A preparation method of ascorbyl tetraisopalmitate

Abstract

The present invention relates to a preparation method of ascorbyl tetraisopalmitate, and belongs to the technical field of bioenzymatic synthesis. The preparation method of the present invention comprises the following steps: S1 fixes a fat synthase complex containing a mixture of malonyl-CoA, β-ketoacyl-ACP synthase and palmitoyl-ACP thioesterase in a fixative to obtain a solid biological fat synthase complex; malonic acid and the solid biological fat synthase complex are catalyzed in a saturated sodium bicarbonate aqueous solution to obtain palmitic acid; S2 esterifies a solution of palmitic acid with ascorbyl glyceride, methyl silicate and immobilized lipase to obtain ascorbyl tetraisopalmitate. The present invention uses sustainable and recyclable malonate as a substrate and utilizes an immobilized fat synthase complex to prepare palmitic acid more quickly and sustainably; ascorbyl tetraisopalmitate is prepared by esterifying ascorbyl glyceride with palmitic acid, providing a preparation method with high product purity and sustainable raw material sources.

Description

Technical Field

[0001] The invention relates to the technical field of bioenzymatic synthesis, in particular to a preparation method of ascorbic acid tetraisopalmitate. Background Art

[0002] Ascorbyl tetraisopalmitate (VCIP) is a fat-soluble, modified ascorbic acid derivative. Ascorbic acid (VC) has six hydrogen bond receptors in its structure, with all four hydroxyl groups replaced by isopalmitoyl groups to form ascorbyl tetraisopalmitate. The long fatty acid in VCIP binds to the electron-loss site of VC, imparting lipid solubility and improving ascorbic acid's transdermal penetration and stability. A neutral pH range is generally recommended for its activity, addressing the organic acid properties of ascorbic acid and demonstrating promising market prospects.

[0003] The application and preparation of ascorbic acid tetraisopalmitate are described in existing domestic patents and literature. For example, invention patent CN115287312A, "A Synthesis Method of Ascorbic Acid Tetraisopalmitate," discloses a preparation method for ascorbic acid tetraisopalmitate, using methyl palmitate and an L-ascorbic acid derivative as substrates and lipase as a catalyst in an organic solvent to cause an ester exchange reaction between methyl palmitate and an L-ascorbic acid derivative to produce ascorbic acid tetraisopalmitate. The lipase in this preparation method is a mixture of one or more of animal lipase, plant lipase, or microbial lipase. This method is theoretically groundbreaking, but the data do not provide sufficient information on side reaction products and reaction conditions, and the type of lipase is not fully described. In addition, the enzymatic oxidation and ester exchange synthesis of lipase are not economically efficient, with long reaction times and low reaction purity.

[0004] The current synthesis methods of ascorbyl tetraisopalmitate involve the use of strong acids, strong bases, corrosive acyl chloride compounds, or strong oxidants, which lead to many side reactions, harsh reaction conditions, and complicated purification and separation. The process for preparing ascorbyl tetraisopalmitate by reacting palmitoyl chloride or 2-hexyldecanol, 2-hexyldecanoic acid with ascorbic acid also belongs to the field of chemical synthesis and does not have the potential for sustainable development and the market demand for pure skin care.

[0005] Therefore, it is very necessary to develop a new, green, mild, high-purity preparation process of ascorbic acid tetraisopalmitate with sustainable raw material sources. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a new, green, mild, high-purity method for preparing ascorbic acid tetraisopalmitate with sustainable raw material sources.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for preparing ascorbyl tetraisopalmitate, comprising the following steps:

[0009] S1. A fat synthase complex obtained by mixing malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase is fixed in a fixative to obtain a solid biological fat synthase complex; and malonate and the solid biological fat synthase complex are then added to a saturated sodium bicarbonate aqueous solution for a catalytic reaction to obtain palmitic acid.

[0010] The fixing agent is an organic cross-linked copolymer complex with physical adsorption effect;

[0011] The reaction conditions of the catalytic reaction are: temperature 35-45°C, rotation speed 160-400 rpm, time 6-7h;

[0012] S2. Under inert gas protection, the palmitic acid prepared in step S1 is dissolved in an organic solvent to prepare a palmitic acid solution, and then ascorbyl glyceride, methyl silicate, and immobilized lipase are uniformly mixed with the palmitic acid solution. The resulting mixture is subjected to an ester exchange reaction, and the product purity is monitored during the reaction. When the product purity is not less than 98%, the immobilized lipase in the mixture is removed, and then water is added for extraction. The organic layer is dried to obtain ascorbyl tetraisopalmitate.

[0013] The invention first uses malonate as a substrate and performs synthesis and condensation in a saturated sodium bicarbonate aqueous solution by using a fat synthase complex composed of immobilized malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase to prepare palmitic acid. Then, ascorbyl glyceride is subjected to an ester exchange reaction with palmitic acid to prepare ascorbyl tetraisopalmitate, thereby providing a new preparation method of ascorbyl tetraisopalmitate.

[0014] The palmitic acid preparation substrate of the present invention is sustainable and recyclable, and can sustainably prepare palmitic acid; the existing palmitic acid on the market is mostly derived from palm plants and chemical synthesis, and the sources are limited, making it impossible to provide palmitic acid sustainably.

[0015] The present invention fixes a fat synthase complex composed of three enzymes, malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase, in a fixing agent such as a resin (through physical adsorption) to obtain a solid biological fat synthase complex. The solid-state enzymatic method is used to carry out a catalytic reaction, which has a faster reaction rate than the conventional bioenzyme fermentation process, greatly improving the demand for the industrialization of biosynthesis by the bioenzymatic method, and providing a green and sustainable raw material for the preparation of ascorbic acid tetraisopalmitate. At the same time, the method is used to prepare ascorbic acid tetraisopalmitate with a reaction time of 7-8h, which is shorter than the reaction time of the conventional bioenzymatic method. During the transesterification reaction, 300 microliters of sample are extracted every 10 minutes in the reaction and analyzed by high performance liquid chromatography to detect the product purity of ascorbic acid tetraisopalmitate, thereby monitoring the product purity.

[0016] As a preferred embodiment of the preparation method of the present invention, in step S1, the concentration of the saturated sodium bicarbonate aqueous solution is 50 mM.

[0017] As a preferred embodiment of the preparation method of the present invention, in step S1, the mass ratio of the malonic acid to the saturated sodium bicarbonate aqueous solution is 1:10. The concentration of the saturated sodium bicarbonate aqueous solution is 50 mM.

[0018] As a preferred embodiment of the preparation method of the present invention, in step S1, the fat synthase complex is obtained by mixing malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase in a mass ratio of 1:1:1. At this mass ratio, the three enzymes can more fully exert their respective functions, resulting in higher catalytic efficiency.

[0019] As a preferred embodiment of the preparation method of the present invention, in step S1, the fixing agent is a resin.

[0020] As a preferred embodiment of the preparation method of the present invention, in step S1, the resin is a polyacrylic acid / polymethyl methacrylate cross-linked copolymer.

[0021] As a preferred embodiment of the preparation method of the present invention, in step S1, the mass ratio of malonate to the fat synthase complex in the solid biological fat synthase complex is 40:1. At this mass ratio, malonate can react more fully and be more efficiently converted into palmitic acid.

[0022] As a preferred embodiment of the preparation method of the present invention, in step S1, the fixing agent is pretreated before fixation, and the pretreatment includes removing impurities in the fixing agent and allowing the fixing agent to absorb water and swell.

[0023] As a preferred embodiment of the preparation method of the present invention, the pretreatment is to soak the fixative in water for 24 hours, then soak it in 95% ethanol for 24 hours, and then rinse it with water for at least three times.

[0024] As a preferred embodiment of the preparation method of the present invention, in step S1, the fixing step is: first dispersing the fixing agent into the biological buffer solution, then adding the fat synthase complex and completely dispersing it, placing the resulting mixture in a water bath at 40°C and 180 rpm for 2 hours, filtering, and drying the resulting solid to obtain the solid biological fat synthase complex;

[0025] The mass volume ratio of the fat synthase complex, fixative, and biological buffer solution is 0.2 g:0.6 g:12 ml;

[0026] The biological buffer solution is an aqueous solution with a molar concentration of 50 mM disodium hydrogen phosphate, a molar concentration of 25 mM citric acid, and a pH of 5.0.

[0027] As a preferred embodiment of the preparation method of the present invention, in step S1, the reaction conditions of the catalytic reaction are: temperature 35° C., rotation speed 160 rpm, and time 6 h.

[0028] As a preferred embodiment of the preparation method of the present invention, in step S1, the reactants of the catalytic reaction are extracted using a low-boiling point organic solvent (dimethyl sulfoxide, methanol, etc.), and the organic layer is evaporated to dryness to obtain the fat-soluble active ingredient, namely palmitic acid.

[0029] As a preferred embodiment of the preparation method of the present invention, in step S2, the organic solvent includes dichloromethane, tetrahydrofuran or N-methylpyrrolidone.

[0030] As a preferred embodiment of the preparation method of the present invention, in step S2, the concentration of the palmitic acid solution is 2.5 mol / L.

[0031] As a preferred embodiment of the preparation method of the present invention, in step S2, the molar ratio of ascorbyl glyceride, palmitic acid, and methyl silicate is 1:4:4, and the concentration of the immobilized lipase in the mixture is 0.03 g / mL.

[0032] As a preferred embodiment of the preparation method of the present invention, in step S2, the conditions of the transesterification reaction are 25-35° C., 180-500 rpm, and the time of the transesterification reaction is 5-6 h.

[0033] As a preferred embodiment of the preparation method of the present invention, the conditions of the transesterification reaction are 30° C., 180 rpm, and 6 h.

[0034] As a preferred embodiment of the preparation method of the present invention, in step S2, the filtration is performed using a vacuum filter.

[0035] As a preferred embodiment of the preparation method of the present invention, in step S2, the mixture is cooled to 10-15°C before adding water for extraction.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The invention uses sustainable and recyclable malonate as a substrate, performs synthesis and condensation in a saturated sodium bicarbonate aqueous solution by using a fat synthase complex composed of immobilized malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase, thereby rapidly preparing palmitic acid and sustainably providing palmitic acid. Then, ascorbyl glyceride is used to carry out an ester exchange reaction with palmitic acid to prepare ascorbyl tetraisopalmitate, with a short reaction time. This provides a new, green, mild, high-purity, and sustainable raw material source preparation method for ascorbyl tetraisopalmitate. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in Example 2 of the present invention;

[0039] Figure 2 HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in Example 3 of the present invention;

[0040] Figure 3 HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in Example 4 of the present invention;

[0041] Figure 4 HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in Comparative Example 4 of the present invention;

[0042] Figure 5 HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in Comparative Example 5 of the present invention;

[0043] Figure 6 This is the HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in Comparative Example 6 of the present invention. DETAILED DESCRIPTION

[0044] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0045] Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial sources.

[0046] Example 1 Preparation of palmitic acid

[0047] The present invention uses malonate as a substrate and performs synthesis and condensation in a saturated sodium bicarbonate aqueous solution using a fat synthase complex consisting of immobilized malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase, thereby preparing palmitic acid. The prepared substrate is sustainable and recyclable, and the solid-state enzymatic method is used for the catalytic reaction, which has a faster reaction rate than a conventional bioenzyme fermentation process. Different macromolecular immobilizers (such as polyacrylic acid copolymers) have different reaction conditions and reaction performances, which greatly improves the demand for the industrialization of biosynthesis using the bioenzymatic method.

[0048] The preparation method of palmitic acid comprises the following steps:

[0049] A. Pre-treat (immerse) the polyacrylic acid / polymethyl methacrylate cross-linked copolymer in distilled water for 24 hours to allow hydration and water absorption, then soak it in 95% ethanol for 24 hours to dissolve any remaining short-chain organic benzene and acrylic acid impurities. Rinse the ethanol-soaked resin three times with distilled water to obtain a pre-treated resin, which is then stored in water at 25°C before use.

[0050] B. Mixing malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase in a mass ratio of 1:1:1 to obtain a fat synthase complex, and then adding 0.2 g of the fat synthase complex to a mixture of 0.6 g of the pretreated resin obtained in step A and 12 mL of a biological buffer solution (the biological buffer solution is an aqueous solution having a molar concentration of 50 mM disodium hydrogen phosphate, a molar concentration of 25 mM citric acid, and a pH of 5.0), mixing uniformly, and incubating the resulting mixture in a reciprocating water bath at 180 rpm at 40° C. for 2 hours. The reacted mixture is filtered through a vacuum filter, and the filtered solid is dried at 25° C. to obtain a solid biological fat synthase complex, which is then stored at 4° C.;

[0051] C. Add malonic acid and the solid biological fat synthase complex prepared in step B to a saturated sodium bicarbonate aqueous solution (concentration of 50 mM) for catalytic reaction. The mass ratio of malonic acid to the saturated sodium bicarbonate aqueous solution is 1:10, and the mass ratio of malonic acid to the fat synthase complex in the solid biological fat synthase complex is 40:1. The reaction conditions are controlled as follows: temperature 35°C, rotation speed 160 rpm, and time 6 h.

[0052] D. When a certain product yield is reached, use a low-boiling point organic solvent for extraction (dimethyl sulfoxide, methanol, etc.), and rotary evaporate to obtain the fat-soluble active ingredient, set aside, and determine the product purity.

[0053] Comparative Example 1 Preparation of palmitic acid

[0054] The method for preparing palmitic acid in this comparative example is the same as that in Example 1, except that the reaction temperature in step S3 is adjusted to 50°C.

[0055] Comparative Example 2 Preparation of Palmitic Acid

[0056] The method for preparing palmitic acid in this comparative example is the same as that in Example 1, except that the rotation speed in step S3 is adjusted to 100 rpm.

[0057] Comparative Example 3 Preparation of Palmitic Acid

[0058] The method for preparing palmitic acid in this comparative example is the same as that in Example 1, except that the reaction time in step S3 is adjusted to 5.5 h.

[0059] Test Case

[0060] The purity of palmitic acid prepared in Example 1 and Comparative Examples 1-3 was determined by gas chromatography, and the actual yield of palmitic acid was calculated. The yield was the mass ratio of the actual yield of palmitic acid to the target yield of palmitic acid prepared from malonic acid, that is, the final product yield of palmitic acid. The experimental scheme was based on a target yield of 500 g and was converted by calculating the actual yield under different experimental conditions.

[0061] The conditions for determination were as follows:

[0062] 1. Test conditions

[0063] A capillary column with polyethylene glycol as the stationary liquid was used; the starting temperature was 170°C, the temperature was increased to 230°C at a rate of 3°C per minute, and maintained for 5 minutes; the injection port temperature was 230°C; and the detector temperature was 250°C.

[0064] 2. Test methods

[0065] Accurately weigh approximately 0.1 g of this product and place it in a reflux flask. Add 2 ml of a 14% boron trifluoride methanol solution, shake to dissolve, and reflux in a water bath for 30 minutes. Add 4 ml of n-heptane and continue to reflux for 5 minutes. Cool, add 10 ml of a saturated sodium chloride solution, shake, and allow to separate. Take the supernatant and wash it three times with 2 ml of water each time. Dry the supernatant over anhydrous sodium sulfate, accurately measure 1 μl, inject it into a gas chromatograph, record the chromatogram, and calculate the purity by area normalization.

[0066] The conversion rates and purities of palmitic acid prepared in Example 1 and Comparative Examples 1-3 are shown in Table 1 below.

[0067] Table 1

[0068] Yield (%) purity(%) Example 1 80 99.2 Comparative Example 1 62 89.3 Comparative Example 2 70 95.4 Comparative Example 3 68 92.6

[0069] The results in Table 1 show that in the preparation method of palmitic acid, if the reaction temperature is too high (higher than 45°C), the reaction speed is too slow (lower than 150 rpm), or the reaction time is too short (shorter than 6 h), the conversion rate or purity of palmitic acid will be reduced.

[0070] Example 2 Preparation of ascorbyl tetraisopalmitate (VCIP)

[0071] The preparation of ascorbyl tetraisopalmitate (VCIP) of the present invention, the specific reaction formula is as follows:

[0072]

[0073] The preparation method of ascorbyl tetraisopalmitate (VCIP) comprises the following steps:

[0074] a. Under inert gas protection, the palmitic acid prepared in Example 1 was dissolved in dichloromethane to prepare a palmitic acid solution having a palmitic acid concentration of 2.5 mol / L, and then ascorbyl glyceride, methyl silicate condensing agent, and immobilized lipase (commercially available) were added to the palmitic acid solution and mixed to obtain a mixture, wherein the molar ratio of ascorbyl glyceride, palmitic acid, and methyl silicate was 1:4:4, and the concentration of immobilized lipase in the mixture was 0.03 g / mL;

[0075] b. The mixture obtained in step a is placed in a reciprocating water bath at 25-35° C. and 180-300 rpm for a transesterification reaction for 5-6 hours. 300 microliters of sample is extracted every 10 minutes and analyzed by high performance liquid chromatography to detect the product purity of ascorbyl tetraisopalmitate, thereby monitoring the product purity and determining the progress of the reaction. When the product purity is ≥98%, the immobilized lipase is removed by vacuum filtration, the mixture is cooled to 10-15° C., washed with deionized water, extracted and separated, the organic layer is dried, and rotary evaporated until the dichloromethane is completely volatilized to obtain a colorless oily liquid, namely, ascorbyl tetraisopalmitate (VCIP). The aqueous layer solution is recovered and the immobilized lipase is recovered by the adsorption capacity of a macromolecular resin for repeated use.

[0076] The determination was carried out according to the "High Performance Liquid Chromatography Operating Procedures", as follows:

[0077] 1. Injection conditions

[0078] Octadecylsilane bonded silica gel was used as the filler (4.6×250 mm); acetonitrile-methanol (55:45) was used as the mobile phase; the flow rate was 1.2 ml / min, the column temperature was 30° C., and the detection wavelength was 236 nm.

[0079] 2. Test methods

[0080] Weigh the sample to make a mobile phase solution containing 1.0 mg in 1 ml, filter it through a PTFE membrane, and take the filtrate as the test solution. Pipette 20 μl of the test solution into the liquid chromatograph, record the chromatogram, and calculate the peak area percentage by area normalization method. The product purity is obtained, such as Figure 1 、 2 , and the area percentage at the retention time of around 17 minutes in 3.

[0081] The conversion rate of the product is calculated. The conversion rate is the mass ratio of the actual output of ascorbic acid tetraisopalmitate prepared from palmitic acid to the target output of ascorbic acid tetraisopalmitate, that is, the final product conversion rate of ascorbic acid tetraisopalmitate (VCIP). This step can be converted by the actual feed amount. The experimental plan is based on the target product output of 100 g, and the conversion rate of the final prepared VCIP product is converted.

[0082] The ascorbic acid tetraisopalmitate (VCIP) prepared in this embodiment is a colorless to yellow liquid with a slight special odor. The product purity is 99.08%, and the calculated product conversion rate is ≥78%. The HPLC chart of the ascorbic acid tetraisopalmitate (VCIP) prepared in this embodiment is as follows: Figure 1 The peak area percentage of this embodiment is the peak corresponding to the retention time of 17.148.

[0083] Example 3 Preparation of ascorbyl tetraisopalmitate (VCIP)

[0084] The preparation method of VCIP in this embodiment is the same as that in Example 2, except that dichloromethane is replaced with tetrahydrofuran. After high performance liquid chromatography, the purity of ascorbic acid tetraisopalmitate (VCIP) prepared using the preparation conditions of this embodiment is 98.81%, and the product conversion rate is 80%. The HPLC chart of ascorbic acid tetraisopalmitate (VCIP) prepared in this embodiment is as follows: Figure 2 The peak area percentage of this embodiment is the peak corresponding to the retention time 17.162.

[0085] Example 4 Preparation of Ascorbyl Tetraisopalmitate (VCIP)

[0086] The preparation method of VCIP in this embodiment is the same as that in Example 2, except that dichloromethane is replaced with N-methylpyrrolidone. After high performance liquid chromatography, the purity of ascorbyl tetraisopalmitate (VCIP) prepared using the preparation conditions of this embodiment is 98.99%, and the product conversion rate is 78.2%. The HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in this embodiment is as follows: Figure 3 The peak area percentage of this embodiment is the peak corresponding to the retention time 17.196.

[0087] Comparative Example 4 Preparation of Ascorbyl Tetraisopalmitate (VCIP)

[0088] The preparation method of VCIP in this comparative example is the same as that in Example 2, except that the molar ratio of ascorbyl glyceride, palmitic acid, and methyl silicate is 1:3:4. After high performance liquid chromatography, the purity of ascorbyl tetraisopalmitate (VCIP) prepared under the preparation conditions of this comparative example is 92.64%, and the product conversion rate is 56%. 。 The HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in this comparative example is as follows: Figure 4 The peak area percentage of this comparative example is the peak corresponding to the retention time of 16.335.

[0089] Comparative Example 5 Preparation of Ascorbyl Tetraisopalmitate (VCIP)

[0090] The preparation method of VCIP in this comparative example is the same as that in Example 2, except that the molar ratio of ascorbyl glyceride, palmitic acid, and methyl silicate is 1:4:3. After high performance liquid chromatography, the purity of ascorbyl tetraisopalmitate (VCIP) prepared under the preparation conditions of this comparative example is 90.96%, and the product conversion rate is 60%. The HPLC chart of ascorbyl tetraisopalmitate (VCIP) prepared in this comparative example is as follows: Figure 5 The peak area percentage of this comparative example is the peak corresponding to the retention time of 16.950.

[0091] Comparative Example 6 Preparation of Ascorbyl Tetraisopalmitate (VCIP)

[0092] The preparation method of VCIP in this comparative example is the same as that in Example 2, except that in step b, an ester exchange reaction is carried out and the reaction time is 4 hours. After high performance liquid chromatography, the purity of ascorbic acid tetraisopalmitate (VCIP) prepared under the preparation conditions of this comparative example is 90.80%, and the product conversion rate is 63%. The HPLC chart of ascorbic acid tetraisopalmitate (VCIP) prepared in this comparative example is as follows: Figure 6 The peak area percentage of this comparative example is the peak corresponding to the retention time of 16.950.

[0093] The experiments in Example 2 and Comparative Examples 4 and 5 above show that a molar ratio of ascorbyl glyceride, palmitic acid, and methyl silicate of 1:4:4 results in higher product purity and yield. However, the reduced molar mass leads to insufficient substrate, resulting in a lower conversion rate. Furthermore, too little condensing agent can lead to incomplete transesterification. The partial deviations in the retention times described above represent theoretical errors and do not affect the purity determination of ascorbyl tetraisopalmitate.

[0094] It can be concluded from Example 2 and Comparative Example 6 that if the transesterification reaction time is too short, the conversion rate of ascorbic acid tetraisopalmitate will be affected. Only when the transesterification reaction time is 5 h or longer can relatively good purity and conversion rate be achieved.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing ascorbyl tetraisopalmitate, characterized in that: The following steps are involved: S1. A fat synthase complex obtained by mixing malonyl-CoA, β-ketoacyl-ACP synthase, and palmitoyl-ACP thioesterase in a mass ratio of 1:1:1 is fixed in a fixative to obtain a solid biological fat synthase complex; and malonate and the solid biological fat synthase complex are then added to a saturated sodium bicarbonate aqueous solution for a catalytic reaction to obtain palmitic acid. The fixing agent is an organic cross-linked copolymer complex with physical adsorption effect; The reaction conditions of the catalytic reaction are: temperature 35-45°C, rotation speed 160-400 rpm, time 6-7h; S2. Under inert gas protection, the palmitic acid prepared in step S1 is dissolved in an organic solvent to prepare a palmitic acid solution, and then ascorbyl glyceride, methyl silicate, and immobilized lipase are uniformly mixed with the palmitic acid solution. The resulting mixture is subjected to an ester exchange reaction, and the product purity is monitored during the reaction. When the product purity is not less than 98%, the immobilized lipase in the mixture is removed, and then water is added for extraction. The organic layer is dried to obtain ascorbyl tetraisopalmitate.

2. The preparation method according to claim 1, wherein In step S1, the mass ratio of the malonic acid to the fat synthase complex in the solid biological fat synthase complex is 40:

1.

3. The preparation method according to claim 1, wherein In step S1, the fixing agent is pretreated before fixation, and the pretreatment includes removing impurities in the fixing agent and allowing the fixing agent to absorb water and swell.

4. The preparation method according to claim 1, wherein In step S1, the fixing step is as follows: first, dispersing the fixing agent into the biological buffer solution, then adding the fat synthase complex and completely dispersing it, placing the obtained mixture in a water bath at 40°C and 180 rpm for 2 hours, filtering, and drying the obtained solid to obtain the solid biological fat synthase complex; The mass volume ratio of the fat synthase complex, the fixative, and the biological buffer solution is 0.2 g:0.6 g:12 mL; The biological buffer solution is an aqueous solution with a molar concentration of 50 mM disodium hydrogen phosphate, a molar concentration of 25 mM citric acid, and a pH of 5.

0.

5. The preparation method according to claim 1, wherein In step S2, the organic solvent includes dichloromethane, tetrahydrofuran or N-methylpyrrolidone.

6. The preparation method according to claim 1, wherein In step S2, the concentration of the palmitic acid solution is 2.5 mol / L.

7. The preparation method according to claim 1, wherein In step S2, the molar ratio of ascorbyl glyceride, palmitic acid, and methyl silicate is 1:4:4; And / or, the concentration of the immobilized lipase in the mixture is 0.03 g / mL.

8. The preparation method according to claim 1, wherein In step S2, the transesterification reaction is carried out at 25-35° C. and 180-500 rpm; And / or, the transesterification reaction time is 5-6 hours.

9. The preparation method according to claim 1, wherein In step S2, the mixture is cooled to 10-15°C before adding water for extraction.

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