| Sugar esters are significant nonionic surfactants, which are nontoxic, nonirritant and degradable. They are widely used as emulsifier in the food, cosmetic, pharmaceutical industries. Sugar esters can be produced by enzymatic or chemical methods. However, comparing with the chemical method, enzymatic synthesis can be performed under milder reaction conditions with a higher regioselectivity.In this paper, the thin layer chromatography (TLC) to qualitatively analyze fructose ester was developed. The optimal conditions of thin layer chromatography were as follow: silica gel G as stationary phase, toluene - ethyl acetate - methanol -water(10:5:4.5:0.2,v/v/v/v)) as the mobile phase, coloration by spraying α -naphthol- sulfuric acid. Under these conditions, a good separation was obtained, and the Rf values of fructose mono-palmitate and fructose di-palmitete were 0.20 and 0.56. respectively.A method of the high performance liquid chromatography - evaporative light scattering detector (HPLC-ELSD) quantitative analysis of fructose ester was developed. This method has not been reported before. The optimal HPLC conditions were as follow: Prevail C18 column as the stationary phase, A (99% ethyl acetate and 1% water, v/v)—B (75% ethanol and 25% water, v/v) (60:40, v/v) as the mobile phase, 1mL/min of flow rate, 30℃ of column temperature, ELSD detector. For the fructose mono-palmitate, the linear range is 5 μ g to 40 μ g, the calibration equation isM =2×10-6A-2.3924, the related coefficient is 0. 9948, the RSD is 2. 01%, and the recovery rate is 96. 4%-98. 6%. For the fructose di-palmitate, the linear range is 5 μ g to 40 μ g, the calibration equation is M=1×10-6A - 2.0152 , the related coefficient is 0. 9984, the RSD is 1. 62%, and the recovery rate is 95. 0%-98. 3%.The solvent and water content are key parameters of enzymatic synthesis of sugar ester. In this paper, an excellent strategy was reported, which included thesolvent methyl ethyl ketone and the water removal method using molecular sieve. Molecular sieve can rapidly absorb water in methyl ethyl ketone and assure lower water content in reaction system. Thereby, the chemical equilibrium is impelled toward the synthesis direction and the product concentration increases. Lipase is stable in methyl ethyl ketone.Such parameters of enzymatic synthesis of sugar ester as the quantity of molecular sieve, the quantity of enzyme, substrates and their proportion and temperature were studied. When the quantity of molecular sieve increased to 85g/L, the conversion yield of fructose had reached 95%. The optimum acyl donor is stearic acid. When the acid/fructose rate was 1.5, both substrates had a higher conversion yield. Increasing this rate could result in a faster reaction speed and enhanced the proportion of di-ester in product. However, the conversion yield of acyl donor declined as a result. Higher reaction temperature brought faster reaction speed and different equilibrium constant, and, enzyme's worse stability. At 60 °C, the lipase had a good stability in methyl ethyl ketone.Under the optimum conditions, namely within a methyl ethyl ketone, lOg/L enzyme, 85g/L molecular sieve, 50mmol/L fructose and 75mmol/L stearic acid, the reaction can reach equilibrium in 10 hours, in which the conversion yield of fructose is 97.5% and that of stearic acid is 86%. After ten runs of such reaction, the enzyme's activity only declines 19%.The products are identified by FT-IR and ESI-MS.
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