Synthesis And Kinetics Of Plant Sterol Esters Catalyzed By Lipase In Non-aqueous Media

As a novel food with significantly physiological value, plant sterol ester has been widely used in food, medicine and cosmetic. Currently, there were bio-synthesis method and chemical method for producing plant sterol ester. The bio-synthesis method has some advantages, such as high efficiency, mild conditions, less by-products and high safety of the product. In this paper, synthesis of plant sterol ester with lipase in non-aqueous media and its kinetics mechanism were investigated.Proper lipase and the non-aqueous solvent were screened and the result showed that the lipase from candida rugosa could efficiently catalyze the synthesis of plant sterol ester. In the solvent of n-hexane with logP 3.5, the esterification degree was higher than other solvents. The effect of water activity on the esterification degree was investigated, Na2SO4/ Na2SO4·10H2O was selected to control water activity of the reaction system, the amount of adding was 25 mg/mL. Water activity of the system was 0.86, which was the optimum parameter for lipase catalyzed synthesis of plant sterol ester.The effects of reaction temperature, reaction time, lipase load, the ratio of fatty acid to plant sterol and adding amount of solvent on the esterification degree were investigated. Through single factor test and orthogonal design experiment, the optimum condition for lipase catalyzed synthesis of plant sterol ester was obtained. The optimum condition was amount of catalyst 11%, reaction time 48 h, reaction temperature 45℃, adding amount of solvent 6 mL/g, the ratio of fatty acid to plant sterol 3:1, the degree of esterification reached 94.62%.The product of the reaction was purified, and the structure of purified product was identified. After surplus fatty acid was removed, the sample was fractionated by the silica gel chromatography. The separation conditions were as followed: mobile phase cyclohexane/ ethyl ether (19:2 V/V), flow rate 30 mL/h, eluent collect 10 min/tube. After separation and purification, the purity of the product reached 98.2% and the purified product was identified as plant sterol esters by TLC, HPLC, LC-MS and IR.The kinetics and mechanism were studied by fixing one substrate concentration and varying another substrate concentration, and the change of initial reaction rate with different substrate concentration was determined. The experiment results supported the Ping-Pong Bi-Bi mechanism with double competitive. The initial reaction rates obtained at various acid and sterol concentrations were fitted to Michaelis - Menten equation by non-linear regression, the kinetic parameter values were obtained, and the kinetic equation for lipase catalyzed synthesis of plant sterol ester in non-aqueous was established.