A method for enzymatic preparation of l-ascorbyl fatty acid esters and products thereof

Abstract

The application discloses a method for preparing L-ascorbyl fatty acid ester by using an enzyme and a product thereof, and comprises the following steps: (1) a one-step method is used to prepare a microarray carrier with a hydrophobic and mesoporous structure, free enzymes are dissolved in a PBS buffer solution, and the enzymes are fixed on the microarray carrier by using a multiple adsorption method and are freeze-dried, so that a lipase microarray is obtained, the immobilization amount of the lipase microarray is between 100-300 mg / g, and the activity is 80-180 U / g; (2) L-ascorbic acid and fatty acid are added into a reaction solvent, the L-ascorbic acid is dissolved by stirring for a certain period of time, then the lipase microarray obtained in the step (1) is added, and the esterification reaction is carried out by stirring for a period of time, nitrogen is combined and vacuum is extracted to remove water, so that a crude L-ascorbyl fatty acid ester product is obtained; and (3) the crude L-ascorbyl fatty acid ester product obtained in the step (2) is separated and purified, and high-purity L-ascorbyl fatty acid ester is obtained. The application has the advantages of high catalytic efficiency, strong fat-solubility and antioxidant property of the product, simple operation, green environmental protection, simple product separation, and suitability for scale-up production.

Description

Technical Field

[0001] This invention belongs to the field of oil modification and lipid deep processing, specifically relating to a method for synthesizing L-ascorbic acid fatty acid esters using lipase microarray catalysis and the resulting product. Background Technology

[0002] L-Ascorbate palmitate (L-AP) is a highly effective fat-soluble antioxidant, recognized as a safe food additive by the World Health Organization, and is the only antioxidant permitted in Chinese infant formula. However, L-AP has a high melting point and poor solubility, and it easily crystallizes in fat-soluble products, limiting its addition amount and application range in oils. Preparing L-ascorbic acid unsaturated fatty acid esters by esterifying unsaturated fatty acids with L-ascorbic acid can lower the melting point, improve fat solubility, expand application areas, and enhance functional activity.

[0003] Currently, chemical methods are commonly used to synthesize L-ascorbic acid fatty acid esters. However, this method suffers from drawbacks such as high energy consumption, significant pollution, equipment corrosion, numerous byproducts, and difficult separation. CN 102260231A discloses a method for preparing ascorbate palmitate using concentrated sulfuric acid as a catalyst, but this method involves cumbersome product and catalyst separation steps and generates large amounts of acidic wastewater, failing to meet environmental protection and sustainability requirements. In contrast, enzymatic synthesis has attracted considerable attention due to its mild reaction conditions, high selectivity, and fewer byproducts. Generally, free lipases exhibit low activity, poor stability, and difficulty in reusing, while immobilized lipases show better stability and catalytic activity in harsh environments. Commonly used immobilized lipases for catalysis are commercially available enzymes, such as Novozym 435, RMIM, and TLIM. These enzymes are expensive, and the resin carriers easily swell in organic solvents, limiting enzyme reusability. CN 103667384A discloses an enzymatic method for synthesizing ascorbate palmitate, but the method uses a mixed solvent containing 2-methyltetrahydrofuran, which is a moderately toxic organic solvent and is not suitable for use in the preparation of food additives. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an enzymatic method for preparing L-ascorbic acid fatty acid esters, which addresses the shortcomings of the prior art. This method has the advantages of high catalytic efficiency, strong product lipophilicity and antioxidant properties, simple operation, green and environmentally friendly operation, easy product separation, and suitability for large-scale production.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0006] A method for preparing L-ascorbic acid fatty acid esters by enzymatic method and the product thereof, comprising the following steps:

[0007] (1) The free enzyme was dissolved in PBS buffer and immobilized on a microarray carrier by multiple adsorption method, and then freeze-dried to obtain a lipase microarray with an immobilization load of 100-300 mg / g and an activity of 80-180 U / g; the microarray carrier was a hydrophobic mesoporous silica.

[0008] (2) Add L-ascorbic acid and fatty acid to the reaction solvent, stir at constant temperature for a certain time to dissolve L-ascorbic acid, then add the lipase microarray obtained in step (1), stir for a period of time to carry out esterification reaction, and obtain crude L-ascorbic acid fatty acid ester product; wherein, dry nitrogen gas is continuously introduced during the reaction process, and the water generated by the reaction is continuously removed by vacuum.

[0009] (3) The crude L-ascorbic acid fatty acid ester obtained in step (2) is separated and purified. First, the immobilized lipase and molecular sieve are removed by filtration, the reaction solvent is recovered by vacuum distillation, and then the unreacted L-ascorbic acid is removed by washing with pure water. The crude product L-ascorbic acid fatty acid ester is obtained by solvent extraction and crystallization. Finally, the crude product L-ascorbic acid fatty acid ester is purified by column chromatography, and then the solvent is removed by rotary evaporation to obtain pure L-ascorbic acid fatty acid ester.

[0010] According to the above scheme, in step (1), the hydrophobic mesoporous silicon is a monodisperse, uniformly sized hexagonal particle with an average diagonal length of 8-10 μm and a specific surface area of ​​150-300 m². 2 / g, thickness of 2-3μm, mesopore cavity size of 15-25nm, mesopore window size of 10-17nm, contact angle between 105° and 150°.

[0011] According to the above scheme, the preparation method of the microarray carrier is as follows: In HCl solution, a template agent and a pore-expanding agent are added and stirred vigorously for 1-3 hours. Then, a mixture of hydrophobic silane and tetraethoxysilane (TEOS) is added, stirred evenly, and allowed to stand at low temperature for 18-30 hours to obtain a mixed solution. Then, the mixed solution is transferred to a hydrothermal reactor and aged at 140-160℃ for 18-30 hours. The solid product is filtered out, washed with ethanol, and dried to obtain a dry powder. Finally, the dried powder is placed in an acidic ethanol aqueous solution and stirred at 50-70℃ to remove the pore-forming agent and the pore-expanding agent to obtain hydrophobic mesoporous silica, which is the microarray carrier.

[0012] Furthermore, the template agent is one or more of block polyether F-127, block copolymer P123, polyethylene glycol PEG, polyoxyethylene PEO, etc.; the pore-expanding agent is one or more of mesitylene, Span 80, decane, dodecane, etc.; and the hydrophobic silane is one or more of ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, polymethylhydrosiloxane, butyltriethoxysilane, octyltriethoxysilane, dodecyltriethoxysilane, octadecyltriethoxysilane, etc.

[0013] Furthermore, the concentration of the HCl solution is 0.5-1.5M, and the amount of template agent and pore-expanding agent added to the HCl solution is 0.01-0.05 g / mL; the total amount of hydrophobic silane and TEOS added to the HCl solution is 100-400 mmol / L, and the molar ratio of hydrophobic silane to TEOS is 1:3-1:10.

[0014] According to the above scheme, in step (1), the free enzyme is one or more of the following: Candida pleurisy lipase (CRL), Candida antarcticis lipase (CALB), NS40086 lipase, Thermophilic filamentosa (TLL), Candida lipolyticis lipase (CLL).

[0015] According to the above scheme, in step (1), the pH of PBS buffer (50mM) is 6.0-8.0, the enzyme immobilization temperature is 10-40℃, and the time is 10-60min; the protein content of the enzyme solution is 1.5-9.0mg / mL, and the ratio of carrier to enzyme solution is 1:20-1:60 (w / v, g / mL).

[0016] According to the above scheme, in step (2), the fatty acids are selected from one or more of linolenic acid, nervonic acid, oleic acid, linoleic acid, DHA, EPA, ARA, lauric acid, palmitic acid, stearic acid, etc., and are derived from flaxseed oil, perilla oil, hemp seed oil, evening primrose oil, peony seed oil, sunflower seed oil, soybean oil, rapeseed oil, conjugated linoleic acid glycerides, algae oil, fish oil, garlic fruit oil, and Chinese tallow tree fruit oil, etc.

[0017] According to the above scheme, in step (2), the reaction solvent is one or more of acetonitrile, acetone, tert-butanol, tert-amyl alcohol, dimethyl sulfoxide, etc.

[0018] According to the above scheme, in step (2), the molar ratio of L-ascorbic acid and fatty acid is 1:1 to 1:9, and the concentrations of L-ascorbic acid and lipase microarray in the reaction solvent are 0.04 to 0.2 mol / L and 4 to 20 g / L, respectively.

[0019] According to the above scheme, in step (2), the esterification reaction temperature is 50-80℃, the time is 4-10h, and the conversion rate is greater than 70%. During the esterification reaction, the reactor is equipped with a vacuum reflux dehydration device to introduce dry nitrogen into the reaction system while simultaneously drawing a vacuum to remove the water produced in the reaction. The gas flow rate is 20-100m³. 3 / h, vacuum degree is 1000~5000Pa.

[0020] According to the above scheme, in step (3), the immobilized lipase separated by filtration is washed with solvent and then freeze-dried for reuse; the organic solvents used for extraction and crystallization are hexane, chloroform, etc.

[0021] According to the above scheme, the pure L-ascorbic acid fatty acid ester obtained in step (3) has a content greater than 95%, a melting point between -13.3 and 116.6℃, a melting enthalpy between 10.1 and 130.6 J / g, and a solubility in edible oil between 243.5 and 4102.2 mg / kg.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. The L-ascorbic acid unsaturated fatty acid ester prepared by this invention has a lower melting point and better lipid solubility than commercially available L-ascorbic acid palmitate (L-AP), and its antioxidant properties are comparable to those of L-AP, which can significantly expand the application range of L-ascorbic acid fatty acid ester in food and daily chemical fields.

[0024] 2. This invention is widely applicable to catalyzing the esterification reaction of fatty acids with different structures with L-ascorbic acid, preparing L-ascorbic acid fatty acid esters with different structures such as L-ascorbic acid oleate, linoleate, linolenic acid ester, DHA ester, ARA ester and nervate, which can meet the needs of different groups of people and application scenarios.

[0025] 3. This invention synthesizes a microarray carrier with hydrophobic and mesoporous structures in one step, avoiding cumbersome post-hydrophobic modification steps and the use of large amounts of organic solvents such as toluene. Furthermore, the pore size and hydrophobicity are controllable. The activity and stability of lipase immobilized on this carrier are higher than those of free enzymes and commercial enzymes. Moreover, the cage-like structure of this carrier facilitates the entry of lipase, while the smaller window prevents enzyme leakage during use, which is conducive to efficient mass transfer of reactants in the system and thus improves enzyme catalytic efficiency.

[0026] 4. The entire process of this invention is carried out under mild reaction conditions and with a short reaction time. Compared with traditional chemical catalysis methods, it does not use strong acidic chemical catalysts and does not generate a large amount of acidic wastewater, effectively saving production costs and reducing environmental pollution. In addition, the enzyme microarray can be regenerated and reused multiple times, which has strong prospects for industrial application. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the enzymatic synthesis of L-ascorbic acid fatty acid esters.

[0028] Figure 2 The images shown are: scanning electron microscope (a), transmission electron microscope (b), nitrogen isothermal adsorption-desorption diagram (c), and pore size distribution diagram (d) of the hydrophobic mesoporous silica support of Example 1.

[0029] Figure 3 The contact angles are those of a conventional mesoporous silica carrier (b) and a hydrophobic mesoporous silica carrier (a) in Example 5.

[0030] Figure 4 Mass spectra of three L-ascorbic acid fatty acid esters synthesized by enzymatic method.

[0031] Figure 5 The reusability of synthesizing three L-ascorbic acid fatty acid esters using a lipase microarray. Detailed Implementation

[0032] To better understand the present invention, the following specific embodiments further illustrate the content of the present invention, but the present invention is not limited to the following embodiments.

[0033] In the following examples, the preparation process of the hydrophobic microarray carrier is as follows: 0.05 g / mL of block polyether F-127 was dissolved in 1 L of 1.0 M HCl solution, and then 0.05 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 2 h to obtain mixture A. Then, a mixture of 100 mmol octyltrimethoxysilane (C8) and 300 mmol tetraethoxysilane (TEOS) was slowly added to mixture A, and stirring was continued for 15 min, followed by standing for 24 h to obtain mixture B. Mixture B was then transferred to a hydrothermal reactor and aged at 150 °C for 24 h. After the reactor had completely cooled, the mixture was removed from the reactor, filtered, washed with ethanol, and dried. Finally, the dried powder is placed in an acidic ethanol-water solution (HCl:ethanol:water = 10:20:70 (v / v / v)) and stirred at 60°C to remove the pore-forming and expanding agents, thus preparing a SiO2 support with a mesoporous structure, namely a hydrophobic microarray support (OMMs-C8), as shown below. Figure 2 As shown, the particles are monodisperse, uniformly sized hexagonal prisms with a diameter of 8–10 μm and a thickness of 2–3 μm; through… Figure 2 Based on nitrogen adsorption-desorption isotherms and pore size distribution data, the mesopore cavity size is calculated to be 15–25 nm, the mesopore window size is 10–17 nm, and the measured specific surface area is approximately 200–300 m². 2 / g; such as Figure 3 As shown, the contact angle is 110°.

[0034] In the following examples, the content of L-ascorbic acid fatty acid esters was detected by liquid chromatography under the following conditions: a Venusil XBP Silica 5um 4.6×250mm column; a PDA detector; an injection volume of 10μL; a column temperature of 35℃; a mobile phase of acetonitrile / 0.05% acetic acid in water (70:30, v:v); isocratic elution; a flow rate of 1mL / min; and a detection wavelength of 254nm.

[0035] Structure determination of L-ascorbic acid fatty acid esters, high-resolution mass spectrometry: Thermo Scientific Q Exactive, ESI ion source, negative mode. Mass spectrometry conditions were as follows: capillary voltage set to 3.2 kV; sheath gas and drying gas temperatures adjusted to 350 °C and 150 °C, respectively; nitrogen flow rates of sheath gas and drying gas at 12 L / min and 15 L / min, respectively.

[0036] The melting point, lipid solubility, and antioxidant properties of L-ascorbic acid fatty acid esters were determined by differential scanning calorimetry (DSC), equilibrium solubility method, and oxidation induction time method, respectively.

[0037] Example 1

[0038] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0039] (1) Preparation of hydrophobic enzyme microarray: 2g of OMMs-C8 was added to 100mL of 9mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 60min at 10℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the enzyme microarray CALB@OMMs-C8 with a loading capacity of 300mg / g and an activity of 180U / g.

[0040] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.18 mol oleic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 70 °C for 20 min. Then, 1.0 g CL@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 70 °C. At the same time, dry nitrogen gas was continuously introduced into the reaction system for 20 min. 3 The reaction proceeded at a rate of 1000–5000 Pa per hour, while simultaneously applying a vacuum of 1000–5000 Pa to remove the water produced during the reaction. After 4 hours, the product was removed and analyzed by HPLC, revealing a conversion rate of 80.5% for L-ascorbic acid oleate.

[0041] (3) Separation and purification: The product obtained in step (2) was first filtered to remove the enzyme and molecular sieve, and the reaction solvent was recovered by vacuum distillation; unreacted L-ascorbic acid was removed by washing with pure water several times; crude L-ascorbic acid fatty acid ester was obtained by extraction and crystallization with n-hexane; then purified by silica gel column chromatography using ethyl acetate:petroleum ether (1:1, v:v) as the eluent, and finally the solvent was removed by rotary evaporation to obtain the final product. The purity of the product was determined to be 97.2% L-ascorbic acid oleate.

[0042] Comparison Example

[0043] Compared to Example 1, no hydrophobic silane was added during the preparation process. A hydrophilic mesoporous SiO2 support (OMMS) was prepared, and a hydrophilic lipase microarray CALB@OMMS was obtained using the same immobilization method. The immobilization load was 40 mg / g, the activity was 30 U / g, and the conversion rate of L-ascorbic acid oleate was 20.6%. Compared with the hydrophobic lipase microarray in Example 1, the immobilization load and catalytic efficiency were significantly reduced.

[0044] Example 2

[0045] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0046] (1) Preparation of hydrophobic enzyme microarray: 2g of OMMs-C8 was added to 100mL of 6mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 50min at 20℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 230mg / g and an activity of 100U / g.

[0047] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.10 mol linoleic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 70 °C for 20 min. Then, 0.8 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 70 °C. At the same time, 100 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 4h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid linoleate was 85.1%;

[0048] (3) Separation and purification: Same as in Example 1, the purity of L-ascorbic acid linoleate is 96.2%.

[0049] Example 3

[0050] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0051] (1) Add 2g of OMMs-C8 to 100mL of 9mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH 7.0 PBS), mix at 20℃ and 220rpm for 50min in a shaker, and freeze-dry the precipitate after centrifugation to obtain the lipase microarray CALB@OMMs-C8 with a loading of 220mg / g and an activity of 160U / g;

[0052] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.10 mol linolenic acid were added to 100 mL of organic solvent tert-butanol and stirred in a magnetic stirrer at 70 °C for 20 min. Then, 2.0 g of CALB@OMMs-C was added. p Then place it in a 70℃ magnetic stirrer and stir, while continuously introducing 60m of dry nitrogen gas into the reaction system. 3 / h, removing the water produced in the reaction. After 4h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid linolenic acid ester was 86.8%;

[0053] (3) Separation and purification: Same as in Example 1, the purity of L-ascorbic acid linolenic acid ester was 96.2%.

[0054] Example 4

[0055] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0056] (1) Preparation of hydrophobic enzyme microarray: 1.2g OMMs-C8 was added to 100mL of 9mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 60min at 10℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 230mg / g and an activity of 110U / g.

[0057] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.16 mol DHA were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 70 °C for 20 min. Then, 1.6 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 70 °C. At the same time, 60 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 4 hours, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid DHA ester was 82.5%;

[0058] (3) Separation and purification: Same as in Example 1, the purity of L-ascorbic acid DHA ester is 95.8%.

[0059] Example 5

[0060] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0061] (1) Preparation of hydrophobic enzyme microarray: 2.0g OMMs-C8 was added to 100mL of 5.0mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH6.0 PBS), and mixed in a shaker at 220rpm for 40min at 20℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 100mg / g and an activity of 90U / g.

[0062] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.18 mol palmitic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 70 °C for 20 min. Then, 1.4 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 70 °C. At the same time, 60 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 4h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid palmitate was 80.7%;

[0063] (3) Separation and purification: Same as in Example 1, the purity of L-ascorbate palmitate (L-AP) was 95.1%.

[0064] Table 1 compares the melting points and solubilities of the five L-ascorbic acid esters prepared in Examples 1-5. Table 2 compares the antioxidant properties of the L-ascorbic acid esters prepared in Examples 1-5, showing that their antioxidant properties are comparable to those of L-AP.

[0065] Table 1 Melting point and solubility of L-ascorbic acid fatty acid esters

[0066]

[0067] Table 2 Comparison of antioxidant properties of L-ascorbic acid ester

[0068]

[0069] Note: In the antioxidant performance test of L-ascorbic acid ester in Table 2, the amount of L-ascorbic acid ester prepared in each example added to the oil sample was 200 ppm.

[0070] Example 6

[0071] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0072] (1) Preparation of hydrophobic enzyme microarray: 1.5g OMMs-C8 was added to 100mL of 9mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 50min at 20℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 200mg / g and an activity of 90U / g.

[0073] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.18 mol nervonic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 60 °C for 20 min. Then, 0.4 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 60 °C. At the same time, 50 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 10h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid nervate was 72.3%;

[0074] (3) Isolation and purification of L-ascorbic acid fatty acid esters: Same as in Example 2, the purity of the products was determined to be 97.2% for L-ascorbic acid nervate.

[0075] Example 7

[0076] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0077] (1) Preparation of hydrophobic enzyme microarray: 2g of OMMs-C8 was added to 100mL of 1.5mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 20min at 30℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 120mg / g and an activity of 80U / g.

[0078] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.18 mol linoleic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 70 °C for 20 min. Then, 1.5 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 70 °C. At the same time, 50 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 10h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid linoleate was 71.3%;

[0079] (3) Isolation and purification of L-ascorbic acid fatty acid esters: Same as in Example 2, the purity of the products was determined to be 97.5% for L-ascorbic acid linoleate.

[0080] Example 8

[0081] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0082] (1) Preparation of hydrophobic enzyme microarray: 2g of OMMs-C8 was added to 100mL of 5mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 30min at 30℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 90mg / g and an activity of 120U / g.

[0083] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.02 mol linolenic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 50 °C for 20 min. Then, 2 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 50 °C. At the same time, 100 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 10h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid linolenic acid ester was 82.3%;

[0084] (3) Isolation and purification of L-ascorbic acid fatty acid esters: Same as in Example 3, the purity of the products was determined to be 97.8% for L-ascorbic acid linolenic acid esters.

[0085] Example 9

[0086] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0087] (1) Preparation of hydrophobic enzyme microarray: 0.5g OMMs-C8 was added to 100mL of 5mg / mL CALB enzyme solution (dissolved in 0.1mol / mL, pH7.0 PBS), and mixed in a shaker at 220rpm for 30min at 30℃. The precipitate after centrifugation was freeze-dried in a freeze dryer to obtain the lipase microarray CALB@OMMs-C8 with a loading capacity of 90mg / g and an activity of 100U / g.

[0088] (2) Enzymatic preparation of L-ascorbic acid fatty acid ester: 0.02 mol L-ascorbic acid and 0.18 mol linolenic acid were added to 100 mL of the organic solvent tert-butanol and stirred in a magnetic stirrer at 50 °C for 20 min. Then, 1.5 g of CALB@OMMs-C8 was added, and the mixture was stirred again in a magnetic stirrer at 50 °C. At the same time, 100 mL of dry nitrogen gas was continuously introduced into the reaction system. 3 / h, removing the water produced in the reaction. After 4h, the sample was taken out for HPLC content determination, and the conversion rate of L-ascorbic acid linolenic acid ester was 75.0%;

[0089] (3) Isolation and purification of L-ascorbic acid fatty acid esters: Same as in Example 3, the purity of the products was determined to be 96.4% for L-ascorbic acid linolenic acid esters.

[0090] Example 10

[0091] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0092] It is basically the same as Example 1, except that the organic solvent in step (2) is acetone, and the conversion rate of L-ascorbic acid oleate was determined to be 74.9%.

[0093] Example 11

[0094] An enzymatic method for preparing L-ascorbic acid fatty acid esters and the resulting product, specifically comprising the following steps:

[0095] The results are basically the same as in Examples 1-3, except that after step (2), an experiment was added to recycle the synthesis of three L-ascorbic acid fatty acid esters using a lipase microarray catalysis. Specifically, after the reaction, the lipase microarray was rinsed with the reaction solvent and dried to remove the raw materials, products, and moisture adhering to the enzyme, resulting in activated and regenerated immobilized enzymes that were then subjected to the same reaction. Figure 5 As shown, the lipase microarray still exhibits high activity after being reused 10 times.

[0096] All the raw materials listed in this invention, as well as the upper and lower limits and ranges of the amounts of raw materials and enzymes, and the upper and lower limits and ranges of process parameters (such as temperature, time, and homogenization speed), can achieve this invention. Examples are not listed here. It should be noted that those skilled in the art can make various improvements and modifications without departing from the inventive concept of this invention, and these all fall within the scope of protection of this invention.

Claims

1. A method for preparing L-ascorbic acid fatty acid esters by enzymatic method, characterized in that... Includes the following steps: (1) The free enzyme was dissolved in PBS buffer and immobilized on the microarray carrier by multiple adsorption method and freeze-dried to obtain the lipase microarray with an immobilization load of 100-300 mg / g and an activity of 80-180 U / g; the microarray carrier was monodisperse, uniformly sized hydrophobic mesoporous silica hexagonal particles. (2) Add L-ascorbic acid and fatty acids to the reaction solvent and stir for a certain time to dissolve L-ascorbic acid. Then add the lipase microarray obtained in step (1) and stir for a period of time to carry out the esterification reaction to obtain crude L-ascorbic acid fatty acid ester. During the reaction, nitrogen gas is introduced and vacuum is used to remove the water generated in the reaction. The gas flow rate is 20-100 m³ / h. 3 / h, with a vacuum degree of 1000-5000 Pa; the fatty acids are selected from one or more of linolenic acid, nervonic acid, linoleic acid, DHA, EPA, ARA, lauric acid, palmitic acid, and stearic acid; (3) The crude L-ascorbic acid fatty acid ester obtained in step (2) is separated and purified. First, the lipase microarray is removed by filtration, the reaction solvent is recovered by vacuum distillation, and then the unreacted L-ascorbic acid is removed by washing with pure water. The crude product L-ascorbic acid fatty acid ester is obtained by solvent extraction and crystallization. Finally, the crude product L-ascorbic acid fatty acid ester is purified by column chromatography, and then the solvent is removed by rotary evaporation to obtain high-purity L-ascorbic acid fatty acid ester. The microarray carrier is prepared as follows: In an HCl solution, a template agent (block polyether F-127) and a pore-expanding agent (trimethylbenzene) are added and stirred vigorously for 1–3 h. Then, a mixture of octyltrimethoxysilane and tetraethoxysilane is added, stirred until homogeneous, and allowed to stand at a low temperature for 18–30 h to obtain a mixed solution. This mixed solution is then transferred to a hydrothermal reactor and aged at 140–160 °C for 18–30 h. The solid product is filtered out, washed with ethanol, and dried to obtain a dry powder. Finally, the dried powder is placed in an acidic ethanol aqueous solution and stirred at 50–70 °C to remove the pore-forming and pore-expanding agents, yielding hydrophobic mesoporous silica, which is the microarray carrier. The concentration of the HCl solution is 0.5–1.5 M, and the amount of template agent and pore-expanding agent added to the HCl solution is 0.01–0.05 g / mL. The total amount of octyltrimethoxysilane and tetraethoxysilane added to the HCl solution is 100–400 g / mL. The molar ratio of octyltrimethoxysilane to tetraethoxysilane is 1:3 to 1:10 (mmol / L).

2. The method for preparing L-ascorbic acid fatty acid esters by enzymatic method according to claim 1, characterized in that... In step (1), each particle of the hydrophobic mesoporous silicon hexagon has an average diagonal length of 8–10 μm and a specific surface area of ​​150–300 m². 2 / g, with a thickness of 2–3 μm, a mesopore cavity size of 15–25 nm, a mesopore window size of 10–17 nm, and a contact angle between 105° and 150°.

3. The method for preparing L-ascorbic acid fatty acid esters by enzymatic method according to claim 1, characterized in that... In step (1), the free enzyme is one or more of the following: Candida pleurisy lipase, Candida antarcticis lipase, NS40086 lipase, Thermophilus sparsely cottony, and Candida lipolyticis lipase.

4. The method for preparing L-ascorbic acid fatty acid esters by enzymatic method according to claim 1, characterized in that... In step (1), the pH of the PBS buffer is 6.0 to 8.0, the enzyme immobilization temperature is 10 to 40 °C, and the time is 10 to 60 min; the protein content of the enzyme solution is 1.5 to 9.0 mg / mL, and the ratio of carrier to enzyme solution is 1:20 to 1:60 g / mL (w / v).

5. The method for preparing L-ascorbic acid fatty acid esters by enzymatic method according to claim 1, characterized in that... In step (2), the reaction solvent is one or more of acetonitrile, acetone, tert-butanol, tert-amyl alcohol, and dimethyl sulfoxide.

6. The method for preparing L-ascorbic acid fatty acid esters by enzymatic method according to claim 1, characterized in that... In step (2), the molar ratio of L-ascorbic acid to fatty acid is 1:1 to 1:9, and the concentrations of L-ascorbic acid and lipase microarray in the reaction solvent are 0.04 to 0.2 mol / L and 4 to 20 g / L, respectively; the stirring and esterification reaction temperature is 50 to 80 ℃, the time is 4 to 10 h, and the conversion rate is greater than 70%.

7. The high-purity L-ascorbic acid fatty acid ester prepared by the method of claim 1, characterized in that... The high-purity L-ascorbic acid fatty acid ester has a content greater than 95%, a melting point between -13.3 and 116.6 °C, a melting enthalpy between 10.1 and 130.6 J / g, and a solubility in edible oil between 243.5 and 4102.2 mg / kg.

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