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研究生:楊淳皓
研究生(外文):Chun-Hao Yang
論文名稱:以流式細胞儀及系統生物學分析山胡椒超臨界流體萃取物之抑菌機制
論文名稱(外文):Use Flow Cytometry and Systemic Biology Approach to Analyze the Bacterial Inactivation Mechanism of Litsea cubea
指導教授:徐源泰徐源泰引用關係
指導教授(外文):Yuan-Tay Shyu
口試委員:林長平曾文聖吳思節
口試委員(外文):Chan-Pin Lin
口試日期:2013-07-11
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:148
中文關鍵詞:Bacillus subtilis ATCC 6633Escherichia coli ATCC 8739流式細胞儀STITCH 3.1Litsea cubeba
外文關鍵詞:Bacillus subtilis ATCC 6633Escherichia coli ATCC 8739Flow cytometerSTITCH 3.1Litsea cubeba
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山胡椒Litsea cubeba,為台灣原住民常用之食用香料,添加於飲食中增加風味並且延長保存時間,其抑菌及抗氧化效果具有研究潛力。本實驗使用蒸餾萃取法與超臨界流體萃取法萃取山胡椒中之物質。結果顯示,超臨界流體萃取物 DPPH 自由基清除率 31.51%;總酚類含量達 22.17 mg of GAE/g ,高於其他市售精油。精油與超臨界流體萃取物之最低抑菌濃度皆為 312.5 ppm,低於食品防腐劑。利用流式細胞儀比較二種山胡椒萃取物與四種已知機制之抗生素Penicillin、Tetracycline、Rafipincin、Trimethoprim 在五種濃度下抑制 Bacillus subtilis ATCC 6633、Escherichia coli ATCC 8739 之螢光變化,三種螢光染劑分別為 Propidium iodide (PI)、Bis (1,3-dibarbituric acid)-trimethine oxanol (BOX)、Carboxyfluorescein diacetate (cFDA) 。顯示二種山胡椒萃取物之抑菌方法為破壞細胞外壁,改變通透性與電位來抑制細菌生長,但較無法明顯分辨從內部抑制之原因。進一步利用氣相層析質譜儀分析二萃取物組成,山胡椒精油與超臨界流體萃取物中可分析出30與44種物質;二者共同組成比例佔精油之92.17% ,僅佔超臨界流體萃取物 73.94%,顯示後者能夠取得更多種化合物,這些化合物大部分屬於揮發性較低之長碳鏈有機物;精油組成分與前人研究類似。以 STITCH 3.1 模擬分析;發現山胡椒精油之成分與 B. subtilis 及 E. coli 內部多種蛋白相關聯,影響細胞之氧氣運輸感知與催化、DNA損傷偵測與移除、胺基酸與乳酸之代謝等;山胡椒超臨界流體萃取物則與脂肪酸之生合成與代謝相關,此結果與流式細胞儀觀察相符合。結合流式細胞儀比對與 STITCH 等創新方法可以快速掌握複雜組成物之植物萃取物功用,嘗試解決植物組成複雜且不易比較之問題;未來應用此方法可減少探索成本,增加尋找有效物質之機會。

Litsea cubeba is a plant frequently used as spice by Taiwanese aborigines as flavoring agent or food preservatives. Recently, its noticeable antibiotic and antioxidant properties have attracted much attention for scientific research. In this study, we employed steam distillation and supercritical fluid extraction techniques to extract compounds from L. cubeba. The supercritical extract of L. cubeba has a DPPH clearance rate of 31.51% and a total phenol content up to 22.17mg GAE/g, a value higher than other essential oil on the market. The minimum concentration of both essential oil and supercritical extract of L. cubeba capable of inhibiting cell growth is 312.5ppm. Flow cytometer is used to measure the effectiveness of L. cubeba extracts on the inhibition of Bacillus subtilis ATCC 6633 and Escherichia coli ATCC8739, the results were then compared in together with Penicillin, Tetracycline, Rafapincin, and Trimethoprim, at 5 different concentrations. Associated fluorescent signal changes were detected by fluorescence-based cell counter. The three fluorescent molecules used in this study were Propidium iodide (PI), Bis (1,3-dibarbituric acid)-trimethine oxanol (BOX), and Carboxyfluorescein diacetate (cFDA). Experiments have confirmed that both extracts of L. cubeba may disrupt the integrity of cell wall and further on change cell permeability and electrical potential, thereby inhibiting cell growth. Yet, it is unclear how such growth inhibition occurs at the molecular level. Further analysis of L. cubeba extracts by Gas-Chromatography-Mass Spectrophotometer, followed by simulation analysis using STITCH 3.1, has identified 30 and 44 compounds in L. cubeba essential oil and supercritical fluid, respectively. The common components of both extracts account for 92.17% of essential oil but only 73.94% of supercritical fluid extract, clearly demonstrating that the later method extracts far more compounds than the former. Based on possible interactions between of the chemical components of L. cubeba and endogenous proteins in B. subtilis and E. coli, it can be postulated that L. cubeba affects cell’s oxygen transport and sensing, enzyme catalysis and electron transfer, DNA damage detection and removal, and metabolism of amino acids and lactic acid. Supercritical extract of L. cubeba was found closely associated with both the synthesis and metabolism of fatty acids and lipids. Further application of flow cytometer, it is now workable for rapid identification of useful chemical compounds in plants.

中文摘要............................................ i
英文摘要............................................ ii
目錄............................................... iv
圖目錄.............................................. viii
表目錄.............................................. xi
第一章 前言.......................................... 1
第二章 前人研究……….................................... 3
第一節 山胡椒介紹..................................... 3
第二節 超臨界流體萃取技術.............................. 4
一、超臨界流體萃取原理................................. 4
二、超臨界流體萃取技術的利基............................ 4
三、超臨界流體萃取技術的應用............................ 5
第三節 流式細胞儀..................................... 6
一、流式細胞儀發展.................................... 6
二、流式細胞儀原理..................................... 7
三、流體學系統........................................ 8
四、光學系統.......................................... 8
五、訊號分析與展示..................................... 9
第四節 流式細胞儀與植物萃取物抑制微生物之研究............... 10
一、探討植物精油抑菌之研究.............................. 10
二、流式細胞儀於微生物抑制偵測之研究...................... 10
三、模式菌株......................................... 11
四、抗生素........................................... 12
五、螢光染劑.......................................... 13
第五節 系統生物學工具STITCH ........................... 14
第六節 研究目的....................................... 14
第三章 實驗材料與方法.................................. 34
第一節 菌種之活化與保存................................ 34
一、菌株活化與保存.................................... 34
二、生長曲線......................................... 36
第二節 山胡椒抑菌物質萃取.............................. 37
一、山胡椒精油萃取.................................... 37
二、山胡椒超臨界流體萃取物萃取.......................... 38
第三節 山胡椒萃取物分析................................ 39
一、密度測量......................................... 39
二、色彩空間......................................... 39
三、清除DPPH自由基之能力.............................. 40
四、總酚類含量測定.................................... 41
第四節 傳統抗菌能力檢測................................ 42
一、紙片擴散測試...................................... 42
二、最低抑制濃度...................................... 43
三、最低殺菌濃度...................................... 44
第五節 流式細胞儀分析................................. 45
一、抑菌物質接觸..................................... 45
二、PI螢光染色...................................... 46
三、BOX螢光染色...................................... 47
四、cFDA螢光染色..................................... 47
五、統計分析......................................... 48
第六節 氣相層析質譜鑑定萃取組成物…....................... 50
一、樣本前處理........................................ 50
二、分析條件......................................... 50
三、資料解讀與整理.................................... 51
第七節 系統生物學方法................................. 53
第八節 穿透式電子顯微鏡............................... 55
一、細菌處理與固定.................................... 55
二、 樣本脫水與滲透................................... 57
三、 樣本包埋與聚合................................... 58
四、 樣本後續處理與電子顯微鏡拍攝........................ 59
第四章 結果與討論.................................... 66
第一節 菌株培養條件................................... 66
一、生長曲線......................................... 66
第二節 山胡椒萃取..................................... 67
一、山胡椒精油產出結果................................. 67
二、山胡椒超臨界流體萃取物產出結果....................... 67
第三節 山胡椒萃取物分析................................ 68
一、密度測量......................................... 68
二、色彩空間......................................... 68
三、清除DPPH自由基之能力............................... 68
四、總酚類含量測定.................................... 69
第四節 傳統抗菌能力檢測............................... 70
一、紙片擴散測試..................................... 70
二、最低抑制濃度/最低殺菌濃度........................... 71
第五節 流式細胞儀分析................................. 73
一、PI螢光染色....................................... 74
二、BOX螢光染色...................................... 75
三、cFDA 螢光染色.................................... 77
四、統計分析......................................... 79
第六節 氣相層析質譜鑑定結果............................ 81
第七節 系統生物學分析................................. 83
一、共同成分......................................... 83
二、山胡椒精油及其特有成分.............................. 84
三、山胡椒超臨界流體萃物及其特有組成分.................... 85
四、整合歸納........................................ 87
第八節 穿透式電子顯微鏡............................... 89
第五章 結論......................................... 136
參考文獻............................................ 137



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