A 3-o-ethyl ascorbic acid composition and its preparation method and application

Abstract

The present invention proposes a 3-o-ethyl ascorbic acid composition and a preparation method and application thereof, and belongs to the technical field of cosmetics. The method comprises the following steps: S1. preparation of a culture medium; S2. activation of bacterial strains; S3. enzymatic fermentation; S4. hydrophobic modification; S5. preparation of a 3-o-ethyl ascorbic acid composition. The 3-o-ethyl ascorbic acid composition prepared by the present invention has high bioavailability and good transdermal absorption rate, can be used as a reducing agent, ultraviolet absorber and melanin formation inhibitor, has high heat resistance and antioxidant properties, hydrolyzes to release ascorbic acid, continuously provides ascorbic acid in the body, can promote the synthesis of collagen, has good safety, is a good cosmetic raw material, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a 3-o-ethyl ascorbic acid composition and a preparation method and application thereof. Background Art

[0002] In the field of cosmetics and skin care products, 3-o-ethyl ascorbic acid (structure as Formula I) is commonly used as an antioxidant and whitening agent, which can help reduce oxidative damage and pigmentation in the skin. When this ingredient is added to skin care products, the skin care products need to go through high-temperature production processes, such as mixing, heating, sterilization, etc. In this case, thermal stability is very important for the effectiveness of 3-o-ethyl ascorbic acid and maintaining its activity. If 3-o-ethyl ascorbic acid is unstable under high temperature conditions, it may decompose or lose its antioxidant properties, thereby affecting the efficacy of the product.

[0003]

[0004] Therefore, when manufacturing cosmetics and skin care products, it is necessary to select 3-o-ethyl ascorbic acid with good thermal stability to ensure its stability and activity under high temperature conditions. However, the thermal stability of 3-o-ethyl ascorbic acid crystals purified by traditional purification methods is not high. Summary of the invention

[0005] The purpose of the present invention is to provide a 3-o-ethyl ascorbic acid composition and a preparation method and application thereof. The composition has high bioavailability, good transdermal absorption rate, can be used as a reducing agent, ultraviolet absorber and melanin formation inhibitor, has high heat resistance and antioxidant properties, releases ascorbic acid through hydrolysis, continuously provides ascorbic acid for the body, can promote collagen synthesis, has good safety, is a good cosmetic raw material, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides a method for preparing a 3-o-ethyl ascorbic acid composition, comprising the following steps:

[0008] S1. Preparation of culture medium: adding glucose, sugar cane juice, ascorbic acid, amino acids, and inorganic salts to water and sterilizing to prepare a culture medium;

[0009] S2. Activation of bacterial strains: inoculating thermophilic Bacillus subtilis and Aspergillus niger into Gould's medium respectively, activating and culturing, and obtaining bacterial strain seed liquid;

[0010] S3. Enzymatic fermentation: adding the immobilized enzyme to the culture medium, inoculating the seed solution of Bacillus subtilis and Aspergillus niger, enzymatic fermentation, separating the immobilized enzyme with a magnet, filtering the product, dialyzing the filtrate, and freeze-drying the dialysate to obtain an ascorbic acid derivative composition;

[0011] S4 hydrophobic modification: adding an ascorbic acid derivative composition and a base to ethanol, adding a long-chain alkyl halide, heating and stirring the reaction, filtering, washing, and drying to obtain a modified ascorbic acid derivative composition;

[0012] S5. Preparation of 3-o-ethyl ascorbic acid composition: uniformly mix the modified ascorbic acid derivative composition and 3-o-ethyl ascorbic acid to prepare the 3-o-ethyl ascorbic acid composition.

[0013] As a further improvement of the present invention, the mass ratio of glucose, sugarcane juice, ascorbic acid, amino acid, inorganic salt and water in step S1 is 5-7:15-20:8-12:3-5:2-3:100, the sugar content of the sugarcane juice is 15-17%, the amino acid is selected from at least one of threonine, lysine, methionine, phenylalanine, valine, leucine, isoleucine, glycine and alanine, and the inorganic salt is sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride, and the mass ratio is 10:1-2:1.5-2:0.7-1.2:1-2.

[0014] As a further improvement of the present invention, the activation culture conditions in step S2 are 40-45°C, 100-200r / min, activation culture 24-30h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

[0015] As a further improvement of the present invention, the mass ratio of the immobilized enzyme and the culture medium in step S3 is 3-5:100, the inoculation amounts of the thermophilic Bacillus subtilis and Aspergillus niger seed liquids are 1-2 v / v% and 2-3 v / v%, respectively, the conditions of the enzymatic fermentation are 40-45° C., 100-200 r / min, and the enzymatic fermentation culture is 36-48 h. The preparation method of the immobilized enzyme is as follows:

[0016] T1. Preparation of porous silica spheres: adding alkyl orthosilicate to ethanol, adding ammonia water and a porogen, stirring the reaction, centrifuging, washing, drying, and calcining to obtain porous silica spheres;

[0017] T2. Magnetic modification: adding porous silica balls into water, adding cobalt nitrate and ferric nitrate, adjusting the pH value of the solution, centrifuging, washing, drying, and calcining to obtain magnetic silica balls;

[0018] T3. Tannic acid modification: adding magnetic silica balls to water, adding tannic acid and a catalyst, heating and stirring to react, centrifuging, washing, and drying to obtain modified magnetic silica balls;

[0019] T4. Adsorption of complex enzyme: Dissolve α-glucosidase, cyclomaltodextrin glucanotransferase and α-amylase in water, add modified magnetic silica balls, stir and mix evenly, evaporate the solvent to obtain immobilized enzyme.

[0020] Preferably, the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate.

[0021] Preferably, the porogen is selected from at least one of hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, hexadecyldimethylbenzylammonium chloride and hexadecyldimethylbenzylammonium bromide.

[0022] As a further improvement of the present invention, the mass ratio of the alkyl orthosilicate, ethanol, ammonia water and porogen is 10-15:80-100:7-10:1-2, the stirring reaction time is 10-12h, the mass ratio of the porous silica balls, cobalt nitrate and ferric nitrate is 100:4.7-4.9:1.8-1.9, the pH value of the adjustment solution is 8-9, the mass ratio of the magnetic silica balls, tannic acid and catalyst is 10:2-3:0.1-0.2, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heated stirring reaction is 40-50°C, the time is 1-3h, and the mass ratio of the α-glucosidase, cyclomaltodextrin glucanotransferase, α-amylase and modified magnetic silica balls is 1-2:3-5:2-4:100.

[0023] As a further improvement of the present invention, the mass ratio of the ascorbic acid derivative composition, the base, and the long-chain fatty amine in step S4 is 10:3-5:2-3, the long-chain fatty amine is selected from at least one of 1-chlorododecane, 1-chlorotetradecane, 1-chlorohexadecane, 1-chlorooctadecane, 1-bromododecane, 1-bromotetradecane, 1-bromohexadecane, and 1-bromooctadecane, and the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h.

[0024] As a further improvement of the present invention, the mass ratio of the modified ascorbic acid derivative composition to 3-o-ethyl ascorbic acid in step S5 is 3-5:8.

[0025] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0026] S1. Preparation of a culture medium: 5-7 parts by weight of glucose, 15-20 parts by weight of sugar cane juice having a sugar content of 15-17%, 8-12 parts by weight of ascorbic acid, 3-5 parts by weight of amino acids, 2-3 parts by weight of inorganic salts are added to 100 parts by weight of water and sterilized to obtain a culture medium;

[0027] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride, and the mass ratio is 10:1-2:1.5-2:0.7-1.2:1-2;

[0028] S2. Activation of bacteria: Thermomyces stearothermophilus Bacillus subtilis and Aspergillus niger were inoculated into Gould's medium respectively, and the culture was activated for 24-30 hours at 40-45°C and 100-200 r / min to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0029] S3. Enzymatic fermentation: 3-5 parts by weight of the immobilized enzyme was added to 100 parts by weight of the culture medium, inoculated with thermophilic Bacillus subtilis and Aspergillus niger seed solution, the inoculation amount was 1-2v / v% and 2-3v / v%, respectively, 40-45°C, 100-200r / min, enzymatic fermentation was cultured for 36-48h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0030] S4. Hydrophobic modification: 10 parts by weight of an ascorbic acid derivative composition, 3-5 parts by weight of a base are added to ethanol, 2-3 parts by weight of a long-chain alkyl halide are added, heated to 40-50 ° C, stirred for 2-4h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0031] S5. Preparation of 3-o-ethyl ascorbic acid composition: 3-5 parts by weight of the modified ascorbic acid derivative composition and 8 parts by weight of 3-o-ethyl ascorbic acid are uniformly mixed to prepare a 3-o-ethyl ascorbic acid composition.

[0032] The present invention further protects a 3-o-ethyl ascorbic acid composition obtained by the above preparation method.

[0033] The present invention further protects the use of the above 3-o-ethyl ascorbic acid composition in the preparation of cosmetics.

[0034] The present invention has the following beneficial effects:

[0035] 3-o-ethyl ascorbic acid is a lipophilic, water-soluble vitamin C derivative, which can easily penetrate the stratum corneum and enter the dermis. Once in the skin, it is easily decomposed by biological enzymes to play the role of vitamin C, thereby improving its bioavailability. It has extremely high stability and has better bioavailability and stability than ordinary vitamin C, so it can play a better role.

[0036] In the ascorbic acid derivative composition of the present invention, ascorbic acid and sugarcane juice are used as raw materials. Under the fermentation action of fermentation bacteria thermophilic Bacillus subtilis and Aspergillus niger, the D-pyranose glucopyranosyl part of maltose can be transferred to the hydroxyl group at the 2-carbon position of ascorbic acid for dehydration to generate 2-oxy-α-D-pyranose glucopyranosyl-L-ascorbic acid, or react to generate 5-oxy-α-D-pyranose glucopyranosyl-L-ascorbic acid, which significantly improves the antioxidant and UV resistance, has strong acid resistance, light resistance and thermal stability, and has good antibacterial and whitening effects. Under the enzymatic fermentation of the immobilized enzyme, 6-oxy-α-D-pyranose-glucopyranose-L-ascorbic acid, 6-oxy-acyl-2-O-α-D-pyranose-glucopyranose-L-ascorbic acid, etc. are easily produced, and the thermal stability and antioxidant properties are significantly enhanced. The prepared ascorbic acid derivative composition can inhibit tyrosinase activity and melanin formation; reduce melanin, lighten spots and whiten the skin, effectively remove free radicals, and is resistant to light, heat, acid and alkali, and air oxidation. It has high bioavailability, prevents skin inflammation caused by sunlight, and can promote collagen production and increase skin elasticity.

[0037] The immobilized enzyme uses porous silica nanospheres as carriers, and cobalt ferrite is deposited on the surface. The added amount is small but has high magnetism. It can not only cooperate with the enzyme to carry out enzymatic catalysis, but also help to separate the immobilized enzyme with a magnet, so that the enzyme can be used repeatedly and its economic value is improved. The modification of tannic acid increases the loading capacity of the enzyme, thereby improving the catalytic efficiency of the enzymatic reaction.

[0038] However, the fat solubility of the ascorbic acid derivative composition prepared above is not strong, and it is not easy to penetrate the skin phospholipid bilayer into the dermis, thereby exerting a skin regulating effect. Therefore, the present invention connects the derivative to the long-chain alkyl group through a nucleophilic substitution reaction with a long-chain alkyl halide, thereby improving its hydrophobicity, thereby making it more lipophilic, improving its skin permeability, and exerting a better skin regulating effect. It can penetrate the stratum corneum of the skin to reach the basal layer, inhibit tyrosinase activity, inhibit the formation of melanin, and reduce melanin to colorless, effectively whitening and removing spots. After entering the dermis, it directly participates in the synthesis of collagen, increases collagen, and thus makes the skin full and elastic.

[0039] The 3-o-ethyl ascorbic acid composition prepared by the invention has high bioavailability and good transdermal absorption rate, can be used as a reducing agent, an ultraviolet absorber and a melanin formation inhibitor, has high heat resistance and antioxidant properties, releases ascorbic acid through hydrolysis, continuously provides ascorbic acid to the body, can promote the synthesis of collagen, has good safety, is a good cosmetic raw material, and has broad application prospects. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] α-Glucosidase, 50 U / mg, purchased from McLean Chemical Reagent Co., Ltd.; cyclomaltodextrin glucanotransferase, CAS=10016-20-3, 10,000 U / g, purchased from Shenzhen Jinfuyuan Biotechnology Co., Ltd.; α-amylase, 50 U / mg, purchased from McLean Chemical Reagent Co., Ltd.

[0042] Bacillus stearothermophilus, 100 billion cfu / g, was purchased from Shandong Pingju Biotechnology Co., Ltd.; Aspergillus niger, 20 billion cfu / g, was purchased from Shandong Changtai Biotechnology Co., Ltd.

[0043] Preparation Example 1 Preparation of immobilized enzyme

[0044] Here’s how:

[0045] T1. Preparation of porous silica spheres: 10 g of methyl orthosilicate was added to 80 g of ethanol, 7 g of ammonia water and 1 g of hexadecyltrimethylammonium chloride were added, the reaction was stirred for 10 h, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain porous silica spheres;

[0046] T2. Magnetic modification: 100 g of porous silica spheres were added to 500 mL of water, 4.7 g of cobalt nitrate and 1.8 g of ferric nitrate were added, the pH value of the solution was adjusted to 8, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain magnetic silica spheres;

[0047] T3. Tannic acid modification: 10 g of magnetic silica spheres were added to 100 mL of water, 2 g of tannic acid and 0.1 g of catalyst, heated to 40 ° C, stirred for 1 h, centrifuged, washed, and dried to obtain modified magnetic silica spheres;

[0048] The catalyst is a Tris-HCl solution with a pH of 8.5;

[0049] T4. Adsorption of complex enzyme: 1g α-glucosidase, 3g cyclomaltodextrin glucanotransferase, and 2g α-amylase were dissolved in 200mL water, 100g modified magnetic silica balls were added, and the mixture was stirred for 20min. The solvent was evaporated to obtain the immobilized enzyme.

[0050] Preparation Example 2 Preparation of immobilized enzyme

[0051] Here’s how:

[0052] T1. Preparation of porous silica spheres: 15 g of tetraethyl orthosilicate was added to 100 g of ethanol, 10 g of ammonia water and 2 g of hexadecyldimethylbenzyl ammonium chloride were added, the mixture was stirred for 12 h, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain porous silica spheres;

[0053] T2. Magnetic modification: 100 g of porous silica spheres were added to 500 mL of water, 4.9 g of cobalt nitrate and 1.9 g of ferric nitrate were added, the pH value of the solution was adjusted to 9, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain magnetic silica spheres;

[0054] T3. Tannic acid modification: add 10 g of magnetic silica spheres to 100 mL of water, add 2-3 g of tannic acid and 0.2 g of catalyst, heat to 50 ° C, stir and react for 3 h, centrifuge, wash, and dry to obtain modified magnetic silica spheres;

[0055] The catalyst is a Tris-HCl solution with a pH of 9.5;

[0056] T4. Adsorption of complex enzyme: 2g α-glucosidase, 5g cyclomaltodextrin glucanotransferase, and 4g α-amylase were dissolved in 200mL water, 100g modified magnetic silica balls were added, and the mixture was stirred for 20min. The solvent was evaporated to obtain the immobilized enzyme.

[0057] Preparation Example 3 Preparation of immobilized enzyme

[0058] Here’s how:

[0059] T1. Preparation of porous silica spheres: 12 g of tetraethyl orthosilicate was added to 90 g of ethanol, 8.5 g of aqueous ammonia and 1.5 g of hexadecyltrimethylammonium bromide were added, the reaction was stirred for 11 h, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain porous silica spheres;

[0060] T2. Magnetic modification: 100 g of porous silica spheres were added to 500 mL of water, 4.82 g of cobalt nitrate and 1.84 g of ferric nitrate were added, the pH value of the solution was adjusted to 8.5, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain magnetic silica spheres;

[0061] T3. Tannic acid modification: 10 g of magnetic silica spheres were added to 100 mL of water, 2.5 g of tannic acid and 0.15 g of catalyst were added, heated to 45 ° C, stirred for 2 h, centrifuged, washed, and dried to obtain modified magnetic silica spheres;

[0062] The catalyst is a Tris-HCl solution with a pH of 9;

[0063] T4. Adsorption of complex enzyme: 1.5 g α-glucosidase, 4 g cyclomaltodextrin glucanotransferase, and 3 g α-amylase were dissolved in 200 mL of water, 100 g of modified magnetic silica balls were added, and the mixture was stirred for 20 min. The solvent was evaporated to obtain the immobilized enzyme.

[0064] Comparative Preparation Example 1

[0065] Compared with Preparation Example 3, the difference is that hexadecyltrimethylammonium bromide is not added in step T1.

[0066] The details are as follows:

[0067] T1. Preparation of silica spheres: 12 g of ethyl orthosilicate was added to 90 g of ethanol, 8.5 g of aqueous ammonia was added, the reaction was stirred for 11 h, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain silica spheres;

[0068] T2. Magnetic modification: 100 g of porous silica spheres were added to 500 mL of water, 4.82 g of cobalt nitrate and 1.84 g of ferric nitrate were added, the pH value of the solution was adjusted to 8.5, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain magnetic silica spheres;

[0069] T3. Tannic acid modification: 10 g of magnetic silica spheres were added to 100 mL of water, 2.5 g of tannic acid and 0.15 g of catalyst were added, heated to 45 ° C, stirred for 2 h, centrifuged, washed, and dried to obtain modified magnetic silica spheres;

[0070] The catalyst is a Tris-HCl solution with a pH of 9;

[0071] T4. Adsorption of complex enzyme: 1.5 g α-glucosidase, 4 g cyclomaltodextrin glucanotransferase, and 3 g α-amylase were dissolved in 200 mL of water, 100 g of modified magnetic silica balls were added, and the mixture was stirred for 20 min. The solvent was evaporated to obtain the immobilized enzyme.

[0072] Comparative Preparation Example 2

[0073] Compared with Preparation Example 3, the difference is that step T2 is not performed.

[0074] The details are as follows:

[0075] T1. Preparation of porous silica spheres: 12 g of tetraethyl orthosilicate was added to 90 g of ethanol, 8.5 g of aqueous ammonia and 1.5 g of hexadecyltrimethylammonium bromide were added, the reaction was stirred for 11 h, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain porous silica spheres;

[0076] T2. Tannic acid modification: 10 g of porous silica balls were added to 100 mL of water, 2.5 g of tannic acid and 0.15 g of catalyst were added, heated to 45 ° C, stirred for 2 h, centrifuged, washed, and dried to obtain modified silica balls;

[0077] The catalyst is a Tris-HCl solution with a pH of 9;

[0078] T3. Adsorption of composite enzyme: 1.5 g α-glucosidase, 4 g cyclomaltodextrin glucanotransferase, and 3 g α-amylase were dissolved in 200 mL of water, 100 g of modified silica balls were added, and the mixture was stirred for 20 min. The solvent was evaporated to obtain the immobilized enzyme.

[0079] Comparative Preparation Example 3

[0080] Compared with Preparation Example 3, the difference is that step T3 is not performed.

[0081] The details are as follows:

[0082] T1. Preparation of porous silica spheres: 12 g of tetraethyl orthosilicate was added to 90 g of ethanol, 8.5 g of aqueous ammonia and 1.5 g of hexadecyltrimethylammonium bromide were added, the reaction was stirred for 11 h, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain porous silica spheres;

[0083] T2. Magnetic modification: 100 g of porous silica spheres were added to 500 mL of water, 4.82 g of cobalt nitrate and 1.84 g of ferric nitrate were added, the pH value of the solution was adjusted to 8.5, centrifuged, washed, dried, and calcined at 500 ° C for 2 h to obtain magnetic silica spheres;

[0084] T3. Adsorption of complex enzyme: 1.5 g α-glucosidase, 4 g cyclomaltodextrin glucanotransferase, and 3 g α-amylase were dissolved in 200 mL of water, 100 g of magnetic silica balls were added, and the mixture was stirred for 20 min. The solvent was evaporated to obtain the immobilized enzyme.

[0085] Example 1

[0086] This embodiment provides a method for preparing a 3-o-ethyl ascorbic acid composition, which specifically comprises the following steps:

[0087] S1. Preparation of culture medium: 5 g of glucose, 15 g of sugar cane juice with a sugar content of 15%, 8 g of ascorbic acid, 2 g of threonine, 1 g of lysine, and 2 g of inorganic salt were added to 100 mL of water and sterilized to prepare a culture medium;

[0088] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride, with a mass ratio of 10:1:1.5:0.7:1;

[0089] S2. Activation of bacteria: Thermomyces stearothermophilus Bacillus subtilis and Aspergillus niger were inoculated into Gould's medium respectively, and activated and cultured for 24 h at 40°C and 100 r / min to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0090] S3. Enzymatic fermentation: 3 g of the immobilized enzyme prepared in Preparation Example 1 was added to 100 g of culture medium, inoculated with thermophilic Bacillus subtilis and Aspergillus niger seed solution, the inoculation amount was 1 v / v% and 2 v / v%, respectively, at 40 ° C, 100 r / min, enzymatic fermentation culture for 36 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0091] S4. Hydrophobic modification: 10 g of the ascorbic acid derivative composition and 3 g of triethylamine were added to 200 mL of ethanol, 2 g of 1-chlorododecane was added, heated to 40 ° C, stirred for 2 h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0092] S5. Preparation of 3-o-ethyl ascorbic acid composition: 3 g of the modified ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0093] Example 2

[0094] This embodiment provides a method for preparing a 3-o-ethyl ascorbic acid composition, which specifically comprises the following steps:

[0095] S1. Preparation of culture medium: 7 g of glucose, 20 g of sugar cane juice with a sugar content of 17%, 12 g of ascorbic acid, 3 g of threonine, 1 g of alanine, 1 g of glycine, and 23 g of inorganic salts were added to 100 mL of water and sterilized to obtain a culture medium;

[0096] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:2:2:1.2:2;

[0097] S2. Activation of bacteria: Thermomyces stearothermophilus Bacillus subtilis and Aspergillus niger were inoculated into Gould's medium respectively, and the culture was activated at 45°C, 200 r / min, for 30 h to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0098] S3. Enzymatic fermentation: 5 g of the immobilized enzyme prepared in Preparation Example 2 was added to 100 g of the culture medium, inoculated with thermophilic Bacillus subtilis and Aspergillus niger seed solution, the inoculation amount was 2 v / v% and 3 v / v%, respectively, 45 ° C, 200 r / min, enzymatic fermentation culture for 48 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0099] S4. Hydrophobic modification: 10 g of the ascorbic acid derivative composition and 5 g of triethylamine were added to 200 mL of ethanol, 3 g of 1-chlorotetradecane was added, heated to 50 ° C, stirred for 4 h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0100] S5. Preparation of 3-o-ethyl ascorbic acid composition: 5 g of the modified ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0101] Example 3

[0102] This embodiment provides a method for preparing a 3-o-ethyl ascorbic acid composition, which specifically comprises the following steps:

[0103] S1. Preparation of culture medium: 6 g of glucose, 17 g of sugar cane juice with a sugar content of 16%, 10 g of ascorbic acid, 2 g of threonine, 1 g of phenylalanine, 1 g of glycine, and 2.5 g of inorganic salt were added to 100 mL of water and sterilized to prepare a culture medium;

[0104] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:1.5:1.7:1:1.5;

[0105] S2. Activation of bacteria: Thermomyces stearothermophilus Bacillus subtilis and Aspergillus niger were inoculated into Gould's medium respectively, and the culture was activated at 42°C, 150 r / min for 27 h to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0106] S3. Enzymatic fermentation: 4 g of the immobilized enzyme prepared in Preparation Example 3 was added to 100 g of the culture medium, inoculated with thermophilic Bacillus subtilis and Aspergillus niger seed solution, the inoculation amounts were 1.5 v / v% and 2.5 v / v%, 42 ° C, 150 r / min, enzymatic fermentation culture for 42 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0107] S4 hydrophobic modification: 10 g of the ascorbic acid derivative composition, 4 g of triethylamine were added to 200 mL of ethanol, 2.5 g of 1-bromohexadecane was added, heated to 45 ° C, stirred for 3 h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0108] S5. Preparation of 3-o-ethyl ascorbic acid composition: 4 g of the modified ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0109] Comparative Examples 1-3

[0110] Compared with Example 3, the difference is that the immobilized enzyme is prepared by Comparative Preparation Examples 1-3.

[0111] Comparative Example 4

[0112] Compared with Example 3, the difference is that no immobilized enzyme is added in step S3.

[0113] The details are as follows:

[0114] S1. Preparation of culture medium: 6 g of glucose, 17 g of sugar cane juice with a sugar content of 16%, 10 g of ascorbic acid, 2 g of threonine, 1 g of phenylalanine, 1 g of glycine, and 2.5 g of inorganic salt were added to 100 mL of water and sterilized to prepare a culture medium;

[0115] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:1.5:1.7:1:1.5;

[0116] S2. Activation of bacteria: Thermomyces stearothermophilus Bacillus subtilis and Aspergillus niger were inoculated into Gould's medium respectively, and the culture was activated at 42°C, 150 r / min for 27 h to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0117] S3. Enzymatic fermentation: 100 g of culture medium was inoculated with thermophilic Bacillus subtilis and Aspergillus niger seed solution, the inoculation amount was 1.5 v / v% and .5 v / v%, respectively, at 42 ° C, 150 r / min, enzymatic fermentation was cultured for 42 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0118] S4 hydrophobic modification: 10 g of the ascorbic acid derivative composition, 4 g of triethylamine were added to 200 mL of ethanol, 2.5 g of 1-bromohexadecane was added, heated to 45 ° C, stirred for 3 h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0119] S5. Preparation of 3-o-ethyl ascorbic acid composition: 4 g of the modified ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0120] Comparative Example 5

[0121] Compared with Example 3, the difference is that the seed liquid of Bacillus stearothermophilus subtilis is not inoculated in step S3.

[0122] The details are as follows:

[0123] S1. Preparation of culture medium: 6 g of glucose, 17 g of sugar cane juice with a sugar content of 16%, 10 g of ascorbic acid, 2 g of threonine, 1 g of phenylalanine, 1 g of glycine, and 2.5 g of inorganic salt were added to 100 mL of water and sterilized to prepare a culture medium;

[0124] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:1.5:1.7:1:1.5;

[0125] S2. Activation of bacteria: Aspergillus niger was inoculated into Goulburn medium and cultured at 42°C, 150 r / min for 27 h to obtain a culture medium with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0126] S3. Enzymatic fermentation: 4 g of the immobilized enzyme prepared in Preparation Example 3 was added to 100 g of the culture medium, inoculated with a seed solution of Aspergillus niger, the inoculation amount was 4 v / v%, 42 ° C, 150 r / min, enzymatic fermentation was cultured for 42 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0127] S4 hydrophobic modification: 10 g of the ascorbic acid derivative composition, 4 g of triethylamine were added to 200 mL of ethanol, 2.5 g of 1-bromohexadecane was added, heated to 45 ° C, stirred for 3 h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0128] S5. Preparation of 3-o-ethyl ascorbic acid composition: 4 g of the modified ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0129] Comparative Example 6

[0130] Compared with Example 3, the difference is that the Aspergillus niger seed liquid is not inoculated in step S3.

[0131] The details are as follows:

[0132] S1. Preparation of culture medium: 6 g of glucose, 17 g of sugar cane juice with a sugar content of 16%, 10 g of ascorbic acid, 2 g of threonine, 1 g of phenylalanine, 1 g of glycine, and 2.5 g of inorganic salt were added to 100 mL of water and sterilized to prepare a culture medium;

[0133] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:1.5:1.7:1:1.5;

[0134] S2. Activation of bacteria: The thermophilic Bacillus subtilis was inoculated into Goulburn medium and cultured at 42°C, 150 r / min for 27 h to obtain a culture medium with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0135] S3. Enzymatic fermentation: 4 g of the immobilized enzyme prepared in Preparation Example 3 was added to 100 g of the culture medium, inoculated with a seed solution of Bacillus subtilis, the inoculation amount was 4 v / v%, 42 ° C, 150 r / min, the enzymatic fermentation culture was 42 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0136] S4 hydrophobic modification: 10 g of the ascorbic acid derivative composition, 4 g of triethylamine were added to 200 mL of ethanol, 2.5 g of 1-bromohexadecane was added, heated to 45 ° C, stirred for 3 h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition;

[0137] S5. Preparation of 3-o-ethyl ascorbic acid composition: 4 g of the modified ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0138] Comparative Example 7

[0139] Compared with Example 3, the difference is that step S4 is not performed.

[0140] The details are as follows:

[0141] S1. Preparation of culture medium: 6 g of glucose, 17 g of sugar cane juice with a sugar content of 16%, 10 g of ascorbic acid, 2 g of threonine, 1 g of phenylalanine, 1 g of glycine, and 2.5 g of inorganic salt were added to 100 mL of water and sterilized to prepare a culture medium;

[0142] The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:1.5:1.7:1:1.5;

[0143] S2. Activation of bacteria: Thermomyces stearothermophilus Bacillus subtilis and Aspergillus niger were inoculated into Gould's medium respectively, and the culture was activated at 42°C, 150 r / min for 27 h to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0144] S3. Enzymatic fermentation: 4 g of the immobilized enzyme prepared in Preparation Example 3 was added to 100 g of the culture medium, inoculated with thermophilic Bacillus subtilis and Aspergillus niger seed solution, the inoculation amount was 1.5 v / v% and .5 v / v%, respectively, at 42 ° C, 150 r / min, enzymatic fermentation culture for 42 h, the immobilized enzyme was separated by a magnet, the product was filtered, the filtrate was dialyzed to remove inorganic salts, and the dialyzate was freeze-dried to obtain an ascorbic acid derivative composition;

[0145] S4. Preparation of 3-o-ethyl ascorbic acid composition: 4 g of ascorbic acid derivative composition and 8 g of 3-o-ethyl ascorbic acid were stirred and mixed for 15 minutes to prepare a 3-o-ethyl ascorbic acid composition.

[0146] Test Example 1

[0147] Using anhydrous ethanol as solvent, the sample (3-o-ethyl ascorbic acid composition prepared in Example 1-3 or Comparative Example 1-7) or the reference substance (Vc) was prepared into solutions of different concentrations; using anhydrous ethanol as solvent, the o-phenanthroline solution was prepared into a solution with a concentration of 7.5 mmol / L; using deionized water as solvent, FeSO4 was prepared into a solution with a concentration of 7.5 mmol / L.

[0148] Add 100 μL of o-phenanthroline solution, 100 μL of FeSO4 solution, 100 μL of sample solution and 100 μL of 0.1% H2O2 solution to a centrifuge tube, and make up to 1.0 mL with phosphate buffer (50 mmol / L, pH = 7.4). Incubate at 37°C for 1 hour, measure the absorbance (A) at 536 nm, and calculate the hydroxyl radical scavenging rate. Calculate EC 50 (mmol / L). The results are shown in Table 1.

[0149] Hydroxyl radical scavenging rate = [(A 样品 -A 空白 ) / (A0-A 空白 )]×100%

[0150] Among them, A0 is the absorbance of the solution when no sample and H2O2 are added.

[0151] Table 1

[0152] Group <![CDATA[EC 50 (mmol / L)]]> Vitamin C 3.94 Example 1 0.62 Example 2 0.64 Example 3 0.60 Comparative Example 1 2.17 Comparative Example 2 1.09 Comparative Example 3 2.35 Comparative Example 4 2.78 Comparative Example 5 2.91 Comparative Example 6 2.87 Comparative Example 7 0.87

[0153] It can be seen from the above table that the 3-o-ethyl ascorbic acid compositions prepared in Examples 1-3 of the present invention have good antioxidant properties.

[0154] Test Example 2

[0155] 100 subjects were selected and randomly divided into 12 groups, 10 in each group, and 3-o-ethyl ascorbic acid composition solutions (concentration of 1 mg / mL) prepared by Examples 1-3 and Comparative Examples 1-7 were used respectively. The average age of the subjects was 32 (25-37) years old, and the test site was the cheek. All subjects had no abnormalities in the test site skin, excluding sensitive muscle subjects. The subjects applied this product every day for 4 weeks, once in the morning and evening, 2 mL each time. Before and after use, at the same time, the subjects washed the test site with clean water and wiped it dry, exposed the test site, and sat for 30 minutes in an environment with a temperature of 23°C ± 2°C and a relative humidity of 55 ± 5%. Then, the colorimeter instrument and melanin skin detector were used to measure the glossiness and melanin coefficient of the skin of the subject's test part, 3 times each time, and the average value was taken, and then the average value of each subject in the whole group was taken to obtain the skin glossiness and melanin coefficient change value of each group. The results are shown in Table 2.

[0156] Glossiness change rate (%) = (glossiness after use - glossiness before use) / glossiness before use × 100%

[0157] Melanin coefficient change rate (%) = (glossiness after use - glossiness before use) / glossiness before use × 100%

[0158] Table 2

[0159] Group Glossiness change rate (%) Melanin coefficient change rate (%) Example 1 38.12 50.27 Example 2 38.54 50.19 Example 3 39.17 50.45 Comparative Example 1 32.42 44.12 Comparative Example 2 36.79 47.53 Comparative Example 3 30.12 41.29 Comparative Example 4 28.45 39.45 Comparative Example 5 29.14 40.02 Comparative Example 6 27.59 38.57 Comparative Example 7 31.02 42.11

[0160] It can be seen from the above table that the 3-o-ethyl ascorbic acid compositions prepared in Examples 1-3 of the present invention have good skin lightening and whitening effects.

[0161] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a 3-o-ethyl ascorbic acid composition, characterized in that: The following steps are involved: S1. Preparation of culture medium: adding glucose, sugar cane juice, ascorbic acid, amino acids, and inorganic salts to water, sterilizing, and preparing a culture medium; S2. Activation of bacterial strains: inoculate thermophilic Bacillus subtilis and Aspergillus niger into Gould's medium respectively, activate and culture them, and prepare bacterial strain seed liquid; S3. Enzymatic fermentation: adding the immobilized enzyme to the culture medium, inoculating the seed solution of thermophilic Bacillus subtilis and Aspergillus niger, enzymatic fermentation, separating the immobilized enzyme with a magnet, filtering the product, dialyzing the filtrate, and freeze-drying the dialyzate to obtain an ascorbic acid derivative composition; The preparation method of the immobilized enzyme is as follows: T1. Preparation of porous silica spheres: adding alkyl orthosilicate to ethanol, adding ammonia water and hexadecyltrimethylammonium chloride, stirring the reaction, centrifuging, washing, drying, and calcining to obtain porous silica spheres; T2. Magnetic modification: adding porous silica balls into water, adding cobalt nitrate and iron nitrate, adjusting the pH value of the solution, centrifuging, washing, drying, and calcining to obtain magnetic silica balls; T3. Tannic acid modification: adding magnetic silica balls to water, adding tannic acid and a catalyst, heating and stirring to react, centrifuging, washing, and drying to obtain modified magnetic silica balls; T4. Adsorption of compound enzyme: α-glucosidase, cyclomaltodextrin glucanotransferase and α-amylase are dissolved in water, modified magnetic silica balls are added, stirred and mixed evenly, and the solvent is evaporated to obtain immobilized enzyme; S4 hydrophobic modification: adding an ascorbic acid derivative composition and triethylamine to ethanol, adding a long-chain alkyl halide, heating and stirring the reaction, filtering, washing, and drying to obtain a modified ascorbic acid derivative composition; S5. Preparation of 3-o-ethyl ascorbic acid composition: The modified ascorbic acid derivative composition and 3-o-ethyl ascorbic acid are uniformly mixed to prepare a 3-o-ethyl ascorbic acid composition.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of glucose, sugarcane juice, ascorbic acid, amino acid, inorganic salt and water is 5-7:15-20:8-12:3-5:2-3:100, the sugar content of the sugarcane juice is 15-17%, the amino acid is selected from at least one of threonine, lysine, phenylalanine, glycine and alanine, and the inorganic salt is sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride in a mass ratio of 10:1-2:1.5-2:0.7-1.2:1-2.

3. The preparation method according to claim 1, characterized in that: The activation culture conditions in step S2 are 40-45°C, 100-200r / min, activation culture for 24-30h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the immobilized enzyme and the culture medium in step S3 is 3-5:100, the inoculation amounts of the thermophilic Bacillus subtilis and Aspergillus niger seed liquids are 1-2 v / v% and 2-3 v / v%, respectively, the conditions of the enzymatic fermentation are 40-45°C, 100-200 r / min, and the enzymatic fermentation culture is 36-48h.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the alkyl orthosilicate, ethanol, ammonia water and hexadecyltrimethylammonium chloride is 10-15:80-100:7-10:1-2, the stirring reaction time is 10-12h, the mass ratio of the porous silica balls, cobalt nitrate and ferric nitrate is 100:4.7-4.9:1.8-1.9, the pH value of the adjustment solution is 8-9, the mass ratio of the magnetic silica balls, tannic acid and the catalyst is 10:2-3:0.1-0.2, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heated stirring reaction is 40-50°C, the time is 1-3h, and the mass ratio of the α-glucosidase, cyclomaltodextrin glucanotransferase, α-amylase and modified magnetic silica balls is 1-2:3-5:2-4:

100.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the ascorbic acid derivative composition, triethylamine and long-chain fatty alkane in step S4 is 10:3-5:2-3, the long-chain fatty alkane is selected from at least one of 1-chlorododecane, 1-chlorotetradecane and 1-bromohexadecane, the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h.

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

8.

8. The preparation method according to claim 1, characterized in that: The specific steps include: S1. Preparation of a culture medium: 5-7 parts by weight of glucose, 15-20 parts by weight of sugar cane juice having a sugar content of 15-17%, 8-12 parts by weight of ascorbic acid, 3-5 parts by weight of amino acids, 2-3 parts by weight of inorganic salts are added to 100 parts by weight of water and sterilized to obtain a culture medium; The inorganic salts are sodium chloride, magnesium chloride, zinc chloride, copper chloride and ferric chloride, and the mass ratio is 10:1-2:1.5-2:0.7-1.2:1-2; S2. Activation of bacteria: Inoculate thermophilic Bacillus subtilis and Aspergillus niger in Gould's medium respectively, at 40-45°C, 100-200r / min, activate and culture for 24-30h, and obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution; S3. Enzymatic fermentation: adding 3-5 parts by weight of the immobilized enzyme to 100 parts by weight of the culture medium, inoculating the seed solution of thermophilic Bacillus subtilis and Aspergillus niger, the inoculation amount is 1-2v / v% and 2-3v / v%, respectively, 40-45°C, 100-200r / min, enzymatic fermentation culture for 36-48h, separating the immobilized enzyme with a magnet, filtering the product, dialyzing the filtrate to remove inorganic salts, and freeze-drying the dialyzate to obtain an ascorbic acid derivative composition; S4. Hydrophobic modification: 10 parts by weight of the ascorbic acid derivative composition, 3-5 parts by weight of triethylamine are added to ethanol, 2-3 parts by weight of a long-chain alkyl halide are added, heated to 40-50 ° C, stirred for 2-4h, filtered, washed, and dried to obtain a modified ascorbic acid derivative composition; S5. Preparation of 3-o-ethyl ascorbic acid composition: 3-5 parts by weight of the modified ascorbic acid derivative composition and 8 parts by weight of 3-o-ethyl ascorbic acid are uniformly mixed to prepare a 3-o-ethyl ascorbic acid composition.

9. A 3-o-ethyl ascorbic acid composition obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the 3-o-ethyl ascorbic acid composition according to claim 9 in the preparation of cosmetics.

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