The Application Of Phospholipase A₂ And The Establishment Of A Novel Method For Extraction Of Statins

This paper including two aspects:1.High-yield fermentation of phospholipase A₂ by Streptomyces and its application in the oil degumming and phospholipid modification;2.a novel dispersive liquid-phase extraction for the determination of statins.1.The fermentation conditions of Streptomyces phospholipase A₂ was optimized in the 50 L fermentor.The results showed that ventilation 0.28 vvm, rotation speed 300 rpm before the 16h,and adjusted speed to 50 r/min after 16 h,the fermentation cycle was 36 h,the highest activity was obtained,and the fermentation period was shorter.The experiments of vegetable oil degumming by phospholipase A₂ throw the design of single factor and orthogonal experimental.And the results showed that added 0.065%of citric acid in 80℃,500r/min handle 0.5 h in the early period.Then enzyme was added 90 uL/kg,pH6.0,the water content of 2%,reaction temperature 40℃,reaction time 3 h,the final phosphorus content was less than 10 mg/kg.In the pilot study,the vegetable oil through the enzyme treatment,decolorization and deodorization to reach the standard level of edible oil.Enzymatic degumming was more environmentally friendly and economic.The best hydrolysis of soybean phospholipid conditions were as follows: substrate concentration at 10%,reaction temperature 40℃,initial pH value of 7.0, Ca²⁺ concentration of 5 mM,reaction time 20 h.At the best reaction conditions,the release of fatty acids was 134.7μmol/g phospholipid.After that,lysophospholipids emulsifying capacity,emulsion stability significantly stronger than phospholipid.2.A fast and novel sample preparation procedure:ultrasound assisted ionic liquid dispersive liquid microextraction for concentration of lovastatin and simvastatin in aqueous samples was developed.An ionic liquid（[C₆MIM][PF₆]） was used as the extraction solvent,and the factors influencing the extraction efficiency such as initial temperature,the volume of ionic liquid,pH of water samples,cooling time and salt concentration were optimized.In combination with HPLC-UVD,both lovastatin and simvastatin exhibited good linear range of 1-100 ng mL^-1.The limits of detection were 0.17ng mL^-1 and 0.29ng mL^-1,respectively.Precisions of proposed method（RSDs,n=9） were 4.12%and 4.52%,respectively.This method was successfully applied in the determination of three real water samples,and good spiked recoveries were obtained at the range of 90.0-102.2%for lovastatin and 80.5-112.0%for simvastatin.These results indicated that ultrasound assisted ionic liquid dispersive liquid phase microextraction would have good application prospect in pretreatment of environmental samples.