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研究生:謝焸任
研究生(外文):Ku-Ren Hsieh
論文名稱:木屑水解糖化產製靈菌紅素之可行性評估
論文名稱(外文):Feasibility study on the production of prodigiosin using wood dust hydrolysate
指導教授:魏毓宏
指導教授(外文):Yu-Hong Wei
口試委員:朱一民張嘉修徐敬衡陳博彥張煜光
口試委員(外文):I-Ming ChuJo-Shu ChangChin-Heng ShuBor-Yann ChenYu-Kaung Chang
口試日期:20170620
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:56
中文關鍵詞:靈菌紅素Serratia marcescens SMΔR木屑水解
外文關鍵詞:prodigiosinSerratia marcescens SMΔRwood dust hydrolysate
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靈菌紅素(Prodigiosin)為一種可由微生物醱酵合成之紅色色素,屬於次級代謝產物。目前已知可由Serratia, Pseudomonas, Vibrio和Streptomyces等微生物生產。文獻指出,靈菌紅素除了具有抗生素之特性外,亦有抑制癌細胞轉移及免疫抑制之功能,為新興之抗癌藥物。本研究有鑑於prodigiosin的高製造成本,首先進行SG培養基配方的修正,接下來利用農業廢棄物做為替代碳源冀望降以低prodigiosin之生產成本,並將綠色科技的概念導入製藥產業。本研究先針對不同濃度soytone作探討,經研究結果顯示單獨以葡萄糖或soytone培養Serratia marcescens SMΔR,菌體雖生長,但prodigiosin之產量很少,而在同時添加葡萄糖及soytone培養,prodigiosin產量為79 mg/L。接下來以固定葡萄糖濃度為24 g/L的條件下培養Serratia marcescens SMΔR,發現在添加8 g/L的soytone可得最高靈菌紅素產量為64 mg/L,之後將其探討最佳C/N比進行篩選,發現在碳氮比為6:4的條件(相當於葡萄糖濃度為12 g/L)下可得最大菌體生長量及最大靈菌紅素產量為83 mg/L,之後將選用Serratia marcescens和木屑水解液進行測試。為了能有效取得木屑中之糖類。本研究以蒸氣蒸爆法先將木屑進行前處理後,再利用商業酵素Ctec 2進行水解,最後以水解液培養Serratia marcescens進行prodigiosin生產評估。在水解率之評估結果發現,在20%木屑添加10%酵素的條件下可得水解液最大glucose濃度為26.06 g/L,水解率為12.11%。進一步以水解液培養Serratia marcescens進行prodigiosin生產評估,結果顯示處理後之水解液生成抑制菌體生長之抑制物對S. marcescens生產prodigiosin之負面影響。因此進一步將水解液稀釋成不同濃度進行prodigiosin生產評估,研究結果發現將水解液稀釋成30%可得靈菌紅素最大產量為27 mg/L。基於上述之研究結果,稀釋後之水解液可用於生產prodigiosin,然而其水解液尚需額外處理,才能有效達到做為營養來源之目的。
Prodigiosin is a red pigment that can be synthesized by microorganisms and belongs to secondary metabolites. It is known to be produced by microorganisms such as Serratia, Pseudomonas, Vibrio and Streptomyces. The literature pointed out that the addition of antibiotics in addition to the characteristics of antibiotics, but also inhibit the transfer of cancer cells and immunosuppressive function, for the emerging anti-cancer drugs. In view of the high manufacturing costs of prodigiosin, the first revision of the SG medium formulation was followed by the use of agricultural waste as an alternative carbon source to reduce the production costs of low prodigiosin and to introduce the concept of green technology into the pharmaceutical industry. In this study, first we explored Serratia marcescens SMΔR with glucose or soytone, but the yield of prodigiosin was very few, and glucose and soytone were added at the same time. The yield of prodigiosin was 79 mg/L. Then, Serratia marcescens SMΔR was cultured under the condition of glucose of 24 g/L, and it was found that the highest yield of 8 mg/L of soytone was 64 mg/L, and then the optimum C/N ratio, it was found that the maximum cell growth and maximum yield of prodigiosin were 83 mg/L at the C/N ratio of 6: 4 (glucose of 12 g/L), and then Serratia marcescens and hydrolysate liquid were tested. In order to be able to effectively obtain the glucose by wood dust. In this study, the wood dust was pretreated by steam explosion, then hydrolyzed by Ctec2, and finally Serratia marcescens was cultured with hydrolysate liquid to evaluate prodigiosin production. The results showed that the maximum glucose concentration of the hydrolysate was 26.06 g/L and the hydrolysis rate was 12.11% under the condition of 20% wood dust and with 10% enzyme. Further evaluation of prodigiosin production by Serratia marcescens in hydrolysate liquid showed a negative effect on the production of prodigiosin by S. marcescens, an inhibitor of excess hydrolysate after treatment and inhibition of cell growth. dilution of the hydrolysate liquid into different concentrations for prodigiosin production evaluation, the study found that the hydrolysate liquid diluted to 30% can get the highest yield of prodigiosin 27 mg/L. But the hydrolysate liquid need additional treatment.
目錄
書名頁 I
審定書 II
中文摘要 III
英文摘要 V
致謝 VII
目錄 VIII
表目錄 XI
圖目錄 XII
第一章 研究動機與目的 1
第二章 文獻回顧 3
2-1 Serratia屬及S. marcescens之生化特性 3
2-2 靈菌紅素(prodigiosin) 6
2-3 靈菌紅素之醱酵工程 9
2-4 農業廢棄物 12
2-5 木質纖維素(Lignocellulose) 13
2-5-1 纖維素 15
2-5-2 半纖維素 16
2-5-3 木質素 16
2-6 纖維分解酵素 17
第三章 材料與方法 18
3-1 實驗藥品與分析儀器 18
3-1-1 實驗藥品 18
3-1-2 分析儀器 19
3-2 菌株及培養基 20
3-2-1 菌株 20
3-2-2 Serratia marcescens SMΔR培養基配方 20
3-3 靈菌紅素之分離與純化 21
3-4 靈菌紅素之HPLC定量 21
3-5 微生物生長之分析 23
3-6 醣類之HPLC定量 24
3-7副產物移除 25
第四章 結果與討論 26
4-1 論文研究架構 26
4-2探討葡萄糖對S. marcescens SMΔR生產靈菌紅素之影響 27
4-3探討soytone對S. marcescens SMΔR生產靈菌紅素之影響素 29
4-4探討氮源最佳添加濃度 32
4-5探討培養基之最適碳氮比 37
4-6經蒸氣蒸爆後木屑基質組成探討培養基之最適碳氮比 42
4-7木屑基質之酵素水解 43
4-8木屑水解液醱酵測試 44
第五章 結論 48
第六章 未來展望 49
參考文獻 50


表目錄
表2-1 Serratia菌屬之生化特性 5
表3-1 本實驗使用之培養基配方 20

圖目錄
圖2-1 Prodiginines家族分類 8
圖2-2 Prodigiosin的代謝路徑 9
圖2-3木質纖維素之組成 14
圖2-4維素之結構 15
圖2-5非結晶和結晶型纖維素水解作用 17
圖3-1靈菌紅素之校正曲線 22
圖3-2菌體乾重對應光學密度(OD600)之校正曲線 23
圖3-3 Glucose之校正曲線 24
圖4-1論文研究架構 26
圖4-2在不同條件下培養所得靈菌紅素之產量 28
圖4-3單獨添加不同濃度soytone對培養S. marcescens菌體生長之影響 30
圖4-4單獨添加不同濃度soytone對培養S. marcescens之pH值變化 31
圖4-5在固定glucose為24 g/L及不同濃度soytone對培養S. marcescens菌體生長之影響 33
圖4-6在固定glucose為24 g/L及不同濃度soytone對培養S. marcescens之pH值變化……………………………………………....34
圖4-7在固定glucose為24 g/L及不同濃度soytone對培養S. marcescens生產靈菌紅素產量影響 35
圖4-8在固定glucose為24 g/L及不同濃度soytone對培養S. marcescens之最大靈菌紅素產量之比較 36
圖4-9固定soytone為8 g/L測試不同C/N ratio培養S. marcescens菌體生長之影響 38
圖4-10固定soytone為8 g/L測試不同C/N ratio培養S. marcescens之pH值變化 39
圖4-11固定soytone為8 g/L測試不同C/N ratio培養S. marcescens生產靈菌紅素產量影響 40
圖4-12固定soytone為8 g/L測試不同C/N ratio培養S. marcescens最大靈菌紅素產量之比較 41
圖4-13經蒸氣蒸爆處理之木屑基質成分組成圖 42
圖4-14蒸氣蒸爆後木屑基質經Ctec2水解之影響 43
圖4-15不同比例之水解液對培養S. marcescens菌體生長之影響 45
圖4-16不同比例之水解液對培養S. marcescens之pH值變化 46
圖4-17 不同比例之水解液對培養S. marcescens生產靈菌紅素之影響 47
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