A process for the preparation of 3-o-ethyl ascorbic acid

Abstract

The application discloses a preparation method of 3-o-ethyl ascorbic acid. The method comprises the following key steps: first, a culture medium containing glucose, sugarcane juice, ascorbic acid, compound amino acid and inorganic salt is prepared, and after sterilization treatment, the culture medium is reserved; Bacillus stearothermophilus and Aspergillus niger are activated in Gause culture medium respectively, and a seed liquid of bacteria with a bacteria content of 10 8 ‑10 9 cfu / mL is prepared; the immobilized compound enzyme is added into the culture medium, the above two kinds of seed liquid of bacteria are inoculated, and enzymatic fermentation is carried out at 40-45 DEG C and 100-200 r / min for 36-48 h; the immobilized enzyme is recovered by a magnet; the fermentation product is filtered, dialyzed and freeze-dried to obtain an ascorbic acid derivative composition; the derivative composition is added into an ethanol system with alkali, long-chain alkyl halogenated alkane is added, and stirring reaction is carried out at 40-50 DEG C for 2-4 h; filtration, washing and drying are carried out to complete hydrophobic modification; finally, the modified derivative composition is mixed with 3-o-ethyl ascorbic acid in proportion, and the target product is prepared.

Description

Technical Field

[0001] This invention belongs to the field of biochemical and cosmetic raw material preparation technology, specifically relating to a method for preparing 3-o-ethyl ascorbic acid, the product obtained by the method, and its application in cosmetic preparation. Background Technology

[0002] 3-o-Ethyl ascorbic acid is an important ascorbic acid derivative. Compared with natural ascorbic acid, it has certain advantages in terms of lipid solubility and stability. It can effectively inhibit melanin formation and promote skin collagen synthesis, and is a commonly used whitening and anti-wrinkle raw material in the cosmetics industry.

[0003] Currently, the main methods for preparing 3-o-ethyl ascorbic acid include chemical synthesis and biotransformation. Chemical synthesis typically uses ascorbic acid as a raw material, proceeding through esterification, alkylation, and other reactions. This method suffers from harsh reaction conditions, numerous byproducts, low product purity, and the use of organic solvents that can cause environmental pollution. Subsequent separation and purification processes are complex, resulting in high production costs. Biotransformation methods often utilize a single bacterial strain or enzyme for catalytic reaction. While the reaction conditions are relatively mild, the product suffers from poor thermal stability and low bioavailability. Furthermore, the enzyme preparation is difficult to recover and reuse, limiting its industrial application.

[0004] Furthermore, the transdermal absorption performance of 3-o-ethyl ascorbic acid obtained by existing methods still needs improvement, and some products are easily decomposed and inactivated in cosmetic formulations, affecting their whitening and anti-wrinkle effects. Therefore, developing a mild and environmentally friendly method for preparing 3-o-ethyl ascorbic acid with high product purity, excellent thermal stability and bioavailability, and ease of industrial production, has significant practical significance and application value. Summary of the Invention

[0005] This invention aims to solve the technical problems of poor thermal stability, low bioavailability, complex process, serious environmental pollution, and difficulty in reusing enzyme preparations in traditional methods for preparing 3-o-ethyl ascorbic acid. It provides a method for preparing 3-o-ethyl ascorbic acid with a mild and environmentally friendly process, high product purity, and excellent heat resistance, antioxidant properties, and transdermal absorption performance. It also provides the product obtained by this method and its application in cosmetics.

[0006] A method for preparing 3-o-ethyl ascorbic acid includes the following steps: S1: Prepare and sterilize the culture medium, which contains glucose, sugarcane juice, ascorbic acid, complex amino acids and inorganic salts; S2: Strain activation. The thermophilic Bacillus subtilis and Aspergillus niger were activated separately in Gao's medium to obtain strains with a bacterial count of 10... 8 -10 9CFU / mL bacterial seed solution; S3: Enzymatic fermentation and product pretreatment: Immobilized complex enzyme is added to the culture medium sterilized in step S1, and the two bacterial seed liquids prepared in step S2 are inoculated. Enzymatic fermentation is carried out at 40-45℃ and 100-200r / min for 36-48h. The immobilized complex enzyme is separated and recovered by magnet. The fermentation product is filtered, dialyzed and freeze-dried to obtain ascorbic acid derivative composition. S4: Hydrophobic modification: The ascorbic acid derivative composition obtained in step S3 is added to an ethanol system with an alkali, and then a long-chain alkyl haloalkane is added. The mixture is stirred at 40-50℃ for 2-4 hours. After filtration, washing and drying, the hydrophobic modification is completed, and the modified derivative composition is obtained. S5: Preparation of target product: The modified derivative composition obtained in step S4 is mixed with 3-o-ethyl ascorbic acid in a certain proportion to obtain the target product of 3-o-ethyl ascorbic acid; The immobilized complex enzyme is prepared by using magnetic porous silica spheres as carriers, which are modified with tannic acid and then adsorbed with α-glucosidase, cyclomaltodextrin glucan transferase and α-amylase.

[0007] Beneficial effects (1) The present invention adopts a co-fermentation method of compound strains (thermophilic Bacillus subtilis and Aspergillus niger) and immobilized compound enzymes. The fermentation conditions are mild (40-45℃), no violent reaction conditions are required, and the use of a large amount of organic solvents is avoided. The process is environmentally friendly and conforms to the concept of green production. At the same time, the synergistic effect of compound strains and compound enzymes can significantly improve the conversion rate of ascorbic acid derivatives and the purity of the product is high.

[0008] (2) The immobilized composite enzyme of the present invention uses magnetic porous silica spheres as carriers, which adsorb various enzymes after being modified with tannic acid. The carrier has a large specific surface area and strong adsorption capacity, resulting in a high enzyme immobilization rate. Moreover, the magnetic carrier is easy to separate and recover quickly by magnets and can be reused multiple times, reducing production costs and solving the problems of traditional enzyme preparations being difficult to recover and having low utilization rates.

[0009] (3) The present invention hydrophobically modifies the ascorbic acid derivative composition obtained by fermentation, and enhances the lipophilicity of the product by grafting long-chain alkyl groups, which significantly improves its transdermal absorption performance, so that the product can enter the deep layer of the skin more efficiently to exert its effect; at the same time, the modified derivative composition can form a synergistic protective effect when mixed with 3-o-ethyl ascorbic acid, improving the thermal stability and storage stability of the target product and preventing it from decomposing and failing in the cosmetic formulation system.

[0010] (4) The bioavailability of the 3-o-ethyl ascorbic acid prepared by this invention is significantly improved. After hydrolysis, it can continuously release ascorbic acid, effectively inhibit melanin formation and promote collagen synthesis. It is also safe and non-irritating, and is especially suitable for cosmetic raw materials. The preparation process is simple and easy to scale up, and has broad prospects for industrial application. Detailed Implementation

[0011] An embodiment of the present invention provides a method for preparing 3-o-ethyl ascorbic acid, comprising the following steps: S1: Prepare and sterilize the culture medium, which contains glucose, sugarcane juice, ascorbic acid, complex amino acids, and inorganic salts. The complex amino acids are composed of the following components in the following mass ratio: glycine: alanine: leucine: lysine: aspartic acid: glutamic acid = 3-4: 2-4: 2-3: 1-2: 1-2: 1-2. The inorganic salts are composed of the following components in the following mass ratio: potassium dihydrogen phosphate: magnesium sulfate: calcium chloride: zinc sulfate = 3-5: 2-4: 1-2: 0.5-1.5; the mass concentrations of each component in the culture medium are: glucose 20-40 g / L, sugarcane juice 10-20 g / L, ascorbic acid 5-10 g / L, complex amino acids 3-8 g / L, and inorganic salts 2-5 g / L. The mass concentration of each component is controlled within a reasonable range to ensure the needs of bacterial growth and enzymatic reaction. The prepared culture medium is sterilized by high-pressure steam at 121℃ for 20-30 minutes, and then cooled to room temperature for use.

[0012] S2: Strain activation. The thermophilic Bacillus subtilis and Aspergillus niger were activated separately in Gao's medium to obtain strains with a bacterial count of 10... 8 -10 9 CFU / mL bacterial seed culture. The synergistic effect of the two bacterial strains can improve the conversion rate of ascorbic acid derivatives; the two strains were separately inoculated into Gao's medium and activated by incubation at 37-40℃ for 24-36 h to obtain a bacterial count of 10. 8 -10 9 Prepare a bacterial seed culture solution of CFU / mL for later use.

[0013] S3: Enzymatic fermentation and product pretreatment. Immobilized complex enzyme is added to the culture medium sterilized in step S1, and the two bacterial seed liquids prepared in step S2 are inoculated. Enzymatic fermentation is carried out at 40-45℃ and 100-200r / min for 36-48h. The immobilized complex enzyme is separated and recovered by magnet. The fermentation product is filtered, dialyzed and freeze-dried to obtain an ascorbic acid derivative composition. This immobilized complex enzyme uses magnetic porous silica spheres as a carrier, which are modified with tannic acid and then adsorbed with α-glucosidase, cyclomaltodextrin glucan transferase, and α-amylase. It has the advantages of convenient separation and reusability. Subsequently, the seed culture of the above two strains is inoculated and enzymatic fermentation is carried out at 40-45℃ and 100-200 r / min for 36-48 h. During the fermentation process, the enzymes produced by the metabolism of the strains and the immobilized complex enzyme synergistically catalyze each other, improving the product conversion rate. After fermentation, the immobilized complex enzyme is separated and recovered using a magnet for subsequent reuse. The fermentation product is first filtered to remove the cells and impurities, then dialyzed to remove small molecule impurities, and then freeze-dried to obtain the ascorbic acid derivative composition.

[0014] Optionally, the preparation method of the immobilized complex enzyme includes: soaking magnetic porous silica spheres in tannic acid solution for 2-4 hours, filtering, washing, and drying to obtain tannic acid-modified magnetic porous silica spheres; adding the modified magnetic porous silica spheres to a mixed enzyme solution containing α-glucosidase, cyclomaltodextrin glucan transferase, and α-amylase, shaking and adsorbing at 25-30℃ for 8-12 hours, filtering, washing, and refrigerating to obtain the immobilized complex enzyme; the mass ratio of the three enzymes in the mixed enzyme solution is 2-3:1-2:1-2, and the total enzyme concentration is 10-20 mg / mL. The amount of immobilized complex enzyme added in S3 is 0.5-1.5% of the culture medium mass; the inoculation amount of the two bacterial seed solutions is 2-5% of the culture medium volume. S4: Hydrophobic modification. The ascorbic acid derivative composition obtained in step S3 is added to an ethanol system with an alkali, followed by the addition of a long-chain alkyl haloalkane. The mixture is stirred and reacted at 40-50°C for 2-4 hours. After filtration, washing, and drying, hydrophobic modification is completed, yielding the modified derivative composition. The alkali promotes the dissociation of the derivative and improves its reactivity. The addition of a long-chain alkyl haloalkane as a hydrophobic modifier, followed by stirring and reacting at 40-50°C for 2-4 hours, allows the long-chain alkyl groups to be grafted onto the ascorbic acid derivative molecules, enhancing the product's lipophilicity and transdermal absorption properties. After the reaction, unreacted raw materials and byproducts are removed by filtration and washing, and the modified derivative composition is obtained after drying. The alkali is sodium hydroxide or potassium hydroxide, and the amount of alkali added is 5-10% of the mass of the ascorbic acid derivative composition; the volume fraction of ethanol in the ethanol system is 70-90%; the long-chain alkyl haloalkane is hexadecyl bromide or octadecyl chloroalkane, and the molar ratio of its added amount to the ascorbic acid derivative composition is 1.2-1.5:1.

[0015] S5: Preparation of the target product: The modified derivative composition obtained in step S4 is mixed with 3-o-ethyl ascorbic acid in a certain proportion to obtain the 3-o-ethyl ascorbic acid target product. In this target product, the modified derivative composition protects 3-o-ethyl ascorbic acid from oxidative decomposition, improves its thermal stability and storage stability, and synergistically enhances its transdermal absorption performance and bioavailability. Optionally, the mass ratio of the modified derivative composition to 3-o-ethyl ascorbic acid is 1:3-5.

[0016] Another embodiment of the present invention provides 3-o-ethyl ascorbic acid prepared according to the above preparation method. This product has high purity, excellent heat resistance, antioxidant properties, and transdermal absorption performance, significantly improved bioavailability, and can continuously release ascorbic acid after hydrolysis. It can effectively inhibit melanin formation, promote collagen synthesis, and has good safety.

[0017] Another embodiment of the present invention provides the application of 3-o-ethyl ascorbic acid in the preparation of cosmetics, including whitening essence, moisturizing lotion, anti-wrinkle cream and toner.

[0018] Optionally, the mass fraction of 3-o-ethyl ascorbic acid added to cosmetics is 0.5-5%.

[0019] Another embodiment of the present invention provides a cosmetic containing 3-o-ethyl ascorbic acid, comprising the above-mentioned 3-o-ethyl ascorbic acid, and cosmetically acceptable excipients.

[0020] The following are examples and comparative examples.

[0021] Example 1 A method for preparing 3-o-ethyl ascorbic acid includes the following steps: S1: Culture medium preparation and sterilization. Prepare the culture medium according to the following mass concentrations: glucose 30g / L, sugarcane juice 15g / L, ascorbic acid 8g / L, compound amino acids 5g / L (compound amino acids are composed of the following components in the following mass ratio: glycine: alanine: leucine: lysine: aspartic acid: glutamic acid = 3:2:2:1:1:1), and inorganic salts 3g / L (potassium dihydrogen phosphate: magnesium sulfate: calcium chloride: zinc sulfate = 4:3:1.5:1). Place the culture medium in an autoclave and sterilize at 121℃ for 25min. Cool to room temperature for later use.

[0022] S2: Strain activation: Bacillus subtilis thermophilus and Aspergillus niger were inoculated into Gao's medium and cultured at 38°C for 30 h to obtain strains with a bacterial count of 5 × 10⁻⁶. 8 CFU / mL bacterial seed solution.

[0023] S3: Enzymatic fermentation and product pretreatment, preparation of immobilized composite enzyme: Magnetic porous silica spheres were soaked in a 5% (w / w) tannic acid solution for 3 hours, filtered, washed, and dried at 60°C for 2 hours to obtain tannic acid-modified magnetic porous silica spheres; these modified magnetic porous silica spheres were added to a mixed enzyme solution (α-glucosidase: cyclomaltodextrin glucan transferase: α-amylase mass ratio = 2.5:1.5:1.5, total enzyme concentration 15 mg / mL), shaken and adsorbed at 28°C for 10 hours, filtered, washed, and stored at 4°C to obtain the immobilized composite enzyme; [The text abruptly ends here, likely due to an incomplete translation or missing information.] 0.8% (relative to the mass of the culture medium) of immobilized complex enzyme was added to the culture medium, and 3% (relative to the volume of the culture medium) of two bacterial seed solutions were inoculated separately. Enzymatic fermentation was carried out at 42℃ and 150 r / min for 42 h. After fermentation, the immobilized complex enzyme was separated and recovered by magnet. The fermentation product was filtered through a filter cloth (100 mesh) to remove bacterial cells and impurities, and then dialyzed for 24 h using a dialysis bag (molecular weight cutoff 8000-10000 Da). After dialysis, it was freeze-dried at -50℃ and 0.09 MPa for 12 h to obtain the ascorbic acid derivative composition.

[0024] S4: Hydrophobic modification: 10g of the ascorbic acid derivative composition obtained in step S3 and 0.7g of sodium hydroxide were added to 80mL of 80% ethanol system. After stirring and dissolving, hexadecyl bromide (molar ratio of 1.3:1 with the ascorbic acid derivative composition) was added, and the mixture was stirred and reacted at 45℃ for 3h. After the reaction was completed, the solid product was collected by filtration, washed 3 times with ethanol, and dried at 60℃ for 4h to obtain the modified derivative composition.

[0025] S5: Preparation of the target product: 2g of the modified derivative composition was mixed with 8g of 3-o-ethyl ascorbic acid and stirred in a high-speed mixer for 30min (2000r / min) to ensure thorough mixing and obtain the target product of 3-o-ethyl ascorbic acid.

[0026] Example 2 A method for preparing 3-o-ethyl ascorbic acid includes the following steps: S1: Culture medium preparation and sterilization. Prepare the culture medium according to the following mass concentrations: glucose 20g / L, sugarcane juice 10g / L, ascorbic acid 5g / L, compound amino acids 3g / L (compound amino acids are composed of the following components in the following mass ratio: glycine: alanine: leucine: lysine: aspartic acid: glutamic acid = 3:2:2:1:1:1), and inorganic salts 2g / L (potassium dihydrogen phosphate: magnesium sulfate: calcium chloride: zinc sulfate = 3:2:1:0.5). Place the culture medium in an autoclave and sterilize at 121℃ for 20min. Cool to room temperature for later use.

[0027] S2: Strain activation: Bacillus subtilis thermophilus and Aspergillus niger were inoculated into Gao's medium and cultured at 37°C for 24 hours to obtain strains with a bacterial count of 1×10⁻⁶. 8 CFU / mL bacterial seed solution.

[0028] S3: Enzymatic fermentation and product pretreatment, preparation of immobilized composite enzyme: Magnetic porous silica spheres were soaked in a 4% (w / w) tannic acid solution for 2 hours, filtered, washed, and dried at 60℃ for 2 hours to obtain tannic acid-modified magnetic porous silica spheres; these modified magnetic porous silica spheres were added to a mixed enzyme solution (α-glucosidase: cyclomaltodextrin glucan transferase: α-amylase mass ratio = 2:1:1, total enzyme concentration 10 mg / mL), shaken and adsorbed at 25℃ for 8 hours, filtered, washed, and stored at 4℃ to obtain the immobilized composite enzyme; the culture medium from step S1 was then added... Add 0.5% (relative to the mass of the culture medium) of immobilized complex enzyme to the medium, and inoculate with 2% (relative to the volume of the culture medium) of two bacterial seed solutions respectively. Ferment for 36 h at 40℃ and 100 r / min. After fermentation, the immobilized complex enzyme is separated and recovered by magnet. The fermentation product is filtered through a filter cloth (100 mesh) to remove bacterial cells and impurities, and then dialyzed for 20 h using a dialysis bag (molecular weight cutoff 8000-10000 Da). After dialysis, freeze-dry for 10 h at -50℃ and 0.09 MPa vacuum to obtain the ascorbic acid derivative composition.

[0029] S4: Hydrophobic modification: 10g of the ascorbic acid derivative composition obtained in step S3 and 0.5g of potassium hydroxide were added to 80mL of 70% ethanol system. After stirring and dissolving, hexadecyl bromide (molar ratio of 1.2:1 with the ascorbic acid derivative composition) was added, and the mixture was stirred and reacted at 40℃ for 2h. After the reaction was completed, the solid product was collected by filtration, washed 3 times with ethanol, and dried at 60℃ for 4h to obtain the modified derivative composition.

[0030] S5: Preparation of the target product: 2g of the modified derivative composition was mixed with 6g of 3-o-ethyl ascorbic acid and stirred in a high-speed mixer for 30min (2000r / min) to ensure thorough mixing and obtain the target product of 3-o-ethyl ascorbic acid.

[0031] Example 3 A method for preparing 3-o-ethyl ascorbic acid includes the following steps: S1: Culture medium preparation and sterilization. Prepare the culture medium according to the following mass concentrations: glucose 40g / L, sugarcane juice 20g / L, ascorbic acid 10g / L, compound amino acids 8g / L (compound amino acids are composed of the following components in the following mass ratio: glycine: alanine: leucine: lysine: aspartic acid: glutamic acid = 3:2:2:1:1:1), and inorganic salts 5g / L (potassium dihydrogen phosphate: magnesium sulfate: calcium chloride: zinc sulfate = 5:4:2:1.5). Place the culture medium in an autoclave and sterilize at 121℃ for 30min. Cool to room temperature for later use.

[0032] S2: Strain activation: Bacillus subtilis thermophilus and Aspergillus niger were inoculated into Gao's medium and cultured at 40℃ for 36 h to obtain strains with a bacterial count of 1×10⁻⁶. 9 CFU / mL bacterial seed solution.

[0033] S3: Enzymatic fermentation and product pretreatment, preparation of immobilized composite enzyme: Magnetic porous silica spheres were soaked in a 6% (w / w) tannic acid solution for 4 hours, filtered, washed, and dried at 60℃ for 2 hours to obtain tannic acid-modified magnetic porous silica spheres; these modified magnetic porous silica spheres were added to a mixed enzyme solution (α-glucosidase: cyclomaltodextrin glucan transferase: α-amylase mass ratio = 3:2:2, total enzyme concentration 20 mg / mL), shaken and adsorbed at 30℃ for 12 hours, filtered, washed, and stored at 4℃ to obtain the immobilized composite enzyme; the culture medium from step S1 was then added... 1.5% (relative to the mass of the culture medium) of immobilized complex enzyme was added to the culture medium, and 5% (relative to the volume of the culture medium) of two bacterial seed solutions were inoculated separately. Enzymatic fermentation was carried out at 45℃ and 200 r / min for 48 h. After fermentation, the immobilized complex enzyme was separated and recovered by magnet. The fermentation product was filtered through a filter cloth (100 mesh) to remove bacterial cells and impurities, and then dialyzed through a dialysis bag (molecular weight cutoff 8000-10000 Da) for 28 h. After dialysis, it was freeze-dried at -50℃ and 0.09 MPa for 14 h to obtain the ascorbic acid derivative composition.

[0034] S4: Hydrophobic modification: 10g of the ascorbic acid derivative composition obtained in step S3 and 1.0g of sodium hydroxide were added to 80mL of 90% ethanol system. After stirring and dissolving, octadecyl chloride (molar ratio of 1.5:1 with the ascorbic acid derivative composition) was added, and the mixture was stirred and reacted at 50℃ for 4h. After the reaction was completed, the solid product was collected by filtration, washed 3 times with ethanol, and dried at 60℃ for 4h to obtain the modified derivative composition.

[0035] S5: Preparation of the target product: 2g of the modified derivative composition was mixed with 10g of 3-o-ethyl ascorbic acid and stirred in a high-speed mixer for 30min (2000r / min) to ensure thorough mixing and obtain the target product of 3-o-ethyl ascorbic acid.

[0036] Comparative Example 1 (fermentation with a single strain without immobilized complex enzymes) A method for preparing 3-o-ethyl ascorbic acid, comprising the following steps: S1: Culture medium preparation and sterilization, same as step S1 in Example 1.

[0037] S2: Strain activation: Only *Bacillus subtilis* thermophilic was inoculated into Gao's medium and cultured at 38°C for 30 hours to obtain a bacterial count of 5 × 10⁻⁶. 8 CFU / mL bacterial seed solution.

[0038] S3: Fermentation and product pretreatment: 3% (relative to the volume of the culture medium) of thermophilic Bacillus subtilis seed liquid was inoculated into the culture medium of step S1, and fermented at 42℃ and 150r / min for 42h; after fermentation, the bacterial cells and impurities were removed by filtration, dialyzed and freeze-dried to obtain the ascorbic acid derivative composition.

[0039] S4-S5: Same as steps S4-S5 in Example 1, to obtain the 3-o-ethyl ascorbic acid product.

[0040] Comparative Example 2 (without hydrophobic modification step) A method for preparing 3-o-ethyl ascorbic acid, comprising the following steps: S1-S3: Same as steps S1-S3 in Example 1, to obtain the ascorbic acid derivative composition.

[0041] S4: Preparation of the target product: 2g of the ascorbic acid derivative composition obtained in step S3 is mixed with 8g of 3-o-ethyl ascorbic acid and stirred evenly to obtain the 3-o-ethyl ascorbic acid product.

[0042] Comparative Example 3 (Traditional Chemical Synthesis Method) Using ascorbic acid as a raw material, sulfuric acid was added as a catalyst, and bromoethane was added as an alkylating agent. The reaction was carried out at 80°C for 6 hours. After the reaction was completed, the product was neutralized with sodium hydroxide solution, filtered and collected as a solid product. The product was recrystallized three times with ethanol and dried to obtain 3-o-ethyl ascorbic acid.

[0043] Test metrics and test methods The 3-o-ethyl ascorbic acid products obtained in Examples 1-3 and Comparative Examples 1-3 were tested for the following indicators, and the test results are shown in Table 1.

[0044] 1. Product purity test High-performance liquid chromatography (HPLC) was used for testing. Instrument model: Agilent 1260; chromatographic column: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-water (v / v 30:70); flow rate: 1.0 mL / min; detection wavelength: 245 nm; column temperature: 30 ℃; injection volume: 20 μL; product purity was calculated by external standard method.

[0045] 2. Thermal stability test Each product was placed in a 60℃ constant temperature drying oven for 10 days. The content of 3-o-ethyl ascorbic acid in the product was tested by HPLC before (0 days) and after (10 days) the product was heated. The retention rate was calculated. The higher the retention rate, the better the thermal stability.

[0046] Retention rate (%) = (3-o-ethyl ascorbic acid content after heat preservation / 3-o-ethyl ascorbic acid content before heat preservation) × 100%.

[0047] 3. Antioxidant performance test (DPPH free radical scavenging rate) Prepare a 0.1 mmol / L DPPH ethanol solution. Mix 2 mL of DPPH solution with 2 mL of ethanol solutions of different products (mass concentration 0.1 mg / mL), let stand in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm (A1). At the same time, measure the absorbance of the mixture of 2 mL DPPH solution and 2 mL ethanol (A0), and the absorbance of the mixture of 2 mL product ethanol solution and 2 mL ethanol (A2). The higher the DPPH free radical scavenging rate, the better the antioxidant activity.

[0048] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100% 4. Transdermal absorption performance test (in vitro transdermal experiment) In vitro transdermal experiments were conducted using a Franz diffusion cell, with mouse abdominal skin as the transdermal barrier. Each product was prepared as a 1% (w / w) aqueous solution and added to the supply cell. Physiological saline was added to the receiving cell. The temperature was controlled at 37℃, and the stirring speed was 500 r / min. The receiving cell solution was collected at 2h, 4h, 8h, 12h, and 24h, and the content of 3-o-ethyl ascorbic acid was determined by HPLC. The cumulative transdermal amount over 24h was calculated. The higher the cumulative transdermal amount, the better the transdermal absorption performance.

[0049] 5. Bioavailability test (in vivo rat experiment) Healthy SD rats (weighing 200-220g) were randomly divided into 6 groups of 6 rats each. After fasting for 12 hours, each rat was administered physiological saline solution of the product (dose 50mg / kg) by gavage. Blood samples were collected from the orbital sinus at 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after gavage. Serum was separated by centrifugation, and the concentration of 3-o-ethyl ascorbic acid in the serum was tested by HPLC. The area under the curve (AUC0-24h) was calculated. The higher the AUC value, the higher the bioavailability.

[0050] 6. Melanin Inhibition Rate Test Using a B16 melanoma cell in vitro culture model, B16 cells were seeded in 96-well plates and cultured for 24 hours. Then, culture media containing different products (0.1 mg / mL) were added, and the cells were cultured for another 48 hours. Cells were collected, centrifuged, washed, and then treated with sodium hydroxide solution (1 mol / L). The cells were then incubated at 80°C for 30 minutes to dissolve melanin. The absorbance was measured at 475 nm. A blank control group (culture media without added products) was also set up. The melanin inhibition rate was calculated; a higher inhibition rate indicated a better whitening effect.

[0051] Inhibition rate (%) = [1 - (Experimental group absorbance / Control group absorbance)] × 100% The test results are shown in the table below.

[0052]

[0053] As shown in Table 1, the 3-o-ethyl ascorbic acid products obtained in Examples 1-3 of this invention are significantly superior to those in Comparative Examples 1-3 in all aspects: 1. Regarding product purity, the purity of the products in Examples 1-3 is higher than that in Comparative Examples 1-3, indicating that the co-fermentation process of the composite strain and immobilized composite enzyme used in this invention can effectively improve product purity and reduce the generation of by-products.

[0054] 2. Regarding thermal stability, the retention rates of the products in Examples 1-3 after being kept at 60°C for 10 days were all above 89%, with the highest reaching 92.5%, while the retention rates of Comparative Examples 1-3 were all below 79%, especially Comparative Example 3, which was only 65.4%. This indicates that the hydrophobic modification step of the present invention and the synergistic effect of the modified derivative and the target product can significantly improve the thermal stability of 3-o-ethyl ascorbic acid.

[0055] 3. Regarding antioxidant properties, the DPPH free radical scavenging rate of the products in Examples 1-3 is all above 88%, which is higher than that of the comparative examples. This indicates that the products prepared by the present invention have excellent antioxidant properties, can effectively scavenge free radicals, and exert anti-aging effects.

[0056] 4. Regarding transdermal absorption performance, the cumulative transdermal absorption rate of the products in Examples 1-3 was all around 78 μg / cm³ over 24 hours. 2 The results above are higher than those of comparative examples 1-3, indicating that the hydrophobic modification step significantly improves the product's lipid solubility and transdermal absorption performance, making it easier for it to penetrate deep into the skin to exert its effects.

[0057] 5. Regarding bioavailability, the AUC0-24h of the products in Examples 1-3 were all above 1120 μg·h / mL, which was higher than that of the comparative examples, indicating that the bioavailability of the products prepared by the present invention was significantly improved and could be absorbed and utilized by the body more efficiently.

[0058] 6. Regarding the melanin inhibition rate, the melanin inhibition rate of the products in Examples 1-3 is all above 85%, with the highest reaching 88.6%, which is higher than that of the comparative examples. This indicates that the products prepared by the present invention can effectively inhibit melanin formation and have excellent whitening effects.

[0059] Furthermore, the immobilized composite enzyme in Example 1 of this invention was recycled and reused 5 times, and its enzyme activity still maintained more than 85% of the initial activity, indicating that the immobilized composite enzyme has good reusability, can effectively reduce production costs, and is suitable for industrial production.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0061] For those skilled in the art, based on the ideas of this invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A method for preparing 3-o-ethyl ascorbic acid, characterized in that, Includes the following steps: S1: Prepare and sterilize the culture medium, which contains glucose, sugarcane juice, ascorbic acid, complex amino acids and inorganic salts; S2: Strain activation. The thermophilic Bacillus subtilis and Aspergillus niger were activated separately in Gao's medium to obtain strains with a bacterial count of 10... 8 -10 9 CFU / mL bacterial seed solution; S3: Enzymatic fermentation and product pretreatment: Immobilized complex enzyme is added to the culture medium sterilized in step S1, and the two bacterial seed liquids prepared in step S2 are inoculated. Enzymatic fermentation is carried out at 40-45℃ and 100-200r / min for 36-48h. The immobilized complex enzyme is separated and recovered by magnet. The fermentation product is filtered, dialyzed and freeze-dried to obtain ascorbic acid derivative composition. S4: Hydrophobic modification: The ascorbic acid derivative composition obtained in step S3 is added to an ethanol system with an alkali, and then a long-chain alkyl haloalkane is added. The mixture is stirred at 40-50℃ for 2-4 hours. After filtration, washing and drying, the hydrophobic modification is completed, and the modified derivative composition is obtained. S5: Preparation of target product: The modified derivative composition obtained in step S4 is mixed with 3-o-ethyl ascorbic acid in a certain proportion to obtain the target product of 3-o-ethyl ascorbic acid; The immobilized complex enzyme is prepared by using magnetic porous silica spheres as carriers, which are modified with tannic acid and then adsorbed with α-glucosidase, cyclomaltodextrin glucan transferase and α-amylase.

2. The preparation method according to claim 1, characterized in that, The inorganic salts mentioned in step S1 are composed of the following components in the following mass ratio: potassium dihydrogen phosphate: magnesium sulfate: calcium chloride: zinc sulfate = 3-5: 2-4: 1-2: 0.5-1.5; the mass concentrations of each component in the culture medium are: glucose 20-40 g / L, sugarcane juice 10-20 g / L, ascorbic acid 5-10 g / L, complex amino acids 3-8 g / L, and inorganic salts 2-5 g / L. The composite amino acid is composed of the following components in the following mass ratio: glycine: alanine: leucine: lysine: aspartic acid: glutamic acid = 3-4: 2-4: 2-3: 1-2: 1-2: 1-2.

3. The preparation method according to claim 1, characterized in that, The preparation method of the immobilized composite enzyme in step S3 includes: soaking magnetic porous silica balls in tannic acid solution for 2-4 hours, filtering, washing, and drying to obtain tannic acid modified magnetic porous silica balls; adding the modified magnetic porous silica balls to a mixed enzyme solution containing α-glucosidase, cyclomaltodextrin glucan transferase, and α-amylase, shaking and adsorbing at 25-30℃ for 8-12 hours, filtering, washing, and refrigerating to obtain the immobilized composite enzyme; the mass ratio of the three enzymes in the mixed enzyme solution is 2-3:1-2:1-2, and the total enzyme concentration is 10-20 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step S3, the amount of immobilized complex enzyme added is 0.5-1.5% of the culture medium mass; the inoculation amount of the two bacterial seed solutions is 2-5% of the culture medium volume.

5. The preparation method according to claim 1, characterized in that, The alkali mentioned in step S4 is sodium hydroxide or potassium hydroxide, and the amount of alkali added is 5-10% of the mass of the ascorbic acid derivative composition; the volume fraction of ethanol in the ethanol system is 70-90%; the long-chain alkyl haloalkane is hexadecyl bromide or octadecyl chloroalkane, and the molar ratio of its added amount to the ascorbic acid derivative composition is 1.2-1.5:

1.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the modified derivative composition to 3-o-ethyl ascorbic acid in step S5 is 1:3-5.

7. 3-o-ethylascorbic acid prepared by any one of the preparation methods according to claims 1-6.

8. The application of 3-o-ethyl ascorbic acid according to claim 7 in the preparation of cosmetics, characterized in that, The cosmetics include whitening essence, moisturizing lotion, anti-wrinkle cream, and toner.

9. The application according to claim 8, characterized in that, The mass fraction of 3-o-ethyl ascorbic acid added to cosmetics is 0.5-5%.

10. A cosmetic containing 3-o-ethyl ascorbic acid, characterized in that, It includes the 3-o-ethyl ascorbic acid as described in claim 7, and cosmetic excipients.

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