1 vvm。結果發現發酵7天後，以氣舉式發酵槽，0.1 vvm通氣量操作下，有最多菌絲體產量(3.13 g/L)。 第二階段為探討可以產出最多粗三萜的發酵條件，並以第一階段之最適培養樟芝的氣舉式發酵槽進行實驗，利用三種不同溫度22℃、25℃以及28℃進行六週長時間發酵，並每隔7天取樣一次，實驗結果發現，當氣舉式發酵槽以25℃發酵42天之菌絲體產量(5.02 g/L)及粗三萜含量較22℃及 28℃多。 第三階段進行500L大型發酵槽放大實驗，利用第二階段之最適培養樟芝的氣舉式發酵槽來進行實驗，結果發現在發酵達第28天時菌絲體產量已達到最大量(5.5g/L)。 第二階段與第三階段細胞實驗部分中，均以樟芝發酵濾液及菌絲體乙醇萃取物進行抑制Hep G2及HeLa細胞生長之實驗，發現半致死率IC50皆隨著發酵時間增加而降低。整體來說，以25℃抑制效果較佳，其發酵21天之菌絲體乙醇萃取物，對Hep G2及HeLa cells之IC50皆可達20 μg/ml。The objectives of this research were to study the optimal condition for cultivating Antrodia cinnamomea in airlift reactors for inhibiting the growth of human hepatocellular carcinoma cell (Hep G2) and human cervical epithelioid carcinoma cell (HeLa cell). The experimental design divided into three parts. In the first part, various airlift reactors including bubble column reactor, airlift reactor with solid draft tube and airlift reactor with net draft tube were investigated for their suitability for cultivating A. cinnamomea. And the stirred tank reactor was used as control. The results indicated that the airlift reactor with solid draft tube operating at aeration rate 0.1 vvm could yield the highest amount of the mycelium (3.13 g/L after 7 days of fermentation). The second part of this research investigated the optimal fermentation conditions for A. cinnamomea for producing the highest amount of triterpenes. Using the airlift reactor with solid draft tube, it was found that when the reactor was operated at 0.1 vvm and 25℃ for 42 days, the mycelium concentration (5.02 g/L) and the crude triterpene content were more than that at 22 and 28℃. The operating scale was increased from 5L to 500L airlift reactor for the third part of this study. It was found that under the optimal fermentation condition, the mycelium in the fermentation broth reached 5.5 g/L in only 28 days, as compared to the 42 days of the 5L reactor. During the second and third part of this study, the inhibition effect on the growth of Hep G2 and HeLa cell by the filtrate and the ethanol extract of the mycelium of A. cinnamomea was investigated. The results indicated that the growth inhibitory effect of the filtrate and the ethanol extract of the mycelium increased with fermentation time. Moreover, the inhibitory effect of the fermentation products cultivated at 25℃ was higher than that at 22 or 28℃. The IC50 of the ethanol extract of the mycelium cultivated at 25℃ for 21 days was 20μg/ml.頁碼 中文摘要 ………………………...……………………………………………………i 英文摘要 …………………………………………………………………...……......iii 壹、文獻整理 …………………………………………………………………………1 一. 樟芝之介紹 ……………………….…………………………………..………1 二. 生化反應器之介紹…………………………………………………..……….15 貳、實驗動機及架構 …………………….………………………………………….21 一. 實驗動機…………………......……………………………………..………....21 二. 實驗架構………………………......……………………………………..……24 叁、材料與方法 …………………….…………………………………………….…27 一、實驗材料……………………..……………………………………….………27 (1) 實驗菌株 ……………………………………………………...………27 (2) 實驗細胞株…………...………………………………..…...…………27 (3) 藥品 …………………...……………………...………………………27 (4) 器材與儀器………………...………………...………………………..28 二、實驗方法………………………………………………………………………31 (1) 樟芝之培養……..…...…………………………………………………….31 a. 培養皿平板培養 …………….…………………………………….31 b. 菌種的保存與活化 …………………………………………………32 c. 菌酉元之液態培養…...………….…………………..…………………32 d. 利用發酵槽培養樟芝...…………...……………………..…..………33 (2) 樟芝發酵產物芝分離萃取與乾燥………...……………........……..…….35 a. 樟芝菌絲體之分離...…...……………………..………………...…….35 b. 樟芝發酵濾液之製備……………………………….…..…………….35 c. 樟芝菌絲體乙醇萃取物之製備………………………...…………….35 (3) 樟芝發酵產品對癌細胞生長之影響實驗.………………………………35 a. 細胞株培養 …………………………………...…………………….35 b. 細胞株之繼代培養……………………….…...……………….….…36 c. 細胞株之冷凍保存………...………………..…………………….…36 d. 細胞株之解凍活化………...…….………………………………..…37 e. 細胞計數 ………………...………………………………………….37 f. 樟芝發酵產品對細胞存活率影響之試驗－MTT assay………........37 (4) 化學分析方法 …………………………………………………………...38 a pH 值檢測 …………..………...…………………...…...……….…38 b 菌絲球粒徑 ……………………………...……………...………….38 c 還原醣測定 ………….…………………...………...……………....39 d 粗三萜分離 ……………………………………...……..……….….40 e High-performance liquid chromatograph 分析.........…....…….........40 肆、結果與討論 ………………..……………………………………………………42 第一階段 1. 不同發酵槽、通氣量及培養時間對樟芝發酵液pH值之影響………42 2. 不同發酵槽、通氣量及培養時間對樟芝菌絲體平均粒徑之影響…..45 3. 不同發酵槽、通氣量及培養時間對樟芝發酵濾液固形物之影響…..48 4. 不同發酵槽、通氣量及培養時間對樟芝發酵液還原醣含量之影響..51 5. 不同發酵槽、通氣量及培養時間對樟芝菌絲體含量之影響………..54 第二階段 (一) 物化性質部分 1. 溫度對樟芝菌絲體生長之影響…..……...………..…………..………58 2. 溫度對樟芝發酵液pH值之影響…………………….……………..…62 3. 溫度對樟芝發酵液菌絲體平均粒徑之影響.......…...……..….………62 4. 溫度對樟芝發酵濾液固形物含量之影響…………...…...….……..…65 5. 溫度對樟芝發酵液還原醣之影響...........…......………..………..……65 6. 溫度對樟芝發酵液菌絲體含量之影響……………..…..………….…68 7. 溫度對樟芝菌絲體乙醇萃取物固形物含量之影響..…………......….68 8. 溫度對樟芝發酵液粗三萜含量之影響…………….….....……..….…71 (二) 細胞實驗部分 1. 不同溫度培養之樟芝發酵濾液抑制Hep G2細胞生長情形…..…….73 2. 不同溫度培養之樟芝菌絲體乙醇萃取物抑制Hep G2細胞生長情形……………………………………………………………………….78 3. 不同溫度發酵42天之樟芝菌絲體粗三萜及濾液粗三萜抑制Hep G2細胞存活率之情形………………..….…………………………..……84 4. 不同溫度培養之樟芝發酵濾液抑制HeLa細胞生長情形………...…87 5. 不同溫度培養之樟芝菌絲體乙醇萃取物抑制HeLa細胞生長情形....92 6. 不同溫度發酵42天之樟芝菌絲體粗三萜及濾液粗三萜抑制HeLa細胞存活率之情形…………………………………………………...…..98 (三) HPLC分析 1. 不同溫度培養樟芝發酵濾液之HPLC分析……………………..….101 2. 不同溫度培養樟芝菌絲體乙醇萃取物之HPLC分析..……….…....112 3. 不同溫度培養樟芝菌絲體粗三萜及發酵濾液粗三萜之HPLC分析……………………………………………………………………...123 第三階段 (一) 利用5L及500L氣舉式發酵槽之樟芝之比較 1. 發酵液菌絲體及還原醣含量………………………...………………129 2. 樟芝發酵液pH之變化……………………………….……..…….….131 3. 樟芝發酵液濾液固形物含量……………………………………..….131 4. 樟芝發酵液粗三萜含量……………………………….……….…….133 5. 利用5L及500L氣舉式發酵槽之樟芝菌絲體乙醇萃取物含量…...133 (二)細胞實驗部分 1. 利用500L氣舉式發酵槽培養之樟芝發酵濾液抑制Hep G2細胞生長之情形…………………………………………………………...……135 2. 利用500L氣舉式發酵槽培養之樟芝菌絲體乙醇萃取物抑制Hep G2細胞生長之情形………………………………………...……..……..138 3. 利用500L氣舉式發酵槽培養發酵42天之樟芝菌絲體粗三萜及濾液粗三萜抑制Hep G2細胞生長之情形……………………………….141 4. 利用500L氣舉式發酵槽培養之樟芝發酵濾液抑制HeLa細胞生長之情形………………………………………………...………….……...143 5. 利用500L氣舉式發酵槽培養之樟芝菌絲體乙醇萃取物抑制HeLa細胞生長之情形………………………………………………………...146 6. 利用500L氣舉式發酵槽培養發酵42天之樟芝菌絲體粗三萜及濾液粗三萜抑制HeLa細胞生長之情形………….…………….………..149 (三) HPLC分析 1. 利用500L氣舉式發酵槽培養樟芝發酵濾液之HPLC分析….….….151 2. 利用500L氣舉式發酵槽培養樟芝菌絲體乙醇萃取物之HPLC分析.155 3. 利用500L氣舉式發酵槽培養42天樟芝菌絲體粗三萜及濾液粗三萜之HPLC分析……………………………………………………….…...159 伍、結論……………………………………….……………….………………...….164 陸、參考文獻……………………………………………………..…………………1663246131 bytesapplication/pdfen-US樟芝氣舉式發酵槽抗癌Antrodia cinnamomeaAirlift ReactorsAnti-tumorthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56366/1/ntu-94-R92641002-1.pdf