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標題: 以鹽輔助均相液液微萃取技術結合HPLC-UV快速偵測微生物發酵培養液中的表面素
Rapid Determination of Surfactin in Microbial Fermentation Broths using Salt-Assisted Homogeneous Liquid-Liquid Microextraction Technique combined with HPLC-UV
作者: 杜怡真
Du, Yi-Chen
關鍵字: Surfactin
Microbial fermentation broth samples
Salt-assisted homogeneous liquid-liquid microextraction
出版社: 化學系所
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摘要: 本研究開發出以鹽輔助均相液液微萃取之樣品前處理技術,並以HPLC-UV分離偵測bacillius subtilis CWS1微生物發酵培養液中的表面素(surfactin)。表面素為一種生物界面活性劑,具有高表面活性、易被生物降解暨對環境友善等優點,因此在未來有極大潛力取代合成的化學界面活性劑成為產品化商品。在產品量化生產過程中,產品的穩定性影響其有效性,為了能監測微生物培養液中表面素的濃度,快速地分析表面素技術是十分重要的。本研究使用實驗室自行設計的玻璃萃取裝置,將與水互溶的有機溶劑及稀釋過的培養液放入萃取裝置之後,加入鹽類並攪拌使溶液達飽和,之後將裝置至於離心機離心,最後取上層液打入HPLC進行分析。在鹽輔助均相液液微萃取技術中,針對萃取溶劑、萃取溶劑體積、添加的鹽類、硫酸銨鹽添加量、及水樣的pH值等參數進行探討。實驗結果顯示,5毫升經pH2的緩衝溶液稀釋的培養液加入300μL的Acetonitrile及4克硫酸銨鹽,攪拌之後離心1分鐘會有最好的萃取效果。本方法測得培養液中表面素的線性範圍為2-250 mg/L ,線性相關係數介於0.9970-0.9994之間,應用培養液在真實樣品中的回收率為77.7%~106.7%。此方法對於偵測微生物發酵培養液中的表面素是一個簡單、快速、省錢、對環境友善、及少基質干擾的分析方法。
In this study, a simple, rapid and efficient sample pretreatment method has been developed, termed new in-tube salt-assisted homogeneous liquid-liquid microextraction (SHLLME) followed by high performance liquid chromatography/ultra-violet detection (HPLC/UV) for the determination of surfactin in microbial fermentation broths of bacterium bacillius subtilis CWS1. Surfactin is one of a biosurfactant and it has the potential to replace synthetic chemical surfactant in the near future because of its high surface activity, biodegradability and environmental-friendly nature. In order to characterize the amount of surfactin for the microbial monitoring of fermentation production process, we have demonstrated a new method to analyze surfactin using a home-made extraction device. In this method, an extractant (water-miscible organic solvents) and salt were added to broth sample in an extraction tube. Then the device was sealed with plunger cork and taken for stirring and centrifugation processes. After that, the amount of separated extractant was measured using self-scaled glass capillary tube which was in-printed in the extraction device. Then the collected extractant was injected directly into HPLC-UV for analysis. Parameters influencing the extraction efficiency of SHLLE-HPLC/UV, such as the extraction solvent, solvent volume, pH, and salt-addition were thoroughly investigated and optimized. Under the selected experimental conditions, surfactin was extracted from 5 mL broth sample (at pH 2) by the addition of 300 μL acetonitrile (extractant) and 4 g ammonium sulfate, and followed by stirring for 5 min, and then centrifugation for 1 min at 3200 rpm, yields the maximum extraction efficiency. The proposed method provides a simple, rapid, inexpensive, eco-friendly and less matrix interferences process for the determination surfactin in microbial fermentation broth samples.
其他識別: U0005-0607201217253500
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