Rapid Determination of Surfactin in Microbial Fermentation Broths using Salt-Assisted Homogeneous Liquid-Liquid Microextraction Technique combined with HPLC-UV

• Main Authors: Yi-Chen Du, 杜怡真
• Other Authors: Jen-Fon Jen
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/14151499896067052723

碩士 === 國立中興大學 === 化學系所 === 100 === 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.