Enzymatic Synthesis Of Carboxylic Ester’ Fragrance And Sugar Ester With Immobilized Candida Sp. 99-125

Food additives are important in food industries, they can improve the color, flavor, and taste of the food. Food additives can be divided into many types through different rules. Nowadays, enzymatic catalyzed food additives are studied widely because the products synthesized by chemical way have some shortcoming. In this study, self-established immobilized lipase Candida sp.99-125 was employed to catalyze the production of carboxylic ester and sugar ester, and the factors during the catalyzed process including solvent, enzyme amount, molecular sieves and pretreatment of lipase were investigated, the purification of the sugar ester was also studied. The results were as follows:1、The immobilized lipase Candida sp.99-125 catalyzed synthesis of ethyl hexanoate in solvent was studied. The effects of some factors on the conversion of hexanoic acid were investigated, the solvent, the amount of lipase, the concentration of acid, temperature. The reaction was performed in 5ml solvent system, the optimal reaction conditions were reaction temperature 40℃, shake speed 150r/min, hexanoic acid concentration 0.3 M, the hexanoic acid/ethanol ratio 1:1.6 and the amount of lipase 0.123g, certain amount of silica gel was added to the reaction medium as the water absorbent. The conversion of the acid reached 96% after reaction for 8 h under the optimal conditions. The stability of the immobilized lipase was also studied, and the conversion of the acid remained above 75% after 3 batches.The control to the reaction was studied, such as stepwise added the ethanol and pretreated the enzyme with substrate. The results showed that it’s effective to stepwise add the ethanol in high acid concentration, but the effect of pretreatment to enzyme was not obvious.2、In the synthesis of short alcohol and short acid, the conversion of the acid increased with the carbon number of the acid. When the acid carbon number was less than three, the effect of the acid to the reaction was more significant than the alcohol, however, when the acid carbon number was more than three, the effect of alcohol to the reaction was more important than the acid, acetic acid and ethanol had inhibition to enzymes. In solvent-free system, propyl caproate, butyl hexanoate, amyl caproate and hexyl caproate can be synthesized.3、In this paper, analytical method including TLC、GC、HPLC-ELSD was studied to find a proper method for sugar ester. The result showed that TLC was a quick qualitative method, toluene:ethyl acetate:methanol: water=10:5:4.5:0.2 (V/V/V/V) was preferred developing solvent. GC method was relatively complicated, sugar ester must be derivated before being analyzed. HPLC-ELSD was fit for sugar ester quantitative analysis, the mobile phase was methanol, flow rate 1mL/min, drift temperature of ELSD was 40℃, carrier gas flow rate 2L/min. Under the condition, every component in the reaction including fructose, fructose monolaurate, lauric acid, fructose dilaurate could be divided effectively.4、In the present work, immobilized lipase catalyzed fructose monolaurate in organic solvent was studied. Using immobilized lipase Candida sp.99-125, the effects of different solvents, temperature, enzyme amount, molar ratio of acid/fructose, molecular sieves on reaction, and different kinds of lipase pretreatment were investigated. T-butanol was selected as the reaction medium. The optimal conditions for fructose monolaurate synthesis were:reaction temperature 45℃, enzyme amount 25 g/L, molar ratio of acid/fructose 4:1, molecular sieves 60 g/L. The concentration of the fructose monolaurate was above 30 g/L. The effect of salt solutions on the lipase activity showed that (NH4)2SO4 (1mM) solution was helpful to the immobilized lipase.5、The separation and purification conditions were studied, two methods were used:extraction and column chromatography. The extraction conditions were optimized:24% NaCl solution was used to remove the residual sugar in the reaction system under 40℃, saturated solution was used to remove residual lauric acid. The mobile phase of column chromatography was toluene:ethyl acetate:methanol: water=10:5:4.5:0.2 (V/V/V/V), the optimal load was 3g.