Cordyceps militaris fermentation product with moisturizing and anti-wrinkle effects

Abstract

The invention provides a cordyceps militaris fermentation product with moisturizing and anti-wrinkle effects, which is characterized in that cordyceps militaris is adopted to ferment wheat germ culture solution, the fermentation solution is subjected to filtration, concentration, ethanol precipitation, precipitate filtration, drying and crushing treatment to obtain the cordyceps militaris fermentation product, and the fermentation product is a soluble protein and polysaccharide compound. The soluble proteoglycan compound prepared by the invention is externally applied to skin, and has remarkable moisturizing, anti-wrinkle and tightening effects.

Description

Technical Field

[0001] The present invention relates to the technical fields of biological fermentation and skin care raw materials, and particularly relates to a preparation method of a Cordyceps militaris fermentation product with moisturizing and anti-wrinkle effects. Background Art

[0003] As a medicinal and edible fungus, Cordyceps militaris contains 18 kinds of amino acids, various vitamins, more than 30 trace elements, rich protein, polysaccharide and fat basic nutrients, and also contains various functional components. At present, it has been found that Cordyceps militaris polysaccharide and cordycepin have pharmacological effects such as antibacterial, anti-fatigue, anti-tumor, anti-infection, immune regulation, blood sugar lowering, and anti-aging. Compared with solid fermentation to obtain Cordyceps militaris fruiting bodies, the advantages of liquid fermentation of Cordyceps militaris mycelia in terms of fermentation scale, mycelial growth rate, growth density, and controllability enable the rapid and efficient acquisition of metabolically active substances of Cordyceps militaris mycelia by liquid deep culture. Existing patents such as CN118064507A, CN111172216A, CN104497160A, CN113462725B, etc. disclose the use of Cordyceps militaris liquid fermentation methods to obtain intracellular or extracellular products with different activities by using different fermentation raw materials and extraction processes. Among them, CN118064507A discloses adding licorice to the Cordyceps militaris fermentation medium, and the obtained Cordyceps militaris two-way fermentation broth has significantly improved moisturizing, anti-aging, and repair effects on the skin compared with the fermentation broth without adding licorice; CN111172216A discloses a liquid fermentation medium formula: 50 g of sucrose, 4 g of KNO3, 1 g of KH2PO4, 1 g of MgSO4·7H2O, 0.05 g of vitamin B1, 1000 mL of water, and a pH value of 6.5, and prepares Cordyceps militaris polysaccharide with the function of inhibiting macrophages from secreting NO as Cordyceps militaris intracellular polysaccharide and Cordyceps militaris extracellular polysaccharide; CN113462725B discloses inoculating Cordyceps militaris strains into Artemisia annua L. medium for cultivation, and extracting Cordyceps militaris secondary metabolites with skin acne-removing, repairing, and soothing effects from the mycelia; CN104497160A discloses a liquid fermentation medium formula: 50 g of sucrose, 4 g of KNO3, 1 g of KH2PO4, 1 g of MgSO4·7H2O, 0.05 g of vitamin B1, 1000 mL of water, and a pH value of 6.5, and prepares a Cordyceps militaris polysaccharide extract applied to lipid-lowering drugs or health foods.

[0004] Wheat germ is the life source of wheat grains and contains extremely rich and high-quality proteins, fats, various vitamins, and some trace physiological active ingredients. Currently, the functional components extracted from wheat germ are mostly used in various health foods. There are few reports on using fermentation technology to enhance the skin care efficacy of the active ingredients in wheat germ. CN118374366A discloses fermenting a wheat germ enzymolysis solution with Kluyveromyces marxianus at 42°C - 45°C, and the resulting fermentation broth has stronger skin moisturizing and skin elasticity enhancing abilities. Currently, no application research has been found on fermenting and transforming wheat germ with Cordyceps militaris to prepare fermentation products for the fields of moisturizing and anti-wrinkle skin care. Summary of the Invention

[0005] The object of the present invention is to provide a Cordyceps militaris fermentation product with moisturizing and anti-wrinkle effects in view of the deficiencies of the above-mentioned existing technologies. This fermentation product has significant moisturizing, anti-wrinkle, and skin tightening effects. The Cordyceps militaris fermentation product with moisturizing and anti-wrinkle effects of the present invention is prepared by fermenting wheat germ culture solution with Cordyceps militaris. The fermentation broth is filtered, concentrated, ethanol-precipitated, filtered after precipitation, and then dried and pulverized to obtain a soluble protein-polysaccharide complex fermentation product.

[0006] The technical solution of the present invention is as follows:

[0007] A Cordyceps militaris fermentation product with moisturizing and anti-wrinkle effects, characterized in that it is prepared by the following method:

[0008] (1) Prepare a Cordyceps militaris strain slant: Inoculate the Cordyceps militaris mother strain on a solid slant medium and perform activation culture at 22 - 32°C for 8 - 10 days to obtain a Cordyceps militaris strain slant;

[0009] (2) Prepare a wheat germ culture solution: By weight percentage: 2 - 5% glucose, 0.4 - 0.6% potassium dihydrogen phosphate, 0.2 - 0.6% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymolysis solution. After preparation, sterilize at 105°C - 121°C for 20 - 30 minutes and cool to 25 - 40°C;

[0010] (3) Fermentation and transformation: Pick a Cordyceps militaris solid slant strain with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 30 - 32°C, control the rotation speed at 150 - 220 rpm / min, and perform shaking culture for 5 - 8 days. Boil the fermentation broth to sterilize and cool it to room temperature, then centrifuge at 8000 - 10000 rpm / min for 20 minutes to remove hyphae and insoluble substances to obtain a clarified fermentation broth;

[0011] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 4 - 5 times its weight of anhydrous ethanol for ethanol precipitation; dry the precipitated protein-polysaccharide at 60 - 80°C for 4 - 6 hours, then pulverize it through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0012] Furthermore, the formula of the solid slant medium in step (1) is by weight ratio: 1-2% glucose, 0.4-0.6% potassium dihydrogen phosphate, 0.2-0.6% magnesium sulfate heptahydrate, 0.3-0.8% peptone, 2.5-3.0% agar, and the balance is potato decoction; the preparation method of the potato decoction is: put 100 g of potatoes into 900 mL of water, boil for 30-50 min, filter, and the filtrate is the potato decoction.

[0013] Furthermore, the preparation method of the wheat germ enzymolysis solution in step (2) is: ultramicro-pulverize defatted wheat germ to 800-1000 mesh, add 20 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin, the addition amount of cellulase is 0.2-0.6% of the weight of wheat germ, the addition amount of trypsin is 0.1-0.5% of the weight of wheat germ, adjust the pH to 6.8-7.0 with sodium hydroxide, and treat and enzymolyze at 35-50 °C for 3-5 hours to obtain wheat germ enzymolysis solution. Defatting wheat germ can prevent the presence of grease in the extract, which will hinder the contact and combination of hyphae with oxygen and reduce the fermentation efficiency. Defatted wheat germ can be obtained by low-temperature pressing method, solvent extraction method, or supercritical CO2 extraction method. Wheat germ is rich in protein, colloid, polysaccharide, cellulose, etc., which are distributed in the germ tissue. Hydrolysis with a composite enzyme of cellulase and trypsin can effectively release them for Cordyceps militaris fermentation and metabolism conversion. The advantage of ultramicro-pulverization is that after micronization, the enzymolysis efficiency of the composite enzyme is improved.

[0014] Furthermore, the prepared soluble protein and polysaccharide complex has significant moisturizing and anti-wrinkle effects, and acceptable excipients for cosmetics can be added to make cosmetics.

[0015] Furthermore, the cosmetics include any one of cream, essence, lotion, facial cleanser, and shampoo.

[0016] Furthermore, for the said cosmetics, calculated by mass percentage, the dosage of the soluble protein polysaccharide is 0.1%-5.0% of the cosmetics.

[0017] The beneficial effects of the present invention:

[0018] (1) The present invention compares the differences in the activity of fermentation products of different strains and different culture medium matrices through theoretical research and experimental testing, and finally finds that wheat germ enzymatic hydrolysate is used as the main culture medium component, and Cordyceps militaris is used to ferment the culture medium. On the one hand, the nutrients of wheat germ culture medium can provide rich nutrition for the accumulation of metabolites by liquid fermentation of Cordyceps militaris hyphae, and produce diversified active metabolites and enzymes. On the other hand, Cordyceps militaris produces a variety of special enzymes, which modify the structure of certain material components in wheat germ and improve its functionality. Finally, soluble proteins and polysaccharide complexes with moisturizing, anti-wrinkle and firming effects are formed in the fermentation liquid, and the activity is significantly better than the soluble proteins and polysaccharide complexes obtained by combining other strains and fermentation culture medium matrices. The present invention ferments germ culture liquids from different crop sources by Cordyceps militaris, including corn germ, wheat germ, and rice germ. It is found that Cordyceps militaris ferments wheat germ culture liquid, and the obtained soluble protein and polysaccharide complex is used for skin external use, and has significant moisturizing and anti-wrinkle effects.

[0019] (2) The soluble protein and polysaccharide complex prepared by the present invention has significant moisturizing and anti-wrinkle effects, is safe and non-irritating, and can be added with cosmetically acceptable excipients to make various cosmetics including facial creams, essences, lotions, toners, cleansers, and shampoos.

[0020] (3) The process of the present invention is simple, the process is green and environmentally friendly, and there is no pollution to the environment. In addition, the product extracted by this process has a high extraction rate, is easy to operate, and is easy to industrialize. DETAILED DESCRIPTION

[0021] The present invention is further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0022] Example 1.

[0023] (1) Preparation of Cordyceps militaris slant: Prepare a solid culture medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.35% peptone, 2.8% agar, and the remaining amount is potato boiling liquid. After preparation, sterilize at 105°C for 25 minutes and cool to 25°C; the preparation method of the potato boiling liquid is: place 100g of potatoes in 900mL of water and boil for 30 minutes, filter, and the filtrate is the potato boiling liquid. Take the Cordyceps militaris strain and inoculate it in the solid culture medium, activate and culture it at 28°C for 9 days to obtain the Cordyceps militaris strain slant;

[0024] (2) Preparation of wheat germ culture liquid: Ultrafinely pulverize defatted wheat germ to 800 mesh, add 20 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin, the addition amount of cellulase is 0.4% of the weight of wheat germ, the addition amount of trypsin is 0.5% of the weight of wheat germ, adjust the pH to 6.8 with sodium hydroxide, treat and enzymolyze at 45°C for 4 hours to obtain wheat germ enzymolyzate. By weight percentage: 2% glucose, 0.5% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymolyzate. After preparing the wheat germ culture liquid, sterilize it at 105°C for 30 min and cool it to 25°C;

[0025] (3) Fermentation and conversion: Pick a solid slant strain of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 30°C, control the rotation speed at 150 rpm / min, shake-flask culture for 6 d, boil and sterilize the fermentation broth and cool it to room temperature, centrifuge at 8000 rpm / min to remove mycelia and insoluble substances to obtain a clarified fermentation broth;

[0026] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times its weight of absolute ethanol for alcohol precipitation; after drying the alcohol-precipitated protein polysaccharide at 75°C for 6 hours, pulverize it and pass it through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0027] Example 2.

[0028] (1) Preparation of Cordyceps militaris strain slant: Prepare solid medium: 2% glucose, 0.6% potassium dihydrogen phosphate, 0.6% magnesium sulfate heptahydrate, 0.3% peptone, 2.5% agar, and the remaining amount is potato boiling solution. After preparation, sterilize it at 121°C for 20 min and cool it to 25°C; The preparation method of the potato boiling solution is: Place 100 g of potatoes in 900 mL of water, boil for 30 min, filter, and the filtrate is the potato boiling solution. Inoculate Cordyceps militaris strain into the solid medium and perform activation culture at 25°C for 8 d to obtain Cordyceps militaris strain slant;

[0029] (2) Preparation of wheat germ culture liquid: Ultrafinely pulverize defatted wheat germ to 1000 mesh, add 15 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin, the addition amount of cellulase is 0.2% of the weight of wheat germ, the addition amount of trypsin is 0.1% of the weight of wheat germ, adjust the pH to 6.8 with sodium hydroxide, treat and enzymolyze at 35°C for 3 hours to obtain wheat germ enzymolyzate. By weight percentage: 4% glucose, 0.6% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymolyzate. After preparing the wheat germ culture liquid, sterilize it at 105°C for 30 min and cool it to 25°C;

[0030] (3) Fermentation and conversion: Pick a solid slant culture of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 32°C, the rotation speed is controlled at 220 rpm / min, and it is cultured on a shaker for 5 days. The fermentation broth is boiled for sterilization and cooled to room temperature, and then centrifuged at 8000 rpm / min for 20 min to remove mycelia and insoluble substances, obtaining a clarified fermentation broth;

[0031] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 4 times its weight of absolute ethanol for alcohol precipitation; after the alcohol-precipitated protein polysaccharide is dried at 80°C for 4 hours, it is pulverized and passed through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0032] Example 3.

[0033] (1) Preparation of Cordyceps militaris slant culture: Prepare a solid medium: 2% glucose, 0.4% potassium dihydrogen phosphate, 0.5% magnesium sulfate heptahydrate, 0.8% peptone, 3.0% agar, and the remaining amount is potato decoction. After preparation, it is sterilized at 121°C for 20 min and cooled to 30°C; the preparation method of the potato decoction is: Place 100 g of potatoes in 900 mL of water, boil for 50 min, and filter. The filtrate is the potato decoction. Pick Cordyceps militaris culture and inoculate it into the solid medium, and carry out activation culture at 30°C for 10 days to obtain a Cordyceps militaris slant culture;

[0034] (2) Preparation of wheat germ culture liquid: Ultra-finely pulverize defatted wheat germ to 800 meshes, add 30 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.6% of the weight of wheat germ, and the addition amount of trypsin is 0.2% of the weight of wheat germ. Adjust the pH to 7.0 with sodium hydroxide and treat it with enzymatic hydrolysis at 50°C for 5 hours to obtain a wheat germ enzymatic hydrolysate. By weight percentage: 5% glucose, 0.4% potassium dihydrogen phosphate, 0.6% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymatic hydrolysate. After preparing the wheat germ culture liquid, it is sterilized at 121°C for 25 min and cooled to 40°C;

[0035] (3) Fermentation and conversion: Pick a solid slant culture of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 30°C, the rotation speed is controlled at 200 rpm / min, and it is cultured on a shaker for 7 days. The fermentation broth is boiled for sterilization and cooled to room temperature, and then centrifuged at 10000 rpm / min to remove mycelia and insoluble substances, obtaining a clarified fermentation broth;

[0036] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times its weight of absolute ethanol for alcohol precipitation; after the alcohol-precipitated protein polysaccharide is dried at 80°C for 6 hours, it is pulverized and passed through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0037] Example 4

[0038] (1) Preparation of Cordyceps militaris slant culture: Preparation of solid medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.5% magnesium sulfate heptahydrate, 0.5% peptone, 2.5% agar, and the remaining amount is potato decoction. After preparation, sterilize at 115 °C for 25 min and cool to 30 °C. The preparation method of the potato decoction is as follows: Place 100 g of potatoes in 900 mL of water, boil for 30 min, filter, and the filtrate is the potato decoction. Inoculate Cordyceps militaris strain into the solid medium and perform activation culture at 28 °C for 8 d to obtain Cordyceps militaris slant culture;

[0039] (2) Preparation of wheat germ culture liquid: Ultra - micro - pulverize defatted wheat germ to 800 - 1000 mesh, add 18 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.2% of the weight of wheat germ, and the addition amount of trypsin is 0.5% of the weight of wheat germ. Adjust the pH to 6.8 with sodium hydroxide and treat at 40 °C for enzymatic hydrolysis for 4 h to obtain wheat germ enzymatic hydrolysate. By weight percentage: 5% glucose, 0.6% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymatic hydrolysate. After preparing the wheat germ culture liquid, sterilize at 105 °C for 20 min and cool to 40 °C;

[0040] (3) Fermentation and transformation: Pick a Cordyceps militaris solid slant culture with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 32 °C, control the rotation speed at 220 rpm / min, shake - flask culture for 8 d. Boil the fermentation broth for sterilization and cool to room temperature, centrifuge at 8000 rpm / min to remove mycelium and insoluble substances to obtain a clarified fermentation broth;

[0041] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 4 - 5 times the weight of absolute ethanol for alcohol precipitation. After drying the alcohol - precipitated protein polysaccharide at 60 °C for 6 h and pulverizing it through a 100 - mesh sieve, obtain a soluble protein and polysaccharide complex;

[0042] Example 5.

[0043] (1) Preparation of Cordyceps militaris slant culture: Preparation of solid medium: 1.5% glucose, 0.4% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, 0.3% peptone, 2.5% agar, and the remaining amount is potato decoction. After preparation, sterilize at 121 °C for 30 min and cool to 25 °C. The preparation method of the potato decoction is as follows: Place 100 g of potatoes in 900 mL of water, boil for 30 min, filter, and the filtrate is the potato decoction. Inoculate Cordyceps militaris strain into the solid medium and perform activation culture at 22 °C for 10 d to obtain Cordyceps militaris slant culture;

[0044] (2) Preparation of wheat germ culture liquid: Ultrafinely pulverize defatted wheat germ to 800 mesh, add water at 30 times the weight of wheat germ, add a composite enzyme of cellulase and trypsin, with the addition amount of cellulase being 0.2% of the weight of wheat germ and the addition amount of trypsin being 0.25% of the weight of wheat germ. Adjust the pH to 6.8 with sodium hydroxide, and treat it enzymatically at 35°C for 3 hours to obtain wheat germ enzymatic hydrolysate. By weight percentage: 3% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymatic hydrolysate. After preparing the wheat germ culture liquid, sterilize it at 105°C for 30 min and cool it to 23°C;

[0045] (3) Fermentation and conversion: Pick a solid slant strain of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 30°C, control the rotation speed at 180 rpm / min, and culture it on a shaker for 8 d. Boil and sterilize the fermentation broth and cool it to room temperature, then centrifuge at 8000 rpm / min or above to remove hyphae and insoluble substances to obtain a clarified fermentation broth;

[0046] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 4 - 5 times its weight of absolute ethanol for alcohol precipitation; after drying the alcohol-precipitated protein polysaccharide at 60°C for 6 hours, pulverize it and pass it through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex.

[0047] During specific implementation, the endpoint parameters of Examples 1 - 5 can be adjusted by oneself according to the settings of the invention content, and the obtained products can all meet the set requirements and achieve the corresponding technical effects.

[0048] Comparative Example 1.

[0049] Use 2% glucose, 0.6% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and 3% peptone to replace the wheat germ culture liquid as the fermentation culture medium, and the other steps are the same as in Example 1.

[0050] (1) Preparation of Cordyceps militaris strain slant: Prepare a solid medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.35% peptone, 2.8% agar, and the remaining amount is potato boiling solution. After preparation, sterilize it at 105°C for 25 min and cool it to 25°C; The preparation method of the potato boiling solution is: Place 100 g of potatoes in 900 mL of water, boil for 30 min, and filter. The filtrate is the potato boiling solution. Inoculate Cordyceps militaris strain into the solid medium and activate it at 28°C for 9 d to obtain Cordyceps militaris strain slant;

[0051] (2) Preparation of fermentation culture medium: By weight percentage: Use 2% glucose, 0.6% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and 3% peptone to prepare the fermentation culture medium, sterilize it at 105°C for 30 min, and cool it to 25°C;

[0052] (3) Fermentation and conversion: Pick a solid slant culture of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of fermentation culture medium. The fermentation temperature is 30 °C, the rotation speed is controlled at 150 rpm / min, and it is cultured on a shaker for 6 days. The fermentation broth is boiled for sterilization and cooled to room temperature, and then centrifuged at 8000 rpm / min to remove mycelia and insoluble substances, obtaining a clarified fermentation broth;

[0053] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its original weight, and add 5 times its weight of absolute ethanol for ethanol precipitation; After the ethanol-precipitated protein polysaccharide is dried at 75 °C for 6 hours, it is pulverized and passed through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex.

[0054] Comparative Example 2.

[0055] Use 2% glucose, 0.6% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and 3% yeast extract to replace the wheat germ culture liquid as the fermentation culture medium, and the other steps are the same as in Example 1

[0056] (1) Preparation of Cordyceps militaris slant culture: Prepare a solid medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.35% peptone, 2.8% agar, and the remaining amount is potato cooking liquid. After preparation, it is sterilized at 105 °C for 25 min and cooled to 25 °C; The preparation method of the potato cooking liquid is: Place 100 g of potatoes in 900 mL of water, boil for 30 min, and filter. The filtrate is the potato cooking liquid. Inoculate Cordyceps militaris strain into the solid medium and activate it at 28 °C for 9 days to obtain Cordyceps militaris slant culture;

[0057] (2) Preparation of fermentation culture medium: By weight percentage: Use 2% glucose, 0.6% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and 3% yeast extract to prepare the fermentation culture medium, sterilize it at 105 °C for 30 min, and cool to 25 °C;

[0058] (3) Fermentation and conversion: Pick a solid slant culture of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of fermentation culture medium. The fermentation temperature is 30 °C, the rotation speed is controlled at 150 rpm / min, and it is cultured on a shaker for 6 days. The fermentation broth is boiled for sterilization and cooled to room temperature, and then centrifuged at 8000 rpm / min to remove mycelia and insoluble substances, obtaining a clarified fermentation broth;

[0059] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its original weight, and add 5 times its weight of absolute ethanol for ethanol precipitation; After the ethanol-precipitated protein polysaccharide is dried at 75 °C for 6 hours, it is pulverized and passed through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex.

[0060] Comparative Example 3.

[0061] Use defatted corn germ to replace defatted wheat germ to prepare corn germ culture medium, and the other steps are the same as in Example 1

[0062] (1) Preparation of Cordyceps militaris strain slant: Prepare solid medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.35% peptone, 2.8% agar, and the remaining amount is potato cooking liquid. After preparation, sterilize at 105°C for 25 min and cool to 25°C. The preparation method of the potato cooking liquid is: Place 100 g of potatoes in 900 mL of water, boil for 30 min, filter, and the filtrate is the potato cooking liquid. Inoculate Cordyceps militaris strain into the solid medium and conduct activation culture at 28°C for 9 d to obtain Cordyceps militaris strain slant;

[0063] (2) Preparation of corn germ culture liquid: Ultra-finely pulverize defatted corn germ to 800 mesh, add 20 times the weight of water of the corn germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.4% of the weight of the corn germ, and the addition amount of trypsin is 0.5% of the weight of the corn germ. Adjust the pH to 6.8 with sodium hydroxide and treat with enzymatic hydrolysis at 45°C for 4 h to obtain corn germ enzymatic hydrolysate. By weight percentage: 2% glucose, 0.5% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, and the remaining amount is corn germ enzymatic hydrolysate. Prepare wheat germ culture liquid and sterilize at 105°C for 30 min and cool to 25°C;

[0064] (3) Fermentation and conversion: Pick a Cordyceps militaris solid slant strain with an area the size of a fingernail and inoculate it into 200 ml of fermentation culture medium. The fermentation temperature is 30°C, control the rotation speed at 150 rpm / min, and shake culture for 6 d. Boil and sterilize the fermentation broth and cool to room temperature, centrifuge at 8000 rpm / min to remove mycelia and insoluble substances to obtain a clarified fermentation broth;

[0065] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times the weight of absolute ethanol for alcohol precipitation; after drying the alcohol-precipitated protein polysaccharide at 75°C for 6 h, pulverize and pass through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex.

[0066] Control Example 4.

[0067] Replace defatted wheat germ with defatted rice germ to prepare rice germ culture solution, and the other steps are the same as in Example 1

[0068] (1) Preparation of Cordyceps militaris strain slant: Prepare solid medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.35% peptone, 2.8% agar, and the remaining amount is potato cooking liquid. After preparation, sterilize at 105°C for 25 min and cool to 25°C. The preparation method of the potato cooking liquid is: Place 100 g of potatoes in 900 mL of water, boil for 30 min, filter, and the filtrate is the potato cooking liquid. Inoculate Cordyceps militaris strain into the solid medium and conduct activation culture at 28°C for 9 d to obtain Cordyceps militaris strain slant;

[0069] (2) Prepare the rice germ culture liquid: Ultra - micro - pulverize defatted rice germ to 800 meshes, add 20 times the weight of water of the rice germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.4% of the weight of the rice germ, and the addition amount of trypsin is 0.5% of the weight of the rice germ. Adjust the pH to 6.8 with sodium hydroxide, and treat it enzymatically at 45°C for 4 hours to obtain the enzymolysis liquid of rice germ. By weight percentage: 2% glucose, 0.5% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, and the remaining amount is the enzymolysis liquid of rice germ. After preparing the rice germ culture liquid, sterilize it at 105°C for 30 min and cool it to 25°C;

[0070] (3) Fermentation and conversion: Pick a solid slant strain of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of fermentation culture medium. The fermentation temperature is 30°C, control the rotation speed at 150 rpm / min, and culture it on a shaker for 6 d. Boil and sterilize the fermentation broth and cool it to room temperature, then centrifuge it at 8000 rpm / min to remove hyphae and insoluble substances to obtain a clarified fermentation broth;

[0071] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times the weight of absolute ethanol for alcohol precipitation; After drying the alcohol - precipitated protein - polysaccharide at 75°C for 6 hours, pulverize it and pass it through a 100 - mesh sieve to obtain a soluble protein and polysaccharide complex.

[0072] Control example 5.

[0073] The product in step (4) of Example 1 is the supernatant extract after alcohol precipitation of the fermentation broth, and the other steps are the same as those in Example 1

[0074] (1) Prepare the Cordyceps militaris strain slant: Prepare a solid medium: 1% glucose, 0.4% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.35% peptone, 2.8% agar, and the remaining amount is the potato boiling solution. After preparation, sterilize it at 105°C for 25 min and cool it to 25°C; The preparation method of the potato boiling solution is: Place 100 g of potatoes in 900 mL of water, boil for 30 min, and filter. The filtrate is the potato boiling solution. Inoculate the Cordyceps militaris strain into the solid medium and activate it at 28°C for 9 d to obtain the Cordyceps militaris strain slant;

[0075] (2) Preparation of rice germ culture liquid: Ultra - finely pulverize defatted rice germ to 800 mesh, add 20 times the weight of water of rice germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.4% of the weight of rice germ, and the addition amount of trypsin is 0.5% of the weight of rice germ. Adjust the pH to 6.8 with sodium hydroxide, and treat it at 45°C for enzymatic hydrolysis for 4 hours to obtain rice germ enzymatic hydrolysate. By weight percentage: 2% glucose, 0.5% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate, and the remaining amount is rice germ enzymatic hydrolysate. After preparing the rice germ culture liquid, sterilize it at 105°C for 30 min and cool it to 25°C;

[0076] (3) Fermentation and conversion: Pick a solid slant strain of Cordyceps militaris with an area the size of a fingernail and inoculate it into 200 ml of fermentation culture medium. The fermentation temperature is 30°C, control the rotation speed at 150 rpm / min, and culture it on a shaker for 6 days. Boil and sterilize the fermentation broth and cool it to room temperature, then centrifuge it at 8000 rpm / min to remove mycelia and insoluble substances to obtain a clarified fermentation broth;

[0077] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times the weight of absolute ethanol for ethanol precipitation; after freeze - drying the supernatant of ethanol precipitation and pulverizing it through a 100 - mesh sieve, obtain a soluble powder.

[0078] Control example 6.

[0079] Directly extract wheat germ enzymatic hydrolysate, without Cordyceps militaris fermentation, and directly prepare a soluble protein and polysaccharide complex

[0080] (1) Preparation of wheat germ enzymatic hydrolysate: Ultra - finely pulverize defatted wheat germ to 800 mesh, add 20 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.4% of the weight of wheat germ, and the addition amount of trypsin is 0.5% of the weight of wheat germ. Adjust the pH to 6.8 with sodium hydroxide, and treat it at 45°C for enzymatic hydrolysis for 4 hours to obtain wheat germ enzymatic hydrolysate.

[0081] (2) Product separation: Centrifuge the wheat germ enzymatic hydrolysate at 8000 rpm / min to remove insoluble substances to obtain a clarified liquid; concentrate it to 1 / 20 of its weight, add 5 times the weight of absolute ethanol for ethanol precipitation; dry the ethanol - precipitated protein - polysaccharide at 75°C for 6 hours and then pulverize it through a 100 - mesh sieve to obtain a soluble protein and polysaccharide complex;

[0082] Control example 7.

[0083] Use Aspergillus oryzae to replace Cordyceps militaris, ferment wheat germ culture liquid, and prepare a soluble protein and polysaccharide complex. (The steps of preparing wheat germ enzymatic hydrolysate and product separation are the same as in Example 3)

[0084] (1) Preparation of Aspergillus oryzae strain slant: Prepare solid medium: 2% sucrose, 3.0% agar, and the remaining amount is potato boiling solution. After preparation, sterilize at 121 °C for 20 min and cool to 30 °C. The preparation method of the potato boiling solution is as follows: Place 100 g of potatoes in 900 mL of water, boil for 50 min, filter, and the filtrate is the potato boiling solution. Inoculate Cordyceps militaris strain into the solid medium and perform activation culture at 30 °C for 7 d to obtain Aspergillus oryzae strain slant;

[0085] (2) Preparation of wheat germ culture liquid: Ultra-finely pulverize defatted wheat germ to 800 mesh, add 30 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin. The addition amount of cellulase is 0.6% of the weight of wheat germ, and the addition amount of trypsin is 0.2% of the weight of wheat germ. Adjust the pH to 7.0 with sodium hydroxide and treat with enzymatic hydrolysis at 50 °C for 5 h to obtain wheat germ enzymatic hydrolysate. By weight percentage: 5% glucose, 0.4% potassium dihydrogen phosphate, 0.6% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymatic hydrolysate. After preparing the wheat germ culture liquid, sterilize at 121 °C for 25 min and cool to 40 °C;

[0086] (3) Fermentation and conversion: Pick an Aspergillus oryzae solid slant strain with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 30 °C, control the rotation speed at 200 rpm / min, perform shaking culture for 7 d, boil and sterilize the fermentation broth and cool it to room temperature, centrifuge at 10000 rpm / min to remove hyphae and insoluble substances to obtain a clarified fermentation broth;

[0087] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times the weight of absolute ethanol for alcohol precipitation; after drying the alcohol-precipitated protein polysaccharide at 80 °C for 6 h, pulverize and pass through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0088] Control example 8.

[0089] Use Lactobacillus plantarum to replace Cordyceps militaris, ferment wheat germ culture liquid, and prepare soluble protein and polysaccharide complex. (The steps of preparing wheat germ enzymatic hydrolysate and product separation are the same as in Example 3)

[0090] (2) Preparation of Lactobacillus plantarum strain slant: 1% peptone, 1% beef extract, 0.5% yeast powder, 2% glucose, 0.5% sodium acetate, 0.2% ammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.1% Tween 80, 0.05‰ manganese sulfate, 0.1‰ magnesium sulfate, 2.5% agar powder, make up to 1000 mL with distilled water, sterilize at 121 °C for 20 min, and cool the temperature to 30 °C. Inoculate Lactobacillus plantarum strain into the solid medium and perform activation culture at 30 °C for 2 d to obtain Lactobacillus plantarum strain slant;

[0091] (2) Preparation of wheat germ culture liquid: Ultrafinely pulverize defatted wheat germ to 800 mesh, add 30 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin, the addition amount of cellulase is 0.6% of the weight of wheat germ, and the addition amount of trypsin is 0.2% of the weight of wheat germ. Adjust the pH to 7.0 with sodium hydroxide, and treat it enzymatically at 50 °C for 5 hours to obtain wheat germ enzymatic hydrolysate. By weight percentage: 5% glucose, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymatic hydrolysate. Adjust the pH to 5.5 with dilute hydrochloric acid, and after preparing the wheat germ culture liquid, sterilize it at 121 °C for 25 min and cool it to 40 °C;

[0092] (3) Fermentation conversion: Pick a solid slant strain of Lactobacillus plantarum with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 34 °C, control the rotation speed at 200 rpm / min, and culture it on a shaker for 3 d. Boil and sterilize the fermentation broth and cool it to room temperature, and centrifuge it at 10000 rpm / min to remove bacteria and insoluble substances to obtain a clear fermentation broth;

[0093] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times the weight of absolute ethanol for ethanol precipitation; after drying the ethanol-precipitated protein polysaccharide at 80 °C for 6 hours, pulverize it and pass it through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0094] Control example 9.

[0095] Use Saccharomyces cerevisiae to replace Cordyceps militaris, ferment the wheat germ extract, and prepare a soluble protein and polysaccharide complex. (The steps of preparing wheat germ enzymatic hydrolysate and product separation are the same as in Example 3)

[0096] (3) Preparation of yeast strain slant: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 1 L distilled water, 2.5% agar powder, sterilize at 115 °C for 25 min, and cool to 30 °C;. Inoculate the yeast strain into the solid medium and activate it at 30 °C for 2 d to obtain a yeast strain slant;

[0097] (2) Preparation of wheat germ culture liquid: Ultrafinely pulverize defatted wheat germ to 800 mesh, add 30 times the weight of water of wheat germ, add a composite enzyme of cellulase and trypsin, the addition amount of cellulase is 0.6% of the weight of wheat germ, and the addition amount of trypsin is 0.2% of the weight of wheat germ. Adjust the pH to 7.0 with sodium hydroxide, and treat it enzymatically at 50 °C for 5 hours to obtain wheat germ enzymatic hydrolysate. By weight percentage: 5% glucose, 0.4% potassium dihydrogen phosphate, 0.6% magnesium sulfate heptahydrate, and the remaining amount is wheat germ enzymatic hydrolysate. Prepare the wheat germ culture liquid and sterilize it at 121 °C for 25 min and cool it to 40 °C;

[0098] (3) Fermentation conversion: Pick a solid slant culture of Saccharomyces cerevisiae with an area the size of a fingernail and inoculate it into 200 ml of wheat germ liquid medium. The fermentation temperature is 30 °C, the rotation speed is controlled at 200 rpm / min, and it is cultured on a shaker for 7 days. The fermentation broth is boiled and sterilized, cooled to room temperature, and centrifuged at 10,000 rpm / min to remove the bacteria and insoluble substances, obtaining a clarified fermentation broth;

[0099] (4) Product separation: Concentrate the fermentation filtrate to 1 / 20 of its weight, add 5 times its weight of absolute ethanol for alcohol precipitation; after the alcohol-precipitated protein polysaccharide is dried at 80 °C for 6 hours, it is pulverized and passed through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex;

[0100] Test Example 1: Determination of the polysaccharide and protein contents of the soluble protein and polysaccharide complex

[0101] The traditional Coomassie brilliant blue method is used, with glucose as the standard, for the determination of protein content; the phenol-sulfuric acid colorimetric method is used, with bovine serum albumin as the standard, for the determination of polysaccharide content.

[0102] The polysaccharide and protein contents of the fermentation products prepared in Examples 1-5 and Comparative Examples 1-9 were determined, and the results are shown in Table 1.

[0103] Table 1 Polysaccharide, protein contents and product yields of fermentation products

[0104] Group Polysaccharide content Protein content Yield Example 1 52.21％ 10.24％ 8.21％ Example 2 51.47％ 9.25％ 9.65％ Example 3 53.91％ 11.63％ 9.02％ Example 4 50.23％ 8.92％ 10.21％ Example 5 52.86％ 9.63％ 9.21％ Comparative Example 1 45.32％ 2.45％ 5.12％ Comparative Example 2 43.61％ 2.96％ 3.65％ Comparative Example 3 46.65％ 10.85％ 10.65％ Comparative Example 4 50.28％ 8.04％ 8.02％ Comparative Example 5 5.45％ 6.35％ 6.23％ Comparative Example 6 45.45％ 16.83％ 17.65％ Comparative Example 7 44.27％ 12.35％ 6.12％ Comparative Example 8 46.90％ 10.64％ 7.08％ Comparative Example 9 51.86％ 11.83％ 6.54％

[0105] It can be seen from the data in Table 1 that the main components of the products precipitated by ethanol after fermentation in the examples of the present invention are polysaccharides and proteins.

[0106] Test Example 2: Safety test of the fermentation product.

[0107] Chorioallantoic membrane test of chicken embryos: The sample is a 5% aqueous solution of the soluble protein and polysaccharide complex prepared in Examples 1-5; the negative control is physiological saline; the positive control is 0.1 mol / L NaOH solution.

[0108] Test operation steps: In this test, 6 chicken embryos are selected for each group, and the situation of the chorioallantoic membrane is recorded with a photographing device. Place it in a polytetrafluoroethylene resin ring, record the time of adding the sample and cover the air chamber with a moistened plastic wrap, move the chicken embryos to a constant temperature and humidity incubator for culture, and observe the degree of change in each toxic effect.

[0109] Calculation results: The endpoint evaluation method is used for the test, and the endpoint evaluation (ES) is calculated, with the result reserved to two decimal places; the score of each chicken embryo = the sum of the bleeding, blood coagulation and vascular lysis degrees observed in each chicken embryo; calculate the average ES score according to the following formula:

[0110]

[0111] Result judgment criteria:

[0112] ES ≤ 4, non-irritating; 4 < ES ≤ 12, slightly irritating; 12 < ES < 16, moderately irritating; ES > 16, strongly irritating / corrosive.

[0113] The test results of the chicken embryo chorioallantoic membrane test are shown in Table 2:

[0114] Table 2 Test results of the chicken embryo chorioallantoic membrane test

[0115] ES value Result Example 1 4.0 Non-irritating Example 2 3.81 Non-irritating Example 3 3.66 Non-irritating Example 4 3.62 Non-irritating Example 5 3.86 Non-irritating

[0116] The results show that the soluble protein and polysaccharide complex prepared by the present invention has good safety and no irritation.

[0117] Test Example 3: Stability test of the soluble protein and polysaccharide complex.

[0118] Take 2.5 g each of the fermented product polysaccharide and protein product prepared in Examples 1 - 5, add 100 ml of water, stir and dissolve at room temperature to prepare a 2.5% solution, encapsulate it in a test sample bottle, and place it under the experimental conditions of -18°C, 25°C, and 54°C respectively. Continuously test the appearance, pH value, viscosity and other items for 30 days. After returning to room temperature, observe whether there are any adverse phenomena such as clarification, precipitation, discoloration, etc. The specific test results are shown in Table 3.

[0119] Table 3 Stability test of the soluble protein and polysaccharide complex

[0120]

[0121]

[0122] From the data in the above table, it can be seen that the samples can remain stable at -18°C, 25°C, and 54°C. The solution is clear, without precipitation and discoloration.

[0123] Performance test.

[0124] Take the soluble protein and polysaccharide complexes obtained in Examples 1 - 5 and Comparative Examples 1 - 9 as test objects for testing:

[0125] 1) Inhibition rate of elastase.

[0126] Skin relaxation and wrinkling are mainly related to elastic fibers, and elastic fibers will be decomposed by elastase, resulting in the loss of collagen and thus causing skin relaxation and wrinkling. Therefore, by measuring the inhibition rate of the firming and anti-wrinkle plant composition on elastase, the purpose of detecting the firming and anti-wrinkle effect can be achieved.

[0127] The specific operation is to mix 100 μL of 0.2 mol / L tris-HCl buffer solution, 25 μL of 10 mmol / L N-methoxysuccinyl-alanine-proline-valine-4-nitroaniline, and 30 mg of soluble protein and polysaccharide complex, let it stand for 10 min at 25 °C, add 25 μL of 0.3 U / mL elastase and continue to let it stand for 10 min. Finally, measure the OD value under the condition of 410 nm, and use 0.3 mL of deionized water to replace the soluble protein and polysaccharide complex as a blank control.

[0128] The inhibition rate of soluble protein and polysaccharide complex on elastase (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group × 100%.

[0129] 2) Antioxidant performance test.

[0130] The specific operation is to take 30 mg of soluble protein and polysaccharide complex and place it in a test tube, add 5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol, shake for 10 s, then place it at room temperature for reaction for 20 min. After the reaction is completed, measure the OD value of the reaction mixture under the condition of 517 nm, and use 0.3 mL of deionized water to replace the soluble protein and polysaccharide complex as a blank control.

[0131] The scavenging rate of soluble protein and polysaccharide complex on DPPH free radicals (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group × 100%.

[0132] The test results of the inhibition rate of elastase and antioxidant performance are recorded in Table 4 below.

[0133] Table 4 Test results of the inhibition rate of elastase and antioxidant performance.

[0134]

[0135]

[0136] It can be concluded from the data in Table 4 that the soluble protein and polysaccharide complex prepared in Examples 1-5 has significantly higher inhibition rate on elastase and scavenging rate on DPPH free radicals compared with the fermentation products prepared in Comparative Examples 1-9.

[0137] Application examples.

[0138] Use the fermentation products prepared in Examples 1-5 and Comparative Examples 1-9 to prepare skin care essence and blank control skin care essence respectively. The blank group is the skin care essence base formula without fermentation products. The basic formula of the skin care essence is shown in Table 5.

[0139] Table 5 Skin care essence formula

[0140]

[0141] According to the above formula, add water, glycerol, 1,3-butanediol, acryloyldimethyltauramide / VP copolymer into the emulsifying pot, heat to 85°C, stir evenly, then cool down to 45°C, and successively add ethylhexylglycerin, hydroxyacetophenone, fermentation product - soluble protein and polysaccharide complex, stir at 30 rpm / min for 10 min to obtain the skin care essence.

[0142] Recruit 90 subjects, and finally 90 valid data are obtained. The subjects are healthy Chinese men and women with sensitive skin, dry or combination dry skin, and wrinkle grade above level 2. In the examples and comparative examples, each group has 9 people. The volunteers use the skin care essence and blank essence twice a day. The usage amount is to take a soybean-sized amount of essence after each facial cleansing and evenly apply it on the face. After 4 hours and 4 weeks, measure the water content in the epidermal stratum corneum (the water content in the stratum corneum is measured 5 times and the average value is taken). The VISIA-CR facial skin image analyzer is used to test the improvement of wrinkles. Each volunteer has the initial wrinkle area measured before using the essence. After 4 weeks of use, it is compared with the initial value before use to analyze the wrinkle reduction rate and measure the change rate after 28 days. The test results are shown in Table 6 below.

[0143] Table 6 Changes in water content of the stratum corneum and wrinkles of the skin using the essence formula

[0144]

[0145] It can be seen from the test results that the essence milks prepared in Examples 1-5 all have good moisturizing and anti-wrinkle effects; while the moisturizing and anti-wrinkle effects of Comparative Examples 1-9 and the blank example are significantly weaker.

[0146] In summary, through the test examples and application examples, the fermentation products of Examples 1-5 and Comparative Examples 1-9 are tested and compared, indicating that using the cordyceps militaris strain and wheat germ of the present invention for fermentation has significant advantages, and the obtained fermentation products have significant moisturizing, anti-wrinkle and firming effects.

[0147] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

[0148] The parts not involved in the present invention are the same as or can be implemented by the prior art.

Claims

1. A Cordyceps militaris fermentation product having moisturizing and anti-wrinkle effects, characterized in that: Prepared by the following method: The method comprises the following steps: (1) Preparing a Cordyceps militaris slant: taking a Cordyceps militaris mother strain and inoculating it into a solid slant culture medium, and performing activation culture at 22 to 32° C. for 8 to 10 days to obtain a Cordyceps militaris slant; (2) preparing wheat germ culture liquid: by weight percentage: 2-5% glucose, 0.4-0.6% potassium dihydrogen phosphate, 0.2-0.6% magnesium sulfate heptahydrate, and the remainder is wheat germ enzymatic hydrolysate, sterilizing at 105° C. to 121° C. for 20-30 min, and cooling to 25-40° C.; (3) Fermentation transformation: Pick a solid slant of Cordyceps militaris about the size of a fingernail and inoculate it into 200 ml of wheat germ liquid culture medium. The fermentation temperature is 30-32°C, the rotation speed is controlled at 150-220 rpm / min, and the culture is carried out on a shaker for 5-8 days. The fermentation liquid is boiled and sterilized, cooled to room temperature, and centrifuged at 8000-10000 rpm / min for 20 minutes to remove mycelium and insoluble matter to obtain a clarified fermentation liquid. (4) Product separation: The fermentation filtrate is concentrated to 1 / 20 of its weight, and 4 to 5 times the weight of anhydrous ethanol is added for alcohol precipitation; the alcohol-precipitated proteoglycan is dried at 60 to 80° C. for 4 to 6 hours, and then crushed through a 100-mesh sieve to obtain a soluble protein and polysaccharide complex.

2. The Cordyceps militaris fermentation product according to claim 1, characterized in that: The solid slant culture medium of step (1) comprises, by weight, 1-2% glucose, 0.4-0.6% potassium dihydrogen phosphate, 0.2-0.6% magnesium sulfate heptahydrate, 0.3-0.8% peptone, 2.5-3.0% agar, and the remainder is potato cooking liquid; the preparation method of the potato cooking liquid is as follows: 100 g of potatoes are placed in 900 mL of water and boiled for 30-50 min, filtered, and the filtrate is the potato cooking liquid.

3. The Cordyceps militaris fermentation product according to claim 1, characterized in that: The preparation method of the wheat germ enzymatic hydrolysate described in step (2) is as follows: ultrafinely pulverizing defatted wheat germ to 800-1000 meshes, adding 15-30 times of water by weight of the wheat germ, adding cellulase and trypsin complex enzyme, wherein the addition amount of cellulase is 0.2-0.6% by weight of the wheat germ, and the addition amount of trypsin complex enzyme is 0.1-0.5% by weight of the wheat germ, adjusting the pH to 6.8-7.0 with sodium hydroxide, and treating the enzymatic hydrolysate at 35-50° C. for 3-5 hours to obtain the wheat germ enzymatic hydrolysate.

4. The Cordyceps militaris fermentation product according to claim 1, characterized in that The prepared soluble protein and polysaccharide complex has significant moisturizing and anti-wrinkle effects and can be added as an acceptable auxiliary material for cosmetics to prepare cosmetics.

5. The Cordyceps militaris fermentation product according to claim 4, characterized in that: The cosmetics include any one of facial cream, essence, lotion, skin softener, facial cleanser and shampoo.

6. The Cordyceps militaris fermentation product according to claim 5, characterized in that: Calculated by mass percentage, the amount of the soluble protein and polysaccharide complex is 0.1% to 5.0% of the cosmetics.

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