Fermented material of trametes versicolor and preparation method and application thereof

Abstract

The present application provides a kind of fermentation of Tricholoma mongolicum and Camellia nitidissima and its preparation method and application.The preparation method of the fermentation of Tricholoma mongolicum and Camellia nitidissima includes inoculating Tricholoma mongolicum liquid into fermentation substrate for fermentation culture, and then taking supernatant to obtain the fermentation of Tricholoma mongolicum and Camellia nitidissima.The fermentation substrate includes appropriately proportioned potato glucose liquid medium and Camellia nitidissima powder.The present application utilizes liquid fermentation of Tricholoma mongolicum and composition containing Camellia nitidissima powder with appropriate proportioning to achieve synergistic effect, especially selecting newly collected Tricholoma mongolicum strain with preservation number CGMCC No.23250, and under suitable conditions, fermentation of composite substrate mainly composed of fresh potato homogenate or filtrate or potato glucose water, Camellia nitidissima powder, soybean peptide and appropriate amount of glucose, to obtain well-performing composite fermentation product, which has good effects of promoting cell proliferation and anti-inflammation, is safe and has no side effects for cosmetics, has good skin feel, has good repair effect, can enhance skin elasticity, and can also be absorbed for a long time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a fermentation product of Agaricus blazei and a preparation method and application thereof. BACKGROUND

[0002] Agaricus blazei Murill, also known as Agaricus blazei, is a kind of fungus belonging to Basidiomycota, Hymenomycetes, Agaricales, Agaricaceae and Agaricus. The fungus belongs to medium-high temperature mushroom, and the mycelium growth temperature is 10-33℃, the mycelium cell dies at 37℃, the optimum growth temperature is 22-27℃, the temperature range of fruiting body occurrence is about 20-33℃, and the optimum temperature is 22-25℃.

[0003] Agaricus blazei is a kind of precious fungus for food and medicine. The fungus has a crisp taste and a delicious almond flavor, is rich in nutrients, has high anticancer activity and can enhance the immune function of cells, reduce blood sugar and blood pressure, reduce cholesterol, prevent arteriosclerosis, improve osteoporosis, beautify, and calm the nerves. The Agaricus blazei polysaccharide extracted therefrom also has outstanding effects of anti-tumor, immune regulation, enhancement of bone marrow hematopoietic function, and bacteriostasis. Therefore, there are various deep processing products of Agaricus blazei. The research on Agaricus blazei polysaccharide is mainly concentrated in Japan. In China, Agaricus blazei was introduced from Japan by the Fujian Academy of Agricultural Sciences in 1992, and the research thereon has gradually deepened over the years. However, the market still expects more new strains of Agaricus blazei with excellent performance and their extracts and products.

[0004] Camellia petelotii (Merr.) Sealy is a woody plant of Theaceae, and is a unique tea flower variety in China, which is also known as "Queen of Tea", and is distributed in Guangxi, Guangdong and other places. Camellia petelotii is a traditional medicine of Guangxi Zhuang nationality, and is recorded in Guangxi Chinese Herbal Medicine Standard, Guangxi National Medicine Concise, Guangxi Medicinal Plant Garden Medicinal Plant Catalogue, Modern Compendium of Materia Medica, and Guangxi Medicinal Plant Catalogue. The traditional medicinal value of Camellia petelotii leaf is clearing heat and resolving toxicity, diuresis and detumescence, and is used for treating pharyngolaryngeal inflammation, nephritis, dysentery, edema, urinary tract infection, hypertension, and preventing tumors (water decoction or tea drinking), and the flower is used for treating hematochezia and irregular menstruation. The exploration of the application of Camellia petelotii extract in cosmetics mainly focuses on the water extract of Camellia petelotii flower and leaf, and the effect of the water extract of Camellia petelotii flower is to moisturize, and the effect of the water extract of Camellia petelotii leaf is to whiten. For example, see the following patent documents: CN201510266489.1 Application of Camellia petelotii leaf extract, whitening skin care composition and preparation method thereof, CN201510266465.6 Application of Camellia petelotii flower extract, moisturizing skin care composition and preparation method thereof, and CN201510266489.1 Application of Camellia petelotii leaf extract, whitening skin care composition and preparation method thereof. However, there are few reports on Camellia petelotii fermented extract.

[0005] The present applicant finds that the synergistic effect can be achieved by using the Tricholoma matsutake liquid fermentation of a composition containing Camellia petelotii powder (leaf) in a proper ratio, and the obtained fermented product has better performance, especially the fermented product obtained by using the Tricholoma matsutake strain JSR-1 collected and screened by the present applicant in Dali, Yunnan has more considerable application prospect in cosmetics. SUMMARY

[0006] In order to solve the above problems, the present application provides a Tricholoma matsutake Camellia petelotii fermented product and a preparation method and application thereof.

[0007] To solve the above technical problems, the technical scheme provided by the present application is as follows:

[0008] A preparation method of a Tricholoma matsutake Camellia petelotii fermented product, wherein the Tricholoma matsutake liquid is inoculated into a fermentation substrate to perform fermentation culture, and then the supernatant is obtained; the fermentation substrate comprises a proper ratio of potato dextrose liquid medium and Camellia petelotii powder. The Camellia petelotii powder in the present application refers to the powder material obtained by crushing the dried leaf of Camellia petelotii (Merr.) Sealy of Theaceae.

[0009] As a preferred embodiment, the preparation method of the fermentation product of the Camptotheca acuminata and the Camellia nitidissima, the fermentation substrate includes, based on 100 parts by weight of the water addition amount, 0.5-2 parts (such as 0.8 parts, 1 part, 1.2 parts, 1.5 parts, 1.8 parts, etc.) of glucose, 0-0.5 parts (such as 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.45 part, etc.) of soybean peptide, 0.5-5 parts (such as 0.8 part, 1 part, 2 parts, 3 parts, 4 parts, etc.) of fresh potatoes, and 0.1-3.0 parts (such as 0.1 part, 1.5 parts, 2 parts, 2.5 parts, etc.) of Camellia nitidissima powder. For example, the fermentation substrate can be prepared by adding 0.5%-2% glucose, 0-0.5% soybean peptide (DD), 0.5%-5% potatoes, and 0.1%-3.0% Camellia nitidissima powder into water, homogenizing, and sterilizing to obtain the fermentation substrate.

[0010] As a preferred embodiment, the preparation method of the fermentation product of the Camptotheca acuminata and the Camellia nitidissima specifically includes the following steps:

[0011] 1) Preparation of the Camptotheca acuminata liquid: inoculate the Camptotheca acuminata strain into a glucose potato agar culture medium for activation, inoculate the obtained single colony into a glucose potato liquid culture medium for expansion culture to obtain the Camptotheca acuminata liquid;

[0012] 2) Preparation of the fermentation substrate: add appropriate amounts of glucose, soybean peptide, fresh potatoes, and Camellia nitidissima powder into water in proportion, homogenize, sterilize to obtain the fermentation substrate;

[0013] 3) Fermentation culture: inoculate the Camptotheca acuminata liquid into the fermentation substrate, after the fermentation culture, take the supernatant to obtain the fermentation product of the Camptotheca acuminata and the Camellia nitidissima.

[0014] As a preferred embodiment, the preparation method of the fermentation product of the Camptotheca acuminata and the Camellia nitidissima, the Camptotheca acuminata strain is the Camptotheca acuminata strain JSR-1 with the preservation number of CGMCC No.23250. The applicant collected wild fungal fruiting bodies from Dali City (100.2305, 25.59238) in Yunnan Province, picked the tissue at the junction of the cap and the stem of the fungus, and then placed it on a slant medium with PDA rich medium, and cultured it in the dark at 25°C for 5 days to obtain mycelium. The mycelium was picked and purified twice to obtain a purified strain of the fungus, and the above-mentioned Camptotheca acuminata strain JSR-1 was obtained and submitted for preservation, with the preservation number of CGMCC No.23250.

[0015] As a preferred embodiment of the preparation method of the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation product, the preparation method of the Agaricus blazei Murrill liquid culture includes the following steps: inoculating the Agaricus blazei Murrill strain into a glucose potato agar medium to activate, and then inoculating the obtained mycelium into a glucose potato liquid medium to expand the culture to a state where a large number of mycelial pellets are dispersed, and obtaining the Agaricus blazei Murrill liquid culture when the liquid volume is about one third to one half of the total volume.

[0016] As a preferred embodiment of the preparation method of the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation product, the Agaricus blazei Murrill liquid culture is inoculated into the fermentation substrate at a proportion of 0.5-20%.

[0017] The preparation method of the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation product, the preparation method of the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation product, and the temperature of the fermentation culture is 20-30°C (such as 22°C, 25°C, 28°C, etc.), and the time is 3-10 days (such as 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, etc.).

[0018] An Agaricus blazei Murrill and Camellia sinensis fermentation product, which is the Agaricus blazei Murrill and Camellia sinensis fermentation liquid prepared according to the above-mentioned method.

[0019] An Agaricus blazei Murrill and Camellia sinensis fermentation product, which is the Agaricus blazei Murrill and Camellia sinensis fermentation freeze-dried powder prepared by freeze-drying the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation liquid.

[0020] Application of the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation product in the manufacture of cosmetics.

[0021] The above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation liquid and other raw materials are formulated into Agaricus blazei Murrill and Camellia sinensis fermentation skin care water, and the content of the above-mentioned Agaricus blazei Murrill and Camellia sinensis fermentation liquid is more than 50% (such as 55%, 60%, 70%, 80%, 90%, 95%, etc.).

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] 1. The application utilizes liquid fermentation of Agaricus blazei Murrill and a composition containing appropriate proportion of Camellia sinensis powder (leaves) to achieve synergistic effect, especially by selecting newly collected Agaricus blazei Murrill strain with preservation number of CGMCC No.23250, and under suitable conditions, fermenting a complex substrate mainly composed of fresh potato homogenate (0.5%-5%), or filtrate, or potato dextrose water (trade name), Camellia sinensis powder, soybean peptide (DD), and appropriate amount of glucose to prepare a complex fermentation product with good performance, which has good effects of promoting cell proliferation and anti-inflammation.

[0024] 2. The Agaricus blazei Murrill and Camellia sinensis fermentation product obtained by the method of the application is safe and has no side effects for cosmetics, has good skin feel, good repair effect, can enhance skin elasticity, and can be absorbed for a long time.

[0025] The new strain of Agaricus blazei of the present application is preserved on September 13, 2021, with a preservation number of CGMCC No.23250, and is classified and named as Agaricus blazei JSR-1, also known as Agaricus blazei JSR-1, and is preserved by the China General Microbiological Culture Collection Center (CGMCC) located at No.3, Beichen West Road, Haidian District, Beijing, China, with a postal code of 100101. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The in vitro free radical scavenging capacity determination results of the Agaricus blazei polysaccharide extracted from the strains No.21-25 in Example 2 are shown;

[0027] Figure 2 The detection results of intracellular proteins (FLG, AQP3) in the UVB damage protection experiment of Example 2 are shown;

[0028] Figure 3 The detection results of intracellular inflammatory factors (KLK7, IL-1β, TNF-α, IL-6, IL-8) in the UVB damage protection experiment of Example 2 are shown;

[0029] Figure 4 The detection results of intracellular inflammatory factors (KLK7, IL-1β, TNF-α, IL-6, IL-8) in the UVB damage repair experiment of Example 2 are shown;

[0030] Figure 5 The detection results of intracellular proteins (FLG, AQP3) in the UVB damage repair experiment of Example 2 are shown;

[0031] Figure 6 The detection results of Col-1, ELN, MMP-1, and GSH-Px in the UVA repair experiment of Example 2 are shown;

[0032] Figure 7 The cell proliferation and toxicity experiment results of the Camellia nitidissima fermentation broth prepared in Example 3 are shown;

[0033] Figure 8 The anti-inflammatory performance test results of the Camellia nitidissima fermentation broth prepared in Example 3 are shown;

[0034] Figure 9 The transdermal absorption performance test results of the four Camellia nitidissima fermentation broths prepared in Example 3 are shown;

[0035] Figure 10 The photos of the Agaricus blazei Camellia nitidissima fermentation skin care water prepared in Example 4 are shown;

[0036] Figure 11The results of sensory test evaluation of two skin care lotions prepared in Example 4 and SK II are shown;

[0037] Figure 12 The effect of the fermented skin care lotion of F. deliciosum and Camellia sinensis (Formula 1) prepared in Example 4 on the amount of skin trans-epidermal water loss (TEWL) is shown;

[0038] Figure 13 The effect of the fermented skin care lotion of F. deliciosum and Camellia sinensis (Formula 1) prepared in Example 4 on the skin elasticity index R2 is shown. DETAILED DESCRIPTION

[0039] The following examples are useful for better understanding of the present application, but do not limit the present application.

[0040] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0041] The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0042] Example 1, Isolation, Identification and Preservation of F. deliciosum JSR-1 (CGMCC No. 23250)

[0043] 1. Isolation of Strains

[0044] The applicant collected wild fungal fruiting bodies from Dali City, Yunnan Province (100.2305, 25.59238), picked tissues at the junction of the cap and stem of the fruiting bodies, and then placed them on slant medium with PDA enriched medium, and cultured at 25°C in the dark for 5 days to obtain mycelium. The mycelium was picked and purified twice to obtain the purified strain of the fungus JSR-1. The steps for each purification were as follows: the mycelium was placed on slant medium with PDA enriched medium, and cultured at 25°C in the dark for 5 days.

[0045] 2. Morphological Identification

[0046] The liquid mycelium was inoculated on PDA medium and cultured at 25°C for 3 days, after which the morphological characteristics of the mycelium were obvious. The mycelium initially grew densely and radially, and then concentrated into white spots as the mycelium grew forward. The mycelium was white on the back, branched, and curved.

[0047] 3. Molecular Phylogenetic Analysis

[0048] Genomic DNA was extracted using Fungal DNA Extraction Kit Type I (centrifugal column type) product number: D301-02. The ITS universal primer sequence was used as the primer for PCR reaction. The reaction program was as follows: 95℃ pre-denaturation for 1 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles, and 72℃ final extension for 5 min. The obtained PCR product was sequenced, and the sequencing results showed that the rDNA-ITS sequence of JSR-1 was homologous to the rDNA-ITS sequence publicly disclosed in the NCBI database. The results showed that the ITS sequence part of the measured JSR-1 had 86.2% homology with the ITS sequence of Agaricus sp. (Agaricus subrufescens strain). It was named Agaricus blazei JSR-1, also known as Agaricus blazei JSR-1.

[0049] 4. Preservation of the strain of Agaricus blazei JSR-1

[0050] JSR-1 was preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2021, and the preservation number was CGMCC No. 23250. The full name of JSR-1 is Agaricus blazei JSR-1 CGMCC No. 23250.

[0051] Example 2, comparison experiment of strain screening

[0052] Agaricus blazei (Agaricus blazei) No. 21-25 (of which No. 21 and No. 22 were collected from Dali City, Yunnan Province; No. 23 and No. 24 were ordinary Agaricus blazei (Agaricus blazei) strains purchased from the General Microbiological Culture Collection Center, with numbers GCMCC5.485 and GCMCC5.787 respectively; No. 25 was from Fujian Academy of Agricultural Sciences; No. 21 was the strain JSR-1), were activated on PDA plates, and single colonies were transferred to potato dextrose liquid medium for expansion culture. The culture conditions were 28℃ and 150 rpm for 5 days, and the fermentation broth was obtained. Subsequently, after the steps of concentration and freeze-drying, five kinds of fermentation freeze-dried powder were obtained and numbered 21-25. The five kinds of fermentation freeze-dried powder were tested and evaluated.

[0053] I. Determination of free radical scavenging capacity in vitro

[0054] 1. Determination of total antioxidant capacity (ABTS method).

[0055] ABTS working solution preparation: 5 mL of 7 mmol / L ABTS aqueous solution and 88 μL of 140 mmol / L potassium persulfate solution were mixed, and the ABTS·+ stock solution was formed by standing overnight at room temperature in the dark. Before use, dilute with anhydrous ethanol to form a working solution, and its absorbance at 734 nm is 0.70±0.02.

[0056] Sample determination: take 4 mL of ABTS working solution, add 40 μL of sample to be tested, shake accurately for 30 s, and measure the absorbance A at 734 nm after 6 min of reaction 样品 . The clearance rate is calculated according to the following formula: ABTS clearance rate / %=(1-A 样品 / 0.700)×100.

[0057] 2. Determine its ability to scavenge 1-diphenyl-2-trinitro radical (DPPH·).

[0058] The experimental method refers to the literature (Wu D, Liu P, Li M, Wang C, Zhao D, Zhang JC. Evaluation of the in vitro antioxidant and anti-aging effects of pueraria water extract and pueraria fermented liquid [J]. Food Industry Science and Technology, 2019, 40(12):285-290+294.), which is: prepare a DPPH· solution with a concentration of 4 mg / mL with anhydrous ethanol, add 2 mL of sample to 2 mL of DPPH· solution, mix well, stand for 30 min, centrifuge at 3000 r / min for 10 min, and measure the absorbance at 517 nm.

[0059] The ability of the test substance to scavenge DPPH· can be represented by the clearance rate, and the greater the clearance rate, the stronger the ability to scavenge. The formula is: clearance rate I=[I-(A i -A j ) / A0]×100%; where: A i -2 mL of DPPH· solution+2 mL of sample; A j -2 mL of DPPH· solution+2 mL of sample; A0-2 mL of DPPH· solution+2 mL of sample.

[0060] 3. Determine its ability to scavenge hydroxyl radicals (·OH).

[0061] The experimental method refers to the literature and is slightly modified (Zhao D, Xu D, Wang D, Zhang JC, Li M, Wang C. Evaluation of the composition of ganoderma lucidum fermentation broth and its whitening and anti-aging effects [J]. Daily chemical industry, 2016, 46(04): 226-230+242.). Specifically: add 6mmol / L FeSO4 solution 1mL to 1mL sample, mix well, then add 6mmol / L H2O2 solution 1mL, mix well, stand for 10min, add 6mmol / L salicylic acid solution 1mL, mix well, then react in 37℃ water bath for 30min, centrifuge at 3000r / min for 10min, take the supernatant to measure the absorbance at 510nm.

[0062] The results of free radical scavenging experiment are shown in Table 1 below and Figure 1 It can be seen that the free radical scavenging ability of the polysaccharide extracted from the No. 25 strain of Armillaria mellea is better.

[0063] Table 1 Free radical scavenging ability of five polysaccharides from Armillaria mellea

[0064]

[0065] II. Anti-inflammatory efficacy: UVB and UVA damage protection, damage repair performance test

[0066] In the damage protection performance test experiment, the blank control group (Control, no sample and no UVB), the model group (Model, no sample and UVB), the sample group (Samples, sample protection before UVB damage), and the positive control group (Positive control, potassium glycyrrhizinate, dose 1%) were set.

[0067] Select well-grown HaCaT cells, control the cell number to be 1.5x10 6 mL -1 , spread in a 6-well plate, 2mL per well, then incubate in a 37℃, 5% CO2 incubator overnight, add No. 21-25 Armillaria mellea polysaccharide sample for protection for 8h, select UVB (damaging dose) of 20mJ / cm 2 to irradiate HaCaT cells, and then add serum-free medium for culture for 24h after irradiation. Collect the cell supernatant, and lyse the adherent cells with lysis solution, then centrifuge and take the supernatant to store in a-20℃ refrigerator for detection of intracellular inflammatory factor expression and protein content.

[0068] In the damage repair performance test experiment, the blank control group (Control: no UVB / UVA and no sample), the model group (Model: UVB / UVA and no sample), the sample group (Samples: add sample for repair after UVB / UVA damage), and the positive control group (Positive control, potassium glycyrrhizinate, dose 1%) were set.

[0069] Select well-growing HaCaT cells and control the cell number to 1.5 x 10⁻⁶. 6 pcs·mL -1 Spread 2 mL of the solution into each well of a 6-well plate, and then incubate overnight at 37°C in a 5% CO2 incubator. Select a concentration of 20 mJ / cm². 2 HaCaT cells were irradiated with UVB (to induce damage first), followed by the addition of Agaricus blazei polysaccharide samples No. 21-25 for 24 hours of repair. Cell supernatant was collected, and adherent cells were lysed with lysis buffer. After centrifugation, the supernatant was stored at -20℃ for the detection of intracellular inflammatory factor expression and protein content.

[0070] 1. UVB damage protection function test

[0071] KLK7, IL-1β, IL-8, TNF-α, and IL-6 are all inflammatory factors; elevated levels indicate pro-inflammatory effects, while decreased levels indicate anti-inflammatory effects. The results of intracellular inflammatory factors (KLK7, IL-1β, IL-8, TNF-α, and IL-6) in the UVB damage protection model are shown in Table 2. Figure 3 It can be seen that the polysaccharides extracted from strains 21 and 25 have superior anti-inflammatory protective properties.

[0072] Table 2 compares the UVB damage protection results of five Agaricus blazei polysaccharides.

[0073]

[0074] Filaggrin (FLG) is an important molecule in the stratum corneum of human skin that connects keratin fibers. With the assistance of FLG monomers, keratin fibers aggregate regularly, forming a strong physical barrier on the outermost layer of the epidermis, preventing moisture loss and the invasion of external allergens. AQP3 is aquaporin 3, involved in water molecule transport; elevated levels indicate improved water entry into cells and are associated with moisturizing. Intracellular protein detection results in the UVB damage protection model are shown in Table 3. Figure 2 It can be seen that the Agaricus blazei polysaccharide extracted from strain 21 is superior.

[0075] Table 3 compares the UVB damage protection results of five Agaricus blazei polysaccharides.

[0076]

[0077] 2. UVB damage repair function test

[0078] The results of intracellular inflammatory factor detection in the UVB damage repair model are shown in Table 4 and... Figure 4It can be seen that the polysaccharide extracted from No. 21 Agaricus blazei has better UVB damage repair performance, and the polysaccharides extracted from No. 24 and No. 25 Agaricus blazei also have better UVB damage repair performance.

[0079] Table 4 Comparison of UVB damage repair effects of five Agaricus blazei polysaccharides

[0080]

[0081] The intracellular protein detection results of the UVB damage repair model are shown in Table 5 and Figure 5 It can be seen that the polysaccharides extracted from No. 21-25 Agaricus blazei have better performance.

[0082] Table 5 Comparison of UVB damage repair effects of five Agaricus blazei polysaccharides

[0083]

[0084]

[0085] III. Anti-aging effect: UVA damage protection performance test

[0086] In the UVA damage protection performance test experiment, a blank control group (Control, no sample and no UVA), a model group (Model, no sample and UVA), a sample group (Samples, sample protection before UVA damage), and a positive control group (Positive control, vitamin C, 0.100 mg / mL) were set.

[0087] Human skin fibroblasts (HSFs) were selected for good growth, and the cell number was controlled at 1.5x10 6 mL -1 , plated in a 6-well plate at 2 mL per well, then incubated in a 37℃, 5% CO2 incubator overnight, and then protected by No. 21-25 Agaricus blazei polysaccharide samples for 8h. The HSFs cells were irradiated with UVA (damage dose) at 7J / cm 2 , and then cultured in serum-free medium for 24h after irradiation. The cell supernatant was collected, and the adherent cells were lysed with lysis solution. After centrifugation, the supernatant was stored in a -20℃ refrigerator for detection of intracellular antioxidant enzymes and protein content.

[0088] Detection index: COL-I (collagen I, the higher the value, the better), ELN (elastin, the higher the value, the better), MMP-1 (matrix metalloproteinase 1, which acts to decompose collagen, the lower the value, the better), GSH-Px (glutathione peroxidase, an antioxidant enzyme, the higher the value, the better).

[0089] The intracellular protein detection results of the UVA damage protection model are shown in Table 6 andFigure 6 As can be seen, polysaccharides extracted from No. 21 Tricholoma matsutake have UVA damage protection performance.

[0090] Table 6 compares the UVA damage protection performance of five Tricholoma matsutake polysaccharides

[0091]

[0092] Example 3, Tricholoma matsutake fermentation experiment

[0093] A method for preparing a Tricholoma matsutake and Camellia sinensis fermentation product, comprising the following steps:

[0094] 1) Preparation of Tricholoma matsutake bacterial solution: inoculate Tricholoma matsutake strain JSR-1 with preservation number CGMCC No. 23250 into glucose potato agar medium for activation, then inoculate the obtained mycelium into glucose potato liquid medium for expansion culture until the mycelium balls are dispersed, and when the liquid volume is about one-third to one-half of the total volume after standing, obtain the Tricholoma matsutake bacterial solution for the next step of production;

[0095] 2) Preparation of fermentation substrate: according to the proportion, add 10 g of potato to 1 L of water and homogenize, then continue to add 5 g of Camellia sinensis powder, 20 g of glucose and 2 g of DD (formula: 2% glucose + 0.2% DD + 1% potato + 0.5% Camellia sinensis powder) to the homogenate, mix well, and sterilize to obtain the fermentation substrate.

[0096] 3) Fermentation culture: inoculate the Tricholoma matsutake bacterial solution obtained in step 1) into the fermentation substrate obtained in step 2) at a proportion of 3% for fermentation culture, the fermentation culture temperature is 28°C, the time is 5d, and the rotation speed is 150 rpm, after the end, take the supernatant, and obtain the Tricholoma matsutake and Camellia sinensis fermentation product. Part of it is made into freeze-dried powder for storage.

[0097] The substrate and potato culture medium without fermentation culture are used as controls, and the specific substrate formula and fermentation process are shown in Table 7.

[0098] Table 7 Substrate formula and fermentation process table of Tricholoma matsutake fermentation experiment

[0099]

[0100] The components in the fermentation product are shown in Table 8.

[0101] Table 8 Component detection results

[0102] Experimental group Total sugar (mg / ml) Polypeptide (100 ug / mL) pH Solid content Uninoculated (unfermented) control group 114.78 1.96 6.2 2.5％ JSR-1 alone 120.17 2.19 5.6 2.6％ Camellia sinensis fermentation broth 130.95 2.11 5.7 2.7％

[0103] Example 4, performance test

[0104] The product obtained in Example 3 is subjected to performance test.

[0105] I. Cell proliferation and toxicity experiment

[0106] HaCaT cells in good logarithmic growth phase were inoculated in 96-well culture plates at a density of 1.0 x 10 4 cells per well, and cultured overnight at 37°C in a 5% CO2 environment. Different concentrations of sample solutions were added, with 7-8 different concentrations for each sample, and six replicates for each concentration. The blank group was not treated, the model group and each sample group were first UVA irradiated (44 min, 4 J / cm 2 ), and the unseeded control sample, JSR-1 culture alone, and Camellia sinensis fermentation broth sample were added to the cells and cultured for 24 hours. 10 μL of CCK8 solution was added to each well, and after 2.5 hours of culture, the absorbance value (OD value) of each well was measured at 450 nm. Cell survival rate = (determination hole OD value-blank control OD value) / (cell control group OD value-blank control OD value) * 100%.

[0107] The results are shown in Figure 7 . The cell survival rate of the model group was significantly lower than that of the blank group, indicating that the UVA modeling was successful. The unseeded control sample had no effect on improving cell survival rate, and JSR-1 culture alone and Camellia sinensis fermentation broth could both promote the proliferation of damaged HaCaT cells, with Camellia sinensis fermentation broth having a better proliferation effect than JSR-1 culture alone.

[0108] II. Anti-inflammatory performance test

[0109] Logarithmic phase HaCaT cells were taken, and the cell suspension was inoculated in a 6-well cell culture plate at a density of 1 x 10 5 cells / mL, 2 mL of cell suspension was added to each well, and the incubator was cultured for 24 hours, and the supernatant was carefully aspirated. The blank group was added with 2 mL of DMEM solution; the experimental group was added with 2 mL of DMEM solution containing samples (JSR-1 and Camellia sinensis fermentation broth freeze-dried powder, which were freeze-dried powder of JSR-1 and Camellia sinensis fermentation broth cultured alone after freeze-drying; Camellia sinensis extract was a finished product purchased from Xi'an Yousuo Biological Technology Co., Ltd.); the positive control group was added with 2 mL of DMEM solution containing glycyrrhizic acid dipotassium (50 μg / mL), and was carefully washed twice with PBS, 1 mL of PBS was added, and was placed under UVA irradiation (44 min, 4 J / cm 2). Each group of samples was repeated 3 times in parallel. The culture medium containing the sample or positive drug was continuously added and incubated for 24 h. The cells were treated with cell lysate, centrifuged at 10,000 r / min at 4°C for 10 min, and the supernatant was taken to obtain the cell lysate supernatant. 20 μL of the cell lysate was used to detect the total protein content in the sample by BCA kit; 20 μL of the cell lysate was used to detect the total protein content in the sample by BCA kit; after the experimental operation according to the ELISA kit instruction, the release level of inflammatory factor IL-8 was calculated. The release amount of inflammatory factor needs BCA content correction.

[0110] Results are shown in Figure 8 It can be seen that the IL-8 of the model group is significantly higher than that of the Control, indicating that the modeling is successful. The positive control dipotassium glycyrrhizinate, JSR-1, Camellia nitidissima fermented broth freeze-dried powder, and Camellia nitidissima extract all help to reduce the inflammatory factor IL-8 of the damaged HaCaT cells. And the Camellia nitidissima fermented broth has a more significant effect than the positive control dipotassium glycyrrhizinate in reducing IL-8; compared with JSR-1, the Camellia nitidissima fermented broth freeze-dried powder has a significant decrease, indicating that it has a better anti-inflammatory effect than JSR-1; and it also has a better anti-inflammatory effect than the Camellia nitidissima extract.

[0111] III. Transdermal absorption experiment

[0112] Experimental process: Add appropriate amount of water to the in vitro permeation and diffusion device constant temperature tank. Turn on the power and magnetic stirring of the constant temperature tank, and set the water temperature in the constant temperature tank to 37±0.1℃. The prepared rat skin is fixed between the two chambers of the diffusion cell with iron clips, 5 mL of receiving liquid is added to the receiving pool of the vertical diffusion pool, and it is preheated in the in vitro permeation and diffusion device constant temperature tank, and the stirring speed of the receiving pool is set to 400 r / min. Add the supply liquid to the supply pool, and seal the upper opening with plastic wrap. Start timing, and when the sample permeates 0, 2, 6, and 8 hours, take 500 μl of sample into a plugged centrifuge tube, and supplement an equal amount of receiving liquid to the receiving pool and remove the air bubbles in the pool at the same time each time. The total sugar content of the sample is determined. According to the ratio of the total sugar content of the receiving chamber to the total sugar of the original solution, the permeation rate is recorded, and the permeation rate-time curve is drawn. The receiving liquid is 0.9% NaCl solution (normal saline); the supply liquid is the sample to be detected, which is the Camellia nitidissima fermented broth prepared in Example 3 (this experiment is recorded as Camellia nitidissima fermented broth 1), and the samples obtained by fermenting the same proportion of substrate inoculated with strain No. 22, Ganoderma lucidum strain wG055 (preservation number CGMCC No. 17789), and Schizophyllum commune strain YSC1 (preservation number CGMCC No. 17788) are Camellia nitidissima fermented broth 2, Camellia nitidissima fermented broth 3, and Camellia nitidissima fermented broth 4.

[0113] The state of the fermented broth and the transdermal absorption amount are shown in Table 9, Figure 9 .

[0114] Transdermal absorption results

[0115]

[0116] From Figure 9 As can be seen from Table 9, the permeation rate of the fermented liquid 1 of Camellia nitidissima was only 2.60 mg / cm 2 at 2 h, but as the transdermal test time was prolonged, it reached 5.45 mg / cm 2 at 4 h and 7.27 mg / cm 2 at 8 h. This means that the fermented liquid 1 of Camellia nitidissima has a better cumulative permeation rate and is suitable for long-acting nutrient cosmetics.

[0117] IV. Hyaluronidase inhibition rate experiment

[0118] The fermented liquid 1, the fermented liquid 2, the fermented liquid 3 and the fermented liquid 4 of Camellia nitidissima described above were used as research objects, 1% of potassium glycyrrhizinate was used as a positive control and distilled water was used as a negative control. The experiment was carried out according to the experimental procedure of Table 10 in turn, and the hyaluronidase inhibition rate was calculated according to the following formula. The experimental results are shown in Table 11.

[0119] Table 10 Hyaluronidase inhibition test method

[0120]

[0121] Hyaluronidase inhibition rate (%) = (C-D)-(A-B) / (C-D) x 100%

[0122] In the formula, A is the OD value of the sample solution of (hyaluronidase + sample + sodium hyaluronate); B is the OD value of the sample blank of (acetic acid buffer + sample + acetic acid buffer); C is the OD value of the control solution of (hyaluronidase + deionized water + sodium hyaluronate); and D is the OD value of the control blank of (acetic acid buffer + deionized water + acetic acid buffer).

[0123] Table 11 Hyaluronidase inhibition experiment results

[0124] Sample Strain Hyaluronidase inhibition ability Glycyrrhizic acid dipotassium (1%) —— 82.88％ Positive control Distilled water 1.24％ Negative control Camellia sinensis fermentation broth 1 JSR-1 76.18％ Camellia sinensis fermentation broth 2 No. 22 56.30％ Camellia sinensis fermentation broth 3 Ganoderma lucidum 70.59％ Camellia sinensis fermentation broth 4 Schizophyllum 55.72％

[0125] As can be seen from Table 11, the inhibition ability of the fermented liquid 1 of Camellia nitidissima can reach 76.18%, followed by the fermented liquid 3 of Camellia nitidissima. The inhibition ability of the fermented liquid 2 of Camellia nitidissima obtained from another strain No. 22 of Agaricus blazei is only 56.30%, and the inhibition ability of the fermented liquid 4 of Camellia nitidissima obtained from fermentation of Schizophyllum commune is only 55.72%.

[0126] Example 5, Preparation of skin care water of Agaricus blazei Camellia nitidissima fermentation

[0127] The skin care water was prepared by using the Camellia ptilophylla fermentation broth prepared in Example 3, and the ingredient ratio was shown in Table 12. The operation steps were as follows:

[0128] 1) In a weighed beaker, butanediol, glycerol and transparent xanthan gum were weighed in sequence, and after stirring and dispersing uniformly, the Camellia ptilophylla fermentation broth was added. Stirring and heating to 80-85℃. Stirring for 10-15 minutes, 35-40 revolutions per minute, uniform and transparent, and then stirring and cooling;

[0129] 2) Cooling to 55-60℃, and then adding weighed trehalose, α-mannan, oat β-glucan and EDTA-2Na in sequence, stirring and dissolving until uniform and transparent;

[0130] 3) Stirring and cooling, and at 45℃, adding preservatives and solubilized (solubilizer HPS) fragrances, respectively, stirring and dissolving until transparent;

[0131] 4) After stirring and cooling to room temperature, weighing, adding deionized water to make up the weight, and stirring uniformly.

[0132] Table 12 Formulation of skin care water

[0133]

[0134]

[0135] The product photos of the two obtained Camellia ptilophylla fermentation skin care water products are shown in Figure 10 .

[0136] Example 6, Performance test of Camellia ptilophylla fermentation skin care water

[0137] 1. Safety test

[0138] The skin closed patch test was carried out according to the “Cosmetic Safety Technical Specification (2015 Edition)”. The skin reaction was observed according to the standard in Table 13. The observation results were recorded. Table 14 shows the results of the human closed patch test. Through the test of 30 volunteers, it was found that the three formula samples did not appear positive reaction, indicating that the samples were relatively safe.

[0139] Table 13 Grading standard for skin reaction in skin closed patch test

[0140]

[0141] Table 14 Results of skin closed patch test

[0142]

[0143]

[0144] 2. Sensory test

[0145] Sensory test was conducted on the three products to analyze the differences in sensory properties of the water-based products during use. A commercially available competitive product (SK II water) was used as a reference.

[0146] The analysis results are shown in Table 15, Figure 11 The attribute list is shown in Table 16. Overall, the three toners had obvious differences in sensory properties, but Formulation 2 had no difference from the competitive product. Among them, Formulation 1 had the thickest texture, the strongest stringiness, the thinnest application during use, the smoothest skin after use, the least amount of residue (immediately / 1 min / 2 min), less shiny (immediately / 1 min / 2 min), and faster overall absorption; Formulation 2 was more transparent, with the strongest water sensation, and the skin was shinier after use (immediately); the competitive product was less transparent and had the strongest oil sensation.

[0147] Table 15 Sensory evaluation data table (average value)

[0148]

[0149] Table 16 Attribute list explanation

[0150]

[0151]

[0152] 3. Human efficacy evaluation

[0153] Volunteer selection: 30 volunteers, according to the Helsinki Declaration, the selection of volunteers follows the medical and ethical standards of human testing, and all volunteers must voluntarily participate in the test, and sign the informed consent form before the test. Before signing the informed consent form, the test personnel need to inform the volunteers of the purpose of the test, the possible benefits, potential risks and problems, and the relevant rights and obligations.

[0154] Inclusion criteria: volunteers who meet all the following conditions will be included: 1) volunteers aged 30-45 years old, male and female; 2) skin barrier is fragile, the amount of trans-epidermal water loss TEWL below the cheek of the skin is >15 g / h / m 2 , the elasticity index R2 below the corner of the eye is <0.65; 3) the volunteer is in good health during the test; 4) no other reason considered unsuitable for the test by the clinician; 5) voluntarily participate and sign the informed consent form; 6) can complete the required content according to the test plan.

[0155] Exclusion criteria: those who meet any of the following will be excluded: 1) those who have serious systemic diseases, immune deficiency or autoimmune diseases; 2) those who have allergic diseases or have cosmetic allergies in the past 1-2 years; 3) women who plan to get pregnant or are pregnant or lactating and within six months after delivery; 4) those who are allergic to skin care products or topical drugs; 5) those who have used tretinoin locally or systemically in the test site within the past 8 weeks; 6) those who have used antibiotics, steroids or other drugs locally or systemically in the test site within the past 4 weeks; 7) those who participate in other tests at the test site now or in the past three months; 8) those who have dermatological diseases in the test area or are receiving drug treatment.

[0156] The test requirements are as follows: 1) Test environment: temperature 22±1℃; humidity 50%-60%. 2) Test area: facial skin cheek. 3) Test time points: baseline value, 14 days after use, 28 days after use. 4) Test indicators: trans-epidermal water loss (TEWL), R2.

[0157] Test results:

[0158] TEWL is an indicator of skin barrier, the larger the value, the more damaged the skin barrier; the lower the value, the better the skin barrier damage. See Figure 12 (p<0.05, indicating a significant difference compared with OD) can be seen that after use, the TEWL value increased from 18.01 g / h / m 2 to 16.34 g / h / m 2 (14D), 15.49 g / h / m 2 (28D), indicating that the sample (i.e. the fermented skin care water of Armillaria mellea and Camellia sinensis in Formula 1) has a repair effect.

[0159] R2 value is an elasticity indicator, the closer to 1, the better the elasticity. See Figure 13 (p<0.05, indicating a significant difference compared with OD) can be seen that after use of the sample, the R2 value increased from 0.60 to 0.64 (14D), 0.65 (28D), indicating that the skin elasticity is enhanced, and the sample (i.e. the fermented skin care water of Armillaria mellea and Camellia sinensis in Formula 1) has the effect of improving skin elasticity.

[0160] Comparative Examples 1-4

[0161] According to the process of Reference Example 3, the inventors also prepared cucumber fermentation broth, bitter gourd fermentation broth, rose hip fermentation broth, and mint fermentation broth.

[0162] Cell proliferation and toxicity experiments were performed according to the method in the first part of Reference Example 4. The results are shown in Table 17, which shows that compared with other fermentation substrates, the Camellia sinensis fermentation broth prepared in Example 3 has a better effect of promoting the proliferation of damaged HaCaT cells.

[0163] Table 17 Fermentation broth preparation process and performance test results of Comparative Examples 1-4

[0164]

[0165]

[0166] While the present application has been disclosed by the description of specific embodiments thereof, it is understood that modifications, improvements or equivalents thereof can be resorted to by those skilled in the art within the spirit and scope of the following claims. Such modifications, improvements or equivalents are also to be considered to be included within the scope of the present application.

Claims

1. A method for preparing a fermented material of Agaricus blazei Murrill Camellia sinensis, characterized by, Agaricus blazei ( Agaricus blazei The supernatant is obtained after inoculating the Murill bacterial culture into the fermentation substrate and fermenting. The fermentation substrate includes an appropriate ratio of potato glucose liquid culture medium and Camellia chrysantha powder. The fermentation substrate, based on 100 parts by weight of water, includes 0.5-2 parts of glucose, 0-0.5 parts of soybean peptide, 0.5-5 parts of fresh potato, and 0.1-3.0 parts of Camellia chrysantha powder. The Agaricus blazei strain used in the Agaricus blazei liquid has the preservation number of CGMCC No. 23250, and the Agaricus blazei liquid is inoculated into the fermentation substrate at a proportion of 0.5-20%. The fermentation culture temperature is 20-30℃, and the time is 3-10d.

2. The preparation method of the Agaricus blazei tea ferment according to claim 1, characterized in that, Specifically comprising the following steps: 1) Preparation of Agaricus blazei liquid: inoculate the Agaricus blazei strain into glucose potato agar medium for activation, then inoculate the obtained single colony into glucose potato liquid medium for expansion culture to obtain the Agaricus blazei liquid; 2) Preparation of fermentation substrate: add appropriate amount of glucose, soybean peptide, fresh potato and camellia sinensis powder into water in proportion, homogenize and sterilize to obtain the fermentation substrate; 3) Fermentation culture: inoculate the Agaricus blazei liquid into the fermentation substrate, and after the fermentation culture, take the supernatant to obtain the Agaricus blazei camellia sinensis fermentation product.

3. The preparation method of the Agaricus blazei tea ferment according to claim 1 or 2, characterized in that, The preparation method of the Agaricus blazei liquid comprises: inoculating the Agaricus blazei strain into glucose potato agar medium for activation, then inoculating the obtained colony into glucose potato liquid medium for expansion culture until the mycelial pellets are more dispersed, and after standing for one-third to one-half of the total volume of the liquid, the Agaricus blazei liquid is obtained.

4. A fermented material of Agaricus blazei Murill and Camellia sinensis, characterized in that, The Agaricus blazei camellia sinensis fermentation product is the Agaricus blazei camellia sinensis fermentation supernatant prepared by the method according to any one of claims 1-3 or the Agaricus blazei camellia sinensis fermentation freeze-dried powder prepared by freeze-drying the Agaricus blazei camellia sinensis fermentation supernatant.

5. Use of the Agaricus blazei camellia sinensis fermentation product according to claim 4 in the manufacture of cosmetics.

6. Use according to claim 5, characterized in that, The Agaricus blazei camellia sinensis fermentation supernatant according to claim 4 and other raw materials are formulated into Agaricus blazei camellia sinensis fermentation skin care water, and the Agaricus blazei camellia sinensis fermentation skin care water comprises the Agaricus blazei camellia sinensis fermentation supernatant at 50wt% or more.

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