A Cordyceps militaris mycelium fermentation product and its preparation method and application

Abstract

The invention discloses a Cordyceps militaris mycelium fermentation product and a preparation method and application thereof, and belongs to the technical field of daily cosmetics. A preparation method of a Cordyceps militaris mycelium fermentation product comprises the following steps: using Cordyceps militaris mycelium as a strain, accessing the strain into a liquid culture medium for expansion culture, and obtaining a seed solution; transferring the seed solution to a fermentation tank containing a liquid culture medium, passing oxygen and stirring, and fermenting for a certain period of time to obtain a fermentation liquid; adding an industrial complex enzyme to the fermentation liquid, heating and stirring to obtain an enzymolysis treatment liquid; adding a certain amount of alcohol solution to the enzymolysis treatment liquid, fully mixing, and obtaining an alcohol precipitation liquid; separating the alcohol precipitation liquid, filtering the supernatant, collecting the clarified filtrate, and obtaining a Cordyceps militaris mycelium fermentation product. The present invention adopts a specific complex enzyme that is inexpensive and suitable for industrialization, fully releases the active substances of Cordyceps militaris, and improves the efficacy of the product in cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of daily cosmetics, and particularly relates to a Cordyceps militaris mycelium fermentation product and a preparation method and application thereof. Background Art

[0002] Cordyceps militaris, also known as northern cordyceps, golden grass, and cordyceps flower, belongs to the Cordyceps family with Cordyceps sinensis. In winter, the larvae live in the soil, where fungi parasitize and absorb nutrients. The larvae are filled with mycelium and die. In summer, seedlings grow from the larvae's corpses, which look like grass and are collected around the summer solstice. Cordyceps militaris has high nutritional value and is a precious Chinese herbal medicine in my country. It is very beneficial to human health. Natural wild northern cordyceps was first recorded in the "Xinhua Materia Medica Outline". It is "sweet in taste and neutral in nature" and has the effects of "benefiting the lungs and kidneys, replenishing the essence, stopping bleeding and reducing phlegm". The modern Chinese medicine masterpiece "Chinese Herbal Medicine Sea" records: "Cordyceps militaris, also known as northern cordyceps (Jilin), is sweet and neutral in nature and enters the lung and kidney meridians. In 2009, the Ministry of Health officially approved Cordyceps militaris as a new resource food. Therefore, the research on the use of Cordyceps militaris in health foods and functional beverages has gradually increased, and people's demand for Cordyceps militaris has also increased.

[0003] Cordyceps militaris is rich in a variety of active ingredients such as adenosine, cordycepin, cordycepic acid, cordyceps polysaccharides, etc. Adenosine is an endogenous nucleoside in the human body, which has skin conditioning, anti-aging, anti-wrinkle and other effects; Cordyceps can inhibit the response of NO enzyme and COX-2 enzyme, play an anti-inflammatory role, reduce the production of IL-13 and the expression of genes such as MDM2, play an anti-inflammatory role, block the expression of MMP-1 and MMP-3 produced by ultraviolet rays, and prevent and treat skin photoaging; Cordycepic acid can eliminate peroxynitrite anions, which is beneficial to skin conditioning and anti-aging; Cordyceps polysaccharides have the ability to increase the activity of SOD enzyme, promote the activity of GSH-Px (glutathione peroxidase), and reduce the content of lipid peroxides.

[0004] In the cosmetics industry, the extraction and application of active ingredients are the key to product efficacy. The existing Cordyceps militaris fruiting bodies or mycelium, the commonly used technologies in the cell wall breaking process are dry grinding, heating autolysis, freezing and thawing, etc., and the fruiting bodies also have enzyme hydrolysis methods. These methods can only lyse some cells and release a small amount of active substances. Although there are reports on the use of lytic enzymes, snail enzymes, cellulases, pectinases, various proteases, etc. in the existing Cordyceps militaris fruiting body enzymatic wall breaking methods, the main components of the Cordyceps militaris mycelium cell wall are chitin and glucan, and the lytic enzymes and snail enzymes commonly used in scientific research are the most effective wall breaking enzymes, but the prices of these two enzymes are extremely high, and they are still in the scientific research stage, and there is still a long way to go in their application in industrial production; the use of other cellulases, pectinases, various proteases, etc. is not ideal, and the active ingredients of Cordyceps militaris are not fully released, so that it can be better used in cosmetics.

[0005] Patent document CN107951011A discloses a method for producing Cordyceps militaris enzyme based on enzyme-enzyme coupling technology, which is characterized in that the cell wall of Cordyceps militaris mycelium or powder is first subjected to biological-grade degradation by a composite enzyme, and then coupled with the edible fungus Lactobacillus plantarum for secondary fermentation. The enzymatic hydrolysis process uses chitosanase and cellulase in combination to achieve the effect of wall decomposition and dissolution. The chitosanase preparation and the cellulase preparation are configured into a composite enzyme preparation according to the activity unit of 1:1-10.

[0006] Patent document CN111808901A discloses a preparation method of a Cordyceps militaris fermentation extract, a product obtained therefrom and an application thereof, wherein lactic acid bacteria are fermented and cultured in a fermentation medium with Cordyceps militaris as a nitrogen source to obtain a Cordyceps militaris fermentation extract. Cordyceps militaris as a nitrogen source exists in the form of a fruiting body.

[0007] Patent document CN113462725A discloses a Cordyceps militaris secondary metabolite of an Artemisia annua extract and a preparation method and application thereof. The method comprises fermenting a special culture medium composed of an Artemisia annua extract with Cordyceps militaris strains, and the focus is on using Cordyceps militaris to perform metabolic conversion of the Artemisia annua extract.

[0008] In addition, the deep liquid fermentation liquid of Cordyceps militaris mycelium is rich in polysaccharides, especially insoluble polysaccharides. The high viscosity of the fermentation liquid greatly increases the difficulty of post-processing of the fermentation liquid. The current mainstream method is alcohol precipitation, that is, adding more than 10% ethanol to the fermentation liquid to effectively precipitate the polysaccharides and facilitate the post-processing process. However, there are many problems with the use of ethanol in industrial production. Ethanol is very flammable, and it is necessary to apply for a dangerous goods use permit and the production plant needs to meet the production conditions of dangerous goods, which has high costs and risks. In the field of cosmetics and skin care, high levels of ethanol can cause irritation and dryness to the skin, destroying the skin's natural protective barrier.

[0009] Therefore, there is an urgent need to improve the preparation process of Cordyceps militaris active ingredients to meet the industrialization needs of Cordyceps militaris in the cosmetics field. Summary of the invention

[0010] In view of this, one of the purposes of the present invention is to provide a method for preparing a Cordyceps militaris mycelium fermentation product, abandoning the expensive lytic enzyme and snail enzyme that are currently only used in laboratory experiments, and using a specific complex enzyme that is low-priced and suitable for industrialization to fully release the active substances of Cordyceps militaris and enhance the efficacy of the product in cosmetics.

[0011] The present invention has found that after deep liquid fermentation using Cordyceps militaris mycelium as the strain, a liquid fermentation liquid rich in mycelium is obtained. After adding a specific industrial complex enzyme and incubating for a certain period of time, the active substances in the cells can be fully released. Then, through an alcohol precipitation process different from the conventional process, the fermentation product harvested is clear and transparent, and is rich in cordyceps polysaccharides, cordycepic acid, cordycepin and other active substances. It has excellent repair and anti-aging effects and can be used as a raw material for functional cosmetics.

[0012] To achieve one of the purposes of the present invention, the technical solution adopted by the present invention is:

[0013] A method for preparing a Cordyceps militaris mycelium fermentation product comprises the following steps:

[0014] S1. preparing seed solution: using Cordyceps militaris mycelium as the strain, inoculating the strain into a liquid culture medium for expansion culture to obtain seed solution;

[0015] S2, fermentation: transferring the seed liquid to a fermentation tank containing a liquid culture medium, aerating and stirring, and fermenting for a certain period of time to obtain a fermentation liquid;

[0016] S3, enzymolysis: adding industrial compound enzyme to the fermentation broth, heating and stirring to obtain enzymolysis treatment liquid;

[0017] S4, alcohol precipitation: adding a certain amount of alcohol solution to the enzymatic hydrolysis solution, mixing thoroughly, and obtaining an alcohol precipitation solution;

[0018] S5. Separation and filtration: Separate the alcohol precipitate, filter the supernatant, collect the clarified filtrate, and obtain the Cordyceps militaris mycelium fermentation product.

[0019] Optionally, the liquid culture medium is a modified PDB culture medium composed of: 10-30 g / L potato extract powder, 10-30 g / L glucose, 2-5 g / L dipotassium hydrogen phosphate, 1-3 g / L magnesium sulfate, and 5-10 mg / L vitamin B1.

[0020] Optionally, the fermentation conditions include: fermentation temperature 16-28°C, stirring speed 50-200rpm, and fermentation time 5-10 days.

[0021] Optionally, the industrial complex enzyme consists of enzyme A and enzyme B, wherein enzyme A is cellulase and pectinase, and enzyme B is chitinase or glucanase.

[0022] Optionally, the amount of the cellulase is 0.001-0.1% by weight of the fermentation broth, the amount of the pectinase is 0.05-0.5% by weight of the fermentation broth, the amount of the glucanase is 0.02-0.5% by weight of the fermentation broth, and the amount of the chitinase is 0.005-0.1% by weight of the fermentation broth.

[0023] Optionally, the enzymatic hydrolysis conditions include: enzymatic hydrolysis temperature of 45-65° C., enzymatic hydrolysis time of 1-3 h, and stirring speed of 30-200 rpm.

[0024] Optionally, the alcohol solution is selected from one or more of butanediol, propylene glycol and pentanediol, and the added amount is 10-70% of the volume of the enzymatic treatment solution.

[0025] Cordyceps militaris mycelium liquid fermentation liquid polysaccharide content is high, viscosity is large, is unfavorable for the carrying out of post-processing process.Therefore, the present invention improves alcohol precipitation process, adopts safer butylene glycol, propylene glycol and / or pentanediol, and the enterprise with cosmetics production qualification can be used for production activities.Butanediol and propylene glycol are non-toxic chemicals certified by the U.S. FDA, and the usage amount in cosmetics is generally 0.5%-5%, which is the common wetting agent in cosmetics, and is widely used.The present invention replaces the ethanol in the existing alcohol precipitation process with butylene glycol or propylene glycol or pentanediol, under suitable dosage, polysaccharide is effectively precipitated, and clear alcohol precipitation liquid is obtained, so as to be convenient to post-processing, and the treatment fluid is made to be more in line with the safety and use requirements of cosmetics, both reducing risk, and meeting the production and use requirements of cosmetics simultaneously.

[0026] Optionally, a certain amount of additives is selectively added to the Cordyceps militaris mycelium fermentation product and mixed thoroughly, wherein the additives include preservatives.

[0027] The second object of the present invention is to provide a Cordyceps militaris mycelium fermentation product obtained by the above-mentioned preparation method. The fermentation product is rich in Cordyceps militaris active ingredients, has high activity and enhanced efficacy.

[0028] The third object of the present invention is to provide a Cordyceps militaris mycelium fermentation product or the Cordyceps militaris mycelium fermentation product obtained by the preparation method and use it in cosmetics.

[0029] A Cordyceps militaris mycelium fermentation product or the Cordyceps militaris mycelium fermentation product obtained by the preparation method is used in cosmetics. The cosmetics have anti-aging and repairing effects. The mass percentage of the Cordyceps militaris mycelium fermentation product in the cosmetics is 0.1-10%.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The invention uses Cordyceps militaris mycelium as a strain for deep liquid fermentation, adds a specific industrial compound enzyme to the fermentation liquid containing the mycelium to destroy the structure of the cell wall and degrade a large amount of polysaccharides secreted by the mycelium to a certain extent, fully releases the active substances in the cell, hydrolyzes the macromolecular polysaccharides and reduces the viscosity of the fermentation liquid for post-processing. Then, the residual macromolecular polysaccharides are removed and the viscosity of the fermentation liquid is reduced through an alcohol precipitation process, and then centrifugation and filtration are performed to obtain a Cordyceps militaris fermentation product.

[0032] Firstly, the present invention adds a specific combination of industrial enzymes, including cellulase, pectinase and glucanase, or cellulase, pectinase and chitinase, to the fermentation liquid containing Cordyceps militaris mycelium after deep liquid fermentation for enzymolysis, thereby effectively improving the breakage rate of mycelium cells, significantly reducing the cost of existing enzymolysis treatment, and maximally promoting the dissolution of active ingredients in the deep liquid fermentation liquid of Cordyceps militaris, including cordycepin, adenosine, cordycepic acid and other active substances, thereby improving the efficacy of the product.

[0033] Secondly, compared with ethanol commonly used in traditional alcohol precipitation processes, the present invention replaces it with butanediol, propylene glycol or pentanediol, making the production and use of fermentation products in cosmetics simpler and safer.

[0034] Again, traditional Cordyceps militaris extracts are usually directly extracted from fruiting bodies or extracted by secondary fermentation. The present invention directly uses Cordyceps militaris mycelium as a strain for fermentation, and its active ingredients are not easily damaged and the fermentation density is extremely high. The active ingredients of the fermentation products obtained are also much higher than those of the traditional methods. The deep liquid fermentation product of Cordyceps militaris obtained by the inventive method is rich in active substances such as cordycepic acid, cordycepin, and small molecule Cordyceps polysaccharides, and has excellent repair and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results are for the expression of laminin 332 and collagen-related genes;

[0036] Figure 2 The results are for the expression of genes related to cell autophagy;

[0037] Figure 3 The results of cell scratch test. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods thereof are described in detail below in conjunction with the accompanying drawings. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0039] Example 1

[0040] The solid mycelium of Cordyceps militaris was inoculated into a modified PDB medium, and cultured at 25°C and 150 rpm for 5 days to prepare a seed solution;

[0041] The seed liquid was transferred to a fermentation tank containing a modified PDB medium at an inoculum rate of 2%, and the fermentation temperature was adjusted to 26°C and the rotation speed was 150rpm. The fermentation was carried out aerobically for 7 days to obtain a fermentation liquid. The modified PDB medium formula was 10g / L potato extract powder, 20g / L glucose, 2g / L potassium dihydrogen phosphate, 1g / L magnesium sulfate, and 10mg / L vitamin B1, and sterilized at 121°C for 20 minutes.

[0042] Put the tank, add industrial complex enzyme to the fermentation liquid containing Cordyceps militaris mycelium; the composition of the industrial complex enzyme is 0.01% cellulase, 0.2% pectinase, and 0.01% chitinase, heat to 60°C, stir at 120rpm for 90 minutes, and obtain enzymolysis treatment liquid;

[0043] Add 40% butanediol to the enzymatic hydrolysis solution, stir and mix, and obtain an alcohol precipitate; then, centrifuge to remove most of the solid impurities, and then filter with a plate and frame to obtain a Cordyceps militaris mycelium fermentation product;

[0044] 0.5% p-hydroxyacetophenone and 0.5% hexylene glycol were added as preservatives to the fermentation product of Cordyceps militaris mycelium, and the mixture was mixed to obtain a product, which was recorded as A1.

[0045] Example 2

[0046] The solid mycelium of Cordyceps militaris was inoculated into the modified PDB medium, and cultured at 25°C and 150 rpm for 5 days to prepare a seed solution;

[0047] The seed liquid was transferred to a fermentation tank containing a modified PDB medium at an inoculum rate of 2%, and the fermentation temperature was adjusted to 20°C, the rotation speed was 100 rpm, and aerobic fermentation was carried out for 10 days to obtain a fermentation liquid; the modified PDB medium formula was 20g / L potato extract powder, 10g / L glucose, 3g / L potassium dihydrogen phosphate, 1g / L magnesium sulfate, 5mg / L vitamin B1, and sterilized at 121°C for 20 minutes;

[0048] Put the tank, add industrial complex enzyme to the fermentation liquid containing Cordyceps militaris mycelium, the complex enzyme combination is 0.01% cellulase, 0.2% pectinase, 0.3% glucanase, heat to 55°C, stir at 100 rpm for 120 minutes, and obtain enzymolysis treatment liquid;

[0049] Add 40% butanediol to the enzymatic hydrolysis solution, stir and mix, and obtain an alcohol precipitate; then, centrifuge to remove most of the solid impurities, and then filter with a plate and frame to obtain a Cordyceps militaris mycelium fermentation product;

[0050] 1% phenoxyethanol and 0.25% sodium benzoate were added as preservatives to the fermentation product of Cordyceps militaris mycelium, and the mixture was mixed to obtain a product, which was recorded as A2.

[0051] Example 3

[0052] The difference from Example 1 is that 40% propylene glycol is added to the enzymatic treatment solution;

[0053] The product of this embodiment is recorded as A3.

[0054] Example 4

[0055] The solid mycelium of Cordyceps militaris was inoculated into a modified PDB medium, and cultured at 20°C and 180 rpm for 6 days to prepare a seed solution;

[0056] The seed liquid was transferred to a fermentation tank containing a modified PDB medium at an inoculum rate of 2%, and the fermentation temperature was adjusted to 20°C and the rotation speed was 180 rpm. The fermentation was carried out aerobically for 9 days to obtain a fermentation liquid. The modified PDB medium formula was 25 g / L potato extract powder, 15 g / L glucose, 4 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 7 mg / L vitamin B1. The medium was sterilized at 121°C for 20 minutes.

[0057] Put the tank, add industrial complex enzyme to the fermentation liquid containing Cordyceps militaris mycelium; the composition of the industrial complex enzyme is 0.1% cellulase, 0.05% pectinase, and 0.08% chitinase, heat to 55°C, stir at 170rpm for 120 minutes, and obtain enzymolysis treatment liquid;

[0058] Add 30% pentanediol to the enzymatic hydrolysis solution, stir and mix, and obtain an alcohol precipitate; then, centrifuge to remove most of the solid impurities, and then filter with a plate and frame to obtain a Cordyceps militaris mycelium fermentation product;

[0059] 0.5% p-hydroxyacetophenone and 0.5% hexylene glycol were added as preservatives to the fermentation product of Cordyceps militaris mycelium, and the mixture was mixed to obtain the product.

[0060] Example 5

[0061] The solid mycelium of Cordyceps militaris was inoculated into the modified PDB medium, and cultured at 27°C and 100 rpm for 5 days to prepare a seed solution;

[0062] The seed liquid was transferred to a fermentation tank containing a modified PDB medium at an inoculum rate of 2%, and the fermentation temperature was adjusted to 28°C, the rotation speed was 100 rpm, and aerobic fermentation was performed for 6 days to obtain a fermentation liquid; the modified PDB medium formula was 30 g / L potato extract powder, 10 g / L glucose, 5 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate, 6 mg / L vitamin B1, and sterilized at 121°C for 20 minutes;

[0063] Put the tank, add industrial complex enzyme to the fermentation liquid containing Cordyceps militaris mycelium, the complex enzyme combination is 0.07% cellulase, 0.5% pectinase, 0.1% glucanase, heat to 63°C, stir at 200 rpm for 60 minutes, and obtain enzymolysis treatment liquid;

[0064] Add 50% butanediol to the enzymatic hydrolysis solution, stir and mix, and obtain an alcohol precipitate; then, centrifuge to remove most of the solid impurities, and then filter with a plate and frame to obtain a Cordyceps militaris mycelium fermentation product;

[0065] 1% phenoxyethanol and 0.25% sodium benzoate were added as preservatives to the fermented product of Cordyceps militaris mycelium, and the mixture was mixed to obtain the product.

[0066] Example 6

[0067] Different from Example 1, the composition of the industrial complex enzyme is 0.005% cellulase, 0.3% pectinase, and 0.05% chitinase. The mixture is heated to 58° C. and stirred at 180 rpm for 150 minutes to obtain an enzymatic treatment solution.

[0068] Example 7

[0069] Different from Example 1, the composition of the industrial complex enzyme is 0.001% cellulase, 0.4% pectinase, and 0.1% chitinase. The mixture is heated to 60° C. and stirred at 160 rpm for 100 minutes to obtain an enzymatic treatment solution.

[0070] Example 8

[0071] Different from Example 2, the composition of the industrial complex enzyme is 0.003% cellulase, 0.5% pectinase, and 0.05% glucanase. The mixture is heated to 62° C. and stirred at 130 rpm for 75 minutes to obtain an enzymatic treatment solution.

[0072] Example 9

[0073] Different from Example 2, the composition of the industrial complex enzyme is 0.08% cellulase, 0.3% pectinase, and 0.02% glucanase. The mixture is heated to 48° C. and stirred at 100 rpm for 180 minutes to obtain an enzymatic treatment solution.

[0074] Comparative Example 1

[0075] The fruiting bodies of Cordyceps militaris were broken into pieces, and 6 times the volume of pure water was added to soak for 2 hours, and then the temperature was raised to 80°C for extraction and wall breaking for 2 hours. After cooling, the solid residue was removed by filtration, and the insoluble matter was removed by plate and frame filtration to obtain the fruiting body extract product of Cordyceps militaris, which was recorded as B1.

[0076] Comparative Example 2

[0077] The fruiting bodies of Cordyceps militaris were crushed, and the fruiting body powder of Cordyceps militaris was used as a nitrogen source to prepare the fermentation medium. The medium formula included: Cordyceps militaris fruiting body powder 10g / L, lactose 5g / L, glucose 5g / L, Tween 80 1ml, dipotassium hydrogen phosphate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L. The medium was sterilized at 121℃ for 20 minutes, and after cooling to room temperature, the lactobacillus strain was inoculated at 2% inoculation and anaerobically fermented at 37℃ for 3 days. After the fermentation was completed, the large particles of solid residue were removed by filtering, and then the fermentation supernatant was obtained by centrifugation, and finally sterilized by 0.22μm filter membrane to obtain the fermentation product of Cordyceps militaris fruiting body, which was recorded as B2.

[0078] Comparative Example 3

[0079] A Cordyceps militaris mycelium fermentation product was prepared according to the method of Example 1, except that 2.0% snail enzyme was added to the fermentation liquid containing the Cordyceps militaris mycelium, and the enzymolysis temperature was 30° C. The obtained Cordyceps militaris mycelium fermentation product was recorded as C1.

[0080] Comparative Example 4

[0081] The Cordyceps militaris mycelium fermentation product was prepared according to the method of Example 1, except that 0.01% cellulase and 0.2% pectinase were added to the fermentation liquid containing the Cordyceps militaris mycelium, and the enzymolysis temperature was 60° C. The obtained Cordyceps militaris mycelium fermentation product was recorded as C2.

[0082] Comparative Example 5

[0083] A Cordyceps militaris mycelium fermentation product was prepared according to the method of Example 1, except that 0.1% chitinase was added to the fermentation liquid containing the Cordyceps militaris mycelium, and the enzymolysis temperature was 37° C. The obtained Cordyceps militaris mycelium fermentation product was recorded as C3.

[0084] Comparative Example 6

[0085] A Cordyceps militaris mycelium fermentation product was prepared according to the method of Example 1, except that 0.3% glucanase was added to the fermentation liquid containing the Cordyceps militaris mycelium, and the enzymolysis temperature was 55° C. The obtained Cordyceps militaris mycelium fermentation product was recorded as C4.

[0086] Comparative Example 7

[0087] A Cordyceps militaris mycelium fermentation product was prepared according to the method of Example 1, except that the fermentation liquid containing the Cordyceps militaris mycelium was not subjected to enzymatic hydrolysis, and the enzymatic hydrolysis step was replaced by direct incubation at 60° C. The obtained Cordyceps militaris mycelium fermentation product was recorded as C5.

[0088] Effect evaluation test

[0089] 1. Component detection and analysis

[0090] The products obtained in Examples 1-3 and Comparative Examples 1-7 were tested for the following components:

[0091] The content of adenosine and cordycepin was tested according to NYT2116-2012 "Determination of cordycepin and adenosine in cordyceps products";

[0092] The content of cordycepic acid was tested by referring to the literature "Colorimetric determination of the content of cordycepic acid in mycelium of Cordyceps sinensis";

[0093] The polysaccharide content was determined by referring to the phenol-sulfuric acid method in NY / T 1676-2008 “Determination of crude polysaccharide content in edible fungi”.

[0094] The test results are shown in Table 1:

[0095] Table 1 Component test results

[0096]

[0097] From the test results in Table 1, it can be seen that the contents of adenosine, cordycepin, cordycepic acid and cordyceps polysaccharide in Examples 1-3 (A1, A2, A3) are close to the contents of the components in Control Example 3 (C1), indicating that the enzymatic hydrolysis effect of the industrial composite enzyme used in the present invention is equivalent to that of snail enzyme, indicating that the industrial composite enzyme used in the present invention can well replace snail enzyme, thereby overcoming the disadvantages of the current snail enzyme being expensive, remaining only in the laboratory stage, and being difficult to industrialize and apply, which is beneficial to industrial production and promotion.

[0098] Secondly, the contents of adenosine, cordycepin, cordycepic acid and cordyceps polysaccharide in Examples 1-3 (A1, A2, A3) are much higher than those in Control Examples 1-2 (B1, B2) and Control Examples 4-7 (C2, C3, C4, C5), indicating that the combination of cellulase, pectinase and chitinase used in the industrial composite enzyme of the present invention, as well as the combination of cellulase, pectinase and glucanase, have significant synergistic effects, can significantly overcome the shortcomings of low effect of single enzyme, can effectively promote the lysis of mycelial cells, fully release active ingredients, and achieve good wall-breaking effect.

[0099] 2. Efficacy evaluation

[0100] 2.1 Expression of laminin 332-related genes LAMA3, LAMB3, and type IV and VII collagen-related genes COL4A1, COL7A1

[0101] Human keratinocytes (expressing laminin 332 genes LAMA3 and LAMB3) were cultured at 4 × 10 510 cells / well were seeded on a 6-well cell culture plate and cultured in a CO2 culture incubator at 37°C for 24 hours. Human fibroblasts (expressing type Ⅳ and Ⅶ collagen-related genes COL4A1 and COL7A1) were cultured at 4×10 5 Inoculate on a 6-well cell culture plate and culture in a CO2 culture incubator at 37°C for 24 hours, then add 2% of the product of the embodiment or control example, and do not add the blank control group, and continue to incubate for 24 hours. According to the instructions of the RNA extraction kit, RNA was extracted and its purity and concentration were tested, and RNA was used as a template for reverse transcription into cDNA. Subsequently, the RT-qPCR method was used to detect the expression of the target gene, with GAPDH as the housekeeping gene. The test results are as follows Figure 1 shown.

[0102] Figure 1 The results showed that compared with the blank control, A1, A2, and A3 were able to effectively promote the expression of laminin 332 and type IV and VII collagen-related genes, and all showed comparable efficacy to C1. B1, B2, C2, C3, C4, and C5 showed weaker or no significant effects, indicating that A1, A2, and A3 were able to significantly promote the wall breaking of Cordyceps militaris mycelium compared with the above control examples, and the released active ingredients significantly enhanced the efficacy of the product.

[0103] 2.2 Expression of key autophagy proteins LC3, Beclin1, and p62 genes

[0104] Human dermal fibroblasts were cultured at 4 × 10 5 Inoculate on a 6-well cell culture plate and culture in a CO2 culture incubator at 37°C for 24 hours, then add 2% of the product of the embodiment or the control example respectively, and do not add the blank control group, and continue to incubate for 72 hours. According to the instructions of the RNA extraction kit, RNA is extracted and its purity and concentration are tested, and RNA is used as a template for reverse transcription into cDNA. Subsequently, the RT-qPCR method is used to detect the expression of the target gene, with GAPDH as the housekeeping gene. The test results are as follows Figure 2 shown.

[0105] Figure 2 The results showed that compared with the blank control, A1, A2, and A3 were able to effectively promote the expression of key autophagy genes LC3 and Beclin1, and inhibit the expression of p62, thereby enhancing the autophagy function of cells and exerting a repair effect, and all showed an effect comparable to that of C1. The promotion effects of B1, B2, C2, C3, C4, and C5 were weak or had no obvious effect, so the expression effects of A1, A2, and A3 were more significant than those of these control examples.

[0106] 2.3 Repair efficacy test - cell scratch test

[0107] Human keratinocytes were cultured at 4 × 10 5 Each well was inoculated on a 6-well cell culture plate and cultured in a CO2 culture incubator at 37°C for 24 hours. A scratch was made artificially with a gun tip, and the cells were removed from the scratched area. Then, 2% of the product of the embodiment or the control example was added, and the blank control group was added with an equal amount of PBS buffer and incubated for another 24 hours. The test results are shown in FIG. Figure 3 shown.

[0108] Figure 3 The results showed that compared with the blank control group, A1, A2, and A3 were able to promote cell growth and migration with significant effects, and all showed effects comparable to C1; the effects of B1, B2, C2, C3, C4, and C5 in promoting cell growth and migration were not obvious.

[0109] In summary, the contents of active ingredients adenosine, cordycepin, cordycepic acid and cordyceps polysaccharide in the mycelium fermentation product of Cordyceps militaris of the present invention are high; compared with direct extraction, secondary fermentation extraction, conventional enzymatic hydrolysis or enzymatic hydrolysis without enzyme in Cordyceps militaris fruiting bodies, they can effectively promote the expression of laminin 332 and type IV and VII collagen related genes, and can also effectively promote the expression of key cell autophagy genes LC3 and Beclin1, and inhibit p62 expression, enhance cell autophagy function, exert repair effect, and also have the effect of promoting cell growth and migration, and are expected to be used in repair and anti-aging cosmetics.

[0110] Those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. As long as they are within the spirit of the present invention, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a Cordyceps militaris mycelium fermentation product, characterized in that: The following steps are involved: S1. Using Cordyceps militaris mycelium as the strain, inoculating the strain into a liquid culture medium for expansion culture to obtain a seed solution, wherein the liquid culture medium is a modified PDB culture medium composed of: 10-30 g / L potato extract powder, 10-30 g / L glucose, 2-5 g / L potassium dihydrogen phosphate, 1-3 g / L magnesium sulfate and 5-10 mg / L vitamin B1; S2, transferring the seed liquid to a fermentation tank containing a liquid culture medium, aerating and stirring, and fermenting to obtain a fermentation liquid, wherein the fermentation conditions include: a fermentation temperature of 16-28° C., a stirring speed of 50-200 rpm, and a fermentation time of 5-10 days; S3, adding industrial complex enzyme to the fermentation broth, heating and stirring to obtain an enzymatic treatment liquid, wherein the industrial complex enzyme is composed of enzyme A and enzyme B, wherein enzyme A is cellulase and pectinase, and enzyme B is chitinase or glucanase, wherein the amount of the cellulase is 0.001-0.1% by weight of the fermentation broth, the amount of the pectinase is 0.05-0.5% by weight of the fermentation broth, the amount of the glucanase is 0.02-0.5% by weight of the fermentation broth, and the amount of the chitinase is 0.005-0.1% by weight of the fermentation broth, and the conditions for the enzymatic hydrolysis include: an enzymatic hydrolysis temperature of 45-65°C, an enzymatic hydrolysis time of 1-3h, and a stirring speed of 30-200rpm; S4, adding a certain amount of alcohol solution to the enzymatic hydrolysis solution, mixing thoroughly to obtain an alcohol precipitation solution, wherein the alcohol solution is selected from one or more of butanediol, propylene glycol and pentanediol, and the added amount is 10-70% of the volume of the enzymatic hydrolysis solution; S5. Separating the alcohol precipitate, filtering the supernatant, and collecting the clarified filtrate to obtain a Cordyceps militaris mycelium fermentation product.

2. The method for preparing a Cordyceps militaris mycelium fermentation product according to claim 1, characterized in that: Additives are selectively added to the Cordyceps militaris mycelium fermentation product and mixed thoroughly. The additives include preservatives.

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