Enzymatic Synthesis Of Unsaturated Monoacyl Trehaloses And Their Oxidation In Aqueous Solution

Carbohydrate fatty acid esters is a kind of nonion surfactant, which is made from renewable feedstock and does harmless to the environment. Due to their functional, nontoxic and nonskin-irritant properties, they have been widely used in food, pharmaceutical, cosmetic and detergent industries. Because the obviously defects of chemical synthesis, we adopt bio-catalytic process to synthesize the carbohydrate fatty acid esters. Most carbohydrate fatty acid esters saled on market are from saturated fatty acid and sugar. However, unsaturated fattty acid have many biological activities and physiological functions and other merits, so the research on carbohydrate unsaturated fatty acid esters attract the interest of many researchers. Lipids with high PUFAs content are susceptible to oxidation and it is reported that the oxidation of an unsaturated substrate in aqueous solution is different from that in bulk oil. We studied on the synthesis and properties of carbohydrate unsaturated fatty acid esters firstly, then investigated their oxidation in aqueous solution. The main results are as follows:Five carbohydrate fatty acid esters were synthesized from trehalose and unsaturated fatty acids or their ethyl esters in tert-butanol using microbial lipase as a biocatalyst in a batch stirred tank reactor. We systematically investigated the effect of many factors such as reaction time, molecular sieves, solvent, temperature, lipase, substrate concentration on the synthesis of carbohydrate fatty acid esters. The optimal condition for nonaqueous synthesis of monoacyl trehalose was as follows: trehalose concentration 13.2mmol/l, linoleic acid concentration 41.7 mmol/l, molecular sieves 3.1 g, lipase 0.3 g and tert-butanol 20 mL. The condensation was carryied at 50oC and 150 rpm for 50.4 h.The solvent was dehydrated by contact with molecular sieves (4 ?) for at least 24 h before use.The silica gel column chromatography with mobile phases of hexane-isopropanol- methanol 5:4:1(v/v/v) followed by 4:4:2 (v/v/v) for unsaturated trehalose esters at 1.3 mL/min was applied. The elution was collected and monitored by thin layer chromatography analysis (TLC). The mobile phase for TLC was ethyl acetate/methanol/water (8.5/1/0.5, v/v/v) and iodine was used for development The fractions containing monoacyl trehalose was pooled. Further purification was performed using semi-preparative HPLC. The eluent was a mixture of methanol and water at 35 oC and 8 mL/min. The identities of the pure compounds were confirmed using MS, 1H NMR and 13C NMR as 6-O-oleoyl trehalose, 6-O-linoleoyl trehalose, 6-O-linolenoyl trehalose, 6-O-eicosapentaenoyl trehalose, 6-O-docosahexaenoyl trehalose. Among them, 6-O-linolenoyl trehalose, 6-O-eicosapentaenoyl trehalose, and 6-O-docosahexaenoyl trehalose are new compound.Surface properties of monoacyl trehaloses with different long unsaturated fatty acid chains were investigated at 30, 40, 50 and 60°C. Each monoacyl trehaloses was dispersed in double distilled water and surface tension of aqueous solution was measured using a Doüy ring method. The critical micelle concentrations (CMC), surface tension at the CMC (γ_CMC), surface excess (Г) and residual area per molecule (A) of these amphiphilic compounds was studied and compared with other carbohydrate fatty acid esters. The effect of fatty acid chains length, double bond number and temperature on the surface properties of monoacyl trehaloses was also investigated. The value of CMC decreased as the chain length and temperature increased, while increased with more double bond when the chain length was the same.Oxidation of unsaturated monoacyl trehalose was studied in aqueous solution. Unsaturated monoacyl trehalose exist as monomers below the CMC, whereas they can aggregate spontaneously to form micelles above this concentration. Effect of double bond number, hydrophobic tail group size and concentration on the oxidative stability of unsaturated monoacyl trehalose in aqueous solution was studied. The results indicate that the oxidation of unsaturated monoacyl trehalose in micelles depended on both the degree of unsaturation and hydrophobic chain length. Oxidation increased as the number of double bonds increased in the unsaturated monoacyl trehaloses with identical hydrophobic chain length. However, when the hydrophobic chain length was different, the extent of oxidation decreased with chain length.