Schizophyllum ferments rose and application thereof

Abstract

The present application belongs to the field of microorganisms, and particularly relates to a schizophyllum for fermenting roses and application thereof. Schizophyllum commune The schizophyllum is named schizophyllum (ZR004), preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 42265 and a preservation date of September 18, 2025. The polyphenol content in the schizophyllum rose ferment obtained by fermenting roses with the schizophyllum ZR004 is obviously improved, and the schizophyllum rose ferment has obvious moisturizing, soothing and repairing effects. The present application fills the skin care effect experimental data of the rose filtrate fermented by the schizophyllum, and promotes the large-scale application of the schizophyllum fermentation product in the daily chemical field.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a type of Schizophyllum commune used for fermenting roses and its applications. Background Technology

[0002] Schizophyllum commune ( Schizophyllum commune Also known as white ginseng, tree flower, or eight-dan firewood, *Schizophyllum commune* belongs to the phylum Basidiomycetes, class Agaricales, order Schizophyllum, family Schizophyllum, and genus *Schizophyllum*. It is a large edible fungus with high nutritional and medicinal value. Schizophyllan (SPG) is one of the most important active substances in *Schizophyllum commune*, possessing broad-spectrum antibacterial activity, antioxidant activity, and excellent moisturizing properties, making it a superior moisturizing ingredient in the cosmetics industry.

[0003] Pingyin rose, a deciduous shrub belonging to the genus Rosa, is a unique rose resource in Shandong Province, my country, with a history of rose cultivation in Pingyin spanning over 1300 years. Pingyin roses are rich in flavonoids, terpenes, steroids, tocopherols, and carotene. Modern research shows that Pingyin rose essential oil possesses excellent antioxidant capabilities, reducing melanin synthesis and inhibiting tyrosinase activity. Functional components in roses promote metabolism, repair cells, and have anti-inflammatory and antibacterial effects. Anthocyanins exhibit good antioxidant properties, effectively scavenging various reactive oxygen free radicals and significantly inhibiting the expression of inflammatory factors. Therefore, Pingyin roses have broad development prospects in the cosmetics field.

[0004] Currently, the biotransformation of plant raw materials using microbial fermentation technology has become an important direction for the development of cosmetic functional ingredients. Existing technologies have reported the use of *Schizophyllum commune* to ferment plant raw materials for the preparation of cosmetic functional ingredients. For example, patent CN117821359A uses *Schizophyllum commune* as the starting strain to ferment a culture medium containing *Buddleja officinalis* powder, achieving moisturizing, anti-aging, and whitening effects; patent CN113337545A adds kudzu root powder to the *Schizophyllum commune* culture medium for fermentation, enhancing the antioxidant effects of the fermentation product. Regarding rose fermentation, existing technologies mainly use bacteria such as lactic acid bacteria and acetic acid bacteria, or yeast, to ferment roses; there are no reports of using *Schizophyllum commune* to ferment roses. Summary of the Invention

[0005] This invention obtains a strain of *Schizophyllum commune* through screening. Its fermentation product has certain skin care effects. Using this strain to ferment Pingyin rose, the content of active ingredients in the product after fermentation is significantly higher than that before fermentation, and a specific *Schizophyllum commune* polysaccharide is produced. The fermentation product has multiple effects such as moisturizing, repairing and soothing, and can be widely used in personal care products, cosmetics and skin care products.

[0006] In a first aspect, the present invention provides a fungus called *Schizophyllum commune*. Schizophyllum communeZR004 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42265 and deposit date of September 18, 2025.

[0007] In a second aspect, the present invention provides a microbial culture comprising the aforementioned Schizophyllum commune and / or its fermentation metabolites.

[0008] In a third aspect, the present invention provides a microbial inoculant, the microbial inoculant comprising the aforementioned Schizophyllum commune or the aforementioned microbial culture, and a carrier.

[0009] Further, the microbial agent is a lyophilized powder, granules, or concentrated liquid; the carrier includes at least one of inorganic carriers, organic carriers, and synthetic / composite carriers. Optionally, the carrier includes at least one of skim milk powder, sodium alginate, wheat bran, and sawdust.

[0010] In a fourth aspect, the present invention provides a *Schizophyllum commune* rose ferment, which is obtained by fermenting roses with the *Schizophyllum commune*.

[0011] In a fifth aspect, the present invention provides a composition comprising the aforementioned *Schizophyllum commune*, the aforementioned microbial culture, the aforementioned microbial inoculant, or the aforementioned rose ferment.

[0012] Furthermore, the composition also includes additives acceptable in the pharmaceutical or cosmetic fields.

[0013] Furthermore, the additives include at least one of the following: preservatives, emulsifiers, humectants, thickeners, pH adjusters, surfactants, stabilizers, antioxidants, and moisturizers.

[0014] Furthermore, the composition may also include other substances with moisturizing, antioxidant, and soothing effects.

[0015] In a sixth aspect, the present invention provides the use of the *Schizophyllum commune*, the microbial culture, the *Schizophyllum commune* rose ferment, the microbial agent, or the composition in any of the following aspects: A1. Applications in the preparation of moisturizing products; A2. Applications in the preparation of anti-inflammatory products; A3. Applications in the preparation of soothing products; A4. Applications in the preparation of products that promote skin repair.

[0016] Furthermore, the products include at least pharmaceuticals and cosmetics.

[0017] Optionally, the drug is an anti-inflammatory drug.

[0018] Furthermore, the dosage form of the cosmetic includes at least one of aqueous solution, emulsion, and cream.

[0019] In a seventh aspect, the present invention provides a method for culturing the aforementioned *Schizophyllum commune*, wherein the culture conditions are as follows: Plate culture: Pick mycelia of Schizophyllum commune, inoculate them onto the surface of seed solid culture medium, and culture at 25~30℃ for 4~6 days; Seed culture: Pick mycelia of Schizophyllum commune, inoculate them into seed culture medium, and culture at 25-30℃ and 150-250 rpm for 4-6 days; Fermentation tank culture conditions: temperature 25~30℃, rotation speed 150~250rpm, oxygenation culture, cycle 4-6 days.

[0020] Furthermore, the formula for the seed solid culture medium is as follows: 30g glucose monohydrate, 2g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 20g agar powder, add deionized water to make up to 1L, and sterilize in an autoclave at 118℃ for 15min.

[0021] The formula for the seed liquid culture medium is as follows: 30g glucose monohydrate, 2g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, add deionized water to make up to 1L, and sterilize in an autoclave at 118℃ for 15min.

[0022] In an eighth aspect, the present invention provides a method for fermenting roses using the aforementioned Schizophyllum commune, the method comprising at least the following steps: S1. Preparation of rose extract: Rose powder was taken and stirred in a water bath at 25~35℃ for 1.5~2.5h at a material-to-liquid ratio of 1:20 (g / mL) to obtain rose extract; S2. Add 5%~15% rose extract (by volume) to the fermentation medium, inoculate with 5%~7.5% of the described Schizophyllum commune seed liquid, ferment at 25~30℃ for 4~6 days to obtain Schizophyllum commune rose ferment.

[0023] Furthermore, the method further includes a step of filtering and sterilizing the *Schizophyllum commune* rose ferment obtained in S2, which can be optionally high-temperature sterilization. Optionally, the specific operation involves centrifuging at 14,000-15,000 rpm in a tubular centrifuge, and sterilizing the centrifuged liquid at 105-110°C for 20-30 minutes.

[0024] Furthermore, the method also includes a step of sterilizing the rose extract obtained in step S1, which may be high-temperature sterilization. In one specific embodiment, the sterilization step of the rose extract is as follows: sterilization at 105°C for 20 minutes in an autoclave.

[0025] Optionally, the fermentation medium is formulated as follows: 45g glucose, 3g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, and deionized water is added to bring the volume to 1L. After sterilization in an autoclave at 115℃ for 20 minutes, a common fermentation medium is obtained.

[0026] The beneficial effects of the present invention include, but are not limited to: The Schizophyllum commune described in this invention ( Schizophyllum commune ZR004 fermented roses produce polysaccharides during fermentation, and the polyphenol content in the resulting rose fermentation product is significantly increased. The polyphenol content in the *Schizophyllum commune* rose fermentation product obtained after fermenting roses using the *Schizophyllum commune* ZR004 method described in this invention is significantly increased, exhibiting significant moisturizing, soothing, and repairing effects. This invention fills the gap in experimental data on the skincare efficacy of *Schizophyllum commune* fermented rose filtrate, promoting the large-scale application of *Schizophyllum commune* fermentation products in the daily chemical industry. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a process flow diagram of fermentation of roses by Schizophyllum commune in an embodiment of the present invention.

[0028] Figure 2 This is a schematic diagram illustrating the cytotoxicity experiment results of *Schizophyllum commune* rose ferment filtrate on keratinocytes in an embodiment of the present invention.

[0029] Figure 3 This is a summary diagram of the AQP3 immunofluorescence staining results in the embodiments of the present invention, in which green fluorescence represents AQP3 and blue fluorescence represents the cell nucleus.

[0030] Figure 4 This is a bar chart of the relative integrated optical density (IOD) values ​​of AQP3 in an embodiment of the present invention. The significance of the sample group compared to the BC group is as follows: This means that a p-value < 0.05 indicates that... A p-value less than 0.01 indicates that... .

[0031] Figure 5 This is a schematic diagram illustrating the cytotoxicity experiment results of *Schizophyllum commune* rose ferment filtrate on human fibroblasts in an embodiment of the present invention.

[0032] Figure 6 This is a schematic diagram of the cell migration test results of the filtrate of *Schizophyllum commune* rose fermentation product in an embodiment of the present invention.

[0033] Figure 7This is a bar chart showing the cell migration rate of the *Rosa schizophyllum commune* ferment filtrate in an embodiment of the present invention. Among them, the significant difference between the sample group, PC group, and BC group is... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0034] Figure 8 This is a schematic diagram showing the cytotoxicity experiment results of *Rosa schizophyllum commune* fermentation filtrate on RAW264.7 macrophages in an embodiment of the present invention.

[0035] Figure 9 This is a bar chart showing the TNF-α content in the soothing experiment of *Rosa schizophyllum commune* ferment filtrate in this embodiment of the invention. Among them: NC group compared to BC group, significance is indicated by #, p-value < 0.05 is indicated by #, p-value < 0.01 is indicated by ##. The sample group and PC group compared to NC group are also shown as... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0036] Figure 10 This is a bar chart showing the IL-6 content in the soothing experiment of *Rosa schizophyllum commune* ferment filtrate in this embodiment of the invention. Among them: The significance of the NC group compared to the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. The significance of the sample group and the PC group compared to the NC group is indicated by... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0037] Figure 11 This is a bar chart showing the IL-1α content in the soothing experiment of *Rosa schizophyllum commune* ferment filtrate in this embodiment of the invention. Among them: The significance of the NC group compared to the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. The significance of the sample group and the PC group compared to the NC group is indicated by... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... . Detailed Implementation

[0038] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0039] Materials and Instruments Plant-based ingredients: fresh potatoes, Pingyin roses.

[0040] Reagents: Agar powder (Beijing Solarbio Science & Technology Co., Ltd.), yeast extract powder (Angel Yeast Co., Ltd.), glucose monohydrate (Shandong Xiwang Sugar Industry Co., Ltd.), potassium dihydrogen phosphate (Jinan Dongzhou Weiye Chemical Co., Ltd.), magnesium sulfate heptahydrate (Jinan Dongzhou Weiye Chemical Co., Ltd.), concentrated sulfuric acid (Yantai Yuandong Fine Chemical), phenol (Xilong Scientific), glucose (Sinopharm).

[0041] Strain: Schizophyllum commune ZR004 was obtained through natural environment screening and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42265 and deposit date of September 18, 2025.

[0042] Instruments: 100L automatic fermenter from Shanghai Bailun Biotechnology Co., Ltd., 722 UV-Vis spectrophotometer, constant temperature shaker, electronic balance, forced-air drying oven, biosensor, fume hood.

[0043] Culture medium formulation: Screening medium (PDA): 200g peeled potatoes, 20g glucose, 20g agar, 1L deionized water. Weigh 200g of peeled potatoes, cut them into small pieces, put them in a pot with 1000mL of water, and boil for 20-30 minutes until the potatoes soften. Filter the potato pieces through gauze, collect the filtrate, and discard the residue. Return the filtrate to the pot, add 15-20g of agar, heat over low heat and stir constantly to prevent scorching or overflow, until the agar is completely dissolved. Add 20g of glucose and stir to dissolve. Add water to a total volume of 1000mL, depending on water loss during heating. While still hot, dispense the medium into test tubes or Erlenmeyer flasks, plug the test tubes with cotton plugs, and if necessary, wrap them with kraft paper and secure with twine. Then autoclave (121°C, 15-20 minutes) to ensure sterility.

[0044] Seed culture medium: Seed solid culture medium: 30g glucose monohydrate, 2g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 20g agar powder, add deionized water to make up to 1L, and sterilize in an autoclave at 118℃ for 15min.

[0045] Seed liquid culture medium: 30g glucose monohydrate, 2g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, add deionized water to a final volume of 1L, and sterilize in an autoclave at 118℃ for 15min.

[0046] Fermentation medium: 45g glucose, 3g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, add deionized water to make up to 1L, sterilize in an autoclave at 115℃ for 20min to obtain ordinary fermentation medium.

[0047] Example 1: Method for Screening and Isolating Bacterial Strains Wild mushroom fruiting body samples were collected from a cultivation base (located in Jinan City, Shandong Province, China). After rinsing with sterile water and blotting with sterile filter paper, the samples were placed gill-down on sterile glass plates and incubated at 26°C for 24–48 hours. Once a large number of spores had fallen and formed spore prints, the spores were inoculated onto PDA agar plates and cultured at 26°C. After spore germination and the acquisition of a pure culture strain, the strain was transferred to PDA slant agar and cultured at 26°C until the mycelium filled the tube. The purified strain was then stored at 4°C for later use. The purified strain was then inoculated into a liquid medium containing rose components for shake-flask fermentation to evaluate changes in growth rate, biomass, and bioactive substance content.

[0048] A strain was obtained with white, fluffy colonies. Fermentation with roses can increase the content of its active ingredients. It was named ZR004. A biotechnology company was commissioned to perform fungal ITS gene sequencing and identification. The ITS-free sequence is shown in SEQ ID NO.1. It was identified as Schizophyllum commune and deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42265 and deposit date of September 18, 2025.

[0049] Example 2: Microbial Culture Method Plate culture: Pick mycelia of Schizophyllum commune, inoculate them onto the surface of seed solid culture medium, and culture at 25~30℃ for 5 days.

[0050] Seed culture: Select mycelia of Schizophyllum commune, inoculate them into seed culture medium, and culture at 25~30℃ and 200rpm for 5 days.

[0051] Fermentation tank culture conditions: temperature 25~30℃, rotation speed 200rpm, oxygenation culture, cycle 5 days.

[0052] Example 3: Fermentation of roses by Schizophyllum commune The process of fermenting roses with Schizophyllum commune is as follows: Figure 1 As shown.

[0053] Experimental Group 1: Pretreatment method: Pingyin rose flowers were ground into powder and sieved through a 100-mesh sieve. 50g of rose powder was taken and dissolved in deionized water to 1L. The mixture was stirred in a 30℃ water bath for 2 hours and then sterilized in an autoclave at 105℃ for 20 minutes to obtain rose extract.

[0054] Fermentation method: Add 5% rose extract (accounting for 5% of the total volume of the culture medium) to the fermentation medium, inoculate with 5% Schizophyllum commune ZR004 seed liquid, and ferment at 28℃ for 5 days.

[0055] Post-processing method: The fermentation broth was sterilized by incubating at 105℃ for 20 minutes. After cooling to room temperature, it was filtered through a 100-mesh filter cloth and centrifuged at 14000 r / min in a tubular centrifuge. The centrifuged liquid was sterilized at 105℃ for 20 minutes to obtain the fermentation filtrate of *Rosa schistosomiasis*.

[0056] Experimental Group 2: The amount of rose extract added was 10%, and other aspects were the same as in Experimental Group 1.

[0057] Experimental Group 3: The amount of rose extract added was 15%, and other aspects were the same as in Experimental Group 1.

[0058] Experimental group 4: The inoculum amount of rose extract was 20%, and the rest was the same as in experimental group 1.

[0059] Pretreatment method for Comparative Example 1: as in Experimental Group 1.

[0060] Cultivation method: Add 5% rose extract to the fermentation medium and incubate at 28℃ for 5 days.

[0061] Post-processing method: Performed according to Example 1 to obtain unfermented rose samples.

[0062] Comparative Example 2: The amount of rose extract added was 10%, and other aspects were the same as in Comparative Example 1.

[0063] Comparative Example 3: The amount of rose extract added was 15%, and other aspects were the same as in Comparative Example 1.

[0064] Comparative Example 4: Rose extract was added at 20%, and other parameters were the same as in Comparative Example 1.

[0065] Example 4: Determination of Polyphenol and Polysaccharide Content 1. Polyphenol content determination (Folin-Ciocalten method) Test principle: The Folin-Ciocalteu method is a method for determining polyphenol content using Folin-Ciocalteu as a reagent. Folin-Ciocalteu is a phenolic compound that can undergo an oxidation reaction with polyphenols to produce a dark brown product. The absorbance of this product is directly proportional to the polyphenol content. Therefore, the polyphenol content can be calculated by measuring the absorbance of the dark brown product.

[0066] Reagents and materials: 0.2 mol / L Folin-Ciocalteu reagent, 15% sodium bicarbonate solution, gallic acid standard solution, and sample solution.

[0067] Experimental steps: Preparation of the standard curve: Accurately transfer 0 μL, 100 μL, 200 μL, 300 μL, 400 μL, and 500 μL of gallic acid standard solution with a concentration of 100 μg / L, and add 5.0 mL of 0.2 mol / L Folin-Ciocalteu reagent to each sample. Shake well and let stand for 5 min, then add 2.0 mL of 15% Na₂CO₃ solution and incubate at room temperature in the dark for 1 h. Use the 0 sample as a blank for zeroing (transmittance corrected to 100%), and measure the absorbance at 765 nm. Plot the standard curve with the standard solution concentration (μg / mL) on the x-axis and the absorbance on the y-axis.

[0068] Sample solution determination: Transfer 1 mL of the test solution into a test tube, repeat the above steps, and measure the absorbance at 765 nm. Calculate the content based on the regression equation established by the standard curve.

[0069] Experimental results: The experimental results are shown in Table 1. The table shows the polyphenol content increase rate before and after fermentation as calculated as (experimental group polyphenol content - comparative example polyphenol content) / comparative example polyphenol content × 100% under the same rose inoculum concentration. The results indicate that the rose inoculum concentrations for experimental groups 1-3 were all suitable. With rose extract inoculum concentrations between 5% and 15%, the polyphenol increase rate was approximately 26%. If the rose inoculum concentration was further increased to 20%, the polyphenol increase rate decreased (experimental groups 1-3 vs. experimental group 4).

[0070] Table 1. Increase rate of polyphenol content before and after fermentation under different rose inoculation amounts.

[0071] 2. Determination of total sugar content (sulfuric acid phenol method) Test principle: Under the action of concentrated sulfuric acid, polysaccharides and oligosaccharides in the sample are first hydrolyzed into monosaccharides. These monosaccharides rapidly dehydrate under strong acid and high temperature conditions, generating furfural (from pentose) or its derivative hydroxymethylfurfural (from pentose). The generated furfural or its derivative undergoes a condensation reaction with phenol to produce an orange-yellow compound with characteristic absorption in the visible light region. This orange-yellow compound has a maximum absorption peak near 490 nm (usually between 480-490 nm). Within a certain concentration range, the absorbance of this solution is directly proportional to the total sugar content in the sample; therefore, quantitative analysis can be achieved by plotting a standard curve.

[0072] Reagents and materials: Glucose (analytical grade), concentrated sulfuric acid, phenol.

[0073] Fume hood, UV spectrophotometer, cuvettes; analytical balance; water bath; 100mL volumetric flasks; pipettes (200μL, 1000μL); pipettes (1mL, 5mL); wash bulbs; test tubes.

[0074] Experimental steps: (1) Reagent preparation: ①100μg / mL glucose standard solution: Dry glucose powder at 105℃ to constant weight, accurately weigh 0.0100g, dissolve in deionized water and dilute to 100mL to obtain a glucose standard solution with a concentration of 100μg / mL, which is valid for 2 weeks.

[0075] ②6% phenol solution: Weigh 6g of phenol powder and dilute to 100mL, then store in a brown bottle for later use.

[0076] (2) Preparation of standard curve: Accurately transfer 0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2 mL of 100 μg / L glucose standard solution, add deionized water to a final volume of 2 mL, and add 2 mL of the test solution diluted to an appropriate concentration to each test tube. Then add 1 mL of 6% phenol solution to each tube, shake well, and quickly add 5.0 mL of concentrated sulfuric acid solution. Mix well and let stand for 10 min, then place in a boiling water bath for 15 min. After cooling to room temperature, use the 0 sample as a blank for zeroing (transmittance calibration 100%), and measure the absorbance at 490 nm. Plot the standard curve with the standard solution concentration (μg / mL) on the ordinate and the absorbance on the abscissa.

[0077] (3) Sample solution determination: Transfer 2 mL of the sample solution to be tested into a test tube, repeat the above steps, and calculate its content based on the regression equation established by the standard curve.

[0078] Experimental results: Table 2 shows the polysaccharide content of *Schizophyllum commune* rose fermentation products obtained in each experimental group and comparative group. The polysaccharides were derived from rose polysaccharides and *Schizophyllum commune* polysaccharides. Comparative groups 1-4 all showed rose polysaccharide content, while experimental groups 1-4 showed the combined content of rose polysaccharides and *Schizophyllum commune* polysaccharides. Experimental group 3 had the highest overall polysaccharide content, and experimental group 4 had the highest rose polysaccharide content (as seen in comparative group 4). However, its overall polysaccharide content was lower than that of experimental group 3. This may be because the high concentration of rose polysaccharides inhibited the growth and metabolism of *Schizophyllum commune*, leading to a decrease in its *Schizophyllum commune* polysaccharide yield.

[0079] Table 2 Polysaccharide Content

[0080] Example 5: Optimization of Schizophyllum commune inoculum size Experimental group 5: The inoculum amount of Schizophyllum commune was 2.5%, and the rest was the same as in experimental group 3.

[0081] Experimental group 6: The inoculum amount of Schizophyllum commune was 7.5%, and the rest was the same as in experimental group 3.

[0082] Comparative Example 5: Fermentation Method: *Schizophyllum commune* ZR004 (5% inoculum) was inoculated into 1L of ordinary fermentation medium and fermented at 28℃ for 5 days to obtain *Schizophyllum commune* fermentation broth. The broth was then sterilized at 105℃ for 20 minutes (i.e., the *Schizophyllum commune* fermentation broth and rose extract were physically mixed without fermentation). Post-treatment Method: After cooling to room temperature, 150g of rose extract was added and stirred evenly. The mixture was filtered through a 100-mesh filter cloth and centrifuged at 14000 r / min in a tubular centrifuge. The centrifuged liquid was sterilized at 105℃ for 20 minutes to obtain a physically mixed *Schizophyllum commune* and rose extract sample.

[0083] The experiment of Example 4 was repeated, and the results are shown in Tables 3 and 4. Experimental groups 3, 5-6, and Comparative Example 3 were compared by changing the inoculum amount of Schizophyllum commune fermentation broth to increase the polyphenol yield and polysaccharide yield: the optimal inoculum amount of Schizophyllum commune fermentation broth was determined to be 5%.

[0084] If the inoculum concentration of Schizophyllum commune is 2.5% (experimental group 5), compared with experimental group 3, the polyphenol enhancement rate and polysaccharide production decrease. This may be because low concentration of Schizophyllum commune inoculation is not conducive to the growth of the strain at a concentration of 15% rose extract, and metabolism is inhibited.

[0085] If the inoculum concentration of Schizophyllum commune is 7.5% (experimental group 6), compared with experimental group 3, the polyphenol increase rate remains unchanged, but the polysaccharide production decreases. That is, with high concentration of Schizophyllum commune inoculation, the carbon source and other nutrients in the culture medium may flow more to cell construction rather than polysaccharide synthesis.

[0086] Compared to control group 5, experimental group 3 showed no significant decrease in polysaccharide content, indicating that the concentration of rose added was appropriate and did not inhibit the metabolic synthesis of Schizophyllum commune. Fermentation showed a significant advantage over non-fermentation (increased polyphenol production rate, with no significant difference in polysaccharide yield), meaning that rose participates in the growth and metabolism of Schizophyllum commune, and there is a biotransformation process between Schizophyllum commune and rose, rather than a product of simple physical mixing.

[0087] Table 3. Increase rate of polyphenol content after fermentation under different inoculum amounts of *Schizophyllum commune*.

[0088] Table 4. Polysaccharide content at different *Schizophyllum commune* inoculum amounts

[0089] The polyphenol content increase rate was highest in experimental groups 1-3, while the polysaccharide content was highest in experimental groups 3, 6, and comparative example 5. From the perspective of both polyphenols and polysaccharides, experimental group 3 was better. Subsequent efficacy verifications all used the fermentation filtrate obtained from the process of experimental group 3.

[0090] Example 6: Efficacy Test 1. Moisturizing test experiment Test Objective: Aquaporin 3 (AQP3) is an important subtype of the aquaporin family and one of the most abundant aquaporins expressed in the skin. It is mainly distributed in the epidermis and basal layer, playing a crucial role in skin hydration by maintaining the balance between intercellular and intracellular hydration levels. This test uses human immortalized keratinocytes (HaCaT) as the research subject and employs immunofluorescence technology to detect the AQP3 content in the cells to evaluate the moisturizing efficacy of the test samples.

[0091] Main reagents: high glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), MTT (Sigma), DMSO (Sinopharm), primary antibody (Abcam), secondary antibody (Abcam), BSA (Sigma).

[0092] Main equipment: CO2 incubator (Thermo, 160i), biosafety cabinet (Sujing Antai, BSC-1604ⅡA2), inverted fluorescence microscope (Keyence BZ-X1000), microplate reader (Tecan, Spark).

[0093] Test method: A. Cytotoxicity test 1) Cell seeding: at 1×10 4 Cells were seeded at a density of cells / well into 96-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0094] 2) Experimental Groups: The experiment included a zeroing group, a control group, a positive control group, and a sample group. In the sample group, each sample had 8 concentration gradients, and each concentration gradient had 3 replicate wells.

[0095] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table.

[0096] Table 5 Test Concentration Setting Table

[0097] 4) Sample Loading: Load samples when the cell seeding rate in the 96-well plate reaches 40%–60%. Add 200 μL of culture medium containing 10% PBS to each well of the control group; add 200 μL of culture medium containing 10% DMSO to each well of the positive control group; add 200 μL of culture medium containing the corresponding concentration of the sample to each well of the sample group; add 200 μL of cell culture medium to the zeroing group without cell seeding. After sample loading, place the 96-well plate in an incubator (37℃, 5% CO2) for incubation.

[0098] 5) Detection: After culturing cells for 24 h, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37℃ in the dark for 4 h. After incubation, discard the supernatant, add 100 μL DMSO to each well, and read the OD value at 490 nm.

[0099] 6) Cell viability calculation: Calculated according to the formula. Cell viability = [(sample well OD - zeroing well OD) / (solvent control well OD - zeroing well OD)] × 100%.

[0100] Eight sample loading concentrations were set, and cytotoxicity assays were conducted. The MTT assay results are shown in Table 6.

[0101] Table 6 MTT Test Results

[0102] A cell viability graph was plotted with the eight selected concentrations of the sample on the x-axis and cell viability values ​​on the y-axis (see [link to graph]). Figure 2 ).

[0103] Therefore, according to the MTT results, the filtrate of *Rosa schistosomiasis* fermentation did not show keratinocyte toxicity in the concentration range of 5.0000% (V / V).

[0104] B. Hydration-related protein content test 1) Cell seeding: at 1×10 5 Cells were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0105] 2) Solution preparation: Prepare working solutions of the test substances using HaCaT cell culture medium at the concentrations listed in Table 7 of the experimental design.

[0106] Table 7 Experimental Design

[0107] 3) Sample Loading: According to the experimental design, when the cell deposition rate in the 24-well plate reaches 40%~60%, the original cell culture medium is aspirated, and samples are loaded into groups. For the experimental group, 1.0 mL of sample working solution is added to each well, with 3 replicates for each concentration. For the control group (BC group), 1.0 mL of cell culture medium is added to each well, with 3 replicates. The plates are then incubated in an incubator (37℃, 5% CO2) for 24 h.

[0108] 4) Sample collection: Discard the supernatant and rinse the cells 3 times with PBS.

[0109] 5) Immunofluorescence staining: Perform routine immunofluorescence staining. The main steps are: fixation, blocking, addition of primary antibody, addition of secondary antibody, DAPI counterstaining, and then taking pictures using a fluorescence microscope.

[0110] 6) Results analysis: The fluorescence intensity of AQP3 was quantitatively analyzed using Image Pro Plus software.

[0111] The AQP3 immunofluorescence staining results of the samples are as follows: Figure 3 As shown (green fluorescence represents AQP3, blue fluorescence represents the cell nucleus), the relative IOD values ​​are shown in Table 8 and... Figure 4 As shown.

[0112] Table 8. Results of AQP3 Immunofluorescence Analysis

[0113] Note: The significance of the sample group compared with the BC group is based on... express, p -value < 0.05 means , p -A value less than 0.01 indicates that .

[0114] Compared with the blank control (BC) group, the fluorescence intensity of AQP3 protein in the filtrate of *Rosa schistosomiasis* fermentation product was significantly enhanced at concentrations of 2.5% and 5.0% (V / V) (p < 0.05), with enhancement rates of 19.00% and 39.00%, respectively.

[0115] Conclusion: Based on the human immortalized keratinocyte (HaCaT) model, the fluorescence intensity of aquaporin 3 (AQP3) in the *Rosa schizophyllum commune* ferment filtrate was significantly enhanced at concentrations of 2.5% and 5.0% (V / V), with enhancement rates of 19.00% and 39.00%, respectively, and was statistically different from that in the BC group (p<0.05). This indicates that the *Rosa schizophyllum commune* ferment filtrate can increase the AQP3 content in keratinocytes and has a moisturizing effect.

[0116] 2. Repair test experiment Test Objective: Cell migration refers to the movement of cells after receiving migration signals or sensing stimulation from certain substances. Increased cell migration ability indicates a role in promoting skin barrier repair. The cell migration ability test, also known as the scratch test, involves artificially creating a blank area, called a "scratch," on the monolayer of cells when they have grown to a fused state. Cells at the edge of the scratch gradually move into the blank area, mimicking the process of cell migration in vivo to some extent. Treating cells with different substances can affect their function and may promote cell migration. Therefore, this experiment uses human fibroblasts as the research object to evaluate the repair efficacy of the sample on skin barrier damage by detecting the effect of the sample on cell migration.

[0117] Main reagents: low-glucose DMEM culture medium (Pronosai), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), trypsin (Gibco), DMSO (Sinopharm).

[0118] Main equipment: CO2 incubator (Thermo, 160i), biosafety cabinet (ESCO, LA2-6A1), inverted microscope (Leica DMi8), microplate reader (Tecan, Spark).

[0119] Test method: A. Cytotoxicity test The procedure is the same as in moisturizing experiment A, and the results are as follows: Eight sample loading concentrations were set, and cytotoxicity assays were conducted. The MTT assay results are shown in Table 9.

[0120] Table 9 MTT Test Results

[0121] A cell viability graph was plotted with the eight selected concentrations of the sample on the x-axis and cell viability values ​​on the y-axis (see [link to graph]). Figure 5 ).

[0122] Therefore, according to the MTT results, the filtrate of *Rosa schistosomiasis* fermentation did not show human fibroblast toxicity in the concentration range of 10.0000% (V / V).

[0123] B. Cell migration test 1) Collect cells in the logarithmic growth phase, at a cell density of 2 × 10⁻⁶. 5 Inoculate 1 per well into a 24-well culture plate.

[0124] 2) After culturing in an incubator (37℃, 5% CO2) for 24 h, make horizontal scratches perpendicular to the 24-well plate using a 200 μL pipette tip, wash the cells 3 times with PBS, and remove the scratched cells.

[0125] 3) Place in an incubator (37℃, 5% CO2) and incubate for 24 h. Set up 3 replicates for each group. The specific experimental group settings are shown in Table 10.

[0126] Table 10 Experimental Design

[0127] 4) Take pictures of each group of migrating cells using an inverted microscope, and calculate the average area of ​​the scratches using Image Pro Plus software.

[0128] Migration rate (%) = [(Initial scratch area - Current scratch area) / Initial scratch area] × 100%.

[0129] 5) Experimental results: The test results are shown in Table 11 and Figure 6 and Figure 7 .

[0130] Table 11 Summary of Cell Migration Results

[0131] Note: The significance of the sample group, PC group and BC group is as follows: This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0132] In the cell migration assay, the cell migration level in the PC group was significantly higher than that in the BC group (p<0.01), indicating that the positive control assay was effective. Compared with the BC group, the cell migration rate was significantly increased (p<0.05) after treatment with 10.00% (V / V) concentration of *Schizophyllum commune* fermentation filtrate, with an increase rate of 20.80%.

[0133] Conclusion: Based on the human fibroblast model, after treatment with the *Schizophyllum commune* rose ferment filtrate at a concentration of 10.00% (V / V) for 24 h, the cell migration rate of human fibroblasts was significantly increased, with an increase rate of 20.80%, which was statistically different from that of the BC group (p<0.05). This indicates that the *Schizophyllum commune* rose ferment filtrate can promote the cell migration of human fibroblasts and has a repair effect.

[0134] 3. Relief Test Experiment Test Objective: Skin irritation primarily involves the physiological processes of skin barrier-neurovascular-immune inflammation. Alleviating skin inflammatory responses can improve skin irritation. When cells are stimulated, they secrete factors such as tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-8 (IL-8), and interleukin-6 (IL-6), causing skin irritation. This experiment uses an LPS-stimulated RAW264.7 macrophage model to evaluate the soothing efficacy of test samples by detecting changes in the levels of inflammatory factors (TNF-α, IL-6, and IL-1α) secreted by cells.

[0135] Main reagents: High glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), LPS (Sigma), dexamethasone (Sigma), Mouse TNF-α ELISA kit (Shanghai Enzyme-Linked Biotechnology), Mouse IL-6 ELISA kit (Shanghai Enzyme-Linked Biotechnology), Mouse IL-1α ELISA kit (Shanghai Enzyme-Linked Biotechnology).

[0136] Main equipment: CO2 incubator (Thermo, 160i), biosafety cabinet (Sujing Antai, BSC-1604ⅡA2), microplate reader (Tecan, Spark).

[0137] Test method: A. Cytotoxicity test The procedure is the same as in moisturizing experiment A, and the results are as follows: Eight sample loading concentrations were set, and cytotoxicity assays were conducted. The MTT assay results are shown in Table 12.

[0138] Table 12 MTT Test Results

[0139] A cell viability graph was plotted with the eight selected concentrations of the sample on the x-axis and cell viability values ​​on the y-axis (see [link to graph]). Figure 8 Therefore, according to the MTT results, the *Rosa schistosomiasis* fermentation filtrate did not show macrophage RAW264.7 toxicity in the concentration range of 10.0000% (V / V).

[0140] B. Inflammatory factor content test The specific arrangements for the experimental groups are shown in Table 13.

[0141] Table 13 Experimental Design

[0142] 1) Cell seeding: at 1×10 5 Cells were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0143] 2) Solution preparation: Prepare working solutions of different concentrations of the test substance according to the experimental design table.

[0144] 3) Induction and Sample Loading: When the cell deposition rate in the 24-well plate reaches 40%–60%, add 100 μL of 10×LPS working solution to each well according to the experimental design. Shake the plate left and right to mix the drug. Simultaneously add the sample. The final LPS concentration is 1 μg / mL. Each group has 3 replicates. After loading, incubate in an incubator (37℃, 5% CO2) for 24 h.

[0145] 4) Sample collection: After incubation, collect the cell culture supernatant into EP tubes. After collection, freeze the samples at -80℃.

[0146] 5) TNF-α content detection: The detection was performed according to the instructions of the Mouse TNF-α ELISA kit.

[0147] IL-6 level detection: Perform the detection according to the instructions of the Mouse IL-6 ELISA kit.

[0148] IL-1α level detection: The detection was performed according to the instructions of the Mouse IL-1α ELISA kit.

[0149] 6) TNF-α content test results Based on the experimental method, cell supernatant was collected and TNF-α content was detected. The detection results are shown in Table 14, and the trend is shown in Figure 9.

[0150] Table 14 Summary of TNF-α Data

[0151] Note: Significance between the NC group and the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. Significance between the sample group, PC group, and NC group is indicated by... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0152] Compared with the BC group, the secretion of the inflammatory factor TNF-α by macrophages in the NC group was significantly increased (p<0.01), indicating that LPS stimulation modeling was successful in this experiment. Compared with the NC group, the secretion of the inflammatory factor TNF-α by macrophages in the PC group was significantly decreased at a dexamethasone loading concentration of 100 μg / mL (p<0.01), indicating that the positive control detection was effective. Compared with the NC group, the secretion of the inflammatory factor TNF-α by macrophages in the *Rosa schistosomiasis* fermentation filtrate was significantly decreased at concentrations of 5% and 10% (V / V) (p<0.05), with inhibition rates of 18.07% and 24.50%, respectively.

[0153] C.IL-6 content test results Based on the experimental method, cell supernatant was collected and IL-6 content was detected. The detection results are shown in Table 15, and the trend is shown in Figure 10.

[0154] Table 15 Summary of IL-6 Data

[0155] Note: Significance between the NC group and the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. Significance between the sample group, PC group, and NC group is indicated by... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0156] Compared with the BC group, the secretion of the inflammatory cytokine IL-6 by macrophages in the NC group was significantly increased (p<0.01), indicating that LPS stimulation modeling was successful in this experiment. Compared with the NC group, the secretion of the inflammatory cytokine IL-6 by macrophages in the PC group was significantly decreased at a dexamethasone loading concentration of 100 μg / mL (p<0.01), indicating that the positive control detection was effective. Compared with the NC group, the secretion of the inflammatory cytokine IL-6 by macrophages in the *Rosa schizophyllum commune* fermentation filtrate was significantly decreased at concentrations of 5% and 10% (V / V) (p<0.05), with inhibition rates of 17.64% and 19.24%, respectively.

[0157] D.IL-1α content test results Based on the experimental method, cell supernatant was collected and IL-1α content was detected. The detection results are shown in Table 16, and the trend is shown in Figure 11.

[0158] Table 16 Summary of IL-1α Data

[0159] Note: Significance between the NC group and the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. Significance between the sample group, PC group, and NC group is indicated by... This means that a p-value < 0.05 is considered as... A p-value < 0.01 indicates that... .

[0160] Compared with the BC group, the secretion of the inflammatory cytokine IL-1α by macrophages in the NC group was significantly increased (p<0.01), indicating that LPS stimulation modeling was successful in this experiment. Compared with the NC group, the secretion of the inflammatory cytokine IL-1α by macrophages in the PC group was significantly decreased at a sample loading concentration of dexamethasone 100 μg / mL (p<0.01), indicating that the positive control detection was effective. Compared with the NC group, the secretion of the inflammatory cytokine IL-1α by macrophages in the *Rosa schizophyllum commune* fermentation filtrate was significantly decreased at concentrations of 5% and 10% (V / V) (p<0.01), with inhibition rates of 29.66% and 37.14%, respectively.

[0161] Conclusion: Based on the LPS-stimulated RAW264.7 macrophage model, the secretion of inflammatory factors TNF-α, IL-6, and IL-1α by RAW264.7 macrophages was significantly reduced at concentrations of 5% and 10% (V / V) of the *Schizophyllum commune* rose ferment filtrate. The inhibition rates of TNF-α were 18.07% and 24.50%, respectively; the inhibition rates of IL-6 were 17.64% and 19.24%, respectively; and the inhibition rates of IL-1α were 29.66% and 37.14%, respectively. These results showed statistically significant differences compared with the NC group (p<0.05), indicating that the *Schizophyllum commune* rose ferment filtrate can inhibit the secretion of inflammatory factors TNF-α, IL-6, and IL-1α by RAW264.7 macrophages and has a soothing effect.

[0162] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Schizophyllum commune, characterized in that, The name of the *Schizophyllum commune* is *Schizophyllum*. Schizophyllum commune ZR004 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42265 and deposit date of September 18, 2025.

2. A microbial culture, characterized in that, The microbial culture includes the Schizophyllum commune as described in claim 1 and / or its fermentation metabolites.

3. A microbial inoculant, characterized in that, The microbial agent includes the *Schizophyllum commune* as described in claim 1 or the microbial culture as described in claim 2, and a carrier.

4. A ferment of *Schizophyllum commune* rose, characterized in that, The *Schizophyllum commune* rose ferment is obtained by fermenting roses with the *Schizophyllum commune* as described in claim 1.

5. A composition, characterized in that, The composition comprises *Schizophyllum commune* as described in claim 1, or a microbial culture as described in claim 2, or a microbial agent as described in claim 3, or *Schizophyllum commune* rose ferment as described in claim 4.

6. The use of *Schizophyllum commune* as claimed in claim 1, the microbial culture as claimed in claim 2, the microbial agent as claimed in claim 3, the *Schizophyllum commune* rose ferment as claimed in claim 4, or the composition as claimed in claim 5, in any of the following aspects: A1. Applications in the preparation of moisturizing products; A2. Applications in the preparation of anti-inflammatory products; A3. Applications in the preparation of soothing products; A4. Applications in the preparation of products that promote skin repair.

7. The application according to claim 6, characterized in that, The products mentioned include at least pharmaceuticals and cosmetics.

8. A method for culturing the *Schizophyllum commune* as described in claim 1, characterized in that, The cultivation conditions are as follows: Plate culture: Pick mycelia of Schizophyllum commune, inoculate them onto the surface of seed solid culture medium, and culture at 25~30℃ for 4~6 days; Seed culture: Pick mycelia of Schizophyllum commune, inoculate them into seed culture medium, and culture at 25-30℃ and 150-250 rpm for 4-6 days; Fermentation tank culture conditions: temperature 25~30℃, rotation speed 150~250rpm, oxygenation culture, cycle 4~6 days.

9. A method for fermenting rose petals using the *Schizophyllum commune* as described in claim 1, characterized in that, The method includes at least the following steps: S1. Preparation of rose extract: Rose powder was stirred in a water bath at 25-35℃ for 1.5-2.5 hours at a material-to-liquid ratio of 1:20 g / mL to obtain rose extract; S2. Add 5%~15% rose extract by volume to the fermentation medium, inoculate with 5%~7.5% of the *Schizophyllum commune* seed liquid according to claim 1, ferment at 25~30℃ for 4~6 days to obtain *Schizophyllum commune* rose ferment.

10. The method according to claim 9, characterized in that, The method also includes the step of filtering and sterilizing the *Schizophyllum commune* rose ferment obtained in S2.

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