The Lipase-catalyzed Synthesis Of Fructose Monolaurate And Its Isolation And Surface Activities

Sugar esters are significant non-ionic surfactants which are wildly used in food, cosmetic, pharmaceutical, detergent industries because of high emulsifying capacity, nontoxicity, wide range of HLB value (1-16) and biodegradability.In our previous work, we synthesized fructose monolaurates in mixing solvent (tert-amyl alcohol + DMSO). DMSO was toxic and difficult to separate, which limited the application of product. Due to this, we synthesized fructose monolaurates in the tert-butyl alcohol which is low toxicity and low boiling point. Then, we purified the products and studied its surface properties which lay solid foundations for the industrialized use of fructose monolaurates.In this paper, we used Candida antarctica lipase B which was displayed on the Pichia pastoris surface to catalyze the esterification of fructose and lauric acid in nonaqueous phase. The influential factors on whole-cell synthesis of fructose laurate monoesters were studied, such as the species of enzyme and its freeze-dried protective agent, the type of organic solvent, catalyst dosage, substrate concentration, substrate ratio, temperature, water activity and amount of molecular sieves and other factors on the synthesis of fructose monolaurates. The optimal reaction conditions were: 5 mL total reaction volume, tert-butyl alcohol, 5% (w/w) fructose solution as freeze-dried protective agent, initial water activity of 0.06 GS115/pKNS-CALB whole-cell catalyst 0.3 g, fructose 0.36 g (immobilized by 0.4 g silica-gel powder), lauric acid 0.8 g, 60℃, 200 rpm, The maximum yield could be 60% after 72 h reaction. On the basis of this, we amplified the reaction system and studied the effects of stirring rate and the low perssure on the synthesis reaction of fructose lauric esters. In 200 mL system, the stirring speed 300 rpm was the best. The yield of fructose lauric esters under 200 mbar was nearly 65%, higher than the result which was detected in ordinary pressure.According to the chemical compositions of products and characteristics of fructose monoesters as non-ionic surfactant, a separation process containing two steps was established. Four factors such as n-hexane, crude products, anhydrous ethanol and distilled water in the process of mixed solvent extraction of the first step to remove fructose were studied. The optimum conditions were as follows: crude products 0.7 g, ethanol 0.4 mL, hexane 1 mL, distilled water 0.2 mL, room temperature conditions. The extraction yield of fructose laurate monoesters of was 94%, 98% fructose removed. Based on the first step of extraction, the further separation using mixed solvent to extract fructose monolaurates was studied. The optimal process conditions were as follows: hexane 4 mL, ethanol 4 mL, distilled water 1 mL. The maximum extraction yield of fructose monolaurates was 89%, the extraction purity was 95%.In order to get the pure products, fructose monolaurates were separated and purified by gradient elution column chromatography using hexane / ethyl acetate = 1:1 and ethyl acetate / hexane / methanol = 7:2:1 two mixed solvents. The product purity was more than 99%. Physical and chemical properties of fructose monolaurates were studied. It contained hydrophilic-lipophilic balance value (HLB), the critical micelle concentration (CMC), the maximum adsorption capacity (Γ_max), the minimum cross-sectional area (A_min), micelle free energy (ΔG_m), foaming capacity and foam stability, emulsifying capacity and emulsion stability. The results were as follows: HLB= 5.8, indicating that it more suitable to be water in oil (w/o) emulsifier; CMC =2.5×10^-4 mol / L, critical surface tensionγCMC = 24.2 mN/m, the maximum adsorption capacity (Γ_max)= 0.7 mol m^-2, the smallest cross-sectional area (A_min) 23.4×10^-20 m², micelle free energy (ΔG_m) is -30.5 kJ / mol, showing that they have good surface activities; frutose monolaurates displayed better foaming power and foaming stability at low concentrations as well as similar emulsion power and emulsion stability comparing with that of the commercial surcrose esters.