Research On The Lipase-catalyzed Synthesis Of D-isoascorbyl Fatty Acid Esters

With social progress, economic development and the raising of people’s living standard, the research and development of food antioxidants, which are green, environmental, nutritious and safe, have been paid more and more attention to. In this article, D-isoascorbyl fatty acid esters were synthesized by enzymatic method, the investigation of influencing factors, optimization of process operation, discussion of reaction kinetics and mechanism and analysis of antioxidation activity of products were studied. These basic researches have important realistic and theoretical significance.With D-isoascorbic acid and capric acid, lauric acid, palmitic acid, stearic acid as raw material and immobilized lipase Novozym 435 as catalyst, four D-isoascorbyl fatty acid esters were synthesized from at 45.0 °C for 12 h at a speed of 200 rpm in tert-butanol reaction system. High performance liquid chromatography(HPLC) was adopted to detect the contents of D-isoascorbic acid in reaction system, the standard curve, equation and an expression for calculation the conversion were established, which provided a base for the further research. After reaction, the four purified products D-isoascorbyl caprate, D-isoascorbyl laurate, D-isoascorbyl palmitate and D-isoascorbyl stearate were obtained through washing, solvents extraction and recrystallization. The purities of the four products were all above 95%, which were determined by iodine method. The chemical structure of the four products were confirmed by UV, IR, ESI-MS and 1H NMR analysis.The effects of solvent, reaction temperature, reaction time, immobilized lipase amount, D-isoascorbic acid concentration, substrate molar ratio, 4A molecular sieve addition and shaking speed on the enzymatic synthesis of D-isoascorbyl fatty acid esters were investigated by using the mono-factor method. The optimum conditions were obtained as follows: the proper solvent of t-butanol, reaction temperature 45.0 °C, reaction time 10 h(D-isoascorbyl caprate), 12 h(D-isoascorbyl laurate), 14 h(D-isoascorbyl palmitate), 16 h(D-isoascorbyl stearate), immobilized lipase amount 12.33 g/L, substrate molar ratio 7:1, D-isoascorbic acid concentration 0.14 mol/L, 4A molecular sieve addition 200.0 g/L and shaking speed 200 r/min. Under the optimum condition, the conversion of D-isoascorbic acid was up to 89.2%, 88.9%, 87.6% and 86.8% for the enzymatic synthesis of D-isoascorbyl caprate, D-isoascorbyl laurate, D-isoascorbyl palmitate and D-isoascorbyl stearate, respectively.Response surface method(RSM) was used to design experiments and research on the key factors of lipase-catalyzed synthesis of D-isoascorbyl fatty acid esters by esterification reaction. Response surface models were established with the reaction conversion as target function, the optimized process conditions for producing D-isoascorbyl fatty acid esters were predicted. Based on three parallel experiments to synthesis D-isoascorbyl caprate, D-isoascorbyl laurate, D-isoascorbyl palmitate and D-isoascorbyl stearate, the reaction conversion had good repeatability, and the average values were 95.4%, 94.4%, 92.4%, and 90.6%, respectively. The experimental data were in good agreement with the model predictions.With D-isoascorbic acid and capric acid, lauric acid, palmitic acid, stearic acid as raw material, the kinetic data of different reaction temperature and different substrate concentration for lipase-catalyzed synthesis of D-isoascorbyl fatty acid esters were obtained through experiment. Ping-Pong bi-bi model and Ping-Pong bi-bi model with D-isoascorbic acid inhibition were used to fit the experiment data, and four reaction kinetic models for synthesis of D-isoascorbyl fatty acid esters were established. the model parameters and its relationship with reaction temperature were determined. The kinetic law of lipase-catalyzed synthesis of D-isoascorbyl fatty acid esters followed Ping-Pong bi-bi model with D-isoascorbic acid inhibition. The D-isoascorbic acid was binded with the lipase resulting in the formation of a dead-end complex, which inhibited the activity of enzyme.Based on radical-scavenging assay and oxidation-reduction reaction, the scavenging effects on hydroxyl radical(?OH), 1, 1-diphenyl-2-picrylhydrazyl radical(DPPH?), superoxide anion radical(?O2-) and reducing ability of D-isoascorbyl caprate, D-isoascorbyl laurate, D-isoascorbyl palmitate, D-isoascorbyl stearate, D-isoascorbic acid and conventional antioxidant BHT were evaluated. The ?OH, DPPH?, ?O2- clearance rates and reducing abilities increased rapidly with the increase of products concentration, but the ?OH clearance rates changed little when the products concentration exceeded 3.0 mmol/L, the DPPH? clearance rates were higher than 90% when the products concentration exceeded 0.10 mmol/L, the ?O2- clearance rates remained about the same when the products concentration exceeded 0.12 mmol/L. At the same molal concentration of antioxidants, the effect of eliminating ?OH, DPPH?, ?O2- and reducing abilities of the four products were better than D-isoascorbic acid and BHT. When the mass fraction of antioxidants were 0.2%, the antioxidant capacities of the four products were basically the same and better than BHT in the soybean oil.