Preparation method of armillaria luteo-virens fermentation liquor with anti-aging effect and application of armillaria luteo-virens fermentation liquor in cosmetics

Abstract

The invention discloses a preparation method of armillaria luteo-virens fermentation broth with an anti-aging effect and application of the armillaria luteo-virens fermentation broth in cosmetics, and the preparation method specifically comprises the following steps: inoculating lactobacillus into a fermentation substrate, and carrying out fermentation culture and sterilization to obtain the armillaria luteo-virens fermentation broth. The fermentation substrate comprises armillaria luteo-virens sporocarp and water; wherein the lactobacillus is composed of lactobacillus helveticus and lactobacillus paracasei, and the viable count ratio of the lactobacillus helveticus to the lactobacillus paracasei is 1: (0.5-2). The active ingredients in the armillaria luteo-virens sporocarp are extracted in a lactic acid bacteria fermentation mode, loss of the active ingredients can be reduced, and the skin care effect of the armillaria luteo-virens can be better exerted; the prepared armillaria luteo-virens sporocarp fermentation liquor can be directly used as a cosmetic and can also be added into a cosmetic basic formula for use, the product safety is high, the effects of resisting oxidation and repairing damaged cells are achieved, and a certain cell growth promoting effect is achieved.

Description

Technical Field

[0001] The present application belongs to the field of cosmetics, and specifically relates to a method for preparing a fermentation liquid of Armillaria lutea with anti-aging effects and its application in cosmetics. Background Art

[0002] With the rapid development of science and technology, and the improvement of people's material living conditions, the aging population continues to intensify. Actively exploring and developing antioxidant and anti-aging products is crucial for slowing the aging of society. Based on extensive experimental evidence, the free radical theory holds the greatest weight among modern theories of aging. This theory states that cells produce a significant number of reactive oxygen species (ROS) during normal metabolic processes. Free radicals exist in a dynamic equilibrium of constant production and clearance within the human body. When the body is influenced by both exogenous and endogenous factors, excessive free radical production or insufficient clearance can cause tissue and cell damage, leading to aging and other diseases.

[0003] The chemical composition of Armillaria japonici is very rich, containing a large amount of protein, amino acids, and sugars. Alkaloid components and a small amount of organic acids, flavonoids, steroidal triglycerides, glycosides and other substances have also been detected. Armillaria japonici has high nutritional value and contains a rich variety of vitamins, such as vitamin B1, vitamin B2, and vitamin C. It has also been detected to be rich in 18 kinds of amino acids, among which the higher content is 8 kinds of amino acids necessary for the human body.

[0004] At present, in view of the rich chemical components in Armillaria chrysogenum, the existing technology generally uses water, ethanol and petroleum ether for direct extraction. However, the alkaloids, sugars and other chemical components directly extracted by solvents have a large molecular weight, which makes them unable to be directly absorbed by the skin, thus affecting the efficacy.

[0005] Therefore, there is an urgent need in the art to develop a method that can effectively develop Armillaria lutea, thereby improving its bioavailability and cosmetic efficacy. Summary of the Invention

[0006] The technical problem to be solved by the present application is to overcome the defects in the prior art of the rich chemical components in the yellow-green Armillaria, such as alkaloids, sugars and other chemical components directly extracted by solvents, which have large molecular weights and cannot be directly absorbed by the skin, thus affecting the efficacy, and to provide a method for preparing the yellow-green Armillaria fermentation liquid with anti-aging efficacy and its application in cosmetics.

[0007] This application adopts the following technical solutions to solve the above technical problems:

[0008] The present application provides a method for preparing a fermentation liquid of Armillaria lutea with anti-aging effects, which specifically comprises the following steps: inoculating lactobacilli into a fermentation substrate, fermenting and culturing, and sterilizing; the fermentation substrate comprises Armillaria lutea fruiting bodies and water; and the lactobacilli are composed of Lactobacillus helveticus and Lactobacillus paracasei.

[0009] In some embodiments, the ratio of the viable cell count of the Lactobacillus helveticus to the Lactobacillus paracasei is 1:(0.5-2).

[0010] In some embodiments, the mass ratio of the Armillaria chrysogenum fruiting body to the water is 1:(10-300), preferably 1:(30-200), and more preferably 1:150.

[0011] In some embodiments, the fermentation substrate further comprises crushing and sieving the fruiting bodies of Armillaria luteola to obtain Armillaria luteola fruiting body powder.

[0012] The particle size of the sieved particles is 50-200 meshes, preferably 50-150 meshes.

[0013] In some embodiments, the fermentation substrate further comprises a carbon source and / or a nitrogen source.

[0014] The carbon source includes carbon sources conventionally added in the art, generally including glucose.

[0015] The nitrogen source includes nitrogen sources conventionally added in the art, generally including soybean peptides.

[0016] Wherein, when the carbon source is added, the mass ratio of the carbon source to the water is 1:(100-1000).

[0017] Wherein, when the nitrogen source is added, the mass ratio of the nitrogen source to the water is 1:(100-1000), preferably 1:(200-500).

[0018] In some embodiments, the method for preparing the fermentation substrate comprises: mixing the components of the fermentation substrate until the components are completely dissolved.

[0019] In some embodiments, the fermentation substrate may be further sterilized before use.

[0020] The sterilization method may generally be high-temperature sterilization.

[0021] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0022] When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, such as 0.13 MPa.

[0023] When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 20 to 30 minutes, for example 30 minutes.

[0024] In some embodiments, the Lactobacillus helveticus includes Lactobacillus helveticus deposited in the China Industrial Microorganism Culture Collection Center with a deposit number of CICC 20243.

[0025] In some embodiments, the Lactobacillus paracasei includes the Lactobacillus paracasei deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 20241.

[0026] In some embodiments, the preparation method of the lactic acid bacteria liquid can be conventional in the art, and generally can include the following steps: dissolving the lactic acid bacteria in sterile deionized water.

[0027] In some embodiments, the Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial solution, and the concentration of live bacteria of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial solution can be 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL, for example 10 10 CFU / mL.

[0028] In some embodiments, the Lactobacillus paracasei is added in the form of a Lactobacillus paracasei bacterial solution, and the viable bacteria concentration of the Lactobacillus paracasei in the Lactobacillus paracasei bacterial solution is 10 7 ~10 13 CFU / mL, preferably 10 8 ~10 11 CFU / mL, more preferably 10 10 CFU / mL.

[0029] In some embodiments, the conditions and methods for the fermentation culture may be conventional in the art, and generally may be static fermentation.

[0030] In some embodiments, the fermentation culture temperature is 30-45°C, preferably 35-40°C.

[0031] In some embodiments, the fermentation culture time is 12 to 48 hours, preferably 12 to 24 hours.

[0032] In some embodiments, the sterilization method may be a high-temperature sterilization method conventionally used in the art.

[0033] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0034] When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, such as 0.13 MPa.

[0035] When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 20 to 30 minutes, for example 30 minutes.

[0036] In some embodiments, after the sterilization operation, the process may further include at least one of centrifugation and collection of supernatant, secondary sterilization, and mixing with a preservative.

[0037] The centrifugal speed may be a conventional speed for such operations in the art, preferably 3000-9000 rpm, more preferably 4000-6000 rpm, for example 4800 rpm.

[0038] The centrifugation time may be a conventional time for such operations in the art, preferably 10 to 40 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

[0039] Wherein, the radius of the centrifuge is 8 to 15 cm.

[0040] Among them, the secondary sterilization method can generally be a high-temperature sterilization method. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature can be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example, 121°C. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure can be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example, 0.13 MPa. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be a conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 20 to 30 minutes, for example, 30 minutes.

[0041] Wherein, during the mixing process with the preservative, the mixing temperature can be a conventional temperature for such operations in the art, preferably 50-80°C, more preferably 70-80°C.

[0042] Wherein, in the process of mixing with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol according to conventional practices in the art.

[0043] When the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization can be 0.1% to 0.8%, preferably 0.1% to 0.6%, and more preferably 0.5%.

[0044] When the preservative includes 1,2-hexanediol, the mass fraction of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.3% to 1.5%, preferably 0.5% to 1%, and more preferably 0.5%.

[0045] In some embodiments, the room temperature generally refers to 15-40°C.

[0046] The present application also provides a yellow-green Armillaria fermentation liquid with anti-aging efficacy, which is prepared by the above-mentioned preparation method of yellow-green Armillaria fermentation with anti-aging efficacy.

[0047] The present application also provides a use of the above-mentioned fermentation liquid of Armillaria lutea with anti-aging efficacy directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0048] In some embodiments, the fermentation liquid of Armillaria lutea with anti-aging efficacy is used as the antioxidant active ingredient and anti-aging active ingredient in the skin external preparation.

[0049] Wherein, the antioxidant active ingredient is an antioxidant active ingredient having a DPPH free radical scavenging effect and / or an antioxidant active ingredient having a hydroxyl free radical scavenging effect.

[0050] The anti-aging active ingredient is an anti-aging active ingredient that promotes the production of collagen in cells.

[0051] The present application also provides a skin external preparation, which includes the above-mentioned Armillaria lutea fermentation liquid with anti-aging effect.

[0052] In some embodiments, the skin external preparation may further include active ingredients commonly used in the art, generally including at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0053] In some embodiments, the topical skin preparation may include, but is not limited to, a facial mask, an essence, or a toner according to conventional methods in the art.

[0054] In some embodiments, the mass percentage of the fermentation liquid of the fruiting body of Armillaria lutea can be 5% to 99% of the external skin preparation, preferably 60% to 99%.

[0055] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0056] The reagents and raw materials used in this application are commercially available.

[0057] The positive progress of this application is that: this application extracts the active ingredients in the fruiting bodies of Armillaria luteola through lactic acid bacteria fermentation, which not only reduces the loss of active ingredients, but also better exerts its skin care effect. The obtained fermentation liquid of the fruiting bodies of Armillaria luteola can be used directly as a cosmetic or added to the basic formula of cosmetics. The product is highly safe, has both antioxidant effects and the effect of repairing damaged cells, and also has a certain effect of promoting cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The present application may be better understood by referring to the following description in conjunction with the accompanying drawings, which together with the following detailed description are incorporated into and form a part of this specification and are used to further illustrate the preferred embodiments of the present application and to explain the principles and advantages of the present application.

[0059] in:

[0060] Figure 1 This is a comparison chart of the DPPH free radical scavenging ability of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 4;

[0061] Figure 2A comparison chart of the hydroxyl radical scavenging abilities of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 4;

[0062] Figure 3 This is a comparison chart of the total antioxidant capacity of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 4;

[0063] Figure 4 A comparison chart of the cytotoxicity of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 4;

[0064] Figure 5 This is a comparison chart of the repair ability of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 4 on damaged skin. DETAILED DESCRIPTION

[0065] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. In the following examples, the experimental methods without specific conditions are selected according to conventional methods and conditions or according to the product specifications.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0067] The raw materials in the following examples are all commercially available.

[0068] Example 1

[0069] 2 g of 100-mesh Armillaria lutea fruiting body powder was mixed evenly with 300 g of deionized water, 0.6 g of glucose, and 0.6 g of soybean peptide, and then sterilized at 121° C. and 0.12 MPa for 30 min. After sterilization, the mixture was allowed to stand at room temperature to obtain a fermentation substrate.

[0070] The preparation method of Lactobacillus helveticus CICC 20243 bacterial liquid comprises: dissolving Lactobacillus helveticus CICC 20243 in sterile deionized water to prepare Lactobacillus helveticus CICC 20243 bacterial liquid; the concentration of live bacteria of Lactobacillus helveticus CICC 20243 in the Lactobacillus helveticus CICC 20243 bacterial liquid is 10 10 CFU / mL.

[0071] The preparation method of the Lactobacillus paracasei CICC 20241 bacterial liquid comprises: dissolving the Lactobacillus paracasei CICC 20241 in sterile deionized water to prepare the Lactobacillus paracasei CICC 20241 bacterial liquid; the concentration of live bacteria of the Lactobacillus paracasei CICC 20241 in the Lactobacillus paracasei CICC 20241 bacterial liquid is 10 10 CFU / mL.

[0072] 1.5 mL of Lactobacillus helveticus CICC 20243 bacterial solution and 1.5 mL of Lactobacillus casei CICC20241 bacterial solution were inoculated into the fermentation substrate for mixed fermentation, shaken and then cultured in a constant temperature incubator at 40°C for 15 hours; after the fermentation, the mixture was sterilized at a temperature of 121°C and a pressure of 0.12 MPa for 30 minutes. After the sterilization was completed, the fermentation liquid was cooled to room temperature and centrifuged at a centrifugal speed of 4800 r / min for 30 minutes; after the centrifugation was completed, the supernatant was sterilized at a temperature of 121°C and a pressure of 0.12 MPa for 30 minutes, and a secondary sterilization was performed under the conditions of 121°C and 0.12 MPa; after the secondary sterilization, p-hydroxyacetophenone and 1,2-hexanediol were added at 70°C, wherein the mass percentage of p-hydroxyacetophenone in the supernatant was 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant was 0.5%, and the mixture was evenly mixed to obtain yellow-green honey with anti-aging effect. Cyclobacterial fermentation broth.

[0073] Example 2

[0074] The preparation method is the same as that in Example 1, except that the fermentation time is 20 h, and other conditions and parameters are the same as those in Example 1.

[0075] Example 3

[0076] The preparation method is the same as that in Example 1, except that 1 mL of Lactobacillus helveticus liquid and 2 mL of Lactobacillus paracasei liquid are inoculated, and other conditions and parameters are the same as those in Example 1.

[0077] Example 4

[0078] The preparation method is the same as that in Example 1, except that 2 mL of Lactobacillus helveticus liquid and 1 mL of Lactobacillus paracasei liquid are inoculated, and other conditions and parameters are the same as those in Example 1.

[0079] Comparative Example 1

[0080] Compared with Example 1, the only difference is that fermentation is not performed. The specific operations are as follows:

[0081] 2 g of 100-mesh Armillaria yellow-green fruiting body powder was mixed evenly with 300 g of deionized water, 0.6 g of glucose, and 0.6 g of soybean peptide. The mixture was then sterilized at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min. After sterilization, the mixture was allowed to stand to room temperature, and then centrifuged at a speed of 4800 rpm for 30 min. The supernatant was taken, and p-hydroxyacetophenone and 1,2-hexanediol were added to the supernatant at 70° C., wherein the mass percentage of p-hydroxyacetophenone in the supernatant was 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant was 0.5%.

[0082] Comparative Example 2

[0083] Compared with Example 1, the only difference is that 3 ml of Lactobacillus delbrueckii liquid is inoculated, and other condition parameters are the same as Example 1.

[0084] Comparative Example 3

[0085] Compared with Example 1, the only difference is that 1.5 ml of Bifidobacterium longum bacterial solution and 1.5 ml of Lactobacillus helveticus are inoculated, and other condition parameters are the same as Example 1.

[0086] Comparative Example 4

[0087] Compared with Example 1, the only difference is that 1.5 ml of Lactobacillus bulgaricus bacterial liquid and 1.5 ml of Lactobacillus plantarum bacterial liquid are inoculated, and other condition parameters are the same as in Example 1.

[0088] Effect Example 1 DPPH free radical scavenging rate

[0089] DPPH is an early synthetic organic free radical, often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, appears purple, and has a characteristic absorption peak at 517nm. When exposed to free radical scavengers, DPPH's lone pair electrons are paired, causing it to fade, meaning the absorbance at the maximum absorption wavelength decreases. Therefore, the scavenging effect of a sample on DPPH free radicals can be evaluated by measuring changes in absorbance.

[0090] Test solution: the product prepared in the above examples or comparative examples diluted 5 times.

[0091] The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

[0092] (1) Take an equal volume (1 mL) of the test solution and 2×10 -4 mol / L DPPH solution (tube A1);

[0093] (2) Take an equal volume (1 mL) of anhydrous ethanol and 2×10 -4 mol / L DPPH solution (tube A2);

[0094] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0095] (4) After 30 minutes of reaction in the dark, measure the absorbance of tubes A1, A2, and A3 at 517 nm;

[0096] The calculation formula of DPPH free radical scavenging rate is: DPPH free radical scavenging rate = [(A2 + A3) - A1] / A2 × 100%, the calculation results are shown in Table 1 and Figure 1 .

[0097] Table 1

[0098] serial number DPPH free radical scavenging rate (%) Example 1 87.57±1.48 Example 2 83.94±1.29 Example 3 84.34±1.66 Comparative Example 1 58.77±1.82 Comparative Example 2 66.94±2.6 Comparative Example 3 71.38±1.61 Comparative Example 4 68.01±1.87

[0099] The results showed that the products prepared in Examples 1 to 3 had significantly higher DPPH radical scavenging effects than the products prepared in Comparative Examples 1 to 4, indicating that the products prepared in the present examples have relatively ideal antioxidant efficacy. This indicates that the product preparation method and the fermentation strain have a certain impact on the antioxidant effect.

[0100] Effect Example 2 Hydroxyl Radical Scavenging Rate

[0101] The Fenton reaction generates hydroxyl radicals (·OH). Salicylic acid is added to the reaction system, where ·OH reacts with the acid to produce the colored compound 2,3-dihydroxybenzoic acid, which exhibits characteristic absorption at 510 nm. Using the fixed reaction time method, the absorbance of the reaction solution containing the analyte is measured at 510 nm and compared with a blank solution to determine the analyte's ability to scavenge ·OH.

[0102] The test solution is the product prepared in the above examples and comparative examples diluted 5 times;

[0103] Add reagents to the test tube according to Table 1, and add 9mmol / L FeSO4, 9mmol / L salicylic acid ethanol solution, test solution, appropriate amount of deionized water and 8.8mmol / L H2O2 solution in sequence. Shake well, heat in a 37℃ water bath for 15 minutes, then take out and measure the absorbance of the blank control A0 and the sample group A x and the background absorbance of the solution without H2O2 x0 When determining A0, the reference solution was a system without hydrogen peroxide. Three parallel experiments were performed in each group, and the absorbance values were measured and averaged. The results are shown in Table 2. The hydroxyl radical scavenging rate of each group was calculated according to the following formula. The results are shown in Tables 3 and Figure 2 .

[0104] Hydroxyl radical scavenging rate = (A0-(A x -A x0 )) / A0×100%.

[0105] Table 2

[0106]

[0107] Table 3

[0108] serial number Hydroxyl free radical scavenging rate (%) Example 1 75.04±1.18 Example 2 69.03±1.68 Example 3 69.57±2.56 Comparative Example 1 20.24±1.84 Comparative Example 2 48.6±2.03 Comparative Example 3 52.39±1.51 Comparative Example 4 48.99±2.07

[0109] The results show that the products prepared in Examples 1 to 3 have significantly higher hydroxyl radical scavenging effects than the products prepared in Comparative Examples 1 to 4, indicating that the products prepared in the present examples have relatively ideal antioxidant efficacy. It can be seen that the product preparation method and the fermentation strain have a certain impact on the antioxidant effect.

[0110] Effect Example 3: Determination of total antioxidant capacity by ABTS method

[0111] The total antioxidant capacity of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 4 was tested using the total antioxidant capacity detection kit (ABTS method) with the product number S0119 produced by Biyuntian Biotechnology Co., Ltd. The results are shown in Tables 4 and Figure 3 ( Figure 3 middle, *** p<0.001, indicating that there is an extremely significant statistical difference compared with Example 1, and a very significant decrease; ** p<0.01 indicates that there is a significant statistical difference compared with Example 1, and the amount is significantly reduced; ns indicates that there is no statistical difference compared with Example 1;).

[0112] Table 4

[0113] serial number TEAC / mM Example 1 0.334±0.021 Example 2 0.302±0.022 Example 3 0.281±0.017 Comparative Example 1 0.11±0.011 Comparative Example 2 0.165±0.004 Comparative Example 3 0.173±0.017 Comparative Example 4 0.159±0.011

[0114] The results showed that the TEAC content of the products prepared in Examples 1 to 3 after treatment was significantly higher than that of the products prepared in Comparative Examples 1 to 4 after treatment, indicating that the products prepared in the present examples have relatively ideal antioxidant efficacy. This indicates that the product preparation method and the fermentation strain have a certain impact on the antioxidant effect.

[0115] Effect Example 4: Skin Fibroblast Toxicity Experiment

[0116] This experiment uses human skin fibroblasts from the Chinese Science Cell Bank to verify the cytotoxicity of the products prepared in the above examples and comparative examples.

[0117] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; CCK-8 was produced by Beijing Biode Biotechnology Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Biode Biotechnology Co., Ltd.

[0118] Equipment: The manufacturer of the WJ-80A-Ⅱ CO2 constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of the Sunrise microplate reader is Decan Trading Co., Ltd.; the manufacturer of the TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; and the manufacturer of the NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0119] 1. Experimental steps:

[0120] The products prepared in the above examples and comparative examples were respectively prepared with serum-free DMEM culture medium to prepare test solutions of the experimental group with a volume percentage of 1%.

[0121] Human skin fibroblasts were cultured in a medium containing 10% fetal bovine serum and 1% double-antibody (1×10 5 The cells were grown in DMEM medium supplemented with 100 mg / L penicillin and 100 mg / L streptomycin. The cells were grown in a 37°C, 5% CO2, saturated humidity incubator. When cell confluence reached 85% or higher, the logarithmic growth phase cells were digested with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. The cells were counted using a cell counter and the cell suspension concentration was adjusted to 7 × 10 4 / mL, the cell suspension was inoculated into a 96-well plate at a ratio of 100μL per well, and incubated at 37℃, 5% CO2 for 12h. Remove the old culture medium and wash the cells twice with phosphate buffer. In the experimental group, 100μL of the above-mentioned filtered and sterilized experimental group test solution of different concentrations was added to each well, and 3 replicates were made for each test solution; the control group contained cells and serum-free DMEM culture medium was added; the blank control group had no cells and 100μL of PBS was added. Then incubate at 37℃, 5% CO2 for 24h. Then 10μL of CCK-8 solution was added to each well, and incubated for another 3h. The absorbance value was measured at a wavelength of 450nm, and the cell survival rate of each group was calculated. The results are shown in Tables 5 and Figure 4 .

[0122] The formula for calculating cell viability is as follows:

[0123] Cell survival rate (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0124] Table 5

[0125] serial number Cell survival rate (%) control group 100±4.19 Example 1 114.59±3.69 Example 2 105.56±2.43 Example 3 107.75±0.94 Comparative Example 1 87.41±2.07 Comparative Example 2 94.1±2.37 Comparative Example 3 96.56±3.03 Comparative Example 4 95.13±3.28

[0126] The results showed that the cell survival rates of cells treated with the products prepared in Examples 1 to 3 were significantly higher than those of cells treated with the products prepared in Comparative Examples 1 to 4. This indicates that the products prepared in the examples of this application are relatively safe and have a certain cell growth-promoting effect. It can be seen that the product preparation method and the fermentation strain have a certain impact on the cell proliferation effect.

[0127] Effect Example 5: Determination of Type I Collagen Content

[0128] This experiment verifies the ability of the products prepared in the above examples and comparative examples to repair damaged skin by measuring the COL-I content in human skin fibroblasts.

[0129] Human skin fibroblasts in the logarithmic phase were taken and the cell suspension was inoculated into a 6-well cell culture plate at a density of 250,000 cells / mL. 2 mL of cell suspension was added to each well and cultured for 12 hours. Experimental group, blank group and model group were set up. Among them, the experimental group and model group were treated with 18mJ / cm 2 The cells were irradiated with UVA for 40 minutes. After irradiation, the supernatant in the experimental group, blank group and model group was discarded by aspiration, 2 mL of DMEM solution was added to the blank group and model group, and 2 mL of the test solution was added to the experimental group (the product prepared in the above embodiment or comparative example was configured into a test solution with a volume percentage of 1% using DMEM, and the test solution needed to be filtered through a 0.22 μm sterile filter membrane before use). The cells were added and treated for 24 hours, the supernatant was discarded, and the cells were washed 2 to 3 times with PBS. The cells were then treated with cell lysis solution and transferred to a centrifuge tube. The cells were centrifuged at 10,000 r / min and 4°C for 10 minutes, and the supernatant was taken to obtain cell lysate. 20 μL of cell lysate was taken to detect the total protein content in the sample using a BCA kit; the determination of collagen content was carried out according to the instructions of the ELISA kit, and the OD values were measured at 450 nm. The standard curve equation was, and the collagen content x was calculated according to the standard curve. The results are shown in Tables 6 and Figure 5 ( Figure 5 middle, ### p<0.001, indicating an extremely significant decrease compared with the blank group; p>0.05, ns, indicating no significant increase compared with the model group; *p<0.05, indicating an increase compared with the model group; **p<0.01, indicating a significant increase compared with the model group; ***p<0.001, indicating an extremely significant increase compared with the model group).

[0130] Table 6

[0131] serial number COL-I content (ng / mL) Blank group 7.585±0.066 Model Group 2.014±0.955 Example 1 6.515±0.24 Example 2 5.945±0.092 Example 3 5.843±0.134 Comparative Example 1 3.056±0.071 Comparative Example 2 4.677±0.159 Comparative Example 3 4.86±0.369 Comparative Example 4 4.657±0.245

[0132] The results showed that the COL-I levels in cells treated with the products prepared in Examples 1-3 were significantly higher than those in cells treated with the products prepared in Comparative Examples 1-4. This indicates that the products prepared in these examples have an ideal ability to promote the repair of damaged skin. This indicates that the product preparation method and the fermented bacterial strain have a certain impact on the repair effect on damaged skin.

[0133] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0134] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art may design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached solutions. Such modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A method for preparing a fermentation liquid of Armillaria chrysogenum with anti-aging effect, characterized in that: The method specifically comprises the following steps: inoculating lactobacillus into a fermentation substrate, culturing the substrate through fermentation, and sterilizing the substrate; the fermentation substrate comprises a yellow-green Armillaria fruiting body and water; The lactobacillus is composed of Lactobacillus helveticus and Lactobacillus paracasei, and the ratio of the number of live bacteria of the Lactobacillus helveticus to the Lactobacillus paracasei is 1:(0.5-2).

2. The method for preparing the fermentation liquid of Armillaria chrysogenum with anti-aging effect according to claim 1, wherein: It meets at least one of the following conditions: The mass ratio of the Armillaria lutea fruiting body to the water is 1:(10-300), preferably 1:(30-200), and more preferably 1:150; The fermentation substrate further comprises crushing and sieving the fruiting bodies of Armillaria luteola to obtain Armillaria luteola fruiting body powder; Preferably, the sieved particle size is 50-200 mesh, more preferably 50-150 mesh; The fermentation substrate also includes a carbon source and / or a nitrogen source; Preferably, the carbon source includes glucose; Preferably, the nitrogen source comprises soybean peptide; Preferably, when the carbon source is added, the mass ratio of the carbon source to the water is 1:(100-1000); Preferably, when the nitrogen source is added, the mass ratio of the nitrogen source to the water is 1:(100-1000), preferably 1:(200-500); The method for preparing the fermentation substrate comprises: mixing the components in the fermentation substrate until the components are completely dissolved.

3. The method for preparing the fermentation liquid of Armillaria chrysogenum with anti-aging effect according to claim 1, wherein: The sterilization method is high temperature sterilization; When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110° C. to 125° C., preferably 115° C. to 121° C., for example, 121° C.; When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.14 MPa, preferably 0.1-0.13 MPa, for example 0.13 MPa; When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15 to 35 minutes, preferably 20 to 30 minutes, for example, 30 minutes.

4. The method for preparing the fermentation liquid of Armillaria chrysogenum with anti-aging effect according to claim 1, wherein: It meets at least one of the following conditions: The Lactobacillus helveticus includes Lactobacillus helveticus purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC 20243; The Lactobacillus paracasei includes Lactobacillus paracasei purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC 20241; The preparation method of the lactic acid bacteria liquid comprises the following steps: dissolving the lactic acid bacteria in sterile deionized water; The Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial liquid, wherein the live bacteria concentration of the Lactobacillus helveticus in the Lactobacillus helveticus bacterial liquid is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL, for example 10 10 CFU / mL; The Lactobacillus paracasei is added in the form of a Lactobacillus paracasei bacterial liquid, wherein the viable bacteria concentration of the Lactobacillus paracasei in the Lactobacillus paracasei bacterial liquid is 10 7 ~10 13 CFU / mL, preferably 10 8 ~10 11 CFU / mL, more preferably 10 10 CFU / mL.

5. The method for preparing the fermentation liquid of Armillaria chrysogenum with anti-aging effect according to claim 1, wherein: It meets at least one of the following conditions: The fermentation culture is static fermentation; The fermentation temperature is 30-45°C, preferably 35-40°C; The fermentation culture time is 12 to 48 hours, preferably 12 to 24 hours; The sterilization method is a high-temperature sterilization method. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110°C to 125°C, preferably 115°C to 121°C, for example, 121°C; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1 to 0.14 MPa, preferably 0.1 to 0.13 MPa, for example, 0.13 MPa; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15 to 35 minutes, preferably 20 to 30 minutes, for example, 30 minutes.

6. The method for preparing the fermentation liquid of Armillaria chrysogenum with anti-aging effect according to claim 1, wherein: After the sterilization operation, the method further includes at least one of centrifuging and collecting the supernatant, secondary sterilization, and mixing with a preservative.

7. The method for preparing the fermentation liquid of Armillaria chrysogenum with anti-aging effect according to claim 6, wherein: It meets at least one of the following conditions: The centrifugal speed is 3000-9000 rpm, preferably 4000-6000 rpm, for example 4800 rpm; The centrifugation time is 10 to 40 minutes, preferably 20 to 40 minutes, for example 30 minutes; The radius of the centrifuge is 8 to 15 cm; The secondary sterilization method is a high-temperature sterilization method. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110° C. to 125° C., preferably 115° C. to 121° C., for example, 121° C.; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1 to 0.14 MPa, preferably 0.1 to 0.13 MPa, for example, 0.13 MPa; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15 to 35 minutes, preferably 20 to 30 minutes, for example, 30 minutes; During the mixing process with the preservative, the mixing temperature is 50-80°C, preferably 70-80°C; In the process of mixing with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization is 0.1% to 0.8%, preferably 0.1% to 0.6%, for example 0.5%; when the preservative includes 1,2-hexanediol, the mass fraction of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.3% to 1.5%, preferably 0.5% to 1%, for example 0.5%.

8. A fermentation liquid of Armillaria lutea with anti-aging efficacy, which is prepared by the method for preparing the fermentation liquid of Armillaria lutea with anti-aging efficacy according to any one of claims 1 to 7.

9. A use of the fermentation liquid of Armillaria chrysogenum with anti-aging efficacy as claimed in claim 8 directly as a product, as an additive or as a base in the preparation of a skin external preparation; Preferably, the fermentation liquid of Armillaria lutea with anti-aging effect is used as the antioxidant active ingredient and anti-aging active ingredient in the skin external preparation; More preferably, the antioxidant active ingredient is an antioxidant active ingredient having a DPPH free radical scavenging effect or an antioxidant active ingredient having a hydroxyl free radical scavenging effect; More preferably, the anti-aging active ingredient is an anti-aging active ingredient that promotes collagen production in cells.

10. A skin external preparation comprising the fermentation liquid of Armillaria lutea with anti-aging efficacy as claimed in claim 8; Preferably, the skin external preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; Preferably, the skin external preparation includes a facial mask, essence or toner; Preferably, the mass percentage of the fermented liquid of Armillaria lutea with anti-aging effect in the external skin preparation is 5% to 99%, preferably 60% to 99%.

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