Lipase-catalyzed Synthesis Of Phytosterol Esters

Phytosterols(plant sterols)are active substances with health care functions.The utilizations of free phytosterols were limited because of the poor solubility in water and oil Phytosterol esters are generally prepared from phytosterols and fatty acids by esterification or transesterification reactions.Phytosterol esters have better lipophilicity and better cholesterol-lowering effect than plant sterols,and have nutritional value of phytosterols and fatty acids.Phytosterol esters are active substances with health care functions and used widely.Soybean oil deodorant distillate(SODD)is a by-product of vegetable oil refining process,used as a waste for the feed industry.SODD contains a variety of biological activity of nutrients,such as free fatty acid,natural vitamin E,phytosterols and glyceride et al.Therefore,it is great significance to extract phytosterols and fatty acids from SODD and to synthesize phytosterol fatty acid esters by enzymatic method.In this paper,the reaction system of enzymatic synthesis of phytosterol esters in nonaqueous system was constructed,and the synthesis process was optimized.Phytosterols and fatty acids were extracted from SODD.Phytosterol fatty acid esters were synthesized according to the optimized process and the qualitative analysis was carried out.The main contents are as follows:Firstly,lipase and organic solvent were screened and the result showed that the lipase from candida rugosa could efficiently catalyze the synthesis of phytosterol esters.In the solvent of n-hexane,the esterification degree was higher than that in other solvents.The product was analysis by TLC and FT-IR.The TLC showed that there are new product appeared.The FTIR showed that a absorption peak at the wavelength of 1743 cm-1 was obvious,it was characteristic absorption peak of' ester-bonds.The result showed that the phytosterol esters were obtained.Secondly,molar ratio of oleic acid to phytosterol,enzyme amount,reaction temperature,and reaction time were investigated for the degree of esterification.The reaction conditions were optimized using response surface methods(RSM)measured by the degree of esterification.The results demonstrated that the optimum conditions were substrate molar ratio(oleic acid:phytosterol)of 3:1,catalyst amount of 7.2%,reaction temperature of 45.3? and reaction time of 25h.Under the optimized reaction conditions,the DE of phytosterol with oleic acid reached 71.95%.Thiredly,the lipase from Candida rugosa was immobilized by simple absorbion,the esterification degree and enzyme activity as an index.The results showed when CRL lipase was immobilized in phosphoric acid buffer solution and n-hexane,the former was better.Several kinds of carriers were screened and macroporous adsorptive resin HP20 was selected as the carrier immobilizing lipase.Finally,crystallization of sterols with the solvent has been studied.The optimal conditions for crystallization were as below.The optimum valume ratio of acetone to methanol in mixed solvent was 0.5,the proportion of the solvent to crystallization material was 3(volum to weight),the time for cooling was 18 h.For washing the crude sterol,the washing temperature was 25?,the washing time was 15min.Then,the fatty acids were extracted from the filtrate after extracting phytosterol.The synthesis of phytosterol fatty acid esters were carried out according to the optimized process.The product was analyzed by TLC and FT-IR.The TLC showed the production of new species.The FTIR showed that a absorption peak at the wavelength of 1737 cm-1 was obvious,it was characteristic absorption peak of ester-bond.The result showed that the phytosterol fatty acid esters were obtained.