Preparation method of beta glucan supramolecular polymer, beta glucan supramolecular polymer and application

Abstract

The application relates to a preparation method of a beta glucan supramolecular polymer, the beta glucan supramolecular polymer and application, and belongs to the technical field of supramolecular polymer preparation. The preparation method of the beta glucan supramolecular polymer comprises the following steps: introducing a schizophyllum commune liquid into a culture medium, wherein the culture medium comprises an active substance and a culture medium substrate, the mass ratio of the schizophyllum commune liquid to the active substance is 1-10:1; and the culture medium is fermented at 27-30 DEG C for 90-100 h to obtain the beta glucan supramolecular polymer loaded with the active substance. The physical wrapping and chemical cross-linking methods are avoided, the residue problem of toxic and harmful reagents is avoided, and the loading rate is effectively improved.

Description

Technical Field

[0001] This application relates to the field of supramolecular polymer preparation technology, and particularly to a method for preparing a β-glucan supramolecular polymer, the β-glucan supramolecular polymer, and its applications. Background Technology

[0002] Plant-based active substances, such as anthocyanins, have poor photothermal stability and are prone to decomposition and discoloration when used in products, which limits their application scenarios. Therefore, it is necessary to improve the stability of plant-based active substances through other means.

[0003] Existing methods for enhancing the stability of plant active substances mainly include physical encapsulation and chemical cross-linking, but these methods have problems such as low encapsulation efficiency and residues of toxic and harmful reagents. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the objectives of this application include providing a method for preparing a β-glucan supramolecular polymer, as well as the β-glucan supramolecular polymer and its applications. This method avoids the use of physical encapsulation and chemical cross-linking methods, thus avoiding the problem of toxic and harmful reagent residues, and effectively improving the encapsulation efficiency.

[0005] In a first aspect, embodiments of this application provide a method for preparing a β-glucan supramolecular polymer, comprising: The *Schizophyllum commune* culture was inoculated into a culture medium containing active substances and a substrate, with a mass ratio of *Schizophyllum commune* culture to active substances of 1–10:1. Fermentation was carried out at 27–30 °C for 90–100 h to obtain a β-glucan supramolecular polymer encapsulated with active substances.

[0006] This application uses *Schizophyllum commune* as the starting strain, directly inoculating it into a culture medium containing active substances at a suitable mass ratio, and fermenting it under suitable conditions. During the fermentation process, *Schizophyllum commune* directly encapsulates the active substances in β-glucan, thereby obtaining a β-glucan supramolecular polymer encapsulating the active substances. This avoids the use of physical encapsulation and chemical cross-linking methods for encapsulating active substances, avoids the problem of toxic and harmful reagent residues, and effectively improves the encapsulation rate of active substances.

[0007] In some embodiments of this application, the mass percentage of the active substance is 1% to 5%; the mass percentage of the Schizophyllum commune liquid is 5% to 10%.

[0008] This application uses an appropriate mass percentage of active substances and Schizophyllum commune culture in a culture medium for fermentation, which facilitates the efficient encapsulation of active substances into β-glucan during the β-glucan synthesis process of Schizophyllum commune, thereby forming a β-glucan supramolecular polymer encapsulating active substances.

[0009] In some embodiments of this application, the mass percentage of the active substance is 1% to 3%; the mass percentage of the Schizophyllum commune liquid is 7.5% to 10%.

[0010] This application further employs an appropriate mass percentage of active substances and Schizophyllum commune culture in a culture medium for fermentation, which facilitates the efficient encapsulation of active substances into β-glucan during the β-glucan synthesis process of Schizophyllum commune, thereby forming a β-glucan supramolecular polymer encapsulating active substances.

[0011] In some embodiments of this application, the mass percentage of the active substance is 2.5% to 3%; the mass percentage of the *Schizophyllum commune* culture is 7.5% to 8%.

[0012] This application further employs a more suitable mass percentage of active substances and Schizophyllum commune culture in the culture medium for fermentation, which facilitates more efficient encapsulation of active substances in β-glucan during the β-glucan synthesis process of Schizophyllum commune, thereby forming β-glucan supramolecular polymers encapsulating active substances.

[0013] In some embodiments of this application, the active substance includes anthocyanins.

[0014] This application uses suitable active substances, all of which can be fermented with Schizophyllum commune in a culture medium to obtain β-glucan supramolecular polymers encapsulating the active substances. The encapsulation rate of the active substances is high, which improves the stability of the active substances and facilitates their subsequent applications.

[0015] In some embodiments of this application, the culture medium substrate, by mass percentage, includes: 0.3%~0.5% yeast extract, 0.05%~0.08% magnesium sulfate, 0.1%~0.3% potassium dihydrogen phosphate, 3%~5% glucose, and the remainder is distilled water.

[0016] This application uses a suitable culture medium substrate to ferment the active substance and Schizophyllum commune, which facilitates the efficient encapsulation of the active substance in β-glucan during the β-glucan synthesis process of Schizophyllum commune, thereby forming a β-glucan supramolecular polymer encapsulating the active substance.

[0017] In some embodiments of this application, the method for preparing Schizophyllum commune culture includes: transferring Schizophyllum commune slant culture to liquid culture medium, expanding culture for 3-5 days, and obtaining Schizophyllum commune culture.

[0018] This application obtains Schizophyllum commune broth by transferring Schizophyllum commune slant culture to liquid culture medium for expansion culture, which facilitates subsequent fermentation with active substances. During the fermentation process, Schizophyllum commune efficiently encapsulates active substances in β-glucan to form β-glucan supramolecular polymers encapsulating active substances.

[0019] In some embodiments of this application, the fermentation product obtained by fermentation is further centrifuged and filtered to obtain a β-glucan supramolecular polymer encapsulating active substances.

[0020] This application obtains a stable β-glucan supramolecular polymer with encapsulated active substances by centrifuging and filtering the fermentation products obtained from fermentation to remove unencapsulated active substances.

[0021] Secondly, embodiments of this application provide a β-glucan supramolecular polymer prepared by the preparation method provided in the first aspect.

[0022] The β-glucan supramolecular polymer prepared by the preparation method of the first aspect of this application can efficiently and stably encapsulate active substances, with a high encapsulation rate and effectively improved stability of active substances, which is beneficial for subsequent applications.

[0023] Thirdly, embodiments of this application provide the application of the β-glucan supramolecular polymer as provided in the second aspect in the preparation of cosmetics. The β-glucan supramolecular polymer provided in this application has a high loading rate for active substances and effectively improves the stability of active substances, which is beneficial for its application in the preparation of cosmetics. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The characteristic curve of the β-glucan-anthocyanin supramolecular polymer provided in Experimental Example 1 of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] Currently, plant active substances are mainly encapsulated through physical encapsulation and chemical cross-linking methods to enhance their stability. However, these methods have problems such as low encapsulation efficiency and residues of toxic and harmful reagents.

[0028] Therefore, this application provides a method for preparing a β-glucan supramolecular polymer, comprising: The *Schizophyllum commune* culture was inoculated into a culture medium containing active substances and a substrate, with a mass ratio of *Schizophyllum commune* culture to active substances of 1–10:1. Fermentation was carried out at 27–30 °C for 90–100 h to obtain a β-glucan supramolecular polymer encapsulated with active substances.

[0029] This application uses *Schizophyllum commune* as the starting strain, directly inoculating it into a culture medium containing active substances at a suitable mass ratio. Fermentation is then carried out under suitable conditions. During the fermentation process, *Schizophyllum commune* synthesizes β-glucan de novo, simultaneously synthesizing β-glucan with a hydrophilic cavity triple helix structure from glucose. Simultaneously, it undergoes supramolecular self-assembly with the active substances, directly encapsulating the active substances within the triple helix cavity structure of the β-glucan, thus obtaining a β-glucan supramolecular polymer encapsulating the active substances. This avoids the use of physical encapsulation and chemical cross-linking methods for encapsulating the active substances, preventing the problem of toxic and harmful reagent residues, and effectively improving the encapsulation rate of the active substances.

[0030] For example, the fermentation temperature may be, but is not limited to, 27°C, 28°C, 29°C, or 30°C. The fermentation time may be, but is not limited to, 90 h, 91 h, 92 h, 93 h, 94 h, 95 h, 96 h, 97 h, 98 h, 99 h, or 100 h.

[0031] In some embodiments of this application, the mass percentage of the active substance is 1% to 5%; the mass percentage of the *Schizophyllum commune* bacterial solution is 5% to 10%. For example, the mass percentage of the active substance may be, but is not limited to, 1%, 2%, 3%, 3.5%, 4%, 4.5%, or 5%. The mass percentage of the *Schizophyllum commune* bacterial solution is 5%, 5.5%, 6%, 6.5%, 7%, 8%, 9%, or 10%. Using appropriate mass percentages of active substance and *Schizophyllum commune* bacterial solution in a culture medium for fermentation facilitates efficient encapsulation of the active substance within β-glucan during the β-glucan synthesis process by *Schizophyllum commune*, forming a β-glucan supramolecular polymer encapsulating the active substance.

[0032] In some embodiments of this application, the mass percentage of the active substance is 1% to 3%; the mass percentage of the *Schizophyllum commune* bacterial solution is 7.5% to 10%. For example, the mass percentage of the active substance may be, but is not limited to, 1%, 1.5%, 2%, 2.5%, or 3%. The mass percentage of the *Schizophyllum commune* bacterial solution is 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. Further fermentation using appropriate mass percentages of the active substance and *Schizophyllum commune* bacterial solution in a culture medium facilitates more efficient encapsulation of the active substance within β-glucan during the β-glucan synthesis process of *Schizophyllum commune*, thereby forming a β-glucan supramolecular polymer encapsulating the active substance.

[0033] In some embodiments of this application, the mass percentage of the active substance is 2.5% to 3%; the mass percentage of the *Schizophyllum commune* bacterial solution is 7.5% to 8%. For example, the mass percentage of the active substance may be, but is not limited to, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3%. The mass percentage of the *Schizophyllum commune* bacterial solution is 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, or 8%. Furthermore, using a more suitable mass percentage of active substance and *Schizophyllum commune* bacterial solution in the culture medium for fermentation facilitates more efficient encapsulation of the active substance within β-glucan during the β-glucan synthesis process of *Schizophyllum commune*, thereby forming a β-glucan supramolecular polymer encapsulating the active substance.

[0034] In some embodiments of this application, the active substance includes anthocyanins. Using suitable active substances, β-glucan supramolecular polymers encapsulating the active substances can be obtained by fermentation with *Schizophyllum commune* in a culture medium, exhibiting a high encapsulation rate of the active substances, improving their stability, and facilitating their subsequent applications.

[0035] In some embodiments of this application, the culture medium substrate, by mass percentage, comprises: 0.3%–0.5% yeast extract, 0.05%–0.08% magnesium sulfate, 0.1%–0.3% potassium dihydrogen phosphate, 3%–5% glucose, and the remainder being distilled water. Using a suitable culture medium substrate for fermentation of the active substance and the *Schizophyllum commune* broth facilitates the efficient encapsulation of the active substance within β-glucan during the β-glucan synthesis process by *Schizophyllum commune*, thereby forming a β-glucan supramolecular polymer encapsulating the active substance.

[0036] In some embodiments of this application, the method for preparing *Schizophyllum commune* bacterial suspension includes: transferring *Schizophyllum commune* slant culture to a liquid culture medium and culturing it for 3-5 days to obtain *Schizophyllum commune* bacterial suspension. By transferring *Schizophyllum commune* slant culture to a liquid culture medium for culturing to obtain *Schizophyllum commune* bacterial suspension, it is beneficial for subsequent fermentation with active substances. This allows *Schizophyllum commune* to efficiently encapsulate active substances within β-glucan during the β-glucan synthesis process, forming a β-glucan supramolecular polymer encapsulating the active substances.

[0037] In some embodiments of this application, the fermentation product obtained from fermentation is further centrifuged and filtered to obtain a β-glucan supramolecular polymer encapsulated with active substances. By centrifuging and filtering the fermentation product obtained from fermentation to remove unencapsulated active substances, a stable β-glucan supramolecular polymer encapsulated with active substances is obtained.

[0038] This application provides a β-glucan supramolecular polymer prepared by the preparation method provided in the first aspect.

[0039] The β-glucan supramolecular polymer prepared by the preparation method of the first aspect of this application can efficiently and stably encapsulate active substances, with a high encapsulation rate and effectively improved stability of active substances, which is beneficial for subsequent applications.

[0040] This application provides an application of the β-glucan supramolecular polymer as provided in the second aspect in the preparation of cosmetics. The β-glucan supramolecular polymer provided in this application has a high loading rate for active substances and effectively improves the stability of active substances, which is beneficial for its application in the preparation of cosmetics.

[0041] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0042] The specific equipment used in the embodiments and test examples is as follows: 1. Slant culture of *Schizophyllum commune*: purchased from Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC 5.43.

[0043] 2. Anthocyanins: Blueberry anthocyanin extract, purchased from Shaanxi Taimei Bioengineering Co., Ltd., with an anthocyanin content of 38%.

[0044] Example 1 This embodiment provides a method for preparing β-glucan supramolecular polymers, including: S1: Transfer the Schizophyllum commune slant culture to potato dextrose liquid medium and culture for 3 days to obtain Schizophyllum commune liquid culture; S2: Inoculate the culture medium with 7.5% by weight of Schizophyllum commune and ferment at 28°C and 160 rpm for 96 h; the culture medium consists of the following components by weight: 3% anthocyanins, 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose, and the remainder is distilled water, pH 6.5; S3: After fermentation, the fermentation product is centrifuged and the resulting supernatant is passed through a 100 nm ceramic membrane to remove free anthocyanins. The resulting filtrate is a β-glucan-anthocyanin supramolecular polymer.

[0045] Example 2 This embodiment provides a method for preparing β-glucan supramolecular polymers, the only difference from Example 1 being that the culture medium includes 1% anthocyanins.

[0046] Example 3 This embodiment provides a method for preparing β-glucan supramolecular polymers, the only difference from Example 1 being that the culture medium includes 5% anthocyanins.

[0047] Example 4 This embodiment provides a method for preparing β-glucan supramolecular polymers, the only difference from Example 1 being that 5% by mass of Schizophyllum commune solution is inoculated into the culture medium.

[0048] Example 5 This embodiment provides a method for preparing β-glucan supramolecular polymers, the only difference from Example 1 being that 10% by mass of Schizophyllum commune solution is inoculated into the culture medium.

[0049] Comparative Example 1 This comparative example provides a method for preparing a β-glucan solution, the only difference from Example 1 being that the culture medium does not contain anthocyanins.

[0050] Comparative Example 2 This comparative example provides a method for preparing anthocyanin solution, comprising: mixing 3% anthocyanin with 97% distilled water to prepare an anthocyanin solution.

[0051] Comparative Example 3 This embodiment provides a method for preparing a β-glucan mixture, including: S1: Transfer the Schizophyllum commune slant culture to potato dextrose liquid medium and culture for 3 days to obtain Schizophyllum commune liquid culture; S2: 7.5% by weight of *Schizophyllum commune* culture was inoculated into the culture medium and fermented at 28°C and 160 rpm for 96 h. The culture medium consisted of the following components by weight: 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose, and the remainder was distilled water, pH 6.5. After fermentation, the fermentation product was centrifuged, and the supernatant was passed through a 100 nm ceramic membrane. The resulting filtrate was a β-glucan solution. S3: Mix 97% β-glucan solution and 3% anthocyanin solution, stir at 28℃ and 160 rpm for 96 h, and then pass through a 100 nm ceramic membrane. The resulting effluent is a β-glucan-anthocyanin mixture.

[0052] The preparation methods provided in Examples 1-5 and Comparative Examples 1-3 are shown in Table 1.

[0053] Table 1. Preparation methods provided in Examples 1-5 and Comparative Examples 1-3

[0054] Note: In Table 1, " / " indicates that the component is not included.

[0055] Experimental Example 1 This experiment determined the β-glucan content and β-glucan-anthocyanin binding rate of the β-glucan supramolecular polymers provided in Examples 1-5 and the solutions provided in Comparative Examples 1-3, as follows: (1) Determination of β-glucan content: Take the supernatant after centrifugation of the above fermentation products, centrifuge at 15000×g and 4℃ for 10min, collect the supernatant; add 2 times the volume of anhydrous ethanol for alcohol precipitation, filter the obtained solids, wash 3 times with anhydrous ethanol, dry at 60℃ to constant weight, weigh, and obtain the β-glucan content. (2) Determination of β-glucan-anthocyanin binding rate: The supernatant and filtrate of each fermentation product after centrifugation were taken, and the total anthocyanin content of each supernatant and filtrate was determined according to the instructions of the total anthocyanin content determination kit (purchased from Suzhou Grace Biotechnology Co., Ltd.); the anthocyanin binding rate was calculated according to the following formula: anthocyanin binding rate (%) = total anthocyanin content (filtrate) / total anthocyanin content (supernatant) * 100%. The results are shown in Table 2.

[0056] (3) Determination of characteristic curves of β-glucan-anthocyanin supramolecular polymers: Samples from Experimental Example 1, Comparative Example 1, and Comparative Example 2 were placed in an ELISA reader for scanning across the entire wavelength range of 200–700 nm. The absorbance was recorded and characteristic curves were plotted. The results are as follows: Figure 1 As shown.

[0057] Table 2 Anthocyanin binding rate

[0058] As shown in Table 2, compared with Examples 1-5 and Comparative Example 3, the preparation methods in Examples 1-5 effectively improved the binding rate of β-glucan and anthocyanins, with the binding rate increasing by 85%-270%. This is because during the de novo synthesis of β-glucan from glucose and the formation of a triple-helix cavity in *Schizophyllum commune*, anthocyanins can efficiently enter the cavity and stably bind to the β-glucan side chains through non-covalent bonds, ultimately significantly increasing the anthocyanin binding rate and the amount of β-glucan-anthocyanin supramolecular polymers generated.

[0059] Compared with Examples 1, 2, and 5, the binding rate of β-glucan and anthocyanins in the preparation methods of Examples 3 and 4 was relatively low. This is because Example 3 added 5% anthocyanins, which far exceeded the β-glucan carrying capacity of the Schizophyllum commune fermentation system, resulting in a large amount of free anthocyanins not entering the β-glucan cavity and being filtered out during the ceramic membrane filtration process. In Example 4, only 5% Schizophyllum commune was added, resulting in a low initial cell volume, which was insufficient to produce enough β-glucan (the β-glucan content was significantly lower than in other examples) to bind with anthocyanins within 96 hours of fermentation, resulting in a lower content of the β-glucan-anthocyanin supramolecular polymer.

[0060] In Comparative Example 3, after stirring for 96 hours, some anthocyanins were able to bind with β-glucan, indicating that physical methods such as stirring can open part of the triple helix cavity of β-glucan, allowing some anthocyanins to enter the cavity and form supramolecular polymers. However, the efficiency of opening the triple helix cavity of β-glucan through physical methods is low, resulting in an anthocyanin binding rate of only 18.0%. The above results indicate that this application utilizes Schizophyllum commune and anthocyanins in a culture medium for fermentation. During the de novo synthesis of β-glucan by Schizophyllum commune, anthocyanins can be directly encapsulated in the triple helix cavity structure of β-glucan, effectively improving the encapsulation rate of active substances and the amount of β-glucan-anthocyanin supramolecular polymers generated.

[0061] Depend on Figure 1 The results showed that the 3% anthocyanin in Comparative Example 2 had a significant characteristic absorption peak at 275 nm, while the β-glucan (SPG) in Comparative Example 1 had no absorption peak at this wavelength. The characteristic absorption peak of the β-glucan-anthocyanin supramolecular polymer (SAC) in Example 1 showed a significant blue shift to 272 nm, and the peak shape differed from that of the anthocyanin in Comparative Example 2, indicating that β-glucan and anthocyanin formed a stable supramolecular polymer. This demonstrates that this application utilizes *Schizophyllum commune* and anthocyanin fermentation in a culture medium. During the de novo synthesis of β-glucan by *Schizophyllum commune*, anthocyanin can be directly encapsulated within the triple helix cavity structure of β-glucan to form a stable β-glucan supramolecular polymer.

[0062] Experimental Example 2 This experiment tested the photothermal stability of the β-glucan-anthocyanin supramolecular polymer provided in Example 1 and the anthocyanin solution provided in Comparative Example 2. The results are shown in Table 3.

[0063] Table 3 Photothermal Stability

[0064] As shown in Table 3, the β-glucan-anthocyanin supramolecular polymer provided in Example 1 showed no significant changes in color and viscosity after 3 months of storage under five storage conditions: 25°C, light exposure, 45°C, -18°C, and alternating temperatures from -18°C to 45°C. In contrast, the 3% anthocyanin solution provided in Comparative Example 2 showed discoloration after 14 days of storage under light exposure, 45°C, and alternating temperatures from -18°C to 45°C. After 45 days of storage, the color further changed and obvious precipitates appeared, indicating that the anthocyanins underwent denaturation under high temperature, light exposure, and continuous temperature changes. This application describes the fermentation of Schizophyllum commune and anthocyanins in a culture medium. During the de novo synthesis of β-glucan by Schizophyllum commune, anthocyanins can be directly encapsulated in the triple helix cavity structure of β-glucan to form a stable β-glucan supramolecular polymer. Compared with anthocyanins, the β-glucan-anthocyanin supramolecular polymer prepared in this application has good photothermal stability, which can effectively extend its shelf life and promote its application in cosmetics.

[0065] Experimental Example 3 This experiment tested the diversity and abundance of skin microbiota using the β-glucan supramolecular polymer provided in Example 1 and the solutions provided in Comparative Examples 1 and 2 to evaluate their efficacy in regulating the skin microecology. The specific steps were as follows: Samples from Example 1, Comparative Examples 1 and 3 were filtered through a 0.22 μm microporous membrane (for sterilization) before use. Volunteers were Chinese residents of Guangzhou, aged 27-35 years, without any skin diseases such as atopic dermatitis, and had not undergone any clinical treatment in the three months prior to sampling. Volunteers washed their faces 30 minutes before sampling to initialize their skin condition. Sterile cotton swabs were used to collect samples from four locations on both cheeks. The sample material was placed in 2 ml of LoBindEppendorf solution. The tubes (containing preservation buffer) served as a blank control group; subsequently, volunteers were randomly assigned to apply half of their face with Example 1 (2% β-glucan-anthocyanin supramolecular polymer), Comparative Example 1 (2% β-glucan), Comparative Example 2 (2% anthocyanin), and pure water (solvent control group). After 12 hours of application, the skin samples from the applied half of the face were collected using sterile cotton swabs, and all samples were stored at -80 °C. High-throughput sequencing was used to determine the conserved regions of V3 and V4 in the 16S rRNA gene of skin microbiota from facial swabs. The alpha diversity indices, specifically the Observed Species index and the Shannon index, were calculated using QIIME software. The UCLUST sequence alignment tool in Quantitative Insights Into MicrobialEcology (QIIME, v 2.0) software was used to divide the sequences from valid gene data into one OUT unit with a similarity greater than 97%. The sequence with the highest abundance in each OUT unit was used as the representative species sequence for that OTU for species classification annotation. The representative sequence of each OTU was aligned with the Silva 138.1 database to obtain the taxonomic information and microbiota abundance of all OTUs. The results are shown in Tables 4 and 5.

[0066] Table 4 Alpha Diversity Index

[0067] Table 5 Abundance of Characteristic Microbial Communities

[0068] As shown in Table 4, compared with the blank group, there was no significant increase in the abundance and diversity indices of skin flora on the volunteers' skin after applying pure water for 12 hours (solvent control group). However, after applying Example 1 (2% β-glucan-anthocyanin supramolecular polymer) and Comparative Example 1 (2% β-glucan), the abundance and diversity indices of skin flora on the skin surface increased significantly, with Example 1 showing a greater increase than Comparative Example 1. Comparative Example 2 (2% anthocyanin) had no significant effect on the abundance and diversity indices of skin flora on the skin surface. These results indicate that both β-glucan and β-glucan-anthocyanin supramolecular polymers can enhance the abundance and diversity of skin flora. This demonstrates that the stable β-glucan supramolecular polymer formed by fermenting Schizophyllum commune and anthocyanin in a culture medium has a significant synergistic effect and can enhance the regulatory effect of β-glucan on the skin microecology.

[0069] As shown in Table 5, compared with the blank group, after applying pure water for 12 hours (solvent control group), the abundance of characteristic microbial communities on the volunteers' skin surface changed significantly. For example, the abundance of harmful skin bacteria such as *Propionibacterium acnes*, *Lactosoma spp.*, and *Streptococcus spp.* increased significantly, while the abundance of the skin probiotic *Lachnospiraceae* NK4A136 decreased significantly. After applying *Example 1* (2% β-glucan-anthocyanin supramolecular polymer) and *Comparative Example 1* (2% β-glucan), the abundance of harmful skin bacteria such as *Propionibacterium acnes*, *Lactosoma spp.*, and *Streptococcus spp.* decreased significantly, while the abundance of the skin probiotic *Lachnospiraceae* decreased significantly. NK4A136 was significantly increased; and the inhibitory effect of Example 1 on the abundance of harmful skin bacteria and the promoting effect on the abundance of beneficial bacteria were significantly higher than those of Comparative Example 1; indicating that this application ferments Schizophyllum commune and anthocyanins in a culture medium to form a stable β-glucan supramolecular polymer, which has a significant synergistic effect and can further significantly improve the structure and composition of facial skin flora based on β-glucan, thus having a good effect on regulating the skin microecology.

[0070] Test Example 4 This experimental example comprehensively analyzed and identified changes in lipid metabolites in facial skin using lipidomics in samples from each group of Experiment 3. Details are as follows: 100 μL of each liquid sample was added to a glass centrifuge tube with a PTFE-lined cap. 0.75 mL of pre-chilled methanol was added, and the mixture was vortexed. 2.5 mL of pre-chilled methyl tert-butyl ether was added, and after vortexing, 10 μL of LPLASH™ internal standard was added. The mixture was incubated on a shaker at room temperature for 1 h. 0.625 mL of mass spectrometry-grade water was added and mixed to separate the organic phases. After incubation at room temperature for 10 min, the mixture was centrifuged at 1000 g for 10 min. The upper organic phase was collected, and the lower layer (water and methanol) was extracted again with 1 mL of a mixed solvent (methyl tert-butyl ether / methanol / water (volume ratio 10:3:2.5)). The upper organic phase was collected again. The two collected organic phases were concentrated by nitrogen blowing. The solutions were redissolved in 100 μL of isopropanol and then analyzed by LC-MS / MS. (1) Chromatographic conditions: Flow rate: 0.35 mL / min; Column temperature: 40 ℃; Injection volume: 5 μL. Elution program: The mobile phase was 0.1% formic acid-10mM ammonium acetate-acetonitrile (60%)-aqueous solution (A) and 0.1% formic acid-10mM ammonium acetate-acetonitrile (10%)-isopropanol solution (B). The gradient elution program was: 0~2 min, 30% B; 2~5 min, 30-43% B; 5~5.1 min, 43~55% B; 5.1~11 min, 55-70% B; 11~16 min, 70-99% B; 16~18 min, 99% B; 18~18.1 min, 99~30% B; (2) Mass spectrometry conditions Data were acquired using an electrospray ionization source in both positive and negative ion modes. Specific conditions were as follows: positive ion spray voltage: 3.50 kV; negative ion spray voltage: -3.50 kV; sheath pressure: 40 psi; positive ion auxiliary gas pressure: 10 L / min; negative ion auxiliary gas pressure: 7 L / min; heater temperature: 350 ℃; capillary temperature: 320 ℃; ion introduction RF level: 50. A first-stage full scan was performed at a resolution of 70,000 m / z, with a first-stage ion (m / z) scan range of 114–1700 m / z. Second-stage fragmentation was performed using an HCD with collision voltages of normalized collision energies of 22 eV, 24 eV, and 28 eV (for negative ions: 22 eV, 24 eV, and 28 eV). The second-stage resolution was 17,500 m / z. Unnecessary MS / MS information was removed using a dynamic exclusion method. The raw data from the lipid analyzer was imported into the Lipidsearch database. First, peak extraction was performed by matching the Lipidsearch database with parameters such as a parent ion mass deviation of 5 ppm and a daughter ion mass deviation of 5 ppm to obtain qualitative information about the substances. Then, peak alignment between samples was performed using parameters such as a retention time deviation of 0.05 minutes and a signal-to-noise ratio of 3. Finally, background ions were removed using blank samples, and the quantitative results were normalized to perform qualitative and quantitative analysis of the lipid data. The formula for calculating substance concentration is:

[0071] Among them, C ki C represents the concentration of lipid i in the sample. ls Q represents the concentration of the corresponding internal standard added to the sample; kl Q is the quantitative value of lipid i in the sample; ks The values ​​are the quantitative values ​​of the corresponding internal standards in the sample. The results are shown in Table 6.

[0072] Table 6. Types and Content of Facial Oils

[0073] As shown in Table 6, compared with the blank group, after applying pure water for 12 hours (solvent control group), the content of characteristic oils on the surface of the volunteers' skin changed significantly, such as a significant increase in the content of common skin oils such as diacylglycerol, phosphatidylglycerol, ceramide, and fatty esters. After applying Comparative Example 1 (2% β-glucan), the content of diacylglycerol, phosphatidylglycerol, ceramide, and fatty esters also increased to a certain extent. However, after applying Example 1 (2% β-glucan-anthocyanin supramolecular polymer), the content of diacylglycerol and fatty esters decreased significantly, while the amount of phosphatidylglycerol and ceramide increased only slightly. This indicates that the present application, through the fermentation of Schizophyllum commune and anthocyanins in a culture medium, forms a stable β-glucan supramolecular polymer, which can significantly inhibit the production and secretion of facial skin oil and has a good oil-controlling effect.

[0074] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for preparing a β-glucan supramolecular polymer, characterized in that, include: The *Schizophyllum commune* broth is inoculated into a culture medium containing an active substance and a culture substrate, wherein the mass ratio of the *Schizophyllum commune* broth to the active substance is 1-10:1; fermentation is carried out at 27-30°C for 90-100 h to obtain a β-glucan supramolecular polymer encapsulated with the active substance.

2. The preparation method according to claim 1, characterized in that, The active substance has a mass percentage of 1% to 5%; the Schizophyllum commune has a mass percentage of 5% to 10%.

3. The preparation method according to claim 2, characterized in that, The active substance has a mass percentage of 1% to 3%; the Schizophyllum commune has a mass percentage of 7.5% to 10%.

4. The preparation method according to claim 3, characterized in that, The active substance has a mass percentage of 2.5% to 3%; the Schizophyllum commune has a mass percentage of 7.5% to 8%.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The active substances include anthocyanins.

6. The preparation method according to any one of claims 1 to 4, characterized in that, The culture medium substrate comprises, by weight percentage: 0.3%~0.5% yeast extract, 0.05%~0.08% magnesium sulfate, 0.1%~0.3% potassium dihydrogen phosphate, 3%~5% glucose, and the remainder being distilled water.

7. The preparation method according to any one of claims 1 to 4, characterized in that, The method for preparing the Schizophyllum commune culture includes: transferring the Schizophyllum commune slant culture to a liquid culture medium, and culturing it for 3-5 days to obtain the Schizophyllum commune culture.

8. The preparation method according to any one of claims 1 to 4, characterized in that, The fermentation product obtained from the fermentation is further centrifuged and filtered to obtain the β-glucan supramolecular polymer encapsulating the active substance.

9. A β-glucan supramolecular polymer prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the β-glucan supramolecular polymer as described in claim 9 in the preparation of cosmetics.

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