Studies On The Immoblization Of PEL And Enzymatic Preparation Of1,3-DG

Diacylglycerol (DG) is a kind of structured lipid that hydroxyl replace acyl in the sn-1,2,3position of triacylglycerol(TG). DG is a natural minor component of various edible oils and the endogenetic intermediate metabolite of lipid. Many researches have shown that dietary DG can reduce visceral fats and blood lipid and suppress the increase of body weight. At the end of2000, DG has received GRAS (generally recognized as safe) status from the U.S.Food and Drug Administration (FDA). A kind of analysis method for glyceride comprising DG was set up in this thesis. The method of immobilizing free penicilium expansum lipase(PEL) and the preparation, purification of DG were studied in the present study.Firstly, In this research a method with gradient elution for the separation and determination of diacylglycerol by HPLC-ELSD was developed. In this study, Thermo4.5μ×250mm silica gel column was adopted, Quantitative determination was carried out by using external reference method.The results of0.05～0.25mg/mL samples fit linear relationship very well.The RSD of the results was1.12%. The average recovery rate was99.75%.Secondly, the method of immobilizing free PEL were studied. In this experiment, taking alginate as the carrier, penidilum expansuin lipase (PEL) was immobilized through crosslinking absorption method. The influence of main immobilization elements put on the activity of immobilized enzyme was tested, and the fixed condition was optimized through single-element experiment and orthogonal experiment. The results indicated that, under the density conditions of3%alginate,1%calcium chloride and2.5%glutaric dialdehyde, the highest activity of penidilum expansuin lipase immobilized is2000U/g, with0.03g/mL enzyme density,10.0pH,60℃temperature and then fixed30min.In organic systems, the esterification ability of immobilized PEL is much stonger than that of free lipase. After7times of repeated use, the activity of the immobilized lipase still kept50%of its original activity.The fixed condition which was optimized through single-element experiment and orthogonal experiment after the enzymatic hydrolysis test on bean oil by using immobilized lipase enzyme are as follows:water content of bean oil is2%, dosage of immobilized lipase is80U/g substrate, the spead of stirring is400r/min, reaction temperature is40℃, reaction time is8hours. The1,3-DG and1,2-DG content in the yield were18.5%and12%.By two-stage molecular distillation, the purity of DG product was increased from18.5%to89%and the yield and recovery of DG product were30.4%and88.7%respectively. Moreover, the physiochemical properties of DG product were determined and the results showed that the POV, AV and color of DG product were similar to the national standard of rapeseed salad oil, however, the smoking point of DG product was below to220℃.