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dc.contributorJen-Fon Jenen_US
dc.contributor.authorHsu, Chueh-Hsuanen_US
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dc.description.abstract本研究開發以固相萃取管柱純化bacillius subtilis CWS1微生物發酵培養液中的表面素(Surfactin)之方法,並對表面素之安定性分析進行探討。表面素為一種生物界面活性劑,因為其具有易被生物降解、低臨界微胞濃度暨對環境友善等優點,在未來具有相當大的潛力來取代化學合成的界面活性劑成為商品化產品。在成為商品化產品前,有效地獲得高純度表面素顯得格外的重要。為了能夠穩定且快速地獲得微生物培養液中的表面素,本研究利用商業化的固相萃取管柱,將微生物發酵培養液離心之後,取上層澄清液注入固相萃取管柱中進行吸附,繼以三氟醋酸水溶液進行流洗動作,最後以乙腈有機溶劑進行洗脫動作,將所得洗脫溶液進行濃縮乾燥可得高純度的表面素。研究中使用HPLC-PDA對所得表面素進行純度確效。在以固相萃取純化微生物發酵培養液中的表面素方法中,為獲得最佳純化效率,對固相萃取管柱吸附樣品之體積量、流洗溶液體積、及洗脫溶劑體積等參數進行探討。實驗結果顯示,微生物發酵培養液在4000 rpm 離心5分鐘後,取上層澄清液40 mL,載入固相萃取管柱,使用乙腈 (30%) 和三氟醋酸 (70%) 混合水溶液60 mL進行流洗,再以70 mL乙腈沖提,經濃縮乾燥可得高純度的表面素。以本研究方法進行培養液中添加表面素純化後經計算回收率介於99.4-101.2% 之間,相對標準偏差則介於3.0 -3.6 % 之間。   在表面素安定性分析探討部分,將已純化之表面素溶解於超純水中,對表面素所存在溶液中之pH值穩定性、溫度穩定性、光照穩定性與氧化穩定性進行探討。實驗結果顯示,表面素於pH 6 ~ 8水溶液中處於高穩定的狀態;在溫度穩定性部分表面素中成份B及C於100oC下經過100分鐘後有發生降解現象,表面素A則不受影響;光照實驗中將表面素溶液經紫外光 (254 nm) 光照0.25分鐘即產生降解,經5小時後表面素A已降解殆盡,表面素B及C也分別於6小時後完全分解,然經日光燈光照後表面素濃度並未任何受影響;氧化實驗中添加過氧化氫於表面素溶液中即會造成表面素降解,若灌注空氣於表面素水溶液中則於1週後可發現降解現象。 研究結果顯示,使用固相萃取管柱純化bacillius subtilis CWS1微生物培養液中的表面素,可大幅縮短純化所需時間及去除微生物培養液中生物基質與其他不純物,有效獲取微生物培養液中表面素。且經由一系列表面素安定性分析之探討,有利於未來表面素之產品化。zh_TW
dc.description.abstractSurfactin is a bacterial lipopeptide which is a powerful surfactant and antimicrobial agent. It has the potential to replace the chemical surfactants in the near future because of its biodegradability and environmental-friendly nature. The extraction of surfactin from microbial fermentation broths are the major obstacles to its commercialization. In this study, we have developed a new simple, rapid isolation and purification of bio-surfactin in microbial fermentation broths of bacterium (bacillius subtitus CWS1) using solid phase extraction (SPE) method, and we also studied the isolated surfactins’ stability nature at various conditions. Firstly (in the SPE method), the crude microbial fermentation broth sample was centrifuged to remove the biomass impurities, and then the clear supernatant liquid was taken to SPE column for isolation process, and followed by SPE column rinsing and elution processes. Then, the eluted solvent was concentrated and directly injected into HPLC-PDA for analysis and validation. Parameters influencing the purification and extraction efficiency of SPE process, such as sample pH, sample volume, ratio of organic solvent/rinsing solution, and volume of elution solvent were thoroughly optimized. Under the optimal experimental conditions, the microbial fermentation broth sample (50 mL) were centrifuged for 5 min at 4000 rpm, and then 40 mL of supernatant liquid was taken to solid phase extraction column (C18 SPE) loading and followed by SPE column rising with 60 mL mixture solution (30% ACN and 70% TFA) and then eluted with 70 mL of acetonitrile, which yields maximum isolation of surfactin with high purity. Secondly, the parameters influencing the stability of the isolated surfactins at various conditions, such as pH, temperature, UV-Vis light irradiation, aeration, and oxidation reagents were investigated. Under the experimental conditions, surfactins were showed maximum stability at pH 6~8 aqueous solutions. Surfactin B & C were showed gradual degradation from 100 minutes at 100 oC and surfactin A was stable even after 100 minutes at 100 oC. Under the UV light irradiation, surfactins solution showed gradual degradation (surfactin A showed degradation from 0.01 hour and completed at 5 hours, and surfactin B & C were showed complete degradation at 6 hours). Surfactins solution was not shown any degradation at prolonged light irradiations. Aeration (oxygen,23% and nitrogen) at surfactins solution showed gradual degradation from 1 day to 8 days and kept constant for further increases. The presented method is a simple, rapid, convenient method for the isolation and purification of bio-surfactin in broth sample and also overcomes the interferences of biomass and impurities from the complex microbial fermentation broths. Surfactins stability/degradation studies also prove the possibility of future for surfactins is favorable.en_US
dc.description.tableofcontents謝誌 I 摘要 II Abstract IV 目錄 VI 圖目錄 IX 表目錄 XI 壹、 緒論 1 1.1 前言 1 1.2 表面素的回顧 1 1.2.1 生物界面活性劑 1 1.2.2 表面素 6 1.3 表面素純化方法的回顧 9 1.4 固相萃取 12 1.4.1 C18吸附劑的應用 13 1.5 電化學循環伏安法 15 1.6 研究目的 17 貳、 研究方法 18 2.1 藥品、實驗器材及儀器設備 18 2.1.1 藥品 18 2.2 儀器設備與實驗器材 19 2.2.1 器材 19 2.2.2 培養基和微生物 20 2.2.3 針式玻璃萃取裝置 21 2.3 藥品配製 23 2.3.1 高效能動相沖提液的配製 23 2.3.2 pH2緩衝溶液之配製 23 2.3.3 表面素標準品儲存容易之配製 23 2.3.4 微生物發酵培養液 24 2.4 玻璃器具之矽烷化 24 2.5 儀器操作參數 26 2.5.1 高效能液相層析儀之參數 26 2.5.2 固相萃取管柱純化系統參數 26 2.5.3 微生物發酵培養液中的表面素快速分析系統參數 26 2.5.4 固相萃取管柱純化步驟 27 2.5.5 微生物發酵培養液中的表面素快速分析步驟41 27 2.6 實驗方法 31 2.6.1 固相萃取管柱純化微生物發酵培養液中表面素之條件探討 31 2.6.2 表面素安定性分析之探討 32 2.7 電化學循環伏安法之分析 34 2.8 表面素進行質譜分析 34 2.9 萃取方法之評估 34 參、 結果與討論 35 3.1 微生物發酵培養液中的表面素快速分析 35 3.2 固相萃取管柱純化微生物培養液中的表面素之最佳化探討 35 3.2.1 微生物發酵培養液 pH 值之最佳化 35 3.2.2 流洗溶液體積及溶劑比例之最佳化 37 3.2.3 洗脫溶劑體積之最佳化 39 3.2.4 應證固相萃取管柱純化將不純物與生物基質移除 39 3.3 表面素安定性之探討 41 3.3.1 表面素pH值之穩定性 41 3.3.2 表面素溫度之穩定性 44 3.3.3 表面素光照之穩定性 44 3.3.4 表面素氧化之穩定性 47 3.4 萃取方法之評估 51 3.5 電化學循環伏安法之偵測 51 3.6 固相萃取管柱純化方法與其他純化方法之比較 51 3.7 表面素質譜鑑定 55 肆、 結論 63 伍、 參考文獻 64zh_TW
dc.subjectsolid phase extractionen_US
dc.subjectmicrobial fermentation brothsen_US
dc.subjectthe stability of surfactinen_US
dc.titleRapid Extraction of Surfactin in Microbial Fermentation Broths Using Solid Phase Extraction and Their Stability Studiesen_US
dc.typeThesis and Dissertationzh_TW
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