Medicinal fomitopsis ferments, preparation method and application

Abstract

The application discloses a medicinal Fomes fomentarius fermentation liquor, a preparation method and application thereof. The preparation method comprises the following steps: crushing medicinal Fomes fomentarius and Hibiscus sabdariffa flowers to obtain medicinal Fomes fomentarius powder and Hibiscus sabdariffa flower powder; first, anaerobic fermentation of the medicinal Fomes fomentarius is carried out by using a composite strain, and then aerobic fermentation is carried out by inoculating a Saccharomyces cerevisiae SM004 strain to obtain a medicinal Fomes fomentarius fermentation product; and the medicinal Fomes fomentarius fermentation product is sequentially subjected to coarse filtration, centrifugation, fine filtration and sterilization, and an antiseptic is added to obtain the fermentation liquor. Through the sectional fermentation process, the anaerobic fermentation of the medicinal Fomes fomentarius is first carried out by using the composite strain, and then the aerobic fermentation is carried out by inoculating the Saccharomyces cerevisiae SM004, so that the dissolution rate and the biological conversion efficiency of active ingredients are significantly improved. It is detected that the fermentation liquor is rich in polysaccharides, polyphenols, lactic acid and other active ingredients.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a medicinal fermentation broth of *Polyporus flocculationii*, its preparation method, and its application. Background Technology

[0002] Medicinal Fomitopsis pinnatifida is widely distributed in 16 provinces and regions of China, including Hebei, Gansu, Shanxi, and Inner Mongolia. It primarily grows in coniferous forests at altitudes of 800–3500 meters, mainly parasitizing wounds or dead branches of living coniferous trees such as spruce, larch, and Korean pine. Modern research indicates that this fungus is rich in polysaccharides, triterpenoids, flavonoids, and sterols, among other active ingredients, and possesses pharmacological effects such as immunomodulation, antitumor activity, antioxidant activity, and hypoglycemic activity.

[0003] However, in related technologies, the fermentation method of medicinal fumonis is difficult to achieve the all-round transformation of the complex matrix of medicinal fumonis, and the types and contents of active ingredients are limited.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a medicinal Fertilizer fermentation broth, its preparation method, and its application. This application employs a segmented fermentation process. First, a composite strain consisting of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, and *Lactobacillus casei* SM003 (CGMCC NO. 26537) is used for anaerobic fermentation of the medicinal Fertilizer. Then, *Saccharomyces cerevisiae* SM004 (CGMCC NO. 26536) is inoculated for aerobic fermentation. This significantly improves the dissolution rate and bioconversion efficiency of the active ingredients. Testing shows that the fermentation broth is rich in polysaccharides, polyphenols, lactic acid, and other active ingredients.

[0006] The Lactobacillus casei SM003 strain of this application is deposited at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101), on February 10, 2023, with accession number CGMCC NO:26537, and culture name: Lactobacillus casei SM003. Lactobacillus casei .

[0007] The *Saccharomyces cerevisiae* strain SM004 disclosed in this application is deposited at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101), on February 10, 2023, with accession number CGMCC NO:26536, and the culture name is *Saccharomyces cerevisiae* SM004. Sacchammyces cerevisiae .

[0008] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, a method for preparing a fermentation broth of medicinal *Polyporus floribunda* is provided, the method comprising the following steps: S100, preparation of fermentation culture medium: *Polyporus floribunda* and hibiscus flowers are pulverized and sieved to obtain *Polyporus floribunda* powder and hibiscus flower powder. The *Polyporus floribunda* powder, hibiscus flower powder, carbon source, and water are mixed in a certain proportion and added to a fermenter. After sterilization, the mixture is cooled and controlled, and then heated to 85°C for sterilization for 20 min; S200, continuous stepwise fermentation with mixed strains: first, the *Polyporus floribunda* is anaerobically fermented using a compound strain, and then *Saccharomyces cerevisiae* strain SM004 is inoculated for aerobic fermentation to obtain the fermentation product of *Polyporus floribunda*; the compound strain consists of *Lactobacillus bulgaricus* strain ATCC 11842, *Streptococcus thermophilus* strain ATCC 19258, *Lactobacillus acidophilus* strain ATCC 4356, and *Lactobacillus plantarum* strain ATCC. The culture consisted of strain 14917 and strain Lactobacillus casei SM003, wherein strain SM003 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 10, 2023, with accession number CGMCC NO:26537 and culture name Lactobacillus casei SM003; S300, post-treatment: the fermentation product of medicinal blastocysteine ​​was subjected to coarse filtration through 100-400 mesh, centrifugation, fine filtration through 0.1μm plate and frame filtration, sterilization at 115-121℃ for 20-30 min, and the addition of preservatives to obtain the fermentation broth.

[0009] In some embodiments, step S200 includes: S210, preparing a compound bacterial culture and a yeast culture: The compound bacteria are inoculated from a glycerol culture tube into MRS medium and cultured at 37°C for 12-20 hours to obtain a compound bacterial culture; wherein the mass ratio of the compound bacteria to the MRS medium is 1:100; Saccharomyces cerevisiae SM004 strain is inoculated into a yeast culture medium and cultured at 30°C for 8-16 hours to obtain a yeast culture; wherein the mass ratio of the Saccharomyces cerevisiae SM004 strain to the yeast culture medium is 1:100; wherein the Saccharomyces cerevisiae SM004 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 10, 2023, with accession number CGMCC NO:26536, and the culture name is Saccharomyces cerevisiae SM004. S220, First stage fermentation: Inoculate the compound bacterial solution into the fermentation medium, stir at a low speed of 50-100 rpm, introduce nitrogen gas at an aeration rate of 0.1-0.6 vvm to ensure dissolved oxygen is 0%, and maintain the anaerobic fermentation temperature at 30-45℃. Monitor the pH in real time, and stop the culture when the pH drops to 3.5-4.0 to end the first stage fermentation; S230, Second stage fermentation: Adjust the pH to 6.0-6.5, inoculate the yeast solution, stir at a speed of 200-600 rpm, introduce air at an aeration rate of 0.1-1 vvm, and carry out aerobic fermentation for 25-38 hours at an aerobic fermentation temperature of 24-30℃ to obtain the medicinal cerevisiae fermentation product.

[0010] In some embodiments, the ratio of viable counts of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, and *Lactobacillus casei* SM003 in the composite bacterial solution is (0.2~0.5):(0.25~0.7):(0.25~0.7):(0.3~1):(0.25~0.7), and the viable count concentration in the yeast solution is not less than 1×10⁻⁶. 5 CFU / mL.

[0011] In some embodiments, the total inoculation amount of the compound bacterial solution and yeast solution in the fermentation medium is 5-10%; the mass ratio of the compound bacterial solution to the yeast solution is 1:1.

[0012] In some embodiments, the yeast culture medium consists of water, yeast extract, peptone, and glucose, with addition amounts of 92-97.5%, 0.5-2%, 1-3%, and 1-3%, respectively.

[0013] In some embodiments, the amounts of medicinal Floodioides, roselle flowers, and carbon source added to the fermentation medium are 1-10%, 0.1-2.0%, and 1.5-3%, respectively. The carbon source includes, but is not limited to, maltose, skim milk powder, and glucose. The medicinal Floodioides is Ficus pumila.

[0014] In some embodiments, the preservative is one or more of p-hydroxyacetophenone, 1,2-hexanediol, and 1,2-pentanediol, with a total addition amount of 1-5%.

[0015] According to another aspect of this application, a medicinal fermentation broth of *Polyporus flocculationii* is provided, wherein the filtrate is prepared by the aforementioned preparation method, and the filtrate contains polysaccharide content ≥1.76 mg / g, lactic acid content ≥1.58 mg / g, total sugar content ≥10.45 mg / g, and polyphenol content ≥0.199 mg / g.

[0016] According to another aspect of this application, there is an application of medicinal Fertilizer of Polyporus flocculation in the preparation of skin care products, which have firming and repairing, antibacterial and antiseptic and / or skin microecological regulating effects.

[0017] In some embodiments, the amount of the filtrate added to the skin care product is 0.5%–10%.

[0018] Compared with the prior art, the advantages and positive effects of this application are as follows: This application uses a segmented fermentation process. First, it uses a composite strain composed of Lactobacillus bulgaricus ATCC 11842, Streptococcus thermophilus ATCC 19258, Lactobacillus acidophilus ATCC 4356, Lactobacillus plantarum ATCC 14917, and Lactobacillus casei SM003 (CGMCC NO. 26537) to carry out anaerobic fermentation of medicinal stromalfoss. Then, it inoculates Saccharomyces cerevisiae SM004 (CGMCC NO. 26536) for aerobic fermentation. This significantly improves the dissolution rate and bioconversion efficiency of active ingredients. The fermentation broth is rich in active ingredients such as polysaccharides, polyphenols, and lactic acid.

[0019] Furthermore, this fermentation broth possesses excellent antioxidant capacity, elastase inhibitory activity, Propionibacterium acnes inhibition effect, and keratinocyte scratch repair promotion effect. It can achieve multiple effects such as firming and repairing, antibacterial and antiseptic, and regulating skin microecology. Moreover, it is highly safe and has low irritation, and has broad application prospects in the field of skin care products. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of DPPH radical scavenging ability of Embodiment 1 of this application are shown.

[0022] Figure 2 The image shows a scratch healing experiment of Embodiment 1 of this application.

[0023] Figure 3 The results of the 24-hour scratch healing rate of Example 1 of this application are shown.

[0024] Figure 4 The analysis shows the difference in elastase inhibition rate in Example 1 of this application.

[0025] Figure 5 The diagram shows a comparison of enzyme activity in Example 1 of this application.

[0026] Figure 6 The results of the moisturizing rate of Example 1 of this application are shown.

[0027] Figure 7 The chromatogram of Example 1 of this application is shown. Detailed Implementation

[0028] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0029] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1: S100, Fermentation medium preparation: Mix 5 parts of medicinal blastocystis powder, 2 parts of maltose, 1 part of roselle pollen and 92 parts of water, and put them into a 5L automatic control fermenter for sterilization (115℃, 30 min), then cool and adjust the pH to 6.0.

[0031] The medicinal porphyria used in this embodiment is *Phellinus pini*. *Phellinus pini*, also known as pine needle porphyria, is a fungus belonging to the genus *Phellinus* in the family Phyllostachyaceae, and is a type of medicinal porphyria. Roselle pollen is used as a vitamin in the culture medium.

[0032] S200, Mixed strain fermentation: First, the medicinal *Polyporus* is anaerobically fermented using a compound strain, then *Saccharomyces cerevisiae* strain SM004 is inoculated for aerobic fermentation. Specific steps include S210-S230: S210. Preparation of compound bacterial culture and yeast culture: The compound bacterial culture is inoculated from the glycerol culture tube into MRS medium and cultured at 37℃ for 12-20 hours to obtain the compound bacterial culture; wherein the inoculation amount of the compound bacterial culture is 1%, that is, the mass ratio of the compound bacterial culture to the MRS medium is 1:100; Saccharomyces cerevisiae SM004 strain is inoculated into yeast medium and cultured at 30℃ for 8-16 hours to obtain the yeast culture; wherein the inoculation amount of Saccharomyces cerevisiae SM004 strain is 1%, that is, the mass ratio of Saccharomyces cerevisiae SM004 strain to the yeast medium is 1:100; wherein the Saccharomyces cerevisiae SM004 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 10, 2023, with accession number CGMCC NO:26536, and the culture name is Saccharomyces cerevisiae SM004. Sacchammyces cerevisiae .

[0033] The yeast culture medium consists of 95 parts water, 1 part yeast extract, 2 parts peptone, and 2 parts glucose.

[0034] S220, First stage fermentation: Introduce the compound bacterial solution into a 5L fermenter containing 3L of fermentation medium, stir at a low speed of 50rpm, introduce nitrogen gas at a flow rate of 2NL / min to ensure 0% dissolved oxygen, and maintain the anaerobic fermentation temperature at 35℃. Monitor the pH in real time. Do not control the pH during the fermentation process. Stop the culture when the pH drops to 3.5-4.0 to end the first stage fermentation. Then, heat to 85℃ for sterilization for 20min. S230, Second stage fermentation: Adjust the pH to 6.0, add the yeast liquid, stir at 200-600 rpm, introduce air (stop introducing nitrogen and introduce air instead, i.e., sterile air after air has been filtered by an air compressor and a filter membrane), the aeration rate is 2NL / min, the temperature of aerobic fermentation is 28℃, aerobic fermentation is carried out for 32 hours, and the medicinal Fermentation Product of Fertilizer is obtained.

[0035] The viable cell ratios of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, *Lactobacillus casei* SM003, and *Saccharomyces cerevisiae* SM004 were 0.5:0.25:0.25:0.5:1:2, with the viable cell concentration of *Lactobacillus casei* SM003 at inoculation being 2.5 × 10⁻⁶. 5 CFU / ml.

[0036] The total inoculum amount of the compound bacterial solution and yeast solution in the fermentation medium was 10%; the mass ratio of the compound bacterial solution to the yeast solution was 1:1.

[0037] S300, Post-processing: The fermentation product of medicinal Fertilizer is sequentially subjected to coarse filtration through 400 mesh, centrifugation, fine filtration through 0.1μm plate and frame filtration, sterilization at 115℃ for 30 min, and the filtrate is collected. Then, 0.5% p-hydroxyacetophenone and 1% 1,2-hexanediol at the total mass of the filtrate are added as preservatives, mixed well, and the filtrate is bottled to obtain the fermentation broth of medicinal Fertilizer.

[0038] Example 2: S100, Fermentation medium preparation: Mix 10 parts of medicinal Poria cocos powder, 1.5 parts of skim milk powder, 2 parts of roselle pollen and 86.5 parts of water, and put them into a 5L automatic fermentation tank for sterilization (121℃, 20 min). After cooling, control the pH value to 6.0. S210: This step is the same as S210 in Example 1, and will not be repeated here.

[0039] S220, First stage fermentation: Introduce the compound bacterial solution into a 5L fermenter containing 3L of fermentation medium, introduce nitrogen to ensure 0% dissolved oxygen, and aerate at 2NL / min. Ferment at 35℃ with a rotation speed of 100rpm. Monitor the pH in real time. Do not control the pH during the fermentation process. Stop the culture when the pH drops to 3.5-4.0 to end the first stage fermentation. Then, heat to 85℃ for sterilization for 20min. S230, Second stage fermentation: Adjust the pH to 6.0, add the yeast liquid, stir at 200-600 rpm, introduce air at a rate of 2 NL / min, aerobic fermentation temperature is 26℃, aerobic fermentation is carried out for 35 hours, and medicinal Fermentation product is obtained.

[0040] The viable cell ratios of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, *Lactobacillus casei* SM003, and *Saccharomyces cerevisiae* SM004 were 0.2:0.5:0.5:1:2:4, with the viable cell concentration of *Lactobacillus casei* SM003 at inoculation being 3.2 × 10⁻⁶. 5 CFU / ml.

[0041] The total inoculum amount of the compound bacterial solution and yeast solution in the fermentation medium was 6%; the mass ratio of the compound bacterial solution to the yeast solution was 1:1.

[0042] S300, Post-processing: The fermentation product of medicinal fumonis is sequentially subjected to 400-mesh coarse filtration, centrifugation, 0.22μm plate and frame filtration, sterilization at 121℃ for 30min, preservative is added, and the product is bottled to obtain the fermentation broth.

[0043] Example 3: S100, Fermentation medium preparation: Mix 10 parts of medicinal Poria cocos powder, 2 parts of skim milk powder, 2 parts of roselle pollen and 86 parts of water, and put them into a 5L automatic control fermenter for sterilization (121℃, 20 min), then cool and control the pH value to 6.0. S210: This step is the same as S210 in Example 1, and will not be repeated here.

[0044] S220, First stage fermentation: Introduce the compound bacterial solution into a 5L fermenter containing 3L of fermentation medium, introduce nitrogen to ensure 0% dissolved oxygen, and aerate at 2NL / min. Ferment at 36℃ with a rotation speed of 80rpm. Monitor the pH in real time. Do not control the pH during the fermentation process. Stop the culture when the pH drops to 3.5-4.0 to end the first stage fermentation. Then, heat to 85℃ for sterilization for 20 min. S230, Second stage fermentation: Adjust the pH to 6.0, add the yeast liquid, stir at 200-600 rpm, introduce air at a rate of 2 NL / min, aerobic fermentation temperature is 30℃, aerobic fermentation is carried out for 38 hours, and medicinal Fermentation product is obtained.

[0045] The viable cell ratios of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, *Lactobacillus casei* SM003, and *Saccharomyces cerevisiae* SM004 were 0.3:0.7:0.7:0.3:4:1, and the viable cell concentration of *Lactobacillus casei* SM003 at inoculation was 2.8 × 10⁻⁶. 5 CFU / ml.

[0046] The total inoculum amount of the compound bacterial solution and yeast solution in the fermentation medium was 8%; the mass ratio of the compound bacterial solution to the yeast solution was 1:1.

[0047] S300, Post-processing: The fermentation product of medicinal fumonis is sequentially subjected to 200-mesh coarse filtration, centrifugation, 0.1μm plate and frame fine filtration, sterilization at 121℃ for 20min, preservative is added, and the product is bottled to obtain the fermentation broth.

[0048] I. Component analysis of Examples 1-3

[0049] Table 1 shows the component analysis results of Examples 1-3. The polysaccharide content was determined using SN / T 4260-2015 "Determination of Crude Polysaccharides in Exported Plant-Derived Foods - Phenol-Sulfuric Acid Method"; the lactic acid content was determined using GB 5009.157-2016 "National Food Safety Standard - Determination of Organic Acids in Food"; the total sugar content was determined using GB / T 15672-2009 "Determination of Total Sugar Content in Edible Fungi"; and the polyphenol content was determined using GB / T 8313-2018 "Determination Method for Tea Polyphenols and Catechins in Tea". As shown in Table 1, the polysaccharide content of the medicinal *Polyporus flocculationii* fermentation broth was ≥1.76 mg / g, the lactic acid content was ≥1.58 mg / g, the total sugar content was ≥10.45 mg / g, and the polyphenol content was ≥0.199 mg / g.

[0050] Table 1 Component Analysis of Examples 1-3

[0051] II. Examples 1-3: Acne-removing and antibacterial effects.

[0052] 1. Experimental Objective and Principle Based on the fact that *Propionibacterium acnes* is a normal component of the hair follicle flora, and that *Propionibacterium acnes* proliferates during the inflammatory process of acne, this study aims to evaluate the acne-reducing efficacy of samples by detecting its antibacterial effect on *Propionibacterium acnes*. *Propionibacterium acnes*, a component of the normal hair follicle flora, uses sebum as its active ingredient, partially converting it into free fatty acids. *Propionibacterium acnes* can also activate catalysis, producing at least two chemokines that promote inflammation. Therefore, inhibiting *Propionibacterium acnes* can achieve an acne-reducing effect. The acne-reducing efficacy of test samples is evaluated by detecting their antibacterial effect on *Propionibacterium acnes*.

[0053] 2. Test indicators: Experimental indicator: Antibacterial rate of Propionibacterium acnes.

[0054] Judgment criteria: If the antibacterial rate is >50%~90%, the product has an antibacterial effect; if the antibacterial rate is >90%, the product has a strong antibacterial effect, which proves that the test sample has achieved the effect of removing acne.

[0055] 3. Test materials: 3.1. Test sample: Fermentation broth of medicinal blastocystis from Examples 1-3.

[0056] 3.2. Test strain: Propionibacterium acnes (ATCC11827) was a third-generation strain provided by Guangdong Huankai Technology Co., Ltd.

[0057] 3.3. Instruments and equipment: Biochemical incubator SHP-160, serial number MCJY-002, biosafety cabinet, serial number MCJY-219.

[0058] 4. Test methods: 4.1 Range of bacterial suspensions used in the experiment: 1×10 4 -9×10 4 CFU / mL.

[0059] 4.2 Refer to WS / T650-2019 "Evaluation Methods for Antibacterial and Bacteriostatic Effects" for quantitative suspension inhibition test.

[0060] 4.3 Quantitative test of bacterial suspension: test concentration 1%, reaction time 5 min, test repeated three times.

[0061] 4.4 Prepare approximately 1×10⁻⁶ m³ of fresh slant culture. 4 ~9×10 4 Set aside the CFU / mL bacterial suspension. Take 0.4mL of bacterial suspension and add 20g of sample diluent. Quickly mix and start timing immediately. Inoculate two dishes with the sample mixture and pour in the culture medium. Repeat the experiment three times. Place in a 36℃ biochemical incubator and incubate for 48 hours. Observe the results, count the viable bacteria, and calculate the inhibition rate.

[0062] 4.4 Perform viable bacterial culture and count, and calculate the inhibition rate.

[0063] 4.5 Calculation formula: X = (AB) / A × 100%; Where: X - inhibition rate (%); A - average recovered colonies of control sample; B - average recovered colonies of test sample.

[0064] 5. Test Results: Table 2 Results of Antibacterial Efficacy of Examples 1-3

[0065] 6. Evaluation Conclusion: As shown in Table 2, referring to the evaluation method of antibacterial and bacteriostatic effects in WS / T 650-2019, after adding Propionibacterium acnes (ATCC11827) to the 1% dilution of the sample to be tested and acting for 5 minutes, the antibacterial rates of Propionibacterium acnes (ATCC11827) in Examples 1-3 were 71.60%, 69.09%, and 70.36%, respectively. This indicates that under these conditions, the fermentation broth of Pterocarpus medicata in Examples 1-3 has an antibacterial effect on Propionibacterium acnes (ATCC11827), proving that the fermentation broth of Pterocarpus medicata in Examples 1-3 has acne-removing effects.

[0066] III. Example 1-3: DPPH free radical scavenging ability.

[0067] 1. Experimental Objectives and Principles DPPH radicals are very stable nitrogen-centered free radicals and are one of the important indicators of the antioxidant capacity of a sample. They are widely used in the research of antioxidant foods, pharmaceuticals and cosmetics.

[0068] DPPH free radicals have unpaired electrons, and their alcoholic solution is purple with strong absorption at 515 nm. When antioxidants are present, DPPH free radicals are scavenged, the solution color lightens, and the absorbance at 515 nm decreases. Within a certain range, the absorbance is directly proportional to the degree of free radical scavenging. Therefore, the degree of decrease in absorbance can reflect the sample's ability to scavenge DPPH free radicals.

[0069] 2. Test Indicators Table 3 Test Indicators

[0070] 3. Experimental Materials and Methods: 3.1 Instruments and Equipment: UV-1000 UV-Vis Spectrophotometer (Shanghai Hanyi).

[0071] 3.2 Samples used in the test: DPPH free radical scavenging ability test kit (Solarbio, BC4750); Ganoderma lucidum spore oil.

[0072] 3.3 Test Methods: 3.3.1 Test operation procedures: Preheat the spectrophotometer for 30 minutes, adjust the wavelength to 515 nm, and zero it with anhydrous ethanol.

[0073] Procedure: Add the following reagents to a 1.5ml EP tube respectively.

[0074]

[0075] 4. Experimental Results: 4.1 Calculation formula: DPPH free radical scavenging rate D% = [[A blank - (A determination - A control)] ÷ A blank] × 100%.

[0076] 4.2 Results: In Example 1, when the concentrations of medicinal *Polyporus flocculationii* fermentation broth added were 0.1%, 0.5%, 1%, and 10%, the DPPH free radical scavenging rates reached 50.62%, 75.21%, 95.64%, and 99.35%, respectively. In Example 2, when the concentrations of medicinal *Polyporus flocculationii* fermentation broth added were 0.1%, 0.5%, 1%, and 10%, the DPPH free radical scavenging rates reached 45.83%, 68.66%, 84.63%, and 95.37%, respectively. In Example 3, when the concentrations of medicinal *Polyporus flocculationii* fermentation broth added were 0.1%, 0.5%, 1%, and 10%, the DPPH free radical scavenging rates reached 48.54%, 72.83%, 91.29%, and 95.77%, respectively. Figure 1 The results of DPPH radical scavenging ability of Embodiment 1 of this application are shown.

[0077] IV. Repairing effects of medicinal Fermentation Broth of Porphyromonas medicata in Examples 1-3

[0078] 1. Experimental Objective and Principle: When trauma occurs, keratinocytes are regulated by cytokines and the extracellular matrix, rapidly proliferating and migrating, forming granulation tissue to fill the wound, secreting collagen, and ultimately participating in scar formation. Keratinocytes can be used as a cell model to study the repair capabilities of cosmetics. By comparing the degree of scratch healing of keratinocytes after administration of the test substance with that of the negative control and after administration of the test substance, the efficacy of the test substance in repair can be evaluated.

[0079] 2. Test Indicators: Experimental index: Keratinocyte scratch healing rate; Judgment criteria: The scratch healing rate of the sample cells was higher than that of the negative group cells, indicating that the sample has a certain repair effect.

[0080] 3. Experimental Materials and Methods: 3.1 Instruments and equipment: EVOS inverted fluorescence microscope.

[0081] 3.2 Cells used in the experiment: keratinocytes (Hacat).

[0082] 3.3 Test Methods: 3.3.1 Sample preparation: Sample group: Dilute the sample with anhydrous ethanol to a volume fraction of 5%, then filter it through a 0.22 μm filter, and collect the filtrate as the sample stock solution.

[0083] Negative control: Incomplete culture medium.

[0084] 3.3.2 Test Operation Procedures: After culturing fibroblasts in 6-well plates for 24 hours, a streak was made in the center of each well using a sterile pipette tip, and the culture medium was discarded. Incomplete culture medium (negative control) and the corresponding sample group culture medium were added separately, and cell healing was observed and photographed at 0 h and 24 h.

[0085] 3.3.3 Calculation formula: 24h scratch healing rate (%) = (1 - 24h scratch square pixels / 0h scratch square pixels) × 100.

[0086] 3.3.4 Data Analysis: Quantitative analysis of cell scratch images at 0h and 24h was performed using Image-pro plus (IPP) image analysis software.

[0087] 4. Experimental Results: Table 4 shows the results of the cell scratch assay. Following the laboratory method (HMC-WI-043 cell scratch assay), the negative control showed low cell scratch healing after 24 hours, with a healing rate of 10.56%. In Examples 1-3, at a concentration of 0.05%, the cell scratch healing rates after 24 hours were 19.71%, 16.57%, and 17.06%, respectively. The 0.05% concentration of the sample promoted cell scratch healing, indicating that Examples 1-3 all possessed certain repair efficacy. Figure 2 The image shown is an experimental image of scratch healing according to Embodiment 1 of this application. Figure 3 This is the 24-hour scratch healing rate result in Example 1 of this application.

[0088] Table 4 Results of cell scratch test

[0089] V. Anti-wrinkle and firming effects of medicinal Fermentation Broth of Pterocarpus 1-3

[0090] 1. Testing principle: Elastase is mainly synthesized and secreted by fibroblasts. It is a protein-degrading enzyme with extremely high selectivity and specificity. It catalyzes the hydrolysis of many amino acids, such as glycine, leucine, alanine, and valine, which contain carboxyl groups in their polypeptide bonds. This can cause the breakdown of elastin in connective tissue proteins, leading to skin laxity and wrinkles.

[0091] The principle of the elastase inhibition assay is that elastase reacts with the enzyme substrate, and the absorbance changes after the addition of the active substance. The magnitude of the change in absorbance can reflect the inhibition rate of the elastase inhibitor.

[0092] This test method is an in vitro method, which mainly characterizes the anti-wrinkle and firming efficacy by evaluating the inhibition rate of elastase activity of the test sample. It is suitable for evaluating cosmetics that achieve anti-wrinkle and firming effects by inhibiting elastase.

[0093] 2. Test method: 2.1. Add appropriate amounts of buffer solution, sample solutions or control solutions of different concentrations, and substrate to a 96-well plate, vortex to mix, and incubate for 20 min. Then add elastase solution, vortex immediately, and incubate for 10 min. Measure the absorbance at 410 nm using a microplate reader, analyze and process the data to calculate the inhibition rate of the test sample against elastase.

[0094] 2.2. Result Calculation: Elastase inhibition rate (%) = [1 - (AB) / (CD)] × 100%; In the formula: A is the absorbance of the experimental group (containing substrate, buffer solution, elastase solution, sample solution (sample group) / quercetin solution (positive control group) / water (negative control group); B represents the absorbance of the experimental blank group (containing substrate, containing buffer solution, containing elastase solution, containing sample solution (sample group) / quercetin solution (positive control group) / water (negative control group)). C represents the absorbance of the control group (containing substrate, buffer solution, elastase solution, and no sample solution); D represents the absorbance of the blank group (containing substrate, buffer solution, elastase solution, and sample solution).

[0095] Table 5 shows the experimental groups and test results. As shown in Table 5, the anti-wrinkle and firming effects of the samples were tested according to the laboratory method (XYJC-SOP-CE-019 elastase inhibition rate method). The test results showed that, under the premise of ensuring the effectiveness of the experimental reaction system (positive control elastase inhibition rate > 50%), when the concentration of Examples 1-3 was 1%, the elastase inhibition rates were 14.03%, 13.21%, and 12.70%, respectively, which were significantly different from the negative control (P < 0.05). This indicates that when the concentration of Examples 1-3 was 1%, it had anti-wrinkle and firming effects. Figure 4 The results for elastase inhibition rate in Example 1 of this application are shown.

[0096] Table 5 Experimental Groups and Test Results

[0097] Note: p<0.05 indicates a statistically significant difference compared to the negative control group, while p≥0.05 indicates no statistically significant difference compared to the negative control group.

[0098] VI. Oil-controlling effects of medicinal Fermentation Broth of Pterocarpus medicata in Examples 1-3.

[0099] 1. Experimental Objective and Principle Sebum secretion is closely related to sebaceous gland function, which in turn is closely related to androgen regulation. 5α-reductase is a membrane protease located on microsomes and the cell nucleus. Using the reduced coenzyme NADPH as a hydrogen donor, it catalyzes the conversion of testosterone to dihydrotestosterone (DHT), which induces excessive sebum secretion from the sebaceous glands. Inhibiting 5α-reductase activity reduces DHT production and lowers sebaceous gland lipid secretion levels.

[0100] Through experimental design, the enzyme activity of 5α-reductase in the test sample was lower than that in the negative control (physiological saline), indicating that the catalytic rate of 5α-reductase in the negative control (physiological saline) was significantly faster than that in the test sample. Therefore, the test sample had a certain inhibitory effect on 5α-reductase, thus achieving the oil control effect.

[0101] Test indicators Experimental indicator: Enzyme activity of 5α-reductase.

[0102] Judgment criteria: The enzyme activity of the test sample was compared with that of the negative control (physiological saline). If the enzyme activity of the test sample was lower than that of the negative control (physiological saline), it indicates that the test sample had a significant inhibitory effect on 5α-reductase.

[0103] 3. Test Methods 3.1 The test sample was 1% of the fermentation broth of medicinal blastocystis.

[0104] 3.2 Take the fermentation broth of medicinal blastocysts, add purified water and stir to prepare a 1% test solution.

[0105] 3.3 Sample pretreatment: Take 20 µL of sample solution and negative control (physiological saline) respectively, add them to the enzyme reaction system for catalysis, and measure the absorbance at 340 nm under a UV spectrophotometer. Calculate the enzyme activity by taking the average absorbance value.

[0106] 3.4 Calculation Formula 5α-reductase activity (U) = ΔA·V / ε·L·to (ε = 6220 mol·L) -1 (where L is the molar absorptivity of NADPH, L=1) In the formula, ΔA represents the change in absorbance within ten minutes. 4. Test Results Table 6 shows the enzyme activity results of 5α-reductase in Examples 1-3 of this application. Figure 5 This is a comparison diagram of enzyme activity in Example 1 of this application.

[0107] Table 6. Enzyme activity results of 5α-reductase

[0108] 5. Evaluation Conclusion As shown in Table 6, the enzyme activity of the negative control (physiological saline) was significantly higher than that of the fermentation broth of *Polyporus flocculationii* in Examples 1-3 of this application when the addition amount was 1%. The enzyme activity inhibition rates of the fermentation broth of *Polyporus flocculationii* in Examples 1-3 when the addition amount was 1% were 29.45%, 27.54%, and 32.73% respectively compared with the negative control group. This indicates that the fermentation broth of *Polyporus flocculationii* in Examples 1-3 of this application all exhibited certain enzyme inhibitory activity at a 1% addition amount, and the effect was significant. This demonstrates that the fermentation broth of *Polyporus flocculationii* in Examples 1-3 of this application has a certain oil-controlling effect at a 1% addition amount.

[0109] VII. Moisturizing effect of medicinal Fermentation Broth of Pterocarpus 1-3

[0110] 1. Testing Principles Depending on the differences in the hygroscopic and moisturizing properties of humectants, different humectant molecules have different forces acting on water molecules, resulting in varying abilities to absorb and retain moisture. Humectants with stronger forces have a greater binding affinity to water molecules and can absorb and retain a larger amount of water.

[0111] 2. Instruments, equipment, and reagents: 2.1 Instruments and equipment: electronic balance with an accuracy of 0.1 mg; constant temperature and humidity chamber.

[0112] 2.2 Reagents and consumables: 7.5 cm × 7.5 cm glass plate; medical breathable tape; glycerin.

[0113] 2.3 Test substance: Sample group: 1% medicinal Fermentation broth of Poria cocos; Positive control group: 3% glycerol.

[0114] 3. Testing Method: The moisturizing rate of the sample and the positive control was calculated by measuring the ratio of the sample amount to the initial sample amount after 1 h, 2 h, and 4 h of storage.

[0115] 4. Testing steps: (1) Pre-treat the sample and positive control; (2) Weigh a certain amount of the sample and positive control and place them on the test glass plate; (3) Place them in a constant temperature and humidity chamber (temperature: 20 °C ± 1 °C, relative humidity 50 % ± 5 %); (4) Calculate the moisture retention rate.

[0116] 5. Calculation formula: Moisturizing rate (%) = (Mt - M0) / M × 100% In the formula: Mt: the mass of the sample (positive control) and the plate after 1 h, 2 h, and 4 h; M0: Mass of the empty board; M: Mass of the sample (positive control) taken.

[0117] 6. Results Analysis The moisturizing rate of the test sample is compared with that of the positive control. If the moisturizing rate of the test sample is higher than that of the positive control, it indicates that the test sample has a moisturizing effect.

[0118] III. Test Results Table 7 shows the moisturizing rate results in Examples 1-3 of this application. Figure 6 This is a comparison chart of the moisturizing rate in Example 1 of this application.

[0119] Table 7 Moisturizing Rate Results

[0120] IV. Conclusion As shown in Table 7, the moisturizing rate of the medicinal *Polyporus flocculationii* fermentation broth added in Examples 1-3 of this application was tested according to the laboratory method (XYJC-SOP-CE-022 Test Method for Moisturizing Efficacy of Cosmetics - Weighing Method). The test results showed that the moisturizing rates of the medicinal *Polyporus flocculationii* fermentation broth added in Example 1 of this application at 1 h, 2 h, and 4 h were 62.00%, 40.88%, and 11.55%, respectively; the moisturizing rates of the medicinal *Polyporus flocculationii* fermentation broth added in Example 2 of this application at 1 h, 2 h, and 4 h were 57.19%, 36.27%, and 9.08%, respectively; and the moisturizing rates of the medicinal *Polyporus flocculationii* fermentation broth added in Example 3 of this application at 1 h, 2 h, and 4 h were 64.95%, 39.60%, and 14.48%, respectively. It can be seen that the moisturizing rates of the medicinal *Polyporus flocculationii* fermentation broth added in Example 3 of this application at 1 h, 2 h, and 4 h were 14.95%, 39.60%, and 14.48%, respectively. When the amount of medicinal Fertilizer fermentation broth added to Examples 1-3 of this application was 1%, the moisturizing rate was higher than that of the positive control, indicating that the amount of medicinal Fertilizer fermentation broth added to Examples 1-3 of this application was 1% and had a moisturizing effect.

[0121] VIII. Product safety tests in Examples 1-3 of this application.

[0122] Thirty-two participants were selected according to the inclusion criteria and randomly divided into eight groups (two males and two females per group) to undergo the trial. The medicinal *Polyporus flocculationii* fermentation broth prepared in Examples 1-3 was mixed with pure water to adjust the concentration to 2%. An appropriate amount was placed in a plaque applicator, and the applicator containing the sample was applied to the flexor surface of the participant's forearm with medical tape for 24 hours. Skin reactions were observed 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after removing the plaque applicator, and the results were recorded. The grading criteria for skin reactions are shown in Table 8 below. Table 8 Skin Reaction Grading Standards

[0123] The 2% medicinal Fermentation Broth of *Polyporus thuringiensis* prepared in Examples 1-3 showed negative reactions at 30 min (after the indentation disappeared), 24 h, and 48 h after the test, with no irritation or erythema. Therefore, the *Polyporus thuringiensis* fermentation broth of Examples 1-3 prepared by this invention, when added to cosmetics at a concentration of 2%, does not irritate the skin or produce a positive reaction, proving that the product is highly safe, has low irritation, and is relatively mild.

[0124] IX. High-performance liquid chromatography (HPLC) detection of Example 1 Example 1 was analyzed by high performance liquid chromatography using an Agilent Vensil MP C18(2) column (4.6×250 mm, 5 μm, 110 Å). The chromatographic conditions were: column temperature 30℃, detection wavelength 230 nm, mobile phase methanol:water = 25:75 (v / v), column equilibration was performed by isocratic elution for 30 min, and then the injection was started with an injection volume of 10 μL, a flow rate of 1 mL / min, and an elution time of 60 min. The column pressure was kept stable at 40-42 bar.

[0125] Figure 7 The chromatogram of Embodiment 1 of this application is shown, as follows: Figure 7 As shown, the medicinal Fertilizer fermentation broth of Example 1 exhibited characteristic absorption peaks in 13 specific retention time intervals. Characteristic retention time intervals (Table 8): 2.447–2.987 min, 2.988–3.120 min, 3.128–3.477 min, 3.494–4.158 min, 4.167–4.441 min, 4.640–5.055 min, 6.443–6.758 min, 7.589–7.988 min, 8.204–9.159 min, 9.815–10.803 min, 13.779–18.588 min, 18.671–21.437 min, 21.462–22.608 min.

[0126] Table 9 Feature Retention Time Intervals

[0127] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A method for preparing a medicinal fermentation broth of *Polyporus flocculationii*, characterized in that, The preparation method includes the following steps: S100, Fermentation medium preparation: The medicinal fumonis and roselle flowers are crushed and sieved to obtain medicinal fumonis powder and roselle flower powder. The medicinal fumonis powder, roselle flower powder, carbon source and water are mixed in proportion and added to the fermenter. After sterilization, the mixture is cooled and controlled. Then the temperature is raised to 85℃ for 20 min for sterilization. S200, Continuous stepwise fermentation with mixed strains: First, the medicinal pore fungus is anaerobically fermented using a compound strain, and then aerobic fermentation is carried out by inoculating with Saccharomyces cerevisiae SM004 strain to obtain the fermentation product of medicinal pore fungus. The composite microbial strain consists of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, and *Lactobacillus casei* SM003. The *Lactobacillus casei* SM003 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 10, 2023, with accession number CGMCC NO:26537, and the culture name is *Lactobacillus casei* SM003. Lactobacillus casei ; S300, Post-processing: The fermentation product of medicinal fumonis is sequentially subjected to coarse filtration through 100-400 mesh, centrifugation, fine filtration through 0.1μm plate and frame filtration, sterilization at 115-121℃ for 20-30 min, and preservative is added to obtain fermentation broth.

2. The method for preparing a medicinal Fermentation Broth of *Polyporus flocculationii* according to claim 1, characterized in that, Step S200 includes: S210. Preparation of compound bacterial culture and yeast culture: The compound bacterial culture is inoculated from the glycerol culture tube into MRS medium and cultured at 37℃ for 12-20 hours to obtain the compound bacterial culture; wherein the mass ratio of the compound bacterial culture to the MRS medium is 1:100; Saccharomyces cerevisiae SM004 strain is inoculated into yeast medium and cultured at 30℃ for 8-16 hours to obtain the yeast culture; wherein the mass ratio of Saccharomyces cerevisiae SM004 strain to the yeast medium is 1:100; wherein the Saccharomyces cerevisiae SM004 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 10, 2023, with accession number CGMCC NO:26536, and the culture name is Saccharomyces cerevisiae SM004. Sacchammyces cerevisiae ; S220, First stage fermentation: Inoculate the compound bacterial solution into the fermentation medium, stir at a low speed of 50-100 rpm, introduce nitrogen gas at a rate of 0.1-0.6 vvm to ensure dissolved oxygen is 0%, anaerobic fermentation temperature is 30-45℃, and monitor pH in real time. When the pH drops to 3.5-4.0, stop the culture and end the first stage fermentation. S230, Second stage fermentation: Adjust the pH to 6.0~6.5, add the yeast liquid, stir at 200-600 rpm, introduce air at a rate of 0.1-1 vvm, and perform aerobic fermentation for 25-38 hours at a temperature of 24~30℃ to obtain the medicinal Fermentation Product of Fertilizer.

3. The method for preparing medicinal Fermentation Broth of *Polyporus flocculationensis* according to claim 2, characterized in that, The ratio of viable counts of *Lactobacillus bulgaricus* ATCC 11842, *Streptococcus thermophilus* ATCC 19258, *Lactobacillus acidophilus* ATCC 4356, *Lactobacillus plantarum* ATCC 14917, and *Lactobacillus casei* SM003 in the compound bacterial solution is (0.2~0.5):(0.25~0.7):(0.25~0.7):(0.3~1):(0.25~0.7), and the viable count concentration in the yeast solution is not less than 1×10⁻⁶. 5 CFU / mL.

4. The method for preparing a medicinal Fermentation Broth of *Polyporus flocculationensis* according to claim 2, characterized in that, The total inoculum amount of the compound bacterial solution and yeast solution in the fermentation medium is 5-10%; the mass ratio of the compound bacterial solution to the yeast solution is 1:

1.

5. The method for preparing a medicinal Fermentation Broth of *Polyporus flocculationii* according to claim 2, characterized in that, The yeast culture medium consists of water, yeast powder, peptone, and glucose, with addition amounts of 92-97.5%, 0.5-2%, 1-3%, and 1-3%, respectively.

6. The method for preparing a medicinal Fermentation Broth of *Polyporus flocculationensis* according to claim 1, characterized in that, The fermentation medium contains 1-10% medicinal Fertilizer, 0.1-2.0% roselle flowers, and 1.5-3% carbon source, respectively. The carbon source includes, but is not limited to, maltose, skim milk powder, and glucose.

7. The method for preparing a medicinal Fermentation Broth of *Polyporus flocculationensis* according to claim 1, characterized in that, The preservative is one or more of p-hydroxyacetophenone, 1,2-hexanediol, and 1,2-pentanediol, with a total addition amount of 1-5%.

8. A medicinal fermentation broth of *Polyporus flocculationii*, characterized in that, The filtrate is prepared by the preparation method according to any one of claims 1-7, and the filtrate contains polysaccharide content ≥1.76 mg / g, lactic acid content ≥1.58 mg / g, total sugar content ≥10.45 mg / g, and polyphenol content ≥0.199 mg / g.

9. The application of the medicinal Fermentation Broth of *Polyporus flocculationii* as described in claim 8 in the preparation of skin care products, characterized in that... The skin care products have firming and repairing, antibacterial and antiseptic, and / or skin microecological regulation effects.

10. The application according to claim 9, characterized in that, The amount of the filtrate added to the skin care product is 0.5%–10%.

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