臺灣博碩士論文加值系統

在1975年時，Streptomyces hygroscopicus在復活節島上的土壤被發現，且在菌體的發酵液中分離出Rapamycin。在早期Rapamycin被當作治療真菌之抗生素，近年來Rapamycin在醫學研究及臨床實驗被發現具有越來越多的用途，所以需要提升Rapamycin在規模放大製程的產量。因此，本研究將探討改變接菌量及接菌型態對於Rapamycin濃度之影響。
本研究在發酵策略上，利用Carboxymethyl cellulose改變培養基黏度，以降低菌體顆粒大小，並提升Rapamycin產量。結果顯示，添加4 g/L CMC，相較於未添加CMC之控制組(169 mg/L)，得到較高Rapamycin濃度(323 mg/L)。且利用不同接菌型態進行發酵程序，包括在前培養階段利用液態培養生成之菌絲、將8種不同基質(薏仁、蕎麥、糙米、糯米、白米、紅豆、黃豆、綠豆)進行固態培養，得知以薏仁為基質具有較高之Rapamycin濃度(393 mg/kg)及在洋菜平板上生長之黑色孢子做為後續接種，並使用15 L攪拌式發酵槽進行發酵程序。結果顯示，液態菌絲接菌，得知由5 L攪拌式發酵槽放大至15 L發酵槽且接菌量同步由10 %降低至5 %，兩者Rapamycin濃度分別為394 mg/L及366 mg/L，對於Rapamycin生產並無太大影響。而以孢子型態(菌落數為2x1010 CFU/ml)及固態菌絲(菌落數為3x109 CFU/ml)接種進行發酵程序，Rapamycin均有一定產量，且固態菌絲型態之發酵過程得到最高之Rapamycin濃度(525 mg/L)。後續的回收純化實驗中，將4種不同顆粒大小之樹脂(MAC-3、L- 493、SD-2、HP-20)進行吸附實驗，結果顯示型號為MAC-3之大孔吸附樹脂於溫度為28 ℃，萃取液濃度為700 mg/L及使用0.5 g樹脂為最適合之靜態脫附實驗條件，吸附率可達89 %及吸附量可達25 mg/g。

In 1975, Streptomyces hygroscopicus was found in the soil on Easter Island, and Rapamycin was as an antifungal agent early. Its immunosuppressive effect has recently attracted more attention. Therefore, this study focused on examining the effects of inoculation volume and different inoculation morphologies on Rapamycin production.
In the results of this study, adding carboxymethylcellulose (CMC) to change morphology could increase Rapamycin production. A maximum yield of 323 mg/L of Rapamycin was obtained when 4 g/L of CMC was added, as compared to 165mg/L in the control batch (without the addition of CMC). In the solid state fermentation (SSF), total 8 substrates were adopted to explore the potential being the inoculation substrate for Rapamycin production, and the results indicated that barley can have the max Rapamycin yield (393 mg/kg). While using spore suspensions and solid state mycelium as inoculated medium, both of them could be have cells growth. Especially, it could produce maximum Rapamycin yield (525 mg/L) by using solid barley as inoculated medium in this study.
Finally, the static adsorption tests for the purification of Rapamycin using macroporous adsorption resin, MAC-3, were performed as follows：0.5 g of the pre-weighed resin were put into a flask. The initial Rapamycin in the solutionwas
700 mg rapamycin/L. The flasks were sealed tightly for 30 minutes to reach the maximum adsorption rate (88.6 %) and adsorption capacity (25 mg/g).

目錄
中文摘要 III
Abstract V
誌謝 VII
目錄 VIII
圖目錄 XIII
表目錄 XVII
第一章 緒論 1
第二章 文獻回顧 2
2.1 菌種介紹 2
2.1.1放線菌(Actinobacteria)介紹 3
2.1.2鏈黴菌(Streptomyces)介紹 5
2.2 Rapamycin介紹 7
2.2.1抗真菌 8
2.2.2抗腫瘤 8
2.2.3抗衰老 9
2.2.4 Rapamycin作用機制與合成路徑 11
2.3固態發酵 (Solid-state fermentation，SSF) 14
2.3.1影響固態發酵的重要因素 15
2.3.2固態發酵與液態發酵之差異 16
2.3.3固態發酵之優缺點 17
2.4發酵策略 18
2.4.1攪拌式發酵槽 18
2.4.2旋轉式發酵槽 20
2.5大孔吸附樹脂應用之起源 21
2.5.1大孔吸附樹脂(macroporous absorption resin)介紹 21
2.5.2吸附理論 23
2.5.2靜態吸附等溫吸附模式 24
2.5.3等溫吸附曲線 25
第三章 實驗材料與方法 28
3.1 實驗材料 28
3.1.1 實驗菌株 28
3.1.2實驗藥品 29
3.2實驗儀器 30
3.3分析方法 33
3.3.1菌體重量分析方法 33
3.3.2 葡萄糖濃度分析方法 33
3.3.3 Rapamycin分析方法 34
3.4實驗方法 35
3.4.1原始菌種保存 35
3.4.2培養基組成 36
3.4.2.1 種菌培養基（Seed medium, SM） 36
3.4.2.2 發酵培養基（Fermentation medium, FM） 37
3.4.3接菌 37
3.5實驗架構 38
3.6實驗培養條件 39
3.6.1搖瓶批次發酵程序(Flasks –Batch experiment) 39
3.6.1.1液態培養基-添加CMC造成不同黏度之影響 39
3.6.1.2固態培養基-不同基質之影響 39
3.6.1.3固態培養基-不同起始含水量之影響 40
3.6.2固態發酵槽批次發酵程序 40
3.6.3 5 L攪拌式發酵槽批次發酵程序 41
3.6.4 15 L攪拌式發酵槽批次發酵程序 42
3.6.4.1接菌量之影響 42
3.6.4.2孢子接菌之影響 43
3.6.4.3固態菌絲接菌之影響 44
3.7 Rapamycin純化條件(Purification process) 45
3.7.1.1樹脂前處理 45
3.7.1.2吸附溶液之準備 45
3.7.2大孔樹脂靜態吸附之實驗 46
3.7.2.1選擇靜態吸附最佳之樹脂 46
3.7.2.2選擇靜態吸附達平衡之時間 46
3.7.2.3不同萃取液濃度之影響 47
3.7.2.4不同溫度之影響 47
3.7.3大孔樹脂靜態脫附之實驗 48
第四章 結果與討論 49
4.1搖瓶批次發酵程序 49
4. 1.1 液態培養基-添加CMC造成不同黏度之影響 49
4.1.2固態培養基-不同基質之影響 52
4.1.3固態培養基-不同起始含水量影響 54
4.2 15 L攪拌式發酵槽批次發酵程序 55
4.2.1液態接菌之影響 55
4.2.1.1液態接菌於15 L攪拌式發酵槽之影響 55
4.2.1.2液態接菌放大培養之比較 58
4.2.2孢子接菌之影響 60
4.2.3固態菌絲接菌之影響 65
4.3比較不同接菌型態於攪拌式發酵槽批次發酵程序 69
4.4 Rapmycin純化條件 76
4.4.1選擇於靜態吸附實驗最佳之樹脂 76
4.4.2時間對靜態吸附實驗之影響 79
4.4.3濃度對靜態吸附實驗之影響 80
4.4.4溫度對靜態吸附實驗之影響 82
4.4.5添加不同克數之樹脂對靜態吸附實驗之影響 84
4.4.6靜態脫附之實驗 86
第五章 結論與未來展望 87
5.1結論 87
5.2未來展望 88
參考文獻 89
附錄 95
作者簡介 96

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