Preparation method of lentinus edodes mycelium fermentation liquor with skin barrier repairing function

Abstract

The invention relates to the technical field of cosmetic raw material preparation, in particular to a preparation method of mushroom mycelium fermentation liquor with a skin barrier repairing function, and solves the problems of unstable metabolites and poor batch repeatability caused by unknown mushroom strain sources and random culture parameters in the prior art. The preparation method of the shiitake mushroom mycelium fermentation liquor with the skin barrier repairing function comprises the steps that shiitake mushroom strains are inoculated into a slant culture medium and cultured for 7-10 days at the temperature of 20-25 DEG C, the lawn is obtained, and the slant culture medium is prepared from 18-24 g / L of potato glucose water, 15-20 g / L of agar powder and the balance water. By optimizing a culture medium formula and key process parameters, stable components, high activity and good batch consistency of the fermentation liquor are realized, and the problem of quality fluctuation caused by strain disorder and extensive process is effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic raw material preparation, and particularly relates to a preparation method of a lentinula edodes mycelium fermentation liquor with skin barrier repair function. BACKGROUND

[0002] The lentinula edodes mycelium fermentation liquor refers to a mixed liquor of metabolic products obtained by artificially culturing lentinula edodes mycelium and deep fermentation in a specific liquid culture medium, and is rich in amino acids, polysaccharides, organic acids, trehalose and trace biological active substances (such as ergothioneine). In recent years, it has been widely explored for use in skin care products due to its good moisturizing property, antioxidant property and skin affinity, and has shown application potential in repairing damaged skin barrier.

[0003] Although the method for preparing skin care active ingredients by using edible fungus fermentation has been reported, there are still problems such as unknown source of fungus, random culture medium formula, rough fermentation condition control and non-standard post-processing technology. Many existing technologies only generally mention the use of lentinula edodes fungus without limiting the specific origin, isolation method or preservation generation number, resulting in unclear genetic background and large metabolic capacity difference. In the culture stage, the key parameters such as seed culture time, type and ratio of carbon and nitrogen source, pH, temperature and rotation speed are lack of systematic optimization, causing different batches of mycelium growth state. The best harvest time point is not clear in the fermentation process, and the fermentation is often terminated by experience, resulting in insufficient accumulation of active metabolites or degradation. In the post-processing stage, there are problems such as untimely sterilization, incomplete centrifugation and unreasonable preservative system, which not only leads to residual mycelium fragments or endotoxins in the fermentation liquor, but also easily causes microbial contamination or inactivation of active ingredients.

[0004] Therefore, in view of the deficiencies in the prior art, there is an urgent need for a preparation method of lentinula edodes mycelium fermentation liquor with skin barrier repair function to solve this problem. The new preparation method should start from the source of the fungus, systematically standardize the whole process of seed culture, fermentation control, sterilization and centrifugation, preservative addition and quality detection, significantly improve the content of active ingredients, batch stability and safety of the fermentation liquor, and better meet the production needs of modern cosmetic raw materials in terms of green, natural, efficient and traceable, and provide reliable technical support for the high-quality development of functional skin care products. SUMMARY

[0005] The present application relates to the technical field of cosmetic raw material preparation, and particularly relates to a preparation method of a lentinula edodes mycelium fermentation liquor with skin barrier repair function.

[0006] To achieve the above objectives, the present invention provides a method for preparing a fermentation broth containing shiitake mycelium that repairs the skin barrier, comprising the following steps: S1. Inoculate the shiitake mushroom spawn into an agar slant culture medium and incubate at 20℃-25℃ for 7-10 days to obtain mycelial growth. The agar slant culture medium consists of 18-24 g / L potato glucose solution, 15-20 g / L agar powder, and the remainder is water. S2. Using an inoculation spatula, chop the mycelial growth into fragments with an area of ​​approximately 0.5 cm². Inoculate 2-3 fragments per 150 mL of seed culture medium into a 500 mL Erlenmeyer flask containing the seed culture medium. The seed culture medium consists of: 20-24 g / L potato dextrose solution, 3-5 g / L yeast extract, 0.05-0.15 g / L KH₂PO₄, 0.05-0.15 g / L MgSO₄, and the remainder water, with the pH at its natural setting. Incubate at 20-25℃ and 120-200 r / min on a shaker for 8-10 days to obtain shiitake mushroom mycelial seed solution. S3. Inoculate the seed culture at a volume ratio of 8-12% into a 500mL Erlenmeyer flask containing 150mL of fermentation medium. The fermentation medium consists of 18-20g / L glucose, 1.5-3g / L maltose, 7-10g / L yeast extract, and the remainder water, with a natural pH. Incubate at 20℃-25℃ and 120-200r / min for 3-5 days. S4. Sterilize the fermented broth at 121℃ with high-pressure steam for 20-30 minutes, then centrifuge at 6000rpm for 10-15 minutes and collect the supernatant. S5, add the preservative system to the supernatant, the preservative system consisting of 3-7% butanediol, 0.3-0.8% 1,2-hexanediol and 0.3-0.8% p-hydroxyacetophenone, and stir until homogeneous; S6. The pH value of the fermentation broth is measured by a multi-parameter tester, the conductivity is measured by a DDS-307 conductivity meter, and the odor is judged by sensory evaluation to see if it is fresh and odorless. Once qualified, the shiitake mushroom mycelium fermentation broth is obtained.

[0007] In step S3, the fermentation medium can be replaced with an alternative formulation consisting of 15-20 g / L glucose, 1.5-3 g / L maltose, 8-10 g / L beef extract and the remainder water, while the other process conditions remain unchanged.

[0008] In step S2, the seed culture time is controlled to be 8-10 days to ensure that the mycelium is in the logarithmic growth phase and has the strongest vitality. This avoids insufficient inoculation due to too short a culture time or aging due to too long a culture time, which would affect the subsequent fermentation efficiency and metabolite yield.

[0009] In step S3, the fermentation time is controlled between 3 and 5 days. The peak metabolic period is determined by daily sampling and testing of pH and mycelial density. Fermentation is terminated when the accumulation of active substances is at its maximum to avoid over-fermentation that could lead to product degradation or the growth of miscellaneous bacteria.

[0010] In step 4, sterilization is carried out immediately after fermentation to inactivate endogenous enzymes and residual microorganisms and prevent the active ingredients from being degraded before centrifugation; the centrifugation speed is not less than 6000 rpm and the time is not less than 10 minutes to fully remove mycelial residue and obtain a clear supernatant.

[0011] In step S5, the preservative system uses a combination of polyol and mild preservative. Butylene glycol serves as a moisturizing solubilizer and preservative synergist, while 1,2-hexanediol and p-hydroxyacetophenone synergistically provide broad-spectrum antibacterial capabilities. The total amount of the three added does not exceed 8%.

[0012] The quality control standards in step S6 are as follows: pH value range of 4.8-5.5, conductivity range of 0.5-2.2 mS / cm, and odor of light fermentation fragrance without rancidity or off-odor. Any indicator exceeding the range is deemed unqualified, and the fermentation or sterilization process must be traced back to ensure the consistency of each batch of products.

[0013] This invention discloses a method for preparing a fermentation broth containing shiitake mushroom mycelium that repairs the skin barrier. In step S1, the strain is explicitly defined as originating from tissue isolation, and a standard slant culture medium is used to ensure the purity and consistency of the starting strain. Step S2, through precise control of the seed culture medium components, inoculum size, culture time (strictly 10 days), and shaking parameters (25℃, 150 r / min), overcomes the fermentation fluctuations caused by unstable seed conditions in traditional methods. Step S3 optimizes the ratio of carbon source (glucose + maltose) to nitrogen source (yeast extract) and limits the fermentation time window (3... S4 (5 days) achieves efficient accumulation of active metabolites and batch repeatability; S5 adopts a combination of "immediate sterilization + high-speed centrifugation" to effectively prevent component deterioration caused by enzymatic hydrolysis or microbial contamination after fermentation termination, ensuring the purity of the supernatant; S6 introduces a polyol and mild preservative compound system to replace traditional phenoxyethanol or MIT-type preservatives, improving product safety and skin compatibility while ensuring preservative efficacy; S7 establishes triple quality control standards of pH, conductivity and odor, enabling quantitative judgment of each batch of products and avoiding quality deviations caused by subjective experience. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic flowchart of the preparation process according to an embodiment of the present invention.

[0016] Figure 2 This is a schematic diagram of the fermentation broth of shiitake mushroom mycelium according to an embodiment of the present invention.

[0017] Figure 3 This is a schematic diagram of a microscope image of shiitake mushroom mycelium according to an embodiment of the present invention.

[0018] Figure 4 This is a schematic diagram of the cultivation of shiitake mushroom strains according to an embodiment of the present invention.

[0019] Figure 5 This is a schematic diagram illustrating the scavenging rate of DPPH free radicals by the fermentation broth of shiitake mushroom mycelium in an embodiment of the present invention.

[0020] Figure 6 This is a schematic diagram of a cell scratch test according to an embodiment of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Example 1

[0022] Please see Figure 1 As shown in this embodiment, a method for preparing a fermentation broth containing shiitake mushroom mycelium that repairs the skin barrier includes the following steps: S1. Inoculate the shiitake mushroom spawn into an agar slant culture medium and incubate at 20℃-25℃ for 7-10 days to obtain mycelial growth. The agar slant culture medium consists of: 18-24 g / L potato glucose solution, 15-20 g / L agar powder, and the remainder is water. S2. Using an inoculation spatula, the mycelial moss is chopped into fragments with an area of ​​approximately 0.5 cm². 2-3 fragments are inoculated per 150 mL of seed culture medium into a 500 mL Erlenmeyer flask containing the seed culture medium. The seed culture medium consists of: 20-24 g / L potato dextrose solution, 3-5 g / L yeast extract, 0.1 g / L KH₂PO₄, 0.1 g / L MgSO₄, and the remainder water. The pH is natural. The culture is then incubated for 8-10 days in a shaker at 20-25℃ and 120-200 r / min to obtain shiitake mushroom mycelial seed solution. S3. The seed culture is inoculated into a 500 mL Erlenmeyer flask containing 150 mL of fermentation medium at a volume ratio of 8-12%. The fermentation medium consists of 18-20 g / L glucose, 1.5-3 g / L maltose, 7-10 g / L yeast extract, and the remainder water. The pH is natural. The culture is carried out in a shaker at 20-25°C and 120-200 r / min for 3-5 days. S4. Sterilize the fermented broth at 121℃ with high-pressure steam for 20-30 minutes, then centrifuge at 6000rpm for 10-15 minutes and collect the supernatant. S5, add the preservative system to the supernatant, the preservative system consisting of 3-7% butanediol, 0.3-0.8% 1,2-hexanediol and 0.3-0.8% p-hydroxyacetophenone, and stir until homogeneous; S6. The pH value of the fermentation broth is measured by a multi-parameter tester, the conductivity is measured by a DDS-307 conductivity meter, and the odor is judged by sensory evaluation to see if it is fresh and odorless. Once qualified, the shiitake mushroom mycelium fermentation broth is obtained.

[0023] In practice, the preparation method of this shiitake mushroom mycelium fermentation broth with skin barrier repair function is strictly carried out according to six steps from S1 to S6: First, in step S1, the shiitake mushroom strain obtained by tissue isolation is inoculated into a slant culture medium composed of 24 g / L potato dextrose solution, 20 g / L agar powder, and the remainder water, and cultured at a constant temperature of 25℃ for 7-10 days to obtain a uniformly growing, highly active white mycelial moss, ensuring a clear strain origin and stable genetic background; then, in step S2, the mycelial moss is chopped into fragments with an area of ​​about 0.5 cm² using a sterile inoculation spatula, and 3... The standard inoculum size was transferred to a 500 mL Erlenmeyer flask containing seed culture medium (composed of 24 g / L potato dextrose solution, 5 g / L yeast extract, 0.1 g / L KH2PO4, 0.1 g / L MgSO4, and the remainder water, pH natural). The flask was precisely cultured for 10 days at 25°C and 150 rpm on a shaker to allow the mycelium to fully grow and enter the logarithmic growth phase, obtaining a highly viable seed culture. In step S3, the seed culture was inoculated at a 10% (v / v) ratio into 150 mL of fermentation medium (composed of 20 g / L glucose, 3 g / L maltose, 10 g / L yeast extract, and the remainder water). In the same conditions of 25℃ and 150r / min, deep liquid fermentation was carried out in the natural pH range (composition, pH 60, 0.5%), with the fermentation time strictly controlled at 3-5 days (preferably 4 days) to ensure that metabolites such as amino acids, ergothioneine, and flavonoids accumulate at their peak. Immediately after fermentation, step S4 was performed, in which the fermentation broth was sterilized under high pressure at 121℃ for 20 minutes to rapidly inactivate endogenous enzymes and residual microorganisms and prevent degradation of active ingredients. Subsequently, it was centrifuged at 6000rpm for 10 minutes to completely remove mycelial residues and obtain a clear supernatant. In step S5, 5% butanediol and 0.5% 1,2-dimethylamine were added to the supernatant. A composite preservative system consisting of hexanediol and 0.5% p-hydroxyacetophenone was thoroughly stirred. This system combines moisturizing, solubilizing, and broad-spectrum antibacterial functions, avoiding the use of traditional irritating preservatives. Finally, in step S6, the pH value of the fermentation broth was tested using a multi-parameter tester (target range 4.8-5.5), and the conductivity was measured using a DDS-307 conductivity meter (target 0.5-2.2 mS / cm). Professionals also used sensory evaluation to determine whether the odor was a fresh fermented aroma and free from rancidity or off-odors. Only after all three indicators were qualified could the product be put into storage. The entire process achieved standardized control of the entire chain from inoculum to final product. Example 2

[0024] Please see Figure 1As shown in this embodiment, a method for preparing a fermentation broth containing shiitake mushroom mycelium that repairs the skin barrier is described. In step S3, the fermentation medium can be replaced with an alternative formula consisting of 15-20 g / L glucose, 1.5-3 g / L maltose, 8-10 g / L beef extract, and the remainder water, while keeping the other process conditions unchanged. Specifically, by replacing "20 g / L glucose, 3 g / L maltose, and 10 g / L yeast extract" in the fermentation medium in step S3 with "20 g / L glucose, 3 g / L maltose, and 10 g / L beef extract," fermentation is carried out while keeping other process conditions (such as inoculum size of 10%, temperature of 25°C, rotation speed of 150 r / min, and fermentation time of 3-5 days) completely unchanged. This achieves the effect of regulating the metabolite profile by adjusting the type of nitrogen source without changing the equipment and operating procedures. This allows the fermentation broth to retain the core active ingredients while flexibly optimizing the amino acid composition or organic acid content according to different skincare needs, thus enhancing the adaptability of the process and the diversity of products.

[0025] In step S2, the seed culture time is controlled to be 8-10 days to ensure that the mycelium is in the logarithmic growth phase and has the strongest vitality. This avoids insufficient inoculation due to too short a culture time or aging due to too long a culture time, which would affect the subsequent fermentation efficiency and metabolite yield. Specifically, by strictly controlling the seed culture time to 10 days in step S2, after inoculating 0.5 cm² mycelial fragments into 150 mL of seed culture medium, the culture is precisely carried out at 25℃ and 150 r / min for 10 days. This allows the mycelium to grow fully and remain stable in the logarithmic growth phase, achieving the effect of ensuring the highest seed liquid vitality and the best inoculation consistency. This effectively avoids the problem of low inoculum due to insufficient culture time or mycelial aging due to excessive culture time, thereby significantly improving the start-up efficiency and metabolic stability of subsequent fermentation.

[0026] In step S3, the fermentation time is controlled between 3 and 5 days. The peak metabolic period is determined by daily sampling and testing of pH and mycelial density. This ensures that fermentation is terminated when the accumulation of active substances is at its maximum, avoiding over-fermentation that could lead to product degradation or the growth of contaminating microorganisms. Specifically, by controlling the fermentation time to between 3 and 5 days in step S3 and combining this with daily sampling and testing of pH and mycelial density, and after inoculating with 10% seed liquid, the changes in the physicochemical parameters of the fermentation system are monitored daily. Fermentation is terminated based on the peak metabolic period (usually day 4). This achieves the effect of timely termination of the reaction when the accumulation of active metabolites (such as trehalose and ergothioneine) reaches its peak, effectively preventing the risk of product degradation, sudden pH drop, or contamination by contaminating microorganisms caused by over-fermentation, and ensuring the high activity and high purity of the fermentation liquid.

[0027] The study included LC-MS analysis of the obtained shiitake mushroom mycelium fermentation broth to identify metabolites in the fermentation broth, including sugars such as trehalose, galactose, and fructose; organic acids such as glutaric acid, citric acid, and rhamnic acid; and amino acids such as arginine, proline, glutamic acid, phenylalanine, and ornithine.

[0028] Determination of polysaccharide content in shiitake mushroom mycelium fermentation broth: Take 400 μL of fermentation broth, add 4 times the volume of anhydrous ethanol, shake well, and place in a 4℃ refrigerator to stand for 12 h. Centrifuge at 4000 r / min for 10-20 min, discard the supernatant, add 1 mL of deionized water to the precipitate to resuspend and dissolve, and then determine the polysaccharide content using the phenol-sulfuric acid method. The results showed that the polysaccharide content of shiitake mushroom was 0.106%.

[0029] The total phenol content in the fermentation broth of shiitake mushroom mycelium was determined by diluting the fermentation broth with deionized water to make its OD value less than 1.0. The absorbance was measured at a wavelength of 760 nm using a visible spectrophotometer, and the total phenol content was calculated according to the standard curve. The results showed that the total phenol content was 0.102 mg / g.

[0030] To verify the effective components and their content in the fermentation broth of shiitake mushroom mycelium, component analysis was performed. First, 400 μL of the fermentation broth was taken, and four volumes of anhydrous ethanol were added. After shaking well, the mixture was placed in a 4℃ refrigerator and allowed to stand for 12 h. After centrifugation at 4000 r / min for 10–20 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of deionized water. The polysaccharide content was then determined using the phenol-sulfuric acid method, showing a shiitake mushroom polysaccharide content of 0.106%. Next, the total phenol content of the fermentation broth was determined using a visible spectrophotometer at a wavelength of 760 nm, showing a total phenol content of 0.102 mg / g. Furthermore, the metabolites in the fermentation broth were identified using liquid chromatography-mass spectrometry (LC-MS), revealing sugars such as trehalose, galactose, and fructose; organic acids such as glutaric acid, citric acid, and rhamnic acid; and amino acids such as arginine, proline, glutamic acid, phenylalanine, and ornithine. These components collectively contribute to the excellent biological activity of the fermentation broth.

[0031] The workflow for analyzing the components of the shiitake mushroom mycelium fermentation broth obtained in step S5, including determining the polysaccharide content using ethanol precipitation combined with the phenol-sulfuric acid method, determining the total phenol content using visible spectrophotometry, and identifying metabolites using liquid chromatography-mass spectrometry (LC-MS), is as follows: First, 400 μL of fermentation broth is taken, 4 times the volume of anhydrous ethanol is added, and the mixture is allowed to stand at 4℃ for 12 h. Then, it is centrifuged (4000 r / min, 10–20 min), the supernatant is discarded, and the precipitate is resuspended in 1 mL of deionized water. The polysaccharide content is determined to be 0.106% using the phenol-sulfuric acid method. Simultaneously, the fermentation broth is diluted and the absorbance is measured at a wavelength of 760 nm. The total phenol content is calculated to be 0.102% based on the standard curve. mg / g; and further, LC-MS technology was used to identify sugars such as trehalose, galactose, and fructose, organic acids such as glutaric acid, citric acid, and rhamnic acid, as well as amino acids such as arginine, proline, glutamic acid, phenylalanine, and ornithine, achieving the effect of clarifying the types and contents of key active ingredients in the fermentation broth, thus providing a scientific basis for subsequent efficacy verification and quality control, and confirming that the fermentation broth is rich in a variety of known natural metabolites with moisturizing, antioxidant, and barrier repair functions. Example 3

[0032] Please see Figure 1 As shown in this embodiment, a method for preparing a fermentation broth containing shiitake mushroom mycelium that repairs the skin barrier is described. In step S4, sterilization is performed immediately after fermentation to inactivate endogenous enzymes and residual microorganisms, preventing the degradation of active ingredients before centrifugation. The centrifugation speed is not less than 6000 rpm and the time is not less than 10 minutes to fully remove mycelial residues and obtain a clear supernatant. Specifically, by specifying in step S4 that sterilization must be performed immediately after fermentation and that the centrifugation conditions are not less than 6000 rpm and not less than 10 minutes, the fermentation broth is subjected to high-pressure steam sterilization at 121°C for 20 minutes immediately after fermentation, followed by high-speed centrifugation. This achieves the effect of rapidly inactivating endogenous enzymes and residual microorganisms and thoroughly separating mycelial residues, effectively preventing the active ingredients from undergoing enzymatic hydrolysis or oxidative degradation before post-processing, and ensuring that the obtained supernatant is clear, stable, and has a low impurity content.

[0033] In step S5, the preservative system uses a combination of polyols and mild preservatives. Butylene glycol acts as a moisturizing solubilizer and preservative synergist, while 1,2-hexanediol and p-hydroxyacetophenone synergistically provide broad-spectrum antibacterial capabilities. The total addition amount of the three does not exceed 8%. Specifically, in step S5, a composite preservative system consisting of 5% butylene glycol, 0.5% 1,2-hexanediol, and 0.5% p-hydroxyacetophenone is used. The total addition amount of this composite system, not exceeding 8%, is added to the supernatant and stirred evenly. By utilizing the moisturizing and solubilizing effects of butylene glycol in conjunction with the broad-spectrum antibacterial functions of 1,2-hexanediol and p-hydroxyacetophenone, effective preservative effects are achieved without using traditional irritating preservatives (such as phenoxyethanol and MIT). This ensures the microbial stability of the product and improves its safety and skin compatibility in applications for sensitive skin.

[0034] The quality control standards in step S6 are: pH value range of 4.8-5.5, conductivity range of 0.5-2.2 mS / cm, and a light fermented aroma without any rancidity or off-odor. Any indicator exceeding the range is considered unqualified, requiring tracing back to the fermentation or sterilization process to ensure consistency of each batch of products. Specifically, by setting the quality control standards of pH value range of 4.8-5.5, conductivity range of 0.5-2.2 mS / cm, and a light fermented aroma without any rancidity or off-odor in step S6, each batch of fermentation liquid is systematically tested for these three indicators before the finished product is put into storage. Any indicator exceeding the standard is considered unqualified, and tracing back to step S3 or S4 is required. This achieves the effect of high consistency between batches, quantifiable quality, and traceable problems, fundamentally solving the technical defects of traditional fermentation processes that rely on experience and have large batch fluctuations, and providing a reliable guarantee for stable industrial production.

[0035] Free radical scavenging ability of shiitake mushroom mycelium fermentation broth was tested: The ABTS free radical scavenging activity assay was used to detect the ABTS free radical scavenging rate of the fermentation broth. The results showed that the 5% concentration of fermentation broth had a 63.87% ABTS free radical scavenging rate. The DPPH free radical scavenging activity assay was used to detect the DPPH free radical scavenging rate of fermentation broth at different concentrations. The results showed that the DPPH free radical scavenging rate gradually increased with the increase of fermentation broth concentration.

[0036] Safety assessment of shiitake mushroom mycelium fermentation broth: microbial testing, irritation testing, and human patch testing were conducted.

[0037] Human efficacy evaluation trial: Thirty healthy female volunteers aged 30 to 55 years were randomly divided into two groups and used essence water containing 0% and 1.75% shiitake mushroom mycelium fermentation liquid for 4 consecutive weeks. Follow-up tests were conducted 2 weeks and 4 weeks after product use to analyze changes in skin stratum corneum moisture content, transepidermal water loss, skin elasticity, skin firmness, wrinkle score, and skin redness score before and after product use. The results showed that the essence water with added fermentation liquid had significant effects in moisturizing, repairing, firming, and anti-wrinkle.

[0038] To evaluate the free radical scavenging ability and safety of shiitake mushroom mycelium fermentation broth, several tests were conducted. First, the ABTS free radical scavenging activity assay was used to determine the ABTS free radical scavenging rate of the fermentation broth. The results showed that the 5% concentration of fermentation broth scavenged 63.87% of ABTS free radicals. Next, the DPPH free radical scavenging activity assay was used to determine the DPPH free radical scavenging rate of fermentation broth at different concentrations. The results showed that the DPPH free radical scavenging rate gradually increased with increasing fermentation broth concentration. Furthermore, microbial testing, irritation testing, and human patch testing were conducted according to SN / T2329-2009 "Cosmetic Eye Irritation / Corrosiveness Chicken Embryo Viral Allantoic Membrane Test" and the "Cosmetic Safety Technical Specifications" (2015 edition). The results showed that the fermentation broth met the microbial indicators, and the chicken embryo viral allantoic membrane test verified no irritation.

[0039] The workflow for conducting free radical scavenging ability tests, safety assessments, and human efficacy evaluations on the fermentation broth of shiitake mycelium obtained in step S5 is as follows: First, the free radical scavenging rate of the 5% concentration fermentation broth was measured to be 63.87% using the ABTS method, and its scavenging rate increased with increasing concentration using the DPPH method. Second, microbial testing, chicken embryo chorioallantoic membrane irritation tests, and human patch tests were conducted according to SN / T2329-2009 and the "Cosmetic Safety Technical Specifications" (2015 edition) to confirm its non-irritating properties. Furthermore, the microbiological indicators were qualified. Finally, 30 female volunteers aged 30–55 were recruited and divided into two groups to use the essence water containing 0% and 1.75% fermentation liquid for 4 consecutive weeks. In the second and fourth weeks, the skin stratum corneum moisture content, transepidermal water loss (TEWL), skin elasticity, firmness, wrinkle score and redness score were measured. The results showed that the group with added fermentation liquid was significantly better than the control group in terms of moisturizing, barrier repair, firming and anti-wrinkle. The results achieved the effect of systematically verifying the antioxidant activity, biosafety and actual skin barrier repair efficacy of the fermentation liquid.

[0040] Table 1. Statistics on the rate of change of serum relative to baseline value during the usage period. Example 4

[0041] The promoting effect of shiitake mushroom mycelium fermentation broth on keratinocyte scratch healing This embodiment evaluates the effect of shiitake mushroom mycelium fermentation broth on skin repair through an in vitro cell scratch assay. Human immortalized keratinocytes were used as the cell model in the experiment.

[0042] Before the experiment, the shiitake mushroom mycelium fermentation broth sample was diluted with anhydrous ethanol to a volume fraction of 5%, and then filtered through a 0.22 μm microporous membrane to obtain a sterile stock solution. Before use, the stock solution was further diluted to a final concentration of 0.05% for cell treatment. An incomplete culture medium was used as the negative control group.

[0043] The experimental procedure was as follows: HaCaT cells were seeded at an appropriate density in 6-well plates and cultured for 24 hours until the cells reached more than 90% confluence; the culture medium was discarded, and a straight line was vertically drawn in the center of the cell monolayer in each well using a sterile pipette tip. The cells were gently washed with PBS to remove the detached cells; then, culture medium containing 0.05% shiitake mycelium fermentation broth (sample group) or incomplete culture medium (negative control group) was added respectively; the same position was photographed and recorded under an EVOS inverted fluorescence microscope at 0 h and 24 h after the incision.

[0044] The scratch area was quantitatively analyzed using Image-Pro Plus (IPP) image analysis software. The scratch healing rate was calculated using the following formula: 24 h scratch healing rate (%) = [1 − (24 h scratch area pixel value / 0 h scratch area pixel value)] × 100%.

[0045] The experimental results showed that the scratch healing rate of the negative control group was 23.28% after 24 hours, while the scratch healing rate of the group treated with 0.05% shiitake mycelium fermentation broth reached 30.91%. The cell migration and healing abilities of the sample group were significantly higher than those of the negative control group.

[0046] The above results indicate that at a concentration of 0.05%, the fermentation broth of shiitake mushroom mycelium can significantly promote the migration of keratinocytes and scratch closure, and has the potential to promote skin barrier repair. It can be used to develop cosmetics or skin care products with repair functions.

[0047] In the preparation method of the shiitake mycelium fermentation broth with skin barrier repair function, the entire process is strictly executed in accordance with the six core steps S1 to S6 and the optimized parameters defined in claims 2-7: First, in step S1, the shiitake mushroom strain obtained by tissue isolation is inoculated into a slant culture medium composed of 24 g / L potato dextrose solution, 20 g / L agar powder, and the remainder water, and cultured at a constant temperature of 25°C for 7-10 days to obtain a uniform, dense, and highly active white mycelial moss, ensuring that the strain origin is clear and the genetic background is stable, laying a pure culture foundation for subsequent fermentation; then, in step S2, the mycelial moss is chopped into pieces with an area of ​​approximately 0.5 mm using a sterile inoculation spatula. Fragments of 1 cm² were inoculated strictly according to the standard inoculation rate of 3 fragments per 150 mL of seed culture medium. These fragments were then transferred to a 500 mL Erlenmeyer flask containing seed culture medium composed of 24 g / L potato dextrose solution, 5 g / L yeast extract, 0.1 g / L KH₂PO₄, 0.1 g / L MgSO₄, and the remainder water, with a natural pH. The culture was precisely incubated for 10 days at 25°C and 150 rpm in a shaker (corresponding to claim 3) to allow the mycelium to fully expand and stabilize in the logarithmic growth phase, resulting in a highly viable and dense seed culture. This avoids insufficient inoculation time leading to low inoculation or excessive inoculation causing mycelial aging, thus ensuring the success of subsequent fermentation. The fermentation process is rapid and metabolically stable. Then, step S3 is performed, inoculating the seed culture at a 10% volume ratio into a 500mL Erlenmeyer flask containing 150mL of fermentation medium. The fermentation medium preferably consists of 20g / L glucose, 3g / L maltose, 10g / L yeast extract, and the remainder water (corresponding to claim 1), or can be replaced with an alternative formula containing 10g / L beef extract (corresponding to claim 2). Deep liquid fermentation is carried out at 25℃ and 150r / min, with the fermentation time strictly controlled between 3-5 days (corresponding to claim 4). During this period, daily sampling is used to detect pH and mycelial density to accurately determine the metabolic peak (usually the 4th day). (days), ensuring that the reaction terminates at the accumulation peak of active metabolites such as trehalose, amino acids, organic acids, and ergothioneine, preventing over-fermentation that could lead to product degradation or the growth of contaminating microorganisms; immediately after fermentation, proceed to step S4, where the fermentation broth is sterilized under high pressure steam at 121°C for 20 minutes, and then centrifuged at a speed of not less than 6000 rpm for at least 10 minutes (corresponding to claim 5) to rapidly inactivate endogenous enzymes and residual microorganisms, thoroughly remove mycelial residues, and obtain a clear, impurity-free supernatant, effectively preventing enzymatic hydrolysis or oxidation of active ingredients before post-processing; subsequently, in step S5, 5% butanediol, 0.5% 1,2-hexanediol, and 0.A composite preservative system consisting of 5% p-hydroxyacetophenone (corresponding to claim 6), with the total addition controlled within 8%, is uniformly dispersed by stirring. The moisturizing and solubilizing functions of butylene glycol synergistically enhance the broad-spectrum antibacterial effects of 1,2-hexanediol and p-hydroxyacetophenone, achieving highly effective preservation without the use of irritating preservatives such as phenoxyethanol and MIT, thus improving the safety and skin compatibility of the product in sensitive skin applications. Finally, in step S6, the pH value of the fermentation broth is measured using a multi-parameter tester (target 4.8-5.5), and the conductivity is measured using a DDS-307 conductivity meter (target 0.5-2.2 mS / cm). Professional personnel then use sensory evaluation to determine if the odor is a light fermented fragrance, free from rancidity or off-odors. Only when all three indicators are qualified can the product be considered a finished product. If a product exceeds the standard, the process will be reviewed by tracing back to step S3 or S4 to ensure high consistency in composition, activity, and safety for each batch. The entire method systematically solves the core problems of existing technologies, such as inconsistent strains, arbitrary cultivation, crude processes, non-standard post-processing, strong preservative irritation, and large batch-to-batch fluctuations, by combining the refined definition of key parameters in claims 2-7 with S1 (identifying the source of the microorganisms), S2 (standardizing seed preparation), S3 (optimizing the carbon-nitrogen source ratio and fermentation time), S4 (strengthening sterilization and solid-liquid separation), S5 (adopting a green preservation system), and S6 (establishing quantitative quality control standards). It achieves a balance between high activity, high purity, high safety, and industrially repeatable production of shiitake mushroom mycelium fermentation broth, providing a stable, natural, and efficient active ingredient solution for skin barrier repair cosmetics.

[0048] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for preparing a fermentation broth containing shiitake mushroom mycelium with skin barrier repair properties, characterized in that, Includes the following steps: S1. Inoculate the shiitake mushroom spawn into an agar slant culture medium and incubate at 20℃-25℃ for 7-10 days to obtain mycelial growth. The agar slant culture medium consists of 18-24 g / L potato glucose solution, 15-20 g / L agar powder, and the remainder is water. S2. Using an inoculation spatula, chop the mycelial growth into fragments with an area of ​​approximately 0.5 cm². Inoculate 2-3 fragments per 150 mL of seed culture medium into a 500 mL Erlenmeyer flask containing the seed culture medium. The seed culture medium consists of: 20-24 g / L potato dextrose solution, 3-5 g / L yeast extract, 0.05-0.15 g / L KH₂PO₄, 0.05-0.15 g / L MgSO₄, and the remainder water, with the pH at its natural setting. Incubate at 20-25℃ and 120-200 rpm for 8-10 days to obtain shiitake mushroom mycelial seed solution. S3. Inoculate the seed culture at a volume ratio of 8-12% into a 500mL Erlenmeyer flask containing 150mL of fermentation medium. The fermentation medium consists of 18-20g / L glucose, 1.5-3g / L maltose, 7-10g / L yeast extract, and the remainder water, with a natural pH. Incubate at 20℃-25℃ and 120-200r / min for 3-5 days. S4. Sterilize the fermented broth at 121℃ with high-pressure steam for 20-30 minutes, then centrifuge at 6000rpm for 10-15 minutes and collect the supernatant. S5, add the preservative system to the supernatant, the preservative system consisting of 3-7% butanediol, 0.3-0.8% 1,2-hexanediol and 0.3-0.8% p-hydroxyacetophenone, and stir until homogeneous; S6. The pH value of the fermentation broth is measured by a multi-parameter tester, the conductivity is measured by a DDS-307 conductivity meter, and the odor is judged by sensory evaluation to see if it is fresh and odorless. Once qualified, the shiitake mushroom mycelium fermentation broth is obtained.

2. The method for preparing a fermentation broth containing shiitake mycelium with skin barrier repair properties according to claim 1, characterized in that, The fermentation medium in step S3 can be replaced with an alternative formula consisting of 15-20 g / L glucose, 1.5-3 g / L maltose, 8-10 g / L beef extract and the remainder water, while the other process conditions remain unchanged.

3. The method for preparing a fermentation broth containing shiitake mycelium with skin barrier repair properties according to claim 1, characterized in that, In step S2, the seed culture time is controlled to be 8-10 days to ensure that the mycelium is in the logarithmic growth phase and has the strongest vitality. This avoids insufficient inoculation due to too short a culture time or aging due to too long a culture time, which would affect the subsequent fermentation efficiency and metabolite yield.

4. The method for preparing a fermentation broth containing shiitake mycelium with skin barrier repair properties according to claim 1, characterized in that, In step S3, the fermentation time is controlled between 3 and 5 days. The peak metabolic period is determined by daily sampling and testing of pH and mycelial density. Fermentation is terminated when the accumulation of active substances is at its maximum to avoid over-fermentation that could lead to product degradation or the growth of miscellaneous bacteria.

5. The method for preparing a fermentation broth containing shiitake mycelium with skin barrier repair properties according to claim 1, characterized in that, In step 4, sterilization is carried out immediately after fermentation to inactivate endogenous enzymes and residual microorganisms and prevent the active ingredients from being degraded before centrifugation. The centrifugation speed is not less than 6000 rpm and the time is not less than 10 minutes to fully remove mycelial residue and obtain a clear supernatant.

6. The method for preparing a fermentation broth containing shiitake mycelium with skin barrier repair properties according to claim 1, characterized in that, In step S5, the preservative system uses a combination of polyol and mild preservative. Butylene glycol serves as a moisturizing solubilizer and preservative synergist, while 1,2-hexanediol and p-hydroxyacetophenone synergistically provide broad-spectrum antibacterial capabilities. The total amount of the three added does not exceed 8%.

7. The method for preparing a fermentation broth containing shiitake mycelium with skin barrier repair properties according to claim 1, characterized in that, The quality control standards in step S6 are as follows: pH value range of 4.8-5.5, conductivity range of 0.5-2.2 mS / cm, and odor of light fermentation fragrance without rancidity or off-odor. Any indicator exceeding the range is deemed unqualified, and the fermentation or sterilization process must be traced back to ensure the consistency of each batch of products.

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