The Synthesis Of Phytosterol Acetate Using Enzymatic Method And Structure Characterization

Phytosterols were obtained from by-product of refining vegetable oils, and are activesubstances with health care functions. But the utilizations of free phytosterols were limitedbecause of the insolubility in water and edible oils and fats. The synthesis of phytosterolsacetate can greatly improve their oil-soluble, and the activity have not been changed, the useof phytosterols were facilitate better in food industry, which were studied recently. In thisresearch, with the presence of the lipase lipozyme435catalyst, phytosterol esters (PE) weresynthesized by esterification of phytosterols and pyruvic acid. The yield of phytosterols esters(PE) was taken as the index of investigation. The optimum technology, the separation andpurification, the structural characteristics, the oil-soluble application were also studied in thisdissertation. The main conclusions are shown as follows:The selection of reaction solvent, the reaction temperature, substrate molar ratio ofphytosterols and pyruvic acid, enzyme amount, and reaction time were investigated for theyield of esters. The optimum technological factors were obtain by the orthogonal test asfollows: n-hexane as the better reaction solvent, the reaction temperature55℃, the substratemolar ratio of phytosterols and pyruvic acids1:5, enzyme amount (relative to the quality ofpyruvic acid)5%, molecular sieves amount40mg/mL, the reaction time18h, and the hightestyield93.01%.The optimum solvents of TLC were n-hexane, anhydrous ether, and glacial acetic acid,and the ratio of them were75:25:0.5, in the condition, the Rf of phytosterols and their esterswas0.35and0.78.The separation and purification of phytosterols esters was discussed by thecrystallization with organic solvent and the silica gel column chromatography. The organicsolvent of the crystallization was ethanol, and the optimum solution temperature was66℃and the optimum amount of55mL/g. The crude products were purified by silica gel columnchromatography, flow rate and the quality of the crude products were taken as the index forstudied, the optimum conditions of purification were showed: flow rate0.5mL/min, thequality of the crude products0.8g, the purity and recoveries of phytosterols acetate were97.3and75.2.Phytosterols acetate and its standard products were analyzed by TLC, the synthesis of phytosterols esters was phytosterols acetate by preliminary determination; Phytosterolsacetate was characterized by fourier transform infrared (FT-IR). The result of FT-IR showedthat the characteristic absorption peak of-OH (near3400cm-1) was disappeared, and twostrong absorption peaks were found near1726cm-1and1244cm-1, which were characteristicabsorption peaks of the C=C double bond and C-O-C. The above showed-OH was esterifiedand ester bonds were obtained in the detected products; Then phytosterols acetate wasproved by the results of GC-MS. The signals at m/z396,382,380and394were thecharacteristic fragment of β-sitosterol, vegetable oil sterol, rapeseed sterol and stigmasterol.This characteristic ion fragments were consistent with other relevant reports. Thecharacteristic peak of phytosterols esters can be seen indicated a high level of purity ofphytosterols acetate by separation and purification.The oil-soluble application of phytosterols and phytosterols acetate was discussed atlast. It was studied on different temperature and four kinds of oil and fats, the result was thesolubility of phytosterols acetate in oils was3times higher than that of phytosterols.Phytosterols and phytosterols acetate had different solubility in different oils under the sameconditions, the maximum solubility in corn oil, soybean oil, peanut oil and the minimum insunflower oil.