Schizophyllum, bacterial agent containing the same, oat bran fermentation liquor, preparation method and application thereof

Abstract

This application discloses a *Schizophyllum commune* strain, its inoculum preparation, an oat bran fermentation broth, its preparation method, and its applications. *Schizophyllum commune* Wbsh-01 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with a deposit date of March 9, 2023, and accession number CGMCC No. 40388. This application screens a *Schizophyllum commune* strain and uses this strain in conjunction with a specific fermentation process to fully extract the active ingredients from oat bran. This not only reduces the loss of active ingredients but also better enhances the skin-care effects of oat bran. Compared with existing *Schizophyllum commune* strains, the extract has more ideal cosmetic effects. The extraction process does not require the addition of organic solvents, achieving energy conservation and environmental protection, and effectively avoiding organic solvent residues, thus improving product safety.

Description

Technical Field

[0001] This application belongs to the field of fermentation, specifically relating to a type of Schizophyllum commune, an inoculum containing it, an oat bran fermentation broth, and their preparation methods and applications. Background Technology

[0002] With the development of the times, people advocate green, environmentally friendly, and safe cosmetics, while also placing high hopes on their efficacy. Currently, while complying with relevant laws and regulations, the cosmetic industry increasingly uses chemically synthesized raw materials. Although these materials have advantages such as clearly defined component structures, definite efficacy, and mature quality control methods, people have gradually discovered that chemically synthesized products can cause adverse effects on the human body, such as sensitization, redness, and irritation. Therefore, reducing the adverse reactions caused by chemically synthesized products has become one of the directions for the development of cosmetics.

[0003] Oat bran is a byproduct of oat flour processing. It mainly consists of the outermost layer of hulled oats and part of the endosperm. It is rich in protein and functional components such as beta-glucan. These functional components have been proven to play an important role in anti-inflammation and anti-oxidation. Oat bran has a mild effect and high safety, making it a good plant-based raw material for skin care products.

[0004] Existing technologies generally use water extraction or organic solvent extraction to obtain active ingredients from oat bran, but the cosmetic effects of the extracts are not ideal. On the one hand, the extraction temperature is generally high, which can easily lead to the inactivation of active ingredients; on the other hand, when using organic solvent extraction, organic solvent residues are easily left, reducing the safety of use.

[0005] Therefore, there is an urgent need in this field to develop an extraction method that is simple to operate, reduces the use of organic solvents, can fully utilize the active ingredients in oat bran, and ensures that the obtained oat bran extract has excellent cosmetic effects. Summary of the Invention

[0006] The technical problem this application aims to solve is to overcome the limitations of existing oat bran extraction methods in terms of active ingredient extraction capacity and the tendency for organic solvent residues to result from organic solvent extraction. This application provides *Schizophyllum commune*, its inoculum, oat bran fermentation broth, its preparation method, and its applications. This application successfully screened a specific *Schizophyllum commune* strain, and using this strain in conjunction with a specific fermentation process can fully extract the active ingredients from oat bran. This not only reduces the loss of active ingredients but also better leverages the skin-care effects of oat bran. Compared to existing *Schizophyllum commune* methods, the extract has more ideal cosmetic effects. The extraction process requires no additional organic solvents, truly achieving energy conservation and environmental protection, and effectively avoiding organic solvent residues in the extract, thus improving product safety. The oat bran fermentation broth prepared by this invention can be used directly as a cosmetic or added to basic cosmetic formulations, exhibiting high safety and good anti-allergic, antioxidant, anti-inflammatory, immediate soothing, and nourishing effects on the skin.

[0007] This application adopts the following technical solution to solve the above-mentioned technical problems:

[0008] This application provides a species of Schizophyllum commune, Wbsh-01, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit date is March 9, 2023, and the accession number is CGMCC No. 40388.

[0009] This application also provides a microbial agent, which includes Schizophyllum commune Wbsh-01 as described above.

[0010] In some embodiments, the viable concentration of *Schizophyllum commune* Wbsh-01 in the bacterial agent may be 10⁻⁶. 6 ~10 9 CFU / mL, preferably 10 7 ~10 9 CFU / mL.

[0011] This application also provides a method for preparing the microbial agent as described above, which includes the following steps:

[0012] A) Culture in petri dishes: The above-mentioned Schizophyllum commune Wbsh-01 was inoculated onto a solid culture medium under aseptic conditions, and after culture, single colonies were obtained;

[0013] B) The single colony is picked up with an inoculation loop and placed in a liquid culture medium, and then cultured to obtain the bacterial agent.

[0014] In step A of some embodiments, the solid culture medium may include PDA solid culture medium conventionally used in the art. According to conventional art practice, the PDA solid culture medium comprises 6 g potato, 20 g glucose, 15–20 g agar, and 1000 mL distilled water.

[0015] In step A of some embodiments, the culture temperature may be 20-30°C, preferably 25-30°C, for example 28°C.

[0016] In step A of some embodiments, the culture time may be 30-50 hours, preferably 45-50 hours, for example 48 hours.

[0017] In step A of some embodiments, the culture may be carried out under static conditions in accordance with the art’s conventions.

[0018] In step B of some embodiments, the liquid culture medium may include PDA liquid culture medium conventionally used in the art. According to conventional art practice, the PDA liquid culture medium comprises 6 g potato, 20 g glucose, and 1000 mL distilled water.

[0019] In step B of some embodiments, the culture temperature may be 20–30°C, preferably 25–30°C, for example 28°C.

[0020] In step B of some embodiments, the culture time may be 30-50 hours, preferably 45-50 hours, for example 48 hours.

[0021] In step B of some embodiments, the culture can be carried out under oscillation conditions as is customary in the art, and the oscillation speed can be 150-200 rpm, preferably 175-185 rpm, for example 180 rpm.

[0022] This application also provides the use of the above-mentioned Schizophyllum commune Wbsh-01 as a fermentation strain in the preparation of topical skin agents.

[0023] This application also provides a method for preparing oat bran fermentation broth, which includes the following steps:

[0024] (1) Inoculate the above-mentioned Schizophyllum commune Wbsh-01 and / or the above-mentioned inoculum into fermentation substrate A, culture it in solid fermentation for 4 to 12 days, and sterilize it once to obtain material A; the fermentation substrate A includes oat bran powder and water;

[0025] (2) The above-mentioned Schizophyllum commune Wbsh-01 and / or the above-mentioned inoculum are inoculated into fermentation substrate B, and the mixture is cultured by liquid fermentation and sterilized twice to obtain the oat bran fermentation broth; the fermentation substrate B includes the material A and water.

[0026] In step (1) of some embodiments, the particle size of the oat bran powder can be conventional in the art, generally 100-300 mesh, preferably 150-250 mesh.

[0027] In step (1) of some embodiments, the water in the fermentation substrate A may include deionized water.

[0028] In step (1) of some embodiments, the mass ratio of the oat bran powder and the water in the fermentation substrate A may be 1:(0.5-2), preferably 1:(0.8-1.2), for example 1:1.

[0029] In step (1) of some embodiments, the fermentation substrate A may also be subjected to sterilization operations in accordance with conventional practices in the art before use.

[0030] The conditions and methods for sterilizing the fermentation substrate A can be the conventional conditions and methods for this type of operation in the art, and generally can be high-temperature sterilization.

[0031] When the fermentation substrate A is sterilized using the high-temperature sterilization method, the sterilization temperature can be a temperature conventional for this type of operation in the art, preferably 110-125°C, more preferably 115-125°C, for example 121°C.

[0032] When the fermentation substrate A is sterilized using the high-temperature sterilization method, the sterilization time can be the conventional time for this type of operation in the art, preferably 25-35 min, more preferably 25-30 min.

[0033] When the fermentation substrate A is sterilized using the high-temperature sterilization method, the sterilization pressure can be the pressure conventional for this type of operation in the art, preferably 0.1 to 0.13 MPa.

[0034] According to conventional practice in the art, after sterilizing the fermentation substrate A, a cooling operation may be further included, which is generally cooling to room temperature.

[0035] In step (1) of some embodiments, the number of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of the fermentation substrate A can be conventional in the art, preferably 10. 7 ~10 9 CFU / g, preferably 10 7 ~10 8 CFU / g. In accordance with conventional art, when inoculating the aforementioned microbial agent, the *Schizophyllum commune* Wbsh-01 inoculated refers to the *Schizophyllum commune* Wbsh-01 contained in the inoculated microbial agent.

[0036] In step (1) of some embodiments, the solid fermentation culture time is preferably 5 to 10 days, more preferably 5 to 7 days.

[0037] In step (1) of some embodiments, the temperature of the solid fermentation culture can be 25-30°C, preferably 25-28°C.

[0038] In step (1) of some embodiments, the humidity of the solid fermentation culture can be 50% to 95%, preferably 50% to 65%, for example 55%.

[0039] In some embodiments, step (1) of the solid fermentation culture can be carried out under static conditions in accordance with the art’s conventions.

[0040] In step (1) of some embodiments, the sterilization method can be the high-temperature sterilization method commonly used in the art.

[0041] When the high-temperature sterilization method is used for the first sterilization, the temperature of the first sterilization can be 110-125°C, preferably 115-125°C, for example 121°C.

[0042] When the high-temperature sterilization method is used for the first sterilization, the sterilization time can be 25 to 35 minutes, for example, 30 minutes.

[0043] When the high-temperature sterilization method is used for the first sterilization, the pressure of the first sterilization can be 0.1 to 0.13 MPa, for example, 0.12 MPa.

[0044] In some embodiments, step (1) may further include pulverization and sieving operations after the initial sterilization operation. The particle size of the material A after pulverization and sieving may be 100-300 mesh, preferably 150-250 mesh.

[0045] In step (2) of some embodiments, the mass ratio of material A to water in the fermentation substrate B may be 1:(20-200), preferably 1:(50-150), and more preferably 1:(60-100).

[0046] In step (2) of some embodiments, the water in the fermentation substrate B may include deionized water.

[0047] In step (2) of some embodiments, the fermentation substrate B may further include a carbon source and / or a nitrogen source.

[0048] The carbon source generally refers to a conventionally used carbon source in the art that is added in addition to material A, preferably including at least one of glucose, potato and corn.

[0049] The carbon source accounts for 0.05% to 1% of the mass of the fermentation substrate B, preferably 0.1% to 0.5%, for example 0.2%.

[0050] The nitrogen source generally refers to a nitrogen source conventionally used in the art that is added in addition to material A, preferably including at least one of soybean peptides, soybean meal powder and milk powder.

[0051] The nitrogen source accounts for 0.05% to 1% of the mass of the fermentation substrate B, preferably 0.1% to 0.5%, for example 0.2%.

[0052] In step (2) of some embodiments, the fermentation substrate B may also be sterilized in accordance with conventional practices in the art before use.

[0053] The conditions and methods for sterilizing the fermentation substrate B can be the conventional conditions and methods for this type of operation in the art, and generally can be high-temperature sterilization.

[0054] When the fermentation substrate B is sterilized using the high-temperature sterilization method, the sterilization temperature can be the temperature conventional for this type of operation in the art, preferably 100-125°C, and more preferably 115-121°C.

[0055] When the fermentation substrate B is sterilized using the high-temperature sterilization method, the sterilization time can be the conventional time for this type of operation in the art, preferably 15 to 35 minutes, more preferably 25 to 35 minutes, for example 30 minutes.

[0056] When the fermentation substrate B is sterilized using the high-temperature sterilization method, the sterilization pressure can be the pressure conventional for this type of operation in the art, preferably 0.1 to 0.13 MPa, for example 0.12 MPa.

[0057] According to conventional practice in the art, after sterilizing the fermentation substrate B, a cooling operation may be further included, which is generally cooling to room temperature.

[0058] In step (2) of some embodiments, the number of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of water, based on the water in the fermentation substrate B, can be conventional in the art, preferably 10. 4 ~10 9 CFU / g, preferably 10 5 ~10 8 CFU / g. In accordance with conventional art, when inoculating the aforementioned microbial agent, the *Schizophyllum commune* Wbsh-01 inoculated refers to the *Schizophyllum commune* Wbsh-01 contained in the inoculated microbial agent.

[0059] In step (2) of some embodiments, the liquid fermentation culture time can be 24 to 72 hours, preferably 30 to 50 hours, for example 48 hours.

[0060] In step (2) of some embodiments, the temperature of the liquid fermentation culture can be 25-35°C, preferably 25-30°C, for example 28°C.

[0061] In step (2) of some embodiments, the liquid fermentation culture can be carried out under shaking conditions, which is conventional in the art. The shaking speed can be 150-300 rpm, preferably 150-250 rpm, for example 180 rpm.

[0062] In step (2) of some embodiments, the secondary sterilization method can be the high-temperature sterilization method commonly used in the art.

[0063] When the high-temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization can be 110-125°C, preferably 115-125°C, for example 121°C.

[0064] When the high-temperature sterilization method is used for the secondary sterilization, the secondary sterilization time can be 15 to 35 minutes, preferably 15 to 25 minutes, for example 30 minutes.

[0065] When the high-temperature sterilization method is used for the secondary sterilization, the pressure of the secondary sterilization can be 0.1 to 0.13 MPa, for example, 0.12 MPa.

[0066] In some embodiments, step (2) may further include centrifugation, collection of supernatant, and tertiary sterilization, as is customary in the art, following the secondary sterilization operation.

[0067] The centrifugation speed can be 3000-9000 rpm, preferably 4000-6000 rpm, for example 4800 rpm.

[0068] The centrifugation time can be 10 to 40 minutes, preferably 20 to 40 minutes, for example 30 minutes.

[0069] The radius of the centrifuge can be 8 to 15 cm, preferably 10 to 13 cm, for example 12 cm.

[0070] The three sterilization methods mentioned above can be the high-temperature sterilization methods commonly used in the field.

[0071] When the high-temperature sterilization method is used for the three sterilization processes, the temperature of the three sterilization processes can be 110-125°C, preferably 115-121°C.

[0072] When the high-temperature sterilization method is used for the three sterilization processes, the sterilization time for the three processes can be 15 to 35 minutes, preferably 15 to 25 minutes, for example, 30 minutes.

[0073] When the high-temperature sterilization method is used for the three sterilization processes, the pressure of the three sterilization processes can be 0.1 to 0.13 MPa, for example, 0.12 MPa.

[0074] The process of sterilization may further include cooling to room temperature after the three sterilization operations.

[0075] This application also provides an oat bran fermentation liquid, which is prepared by the oat bran fermentation liquid preparation method described above.

[0076] This application also provides the use of the oat bran fermentation liquid as described above, either directly as a product, as an additive, or as a base in the preparation of topical skin agents.

[0077] In some embodiments, the oat bran fermentation liquid can be used as at least one of the antioxidant active ingredient, anti-allergic active ingredient, repair active ingredient, and anti-inflammatory active ingredient in the topical skin agent.

[0078] Preferably, the antioxidant active ingredient is an antioxidant active ingredient with hydroxyl radical scavenging activity.

[0079] Preferably, the anti-allergic active ingredient is an anti-allergic active ingredient with hyaluronidase inhibitory activity.

[0080] Preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient that can clear the inflammatory factor IL-8.

[0081] This application also provides a topical skin agent comprising the oat bran fermentation liquid as described above.

[0082] In some embodiments, the topical skin agent may further include active ingredients and / or preservatives commonly used in the art.

[0083] The active ingredients may include at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients, and antioxidant active ingredients.

[0084] The preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol.

[0085] When the preservative includes p-hydroxyacetophenone, the p-hydroxyacetophenone may account for 0.3% to 1% of the mass of the topical skin agent.

[0086] When the preservative includes 1,2-hexanediol, the 1,2-hexanediol may account for 0.3% to 1% of the mass of the topical skin agent.

[0087] In some embodiments, the topical skin agent may be, in accordance with the conventions of the art, including but not limited to, face masks, serums, or toners.

[0088] In some embodiments, the oat bran fermentation liquid accounts for 5% to 99% of the mass percentage of the topical skin agent, preferably 60% to 99%.

[0089] In some embodiments, the room temperature generally refers to 24–30°C.

[0090] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0091] All reagents and raw materials used in this application are commercially available.

[0092] The significant advancements of this application are as follows: This application successfully screened a specific strain of *Schizophyllum commune* and used this strain in conjunction with a specific fermentation process to extract active ingredients from oat bran. This not only reduces the loss of active ingredients but also better leverages the skin-care benefits of oat bran. Compared to existing *Schizophyllum commune* methods, the extract exhibits more ideal cosmetic efficacy. The extraction process requires no additional organic solvents, achieving energy conservation and environmental protection, and effectively avoiding organic solvent residues in the product, thus improving product safety. The oat bran fermentation liquid prepared by this invention can be used directly as a cosmetic or added to basic cosmetic formulations, exhibiting high safety and good anti-allergic, antioxidant, anti-inflammatory, immediate soothing, and nourishing effects on the skin. Attached Figure Description

[0093] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain its principles and advantages.

[0094] in:

[0095] Figure 1 This is a schematic diagram of the phylogenetic tree of the ITS sequence of strain Wbsh-01 in Example 1;

[0096] Figure 2 This is a comparison chart of the hydroxyl radical scavenging abilities of the products obtained in Examples 3-6 and Comparative Examples 1-8;

[0097] Figure 3 A comparison chart of the hyaluronidase inhibition rates of the products prepared in Examples 3-6 and Comparative Examples 1-8;

[0098] Figure 4 This is a comparison chart showing the cell viability after cells were treated with the products obtained in Examples 3-6 and Comparative Examples 1-8.

[0099] Figure 5 This is a comparison of the relative expression levels of the inflammatory cytokine IL-8 in cells after treatment with the products prepared using Examples 3-6 and Comparative Examples 1-8. Detailed Implementation

[0100] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0101] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0102] The Lactobacillus helveticus with accession number CICC 20243 used in the following comparative examples was purchased from the China Industrial Microbial Culture Collection Center.

[0103] The Saccharomyces cerevisiae strain CGMCC No. 17452 used in the following comparative examples was deposited on March 27, 2019, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 17452.

[0104] The Schizophyllum commune with accession number CICC 2592 used in the following comparative examples was purchased from the China Industrial Microbial Culture Collection Center.

[0105] In the following examples and comparative examples, the oat bran was purchased from Zhangjiakou Youya Food Co., Ltd.

[0106] In the following examples, the PDA-enriched culture medium was prepared as follows: 6g potato extract powder, 22g glucose, 20g agar and 2g soybean peptide were added to 1000mL of deionized water, mixed evenly, and sterilized in an autoclave at 115℃ for 20min.

[0107] In the following examples, the PDA solid culture medium raw material was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., product number HB0233. The preparation method of PDA solid culture medium is as follows: Take 46g of PDA solid culture medium raw material (including 6g potato extract powder, 20g glucose, and 20g agar), add it to 1000mL of deionized water, mix well, and sterilize in an autoclave at 115℃ for 20min.

[0108] In the following examples, the raw materials for PDA liquid culture medium were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., product number HB0233-4, product name potato glucose solution. The preparation method of PDA liquid culture medium is as follows: take 26g of potato glucose solution (including 6g of potato extract powder and 20g of glucose), add it to 1000mL of deionized water, mix well, and sterilize in an autoclave at 121℃ for 20min, and set aside for later use.

[0109] Example 1

[0110] 1. Isolation of Schizophyllum commune Wbsh-01

[0111] Wild fungal fruiting bodies were collected from dead trees in Yunnan Province. Tissue from the junction of the cap and stipe was selected and placed on a slant agar medium (PDA-enriched medium) and incubated in the dark at 28°C for 5 days to obtain mycelium. The mycelium was purified three times using an inoculation loop to obtain purified strains. The purification steps involved transferring the mycelium to a slant agar medium (PDA-enriched medium) and incubating in the dark at 28°C for 5 days.

[0112] 2. Morphological observation of Schizophyllum commune Wbsh-01

[0113] The mycelium is white, branched, and curved, with a diameter of 1–2 μm. The mycelium grows radially outward from the inoculation point, which is yellow. After 8 days of development, the mycelium intertwines and forms white protrusions on the substrate surface, which are the primordia of the fruiting bodies.

[0114] 3. Molecular biological identification of Schizophyllum commune Wbsh-01 (sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing)

[0115] Genomic DNA was extracted from Schizophyllum commune Wbsh-01 following the operating procedures of the fungal genomic DNA extraction kit (model D3390-02, manufacturer: OMEGA).

[0116] Using the genomic DNA of *Schizophyllum commune* Wbsh-01 as a template, PCR was performed using universal primers for fungal ribosomal rDNA regions: ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGAT ATGC). The PCR instrument was manufactured by Hangzhou Langji Scientific Instruments Co., Ltd., model MG96+. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, followed by cycling at 94℃ for 30 s, annealing at 54℃ for 30 s, and extension at 72℃ for 90 s, for a total of 25 cycles. The obtained PCR products were sequenced, and the obtained rDNA-ITS sequences were directly compared for homology with publicly available rDNA-ITS sequences in the NCBI Genbank database. The full-length ITS sequence was extracted using TS2Database (as shown in Sequence Listing 1) and analyzed using an ITS phylogenetic tree (see [link to phylogenetic analysis]). Figure 1 , Figure 1 The strain 521 is Schizophyllum commune Wbsh-01. The sequence similarity between strain Wbsh-01 and Schizophyllum commune MH539647.1 is 100%, indicating that the bacterium is Schizophyllum commune.

[0117] Based on the combined morphological identification results and DNA sequence analysis results, strain Wbsh-01 was identified as Schizohyllum commune, belonging to the phylum Basidiomycota, order Agaricales, and genus Schizophyllum.

[0118] 4. Preservation of Schizophyllum commune strain Wbsh-01

[0119] The new strain Wbsh-01 was deposited on March 9, 2023, with accession number CGMCC No. 40388, classified as Schizophyllum commune, and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0120] Example 2 Preparation of bacterial agent

[0121] Under aseptic conditions, *Schizophyllum commune* Wbsh-01 was inoculated onto PDA solid medium for streak activation and incubated statically at 28°C for 48 hours to obtain single colonies. Single colonies were then picked up using an inoculation loop and incubated in PDA liquid medium at 28°C and 180 rpm for 48 hours to prepare *Schizophyllum commune* Wbsh-01 inoculum. The viable cell concentration of *Schizophyllum commune* Wbsh-01 inoculum was 10⁻⁶. 9 CFU / mL.

[0122] Example 3: Preparation of Oat Bran Fermentation Broth

[0123] (1) Commercially available oat bran was pulverized and sieved to obtain oat bran powder with a particle size of 100 mesh. 20g of oat bran powder was mixed with 20g of deionized water, and the mixture was placed in a high-temperature sterilizer at 121℃ and 0.13MPa for 30min for sterilization. After sterilization, it was cooled to room temperature to obtain fermentation substrate A.

[0124] 4 mL of the *Schizophyllum commune* Wbsh-01 inoculum prepared in Example 2 above was inoculated into 40 g of fermentation substrate A. The viable count of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of fermentation substrate A was 10. 8 After inoculating with *Schizophyllum commune* Wbsh-01 inoculum at CFU / g, solid-state fermentation was carried out under static conditions at a temperature of 28℃ and a humidity of 55% for 5 days. Following solid-state fermentation, a single sterilization process was performed at 121℃ for 30 minutes at a pressure of 0.13 MPa. The sterilized material was then dried, pulverized, and sieved to obtain material A with a particle size of 100 mesh.

[0125] (2) Mix 3g of material A obtained in step (1), 0.6g of soybean peptide, 0.6g of glucose and 300g of deionized water. After mixing evenly, sterilize at high temperature for 30 minutes in a high-temperature sterilizer at 121℃ and 0.12MPa. After sterilization, cool to room temperature to obtain fermentation substrate B.

[0126] 30 mL of the *Schizophyllum commune* Wbsh-01 inoculum prepared in Example 2 was inoculated into 304.2 g of fermentation substrate B. Using the deionized water in fermentation substrate B as a reference, the viable count of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of deionized water was 10-1. 8The inoculation concentration was CFU / g, followed by liquid fermentation culture at 28℃ for 48 hours using a shaking incubator at 180 rpm. After liquid fermentation, the resulting material was sterilized a second time for 30 minutes at 121℃ and 0.12 MPa. After the second sterilization, the system was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes with a centrifugation radius of 12 cm. The supernatant was collected and subjected to a third sterilization at 121℃ and 0.12 MPa for 30 minutes. After the third sterilization, the mixture was cooled to room temperature to obtain the oat bran fermentation broth.

[0127] Example 4

[0128] Compared with Example 3, the only difference is that the solid fermentation culture time in step (1) is adjusted to 10 days, while other conditions and parameters are the same as in Example 3, and oat bran fermentation liquid is obtained.

[0129] Example 5

[0130] Compared with Example 3, the only difference is that in step (2), 3g of material A is changed to 5g of material A, and other conditions and parameters are the same as in Example 3, so as to obtain oat bran fermentation liquid.

[0131] Example 6

[0132] Compared with Example 3, the only difference is that in step (2), soybean peptides and glucose are not added to fermentation substrate B, and other conditions and parameters are the same as in Example 3, so as to obtain oat bran fermentation liquid.

[0133] Comparative Example 1

[0134] Compared with Example 3, the only difference is that solid-state fermentation culture is not performed, and the liquid fermentation culture time is adjusted to 7 days, specifically including the following steps:

[0135] Commercially available oat bran was pulverized and sieved to obtain oat bran powder with a particle size of 100 mesh. 3g of oat bran powder, 0.6g of soybean peptides, 0.6g of glucose, and 300g of deionized water were mixed thoroughly and then sterilized in a high-temperature sterilizer at 121℃ and 0.12MPa for 30 minutes. After sterilization, the mixture was cooled to room temperature to obtain the fermentation substrate.

[0136] 30 mL of the *Schizophyllum commune* Wbsh-01 inoculum prepared in Example 2 was inoculated into 304.2 g of fermentation substrate. Based on the amount of deionized water in the fermentation substrate, the viable count of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of deionized water was 10-1. 8The inoculation concentration was CFU / g, followed by liquid fermentation culture at 28℃ for 7 days using a shaking incubator at 180 rpm. After liquid fermentation, the resulting material was sterilized for 30 minutes at 121℃ and 0.12 MPa. After sterilization, the system was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes with a centrifugation radius of 12 cm. The supernatant was collected and sterilized for 30 minutes at 121℃ and 0.12 MPa. After sterilization, the supernatant was cooled to room temperature to obtain the oat bran fermentation broth.

[0137] Comparative Example 2

[0138] Commercially available oat bran was pulverized and sieved to obtain oat bran powder with a particle size of 100 mesh. 20g of oat bran powder was mixed with 20g of deionized water, and the mixture was sterilized in a high-temperature autoclave at 121℃ and 0.13MPa for 30 minutes. After sterilization, it was cooled to room temperature to obtain fermentation substrate A.

[0139] 4 mL of the *Schizophyllum commune* Wbsh-01 inoculum prepared in Example 2 above was inoculated into 40 g of fermentation substrate A. The viable count of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of fermentation substrate A was 10. 8 After inoculating with *Schizophyllum commune* Wbsh-01 inoculum at CFU / g, solid-state fermentation was carried out under static conditions at a temperature of 28℃ and a humidity of 55% for 7 days. Following solid-state fermentation, a single sterilization process was performed at 121℃ for 30 minutes at a pressure of 0.13 MPa. The sterilized material was then dried, pulverized, and sieved to obtain material A with a particle size of 100 mesh.

[0140] Mix 3g of material A obtained in step (1), 0.6g of soybean peptide, 0.6g of glucose and 300g of deionized water. After mixing evenly, sterilize in a high-temperature sterilizer at 121℃ and 0.12MPa for 30 minutes. After sterilization, cool to room temperature and then centrifuge at 4800rpm for 30 minutes with a centrifugation radius of 12cm. After centrifugation, take the supernatant and sterilize it in a high-temperature sterilizer at 121℃ and 0.12MPa for 30 minutes. After sterilization, cool to room temperature to obtain oat bran fermentation liquid.

[0141] Comparative Example 3

[0142] Compared with Example 3, the only difference is that liquid fermentation culture is not performed, and the liquid fermentation culture in step (2) is changed to water extraction, specifically including the following steps:

[0143] (1) Commercially available oat bran was pulverized and sieved to obtain oat bran powder with a particle size of 100 mesh. 20g of oat bran powder was mixed with 20g of deionized water, and the mixture was placed in a high-temperature sterilizer at 121℃ and 0.13MPa for 30min for sterilization. After sterilization, it was cooled to room temperature to obtain fermentation substrate A.

[0144] 4 mL of the *Schizophyllum commune* Wbsh-01 inoculum prepared in Example 2 above was inoculated into 40 g of fermentation substrate A. The viable count of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of fermentation substrate A was 10. 8 After inoculating with *Schizophyllum commune* Wbsh-01 inoculum at CFU / g, solid-state fermentation was carried out under static conditions at a temperature of 28℃ and a humidity of 55% for 5 days. Following solid-state fermentation, a single sterilization process was performed at 121℃ for 30 minutes at a pressure of 0.13 MPa. The sterilized material was then dried, pulverized, and sieved to obtain material A with a particle size of 100 mesh.

[0145] (2) Mix 3g of material A obtained in step (1), 0.6g of soybean peptide, 0.6g of glucose and 300g of deionized water, and perform water extraction at 60℃ for 60min; after extraction, centrifuge the material, collect the supernatant, and place the supernatant in a high-temperature sterilizer at 121℃ and 0.12MPa for high-temperature sterilization for 30min to obtain oat bran fermentation liquid.

[0146] Comparative Example 4

[0147] (1) Commercially available oat bran was pulverized and sieved to obtain oat bran powder with a particle size of 100 mesh. 20g of oat bran powder was mixed with 20g of deionized water, and the mixture was placed in a high-temperature sterilizer at 121℃ and 0.13MPa for 30min for sterilization. After sterilization, it was cooled to room temperature to obtain fermentation substrate A.

[0148] Preparation method of Lactobacillus helveticus bacterial suspension: Disperse Lactobacillus helveticus (CICC 20243) in sterile deionized water to prepare a viable bacteria concentration of 10. 9 Prepare a CFU / mL Lactobacillus helveticus culture for later use.

[0149] 4 mL of the prepared Lactobacillus helveticus culture was inoculated into 40 g of fermentation substrate A. The viable count of Lactobacillus helveticus (CICC 20243) inoculated per unit mass of fermentation substrate A was 10-1. 8 After inoculating with *Lactobacillus helveticus* culture at CFU / g, solid-state fermentation was carried out under static conditions at a temperature of 28℃ and a humidity of 55% for 5 days. Following solid-state fermentation, a single sterilization process was performed at 121℃ for 30 minutes at a pressure of 0.13 MPa. The sterilized material was then dried, pulverized, and sieved to obtain material A with a particle size of 100 mesh.

[0150] (2) Mix 3g of material A obtained in step (1), 0.6g of soybean peptide, 0.6g of glucose and 300g of deionized water. After mixing evenly, sterilize at high temperature for 30 minutes in a high-temperature sterilizer at 121℃ and 0.12MPa. After sterilization, cool to room temperature to obtain fermentation substrate B.

[0151] 30 mL of the prepared *Lactobacillus helveticus* culture was inoculated into 304.2 g of fermentation substrate B. Using the deionized water in fermentation substrate B as a reference, the viable count of *Lactobacillus helveticus* (CICC 20243) inoculated per unit mass of deionized water was 10-1. 8 The inoculation concentration was CFU / g, followed by liquid fermentation culture. The fermentation was carried out statically in a 28℃ incubator for 48 hours. After fermentation, the resulting material was sterilized a second time in a high-temperature autoclave at 121℃ and 0.12MPa for 30 minutes. After the second sterilization, the system was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes with a centrifugation radius of 12 cm. The supernatant was collected and placed in a high-temperature autoclave at 121℃ and 0.12MPa for a third sterilization for 30 minutes. After the third sterilization, the mixture was cooled to room temperature to obtain the oat bran fermentation broth.

[0152] Comparative Example 5

[0153] Compared with Example 3, the only difference is that the inoculated agent in step (1) is replaced with an equal amount of brewer's yeast liquid, and the inoculated agent in step (2) is replaced with an equal amount of brewer's yeast liquid. Other conditions and parameters are the same as in Example 3, and oat bran fermentation liquid is obtained.

[0154] Preparation method of Saccharomyces cerevisiae inoculum: A single colony of Saccharomyces cerevisiae (CICC 17452) is inoculated into YPD liquid medium and fermented at 28℃ with a shaking speed of 180 rpm for 48 hours. The fermented material is then prepared into a culture medium with a viable cell concentration of 102.9 Prepare a CFU / mL culture of Saccharomyces cerevisiae for later use.

[0155] Comparative Example 6

[0156] Compared with Example 3, the only difference is that the inoculum in step (1) is replaced with an equal amount of Schizophyllum commune (CICC2592) bacterial solution, and the inoculum in step (2) is replaced with an equal amount of Schizophyllum commune (CICC 2592) bacterial solution. Other conditions and parameters are the same as in Example 3, and oat bran fermentation broth is obtained.

[0157] Preparation method of Schizophyllum commune (CICC 2592) bacterial suspension: Disperse Schizophyllum commune (CICC 2592) in sterile deionized water to prepare a viable cell concentration of 10. 9 Prepare a CFU / mL suspension of Schizophyllum commune (CICC 2592) for later use.

[0158] Comparative Example 7

[0159] Compared with Example 3, the only difference is that in step (1), the solid fermentation culture time is 14 days, and other conditions and parameters are the same as in Example 3, so as to obtain oat bran fermentation broth.

[0160] Comparative Example 8

[0161] Compared with Example 3, the only difference is that in step (1), the solid fermentation culture time is 1 day, and other conditions and parameters are the same as in Example 3, so as to obtain oat bran fermentation liquid.

[0162] Example 1

[0163] Hydroxyl radicals (·OH) are generated using the Fenton reaction. Salicylic acid is added to the reaction system, and the ·OH reacts with the salicylic acid to form a colored compound, 2,3-dihydroxybenzoic acid, which exhibits characteristic absorption at 510 nm. Using a 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 scavenging effect on ·OH.

[0164] Preparation of test solution: Take the products obtained in Examples 3-6 and Comparative Examples 1-8 above and mix them with water to prepare test solutions with a mass percentage of 20%.

[0165] Add reagents to the test tubes according to Table 1, adding 9 mmol / L FeSO4, 9 mmol / L salicylic acid ethanol solution, the test solution, an appropriate amount of deionized water, and 8.8 mmol / L H2O2 solution in sequence. Shake well, heat in a 37℃ water bath for 15 min, and then remove and measure the absorbance A0 of the blank control and the absorbance A of the sample group. x Background absorbance A of the solution without added H2O2 x0When determining A0, the reference solution was a system without hydrogen peroxide; three parallel experiments were performed for each group, the absorbance values ​​were measured and the average value was taken. The results are shown in Table 2. The hydroxyl radical scavenging rate of each group was calculated according to the following formula, and the results are shown in Table 2 and... Figure 2 (Table 2 and) Figure 2 In the figure, ***p<0.001 indicates a highly statistically significant difference compared to Example 3, meaning a highly significant reduction; ** p < 0.01 indicates a statistically significant difference compared to Example 3, representing a significant reduction; ns indicates no statistically significant difference compared to Example 3.

[0166] Hydroxyl radical scavenging rate = (A0 - (A) x -A x0 )) / A0×100%.

[0167] Table 1

[0168]

[0169] Table 2

[0170]

[0171]

[0172] The results showed that the oat bran fermentation broth prepared using the specific *Schizophyllum commune* Wbsh-01 strain described in this application, combined with solid-state and liquid fermentation processes, exhibited ideal antioxidant effects. Furthermore, the research process revealed that the *Schizophyllum commune* Wbsh-01 strain used in this application, compared to existing *Schizophyllum commune* strains, more readily utilizes components in oat bran, resulting in an oat bran fermentation broth with more ideal antioxidant effects. The research also found that process parameters such as fermentation type, fermentation strain type, and solid-state fermentation time significantly influenced the antioxidant efficacy of the fermentation products.

[0173] Example 2: Hyaluronidase Inhibition Test

[0174] Hyaluronidase is a lysosome that breaks down polysaccharides. It hydrolyzes potassium hyaluronate to produce β-N-acetylglucosamine, which condenses with acetylacetone under alkaline conditions to form the chromogen 2-methyl-3-diacetylpyrrole derivative. The chromogen reacts with Ehrlich's reagent in concentrated hydrochloric acid and ethanol to produce a colorimetric reaction. Hyaluronidase is strongly correlated with inflammation and allergies and is involved in type I hypersensitivity reactions. Therefore, in vitro hyaluronidase inhibition assays can serve as a rapid method for assessing anti-allergy levels.

[0175] Reagents: Hyaluronidase, sodium hyaluronate, anhydrous ethanol, sodium hydroxide, anhydrous sodium carbonate, concentrated hydrochloric acid, p-dimethylaminobenzaldehyde, acetylacetone, glacial acetic acid, anhydrous calcium chloride.

[0176] Equipment: The Sunrise microplate reader is manufactured by Diken Trading Co., Ltd.; the digital display constant temperature water bath is manufactured by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0177] Take 0.1 mL of CaCl2 solution (0.25 mmol / L) and 0.5 mL of hyaluronidase solution (100 U / mL) and incubate at 37°C in a water bath for 20 min; add 0.5 mL of the test sample solution (the product prepared in the example or comparative example), and continue incubation for 20 min; then add 0.5 mL of sodium hyaluronate solution (0.5 mg / mL), incubate at 37°C in a water bath for 30 min, remove and place at room temperature for 5 min; add 0.1 mL of... NaOH solution (0.4 mol / L) and 0.5 mL acetylacetone solution (3.5 mL acetylacetone dissolved in 50 mL 1.0 mol / L sodium carbonate solution) were heated in a boiling water bath for 15 min, then immediately transferred to an ice water bath for cooling for 5 min. 1.0 mL Ehrlich reagent (0.8 g p-dimethylaminobenzaldehyde dissolved in 15 mL concentrated hydrochloric acid and 15 mL anhydrous ethanol) was added dropwise, and the solution was diluted with 3.0 mL anhydrous ethanol. The solution was allowed to stand at room temperature for 20 min for color development. The absorbance was measured at 540 nm using a spectrophotometer. Three parallel experiments were performed for each group, and the absorbance values ​​were measured and averaged. The results are shown in Table 3. The calculation formula for the inhibition rate of hyaluronidase by the test sample solution is as follows, and the results are shown in Table 3. Figure 3 (Table 3 and Figure 3 In the figure, ***p<0.001 indicates a highly statistically significant difference compared to Example 3, meaning a highly significant reduction; ** p < 0.01 indicates a statistically significant difference compared to Example 3, representing a significant reduction; ns indicates no statistically significant difference compared to Example 3.

[0178] Hyaluronidase inhibition rate = [(AB) - (CD)] / (AB) × 100%

[0179] Where: A—Absorbance value of control solution (acetic acid buffer solution is used instead of test sample solution); B—Absorbance value of control blank solution (acetic acid buffer solution is used instead of test sample solution and enzyme solution); C—Absorbance value of test sample solution; D—Absorbance value of test sample blank solution (acetic acid buffer solution is used instead of enzyme solution).

[0180] Table 3

[0181]

[0182]

[0183] The results showed that the oat bran fermentation broth prepared using the specific *Schizophyllum commune* Wbsh-01 strain described in this application, combined with solid-state and liquid fermentation processes, exhibited ideal anti-allergic effects. Furthermore, the research process revealed that the *Schizophyllum commune* Wbsh-01 strain used in this application, compared to existing *Schizophyllum commune* strains, more readily utilizes components in oat bran, resulting in an oat bran fermentation broth with stronger hyaluronidase inhibitory activity, indicating a more ideal anti-allergic effect. Moreover, the research process also found that process parameters such as fermentation type, fermentation strain type, and solid-state fermentation time significantly influenced the anti-allergic efficacy of the fermentation product.

[0184] Example 3: Sensory Evaluation

[0185] Sensory evaluations (moisturizing sensation, penetration, hydration, etc.) were conducted on the products prepared in the above embodiments and comparative examples. A total of 48 trained inspectors, 24 men and 24 women, were selected to rate the user experience of the products prepared in the above embodiments or comparative examples (using a 5-point scale: 1: very poor, 2: poor, 3: average, 4: good, 5: very good). The results are shown in Table 4.

[0186] Table 4

[0187] Moisturizing feeling Permeability Moisturizing Example 3 4.82 4.63 4.76 Example 4 4.58 4.29 4.59 Example 5 4.64 4.42 4.61 Example 6 4.42 4.65 4.48 Comparative Example 1 3.74 3.59 3.75 Comparative Example 2 3.19 3.15 3.09 Comparative Example 3 3.28 3.41 3.17 Comparative Example 4 3.96 3.26 3.59 Comparative Example 5 4.07 3.49 3.67 Comparative Example 6 4.13 3.93 4.08 Comparative Example 7 3.94 3.64 3.91 Comparative Example 8 3.61 3.47 3.84

[0188] The skin feel test results show that after the subjects used the products prepared in the embodiments and comparative examples of this application, the evaluation scores of the embodiments in terms of moisturizing, penetration and hydration were higher than those of the comparative examples, indicating that the skin feel of the products prepared in the embodiments of this application is more ideal.

[0189] Example 4: Protection against photodamage (repair)

[0190] Experimental steps:

[0191] The products obtained in the above examples and comparative examples were prepared into experimental test solutions with a volume percentage of 2% using serum-free DMEM culture medium. The experimental test solutions were filtered through a 0.22 μm sterile filter membrane.

[0192] HaCaT cells were cultured in a solution containing 10% fetal bovine serum and 1% penicillin-antibody (1×10⁻⁶). 5 Cells were cultured in DMEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin. Cells were grown in an incubator at 37°C, 5% CO2, and saturated humidity. When cell confluence reached 85% or higher, the logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion was terminated with serum-containing DMEM. Cells were counted using a cell counting chamber, and the cell suspension concentration was adjusted to 7 × 10⁻⁶ cells / mL. 4 Cells / mL were seeded into 96-well plates at a ratio of 100 μL per well and incubated at 37°C with 5% CO2 for 12 h.

[0193] After incubation, the old culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 100 μL of PBS solution was added. Both the model group and the experimental group were irradiated with UVB at a dose of 40 mJ / cm². 2 The negative control group was not irradiated; PBS was discarded, and serum-free DMEM medium was added to the model group and negative control group. 100 μL of the filtered and sterilized experimental test solution was added to each well of the experimental group, with six replicates per test solution. The blank control group was cell-free and contained 100 μL of PBS. The cells were then incubated at 37°C and 5% CO2 for 24 h. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 3 h. The absorbance was measured at 450 nm. The absorbance results are shown in Table 5. The cell viability of each group was calculated using the following formula. The results are shown in Table 5. Figure 4 (Table 5 and Figure 4 middle, ns p > 0.05 indicates no statistical difference compared to the model group; *p < 0.05 indicates a statistical difference compared to the model group; **p < 0.01 indicates a significant statistical difference compared to the model group; ***p < 0.001 indicates an extremely significant statistical difference compared to the model group. ### p < 0.001 indicates a highly statistically significant difference compared to the control group.

[0194] The formula for calculating cell viability is as follows:

[0195] Cell viability = (A experimental group or model group - A blank control group) / (A negative control group - A blank control group) × 100%.

[0196] Table 5

[0197]

[0198]

[0199] The results show that the product prepared in this application has a relatively ideal repair effect on damaged skin. Furthermore, during the research and development process, it was found that the oat bran fermentation broth prepared using *Schizophyllum commune* Wbsh-01, compared with existing *Schizophyllum commune* strains, has a more ideal repair effect on damaged cells. Moreover, the research and development process also revealed that process parameters such as fermentation type, fermentation strain type, and solid-state fermentation time have a significant impact on the repair efficacy of the fermentation product.

[0200] Example 6: Measurement of the inflammatory factor IL-8 (repairing cells)

[0201] Sample preparation: The products prepared in the above examples and comparative examples were prepared into experimental test solutions with a volume percentage of 2% using serum-free DMEM medium.

[0202] HaCaT cells were seeded at a density of 250,000 cells / mL into 6-well cell culture plates, with 2 mL of cell suspension added to each well. After 12 h of culture, the supernatant was discarded. For the control group, 2 mL of DMEM solution was added. For both the experimental and model groups, cells were induced with 800 μg / mL LPS for 6 h, and the supernatant was discarded. For the experimental group, 2 mL of the test sample was added, and for the model group, 2 mL of DMEM solution was added. Cells were treated for 24 h. The supernatant was collected, and 200 μL of cell lysis buffer was added to each well. Cells were centrifuged at 10000 rpm at 4 °C for 10 min, and the supernatant was collected to obtain the cell lysis supernatant.

[0203] 20 μL of cell lysate was used to detect the total protein content in the sample using a BCA kit. Inflammatory factors were measured according to the ELISA kit instructions. The OD value of the cell lysate supernatant from each group was measured at 450 nm. The results were then compared with the standard curve (Y = 0.0023X + 0.077, R0). 2 =0.9985), calculate the expression level A of the inflammatory factor IL-8, correct it with the protein content B to get C = A / B, and then calculate the relative expression level = C / C. 空白组 The results are shown in Table 6 and Figure 5 (Table 6 and Figure 5 In this context, *p < 0.05 indicates a statistically significant difference compared to the model group; **p < 0.01 indicates a statistically significant difference compared to the model group; and ***p < 0.001 indicates an extremely statistically significant difference compared to the model group. ### p < 0.001 indicates a highly statistically significant difference compared to the control group.

[0204] Table 6

[0205] OD value IL-8 relative expression level Blank group 0.114 1.00 Model group 0.131 <![CDATA[1.62±0.01 ### ]]> Example 3 0.099 <![CDATA[0.69±0.03 *** ]]> Example 4 0.096 <![CDATA[0.67±0.06 *** ]]> Example 5 0.096 <![CDATA[0.79±0.05 *** ]]> Example 6 0.102 <![CDATA[0.79±0.06 *** ]]> Comparative Example 1 0.108 <![CDATA[1.18±0.04 ** ]]> Comparative Example 2 0.121 <![CDATA[1.17±0.06 ** ]]> Comparative Example 3 0.123 <![CDATA[1.37±0.03 * ]]> Comparative Example 4 0.122 <![CDATA[1.34±0.08 * ]]> Comparative Example 5 0.126 <![CDATA[1.37±0.07 * ]]> Comparative Example 6 0.128 <![CDATA[1.36±0.13 * ]]> Comparative Example 7 0.108 <![CDATA[1.2±0.15 * ]]> Comparative Example 8 0.11 <![CDATA[1.19±0.06 ** ]]>

[0206] The results showed that the oat bran fermentation broth prepared in this application had ideal anti-inflammatory effects. After treating damaged cells, it significantly reduced the relative expression level of the intracellular inflammatory factor IL-8, which was lower than that of the blank group. Although the product prepared in the comparative proportion could reduce the relative expression level of the intracellular inflammatory factor IL-8 after cell treatment compared with the model group, the reduction was limited and higher than that of the blank group, indicating that the anti-inflammatory effect of the product prepared in the comparative proportion was generally weak.

[0207] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0208] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A strain of Schizophyllum commune ( Schizophyllum commune Wbsh-01, with accession number CGMCC No.40388, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

2. A microbial agent, characterized in that, The microbial agent includes *Schizophyllum commune* Wbsh-01 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The viable bacterial concentration of *Schizophyllum commune* Wbsh-01 in the bacterial agent is 10. 6 ~10 9 CFU / mL.

4. The microbial agent as described in claim 3, characterized in that, The viable bacterial concentration of *Schizophyllum commune* Wbsh-01 in the bacterial agent is 10. 7 ~10 9 CFU / mL.

5. A method for preparing the microbial agent as described in any one of claims 2-4, characterized in that, Includes the following steps: A) Culture in petri dishes: The *Schizophyllum commune* Wbsh-01 as described in claim 1 is inoculated onto a solid culture medium under aseptic conditions, and then cultured to obtain single colonies; B) The single colony is picked up with an inoculation loop and placed in a liquid culture medium, and then cultured to obtain the bacterial agent.

6. The method for preparing the microbial agent as described in claim 5, characterized in that, The preparation method of the bacterial agent satisfies at least one of the following conditions: In step A, the solid culture medium includes PDA solid culture medium; In step A, the culture temperature is 20~30℃; In step A, the culture time is 30-50 hours; In step A, the culture is carried out under static conditions; In step B, the liquid culture medium includes PDA liquid culture medium; In step B, the culture temperature is 20~30℃; In step B, the culture time is 30-50 hours; In step B, the culture is carried out under oscillation conditions, and the oscillation speed is 150~200 rpm.

7. The method for preparing the microbial agent as described in claim 6, characterized in that, The preparation method of the bacterial agent satisfies at least one of the following conditions: In step A, the culture temperature is 25~30℃; In step A, the culture time is 45-50 hours; In step B, the culture temperature is 25~30℃; In step B, the culture time is 45-50 hours; In step B, the rotational speed of the oscillation is 175~185 rpm.

8. The use of the *Schizophyllum commune* Wbsh-01 as described in claim 1 as a fermentation strain in the preparation of topical cosmetic agents.

9. A method for preparing oat bran fermentation broth, characterized in that, Includes the following steps: (1) The Schizophyllum commune Wbsh-01 as described in claim 1 and / or the bacterial agent as described in any one of claims 2-4 are inoculated into fermentation substrate A, and the substrate is cultured in solid state for 4-12 days and sterilized once to obtain material A; the fermentation substrate A includes oat bran powder and water; (2) The oat bran fermentation broth is prepared by inoculating the Schizophyllum commune Wbsh-01 as described in claim 1 and / or the bacterial agent as described in any one of claims 2-4 into the fermentation substrate B, followed by liquid fermentation culture and secondary sterilization; the fermentation substrate B includes the material A and water.

10. The method for preparing oat bran fermentation broth as described in claim 9, characterized in that, The method for preparing the oat bran fermentation broth satisfies at least one of the following conditions: In step (1), the oat bran powder has a particle size of 100-300 mesh; In step (1), the water in the fermentation substrate A includes deionized water; In step (1), the mass ratio of the oat bran powder to the water in the fermentation substrate A is 1:(0.5~2). In step (1), the fermentation substrate A includes a sterilization process before use; In step (1), the number of *Schizophyllum commune* Wbsh-01 inoculated into the fermentation substrate A per unit mass is 10. 7 ~10 9 CFU / g; In step (1), the solid-state fermentation culture time is 5~10 days; In step (1), the temperature of the solid fermentation culture is 25~30℃; In step (1), the humidity of the solid-state fermentation culture is 50%~95%; In step (1), the solid-state fermentation culture is carried out under static conditions; In step (1), the sterilization method is high-temperature sterilization. In step (1), the sterilization operation is followed by crushing and sieving operations; In step (2), the mass ratio of material A to water in the fermentation substrate B is 1:(20~200). In step (2), the water in the fermentation substrate B includes deionized water; In step (2), the fermentation substrate B further includes a carbon source and / or a nitrogen source; In step (2), the fermentation substrate B also includes a sterilization process before use; In step (2), based on the water in the fermentation substrate B, the number of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of the water is 10. 4 ~10 9 CFU / g; In step (2), the liquid fermentation culture time is 24~72h; In step (2), the temperature of the liquid fermentation culture is 25~35℃; In step (2), the liquid fermentation culture is carried out under shaking conditions, and the shaking speed is 150~300 rpm; In step (2), the secondary sterilization method is high-temperature sterilization. In step (2), the secondary sterilization operation is followed by centrifugation, collection of supernatant and tertiary sterilization; the tertiary sterilization method is high-temperature sterilization.

11. The method for preparing oat bran fermentation broth as described in claim 10, characterized in that, The method for preparing the oat bran fermentation broth satisfies at least one of the following conditions: In step (1), the oat bran powder has a particle size of 150-250 mesh; In step (1), the mass ratio of the oat bran powder to the water in the fermentation substrate A is 1:(0.8~1.2). In step (1), the number of *Schizophyllum commune* Wbsh-01 inoculated into the fermentation substrate A per unit mass is 10. 7 ~10 8 CFU / g; In step (1), the solid-state fermentation culture time is 5-7 days; In step (1), the temperature of the solid fermentation culture is 25~28℃; In step (1), the humidity of the solid-state fermentation culture is 50%~65%; In step (1), the particle size of material A after crushing and sieving is 100~300 mesh; In step (2), the mass ratio of material A to water in the fermentation substrate B is 1:(50~150). In step (2), the carbon source includes at least one of glucose, potato, and corn; the carbon source accounts for 0.05% to 1% of the mass of the fermentation substrate B; the nitrogen source includes at least one of soybean peptides, soybean meal, and milk powder; the nitrogen source accounts for 0.05% to 1% of the mass of the fermentation substrate B. In step (2), based on the water in the fermentation substrate B, the number of *Schizophyllum commune* Wbsh-01 inoculated per unit mass of the water is 10. 5 ~10 8 CFU / g; In step (2), the liquid fermentation culture time is 30~50h; In step (2), the temperature of the liquid fermentation culture is 25~30℃; In step (2), the liquid fermentation culture is carried out under shaking conditions, and the shaking speed is 150~250 rpm; In step (2), the centrifugation speed is 3000~9000 rpm; the centrifugation time is 10~40 min; and the centrifugation radius is 8~15 cm.

12. The method for preparing oat bran fermentation broth as described in claim 11, characterized in that, The method for preparing the oat bran fermentation broth satisfies at least one of the following conditions: In step (1), the particle size of material A after crushing and sieving is 150~250 mesh; In step (2), the mass ratio of material A to water in the fermentation substrate B is 1:(60~100). In step (2), the carbon source accounts for 0.1% to 0.5% of the mass of the fermentation substrate B; the nitrogen source accounts for 0.1% to 0.5% of the mass of the fermentation substrate B. In step (2), the centrifugation speed is 4000~6000 rpm; the centrifugation time is 20~40 min; and the centrifugation radius is 10~13 cm.

13. An oat bran fermentation liquid, characterized in that, It is prepared by the method for preparing oat bran fermentation broth as described in any one of claims 9-11.

14. The use of the oat bran fermentation liquid as described in claim 13, directly as a product, as an additive, or as a base in the preparation of topical cosmetic agents.

15. The application of the oat bran fermentation liquid as described in claim 14, directly as a product, as an additive, or as a base in the preparation of topical cosmetic agents, characterized in that... The oat bran fermentation liquid is at least one of the antioxidant active ingredient, anti-allergic active ingredient, repair active ingredient, and anti-inflammatory active ingredient in the topical skin cosmetic agent.

16. The application of the oat bran fermentation liquid as described in claim 15, as a product, as an additive, or as a base in the preparation of topical cosmetic agents, characterized in that... The antioxidant active ingredient is an antioxidant active ingredient with hydroxyl radical scavenging activity; The anti-allergic active ingredient is an anti-allergic active ingredient with hyaluronidase inhibitory activity; The anti-inflammatory active ingredient is an anti-inflammatory active ingredient that can clear the inflammatory factor IL-8.

17. A topical skin agent, characterized in that, Includes the oat bran fermentation liquid as described in claim 13.

18. The topical skin agent as described in claim 17, characterized in that, The topical skin preparation also includes active ingredients and / or preservatives; The topical skin agents include face masks, serums, or toners; The oat bran fermentation liquid accounts for 5% to 99% of the mass of the topical skin agent.

19. The topical skin agent as described in claim 18, characterized in that, The active ingredient includes at least one of moisturizing active ingredient, whitening active ingredient, anti-inflammatory active ingredient, anti-allergic active ingredient, and antioxidant active ingredient; the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone, the p-hydroxyacetophenone accounts for 0.3% to 1% of the mass percentage of the topical skin agent; when the preservative includes 1,2-hexanediol, the 1,2-hexanediol accounts for 0.3% to 1% of the mass percentage of the topical skin agent. The oat bran fermentation liquid accounts for 60% to 99% of the mass of the topical skin agent.

Images