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中文論文名稱 Serratia ureilytica TKU013以烏賊軟骨為唯一碳/氮源發酵生產抗氧化物之研究
英文論文名稱 The study on the antioxidants produced by Serratia ureilytica TKU013 using squid pen as the sole carbon/nitrogen sources
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 97
學期 2
出版年 98
研究生中文姓名 劉高呈
研究生英文姓名 Kao-Cheng Liu
學號 696180271
學位類別 碩士
語文別 中文
口試日期 2009-07-16
論文頁數 53頁
口試委員 指導教授-王三郎
共同指導教授-郭曜豪
委員-郭曜豪
委員-陳佑汲
中文關鍵字 Serratia ureilytica.  抗氧化  細胞毒殺 
英文關鍵字 Serratia ureilytica.  antioxidation  cytotoxicity 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本研究係探討Serratia ureilytica TKU013發酵幾丁質類物質生產抗氧化物質之研究。以烏賊軟骨粉(SPP)、蝦頭粉(SHP)、蟹殼(CSP)粉當作碳/氮源,依不同比例(0.5 %~2 %)之添加濃度,於25℃培養0~4天,發現以1.5 %烏賊軟骨粉培養4天所得發酵上清液之抗DPPH活性最高。乙酸乙酯層粗萃取物具有較高的DPPH清除率(ED50值為8.56 ug/mL)。經矽膠管柱層析分離出8個分液,自第4個分液進一步分離出1個命名為Serranticin的抗氧化化合物。
濃度200 ug/mL的Serranticin對DPPH的清除率達到98 %。在生物活性方面,Serranticin具細胞毒殺之效果,對MCF-7 (Human breast adenocarcinoma)及WiDr(Human colon adenocarcinoma)的ED50分別為14.7 ug/mL及6.52 ug/mL。此外,亦探討發酵烏賊軟骨粉、蝦頭粉、蟹殼粉所得上清液之總酚含量、還原力、亞鐵離子螯合力。
英文摘要 Chitinous materials were used the sole carbon/nitrogen sources for antioxidants produced by Serratia ureilytica TKU013. To investigate the effect of different carbon/nitrogen sources (squid pen powder, shrimp head powder, crab shell powder, 0.5~2 %) on the production of antioxidant materials by TKU013, here incubated Serratia ureilytica TKU013 for 0~4 days at 25℃ and analyzed the antioxidant activity of the cultured supernatant showed using DPPH scavenging ability. Biological test that TKU013 culture supernatant (1.5 % SPP) incubated for four days has the highest antioxidant activity. Moreover, a new compound Serranticin was isolated from ethyl acetate layer.
Serranticin exhibits cytotoxic effects (MCF-7 ED50=14.78 ug/mL, WiDr ED50=6.52 ug/mL), and has strong antioxidative effects assayed by DPPH.
In the second part, Serratia sp. TKU013 was cultured using different carbon source (squid pen powder, shrimp head powder, crab shell powder, 0.5~2 %) and incubated in 50 mL of liquid media in shaking flasks at 25℃ for 0~4days, and the quantity of total polyphenol content, reducing power and Fe2+ ion chelating ability.
論文目次 中文摘要 I
英文摘要III
誌謝 V
目錄 VI
圖目錄 IX
表目錄 XI

第一章 緒論 1
第二章 文獻回顧 4
2.1沙雷氏菌屬之特性 4
2.2 自由基 4
2.3 活性氧 4
2.4 抗氧化劑 5
2.5 抗氧化劑的作用機制 6
2.5.1 自由基終止型 6
2.5.2 還原作用 7
2.5.3 金屬螯合 7
2.6 抗氧化活性 7
2.6.1 DPPH清除率測定 7
2.6.2 亞鐵離子螯合力 8
2.6.3 還原力 9
第三章 材料與方法 10
3.1 實驗材料 10
3.2 實驗儀器 10
3.3 DPPH自由基清除能力測定 11
3.4 癌細胞毒殺分析實驗 11
3.4.1 細胞種類 12
3.4.2 細胞培養 12
3.4.3 測試樣品之配置 12
3.4.4 MTT染色測定 13
3.5 總酚含量測定 13
3.6 還原力測定 13
3.7 亞鐵離子螯合力測定 14
3.8 分離純化 14
第四章 結果與討論 15
4.1 DPPH自由基清除能力之測定 15
4.2 矽膠管柱層析 17
4.3 Serranticin結構解析 21
4.4 癌細胞毒殺分析實驗 35
4.5 總酚含量 35
4.6 亞鐵離子螯合力測定 38
4.7 還原力測定 41
第五章 結論 44
參考文獻 45

圖目錄
圖1.1 幾丁質與幾丁聚醣之結構 3
圖2.1 DPPH與抗氧化劑之反應 8
圖3.1 MTT之結構與反應 12
圖4.1碳/氮源濃度及發酵時間對DPPH自由基清除能力之影響 16
圖4.2分離純化流程圖 18
圖4.3各fraction對DPPH自由基清除能力之影響 19
圖4.4 Siderophore之結構 23
圖4.5 Serranticin HMBC的關聯性 25
圖4.6 Serranticin之1H-NMR圖譜 26
圖4.7 Serranticin之13C-NMR圖譜 26
圖4.8 Serranticin之HMQC圖譜 27
圖4.9 Serranticin之HMBC圖譜 27
圖4.10 Serranticin之COSY圖譜 28
圖4.11 Serranticin之HRMS圖譜 28
圖4.12 Serranticin之IR圖譜 29
圖4.13從Serratia sp.所分離到化合物之化學結構 30
圖4.14不同碳/氮源及發酵時間對總酚含量之影響 36
圖4.15Ferrozine的結構 38
圖4.16不同碳/氮源及發酵時間對亞鐵離子螯合力之影響 39
圖4.17不同碳/氮源及發酵時間對還原力之影響 42

表目錄
表2.1 微生物來源之抗氧化劑 6
表4.1微生物來源之抗DPPH活性比較 17
表4.2各Fraction與抗氧化劑Vitamin E比較 20
表4.3 Serranticin的1H-NMR (400 MHz)及13C-NMR (100 MHz)的光譜資料 24
表4.4 Serranticin細胞毒殺之結果 35
表4.5微生物來源之總酚含量比較 37
表4.6微生物來源之亞鐵離子螯合力比較 40
表4.7微生物來源之還原力比較 43
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