D-isoascorbyl palmitate: lipase-catalyzed synthesis, structural characterization and process optimization using response surface methodology
© Sun et al.; licensee Chemistry Central Ltd. 2013
Received: 9 May 2013
Accepted: 4 July 2013
Published: 8 July 2013
Isoascorbic acid is a stereoisomer of L-ascorbic acid, and widely used as a food antioxidant. However, its highly hydrophilic behavior prevents its application in cosmetics or fats and oils-based foods. To overcome this problem, D-isoascorbyl palmitate was synthesized in the present study for improving the isoascorbic acid’s oil solubility with an immobilized lipase in organic media. The structural information of synthesized product was clarified using LC-ESI-MS, FT-IR, 1H and 13C NMR analysis, and process parameters for high yield of D-isoascorbyl palmitate were optimized by using One–factor-at-a-time experiments and response surface methodology (RSM).
The synthesized product had the purity of 95% and its structural characteristics were confirmed as isoascorbyl palmitate by LC-ESI-MS, FT-IR, 1H, and 13C NMR analysis. Results from “one–factor-at-a-time” experiments indicated that the enzyme load, reaction temperature and D-isoascorbic-to-palmitic acid molar ratio had a significant effect on the D-isoascorbyl palmitate conversion rate. 95.32% of conversion rate was obtained by using response surface methodology (RSM) under the the optimized condition: enzyme load of 20% (w/w), reaction temperature of 53°C and D- isoascorbic-to-palmitic acid molar ratio of 1:4 when the reaction parameters were set as: acetone 20 mL, 40 g/L of molecular sieves content, 200 rpm speed for 24-h reaction time.
The findings of this study can become a reference for developing industrial processes for the preparation of isoascorbic acid ester, which might be used in food additives, cosmetic formulations and for the synthesis of other isoascorbic acid derivatives.
D- isoascorbic acid (synonyms: Erythorbic acid) is a stereoisomer of ascorbic acid (Vitamin C). It is a novel food antioxidant and preservative with excellent safe performance . D- isoascorbic acid can prevent the food oxidation, inhibit the decrease of color, aroma and flavors, and block the production of the carcinogen ammonium nitrite during food manufacturing process. It had been classified as generally recognized as safe (GRAS) additives by US Food and Drug Administration (FDA). Now it can be used in processed foods in accordance with Good Manufacturing Practice (GMP) . D-isoascorbic acid is freely soluble in water. However, its highly hydrophilic behavior similar with ascorbic acid prevents its application in cosmetics or fats and oils-based foods . Esterification process of converting ascorbic acid to its acid esters has been regarded as an effective solution for overcoming such problems. Furthermore, the esterified ascorbic acid products also have bifunctional activity including its original antioxidant activity and the bioactivity of the connected group. For example, the biosynthesized ascorbyl benzoate owned the antioxidant and antimicrobial/ antifungal activities from original ascorbic acid and connected benzoic acid group . And the fatty acid ester of ascorbic acid also has the antioxidant and surfactant functions with its potential application in high-fat food and cosmetics [5–7]. As for the isoascorbic acid, an erythorbyl fatty acid ester of erythorbyl laurate had been recently synthesized for improving the lipophilicity . However, other erythorbyl fatty acid esters are still needed for enlarging its application fields, especially in oil & fat foods.
Oil-soluble ascorbic acid derivatives can be prepared by enzymatic or chemical synthesis [9–11]. For the chemical esterification process, a strongly corrosive acid including hydrogen fluoride or sulfuric acid is used as a catalyst, which results in a series of disadvantages, for example, formation of many side-products and high energy consumption . Enzymatic synthesis is preferred because of its advantages-high catalytic efficiency, mild reaction condition, and inherent selectivity of the natural catalyst [12–15]. As for isoascorbic acid industry, development of its ester products is attractive for enlarging the application fields of oil foods, cosmetics and pharmaceuticals. Furthermore, other erythorbyl fatty acid esters are still needed for increase its application fields. Optimizating the reaction parameters for esterification reaction plays an important role for maximum yield and economical production of isoascorbyl palmitate. Various statistical optimization techniques such as response surface methodology (RSM) with Central Composite Rotatable design (CCRD), Box-Behnken or uniform design method had been applied for ascorbyl palmitate sysnthesis , L-ascorbyl laurate , ascorbyl oleate  and L-ascorbyl lactate . However, there have been no detailed reports on the effects of the reaction parameters on isoascorbic esters production till now.
The objectives of this study were to: (1) synthesize an oil-soluble isoascorbic acid palmitate by enzymatic method in an organic solvent system, (2) clarify the structural information using LC-ESI-MS, FT-IR, 1H and 13C NMR analysis, (3) evaluate the key reaction parameter for D-isoascorbyl palmitate process, and (4) optimize the reaction parameters for maximum conversion rate of D-isoascorbyl palmitate using response surface methodology.
Results and discussion
Identification of isoascorbic acid and its esters by LC-MS
Structural characteristic analysis of the synthesized D-isoascorbyl palmitate
13C NMR(400MHz,DMSO-d6):δ (ppm):(173.21 (C-1=O), 170.60 (C-1'=O), 152.96 (C-2), 118.74 (C-3), 76.62(C-4), 68.07(C-5), 63.84(C-6), 34.11 (C-2'), 33.80 (C-3'), 31.77-28.94 (C-4'-C12'), 24.95 (C-13'), 24.81 (C-14'), 22.57 (C-15'), 14.39 (C-16').
The 13C NMR spectrum of isoascorbyl palmitate showed the carbonyl group at C-1 and double bonds between C-2 and C-3 in isoascorbic moiety were intact which indicated that the enzymatic reaction happened in other position. The C-6’ signal at 65.6 ppm in the synthesized isoascorbyl ester had a down-field shift of 3.9 ppm in comparison with that of isoascorbic acid (61.7 ppm). These results proved the presence of an ester bond on C-6′of the isoascorbyl moiety and correspond with the pattern of chemical shift reported by Park et al.  and Stamatis et al..
One-factor-at-a-time experiments for isoascorbyl palmitate synthesis process
Effect of lipase source on D-isoascorbyl palmitate synthesis
Lipases (E.C. 184.108.40.206) generally catalyze the hydrolysis of oils and fats [20, 21]. Under specific conditions, they also catalyze the hydrolysis reactions in organic solvents by direct esterification with free acid, transesterification, acidolysis, alcoholysis and aminolysis [22, 23]. The lipases sources had the difference in structure including the lid region structure which affected the catalytic activity, regioselectivity and stereoselectivity.
Influence of the lipase source on the synthesis of D- isoascorbyl palmitate
Effective temperature (°C)
Conversion rate (%)a
Macroporous acrylic resin
41.30 ± 2.6
4.30 ± 1.9
Anionic exchange resin
15.20 ± 3.5
Effect of reaction medium source on D-isoascorbyl palmitate synthesis
Influence of the organic solvent on the synthesis of D- isoascorbyl palmitate
Conversion rate (%)a
57.8 ± 1.8
49.6 ± 2.3
25.28 ± 3.9
Influence of enzyme load on D-isoascorbyl palmitate synthesis
Effect of reaction time on D-isoascorbyl palmitate synthesis
Effect of reaction temperature on D-isoascorbyl palmitate synthesis
Effect of substrate molar ratio on D-isoascorbyl palmitate synthesis
Effect of molecular sieves content on D-isoascorbyl palmitate synthesis
Response surface optimization
Variables and experimental design levels for response surface
Enzyme load(%, w/w)
Molar ratio(D-isoascorbic: palmitic acid)
Experimental designs and the results of Box-Behnken design for optimizing reaction conditions for the production of D- isoascorbyl palmitate
Conversion rate (%)
93.28 ± 0.82
85.26 ± 0.68
63.66 ± 1.37
66.07 ± 0.25
84.39 ± 0.51
86.21 ± 0.96
86.62 ± 1.19
50.30 ± 0.42
71.18 ± 0.28
37.07 ± 0.01
72.12 ± 1.07
54.82 ± 1.32
90.73 ± 0.72
84.99 ± 1.44
84.61 ± 0.58
Results of ANOVA analysis of a full second-order polynomial model for reaction conditions for the production of D- isoascorbyl palmitate
Sum of squares
Lack of fit
Mutual effect of parameters and attaining optimum condition
Validation of the model
The availability of the regression model (Eq. (2)) of the conversion rate of isoascorbyl palmitate was tested using the calculated optimal condition, viz. acetone 20 mL, 40 g/L of molecular sieves content, 200 rpm speed, 20% enzyme load, D- isoascorbic-to-palmitic acid molar ratio of 1:4, temperature of 53o for 24-h during the course of optimization experiments. The mean value of the mycelial biomass was 95.32 ± 0.17%, which agreed with the predicted value (96.98%) well that indicated the high validity and adequacy of the model.
D-isoascorbic acid (purity > 99%) was provided from Parchn Sodium Isovitamin C Co., Ltd (Dexing, Jiangxi, China). Palmitic acid (purity > 99.5%) was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Novozym 435 was purchased from Novo Nordisk Co., Ltd (Beijing, China). Lipozyme TLIM, a lipase from Thermomyces lanuginosus immobilized on silica granulation and Lipozyme RMIM, a lipase from Rhizomucor miehei, immobilized on an anionic exchange resin, also purchased from Novo Nordisk Co., Ltd (Beijing, China). Lipase LVK-H100 and LBK-B400, were kindly gifted by Leveking bio-engineering Co., Ltd (Shenzhen, China). The properties of all lipases are shown in Table 1.
2-Methyl-2-butanol, n-hexane, ethanol, chloroform, petroleum ether, acetone and acetic ether were analytical reagent grade purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). HPLC-grade methanol was purchased from Tedia, USA. All reagents were dehydrated by molecular sieve 4 Å (Shanghai world molecular sieve Co., Ltd., Shanghai, China) for at least 24 h and filtered using a membrane filter (0.45 μm) prior to use as a reaction medium.
Procedure for lipase-catalysed esterification
D-isoascorbic acid (2.5 mmol), palmitic acid (10 mmol) and the immobilized lipase (150 mg, about 5% of the substrates amount) were weighed into a 150 mL conical flask. 20 mL of 2-methyl-2-butanol and 1.0 g of molecular sieve 4 Å were then added. The stoppered flasks were shaken at the speed of 200 rpm on a thermo-constant orbital shaker at 50°C for 48 h. The sampled reaction mixture was filtered through a membrane filter (0.45 μm), and 20 μL of each aliquot were injected into the HPLC for further analyzing concentrations of the substrate isoascorbic acid and the produced D-isoascorbyl palmitate.
Purification of produced D- isoascorbyl palmitate
The purification process was conducted according to the method described by Park et al.  and Bradoo et al.  with a slight modification. Briefly, the reaction solution was filtered with a membrane filter (0.45 μm) to remove the lipase and molecular sieve. The mixture solution of D-isoascorbyl palmitate, isoascorbic acid and palmitic acid was obtained by vacuum evaporating the 2-Methyl-2-butanol, and resolved in ethyl acetate. The same quota of deonized water was added for removing the residue isoascorbic acid, and hexane was used to washing out the palmitic acid. The insoluble D- isoascorbyl palmitate was then finally obtained by vacuum drying for 2 h.
Produced D-isoascorbyl palmitate and residual isoascorbic acid was identified by mass spectrometry with a quadrupole ion trap Thermo Finnigan™ LXQ™ LC-ESI-MS (San Jose, CA, USA) equipped with a degasser, LC-20AD binary pumps, a model SIL-20AC autosampler, a model CTO-20A thermostat, an electro-spray ionization (ESI) interface, and a model CBM-20A system controller. FT-IR spectra with Thermo-Nicolet Nexus 670 Fourier Transform Infrared Spectrometer (San Jose, CA, USA), 1H and 13C NMR spectra with a Bruker AVANCE NMR Spectrometer (Switzerland) at 400 MHz.
Produced D-isoascorbyl palmitate and residual isoascorbic acid were quantitatively analyzed by using a Waters Alliance LC-20AT (SHIMADZU, Japan) liquid chromatography connected to a model 2996 (DAD) diode array detector and controlled by LC Driver Ver.2.0 for Waters Empower™ software. The column equipped in the HPLC system was ZORBAX Eclipse XDB-C18 (150 mm×4.6 mm, 5 μm, Torrance, CA, USA). The mobile phase was methanol/water (90:10, v/v) at 1.0 ml/min flow rate for 15 min. Samples of 20 μL were injected automatically. The purity of sample was 95% with a sole peak in the HPLC chromatograph, which could be used as a standard. Purified D-isoascorbyl palmitate had the purity of 95% determining with HPLC (data not shown) as the standards (0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 g/L) were used to obtain the D-isoascorbyl palmitate calibration curve. The conversion rate (%) was calculated by dividing the initial molar amount of D-isoascorbic acid by the produced molar amount of isoascorbyl palmitate.
Experimental design and evaluation
Where Y is the predicted response variable (conversion rate, %); Ao, Ai, Aii, Aij are constant regression coefficients of the model, and Xi, Xj (i=1, 3; j=1, 3, i≠j) represent the independent variables (reaction parameters) in the form of coded values. The accuracy and general ability of the above polynomial model could be evaluated by the coefficient of determination R2.
Isoascorbyl palmitate was successfully synthesized by using lipase-catalysed esterification of isoascorbic acid and palmitic acid under the mild reaction conditions. It structure was characterized by LC-MS, FT-IR, 1H, and 13C NMR. The effect of various parameters on synthesis of D-isoascorbyl palmitate, such as enzyme source, type of organic, enzyme load, reaction time, temperature, molecular sieves content and D-isoascorbic-to-palmitic acid molar ratio were discussed using “one–factor-at-a-time” experiments and Response surface methodology. The optimized condition was obtained as follow: enzyme load of 20% (w/w), reaction temperature of 53°C and D-isoascorbic-to-palmitic acid molar ratio of 1:4. Under these optimal conditions, 95.32% of conversion rate was obtained which was in agreement with the predicted value (96.98%). The results are of a reference for developing industrial processes for the preparation of isoascorbic acid ester, which might be used in food additives, cosmetic formulations and for the synthesis of other isoascorbic acid derivatives.
This work was supported by funding from the National High Technology Research and Development Program (2012AA022103), China Postdoctoral Science special Foundation (2013T60648), China Postdoctoral Science Foundation (2012M511222), 2012 Excellent Key Young Teachers Project of Jiangsu University, Graduate Research and Innovation Projects of Jiangsu Province (CX10B_021X, CXLX12_0670), Advanced Programs of Jiangxi Postdoctoral Science Foundation (195), the Research Foundation for Advanced Talents of Jiangsu University and Science & Technology Platform Construction Program of Jiangxi Province.
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