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系統識別號 U0002-0606201921363100
中文論文名稱 Serratia marcescens TKU011 發酵生產靈菌紅素之研究
英文論文名稱 Studies on the production of prodigiosin by Serratia marcescens TKU011 fermentation
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 107
學期 2
出版年 108
研究生中文姓名 陳善平
研究生英文姓名 Shan-Ping Chen
學號 606180031
學位類別 碩士
語文別 中文
口試日期 2019-06-04
論文頁數 72頁
口試委員 指導教授-王三郎
共同指導教授-阮文邦
委員-王三郎
委員-糜福龍
委員-王全祿
中文關鍵字 Serratia marcescens  靈菌紅素  α-幾丁質  酪蛋白 
英文關鍵字 Serratia marcescens  prodigiosin  α-chitin  casein 
學科別分類 學科別自然科學化學
中文摘要 本研究主要探討篩選自台灣中部土壤之靈菌紅素(prodigiosin)生產菌Serratia marcescens TKU011,生產靈菌紅素之液態發酵條件。研究結果發現,充填0.1% α-chitin、0.06% casein、0.005%K2HPO4、0.01% CaSO4˙2H2O之10毫升液態培養基(pH 6.15)的50mL錐形瓶,於25℃震盪培養箱(150 rpm)連續培養兩天,可得較高靈菌紅素生產量(6.25 μM)。本實驗探討所得靈菌紅素生產條件,比起過去使用烏賊軟骨作為主要碳/氮源所得靈菌紅素產量(3.4 μM)高出1.84倍。分離所得靈菌紅素之分子結構,經由1H-NMR(300MHz)、MASS (MALDI-TOF)、FT-IR等儀器分析獲得確證。
英文摘要 This study focused on the selecting of Serratia marcescens TKU011, a strain of prodigiosin-producing bacteria from the soil of middle Taiwan, to produce liquid fermentation conditions for the production of prodigiosin. The results showed that a 50 mL Erlenmeyer flask fill with 10 mL liquid medium (pH 6.15) containing 0.1% α-chitin, 0.06% casein, 0.005% K2HPO4, 0.01% CaSO4˙2H2O are shaken at 25℃(150 rpm) continuous culture for two days yielded higher prodigiosin production (6.25 μM). In this experiment, the production conditions of obtained prodigiosin are 1.84 times higher than the production of squid pen powder (3.4 μM) which is used as the main carbon / nitrogen in the past. The molecular structure of the obtain prodigiosin is separated and confirmed by 1H-NMR (300MHz), MASS (MALDI-TOF), FT-IR and other instruments.
論文目次 目錄
中文摘要 …............................................................................. I
英文摘要 …............................................................................. II
目錄 ….................................................................................... III
圖目錄 …................................................................................ IV
表目錄 …..................................................................................V
縮寫表 …................................................................................ VI
第一章 緒論 …....................................................................... 1
第二章 文獻回顧 …............................................................... 2
2.1 Serratia marcescens ….................................................... 2
2.1.1 微生物學特性 ….................................................... 2
2.1.2 化學特性 …............................................................ 3
2.2 PDG介紹 …................................................................... 5
2.2.1 生化特性 …............................................................ 5
2.2.2 生產條件 …............................................................ 6
2.2.3 應用 ….................................................................... 6
2.3 培養條件探討 …............................................................... 8
2.3.1培養基種類 …......................................................... 8
2.3.2 培養溫度 …............................................................ 8
2.3.3 培養體積 …............................................................ 9
2.3.4 培養時間 …............................................................ 9
2.3.5 培養酸鹼度 …........................................................ 9
第三章 材料與方法 ….......................................................... 10
3.1 實驗規劃 …................................................................. 10
3.1.1 架構 ….................................................................. 10
3.1.2 流程圖 ….............................................................. 11
3.2實驗菌株 ….................................................................. 11
3.3實驗材料 ….................................................................. 12
3.4實驗儀器 ….................................................................. 12
3.5 PDG濃度測定 …........................................................... 13
3.5.1 OD535之意義 …..................................................... 13
3.5.2 樣品預處理及吸光度換算濃度之方法 ….................. 13
3.6 Serratia marcescens生長 …......................................... 15
3.6.1 OD600之意義 …..................................................... 15
3.6.2 PDG的凋亡曲線 ….............................................. 16
3.7 Prodigiosin最適生產條件 …....................................... 16
3.7.1 培養基種類 …...................................................... 16
3.7.2 氮源種類 ….......................................................... 17
3.7.3 碳源種類 ….......................................................... 17
3.7.4 碳/氮源比例選擇 …............................................. 17
3.7.5 鹽類種類 ….......................................................... 18
3.7.6 鹽類比例選擇 ….................................................. 18
3.7.7 上清液或培養液實驗方法之選擇 ….................. 18
3.7.8 培養液體積大小之影響 ….................................. 19
3.7.9 酸鹼值之影響 ….................................................. 19
3.7.10 活化溫度選擇 …................................................ 19
3.7.11 生產溫度選擇 …................................................ 19
3.7.12 整體培養溫度(活化及生產)選擇 …................. 20
3.7.13 培養時間選擇 …................................................ 20
3.7.14 培養過程之有無接觸空氣探討 ….................... 20
3.8 PDG之結構鑑定 …..................................................... 21
3.8.1 1H-NMR結構鑑定 …............................................ 21
3.8.2 13C-NMR結構鑑定 …........................................... 22
3.8.3 質譜儀(MALDI TOF)結構鑑定 …...................... 22
3.8.4 傅立葉紅外線(FT-IR)光譜儀結構鑑定 ….......... 22
第四章 結果與討論 ….......................................................... 23
4.1 Serratia marcescens生長狀況之探討 …..................... 23
4.2 Prodigiosin最適生產條件之探討 …........................... 24
4.2.1 培養基種類 …...................................................... 24
4.2.2 氮源種類 ….......................................................... 25
4.2.3 碳源種類 ….......................................................... 26
4.2.4 碳/氮源比例選擇 …............................................. 29
4.2.5 鹽類種類 ….......................................................... 31
4.2.6 鹽類比例選擇 ….................................................. 33
4.2.7 上清液或培養液實驗方法之選擇 ….................. 35
4.2.8 培養液體積大小之影響 ….................................. 37
4.2.9 酸鹼值之影響 ….................................................. 40
4.2.10 活化溫度選擇 …................................................ 41
4.2.11 生產溫度選擇 …................................................ 45
4.2.12 整體培養溫度選擇 …........................................ 47
4.2.13 培養時間選擇 …................................................ 52
4.2.14 培養過程之有無接觸空氣探討 ….................... 54
4.3 PDG之結構鑑定 …..................................................... 55
4.3.1 1H-NMR …............................................................. 56
4.3.2 13C-NMR …............................................................. 57
4.3.3質譜儀(MALDI TOF) …........................................ 58
4.3.4傅立葉紅外線(FT-IR) …........................................ 61
4.4 PDG濃度計算方式比較 …......................................... 63
4.5 PDG最適生產條件之整理與比較 …......................... 65
第五章 結論 …...................................................................... 67
第六章 參考文獻 ….............................................................. 68

圖目錄
圖2.1黏質沙雷氏菌存活曲線圖 …................................. 2, 16
圖3.1 Prodigiosin (OD535-濃度)標準曲線 …….................... 15
圖4.1 Prodigiosin之細菌生長與濃度比較 …...................... 23
圖4.2 比較不同碳/氮源比例生產Prodigiosin …................. 30
(A) -Chitin vs. Casein (B) -Chitin vs. Casein
圖4.3 比較不同磷酸氫二鉀比例生產Prodigiosin ….......... 34
圖4.4 比較不同次要鹽類生產比例生產Prodigiosin …...... 35
圖4.5 比較培養液與上清液之選擇生產Prodigiosin …...... 36
(A) -Chitin vs. Casein (B) -Chitin vs. Casein
圖4.6 比較不同體積之 ….................................................... 38
(A) 菌種生長變化 (B) Prodigiosin生產濃度變化
圖4.7 比較不同pH值生產Prodigiosin …........................... 40
圖4.8 比較不同生產溫度生產Prodigiosin之 …................ 46
(A) 菌體生長變化 (B) Prodigiosin生產濃度
圖4.9 比較整體培養溫度生產Prodigiosin之 …................ 50
(A) 菌體生長變化 (B) Prodigiosin生產濃度變化
圖4.10 比較菌體培養時間 .................................................. 53
圖4.11 0.12%/0.04%作為碳/氮源生產Prodigiosin之變化 ....... 53
圖4.12 比較接觸空氣生產Prodigiosin之影響 …............. 55
圖4.13靈菌紅素分子結構 …............................................... 55
圖4.14 α-PDG 1H-NMR(300MHz)圖譜之 …....................... 56
(A) 小範圍(-1) - 7 (B) 大範圍1 - 12
圖4.15 α-PDG 13C-NMR(300MHz)圖譜 ….......................... 58
圖4.16 α-PDG 質譜儀MALDI TOF單一圖譜之 ….......... 59
(A) 小範圍250 - 450 (B)大範圍0 - 1500
圖4.17 PDG 質譜儀MALDI TOF 多種類混合圖譜之 …. 60
(A) 小範圍 305 - 350 (B) 大範圍 0 - 1300
圖4.18 α-PDG 傅立葉轉換紅外線光譜儀圖譜之 ….......... 62
(A) 小範圍 400 - 2000(B) 大範圍 400 - 4000

表目錄
表2.1 Serratia marcescens sp.之化學試驗表 ….................... 4
表2.2 Serratia marcescens sp.發酵之培養基發展 …............ 8
表3.1 TKU011發酵生產靈菌紅素之實驗架構 ….............. 10
表3.2 TKU011發酵生產靈菌紅素之實驗流程圖 ….......... 11
表4.1 OD535轉換為濃度單位之方法 …......................... 14, 64
表4.2 比較不同培養基種類生產PDG …............................ 24
表4.3 比較不同氮源種類生產PDG …................................ 26
表4.4 比較不同碳源生產PDG之 ….................................. 27
(A) 0.12%碳源+0.04%氮源 (B) 0.08%碳源+0.08%氮源
表4.5 比較不同主要鹽類種類生產PDG …........................ 32
表4.6 比較不同次要鹽類之種類及比例生產PDG …........ 33
表4.7 比較其他期刊之溶液體積測試結果 ….................... 39
表4.8比較其他期刊之培養溶液酸鹼值測試結果 …......... 41
表4.9 比較不同活化溫度生產PDG之 ….......................... 43
(A) 菌體生長情形(第三天) (B) PDG生產濃度(第三天)
表4.10 比較整體培養溫度生產PDG之 …........................ 48
(A) 菌體生長情形(第二天) (B) PDG生產濃度(第二天)
表4.11 比較其他期刊之培養溫度測試結果 ….................. 52
表4.12比較其他期刊之培養時間測試結果 …................... 54
表4.13比較不同濃度計算之本實驗結果 …....................... 65
表4.14 生產PDG優化前後條件之比較 …........................ 66

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