Oxidoreductase composition as well as preparation method and application thereof

Abstract

The invention provides an oxidoreductase composition as well as a preparation method and application thereof, and belongs to the technical field of biology. The composition contains various complex enzymes, acetyl hexapeptide-8, a centella asiatica extract, an aesculus chinensis extract, ceramide NP and other components. The centella asiatica extract is obtained by performing common enzymolysis on centella asiatica through cellulase, pectinase and papain, then performing fermentation through lactobacillus plantarum and bifidobacterium adolescentis and performing purification; the aesculus chinensis extract is obtained by fermenting mature aesculus chinensis seeds with lactobacillus reuteri and lactobacillus bulgaricus and purifying. All the components are matched for use, and meanwhile, the composition has the effects of resisting sugar, diminishing swelling, resisting aging, tightening and smoothing skin, brightening and dissolving fat, and is safe and efficient.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an oxidoreductase composition and a preparation method and application thereof. Background Art

[0002] In today's beauty and skin care industry, consumers' demands for cosmetics' efficacy are becoming increasingly diversified and refined. With the accelerated pace of life, increased environmental pressure and changes in dietary structure, the skin is facing many problems, which has prompted the research and development of cosmetics with multiple functions to become the focus of the industry.

[0003] Glycation is one of the main causes of skin aging and dull complexion. Excess sugar in the body combines with collagen in the skin to form advanced glycation end products (AGEs). AGEs can cause collagen to lose elasticity, leading to sagging skin and wrinkles. They can also affect skin metabolism, causing dull skin and lack of luster. Therefore, anti-sugar ingredients have become a key additive in this type of cosmetics.

[0004] Poor water metabolism and obstructed lymphatic circulation often cause skin swelling. In modern life, bad habits such as long-term sitting, staying up late, and high-salt diet make the face, especially around the eyes, prone to edema. To address this problem, cosmetics have added certain ingredients that promote blood circulation and diuresis, which can accelerate the discharge of excess water in the skin and reduce edema.

[0005] Skin aging is accompanied by a decline in cell vitality and loss of collagen and elastic fibers. To achieve anti-aging effects, a variety of peptide active ingredients are widely used to block the release of neurotransmitters, relax facial muscles, reduce the formation of expression lines, and also help improve skin firmness.

[0006] In addition, to improve the smoothness and glossiness of the skin, some acidic ingredients can gently remove aging keratin cells on the skin surface, promote the renewal of skin cells, and make the skin smoother and more delicate.

[0007] In terms of fat dissolving, as people pay more attention to facial contouring, cosmetics containing fat dissolving ingredients have also emerged. For example, some products containing L-carnitine, phosphatidylcholine and other ingredients can promote the decomposition of fat in fat cells and convert fat into substances that can be metabolized by cells, thereby reducing facial fat accumulation, especially in areas such as the jawline, making the facial lines tighter and more stylish.

[0008] There are many kinds of cosmetics with various functions, and the functions vary greatly. There are also problems such as poor ingredient safety, unclear effects, and long use cycles. Therefore, it is necessary to continuously develop safer and more efficient products to provide people with more comprehensive and efficient skin care solutions, so that the skin can stay healthy, young and beautiful in a complex living environment. Summary of the invention

[0009] Aiming at the problems of poor safety of ingredients in existing products, unclear effects and single efficacy, the present invention provides an oxidoreductase composition and its preparation method and application. The composition of the present invention contains various composite enzymes, acetyl hexapeptide-8, Centella asiatica extract, horse chestnut extract and ceramide NP and other ingredients; Centella asiatica extract and horse chestnut extract are prepared by a special extraction method; various ingredients are used in combination, and have the effects of anti-sugar, swelling, anti-aging, firming, smoothing skin, brightening and fat dissolving, which is safe and efficient. The specific technical scheme is as follows: A redox enzyme composition comprises the following raw materials in parts by weight: 1 to 3 parts of lysozyme, 2 to 5 parts of superoxide dismutase, 0.5 to 2 parts of papain, 0.3 to 0.5 parts of trypsin, 0.5 to 1 part of lactoperoxidase, 1 to 3 parts of glucose oxidase, 1 to 3 parts of redox enzyme, 0.5 to 1 part of bromelain, 2 to 5 parts of acetyl hexapeptide-8, 3 to 6 parts of Centella asiatica extract, 3 to 6 parts of horse chestnut extract and 3 to 6 parts of ceramide NP.

[0010] In the above composition, the preparation method of the Centella asiatica extract includes: taking the whole plant of Centella asiatica, crushing, sieving, and taking the powder under the sieve; adding a buffer solution to the powder, adding 0.5% to 1.0% cellulase, 0.3% to 0.8% pectinase and 0.5% to 1.0% papain in accordance with the mass of the powder, enzymolysis, cooling, obtaining a material, inoculating 3% to 5% of the mass of the material with Lactobacillus plantarum and 3% to 5% of the mass of Bifidobacterium adolescentis, anaerobic fermentation, obtaining a fermentation liquid, filtering, concentrating the filtrate, and obtaining a concentrated liquid; extracting the concentrated liquid with ethanol-water solution under hot reflux for multiple times, combining the extracts, adsorbing the extracts through a resin column, washing with water to remove impurities, eluting with ethanol-water solution, collecting the eluate, ultrafiltration, taking the ultrafiltrate and concentrating it under reduced pressure, and drying to obtain the Centella asiatica extract. In the above composition, the preparation method of the Centella asiatica extract comprises the following steps: taking the whole plant of Centella asiatica, crushing it, passing it through a 40-mesh to 60-mesh sieve, taking the sieve to obtain a powder; adding a pH 5.5-6.0 phosphate buffer solution 10 to 15 times the weight of the powder to the powder, and then adding 0.5% to 1.0% of cellulase by weight of the powder, 0.3% to 0.8% of pectinase by weight of the powder, and 0.5% to 1.0% of papain by weight of the powder in sequence, performing enzymolysis at 40°C to 45°C and a speed of 120r / min to 150r / min for 2.5h to 3.0h, cooling to room temperature to obtain a material, inoculating Lactobacillus plantarum with a mass inoculation amount of 3% to 5% of the mass of the material and inoculating Bifidobacterium adolescentis with a mass inoculation amount of 3% to 5%, anaerobically fermenting at 33°C to 37°C for 20h to 25h to obtain a fermentation liquid, and using 2 00-250 mesh sieve, take the filtrate, and concentrate under reduced pressure to 30%-40% of the volume of the fermentation liquid to obtain a concentrated solution; according to the volume ratio of concentrated solution: ethanol aqueous solution = 1: (6-8), the concentrated solution is subjected to hot reflux extraction with 65%-70% volume concentration ethanol aqueous solution for 2-3 times, each time for 1.5h-2.0h, the extracts are combined, the extracts are adsorbed by a D101 macroporous adsorption resin column, and then eluted with 2-3 column volumes of deionized water to remove impurities, and then eluted with 50%-70% volume concentration ethanol aqueous solution at a flow rate of 1mL / min-2mL / min for 3-4 column volumes, the eluate of ethanol aqueous solution is collected, and the eluate is ultrafiltered using a 5kDa ultrafiltration membrane, the ultrafiltrate below 5kDa is taken, concentrated under reduced pressure, and dried to obtain Centella asiatica extract.

[0011] In the preparation method of the Centella asiatica extract, the enzymatic activity of the cellulase is 20,000 U / g to 100,000 U / g, the enzymatic activity of the pectinase is 20,000 U / g to 30,000 U / g, and the enzymatic activity of the papain is 100,000 U / g to 200,000 U / g.

[0012] In the preparation method of the Centella asiatica extract, the bacterial activity of the Lactobacillus plantarum is 5 billion CFU / g to 10 billion CFU / g, and the bacterial activity of the Bifidobacterium adolescentis is 5 billion CFU / g to 10 billion CFU / g.

[0013] In the preparation method of the Centella asiatica extract, the temperature of the reduced pressure concentration is 60°C to 80°C; and the temperature of the drying is 70°C to 80°C.

[0014] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing shelled horse chestnut ripe seeds, sieving, taking the sieve to obtain powder; adding water to the powder to obtain a base liquid; sequentially adding glucose, peptone, 1% to 3% of Lactobacillus reuteri by weight of the powder, and 1% to 3% of Lactobacillus bulgaricus by weight of the powder to the base liquid, performing anaerobic fermentation to obtain a fermentation liquid, filtering, taking the filtrate and freeze-drying to obtain a crude extract; dissolving the crude extract with n-hexane, adding sodium hydroxide aqueous solution for extraction, taking the lower aqueous phase, concentrating under reduced pressure, and drying to obtain the horse chestnut extract.

[0015] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing the shelled ripe horse chestnut seeds, passing through a 40-mesh to 60-mesh sieve, taking the sieve bottom to obtain a powder; adding water 10 to 15 times the mass of the powder to the powder to obtain a base liquid; adding 3% to 5% of glucose by mass of the base liquid, 2% to 3% of peptone by mass of the base liquid, 1% to 3% of Lactobacillus reuteri by mass of the powder, and 1% to 3% of Lactobacillus bulgaricus by mass of the powder to the base liquid in sequence, and performing anaerobically fermentation at 35°C to 40°C for 3 to 4 days to obtain a fermentation liquid, filtering with a 200-mesh to 250-mesh sieve, taking the filtrate, and freeze-drying to obtain a crude extract; dissolving the crude extract with 5 to 6 times the mass of n-hexane by mass of the crude extract, and then adding an equal volume of n-hexane and a 4% to 6% mass concentration of sodium hydroxide aqueous solution for extraction, taking the lower aqueous phase extract, concentrating under reduced pressure, and drying to obtain the horse chestnut extract.

[0016] In the method for preparing the horse chestnut extract, the bacterial activity of the Lactobacillus reuteri is 5 billion CFU / g to 10 billion CFU / g, and the bacterial activity of the Lactobacillus bulgaricus is 5 billion CFU / g to 10 billion CFU / g.

[0017] In the method for preparing the horse chestnut extract, the temperature for the reduced pressure concentration is 60°C to 80°C; and the temperature for the drying is 70°C to 80°C.

[0018] The method for preparing the above-mentioned oxidoreductase composition comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to their mass fractions, and precool them in a dry, sterile environment at a constant temperature of 2°C to 8°C for 30 min to 60 min to maintain the stability of enzyme activity; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 2°C to 8°C by circulating condensed water, and stirred and mixed evenly to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, add the mixture C to the mixed enzyme A, and stir and mix them evenly at 2° C. to 8° C. to obtain a redox enzyme composition.

[0019] Application of the above-mentioned oxidoreductase composition in the preparation of cosmetics.

[0020] The present invention provides an oxidoreductase composition and a preparation method and application thereof, and the beneficial effects are as follows: 1. The composition of the present invention comprises lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase, bromelain, acetyl hexapeptide-8, Centella asiatica extract, horse chestnut extract and ceramide NP. Superoxide dismutase with a relatively small molecular weight has a certain chance to penetrate the stratum corneum and can penetrate into the skin through lipid channels between stratum corneum cells and appendages such as hair follicles and sweat glands; superoxide dismutase can remove superoxide anion free radicals, reduce the damage of free radicals to cells, and resist oxidation to delay aging. Proteases such as papain and trypsin can also play a certain role on the surface of the skin. They can decompose aging and accumulated keratin on the surface of the skin, and the small peptide fragments and amino acids produced will promote the absorption of subsequent ingredients. At the same time, they themselves also slowly enter the upper layer of the skin through intercellular penetration at the superficial part of the skin, where they decompose some pathological and aging proteins. Oxidoreductases such as glucose oxidase and lactoperoxidase mainly play a role by regulating the redox potential on the surface of the skin. The microenvironment on the surface of the skin is conducive to maintaining the activity of these enzymes, and some of the metabolites they produce can diffuse into the upper layer of the skin, affecting the physiological functions of the skin, such as regulating the metabolic state of cells and immune response. Acetyl hexapeptide-8 can inhibit the release of neurotransmitters and reduce muscle contraction, thereby reducing wrinkles and achieving anti-aging effects. Ceramide NP can enhance the skin barrier function, reduce water loss and damage to the skin caused by external stimuli, and help anti-aging.

[0021] Second, lysozyme can destroy bacterial cell walls, superoxide dismutase can remove free radicals, and papain and trypsin can decompose aging and accumulated keratin on the skin surface. Their synergistic effect can maintain the skin microenvironment from multiple aspects. Lysozyme inhibits the growth of harmful microorganisms, superoxide dismutase prevents free radical damage, and papain and trypsin jointly decompose aging proteins and microbial proteins on the skin surface, thereby cleaning the skin, preventing microbial invasion and reducing the accumulation of protein aging products, and jointly achieving the effect of protecting skin health and promoting skin renewal.

[0022] 3. Lactoperoxidase and glucose oxidase participate in the redox reaction. Oxidoreductase can regulate the redox state in cells, and bromelain can decompose surface aging proteins. Their synergistic effect can regulate the redox balance of the skin and reduce the damage of oxidative stress to the skin. At the same time, bromelain decomposes proteins denatured by oxidative damage, promotes skin metabolism, maintains the skin's health, and helps repair and regenerate the skin.

[0023] Fourth, through multiple enzymatic hydrolysis and fermentation steps, additional enzymatic hydrolysis products and fermentation products are produced, so that the effective ingredients in Centella asiatica can be fully released, and the content of active ingredients is increased. These ingredients have better effects on skin repair and anti-inflammatory effects; after multiple fine separation steps such as hot reflux extraction, macroporous adsorption resin column adsorption elution and ultrafiltration membrane filtration, impurities can be effectively removed, the purity of the extract can be improved, and impurities that irritate the skin can be reduced. Microbial fermentation is used in the extraction process. The metabolic activities of microorganisms will produce some new active ingredients and modify the original ingredients, making them have better biological activity and more conducive to skin absorption and utilization.

[0024] 5. After the fermentation process, the effective ingredients in the horse chestnut seeds, such as saponins, are more fully released, the content of active ingredients in the extract is increased, and its efficacy in reducing swelling and improving microcirculation is enhanced. Through steps such as n-hexane dissolution and sodium hydroxide aqueous solution extraction, some impurities such as oil and pigment can be removed, the quality and purity of the extract can be improved, and the adverse effects on the skin can be reduced. The metabolites of microorganisms in the fermentation process can interact with the ingredients in the horse chestnut seeds, enhancing the biological activity of the extract and making it play a better role in skin care. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0026] Example 1 A redox enzyme composition, comprising the following raw materials in parts by weight: 2 parts of lysozyme, 3 parts of superoxide dismutase, 1 part of papain, 0.4 parts of trypsin, 0.6 parts of lactoperoxidase, 2 parts of glucose oxidase, 2 parts of redox enzyme, 0.8 parts of bromelain, 3 parts of acetyl hexapeptide-8, 5 parts of Centella asiatica extract, 5 parts of horse chestnut extract and ceramide NP 5 parts; lysozyme comes from Shandong Jishuo Biotechnology Co., Ltd., and the enzymatic activity of lysozyme is 20,000 U / mg; superoxide dismutase comes from Shanxi Shengdayue Biotechnology Co., Ltd., and the enzymatic activity of superoxide dismutase is 100,000 U / g; papain comes from Zhengzhou Maida Biotechnology Co., Ltd., and the enzymatic activity of papain is 100,000 U / g; trypsin comes from Xi'an Muguo Biotechnology Co., Ltd., and the enzymatic activity of trypsin is 4,000 U / g; lactoperoxidase comes from Shanghai Xinhu Biotechnology Co., Ltd., and the enzymatic activity of lactoperoxidase is 35 U / mg; glucose oxidase comes from Jiangsu Praite Bioengineering Co., Ltd., and the enzymatic activity of glucose oxidase is 100,000 U / g; the oxidoreductase is catalase, which comes from Zhengzhou Wanbo Chemical Products Co., Ltd., and the enzymatic activity of catalase is 200,000 U / g; bromelain comes from Lanli Biotechnology (Xi'an) Co., Ltd., and the enzymatic activity of bromelain is 300,000 U / g.

[0027] In the above composition, the preparation method of the Centella asiatica extract comprises the following steps: taking the whole plant of Centella asiatica, crushing it, passing it through a 40-mesh sieve, and taking the sieve to obtain a powder; adding a pH 5.8 phosphate buffer solution 12 times the weight of the powder to the powder, and then adding 0.8% of the weight of the powder with an enzyme activity of 50,000 U / g of cellulase, 0.5% of the weight of the powder with an enzyme activity of 30,000 U / g of pectinase, and 0.8% of the weight of the powder with an enzyme activity of 100,000 U / g of papain, enzymolysis at 42°C and a speed of 140r / min for 2.5h, cooling to room temperature, inoculating Lactobacillus plantarum with a bacterial activity of 10 billion CFU / g at a 4% weight inoculation amount, inoculating Bifidobacterium adolescentis with a bacterial activity of 10 billion CFU / g at a 4% weight inoculation amount, and anaerobic fermentation at 35°C for 22 h, obtain the fermentation broth, filter with a 250-mesh sieve, take the filtrate, and concentrate under reduced pressure at 80°C to 35% of the volume of the fermentation broth to obtain a concentrate; according to the volume ratio of concentrate: ethanol aqueous solution = 1:7, the concentrate is subjected to hot reflux extraction twice with 68% volume concentration ethanol aqueous solution, each time for 2.0 h, the extracts are combined, the extracts are adsorbed by a D101 macroporous adsorption resin column, and then eluted with 2 column volumes of deionized water to remove impurities, and then eluted with 60% volume concentration ethanol aqueous solution at a flow rate of 2 mL / min for 3 column volumes, the eluate of the ethanol aqueous solution is collected, and the eluate is ultrafiltered with a 5 kDa ultrafiltration membrane, the ultrafiltrate below 5 kDa is taken, concentrated under reduced pressure at 70°C, and dried at 75°C to obtain the Centella asiatica extract.

[0028] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing the shelled ripe horse chestnut seeds, passing through a 40-mesh sieve, taking the sieve bottom to obtain a powder; adding water 12 times the mass of the powder to the powder to obtain a base liquid; adding 4% of the mass of the base liquid glucose, 2.5% of the mass of the base liquid peptone, 2% of the mass of the powder Lactobacillus reuteri with a bacterial activity of 10 billion CFU / g, and 2% of the mass of the powder Lactobacillus bulgaricus with a bacterial activity of 5 billion CFU / g to the base liquid in sequence, anaerobically fermenting at 37°C for 3.5 days to obtain a fermentation liquid, filtering with a 200-mesh sieve, taking the filtrate, and freeze-drying to obtain a crude extract; dissolving the crude extract with 5.5 times the mass of n-hexane of the crude extract, then adding an equal volume of n-hexane and a 5% mass concentration of sodium hydroxide aqueous solution to perform extraction at room temperature for 30 minutes, taking the lower aqueous phase extract, concentrating under reduced pressure at 70°C, and drying at 78°C to obtain a horse chestnut extract.

[0029] The method for preparing the above-mentioned oxidoreductase composition comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to their mass fractions, and precool them in a dry, sterile environment at a constant temperature of 4°C for 40 minutes to maintain the activity of the enzymes; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 4°C by circulating condensed water, and stirred and mixed to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, the mixture C is added to the mixed enzyme A, and the mixture is stirred at 6° C. to obtain a redox enzyme composition.

[0030] Application of the above-mentioned oxidoreductase composition in the preparation of cosmetics.

[0031] Example 2 A redox enzyme composition, comprising the following raw materials in parts by weight: 1 part of lysozyme, 2 parts of superoxide dismutase, 0.5 parts of papain, 0.3 parts of trypsin, 0.5 parts of lactoperoxidase, 1 part of glucose oxidase, 1 part of redox enzyme, 0.5 parts of bromelain, 2 parts of acetyl hexapeptide-8, 3 parts of Centella asiatica extract, 3 parts of horse chestnut extract and ceramide NP 3 parts; lysozyme comes from Shandong Jishuo Biotechnology Co., Ltd., and the enzymatic activity of lysozyme is 20,000 U / mg; superoxide dismutase comes from Shanxi Shengdayue Biotechnology Co., Ltd., and the enzymatic activity of superoxide dismutase is 100,000 U / g; papain comes from Zhengzhou Maida Biotechnology Co., Ltd., and the enzymatic activity of papain is 100,000 U / g; trypsin comes from Xi'an Muguo Biotechnology Co., Ltd., and the enzymatic activity of trypsin is 4,000 U / g; lactoperoxidase comes from Shanghai Xinhu Biotechnology Co., Ltd., and the enzymatic activity of lactoperoxidase is 35 U / mg; glucose oxidase comes from Jiangsu Praite Bioengineering Co., Ltd., and the enzymatic activity of glucose oxidase is 100,000 U / g; the oxidoreductase is catalase, which comes from Zhengzhou Wanbo Chemical Products Co., Ltd., and the enzymatic activity of catalase is 200,000 U / g; bromelain comes from Lanli Biotechnology (Xi'an) Co., Ltd., and the enzymatic activity of bromelain is 300,000 U / g.

[0032] In the above composition, the preparation method of the Centella asiatica extract includes the following steps: taking the whole plant of Centella asiatica, crushing it, passing it through a 40-mesh sieve, and taking the sieve to obtain a powder; adding a pH 5.5 phosphate buffer solution 10 times the weight of the powder to the powder, and then adding 0.5% of the weight of the powder with an enzyme activity of 20,000 U / g of cellulase, 0.3% of the weight of the powder with an enzyme activity of 20,000 U / g of pectinase, and 0.5% of the weight of the powder with an enzyme activity of 100,000 U / g of papain, enzymolysis at 40°C and a speed of 120r / min for 2.5h, cooling to room temperature, inoculating Lactobacillus plantarum with a bacterial activity of 5 billion CFU / g at a 3% weight inoculation amount, inoculating Bifidobacterium adolescentis with a bacterial activity of 5 billion CFU / g at a 3% weight inoculation amount, and anaerobically fermenting at 33°C for 20h , obtain a fermentation broth, filter with a 200-mesh sieve, take the filtrate, and concentrate under reduced pressure at 80°C to 30% of the volume of the fermentation broth to obtain a concentrate; according to the volume ratio of concentrate: ethanol aqueous solution = 1:6, the concentrate is subjected to hot reflux extraction twice with a 65% volume concentration ethanol aqueous solution, each time for 1.5 hours, the extracts are combined, the extracts are adsorbed by a D101 macroporous adsorption resin column, and then eluted with 2 column volumes of deionized water to remove impurities, and then eluted with a 50% volume concentration ethanol aqueous solution at a flow rate of 1 mL / min for 3 column volumes, the eluate of the ethanol aqueous solution is collected, and the eluate is ultrafiltered with a 5 kDa ultrafiltration membrane, the ultrafiltrate below 5 kDa is taken, concentrated under reduced pressure at 60°C, and dried at 70°C to obtain a Centella asiatica extract.

[0033] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing the shelled ripe horse chestnut seeds, passing through a 40-mesh sieve, taking the sieve bottom to obtain a powder; adding water 10 times the mass of the powder to the powder to obtain a base liquid; adding 3% of the mass of the base liquid glucose, 2% of the mass of the base liquid peptone, 1% of the mass of the powder Lactobacillus reuteri with a bacterial activity of 5 billion CFU / g, and 1% of the mass of the powder Lactobacillus bulgaricus with a bacterial activity of 5 billion CFU / g to the base liquid in sequence, anaerobically fermenting at 35°C for 3 days to obtain a fermentation liquid, filtering with a 200-mesh sieve, taking the filtrate, and freeze-drying to obtain a crude extract; dissolving the crude extract with 5 times the mass of the crude extract in n-hexane, then adding an equal volume of n-hexane and a 4% mass concentration of sodium hydroxide aqueous solution to perform extraction at room temperature for 30 minutes, taking the lower aqueous phase extract, concentrating under reduced pressure at 60°C, and drying at 70°C to obtain a horse chestnut extract.

[0034] The method for preparing the above-mentioned oxidoreductase composition comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to their mass fractions, and precool them in a dry, sterile environment at a constant temperature of 2°C for 30 minutes to maintain the activity of the enzymes; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 2° C. with circulating condensed water, and stirred to mix evenly to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, the mixture C is added to the mixed enzyme A, and the mixture is stirred at 2° C. to obtain a redox enzyme composition.

[0035] Application of the above-mentioned oxidoreductase composition in the preparation of cosmetics.

[0036] Example 3 A redox enzyme composition, comprising the following raw materials in parts by weight: 1 part of lysozyme, 5 parts of superoxide dismutase, 0.5 parts of papain, 0.5 parts of trypsin, 0.5 parts of lactoperoxidase, 3 parts of glucose oxidase, 1 part of redox enzyme, 1 part of bromelain, 2 parts of acetyl hexapeptide-8, 6 parts of Centella asiatica extract, 3 parts of horse chestnut extract and ceramide NP 6 parts; lysozyme comes from Shandong Jishuo Biotechnology Co., Ltd., and the enzymatic activity of lysozyme is 20,000 U / mg; superoxide dismutase comes from Shanxi Shengdayue Biotechnology Co., Ltd., and the enzymatic activity of superoxide dismutase is 100,000 U / g; papain comes from Zhengzhou Maida Biotechnology Co., Ltd., and the enzymatic activity of papain is 100,000 U / g; trypsin comes from Xi'an Muguo Biotechnology Co., Ltd., and the enzymatic activity of trypsin is 4,000 U / g; lactoperoxidase comes from Shanghai Xinhu Biotechnology Co., Ltd., and the enzymatic activity of lactoperoxidase is 35 U / mg; glucose oxidase comes from Jiangsu Praite Bioengineering Co., Ltd., and the enzymatic activity of glucose oxidase is 100,000 U / g; the oxidoreductase is catalase, which comes from Zhengzhou Wanbo Chemical Products Co., Ltd., and the enzymatic activity of catalase is 200,000 U / g; bromelain comes from Lanli Biotechnology (Xi'an) Co., Ltd., and the enzymatic activity of bromelain is 300,000 U / g.

[0037] In the above composition, the preparation method of the Centella asiatica extract comprises the following steps: taking the whole plant of Centella asiatica, crushing it, passing it through a 40-mesh sieve, and taking the sieve to obtain a powder; adding a pH 5.5 phosphate buffer solution 15 times the weight of the powder to the powder, and then adding 1.0% of the weight of the powder with an enzyme activity of 20,000 U / g of cellulase, 0.8% of the weight of the powder with an enzyme activity of 20,000 U / g of pectinase, and 1.0% of the weight of the powder with an enzyme activity of 100,000 U / g of papain, enzymolysis at 45°C and a speed of 120r / min for 3.0h, cooling to room temperature, inoculating Lactobacillus plantarum with a bacterial activity of 10 billion CFU / g at a 3% weight inoculation amount, inoculating Bifidobacterium adolescentis with a bacterial activity of 10 billion CFU / g at a 3% weight inoculation amount, and anaerobic fermentation at 33°C for 25 h, obtain the fermentation broth, filter with a 200-mesh sieve, take the filtrate, and concentrate under reduced pressure at 75°C to 40% of the volume of the fermentation broth to obtain a concentrate; according to the volume ratio of concentrate: ethanol aqueous solution = 1:6, the concentrate is subjected to hot reflux extraction twice with 70% volume concentration ethanol aqueous solution, each time for 2.0 h, the extracts are combined, the extracts are adsorbed by a D101 macroporous adsorption resin column, and then eluted with 2 column volumes of deionized water to remove impurities, and then eluted with 70% volume concentration ethanol aqueous solution at a flow rate of 1 mL / min for 4 column volumes, the eluate of the ethanol aqueous solution is collected, and the eluate is ultrafiltered with a 5 kDa ultrafiltration membrane, the ultrafiltrate below 5 kDa is taken, concentrated under reduced pressure at 60°C, and dried at 80°C to obtain the Centella asiatica extract.

[0038] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing the shelled ripe horse chestnut seeds, passing through a 40-mesh sieve, taking the sieve bottom to obtain a powder; adding water 15 times the mass of the powder to the powder to obtain a base liquid; adding 3% of the mass of the base liquid glucose, 3% of the mass of the base liquid peptone, 1% of the mass of the powder Lactobacillus reuteri with a bacterial activity of 10 billion CFU / g, and 1% of the mass of the powder Lactobacillus bulgaricus with a bacterial activity of 10 billion CFU / g to the base liquid in sequence, anaerobically fermenting at 35°C for 4 days to obtain a fermentation liquid, filtering with a 200-mesh sieve, taking the filtrate, and freeze-drying to obtain a crude extract; dissolving the crude extract with n-hexane 6 times the mass of the crude extract, then adding an equal volume of n-hexane and a 4% mass concentration of sodium hydroxide aqueous solution to perform room temperature extraction for 30 minutes, taking the lower aqueous phase extract, concentrating under reduced pressure at 80°C, and drying at 70°C to obtain a horse chestnut extract.

[0039] The method for preparing the above-mentioned oxidoreductase composition comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to mass fraction, and precool them in a dry, sterile environment at a constant temperature of 4°C for 30 minutes to maintain the activity of the enzymes; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 4°C by circulating condensed water, and stirred and mixed to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, the mixture C is added to the mixed enzyme A, and the mixture is stirred at 4° C. to obtain a redox enzyme composition.

[0040] Application of the above-mentioned oxidoreductase composition in the preparation of cosmetics.

[0041] Example 4 A redox enzyme composition, comprising the following raw materials in parts by weight: 3 parts of lysozyme, 5 parts of superoxide dismutase, 2 parts of papain, 0.5 parts of trypsin, 1 part of lactoperoxidase, 3 parts of glucose oxidase, 3 parts of redox enzyme, 1 part of bromelain, 5 parts of acetyl hexapeptide-8, 6 parts of Centella asiatica extract, 6 parts of horse chestnut extract and ceramide NP 6 parts; lysozyme comes from Shandong Jishuo Biotechnology Co., Ltd., and the enzymatic activity of lysozyme is 20,000 U / mg; superoxide dismutase comes from Shanxi Shengdayue Biotechnology Co., Ltd., and the enzymatic activity of superoxide dismutase is 100,000 U / g; papain comes from Zhengzhou Maida Biotechnology Co., Ltd., and the enzymatic activity of papain is 100,000 U / g; trypsin comes from Xi'an Muguo Biotechnology Co., Ltd., and the enzymatic activity of trypsin is 4,000 U / g; lactoperoxidase comes from Shanghai Xinhu Biotechnology Co., Ltd., and the enzymatic activity of lactoperoxidase is 35 U / mg; glucose oxidase comes from Jiangsu Praite Bioengineering Co., Ltd., and the enzymatic activity of glucose oxidase is 100,000 U / g; the oxidoreductase is catalase, which comes from Zhengzhou Wanbo Chemical Products Co., Ltd., and the enzymatic activity of catalase is 200,000 U / g; bromelain comes from Lanli Biotechnology (Xi'an) Co., Ltd., and the enzymatic activity of bromelain is 300,000 U / g.

[0042] In the above composition, the preparation method of the Centella asiatica extract comprises the following steps: taking the whole plant of Centella asiatica, crushing it, passing it through a 60-mesh sieve, and taking the sieve to obtain a powder; adding a pH 6.0 phosphate buffer solution 15 times the weight of the powder to the powder, and then adding 1.0% of the weight of the powder with an enzyme activity of 100,000 U / g of cellulase, 0.8% of the weight of the powder with an enzyme activity of 30,000 U / g of pectinase, and 1.0% of the weight of the powder with an enzyme activity of 200,000 U / g of papain in sequence, enzymolyzing at 45°C and a speed of 150r / min for 3.0h, cooling to room temperature, inoculating Lactobacillus plantarum with a bacterial activity of 10 billion CFU / g at a mass inoculation of 5%, inoculating Bifidobacterium adolescentis with a bacterial activity of 10 billion CFU / g at a mass inoculation of 5%, and anaerobic fermentation at 37°C for 2 5h to obtain the fermentation broth, filter it with a 250-mesh sieve, take the filtrate, and concentrate it under reduced pressure at 60°C to 40% of the volume of the fermentation broth to obtain a concentrate; according to the volume ratio of concentrate: ethanol aqueous solution = 1:8, the concentrate was subjected to hot reflux extraction 3 times with 70% volume concentration ethanol aqueous solution, each time for 2.0h, the extracts were combined, the extracts were adsorbed by a D101 macroporous adsorption resin column, and then eluted with 3 column volumes of deionized water to remove impurities, and then eluted with 70% volume concentration ethanol aqueous solution at a flow rate of 2mL / min for 4 column volumes, the eluate of ethanol aqueous solution was collected, and the eluate was ultrafiltered with a 5kDa ultrafiltration membrane, the ultrafiltrate below 5kDa was taken, concentrated under reduced pressure at 80°C, and dried at 80°C to obtain Centella asiatica extract.

[0043] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing the shelled ripe horse chestnut seeds, passing through a 60-mesh sieve, taking the sieve bottom to obtain a powder; adding water 15 times the mass of the powder to the powder to obtain a base liquid; adding 5% of the mass of the base liquid glucose, 3% of the mass of the base liquid peptone, 3% of the mass of the powder Lactobacillus reuteri with a bacterial activity of 10 billion CFU / g, and 3% of the mass of the powder Lactobacillus bulgaricus with a bacterial activity of 10 billion CFU / g to the base liquid in sequence, anaerobically fermenting at 40°C for 4 days to obtain a fermentation liquid, filtering with a 250-mesh sieve, taking the filtrate, and freeze-drying to obtain a crude extract; dissolving the crude extract with 6 times the mass of the crude extract in n-hexane, then adding an equal volume of n-hexane and a 6% mass concentration of sodium hydroxide aqueous solution to perform extraction at room temperature for 25 minutes, taking the lower aqueous phase extract, concentrating under reduced pressure at 80°C, and drying at 80°C to obtain a horse chestnut extract.

[0044] The method for preparing the above-mentioned oxidoreductase composition comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to their mass fractions, and precool them in a dry, sterile environment at a constant temperature of 8°C for 60 minutes to maintain the activity of the enzymes; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 8° C. with circulating condensed water, and stirred to mix evenly to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, the mixture C is added to the mixed enzyme A, and the mixture is stirred at 8° C. to obtain a redox enzyme composition.

[0045] Application of the above-mentioned oxidoreductase composition in the preparation of cosmetics.

[0046] Example 5 A redox enzyme composition, comprising the following raw materials in parts by weight: 3 parts of lysozyme, 2 parts of superoxide dismutase, 2 parts of papain, 0.3 parts of trypsin, 1 part of lactoperoxidase, 1 part of glucose oxidase, 3 parts of redox enzyme, 0.5 parts of bromelain, 5 parts of acetyl hexapeptide-8, 3 parts of Centella asiatica extract, 6 parts of horse chestnut extract and ceramide NP 3 parts; lysozyme comes from Shandong Jishuo Biotechnology Co., Ltd., and the enzymatic activity of lysozyme is 20,000 U / mg; superoxide dismutase comes from Shanxi Shengdayue Biotechnology Co., Ltd., and the enzymatic activity of superoxide dismutase is 100,000 U / g; papain comes from Zhengzhou Maida Biotechnology Co., Ltd., and the enzymatic activity of papain is 100,000 U / g; trypsin comes from Xi'an Muguo Biotechnology Co., Ltd., and the enzymatic activity of trypsin is 4,000 U / g; lactoperoxidase comes from Shanghai Xinhu Biotechnology Co., Ltd., and the enzymatic activity of lactoperoxidase is 35 U / mg; glucose oxidase comes from Jiangsu Praite Bioengineering Co., Ltd., and the enzymatic activity of glucose oxidase is 100,000 U / g; the oxidoreductase is catalase, which comes from Zhengzhou Wanbo Chemical Products Co., Ltd., and the enzymatic activity of catalase is 200,000 U / g; bromelain comes from Lanli Biotechnology (Xi'an) Co., Ltd., and the enzymatic activity of bromelain is 300,000 U / g.

[0047] In the above composition, the preparation method of the Centella asiatica extract comprises the following steps: taking the whole plant of Centella asiatica, crushing it, passing it through a 60-mesh sieve, and taking the sieve to obtain a powder; adding a pH 6.0 phosphate buffer solution 10 times the weight of the powder to the powder, and then adding 0.5% of the weight of the powder with an enzyme activity of 100,000 U / g of cellulase, 0.3% of the weight of the powder with an enzyme activity of 30,000 U / g of pectinase, and 0.5% of the weight of the powder with an enzyme activity of 200,000 U / g of papain, enzymolysis at 40°C and a speed of 150r / min for 2.5h, cooling to room temperature, inoculating Lactobacillus plantarum with a bacterial activity of 5 billion CFU / g at a mass inoculation of 5%, inoculating Bifidobacterium adolescentis with a bacterial activity of 5 billion CFU / g at a mass inoculation of 5%, and anaerobic fermentation at 37°C for 20 h, obtain the fermentation broth, filter with a 250-mesh sieve, take the filtrate, and concentrate under reduced pressure at 80°C to 30% of the volume of the fermentation broth to obtain a concentrate; according to the volume ratio of concentrate: ethanol aqueous solution = 1:8, the concentrate is subjected to hot reflux extraction 3 times with 65% volume concentration ethanol aqueous solution, each time for 1.5 h, the extracts are combined, the extracts are adsorbed by a D101 macroporous adsorption resin column, and then eluted with 3 column volumes of deionized water to remove impurities, and then eluted with 50% volume concentration ethanol aqueous solution at a flow rate of 2 mL / min for 3 column volumes, the eluate of the ethanol aqueous solution is collected, and the eluate is ultrafiltered with a 5 kDa ultrafiltration membrane, the ultrafiltrate below 5 kDa is taken, concentrated under reduced pressure at 80°C, and dried at 70°C to obtain the Centella asiatica extract.

[0048] In the above composition, the preparation method of the horse chestnut extract comprises the following steps: crushing the shelled ripe horse chestnut seeds, passing through a 60-mesh sieve, taking the sieve bottom to obtain a powder; adding water 10 times the mass of the powder to the powder to obtain a base liquid; adding 5% of the mass of the base liquid glucose, 2% of the mass of the base liquid peptone, 5% of the mass of the powder Lactobacillus reuteri with a bacterial activity of 5 billion CFU / g, and 3% of the mass of the powder Lactobacillus bulgaricus with a bacterial activity of 5 billion CFU / g to the base liquid in sequence, anaerobically fermenting at 40°C for 3 days to obtain a fermentation liquid, filtering with a 250-mesh sieve, taking the filtrate, and freeze-drying to obtain a crude extract; dissolving the crude extract with 5 times the mass of the crude extract in n-hexane, then adding an equal volume of n-hexane and a 6% mass concentration of sodium hydroxide aqueous solution to perform room temperature extraction for 40 minutes, taking the lower aqueous phase extract, concentrating under reduced pressure at 60°C, and drying at 80°C to obtain the horse chestnut extract.

[0049] The method for preparing the above-mentioned oxidoreductase composition comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to their mass fractions, and precool them in a dry, sterile environment at a constant temperature of 3°C for 60 minutes to maintain the activity of the enzymes; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 3° C. with circulating condensed water, and stirred and mixed to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, the mixture C is added to the mixed enzyme A, and the mixture is stirred at 5° C. to obtain a redox enzyme composition.

[0050] Application of the above-mentioned oxidoreductase composition in the preparation of cosmetics.

[0051] Comparative Example 1 Lysozyme, superoxide dismutase, papain and trypsin were not added to the composition; other parameters and methods were the same as in Example 1.

[0052] Comparative Example 2 Lactoperoxidase, glucose oxidase, oxidoreductase and bromelain were not added to the composition; other parameters and methods were the same as in Example 1.

[0053] Comparative Example 3 No Centella asiatica extract was added to the composition; other parameters and methods were the same as in Example 1.

[0054] Comparative Example 4 In the preparation method of Centella asiatica extract, papain enzymolysis is not used, and Lactobacillus plantarum and Bifidobacterium adolescentis fermentation is not used; other parameters and methods are the same as those in Example 1.

[0055] Comparative Example 5 In the preparation method of the Centella asiatica extract, no fermentation with Lactobacillus plantarum and Bifidobacterium adolescentis is used, and no extraction is performed; the enzymatic hydrolyzate is directly filtered through a sieve and an ultrafiltration membrane; and other parameters and methods are the same as those in Example 1.

[0056] Comparative Example 6 No horse chestnut extract was added to the composition; other parameters and methods were the same as in Example 1.

[0057] Comparative Example 7 In the preparation method of the horse chestnut extract, no extraction is performed, and the crude extract is directly the horse chestnut extract; other parameters and methods are the same as in Example 1.

[0058] Comparative Example 8 The preparation method of the horse chestnut extract is as follows: crushing shelled horse chestnut ripe seeds, passing through a 40-mesh sieve, taking the sieve bottom to obtain a powder; using an ethanol aqueous solution with a volume concentration of 60% and a mass of 8 times the mass of the powder for percolation to obtain an ethanol-water extract, concentrating under reduced pressure at 70° C., and drying at 78° C. to obtain the horse chestnut extract. Other parameters and methods are the same as those in Example 1.

[0059] The compositions of the above-mentioned embodiments and comparative examples were tested for safety and efficacy.

[0060] 1. Safety Testing (1) Cytotoxicity test: A number of human skin fibroblasts (HSF) cultured in vitro were inoculated in a 96-well plate, with about 5,000 cells in each well. After culturing with normal cell culture medium for 24 hours, the culture medium containing the oxidoreductase composition was replaced with a cell culture medium containing a 0.1 mg / ml composition concentration. A blank control group (cultured with normal cell culture medium only) was set up at the same time. The culture was continued for 48 hours. The cell viability was detected by the MTT method, and the absorbance value at 570 nm was measured by an enzyme marker to calculate the relative cell survival rate, which was (absorbance value of the test group / absorbance value of the blank control group) × 100%. The results are shown in Table 1 below.

[0061] (2) Skin irritation test: 78 healthy New Zealand white rabbits without skin diseases were selected and randomly divided into 13 groups (6 rabbits in each group) for skin irritation test. The hair on both sides of the back spine was shaved off, with an area of ​​about 3 cm×3 cm. 0.5 mL of the composition sample of each embodiment and comparative example (the concentration of the composition was 0.1 mg / ml, and the solvent was water) was evenly applied on one side of the skin, and the other side was applied with an equal amount of physiological saline as a blank control, and then fixed with medical tape. The skin reaction was observed 24 hours, 48 ​​hours and 72 hours after application, and the symptoms such as erythema and edema were scored according to the Draize scoring standard (0 points: no reaction; 1 point: mild erythema or mild edema; 2 points: moderate erythema or moderate edema; 3 points: severe erythema or severe edema; 4 points: severe erythema, edema and skin necrosis). The results are shown in Table 1 below.

[0062] (3) Skin allergy test: 39 healthy guinea pigs were randomly divided into 13 groups (3 in each group) for guinea pig skin allergy test. The hair on both sides of the spine of the guinea pigs was shaved, with an area of ​​about 2 cm×2 cm. On the 0th day, the 7th day and the 14th day, 0.1 mL of the composition sample of each embodiment and comparative example (the concentration of the composition was 0.02 mg / ml, and the solvent was water) was applied to the shaved area on the left side for sensitization treatment, and the right side was applied with an equal amount of physiological saline as a control and gently massaged. On the 21st day, 0.1 mL of the sample (the concentration of the composition was 0.1 mg / ml) was applied to the shaved area on the right side of the guinea pig for provocation treatment, and the skin reaction was observed after 24 hours, 48 ​​hours and 72 hours, and scored according to the Magnusson-Kligman grading standard (0 points: no allergic reaction; 1 point: mild erythema; 2 points: moderate erythema; 3 points: severe erythema, edema). The results are shown in Table 1 below.

[0063] Table 1 Safety test results

[0064] 2. Beauty efficacy testing (1) Anti-sugar efficacy test: 140 female adult subjects (30-50 years old) with signs of skin glycation, puffy eye bags, and obvious skin aging characteristics (including dull skin, sagging, increased wrinkles, etc.) were selected and randomly divided into 14 groups (10 people in each group), of which one group was a blank control group, using a base without active ingredients (basic lotion, 2wt% Tween-80 emulsifier, 6wt% jojoba oil, 92wt% purified water) as a placebo, and the remaining 13 groups were experimental groups. Each group used a base lotion containing the composition once in the morning and evening. The emulsion (composition mass fraction: 3wt%, 2wt% emulsifier, 6wt% jojoba oil, 89wt% purified water) was applied on the face for 8 consecutive weeks. The changes in the content of advanced glycation end products (AGEs) in the skin were determined by fluorescence spectroscopy before and after use (the sample was the stratum corneum dandruff shed from the skin surface bonded with tape). The anti-sugar effect was expressed by the reduction rate of AGEs fluorescence intensity, AGEs fluorescence intensity reduction rate = (fluorescence intensity before use - fluorescence intensity after use) / fluorescence intensity before use × 100%. The results are shown in Table 2 below.

[0065] (2) Deswelling efficacy test: While conducting the anti-sugar efficacy test, the subjects were smeared with a basic lotion containing the composition around their eyes, and the blank control group was smeared with a basic lotion; the application was carried out once in the morning and evening every day for 2 consecutive weeks; the thickness of the skin at the eye bag site of the subjects was measured with an eye skin profile measuring instrument before and after use, and the deswelling rate = (skin thickness at the eye bag site before use - skin thickness at the eye bag site after use) / skin thickness at the eye bag site before use × 100%. The results are shown in Table 2 below.

[0066] (3) Anti-aging and elasticity efficacy test and brightening efficacy test: Anti-aging and elasticity efficacy test were conducted at the same time as the anti-sugar efficacy test. The product was applied on the face for 8 consecutive weeks. The elastic modulus of the skin on the face (cheeks, forehead, chin, etc.) of the subjects was measured using a skin elasticity tester before and after use. The firming effect was expressed as the increase rate of skin elastic modulus, which was (elastic modulus after use - elastic modulus before use) / elastic modulus before use × 100%. The chromaticity value (L value) of the skin on the face (cheek) of the subjects was measured using a skin colorimeter before and after use. The L value represents skin brightness. The brightening effect was expressed as the increase rate of skin brightness value L, which was (L value after use - L value before use) / L value before use × 100%. The results are shown in Table 2 below.

[0067] (4) Skin smoothing efficacy test: 84 female adult subjects (20-45 years old) with rough skin surface, slight acne pits or rough texture were selected and randomly divided into 14 groups (6 people in each group), of which 1 group was a blank control group, using a basic lotion (same as above) without active ingredients as a placebo, and the remaining 13 groups were experimental groups. Each group used a basic lotion (same as above) containing the composition once in the morning and evening, applied it on the face, and used it continuously for 8 weeks; before and after use, the facial skin of the subjects was imaged in three dimensions using a skin three-dimensional imaging system, and the skin surface roughness parameter was calculated. The smoothing effect was expressed as the skin surface roughness reduction rate, skin surface roughness reduction rate = (roughness parameter before use - roughness parameter after use) / roughness parameter before use × 100%. The results are shown in Table 2 below.

[0068] (5) Lipolysis efficacy test: 84 female adult subjects (20-45 years old) with certain fat accumulation on the face (including baby fat, double chin, etc.) were selected and randomly divided into 14 groups (6 people in each group), of which 1 group was a blank control group, using a basic lotion (same as above) without active ingredients as a placebo, and the remaining 13 groups were experimental groups. Each group used a basic lotion (same as above) containing the composition once in the morning and evening, applied it on the face and jaw, and used it continuously for 12 weeks; the thickness of subcutaneous fat at the mandibular angle of the subjects' faces was measured with an ultrasonic skin fat meter before and after use, and the fat dissolution rate = (subcutaneous fat thickness at the mandibular angle of the face before use / subcutaneous fat thickness at the mandibular angle of the face after use) / subcutaneous fat thickness at the mandibular angle of the face before use × 100%. The results are shown in Table 2 below.

[0069] Table 2 Beauty efficacy test results (average value)

[0070] It can be seen from the above experimental results that the oxidoreductase composition prepared in Examples 1 to 5 has safe ingredients, is non-irritating to the skin and does not cause allergic reactions, can significantly reduce facial advanced glycation end products (AGEs), eliminate eye skin swelling, increase collagen content and skin elasticity, reduce skin surface roughness, and can brighten skin tone and dissolve facial fat. It has comprehensive and efficient skin care advantages, delays skin aging and restores cell vitality. From the results of comparative example 1, it can be seen that lysozyme, superoxide dismutase, papain and trypsin as biologically active substances have the effects of anti-inflammatory, anti-oxidation, enhancing skin immunity, promoting skin metabolism and improving skin texture. Lysozyme, superoxide dismutase, papain and trypsin are not added to the composition, and the effects of repairing rough and aging skin and improving dull skin color are reduced; from the results of comparative example 2, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are biologically active substances, which have the effects of antibacterial, anti-oxidation, promoting skin metabolism and exfoliation. Lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are not added to the composition, and the effects of repairing rough and aging skin, improving dull skin color and anti-sugar are reduced; from the results of comparative example 3, it can be seen that Centella asiatica extract has the effects of anti-inflammatory, soothing, anti-aging and improving skin barrier. Centella asiatica extract is not added to the composition, and the effects of repairing rough and aging skin, improving The effects of dull skin tone, etc. are reduced; from the results of Comparative Examples 4 and 5, it can be seen that in the preparation method of Centella asiatica extract, papain enzymolysis is not used, and Lactobacillus plantarum and Bifidobacterium adolescentis fermentation are not used; or Lactobacillus plantarum and Bifidobacterium adolescentis fermentation are not used, and extraction is not performed, the enzymatic hydrolyzate is directly filtered through a sieve and an ultrafiltration membrane, the composition of the product obtained by extraction changes, and the effect is weakened; from the results of Comparative Example 6, it can be seen that the horse chestnut extract has the effects of improving skin microcirculation, anti-inflammatory and detumescent, and antioxidant. Without adding horse chestnut extract to the composition, the effects of repairing rough and aging skin and improving dull skin tone are reduced; from the results of Comparative Example 7, it can be seen that in the preparation method of horse chestnut extract, no extraction is performed, the purity of the effective product components obtained by extraction decreases, and the effect is weakened; from the results of Comparative Example 8, it can be seen that in the preparation method of horse chestnut extract, 60% ethanol water is used for percolation and then directly concentrated and dried, the composition of the product obtained by extraction changes, and the effect is weakened.

Claims

1. An oxidoreductase composition, characterized in that The invention comprises the following raw materials in parts by weight: 1 to 3 parts of lysozyme, 2 to 5 parts of superoxide dismutase, 0.5 to 2 parts of papain, 0.3 to 0.5 parts of trypsin, 0.5 to 1 part of lactoperoxidase, 1 to 3 parts of glucose oxidase, 1 to 3 parts of oxidoreductase, 0.5 to 1 part of bromelain, 2 to 5 parts of acetyl hexapeptide-8, 3 to 6 parts of Centella asiatica extract, 3 to 6 parts of horse chestnut extract and 3 to 6 parts of ceramide NP.

2. An oxidoreductase composition according to claim 1, characterized in that: The preparation method of Centella asiatica extract comprises: taking the whole plant of Centella asiatica, crushing, sieving, and taking the powder under the sieve; adding a buffer solution to the powder, adding 0.5% to 1.0% cellulase, 0.3% to 0.8% pectinase and 0.5% to 1.0% papain in accordance with the weight of the powder in sequence, performing enzymolysis, cooling, and obtaining a material, inoculating 3% to 5% of the weight of the material with Lactobacillus plantarum and 3% to 5% of the weight of Bifidobacterium adolescentis, performing anaerobic fermentation, obtaining a fermentation liquid, filtering, and concentrating the filtrate to obtain a concentrated liquid; extracting the concentrated liquid with an ethanol-water solution by hot reflux for multiple times, combining the extracts, adsorbing the extracts through a resin column, washing with water to remove impurities, eluting with an ethanol-water solution, collecting the eluate, ultrafiltration, and concentrating the ultrafiltrate under reduced pressure, and drying to obtain the Centella asiatica extract.

3. An oxidoreductase composition according to claim 2, characterized in that: The enzymatic activity of the cellulase is 20,000 U / g to 100,000 U / g, the enzymatic activity of the pectinase is 20,000 U / g to 30,000 U / g, and the enzymatic activity of the papain is 100,000 U / g to 200,000 U / g.

4. The oxidoreductase composition according to claim 2, characterized in that: The bacterial activity of the plant lactobacillus is 5 billion CFU / g to 10 billion CFU / g, and the bacterial activity of the adolescent bifidobacterium is 5 billion CFU / g to 10 billion CFU / g.

5. The oxidoreductase composition according to claim 2, characterized in that: In the preparation method of Centella asiatica extract, the temperature of reduced pressure concentration is 60°C to 80°C; and the temperature of drying is 70°C to 80°C.

6. The oxidoreductase composition according to claim 1, characterized in that: The preparation method of horse chestnut extract comprises the following steps: crushing shelled horse chestnut ripe seeds, sieving, taking the sieve bottom to obtain powder; adding water to the powder to obtain base liquid; sequentially adding glucose, peptone, 1% to 3% of Lactobacillus reuteri by weight of the powder, and 1% to 3% of Lactobacillus bulgaricus by weight of the powder to the base liquid, performing anaerobic fermentation to obtain fermentation liquid, filtering, taking the filtrate and freeze-drying to obtain a crude extract; dissolving the crude extract with n-hexane, adding sodium hydroxide aqueous solution for extraction, taking the lower aqueous phase, concentrating under reduced pressure, and drying to obtain the horse chestnut extract.

7. An oxidoreductase composition according to claim 6, characterized in that: The bacterial activity of the Lactobacillus reuteri is 5 billion CFU / g to 10 billion CFU / g, and the bacterial activity of the Lactobacillus bulgaricus is 5 billion CFU / g to 10 billion CFU / g.

8. The oxidoreductase composition according to claim 6, characterized in that: In the method for preparing the horse chestnut extract, the temperature for reduced pressure concentration is 60°C to 80°C, and the temperature for drying is 70°C to 80°C.

9. A method for preparing an oxidoreductase composition, for preparing an oxidoreductase composition according to claim 1, characterized in that: The preparation method comprises the following steps: S1: Under a sterile environment, take lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain according to their mass fractions, and precool them in a dry, sterile environment at a constant temperature of 2°C to 8°C for 30 min to 60 min to maintain the stability of enzyme activity; S2: Under a sterile environment, the pre-cooled lysozyme, superoxide dismutase, papain, trypsin, lactoperoxidase, glucose oxidase, oxidoreductase and bromelain are added to a sterile stirring container cooled to 2°C to 8°C by circulating condensed water, and stirred and mixed evenly to obtain a mixed enzyme A; S3: Under a sterile environment, acetyl hexapeptide-8, Centella asiatica extract and Aesculus hippocastanum extract are mixed uniformly according to weight proportions to obtain a mixture B; S4: Under a sterile environment, the mixture B and the ceramide NP are mixed evenly to obtain a mixture C; S5: Under a sterile environment, add the mixture C to the mixed enzyme A, and stir and mix them evenly at 2° C. to 8° C. to obtain a redox enzyme composition.

10. Use of the oxidoreductase composition according to any one of claims 1 to 8 in the preparation of cosmetics.

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