Synthesis process of VC (Vitamin C) derivative ascorbyl glyceride

Abstract

The invention belongs to the technical field of ascorbic acid derivative synthesis, and particularly relates to a synthesis process of VC derivative ascorbic acid glyceride. Comprising the following steps: S1, co-loading candida antarctica lipase and glycosyltransferase into hierarchical pore silicon dioxide nanospheres, then dispersing the hierarchical pore silicon dioxide nanospheres into a eutectic solvent, and uniformly dispersing to obtain a reaction system; s2, adding vitamin C and glycerol into the reaction system, heating to 40-50 DEG C, and stirring to react for 10-12 hours; and S3, after the reaction is finished, collecting a product, and recovering the hierarchical pore silicon dioxide nanospheres. According to the method, the limitation of traditional chemical synthesis and single-enzyme catalysis is broken through, and stable ascorbyl glyceride with high antioxidant activity is efficiently synthesized by combining double-enzyme synergistic catalysis with the confinement reaction of the hierarchical porous silicon dioxide nanospheres and a special eutectic solvent system; and a theoretical basis is provided for a process for preparing ascorbyl glyceride through double-enzyme synergy.

Description

Technical Field

[0001] The present invention belongs to the technical field of the synthesis of ascorbic acid derivatives, and particularly relates to a synthesis process of a VC derivative, ascorbyl glycerol ester. Background Art

[0002] Vitamin C is a common water-soluble vitamin and an essential nutrient element in the human body. It plays an important role in various physiological activities in the human body and is applied in industries such as food, cosmetics, medicine, and aquaculture. However, due to its instability and easy oxidation, its application in various fields has been greatly restricted.

[0003] Since the 1970s, in order to overcome the disadvantage of the instability of VC, the research on VC derivatives has been concerned by experts and scholars, aiming to find a new derivative with both good stability and the ability to exert the physiological function of ascorbic acid. At present, a variety of VC derivatives have been developed, mainly including VC salts, VC esters, and VC glycoside derivatives.

[0004] Glycerol (propanetriol) has three hydroxyl groups, and ascorbic acid (vitamin C) has an enediol structure. Under certain conditions, the hydroxyl group in the ascorbic acid molecule can undergo an esterification reaction with the hydroxyl group in the glycerol molecule to form ascorbyl glycerol ester. The current preparation methods mainly include: (1) Chemical synthesis method: The esterification reaction is catalyzed by strong acids (such as sulfuric acid and p-toluenesulfonic acid), which has problems such as many by-products (such as oxidative degradation), deep color of the product (requiring decolorization with activated carbon), and serious equipment corrosion. The purity of the product is usually <85%.

[0005] (2) Single enzyme catalysis method: The transesterification reaction is catalyzed by traditional lipases (such as Novozym 435). However, due to the high water solubility of vitamin C and the large steric hindrance of the glycerol molecule, the conversion rate is low (<50%), and the reusability of the enzyme is poor (the activity is lost by 60% after <5 times).

[0006] The ascorbyl glycerol ester synthesized by the above methods has poor stability, and the ascorbyl glycerol ester is easy to oxidize. The traditional process lacks efficient stabilization means.

[0007] Based on this, we propose a synthesis process of a VC derivative, ascorbyl glycerol ester, hoping to solve the deficiencies in the prior art. Summary of the Invention

[0008] The purpose of the present invention is to provide a synthesis process of a VC derivative, ascorbyl glycerol ester, aiming at the existing problems.

[0009] The present invention is achieved by the following technical solutions: A synthesis process of a VC derivative, ascorbyl glycerol ester, includes the following steps: S1. Co - load Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres, and then disperse them into a deep eutectic solvent. After uniform dispersion, a reaction system is obtained; S2. Add vitamin C and glycerol to the reaction system, heat up to 40 - 50 °C, and stir - react for 10 - 12 h; S3. After the reaction ends, collect the product and recover the hierarchical porous silica nanospheres.

[0010] Further preferably, the specific steps of co - loading Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres in step S1 include: (1) Ultrasonically disperse polylactic acid microspheres into a 70% ethanol solution. After uniform dispersion, add an amphiphilic block copolymer and 1 - butyl - 3 - methylimidazolium tetrafluoroborate, and continue ultrasonic dispersion to form a uniform emulsion; (2) After dropping tetraethyl orthosilicate and stirring evenly, dropwise add ammonia water to adjust the pH to 9 - 10. Under the conditions of 40 - 50 °C and 100 - 200 rpm, stir at a constant temperature for 8 - 10 h, and then perform gradient temperature - rising treatment to obtain pretreated porous silica nanospheres; (3) Ultrasonically disperse the pretreated hierarchical porous silica nanospheres obtained in step (2) into anhydrous ethanol. While stirring, add γ - (2,3 - epoxypropoxy) propyltrimethoxysilane with a mass 0.05 - 0.15 times that of the pretreated hierarchical porous silica nanospheres. Stir at 100 - 200 rpm and 70 - 90 °C for 3 - 4 h, then perform suction filtration. Then wash with anhydrous ethanol 3 times in sequence, then wash with deionized water 3 times, and finally place it in a vacuum drying oven. After drying to a constant weight at 60 - 70 °C, perform plasma treatment in a plasma equipment to obtain hierarchical porous silica nanospheres; (4) Add Candida antarctica lipase and glycosyltransferase to a phosphate - buffered saline (PBS) in a mass ratio of 1:1 - 2 to prepare an enzyme solution. Then add hierarchical porous silica nanospheres, stir and mix evenly, and place it in a constant - temperature shaker at 35 - 37 °C and 100 - 200 rpm for immobilization for 12 - 16 h. Then filter out the immobilized enzyme particles, wash with phosphate - buffered solution 3 - 5 times, and then place it in a vacuum drying oven. After drying at a low temperature, store it for later use.

[0011] Further preferably, the particle size of the polylactic acid microspheres in step (1) is 200 - 300 nm; The addition amount of the amphiphilic block copolymer is 5 wt%, and the addition amount of 1 - butyl - 3 - methylimidazolium tetrafluoroborate is 10 wt%.

[0012] Further preferably, the concentration of tetraethyl orthosilicate in step (2) is 0.8 - 1.2 mol / L; The specific operation of the gradient temperature rise is as follows: first, pyrolyze with nitrogen at 180 - 200 °C for 8 - 10 min, then oxidize with air at 300 - 360 °C for 6 - 8 min, and finally calcine at 500 - 600 °C for 5 - 7 min.

[0013] Further preferably, in step (3), the power of the plasma treatment is 50 - 100 W, and the treatment time is 6 - 8 min.

[0014] Further preferably, in step (4), the mass sum of the Candida antarctica lipase and the glycosyltransferase and the mass - volume ratio of the phosphate - buffered saline (PBS) is 1 mg: 20 - 30 mL; The addition amount of the hierarchically - porous silica nanospheres is 1 - 1.5 times the mass sum of the Candida antarctica lipase and the glycosyltransferase; The specific parameters of the drying under low - temperature conditions are: set the cold trap temperature of the vacuum freeze - dryer to - 40 - - 30 °C, set the vacuum degree to 20 Pa, and the drying temperature to - 30 - - 20 °C.

[0015] Further preferably, the preparation of the deep - eutectic solvent in step S1 includes the following steps: 1) Place choline chloride in a vacuum drying oven and dry it at 60 - 70 °C for 10 - 12 h, and dehydrate glycerol using a molecular sieve. 2) Add the treated choline chloride and glycerol to a reaction kettle in a molar ratio of 1: 2 - 4, then add an ionic liquid to the reaction kettle, raise the temperature to 50 - 60 °C, stir and mix evenly, then raise the temperature to 70 - 80 °C, and apply ultrasonic treatment for 1 - 2 h. 3) Cool down to 40 - 50 °C at a rate of 1 - 2 °C / min, keep it at a constant temperature for 2 - 3 h, then transfer it to a microwave reactor, perform microwave treatment at 300 - 500 W for 10 - 15 min, and then transfer it to a vacuum dryer to dehydrate at 50 - 60 °C for 18 - 20 h.

[0016] Further preferably, the addition amount of the ionic liquid in step 2) is 10 - 20% of the total amount of choline chloride and glycerol, and the preparation method of the ionic liquid is: add sorbitol and citric acid to a reaction kettle in a mass ratio of 1: 2.5 - 3, stir and mix evenly, then raise the temperature to 90 - 100 °C and react for 3 - 5 h, then cool down to 60 - 80 °C, add choline chloride 1.5 - 2 times the amount of sorbitol, stir and react at 200 - 300 rpm for 2 - 3 h, and filter after cooling to room temperature.

[0017] Further preferably, the mass - volume ratio of the hierarchically - porous silica nanospheres to the deep - eutectic solvent in step S1 is 1 - 3 g: 100 mL.

[0018] Further preferably, the molar ratio of vitamin C to glycerol in step S2 is 1:1.5 to 2.

[0019] The present invention has the following advantages compared with the prior art: The present invention breaks through the limitations of traditional chemical synthesis and single-enzyme catalysis. Through the dual-enzyme co-catalysis combined with the confined reaction of hierarchical porous silica nanospheres and a special deep eutectic solvent system, ascorbyl glycerol ester with high antioxidant activity and stability is efficiently synthesized. The ascorbyl glycerol ester prepared by the method of the present invention maintains high antioxidant activity, and the scavenging rates for superoxide anion radicals, hydroxyl radicals and DPPH radicals are 92.39%, 90.89% and 95.61% respectively. It provides a theoretical basis for the process of preparing ascorbyl glycerol ester by dual-enzyme co-catalysis.

[0020] First of all, with the help of gradient pyrolysis technology, the present invention realizes the precise construction of the microporous-mesoporous-macroporous three-level structure of silica. The finally prepared hierarchical porous silica nanospheres have a high specific surface area, hierarchical mass transfer channels and a functionalizable surface. Candida antarctica lipase and glycosyltransferase are co-loaded into the hierarchical porous silica nanospheres to form a "confined catalysis" environment, restricting the reaction within the nanopores, improving the mass transfer efficiency of the substrate, increasing the local concentration of the substrate. Candida antarctica lipase and glycosyltransferase act synergistically to catalyze the glycosylation pre-activation of glycerol and microorganism C, reducing the reaction activation energy, promoting the synthesis of the product, and co-loading Candida antarctica lipase and glycosyltransferase into the hierarchical porous silica nanospheres can prevent enzyme inactivation, and the enzyme can be recycled, reducing costs.

[0021] Secondly, the solvent system of the present invention is a deep eutectic solvent, which is composed of choline chloride and glycerol, and has both the functions of a solvent and a substrate, further promoting the synthesis of the product. In the preparation of the deep eutectic solvent, polar groups such as hydroxyl groups and carboxyl groups in the ionic liquid can form hydrogen bonds with choline chloride and glycerol molecules, playing a bridging role to promote the formation of hydrogen bonds between choline chloride and glycerol. At the same time, the presence of the ionic liquid can also change the microstructure and solvation environment of the system, further enhancing the stability of the hydrogen bonds. With the help of the thermal effect and non-thermal effect of microwaves, the intermolecular interaction is further enhanced, enabling the ionic liquid to play a full role and making the deep eutectic solvent system more stable and uniform. In addition, the deep eutectic solvent of the present invention can be recycled, making the entire synthesis process more environmentally friendly. Description of the Drawings

[0022] Figure 1 It is a diagram of the scavenging ability for superoxide anion radicals; Figure 2 It is a diagram of the scavenging ability for hydroxyl radicals; Figure 3 It is a diagram of the scavenging ability for DPPH radicals. Detailed implementation manners

[0023] For further explanation of the present invention, the following specific embodiments are described below.

[0024] Example 1

[0025] A synthesis process of a VC derivative, ascorbyl glycerol, comprises the following steps: S1. Co-load Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres, and then disperse them into a deep eutectic solvent, and disperse evenly to obtain a reaction system; The mass-volume ratio of the hierarchical porous silica nanospheres to the deep eutectic solvent is 1 g: 100 mL; The specific steps of co-loading Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres include: (1) Ultrasonically disperse polylactic acid microspheres (with a particle size of 200 nm) into a 70% ethanol solution. After dispersing evenly, add an amphiphilic block copolymer (5 wt%) and 1-butyl-3-methylimidazolium tetrafluoroborate (10 wt%), and continue to ultrasonically disperse evenly to form an emulsion; (2) After dropping tetraethyl orthosilicate (0.8 mol / L) and stirring evenly, dropwise add ammonia water to adjust the pH to 9, and keep stirring at 40 °C and 100 rpm for 8 h. First, pyrolyze with nitrogen at 180 °C for 8 min, then oxidize with air at 300 °C for 6 min, and finally calcine at 500 °C for 5 min to obtain pretreated porous silica nanospheres; (3) Ultrasonically disperse the pretreated hierarchical porous silica nanospheres obtained in step (2) into absolute ethanol, add γ-(2,3-epoxypropoxy)propyltrimethoxysilane which is 0.05 times the mass of the pretreated hierarchical porous silica nanospheres while stirring, stir at 100 rpm and 70 °C for 3 h, then filter, and then wash with absolute ethanol three times in sequence, then wash with deionized water three times, and finally place it in a vacuum drying oven, dry to constant weight at 60 °C, and then place it in a plasma equipment, and perform plasma treatment at 50 W for 6 min to obtain hierarchical porous silica nanospheres; (4) Add Candida antarctica lipase and glycosyltransferase into a phosphate buffer solution (PBS) in a mass ratio of 1:1 in sequence to prepare an enzyme solution, then add hierarchical porous silica nanospheres and stir evenly, and place it in a constant temperature shaker at 35 °C and 100 rpm for immobilization for 12 h, then filter out the immobilized enzyme particles, wash them three times with a phosphate buffer solution, and then place them in a vacuum drying oven. Set the cold trap temperature of the vacuum freeze dryer to -40 °C, the vacuum degree to 20 Pa, and the drying temperature to -30 °C, and dry and store for later use under low temperature conditions; The mass sum of the Candida antarctica lipase and the glycosyltransferase and the mass-to-volume ratio of the phosphate buffer solution (PBS) is 1 mg: 20 mL; The addition amount of the hierarchical porous silica nanospheres is 1 time the mass sum of the Candida antarctica lipase and the glycosyltransferase; The preparation of the deep eutectic solvent includes the following steps: 1) Place choline chloride in a vacuum drying oven and dry it at 60 °C for 10 h, and dehydrate glycerol using a molecular sieve; 2) Add the treated choline chloride and glycerol to the reaction kettle according to a molar ratio of 1:2, then add 10% of the ionic liquid based on the total amount of choline chloride and glycerol to the reaction kettle, heat up to 50 °C, stir and mix evenly, then heat up to 70 °C, and apply ultrasonic treatment for 1 h; The preparation method of the ionic liquid is: add sorbitol and citric acid to the reaction kettle according to a mass ratio of 1:2.5, stir and mix evenly, then heat up to 90 °C and react for 3 h, then cool down to 60 °C, add choline chloride 1.5 times that of sorbitol, and stir and react at 200 rpm for 2 h. After cooling to room temperature, filter; 3) Cool down to 40 °C at a rate of 1 °C / min, keep it at a constant temperature and age for 2 h, then transfer it to a microwave reactor, treat it at 300 W for 10 min, and then transfer it to a vacuum dryer to dehydrate at 50 °C for 18 h; S2. Add vitamin C and glycerol with a molar ratio of 1:1.5 to the reaction system, heat up to 40 °C, and stir and react for 10 h; S3. After the reaction is completed, collect the product and recover the hierarchical porous silica nanospheres.

[0026] Example 2

[0027] A synthesis process of a VC derivative, ascorbyl glycerol, includes the following steps: S1. Co-load Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres, and then disperse them into a deep eutectic solvent to obtain a uniformly dispersed reaction system; The mass-to-volume ratio of the hierarchical porous silica nanospheres to the deep eutectic solvent is 2 g: 100 mL; The specific steps of co-loading Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres include: (1) Ultrasonically disperse poly(lactic acid) microspheres (particle size of 250 nm) in a 70% ethanol solution. After uniform dispersion, add an amphiphilic block copolymer (5 wt%) and 1-butyl-3-methylimidazolium tetrafluoroborate (10 wt%), and continue ultrasonic dispersion to form a uniform emulsion; (2)After dropwise adding tetraethyl orthosilicate (1 mol / L) and stirring evenly, ammonia water was added dropwise to adjust the pH to 9.5. After constant temperature stirring at 45 °C and 150 rpm for 9 h, it was first pyrolyzed with nitrogen at 190 °C for 9 min, then oxidized with air at 330 °C for 7 min, and finally calcined at 550 °C for 6 min to obtain pretreated hierarchical porous silica nanospheres; (3)The pretreated hierarchical porous silica nanospheres obtained in step (2) were ultrasonically dispersed in absolute ethanol. While stirring, γ-(2,3-epoxypropoxy)propyltrimethoxysilane, which was 0.1 times the mass of the pretreated hierarchical porous silica nanospheres, was added. After stirring at 150 rpm and 80 °C for 3.5 h, filtration was carried out, and then it was washed 3 times with absolute ethanol and 3 times with deionized water in sequence. Finally, it was placed in a vacuum drying oven and dried to constant weight at 65 °C, and then placed in a plasma equipment for 7 min of plasma treatment at 70 W to obtain hierarchical porous silica nanospheres; (4)Candida antarctica lipase and glycosyltransferase were added to phosphate buffer solution (PBS) in a mass ratio of 1:1.5 in sequence to prepare an enzyme solution. Then, hierarchical porous silica nanospheres were added and stirred evenly, and then placed in a constant temperature shaker at 36 °C and 150 rpm for immobilization for 14 h. Then, the immobilized enzyme particles were filtered out, washed 4 times with phosphate buffer solution, and then placed in a vacuum drying oven. The cold trap temperature of the vacuum freeze dryer was set at -35 °C, the vacuum degree was set at 20 Pa, and the drying temperature was -25 °C. After drying under low temperature conditions, it was stored for standby; The mass volume ratio of the total mass of the Candida antarctica lipase and glycosyltransferase to the phosphate buffer solution (PBS) is 1 mg:25 mL; The addition amount of the hierarchical porous silica nanospheres is 1.25 times the total mass of the Candida antarctica lipase and glycosyltransferase; The preparation of the deep eutectic solvent includes the following steps: 1) Choline chloride was placed in a vacuum drying oven and dried at 65 °C for 11 h, and glycerol was dehydrated with molecular sieves; 2) The treated choline chloride and glycerol were added to a reaction kettle in a molar ratio of 1:3, and then 15% of ionic liquid based on the total amount of choline chloride and glycerol was added to the reaction kettle. The temperature was raised to 55 °C, stirred evenly and then raised to 75 °C, and ultrasonic treatment was applied for 1.5 h; The preparation method of the ionic liquid is as follows: Sorbitol and citric acid were added to a reaction kettle in a mass ratio of 1:2.75, stirred evenly and then heated to 95 °C for reaction for 4 h. Then, after cooling to 70 °C, choline chloride 1.75 times that of sorbitol was added, and stirred at 250 rpm for reaction for 2.5 h. After cooling to room temperature, filtration was carried out; 3) After cooling to 45°C at a rate of 1.5°C / min, keep it at a constant temperature for 2.5 h and then transfer it to a microwave reactor. After treating it at 400 W for 12 min, transfer it to a vacuum dryer and dehydrate it at 55°C for 19 h; S2. Add vitamin C and glycerol with a molar ratio of 1:1.75 to the reaction system, heat up to 45°C, and stir and react for 11 h; S3. After the reaction ends, collect the product and recycle the hierarchical porous silica nanospheres.

[0028] Example 3

[0029] A synthesis process of a VC derivative, ascorbyl glycerol, comprises the following steps: S1. Co-load Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres, and then disperse them into a deep eutectic solvent to obtain a uniformly dispersed reaction system; The mass-volume ratio of the hierarchical porous silica nanospheres to the deep eutectic solvent is 3 g:100 mL; The specific steps of co-loading Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres include: (1) Ultrasonically disperse polylactic acid microspheres (with a particle size of 300 nm) into a 70% ethanol solution. After uniform dispersion, add an amphiphilic block copolymer (5 wt%) and 1-butyl-3-methylimidazolium tetrafluoroborate (10 wt%), and continue ultrasonic dispersion to form a uniform emulsion; (2) After dropping tetraethyl orthosilicate (1.2 mol / L) and stirring evenly, dropwise add ammonia water to adjust the pH to 10. Under the conditions of 50°C and 200 rpm, keep stirring at a constant temperature for 10 h. First, pyrolyze it with nitrogen at 200°C for 10 min, then oxidize it with air at 360°C for 8 min, and finally calcine it at 600°C for 5 - 7 min to obtain pretreated porous hierarchical silica nanospheres; (3) Ultrasonically disperse the pretreated hierarchical porous silica nanospheres obtained in step (2) into absolute ethanol. While stirring, add γ-(2,3-epoxypropoxy)propyltrimethoxysilane, which is 0.15 times the mass of the pretreated hierarchical porous silica nanospheres. Stir and process at 200 rpm and 90°C for 4 h, then filter by suction. Then wash it with absolute ethanol three times in sequence, then wash it with deionized water three times, and finally place it in a vacuum drying oven. After drying to a constant weight at 70°C, place it in a plasma equipment and treat it with 100 W plasma for 8 min to obtain hierarchical porous silica nanospheres; (4)Add Candida antarctica lipase and glycosyltransferase to phosphate buffer solution (PBS) in a mass ratio of 1:2 in sequence to prepare an enzyme solution. Then add hierarchical porous silica nanospheres and stir evenly. Place it in a constant temperature shaker at 37 °C and 200 rpm for immobilization for 16 h. Then filter out the immobilized enzyme particles, wash them 5 times with phosphate buffer, and place them in a vacuum drying oven. Set the cold trap temperature of the vacuum freeze dryer to -30 °C, the vacuum degree to 20 Pa, and the drying temperature to -20 °C. Dry and store for later use under low temperature conditions; The mass-volume ratio of the total mass of the Candida antarctica lipase and glycosyltransferase to the phosphate buffer solution (PBS) is 1 mg:30 mL; The addition amount of the hierarchical porous silica nanospheres is 1.5 times the total mass of the Candida antarctica lipase and glycosyltransferase; The preparation of the deep eutectic solvent includes the following steps: 1) Place choline chloride in a vacuum drying oven and dry it at 70 °C for 12 h. Dehydrate glycerol using molecular sieves; 2) Add the treated choline chloride and glycerol to the reaction kettle in a molar ratio of 1:4. Then add 20% of the ionic liquid based on the total amount of choline chloride and glycerol to the reaction kettle. Heat up to 60 °C, stir evenly, then heat up to 80 °C, and apply ultrasonic treatment for 2 h; The preparation method of the ionic liquid is as follows: Add sorbitol and citric acid to the reaction kettle in a mass ratio of 1:3, stir evenly, heat up to 100 °C and react for 5 h. Then cool down to 80 °C, add choline chloride 2 times the amount of sorbitol, stir and react at 300 rpm for 3 h, and filter after cooling to room temperature; 3) Cool down to 50 °C at a rate of 2 °C / min, keep it at a constant temperature for aging for 3 h, then transfer it to a microwave reactor, treat it at 500 W for 15 min, and then transfer it to a vacuum dryer to dehydrate at 60 °C for 20 h; S2. Add vitamin C and glycerol with a molar ratio of 1:2 to the reaction system, heat up to 50 °C, and stir and react for 12 h; S3. After the reaction is completed, collect the product and recover the hierarchical porous silica nanospheres.

[0030] Comparative Example 1 A synthesis process of a VC derivative ascorbyl glycerol ester includes the following steps: S1. Disperse Candida antarctica lipase and glycosyltransferase in a deep eutectic solvent in a mass ratio of 1:1.5, and disperse evenly to obtain a reaction system; The preparation of the deep eutectic solvent includes the following steps: 1) Place choline chloride in a vacuum drying oven and dry it at 65 °C for 11 h. Dehydrate glycerol using molecular sieves. 2) Add the treated choline chloride and glycerol to a reaction kettle in a molar ratio of 1:3. Then add 15% of the total amount of choline chloride and glycerol as ionic liquid to the reaction kettle. Heat up to 55 °C, stir and mix evenly, then heat up to 75 °C, and apply ultrasonic treatment for 1.5 h. The preparation method of the ionic liquid is as follows: Add sorbitol and citric acid to a reaction kettle in a mass ratio of 1:2.75, stir and mix evenly, then heat up to 95 °C and react for 4 h. Then cool down to 70 °C, add choline chloride 1.75 times that of sorbitol, and stir and react at 250 rpm for 2.5 h. After cooling to room temperature, filter it. 3) Cool down to 45 °C at a rate of 1.5 °C / min, keep it at a constant temperature and age for 2.5 h, then transfer it to a microwave reactor, treat it at 400 W for 12 min, and then transfer it to a vacuum dryer to dehydrate at 55 °C for 19 h. S2. Add vitamin C and glycerol with a molar ratio of 1:1.75 to the reaction system, heat up to 45 °C, and stir and react for 11 h. S3. After the reaction is completed, collect the product and recover the hierarchical porous silica nanospheres.

[0031] Comparative Example 2 A synthesis process of a VC derivative, ascorbyl glycerol, includes the following steps: S1. Load Candida antarctica lipase into hierarchical porous silica nanospheres, and then disperse it into a deep eutectic solvent to obtain a uniformly dispersed reaction system. The mass-to-volume ratio of the hierarchical porous silica nanospheres to the deep eutectic solvent is 2 g:100 mL. The specific steps of loading Candida antarctica lipase into hierarchical porous silica nanospheres include: (1) Ultrasonically disperse poly(lactic acid) microspheres (particle size of 250 nm) in a 70% ethanol solution. After uniform dispersion, add an amphiphilic block copolymer (5 wt%) and 1-butyl-3-methylimidazolium tetrafluoroborate (10 wt%), and continue to ultrasonically disperse evenly to form an emulsion. (2) After dropping tetraethyl orthosilicate (1 mol / L) and stirring evenly, dropwise add ammonia water to adjust the pH to 9.5. Under the conditions of 45 °C and 150 rpm, stir at a constant temperature for 9 h. First, pyrolyze in nitrogen at 190 °C for 9 min, then oxidize in air at 330 °C for 7 min, and finally calcine at 550 °C for 6 min to obtain pretreated porous silica nanospheres. (3) Ultrasonically disperse the pretreated hierarchical porous silica nanospheres obtained in step (2) in absolute ethanol. While stirring, add γ-(2,3-epoxypropoxy)propyltrimethoxysilane, which is 0.1 times the mass of the pretreated hierarchical porous silica nanospheres. Stir at 150 rpm and 80 °C for 3.5 h, then filter by suction. Then wash with absolute ethanol three times in sequence, then wash with deionized water three times. Finally, place it in a vacuum drying oven, dry to constant weight at 65 °C, and then place it in a plasma equipment for 7 min of plasma treatment at 70 W to obtain hierarchical porous silica nanospheres; (4) Add Candida antarctica lipase to phosphate buffer solution (PBS) to prepare an enzyme solution. Then add hierarchical porous silica nanospheres, stir and mix well, and place it in a constant temperature shaker at 36 °C and 150 rpm for immobilization for 14 h. Then filter out the immobilized enzyme particles, wash with phosphate buffer four times, and then place it in a vacuum drying oven. Set the cold trap temperature of the vacuum freeze dryer to -35 °C, the vacuum degree to 20 Pa, and the drying temperature to -25 °C. Dry and store for later use under low temperature conditions; The mass-volume ratio of the Candida antarctica lipase to the phosphate buffer solution (PBS) is 1 mg:25 mL; The addition amount of the hierarchical porous silica nanospheres is 1.25 times the mass of the Candida antarctica lipase; The preparation of the deep eutectic solvent includes the following steps: 1) Place choline chloride in a vacuum drying oven and dry at 65 °C for 11 h. Dehydrate glycerol using molecular sieves; 2) Add the treated choline chloride and glycerol to the reaction kettle according to a molar ratio of 1:3. Then add 15% of the ionic liquid based on the total amount of choline chloride and glycerol to the reaction kettle. Heat up to 55 °C, stir and mix well, then heat up to 75 °C, and apply ultrasonic treatment for 1.5 h; The preparation method of the ionic liquid is as follows: Add sorbitol and citric acid to the reaction kettle according to a mass ratio of 1:2.75, stir and mix well, then heat up to 95 °C and react for 4 h. Then cool down to 70 °C, add choline chloride, which is 1.75 times the amount of sorbitol, and stir and react at 250 rpm for 2.5 h. Filter after cooling to room temperature; 3) Cool down to 45 °C at a rate of 1.5 °C / min, keep it at a constant temperature for 2.5 h, then transfer it to a microwave reactor, treat it at 400 W for 12 min, and then transfer it to a vacuum dryer to dehydrate at 55 °C for 19 h; S2. Add vitamin C and glycerol with a molar ratio of 1:1.75 to the reaction system, heat up to 45 °C, and stir and react for 11 h; S3. After the reaction is completed, collect the product and recover the hierarchical porous silica nanospheres.

[0032] Comparative Example 3 A synthesis process of a VC derivative, ascorbyl glycerol, comprises the following steps: S1. Load glycosyltransferase into hierarchical porous silica nanospheres, and then disperse them into a deep eutectic solvent to obtain a uniformly dispersed reaction system; The mass-volume ratio of the hierarchical porous silica nanospheres to the deep eutectic solvent is 2 g: 100 mL; The specific steps of loading glycosyltransferase into hierarchical porous silica nanospheres include: (1) Ultrasonically disperse polylactic acid microspheres (particle size of 250 nm) into a 70% ethanol solution. After uniform dispersion, add an amphiphilic block copolymer (5 wt%) and 1-butyl-3-methylimidazolium tetrafluoroborate (10 wt%), and continue ultrasonic dispersion to form an emulsion; (2) After dropwise adding tetraethyl orthosilicate (1 mol / L) and stirring evenly, add ammonia water to adjust the pH to 9.5. Under the conditions of 45 °C and 150 rpm, stir constantly for 9 h, first pyrolyze with nitrogen at 190 °C for 9 min, then oxidize with air at 330 °C for 7 min, and finally calcine at 550 °C for 6 min to obtain pretreated porous silica nanospheres; (3) Ultrasonically disperse the pretreated hierarchical porous silica nanospheres obtained in step (2) into absolute ethanol. While stirring, add γ-(2,3-epoxypropoxy)propyltrimethoxysilane which is 0.1 times the mass of the pretreated hierarchical porous silica nanospheres. Stir at 150 rpm and 80 °C for 3.5 h, then filter, and then wash with absolute ethanol 3 times in sequence, then wash with deionized water 3 times, and finally place it in a vacuum drying oven. After drying to constant weight at 65 °C, place it in a plasma equipment and treat it with 70 W plasma for 7 min to obtain hierarchical porous silica nanospheres; (4) Add glycosyltransferase to a phosphate buffer solution (PBS) to prepare an enzyme solution, then add hierarchical porous silica nanospheres and stir evenly. Place it in a constant temperature shaker at 36 °C and 150 rpm for immobilization for 14 h, then filter out the immobilized enzyme particles, wash them 4 times with a phosphate buffer solution, and then place them in a vacuum drying oven. Set the cold trap temperature of the vacuum freeze dryer to -35 °C, the vacuum degree to 20 Pa, and the drying temperature to -25 °C. After drying under low temperature conditions, store for later use; The mass-volume ratio of the glycosyltransferase to the phosphate buffer solution (PBS) is 1 mg: 25 mL; The addition amount of the hierarchical porous silica nanospheres is 1.25 times the mass of the glycosyltransferase; The preparation of the deep eutectic solvent comprises the following steps: 1) Place choline chloride in a vacuum drying oven and dry it at 65 °C for 11 h. Dehydrate glycerol using molecular sieves. 2) Add the treated choline chloride and glycerol to a reaction kettle in a molar ratio of 1:3. Then add 15% of the total amount of choline chloride and glycerol as ionic liquid to the reaction kettle. Heat up to 55 °C, stir and mix evenly, then heat up to 75 °C and apply ultrasonic treatment for 1.5 h. The preparation method of the ionic liquid is as follows: Add sorbitol and citric acid to a reaction kettle in a mass ratio of 1:2.75, stir and mix evenly, then heat up to 95 °C and react for 4 h. Then cool down to 70 °C, add choline chloride 1.75 times that of sorbitol, and stir and react at 250 rpm for 2.5 h. After cooling to room temperature, filter it. 3) Cool down to 45 °C at a rate of 1.5 °C / min, keep it at a constant temperature and age for 2.5 h, then transfer it to a microwave reactor, treat it at 400 W for 12 min, and then transfer it to a vacuum dryer to dehydrate at 55 °C for 19 h. S2. Add vitamin C and glycerol with a molar ratio of 1:1.75 to the reaction system, heat up to 45 °C, and stir and react for 11 h. S3. After the reaction is completed, collect the product and recover the hierarchical porous silica nanospheres.

[0033] Comparative Example 4 A synthesis process of a VC derivative, ascorbyl glycerol, includes the following steps: S1. Co-load Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres, and then disperse them in tert-butanol to obtain a uniformly dispersed reaction system. The mass-volume ratio of the hierarchical porous silica nanospheres to tert-butanol is 2 g:100 mL. The specific steps of co-loading Candida antarctica lipase and glycosyltransferase into hierarchical porous silica nanospheres include: (1) Ultrasonically disperse poly(lactic acid) microspheres (particle size of 250 nm) in a 70% ethanol solution. After uniform dispersion, add an amphiphilic block copolymer (5 wt%) and 1-butyl-3-methylimidazolium tetrafluoroborate (10 wt%), and continue to ultrasonically disperse evenly to form an emulsion. (2) After dropping tetraethyl orthosilicate (1 mol / L) and stirring evenly, dropwise add ammonia water to adjust the pH to 9.5. Under the conditions of 45 °C and 150 rpm, keep stirring at a constant temperature for 9 h. First, pyrolyze in nitrogen at 190 °C for 9 min, then oxidize in air at 330 °C for 7 min, and finally calcine at 550 °C for 6 min to obtain pretreated hierarchical porous silica nanospheres. (3) Ultrasonically disperse the pretreated hierarchical porous silica nanospheres obtained in step (2) in absolute ethanol, and while stirring, add γ-(2,3-epoxypropoxy)propyltrimethoxysilane which is 0.1 times the mass of the pretreated hierarchical porous silica nanospheres. Stir at 150 rpm and 80 °C for 3.5 h, then filter by suction. Then wash with absolute ethanol three times in sequence, then wash with deionized water three times, and finally place it in a vacuum drying oven. After drying to a constant weight at 65 °C, place it in a plasma equipment and perform plasma treatment at 70 W for 7 min to obtain hierarchical porous silica nanospheres; (4) Add Candida antarctica lipase and glycosyltransferase to phosphate buffer solution (PBS) in a mass ratio of 1:1.5 in sequence to prepare an enzyme solution. Then add hierarchical porous silica nanospheres and stir to mix evenly. Place it in a constant temperature shaker at 36 °C and 150 rpm for immobilization for 14 h. Then filter out the immobilized enzyme particles, wash them four times with phosphate buffer solution, and then place them in a vacuum drying oven. Set the cold trap temperature of the vacuum freeze dryer to -35 °C, the vacuum degree to 20 Pa, and the drying temperature to -25 °C. After drying under low temperature conditions, store for later use; The mass volume ratio of the total mass of the Candida antarctica lipase and glycosyltransferase to the phosphate buffer solution (PBS) is 1 mg:25 mL; The addition amount of the hierarchical porous silica nanospheres is 1.25 times the total mass of the Candida antarctica lipase and glycosyltransferase; S2. Add vitamin C and glycerol with a molar ratio of 1:1.75 to the reaction system, heat up to 45 °C, and stir and react for 11 h; S3. After the reaction is completed, collect the product and recover the hierarchical porous silica nanospheres.

[0034] In vitro antioxidant activity evaluation Weigh the ascorbyl glycerol esters prepared by the methods corresponding to Example 2 and Comparative Examples 1-4 above respectively to prepare a stock solution of 10 mg / mL. Then use the extraction solutions of the superoxide anion, hydroxyl radical, and DPPH radical scavenging ability detection kits to dilute them to 1, 2, 3, 4, 5 mg / mL respectively. Use L-ascorbic acid with the same mass concentration gradient as a control to compare the scavenging effects of the samples to be tested and the control solution. Conduct 3 repeated experiments at each mass concentration, and finally calculate the scavenging rates of ascorbyl glycerol esters and L-ascorbic acid on various free radicals.

[0035] The test results are as follows Figures 1 to 3 shown.

[0036] From Figures 1 to 3It can be seen that the in vitro antioxidant analysis shows that the ascorbyl glycerol ester prepared by the method of the present invention maintains a high antioxidant activity, and the scavenging rates for superoxide anion radicals, hydroxyl radicals and DPPH radicals are 92.39%, 90.89% and 95.61% respectively. The present invention breaks through the limitations of traditional chemical synthesis and single-enzyme catalysis, and efficiently synthesizes a stable ascorbyl glycerol ester with high antioxidant activity through the confined reaction of dual-enzyme co-catalysis combined with hierarchical porous silica nanospheres and a special eutectic solvent system.

[0037] As mentioned above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. A synthesis process of VC derivative ascorbyl glyceride, characterized in that: The steps include: S1, co-loading Antarctic Candida lipase and glycosyltransferase into hierarchical silica nanospheres, and then dispersing them into a low eutectic solvent to obtain a uniformly dispersed reaction system; S2. Add vitamin C and glycerol to the reaction system, raise the temperature to 40-50°C, and stir the reaction for 10-12 hours; S3. After the reaction is completed, the product is collected and the multi-level porous silica nanospheres are recovered.

2. The synthesis process of a VC derivative ascorbyl glyceride according to claim 1, characterized in that: The specific steps of co-loading the Antarctic Candida lipase and the glycosyltransferase into the hierarchical porous silica nanospheres in step S1 include: (1) Ultrasonic dispersion of polylactic acid microspheres in 70% ethanol solution, adding amphiphilic block copolymer and 1-butyl-3-methylimidazolium tetrafluoroborate after uniform dispersion, and continuing ultrasonic dispersion to form an emulsion; (2) After adding ethyl orthosilicate and stirring evenly, adding ammonia water to adjust the pH to 9-10, stirring at a constant temperature of 40-50°C and 100-200 rpm for 8-10 hours, and then performing a gradient temperature increase treatment to obtain pretreated porous silica nanospheres; (3) ultrasonically dispersing the pretreated hierarchical porous silica nanospheres obtained in step (2) into anhydrous ethanol, adding γ-(2,3-epoxypropoxy)propyltrimethoxysilane in an amount of 0.05 to 0.15 times the mass of the pretreated hierarchical porous silica nanospheres while stirring, stirring at 100 to 200 rpm and 70 to 90° C. for 3 to 4 hours, filtering, then washing with anhydrous ethanol for 3 times, then washing with deionized water for 3 times, finally placing in a vacuum drying oven, drying at 60 to 70° C. to constant weight, and placing in a plasma device for plasma treatment to obtain hierarchical porous silica nanospheres; (4) Antarctic Candida lipase and glycosyltransferase were added to a phosphate buffer solution in a mass ratio of 1:1-2 to prepare an enzyme solution. The multi-level porous silica nanospheres were then added and stirred to mix well. The solution was then placed in a constant temperature shaker at 35-37°C and 100-200 rpm for immobilization for 12-16 h. The immobilized enzyme particles were then filtered out, washed with phosphate buffer for 3-5 times, and placed in a vacuum drying oven. The particles were dried at low temperature and stored for later use.

3. The synthesis process of a VC derivative ascorbyl glyceride according to claim 2, characterized in that: The particle size of the polylactic acid microspheres described in step (1) is 200-300 nm; The amount of the amphiphilic block copolymer added is 5 wt %, and the amount of 1-butyl-3-methylimidazolium tetrafluoroborate added is 10 wt %.

4. The synthesis process of a VC derivative ascorbyl glyceride according to claim 2, characterized in that: The concentration of ethyl orthosilicate in step (2) is 0.8-1.2 mol / L; The specific operation of the gradient heating is: first pyrolysis with nitrogen at 180-200°C for 8-10 minutes, then oxidation with air at 300-360°C for 6-8 minutes, and finally calcination at 500-600°C for 5-7 minutes.

5. The synthesis process of a VC derivative ascorbyl glyceride according to claim 2, characterized in that: The power of the plasma treatment in step (3) is 50-100 W, and the treatment time is 6-8 min.

6. The synthesis process of a VC derivative ascorbyl glyceride according to claim 2, characterized in that: The mass volume ratio of the total mass of the Antarctic Candida lipase and the glycosyltransferase to the phosphate buffer solution in step (4) is 1 mg: 20-30 mL; The added amount of the multi-level porous silica nanospheres is 1 to 1.5 times the total mass of the Antarctic Candida lipase and the glycosyltransferase; The specific parameters of drying under low temperature conditions are: setting the cold trap temperature of the vacuum freeze dryer to -40~-30°C, the vacuum degree to 20Pa, and the drying temperature to -30~-20°C.

7. The synthesis process of a VC derivative ascorbyl glyceride according to claim 1, characterized in that: The preparation of the deep eutectic solvent in step S1 comprises the following steps: 1) Place choline chloride in a vacuum drying oven at 60-70°C for 10-12 hours, and dehydrate glycerol using molecular sieves; 2) Add the treated choline chloride and glycerol into the reactor in a molar ratio of 1:2-4, then add the ionic liquid into the reactor, heat it to 50-60°C, stir and mix, heat it to 70-80°C, and apply ultrasonic treatment for 1-2h; 3) After cooling to 40~50℃ at a rate of 1~2℃ / min, age at constant temperature for 2~3h and then transfer to a microwave reactor. After microwave treatment at 300~500W for 10~15min, transfer to a vacuum dryer and dehydrate at 50~60℃ for 18~20h.

8. The synthesis process of a VC derivative ascorbyl glyceride according to claim 7, characterized in that: The amount of the ionic liquid added in step 2) is 10-20% of the total amount of choline chloride and glycerol. The preparation method of the ionic liquid is as follows: sorbitol and citric acid are added to a reactor in a mass ratio of 1:2.5-3, stirred and mixed, heated to 90-100°C for reaction for 3-5h, then cooled to 60-80°C, choline chloride 1.5-2 times of sorbitol is added, stirred at 200-300rpm for reaction for 2-3h, cooled to room temperature and filtered.

9. The synthesis process of a VC derivative ascorbyl glyceride according to claim 1, characterized in that: The mass volume ratio of the hierarchical porous silica nanospheres to the low eutectic solvent described in step S1 is 1-3 g:100 mL.

10. The synthesis process of VC derivative ascorbyl glyceride according to claim 1, characterized in that: The molar ratio of vitamin C to glycerol described in step S2 is 1:1.5~2.

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