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系統識別號 U0002-0108201601033300
中文論文名稱 Paenibacillus sp. TKU036 發酵烏賊軟骨所生產抗氧化物之分離與鑑定
英文論文名稱 Purification and identification of antioxidants from Paenibacillus sp. TKU036 fermented with squid pen as the sole carbon/nitrogen source
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 104
學期 2
出版年 105
研究生中文姓名 李欣庭
研究生英文姓名 Hsin-Ting Li
學號 603180083
學位類別 碩士
語文別 中文
口試日期 2016-07-07
論文頁數 63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-王三郎
委員-梁慈雯
中文關鍵字 色胺酸  尿黑酸  抗氧化  烏賊軟骨 
英文關鍵字 Paenibacillus sp.  squid pen  antioxidant  tryptophan  homogentisic acid 
學科別分類 學科別自然科學化學
中文摘要 抗氧化物能夠直接地清除活性氧化物質,基本上區分兩類,天然抗氧化物及合成抗氧化物。合成的抗氧化物如butylated hydroxyanisole(BHA)、butylatedhydroxytoluene(BHT),因便宜而常添加於食品中作抗氧化劑,防止食品脂質過氧化而腐壞,但由於 BHA 及 BHT 這類合成抗氧化物長期服用下有致癌風險,因此遭許多國家限制使用,並致力於從天然資源獲取抗氧化物來添加於食品中。
實驗使用菌株Paenibacillus sp. TKU036以烏賊軟骨粉(SPP)做碳/氮源,於 37 ℃ 培養 3 天有最好 DPPH 自由基清除率,將此離心之上清液經冷凍乾燥後由乙醇在 60 ℃ 下萃取,使用矽膠管住層析分得 14 個分液,在第 4 個分液有最佳的 DPPH 自由基清除活性,接著將此分液進一步以 HPLC 分離純化後能得到色胺酸及尿黑酸。比較 TKU036 發酵不同碳/氮源之上清液,及發酵前後 SPP 培養基上清液的 HPLC 指紋圖譜,發現 TKU036 只有以烏賊軟骨作碳/氮源發酵才能有效生產色胺酸及尿黑酸,而此 2 化合物原本是不存在於未發酵之培養基。
英文摘要 Antioxidants can scavenge reactive oxygen species. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) may be added to food products to retard oxidation reactions. Though synthetic antioxidants are effective and inexpensive compared to natural ones, however, their applications are limited because many are suspected to be carcinogenic.
Paenibacillus sp. TKU036 using squid pen powder (SPP) as carbon/nitrogen source culturing at 37℃ 3 days shows the highest DPPH radical scavenging, this supernatant extracted by ethanol at 60℃, then obtained 14 fraction by silicone chromatography. The 4th fraction exhibited the highest DPPH radical scavenging activity, then the 4th fraction was further purified by HPLC and got tryptophan and homogentisic acid. Comparing the HPLC fingerprint of different carbon/nitrogen source fermented by TKU036, and the result shows that only TKU036 using squid pen as carbon/nitrogen source can effectively produce tryptophan and homogentisic acid, and these 2 compounds are not originally present in the unfermented SPP.
論文目次 目錄
簽名頁
授權書
中文摘要 I
英文摘要 II
目錄 III
表目錄 VI
圖目錄 VII
縮寫表 IX
第一章 緒論 1
第二章 文獻回顧 4
2.1多黏芽孢桿菌(Paenibacillus sp.)之簡介 4
2.2 自由基 5
2.3 抗氧化物 6
2.4 DPPH 自由基清除率測定 8
2.5 幾丁質 9
第三章 材料與方法 11
3.1 實驗材料 11
3.1.1 培養基 11
3.1.2 藥品 11
3.2 實驗儀器 11
3.3 生產菌株之篩選與鑑定 13
3.3.1菌株篩選 13
3.3.2 菌株鑑定 13
3.4 DPPH自由基清除能力之測定 15
3.5 TLC 斑點測定法(Dot-blot DPPH staining method on TLC) 15
3.6.1 碳/氮源對抗氧化活性之影響 16
3.6.2 烏賊軟骨粉濃度對抗氧化活性之影響 16
3.6.3 培養液體積對抗氧化活性之影響 16
3.7 萃取、分離與純化 17
3.7.1 乙酸乙酯、丁醇萃取,薄層層析分離 17
3.7.2 熱乙醇萃取,高效液相層析純化 17
第四章 結果與討論 18
4.1菌種鑑定 18
4.2 DPPH 自由基清除能力之測定 20
4.2.1碳/氮源之探討 22
4.2.2濃度之探討 23
4.2.3培養體積之探討 24
4.3萃取、分離與純化 25
4.3.1 乙酸乙酯、丁醇萃取,TLC 分離 26
4.3.2熱乙醇萃取,HPLC分離 30
4.4 結構解析 40
4.4.1 色胺酸(BuOH-2, 4-5)之結構解析 41
4.4.2 尿黑酸(4-4)之結構解析 47
4.5 抗發炎測定 51
4.6 色胺酸 52
4.7 尿黑酸 54
參考文獻 59


表目錄
表 2.1 微生物來源之抗氧化物 7
表 2.2 微生物以SPP為發酵源之產物 10
表4.1 Paenibacillus sp.TKU036之16S rDNA 部分基因序列 18
表4.2 TKU036之API 50 CHB 鑑定結果 19
表 4.3 比較 TKU036 與其他微生物之抗氧化活性培養條件 21
表4.4 丁醇萃取物與BuOH-1、BuOH-2 DPPH 自由基清除率 29
表 4.5 4-5 NMR 光譜資訊 42
表 4.6 4-4 NMR 光譜資訊 48
表 4.7 4-4、4-5之抗發炎活性 51
表 4.8 微生物來源之色胺酸與應用 53
表 4.9 微生物來源之尿黑酸 54


圖目錄
圖 2.1 DPPH 結構 8
圖 2.2 抗氧化物清除 DPPH 自由基之反應 8
圖 2.3 幾丁質結構圖 10
圖 4.14乙醇萃取物之 HPLC 圖譜(292 nm) 38
圖 4.15 TKU036 發酵不同碳源之 HPLC 指紋圖譜比較 39
圖 4.16 色胺酸結構 40
圖 4.17 尿黑酸結構 40
圖 4.18 4-5 HMBC關聯性 42
圖 4.19 BuOH-2 之1H-NMR 圖譜(methanol-d4, 600 MHz) 43
圖 4.20 4-5之1H-NMR 圖譜(D2O, 600 MHz) 43
圖 4.21 BuOH-2 之13C-NMR 圖譜(methanol-d4, 150 MHz) 44
圖 4.22 4-5之13C-NMR 圖譜(D2O, 150 MHz) 44
圖 4.23 BuOH-2之 HSQC 圖譜(methanol-d4) 45
圖 4.24 4-5之 HSQC 圖譜(D2O) 45
圖 4.25 BuOH-2之 HMBC 圖譜 46
圖 4.26 4-5之 HMBC 圖譜(D2O) 46
圖 4.27 4-5 HMBC關聯性 48
圖 4.28 4-4之1H-NMR 圖譜(methanol-d4, 600 MHz) 49
圖 4.29 4-4之13C-NMR 圖譜(methanol-d4, 150 MHz) 49
圖 4.30 4-4之 HMQC 圖譜(methanol-d4) 50
圖 4.31 4-4之 HMBC 圖譜(methanol-d4) 50
圖 4.32 色胺酸甲氧基引朵代謝途徑 52
圖 5.1 酪胺酸降解途徑 57
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