High-transparency schizophyllum commune polysaccharide as well as preparation method and cosmetics thereof

Abstract

The invention relates to the field of cosmetics, in particular to high-transparency schizophyllum commune polysaccharide, a preparation method thereof and cosmetics. The method comprises the following steps: freezing a schizophyllum commune fermentation mixture containing mycelia under a negative temperature condition to obtain a frozen product; unfreezing the frozen product to obtain an unfrozen mixture; centrifuging the unfrozen mixture, filtering to remove mycelium, and collecting fermentation filtrate; adding absolute ethyl alcohol with the same mass into the fermentation filtrate for alcohol precipitation of polysaccharide to obtain polysaccharide precipitate; collecting polysaccharide precipitates, and repeatedly washing the polysaccharide precipitates with an ethanol solution to obtain washed polysaccharide precipitates; and centrifuging the washed polysaccharide precipitate, collecting the centrifuged polysaccharide precipitate, and redissolving the polysaccharide precipitate in the solvent. According to the technical scheme, the freezing and thawing step is specifically applied to the mycelium-containing schizophyllum commune fermentation mixture, and the colorless schizophyllum commune polysaccharide with excellent transparency is obtained by taking secreted exopolysaccharide as a main target for non-pure mycelium.

Description

Technical Field

[0001] This application relates to the field of cosmetics, specifically to a high-transparency Schizophyllum commune polysaccharide, its preparation method, and cosmetics. Background Technology

[0002] Schizophyllum commune is an edible fungus with important medicinal value. The polysaccharide produced by its metabolism is a β-glucan, which has attracted much attention due to its excellent immunomodulatory, antitumor, and antiviral biological activities, and has broad application prospects in the fields of medicine, functional food and cosmetics.

[0003] In the industrial production of Schizophyllum commune polysaccharides, liquid submerged fermentation technology is typically used to obtain a fermentation mixture containing mycelia and extracellular polysaccharides. Currently, the classic process route for separating and purifying polysaccharides from the fermentation broth is as follows: filtration or centrifugation to remove mycelia and obtain a clear fermentation filtrate; adding a certain multiple of organic solvents such as ethanol or acetone to the filtrate for alcohol precipitation, causing the polysaccharides to precipitate from the solution; collecting the polysaccharide precipitate and removing co-precipitated protein impurities through repeated alcohol washing or traditional methods such as the Sevage method or enzymatic methods; finally, redissolving the preliminarily purified polysaccharides in water for further processing.

[0004] However, the aqueous solution of Schizophyllum commune polysaccharide obtained by the above-mentioned traditional process often appears white to pale yellow or slightly turbid, with poor transparency. This visual deficiency severely limits its application in high-value fields such as high-end cosmetics or injectable pharmaceutical preparations. Summary of the Invention

[0005] The purpose of this application is to provide a high-transparency Schizophyllum commune polysaccharide, its preparation method, and a cosmetic.

[0006] In a first aspect, this application provides a method for preparing high-transparency Schizophyllum commune polysaccharide, comprising: A mixture of Schizophyllum commune fermentation materials containing mycelium was frozen at a negative temperature to obtain a frozen product. The frozen product is thawed to obtain a thawed mixture; Centrifuge and filter the thawed mixture to remove mycelium, and collect the fermentation filtrate. An equal mass of anhydrous ethanol was added to the fermentation filtrate to precipitate the polysaccharide, resulting in a polysaccharide precipitate. Collect the polysaccharide precipitate, and wash the polysaccharide precipitate repeatedly with ethanol solution to obtain the washed polysaccharide precipitate; The washed polysaccharide precipitate was centrifuged, the precipitate was collected, and the precipitate was redissolved in a solvent.

[0007] The above technical solution, by specifically applying the freeze-thaw step to a "Schizophyllum commune fermentation mixture containing mycelium," rather than simply targeting the mycelium, focuses on the secreted extracellular polysaccharides, resulting in colorless and highly transparent Schizophyllum commune polysaccharides. Furthermore, the above technical solution employs a "freeze-thaw-self-digestion" process to rupture the mycelium, releasing proteases that decompose soluble proteins in the fermentation broth. This cleverly utilizes endogenous enzymes within the mycelium for "self-digestion," yielding highly transparent and pure Schizophyllum commune polysaccharides. Furthermore, the above technical solution utilizes Schizophyllum commune's own enzyme system to degrade impurities, avoiding the use of exogenous chemical reagents or enzyme preparations, making it more environmentally friendly and safer. This expands its application in high-value fields such as high-end cosmetics or injectable pharmaceutical preparations. Furthermore, the above technical solution, employing a "freeze-thaw-self-digestion" concept process, requires simple equipment and simplifies the repeated washing process after alcohol precipitation, avoiding the complex processes associated with traditional exogenous reagent additions, making it easy to industrialize.

[0008] In other embodiments of this application, the fermentation mixture of Schizophyllum commune containing mycelium is frozen under negative temperature conditions, including: Freezing is carried out under sub-zero temperature conditions of -20℃ to -80℃; Optionally, the freezing time is 1-3 days.

[0009] In other embodiments of this application, thawing the frozen product includes: Thawing temperature: 10~25℃, thawing time: more than 1~3 days.

[0010] In other embodiments of this application, the polysaccharide precipitate is repeatedly washed with an ethanol solution, including: The polysaccharide precipitate was washed with an ethanol solution with a mass fraction of 0% to 100%.

[0011] In other embodiments of this application, the polysaccharide precipitate is repeatedly washed with ethanol, including: The polysaccharide precipitate was washed with an ethanol solution of 75% to 100% by mass. Optionally, the washing process is repeated 1 to 3 times; optionally, the mass fraction of the ethanol solution gradually decreases during multiple washes. Optionally, the mass ratio of the added polysaccharide precipitate to the ethanol solution is (0.5~2):(0.5~2).

[0012] In other embodiments of this application, redissolving the polysaccharide precipitate in a solvent includes: The polysaccharide precipitate was redissolved in water.

[0013] Secondly, this application provides a high-transparency Schizophyllum commune polysaccharide, which is prepared using any of the preparation methods described in the first aspect; High-transparency Schizophyllum polysaccharide is mainly derived from the extracellular secreted polysaccharide of Schizophyllum through bio-fermentation.

[0014] In other embodiments of this application, when the viscosity of the high-transmittance Schizophyllum commune polysaccharide aqueous solution is >8333 cP, the absorbance OD600 is below 0.11; Optionally, when the viscosity of the high-transmittance Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 5000 cP, the absorbance OD600 is between 0.08 and 0.11.

[0015] In other embodiments of this application, when the polysaccharide content in the high-transparency Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 0.81%, the viscosity of the Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 5000 cP; optionally, when the polysaccharide content in the high-transparency Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 1.10%, the viscosity of the Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 8333 cP.

[0016] In other embodiments of this application, when the viscosity of the high-transparency Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 5000 cP, the polysaccharide content in the high-transparency Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 0.81%; optionally, when the viscosity of the high-transparency Schizophyllum commune polysaccharide aqueous solution is >8333 cP, the polysaccharide content in the high-transparency Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 1.10%.

[0017] Thirdly, this application provides a cosmetic product comprising the high-transparency Schizophyllum commune polysaccharide provided in any of the first aspects; or the cosmetic product comprises the high-transparency Schizophyllum commune polysaccharide prepared by the preparation method of the high-transparency Schizophyllum commune polysaccharide provided in any of the second aspects. Attached Figure Description

[0018] 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.

[0019] Figure 1 This is a flowchart of the preparation process of Schizophyllum commune polysaccharide.

[0020] Figure 2 The diagram shows the appearance of the polysaccharides from *Schizophyllum commune* in Examples 1-2 and Comparative Examples 1-3.

[0021] Figure 3 This is a comparison of the protein content of Schizophyllum commune polysaccharides in Examples 1-2 and Comparative Examples 1-3.

[0022] Figure 4The absorbance values ​​of Schizophyllum commune polysaccharides in Examples 1-2 and Comparative Examples 1-3 are compared.

[0023] Figure 5 This is a schematic diagram showing the appearance comparison of different concentrations of Schizophyllum commune polysaccharides in Experiment Example 2.

[0024] Figure 6 This is a schematic diagram showing the protein content of different concentrations of Schizophyllum commune polysaccharides in Experiment Example 2.

[0025] Figure 7 This is a schematic diagram showing the absorbance values ​​of different concentrations of Schizophyllum commune polysaccharides in Experiment Example 2. 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. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0027] Therefore, the following 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 of the application. 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.

[0028] The inventors discovered through research that the aqueous solution of Schizophyllum commune polysaccharide obtained by traditional alcohol precipitation is white to pale yellow or slightly turbid with poor transparency. The root cause of this problem is that the fermentation broth contains a large amount of soluble proteins. During alcohol precipitation, these proteins co-precipitate with the polysaccharide or form complexes, creating stubborn impurities that are difficult to remove completely by conventional washing. Existing exogenous protein removal methods, such as the Sevage method, introduce the risk of toxic organic solvent residues, while enzymatic methods are costly and may introduce exogenous proteins, both of which have certain limitations.

[0029] Based on the above research, this application provides a method for preparing high-transparency Schizophyllum commune polysaccharide, the method comprising: A mixture of Schizophyllum commune fermentation materials containing mycelium was frozen at a negative temperature to obtain a frozen product. The frozen product is thawed to obtain a thawed mixture; Centrifuge and filter the thawed mixture to remove mycelium, and collect the fermentation filtrate. An equal mass of anhydrous ethanol was added to the fermentation filtrate to precipitate the polysaccharide, resulting in a polysaccharide precipitate. Collect the polysaccharide precipitate, and wash the polysaccharide precipitate repeatedly with ethanol solution to obtain the washed polysaccharide precipitate; The washed polysaccharide precipitate was centrifuged, the precipitate was collected, and the precipitate was redissolved in a solvent.

[0030] The above technical solution, by specifically applying the freeze-thaw step to a "Schizophyllum commune fermentation mixture containing mycelium," rather than simply targeting the mycelium, focuses on the secreted extracellular polysaccharides, resulting in colorless and highly transparent Schizophyllum commune polysaccharides. Furthermore, the above technical solution employs a "freeze-thaw-self-digestion" process to rupture the mycelium, releasing proteases that decompose soluble proteins in the fermentation broth. This cleverly utilizes endogenous enzymes within the mycelium for "self-digestion," yielding highly transparent and pure Schizophyllum commune polysaccharides. Furthermore, the above technical solution utilizes Schizophyllum commune's own enzyme system to degrade impurities, avoiding the use of exogenous chemical reagents or enzyme preparations, making it more environmentally friendly and safer. This expands its application in high-value fields such as high-end cosmetics or injectable pharmaceutical preparations. Furthermore, the above technical solution, employing a "freeze-thaw-self-digestion" concept process, requires simple equipment and simplifies the repeated washing process after alcohol precipitation, avoiding the complex processes associated with traditional exogenous reagent additions, making it easy to industrialize.

[0031] Furthermore, referring to Figure 1 In some embodiments of this application, the preparation method of high-transparency Schizophyllum commune polysaccharide includes the following steps: Step S1: Prepare a fermentation mixture of Schizophyllum commune containing mycelium.

[0032] Furthermore, in some embodiments of this application, the steps for preparing a Schizophyllum commune fermentation mixture containing mycelium include: strain activation, primary inoculation and fermentation, secondary inoculation and fermentation, and fermentation inoculation and fermentation.

[0033] Further optionally, in some embodiments of this application, strain activation includes: under aseptic conditions, after thoroughly sterilizing the inoculation spatula by flaming, preferentially selecting colonies far from the center of the plate, using the inoculation spatula to cut a 1×1 cm solid culture medium containing hyphae, inoculating it into an Erlenmeyer flask containing the culture medium, and culturing it at 28°C and 120±20 r / min for 80±12 h to obtain a shake-flask primary activated seed solution.

[0034] Further optionally, in some embodiments of this application, the primary inoculation and fermentation include: inoculating the activated bacterial solution obtained above into a 20 L fermenter, setting the fermentation parameters to 28°C, tank pressure 0.05 MPa, dissolved oxygen 30%, and fermenting for 30 h to 40 h.

[0035] Further optionally, in some embodiments of this application, the secondary inoculation and fermentation includes: inoculating the aforementioned primary seed into a secondary seed tank, with the fermentation parameters remaining unchanged.

[0036] Further optionally, in some embodiments of this application, the fermentation inoculation and fermentation include: inoculating the aforementioned secondary seed into a fermenter, setting the fermentation parameters to 28°C, tank pressure 0.05 MPa, dissolved oxygen 30%, and fermenting for 60 h to 70 h. After fermentation, the fermentation stock solution is heated to 80°C and kept at that temperature for 30 minutes to inactivate the inoculum.

[0037] Step S2: Freeze the fermentation mixture of Schizophyllum commune containing mycelium under negative temperature conditions to obtain the frozen product.

[0038] In some embodiments of this application, the fermentation mixture of Schizophyllum commune containing mycelium is frozen under negative temperature conditions, including: Freezing is carried out under negative temperature conditions of -20℃ to -80℃.

[0039] For example, in some embodiments of this application, freezing is performed at negative temperatures of -20°C, -25°C, -30°C, -35°C, -40°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, or any two of the aforementioned values.

[0040] In some embodiments of this application, the freezing time is greater than 0 days and less than or equal to 7 days. Optionally, the freezing time is 1 to 3 days.

[0041] For example, in some embodiments of this application, the freezing time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

[0042] In other optional embodiments of this application, the freezing time can be more than 7 days.

[0043] For example, in some embodiments of this application, 2 kg of Schizophyllum commune fermentation broth containing mycelium is taken and frozen at -20°C for 3 days.

[0044] Step S3: Thaw the frozen product to obtain a thawed mixture.

[0045] In some embodiments of this application, thawing the frozen product includes: Thawing temperature greater than 0 ℃, thawing time greater than 0 days.

[0046] Exemplarily, in some embodiments of this application, thawing the frozen product includes: The thawing temperature is 10℃~25℃, and the thawing time is 1 to 2 days.

[0047] For example, in some embodiments of this application, a thawed mixture is obtained by taking out the mixture and allowing it to thaw naturally at room temperature for 1 day.

[0048] Step S4: Centrifuge and filter the thawed mixture to remove mycelium and collect the fermentation filtrate.

[0049] In some embodiments of this application, centrifuging the thawed mixture includes: Centrifuge at 10,000 to 15,000 rpm for 10 to 30 minutes. For example, centrifuge at speeds of 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, 15,000 rpm, or any two of the aforementioned values ​​for 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes, 22 minutes, 23 minutes, 25 minutes, 28 minutes, or 30 minutes, or any two of the aforementioned values.

[0050] The fermentation filtrate after centrifugation is a fermentation filtrate containing extracellular secretory polysaccharides from Schizophyllum commune.

[0051] Step S5: Add an equal mass of anhydrous ethanol to the fermentation filtrate to precipitate the polysaccharide, thus obtaining the polysaccharide precipitate.

[0052] In some embodiments of this application, adding an equal mass of anhydrous ethanol to the fermentation filtrate for polysaccharide precipitation includes: Based on the mass ratio of fermentation filtrate to ethanol solution, add ethanol solution to fermentation filtrate in a 1:1 ratio and stir to allow polysaccharides to be fully extracted.

[0053] Further, optionally, in some embodiments of this application, the polysaccharide precipitate is repeatedly washed with an ethanol solution, including: The polysaccharide precipitate was washed with an ethanol solution with a mass fraction of 0% to 100%.

[0054] For example, the polysaccharide precipitate is washed with an ethanol solution of mass fraction of 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any two of the aforementioned values.

[0055] Further, optionally, in some embodiments of this application, the polysaccharide precipitate is repeatedly washed with an ethanol solution, including: The washing cycle is repeated more than 0 times and less than or equal to 10 times. Optionally, the concentration of ethanol is gradually reduced during repeated washing.

[0056] For example, in some embodiments of this application, the number of times the washing is repeated is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0057] Exemplary, in some embodiments of this application, 95% ethanol is added to the fermentation filtrate at a 1:1 ratio, and the mixture is stirred to allow the polysaccharides to fully precipitate. The precipitated Schizophyllum commune polysaccharide precipitate is then passed through a 100-mesh filter cloth to remove excess ethanol. 60% ethanol is then added to the Schizophyllum commune polysaccharide precipitate at a 1:1 ratio to wash the polysaccharide precipitate, and the washing is repeated twice.

[0058] Step S6: Centrifuge the washed polysaccharide precipitate, collect the centrifuged polysaccharide precipitate, and redissolve the polysaccharide precipitate in a solvent.

[0059] Further optionally, in some embodiments of this application, centrifuging the washed polysaccharide precipitate includes: Centrifuge at 10,000 to 15,000 rpm for 10 to 30 minutes. For example, centrifuge at speeds of 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, 15,000 rpm, or any two of the aforementioned values ​​for 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes, 22 minutes, 23 minutes, 25 minutes, 28 minutes, or 30 minutes, or any two of the aforementioned values.

[0060] Further, optionally, in some embodiments of this application, the polysaccharide precipitate is redissolved in a solvent, including: The polysaccharide precipitate was redissolved in water.

[0061] For example, in some embodiments of this application, the precipitate is collected by centrifugation at 13,000 rpm for 15 min; the precipitate is then redissolved in pure water to obtain an aqueous solution of Schizophyllum commune polysaccharide.

[0062] This application provides a high-transparency Schizophyllum commune polysaccharide, which is prepared using any of the preparation methods provided in the foregoing embodiments; High-transparency Schizophyllum polysaccharide is mainly derived from the extracellular secreted polysaccharide of Schizophyllum through bio-fermentation.

[0063] Further optionally, in some embodiments of this application, when the viscosity of the high-transmittance Schizophyllum commune polysaccharide aqueous solution is >8333 cP, the absorbance OD600 is below 0.11; for example, when the viscosity of the high-transmittance Schizophyllum commune polysaccharide aqueous solution is >8333 cP, the absorbance OD600 is 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 or a range between any two of the aforementioned values.

[0064] Further optionally, in some embodiments of this application, when the viscosity of the high-transmittance Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 5000 cP, the absorbance OD600 is between 0.08 and 0.11. Exemplarily, in some embodiments of this application, when the viscosity of the high-transmittance Schizophyllum commune polysaccharide aqueous solution is greater than or equal to 5000 cP, the absorbance OD600 is within the range of 0.08, 0.09, 0.10, 0.11, or any two of the aforementioned values.

[0065] Further optionally, in some embodiments of this application, when the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 0.81%, the viscosity of the *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 5000 cP. Exemplarily, in some embodiments of this application, when the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 0.81%, the viscosity of the *Schizophyllum commune* polysaccharide aqueous solution is 5000 cP, 5100 cP, 5200 cP, 5500 cP, 6000 cP, 7000 cP, 8000 cP, 8500 cP, or a range between any two of the aforementioned values.

[0066] Further optionally, in some embodiments of this application, when the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 1.10%, the viscosity of the *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 8333 cP. Exemplarily, in some embodiments of this application, when the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 1.10%, the viscosity of the *Schizophyllum commune* polysaccharide aqueous solution is 8333 cP, 8500 cP, 8600 cP, 8700 cP, 8800 cP, 9000 cP, or a range between any two of the aforementioned values.

[0067] Further optionally, in some embodiments of this application, when the viscosity of the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 5000 cP, the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 0.81%. Exemplarily, in some embodiments of this application, when the viscosity of the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 5000 cP, the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is 0.81%, 0.82%, 0.85%, 0.90%, 1.00%, 1.05%, 1.10%, 1.15%, 1.20%, or a range between any two of the aforementioned values.

[0068] Further optionally, in some embodiments of this application, when the viscosity of the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is >8333 cP, the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is greater than or equal to 1.10%. Exemplarily, in some embodiments of this application, when the viscosity of the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is >8333 cP, the polysaccharide content in the high-transparency *Schizophyllum commune* polysaccharide aqueous solution is 1.10%, 1.11%, 1.12%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.20%, or a range between any two of the aforementioned values.

[0069] Some embodiments of this application provide a cosmetic product comprising the high-transparency Schizophyllum commune polysaccharide provided in any of the foregoing embodiments; or the cosmetic product comprises the high-transparency Schizophyllum commune polysaccharide prepared by the preparation method of the high-transparency Schizophyllum commune polysaccharide provided in any of the foregoing embodiments.

[0070] The aforementioned cosmetics contain the aforementioned high-transparency Schizophyllum commune polysaccharide, which can effectively improve the transparency of cosmetics, resulting in higher purity, fewer impurities, and better performance.

[0071] The features and performance of this application will be further described in detail below with reference to embodiments: Example 1 A method for preparing high-transparency Schizophyllum commune polysaccharide is provided, comprising the following steps: (1) Preparation of Schizophyllum commune fermentation broth: Under aseptic conditions, after thoroughly sterilizing the inoculation spatula by flaming, colonies away from the center of the plate were selected preferentially. A 1×1 cm sample of solid culture medium containing mycelia was scraped using the spatula and inoculated into an Erlenmeyer flask containing the medium. The flask was incubated at 28℃ and 120 rpm for 80 h to obtain a primary activated seed culture. The primary activated seed culture was then inoculated into a 20 L fermenter, with fermentation parameters set to 28℃, 0.05 MPa, and 30% dissolved oxygen. Fermentation lasted 30 h to obtain the primary seed culture. The primary seed culture was then inoculated into a secondary seed tank, with the fermentation parameters remaining unchanged to obtain the secondary seed culture. The secondary seed culture was then inoculated into the fermenter, with fermentation parameters set to 28℃, 0.05 MPa, and 30% dissolved oxygen. Fermentation lasted 60 h. After fermentation, the fermentation stock was heated to 80℃ and incubated for 30 minutes to inactivate the mycelia; this yielded a *Schizophyllum commune* fermentation broth containing mycelia.

[0072] (2) Purification of Schizophyllum polysaccharides from Schizophyllum fermentation broth using a freeze-thaw-self-digestion process: Take 2 kg of the *Schizophyllum commune* fermentation broth containing mycelium obtained in step (1), freeze it at -20 ℃ for 3 days, then thaw it naturally at room temperature for 1 day. Centrifuge at 13000 rpm for 15 min to obtain a fermentation filtrate containing extracellular secreted *Schizophyllum commune* polysaccharide. Add 95% ethanol to the fermentation filtrate at a 1:1 ratio and stir to allow the polysaccharide to fully precipitate. Pass the precipitated *Schizophyllum commune* polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol. Add 60% ethanol to the *Schizophyllum commune* polysaccharide precipitate at a 1:1 ratio to wash the polysaccharide precipitate, repeating the washing twice. Centrifuge at 13000 rpm for 15 min and collect the precipitate. Redissolve the precipitate in pure water to obtain an aqueous solution of *Schizophyllum commune* polysaccharide, i.e., high-transparency *Schizophyllum commune* polysaccharide.

[0073] Example 2 A method for preparing high-transparency Schizophyllum commune polysaccharide is provided, which differs from Example 1 in that: step (2) uses a "freeze-thaw-self-digestion" process to purify Schizophyllum commune polysaccharide from the fermentation broth; step (2) of this example is as follows: Take 2 kg of the *Schizophyllum commune* fermentation broth containing mycelium obtained in step (1), freeze it at -80℃ for 1 day, then thaw it naturally at room temperature for 1 day. Centrifuge at 13000 rpm for 15 min to obtain a fermentation filtrate containing extracellular secreted *Schizophyllum commune* polysaccharide. Add 95% ethanol to the fermentation filtrate at a 1:1 ratio and stir to allow the polysaccharide to fully precipitate. Pass the precipitated *Schizophyllum commune* polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol. Add 60% ethanol to the *Schizophyllum commune* polysaccharide precipitate at a 1:1 ratio to wash the polysaccharide precipitate, repeating the washing twice. Centrifuge at 13000 rpm for 15 min and collect the precipitate. Redissolve the precipitate in pure water to obtain an aqueous solution of *Schizophyllum commune* polysaccharide, i.e., high-transparency *Schizophyllum commune* polysaccharide.

[0074] Example 3 A method for preparing high-transparency Schizophyllum commune polysaccharide is provided, which differs from Example 1 in that: step (2) uses a "freeze-thaw-self-digestion" process to purify Schizophyllum commune polysaccharide from the fermentation broth; step (2) of this example is as follows: Take 2 kg of the *Schizophyllum commune* fermentation broth containing mycelium obtained in step (1), freeze it at -40℃ for 3 days, then thaw it naturally at room temperature for 1 day. Centrifuge at 13000 rpm for 15 min to obtain a fermentation filtrate containing extracellular secreted *Schizophyllum commune* polysaccharides. Add 95% ethanol to the fermentation filtrate at a 1:1 ratio and stir to allow the polysaccharides to fully precipitate. Pass the precipitated *Schizophyllum commune* polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol. Add 60% ethanol to the *Schizophyllum commune* polysaccharide precipitate at a 1:1 ratio to wash the polysaccharide precipitate, repeating the washing twice. Centrifuge at 13000 rpm for 15 min and collect the precipitate. Redissolve the precipitate in pure water to obtain an aqueous solution of *Schizophyllum commune* polysaccharides. This is high-transparency *Schizophyllum commune* polysaccharides.

[0075] Example 4 A method for preparing high-transparency Schizophyllum commune polysaccharide is provided, which differs from Example 1 in that: step (2) uses a "freeze-thaw-self-digestion" process to purify Schizophyllum commune polysaccharide from the fermentation broth; step (2) of this example is as follows: Take 2 kg of the *Schizophyllum commune* fermentation broth containing mycelium obtained in step (1), freeze it at -20℃ for 5 days, then thaw it naturally at room temperature for 1 day. Centrifuge at 13000 rpm for 15 min to obtain a fermentation filtrate containing extracellular secreted *Schizophyllum commune* polysaccharides. Add 95% ethanol to the fermentation filtrate at a 1:1 ratio and stir to allow the polysaccharides to fully precipitate. Pass the precipitated *Schizophyllum commune* polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol. Add 60% ethanol to the *Schizophyllum commune* polysaccharide precipitate at a 1:1 ratio to wash the polysaccharide precipitate, repeating the washing twice. Centrifuge at 13000 rpm for 15 min and collect the precipitate. Redissolve the precipitate in pure water to obtain an aqueous solution of *Schizophyllum commune* polysaccharides. This is the high-transparency *Schizophyllum commune* polysaccharide.

[0076] Comparative Example 1 A method for preparing polysaccharide from Schizophyllum commune is provided, which differs from Example 1 in that step (2) is different; step (2) of Comparative Example 1 is as follows: Take 2 kg of the *Schizophyllum commune* fermentation broth containing mycelia obtained in step (1), freeze it at -20℃ for 3 days, then thaw it naturally at room temperature for 1 day; homogenize it at 10,000 rpm for 30 s using a T18 digital ULTRA TURRAX® (IKA) disperser, then centrifuge it at 13,000 rpm for 15 min to obtain a fermentation filtrate containing extracellular secreted *Schizophyllum commune* polysaccharides; add 95% ethanol to the fermentation filtrate at a 1:1 ratio and stir to allow the polysaccharides to fully precipitate. Pass the precipitated *Schizophyllum commune* polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol, add 60% ethanol to the *Schizophyllum commune* polysaccharide precipitate at a 1:1 ratio, wash the polysaccharide precipitate, and repeat the washing 4 times; centrifuge at 13,000 rpm for 15 min and collect the precipitate; redissolve the precipitate in pure water to obtain an aqueous solution of *Schizophyllum commune* polysaccharides.

[0077] Comparative Example 2 A method for preparing polysaccharide from Schizophyllum commune is provided, which differs from Example 1 in that step (2) is different; step (2) of Comparative Example 2 is as follows: Take 2 kg of the *Schizophyllum commune* fermentation broth containing mycelia obtained in step (1), freeze it at -20 ℃ for 3 days, and then thaw it naturally at room temperature for 1 day. Homogenize it at 13000 rpm for 60 s using a T18 digital ULTRA TURRAX® (IKA) disperser, and then centrifuge it at 13000 rpm for 15 min to obtain a fermentation filtrate containing extracellular secreted *Schizophyllum commune* polysaccharides. Add 95% ethanol to the fermentation filtrate at a 1:1 ratio and stir to allow the polysaccharides to fully precipitate. Pass the precipitated *Schizophyllum commune* polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol. Add 60% ethanol to the *Schizophyllum commune* polysaccharide precipitate at a 1:1 ratio to wash the polysaccharide precipitate, and repeat the washing 4 times. Centrifuge at 13000 rpm for 15 min and collect the precipitate. Redissolve the precipitate in pure water to obtain an aqueous solution of *Schizophyllum commune* polysaccharides.

[0078] Comparative Example 3 A method for preparing polysaccharide from Schizophyllum commune is provided, which differs from Example 1 in that step (2) is different; step (2) of Comparative Example 2 is as follows: Add pure water to the fermentation broth obtained in step (1) at a ratio of 1:1 to dilute the fermentation broth, centrifuge at 13000 rpm for 15 min, and obtain a fermentation filtrate containing extracellular secretory Schizophyllum commune polysaccharide. Take 2 kg of Schizophyllum commune fermentation broth containing mycelia. Add 95% ethanol to the fermentation filtrate at a ratio of 1:1 and stir to allow the polysaccharide to fully precipitate. Pass the precipitated Schizophyllum commune polysaccharide precipitate through a 100-mesh filter cloth to remove excess ethanol, add 60% ethanol to the Schizophyllum commune polysaccharide precipitate at a ratio of 1:1, wash the polysaccharide precipitate, and repeat the washing 5 times; centrifuge at 13000 rpm for 15 min, collect the polysaccharide precipitate; redissolve the polysaccharide precipitate in pure water to obtain an aqueous solution of Schizophyllum commune polysaccharide.

[0079] The process parameters for each embodiment and comparative example are detailed in Table 1.

[0080] Table 1

[0081] Experimental Example 1 The physicochemical characteristics of the Schizophyllum commune polysaccharide aqueous solutions prepared in the above embodiments and comparative examples were characterized.

[0082] (1) The polysaccharide content of Schizophyllum commune was determined by the sulfuric acid-phenol method; the polysaccharide content is detailed in Table 2. (2) The viscosity of the polysaccharide sample of Schizophyllum commune was determined using a BROOKFIELD DVS+ viscometer. The measurement conditions were 12 rpm, SO3 rotor, and 25℃. The viscosity details are shown in Table 2. (3) The polysaccharide samples were scanned in the wavelength range of 200~1000nm using a Spark 10M multi-functional microplate reader (TECAN Group, Switzerland); the absorbance was obtained, as detailed in Table 2. (4) The protein content of the polysaccharide samples of Schizophyllum commune was determined using the Micro BCA™ Protein Detection Kit (Thermo Scientific™); the protein content is detailed in Table 2.

[0083] (5) Calculation of relative polysaccharide yield: The polysaccharide yield of Comparative Example 3 was used as a control. The polysaccharide content of the sample prepared by the traditional alcohol precipitation process was recorded as 1. The relative polysaccharide yield of other comparative examples and embodiments relative to Comparative Example 3 (+ indicates increase) was calculated. See Table 2 for details.

[0084] The physicochemical characteristics of the *Schizophyllum commune* polysaccharides prepared in each embodiment and comparative example are shown in Table 2. Figure 2 , Figure 3 and Figure 4 As shown.

[0085] Table 2

[0086] The results in the table above show that: In terms of apparent transparency and brightness, the absorbance OD600 of Examples 1 and 2 is significantly better than that of the comparative examples, which shows that the solution of this application can effectively improve the transparency of Schizophyllum commune polysaccharide.

[0087] Regarding protein residue, Examples 1 and 2 had the least amount of residue, which was significantly better than the comparative examples, indicating that the amount of protein residue was negatively related to the transparency of polysaccharides, thus proving the hypothesis put forward in the early stage of this invention.

[0088] Furthermore, the comparison results between Comparative Examples 1 and 2 and the various embodiments show that homogenized and broken mycelia lead to the release of more intracellular proteins, and the homogenization intensity is negatively correlated with the polysaccharide transparency.

[0089] Experimental Example 2 The polysaccharide precipitates prepared in each of the above Examples 1 were redissolved in pure water at multiple redissolution ratios to obtain polysaccharide aqueous solution samples of different concentrations. The physicochemical characteristics of the polysaccharide aqueous solutions of different concentrations were compared at room temperature.

[0090] The results are shown in Table 3. Figure 5 , Figure 6 and Figure 7 As shown.

[0091] Table 3

[0092] The test results in Table 3 above show that: The polysaccharide prepared in this application still has high transparency under high concentration and high viscosity conditions.

[0093] When the polysaccharide content is 1.14 ± 0.04% and the viscosity is >8333 cP, the absorbance OD600 is below 0.11 ± 0.002.

[0094] When the polysaccharide content is between 0.81% and 1.18% and the viscosity is greater than 5000 cP, the absorbance OD600 is between 0.08 and 0.11.

[0095] In summary, this application specifically applies the freeze-thaw step to a "Schizophyllum commune fermentation mixture containing mycelium," targeting not just the mycelium itself, but primarily the secreted extracellular polysaccharides, resulting in colorless and highly transparent Schizophyllum commune polysaccharides. Furthermore, the above technical solution employs a "freeze-thaw-self-digestion" process to rupture the mycelium, releasing proteases that decompose soluble proteins in the fermentation broth. This cleverly utilizes endogenous enzymes within the mycelium for "self-digestion," yielding highly transparent and pure Schizophyllum commune polysaccharides. Furthermore, this technical solution utilizes Schizophyllum commune's own enzyme system to degrade impurities, avoiding the use of exogenous chemical reagents or enzyme preparations, making it more environmentally friendly and safer. This expands its application in high-value fields such as high-end cosmetics and injectable pharmaceutical preparations. Furthermore, the above technical solution, employing a "freeze-thaw-self-digestion" concept, requires simple equipment and simplifies the repeated washing process after alcohol precipitation, avoiding the complex processes associated with traditional exogenous reagent additions, making it easy to industrialize.

[0096] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing high clarity schizophyllan polysaccharide, characterized by, The method comprises the following steps: frozen mycelium containing Schizophyllum fermentation mixture under negative temperature conditions to obtain a frozen product; thawing the frozen product to obtain a thawed mixture; centrifuging and filtering the thawed mixture to remove mycelium and collect fermentation filtrate; adding ethanol to the fermentation filtrate to precipitate polysaccharides by alcohol to obtain a polysaccharide precipitate; collecting the polysaccharide precipitate and repeatedly washing the polysaccharide precipitate with an ethanol solution to obtain a washed polysaccharide precipitate; centrifuging the washed polysaccharide precipitate, collecting the centrifuged polysaccharide precipitate, and resuspending the polysaccharide precipitate in a solvent.

2. The method for preparing high-transparency Schizophyllum polysaccharide according to claim 1, wherein the step of freezing the Schizophyllum fermentation mixture containing mycelium under negative temperature conditions comprises: freezing at a negative temperature of -20°C to -80°C; optionally, the freezing time is 1-3 days.

3. The method for preparing high-transparency Schizophyllum polysaccharide according to claim 1, wherein the step of thawing the frozen product comprises: thawing at a temperature of 10-25°C for more than 1-3 days.

4. The method for preparing high-transparency Schizophyllum polysaccharide according to claim 1, wherein the step of repeatedly washing the polysaccharide precipitate with ethanol comprises: washing the polysaccharide precipitate with an ethanol solution with a mass fraction of 75%-100%; optionally, the washing is repeated for 1-3 times; optionally, the mass fraction of the ethanol solution gradually decreases during the repeated washing; optionally, the mass ratio of the polysaccharide precipitate to the ethanol solution is (0.5-2):(0.5-2).

5. The method for preparing high-transparency Schizophyllum polysaccharide according to claim 1, wherein the step of resuspending the polysaccharide precipitate in a solvent comprises: resuspending the polysaccharide precipitate in water. The high-transparency Schizophyllum polysaccharide is prepared by the method according to any one of claims 1-5. The high-transparency Schizophyllum polysaccharide is mainly derived from the extracellular secreted polysaccharide of Schizophyllum fermentation.

7. The high-transparency Schizophyllum polysaccharide according to claim 6, wherein when the viscosity of the high-transparency Schizophyllum polysaccharide aqueous solution is greater than 8333 cP, the absorbance OD600 is less than 0.11; optionally, when the viscosity of the high-transparency Schizophyllum polysaccharide aqueous solution is greater than or equal to 5000 cP, the absorbance OD600 is 0.08-0.

11.

6. A high clarity schizophyllan polysaccharide, characterized in that, 8. The high-transparency Schizophyllum polysaccharide according to claim 6, wherein when the polysaccharide content in the high-transparency Schizophyllum polysaccharide aqueous solution is greater than or equal to 0.81%, the viscosity of the Schizophyllum polysaccharide aqueous solution is greater than or equal to 5000 cP; optionally, when the polysaccharide content in the high-transparency Schizophyllum polysaccharide aqueous solution is greater than or equal to 1.10%, the viscosity of the Schizophyllum polysaccharide aqueous solution is greater than or equal to 8333 cP.

9. The high-transparency Schizophyllum polysaccharide according to claim 6, wherein ​ ​ ​ ​ ​ ​ When the viscosity of the high-transparency schizophyllan polysaccharide aqueous solution is greater than or equal to 5000 cP, the polysaccharide content in the high-transparency schizophyllan polysaccharide aqueous solution is greater than or equal to 0.81%; optionally, when the viscosity of the high-transparency schizophyllan polysaccharide aqueous solution is greater than 8333 cP, the polysaccharide content in the high-transparency schizophyllan polysaccharide aqueous solution is greater than or equal to 1.10%.

10. A cosmetic product, characterized by, The cosmetic comprises the high-transparency schizophyllan polysaccharide according to any one of claims 1-4; or the cosmetic comprises the high-transparency schizophyllan polysaccharide prepared by the preparation method according to any one of claims 6-9.

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