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研究生:陳雅君
研究生(外文):Ya-Chun Chen
論文名稱:金柑酵母菌發酵液主成分及其生物活性分析之研究
論文名稱(外文):Study on the main compounds from yeast fermented kumquat solution and their bioactivity
指導教授:林世斌林世斌引用關係
指導教授(外文):Shih-Bin Lin
口試委員:林恩仕陳莉臻陳怡伶曹博宏林世斌
口試委員(外文):En-Shyh LinLi-Chen ChenYi-Lin ChenPo-Hung TsaoShih-Bin Lin
口試日期:2011-02-26
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:食品科學系碩士班
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:96
中文關鍵詞:柑橘類金柑抗菌抗氧化抗發炎
外文關鍵詞:citruskumquatphenolic compoundsantibacterial effectantioxidationanti-inflammation
相關次數:
  • 被引用被引用:5
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:3
已知金柑 (Fortunella margarita Swingle) 經酵母菌發酵後,其總酚及總類黃酮含量會明顯提升,且其抗氧化能力明顯增加。HPLC 分析顯示金柑發酵液含有三個主要的未知成分 (以 UN1-3 命名之),續以 HPLC 半製備型管柱分別收集此三種成分,並以質譜分析得知 UN1-3 分子量分別為 598.18、446.12 和 446.12 Da。在抑菌測試結果發現 UN1-3 皆有顯著抑制 S. aureus (BCRC 10780 及 BCRC 10781) 和 E. coli (BCRC 10314 及 BCRC 10675) 生長的能力。氧自由基吸收能力 (oxygen radical absorbance capacity, ORAC) 結果顯示 UN1-3 皆具有抗氧化活性,此外細胞抗氧化試驗顯示 UN1-3 可保護 RAW264.7 細胞株免於 AAPH (2,2-azobis (2-amidinopropane) di- hydrochloride) 所誘導之氧化傷害。同時,這些成分亦顯著抑制由脂多醣 (lipopolysaccharide, LPS) 誘發之一氧化氮 (nitric oxide, NO) 的生成。Western blotting 及 RT-PCR 分析顯示 UN1-3 皆具有抑制誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 及其 mRNA 轉錄的活性。然而,根據 RAW-Blue 細胞株結果顯示 UN2 和 UN3 在 50 g/mL 濃度下有抑制轉錄因子 NF-B 活性之效果,而 UN1 並無調控能力。
Kumquat (Fortunella margarita Swingle) contains many bioactive phenolic compounds, which have obvious effects on oxidation restraining. Previous study revealed yeast fermented kumquat juice could significantly improved the total contents of polyphenol and flavonoids and also the antioxidant capacity. HPLC analysis demonstrated three unknown compounds, designated as UN1-3, in the fermented kumquat juice, and these compounds, consequently isolated and collected with a semi-preparative column. Their molecular weight were determined to be 598.18, 446.12 and 446.12 Da, respectively, by liquid chromatography mass spectrometry (LC-MS). Antibacterial test showed that UN1-3 had the ability to inhibit S. aureus (BCRC 10780 and BCRC 10781) and E. coli (BCRC 10314 and BCRC 10675). Oxygen radical absorbance ability (ORAC) showed that these compounds had good antioxidant activity. Besides, in vitro cellular test revealed that UN1-3 could protect cells from the oxidant damage caused by AAPH (2,2-azobis (2-amidinopropane) di- hydrochloride). In addition, they also show significant suppression on the LPS-induced NO in RAW264.7 cell. Western blot and RT-PCR analysis exhibited UN1-3 could effectively suppress the expression of iNOS protein and mRNA, respectively. UN2 and UN3, but not UN1, showed down-regulation on NF-B activity at the concentration of 50 g/mL.
摘要...........................................................................I
英文摘要......................................................................II
目錄 ........................................................................III
表目錄......................................................................VIII
圖目錄........................................................................IX
壹、前言.......................................................................1
貳、文獻整理...................................................................3
一、金柑及其機能性成分.........................................................3
二、柑橘類之酚類化合物.........................................................5
(一) 類黃酮與酚酸..............................................................5
(二) 發酵對酚類化合物含量之影響................................................8
三、發炎反應 (inflammation)....................................................9
(一) 巨噬細胞 (macrophage) 與發炎之相關性......................................9
(二) 脂多醣 (lipopolysaccharide, LPS) 誘導發炎之相關模式......................11
(三) 發炎相關介質.............................................................13
1. 一氧化氮 (nitric oxide, NO)................................................13
2. 細胞激素 (cytokines).......................................................15
3. 花生四烯酸衍生物...........................................................17
(四) 抗發炎藥物 (anti-inflammatory agents)....................................18
四、柑橘類中多酚類之生物活性..................................................19
(一) 抗菌效果 (antibacterial effects).........................................19
(二) 抗氧化作用 (antioxidation)...............................................20
(三) 抗發炎活性 (anti-inflammation)...........................................22
五、酚類化合物之萃取、分離及鑑定方法..........................................24
六、抗氧化及抗發炎評估方法....................................................26
(一) 抗氧化評估方法...........................................................26
1. 化學方法...................................................................26
2. 生物方法...................................................................27
(二) 抗發炎評估方法...........................................................27
1. NO 生成測定................................................................27
2. 發炎相關蛋白質及基因表現...................................................28
參、實驗架構..................................................................30
肆、材料與方法................................................................31
一、實驗材料..................................................................31
(一) 金柑.....................................................................31
(二) 生物性材料...............................................................31
1. 細胞株.....................................................................31
2. 菌株.......................................................................31
(三) 藥品試劑.................................................................31
(四) 引子 (primer)............................................................34
二、儀器設備..................................................................34
三、實驗方法..................................................................35
(一) 金柑酵母發酵液之製備.....................................................35
(二) 金柑發酵液中主成分之前處理...............................................36
(三) 高效能液相層析 (high performance liquid chromatography, HPLC)............36
1. 分離成分之 HPLC 分析.......................................................36
2. HPLC 半製備型管柱之分離純化................................................37
(四) 分離成分之質譜分析 (mass spectrometer, MS)...............................38
(五) 生長抑制曲線測試.........................................................38
(六) 氧自由基吸收能力 (oxygen radical absorbance capacity, ORAC)..............39
(七) RAW264.7細胞氧化傷害試驗.................................................39
1. 細胞培養 (cell culture)....................................................39
2. 細胞存活率測定 (MTT 試驗)..................................................40
3. 細胞氧化傷害試驗...........................................................41
(八) NO 生成分析..............................................................41
(九) 反轉錄-聚合連鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR).......................................................................42
1. 總 RNA (total RNA) 之抽取..................................................42
2. 反轉錄酵素反應 (RT-PCR)....................................................43
3. 聚合酶連鎖反應 (polymerase chain reaction, PCR)............................43
4. DNA 膠體電泳 (electrophoresis).............................................44
(十) 西方墨點法 (Western blotting)............................................44
1. 蛋白質之萃取...............................................................44
2. 蛋白質定量.................................................................45
3. SDS 膠體電泳 (SDS-PAGE) 分析...............................................45
4. 轉印及免疫偵測.............................................................46
(十一) NF-B 報導基因 (reporter gene) 表達之分析.............................47
(十二) 統計............................................................................47
伍、結果與討論................................................................49
一、金柑酵母菌發酵液之主成分分離..............................................49
二、分離成分之抗菌能力分析....................................................51
三、分離成分之抗氧化活性......................................................54
四、分離成分之抗發炎活性......................................................56
陸、結論......................................................................61
柒、參考文獻..................................................................62
捌、圖表......................................................................80
玖、附錄......................................................................94
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