Preparation method of tremella polysaccharide and application thereof in cosmetics

Abstract

The application belongs to the technical field of polysaccharide preparation, and particularly relates to a preparation method of tremella polysaccharide and application of the tremella polysaccharide in cosmetics. The preparation method comprises the steps of hot water shearing and crushing, synergistic enzymolysis of propylene carbonate and potassium polyclar, isoelectric point deproteinization, alcohol precipitation, ion exchange chromatography purification, controllable degradation and freeze-drying. The preparation method has high extraction efficiency, completely removes proteins, has low polysaccharide loss rate, and the obtained tremella polysaccharide has the effects of moisturizing, skin barrier repair, anti-inflammatory and antioxidant, and can be used for preparing cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide preparation technology, specifically relating to a method for preparing Tremella fuciformis polysaccharide and its application in cosmetics. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Tremella fuciformis, a fungus used both as food and medicine, is rich in polysaccharides, which are its main active ingredients. Tremella fuciformis polysaccharides possess excellent moisturizing, antioxidant, anti-inflammatory, and immunomodulatory biological activities, and have received widespread attention in the functional food and cosmetic fields in recent years. In cosmetic applications, tremella fuciformis polysaccharides are often used as natural moisturizers, skin barrier repair agents, and anti-aging active ingredients.

[0004] Currently, extraction methods for Tremella fuciformis polysaccharides mainly include hot water extraction, acid / alkali extraction, and enzyme-assisted extraction. Hot water extraction is simple to operate, but suffers from high extraction temperatures, long extraction times, and low efficiency. While acid / alkali extraction can improve yield, it easily leads to polysaccharide degradation and causes significant environmental pollution. Enzyme-assisted extraction has been widely studied due to its mild conditions and high specificity; however, conventional enzymatic hydrolysis is insufficient to fully disrupt the dense structure of Tremella fuciformis cell walls, especially since the fruiting body is rich in glycoprotein complexes tightly bound to polysaccharides, resulting in insufficient release of the target polysaccharides and requiring further improvement in extraction efficiency.

[0005] To improve the extraction efficiency and purity of Tremella fuciformis polysaccharides, researchers have made various attempts. For example, Chinese patent CN115594775A discloses a method for preparing Tremella fuciformis polysaccharides, the steps of which include: first, soaking Tremella fuciformis in an ethanol-acetic acid aqueous solution and filtering; then adding an aqueous solution containing additives (including sodium ascorbate, ascorbic acid, sodium tartrate, and sorbitol) to the filter residue, mixing, slurrying, centrifuging, and collecting the supernatant; then enzymatically hydrolyzing the supernatant with a compound enzyme (a mixture of papain and neutral protease), precipitating with alcohol to obtain a polysaccharide precipitate; finally, redissolving the polysaccharide precipitate, heating to remove the organic solvent, and drying to obtain the final product. Chinese patent CN102603913A discloses a method for extracting polysaccharides from wheat by-products, the method including pretreatment, defatting treatment, microwave extraction, enzymatic destarch removal, alcohol precipitation, isoelectric point method or enzymatic hydrolysis to remove protein, and using hydrogen peroxide to decolorize the polysaccharide to improve sensory properties. This patent involves common deproteinization and hydrogen peroxide treatment steps in polysaccharide extraction. However, it targets polysaccharides from wheat by-products, whose raw material characteristics differ significantly from those of Tremella fuciformis fruiting bodies. Furthermore, its hydrogen peroxide treatment is primarily for decolorization purposes, without precise control over degradation conditions, making it difficult to achieve targeted regulation of polysaccharide molecular weight. The molecular weight of Tremella fuciformis polysaccharides has a significant impact on their skin feel and absorption performance in cosmetic formulations.

[0006] Current technologies still have shortcomings in terms of extraction efficiency and deep removal of protein impurities from Tremella fuciformis polysaccharides. Therefore, developing a method for preparing Tremella fuciformis polysaccharides that can achieve high yield, low loss rate, and produce products with excellent moisturizing, barrier repair, anti-inflammatory, and antioxidant effects, and effectively applying it to cosmetics, has significant market value and potential for technological innovation. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of the prior art and provide a method for preparing Tremella polysaccharide with high yield, low loss and significant efficacy, as well as the Tremella polysaccharide obtained by the method and its application in cosmetics.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing Tremella polysaccharide, characterized by comprising the following steps: (1) Crude extraction: Add water to the fruiting body of Tremella fuciformis at a material-to-liquid ratio of 1:25-1:35 g:mL, steam and soften at 70-90℃, and then perform high-speed shearing and crushing for 10-25 minutes to obtain crude extract; (2) Enzymatic hydrolysis and deproteinization: Add 0.5%-2.0% propylene carbonate and 0.1%-0.5% potassium polycrylene to the crude extract obtained in step (1) by volume, then add papain to a final concentration of 0.1%-0.5%, adjust the pH to 6.0-7.0, and hydrolyze at 40-60℃ for 1-4 hours; after enzymatic inactivation, adjust the pH to 2.5-3.0, let stand at 4℃ to precipitate, centrifuge to remove protein precipitate, and obtain purified solution; (3) Alcohol precipitation and chromatography purification: Add 3-5 times the volume of anhydrous ethanol to the purified solution obtained in step (2), precipitate at 4℃ for 10-15 hours, and collect the crude polysaccharide by centrifugation; after redissolving the crude polysaccharide, load it onto a DEAE-52 ion exchange chromatography column, and perform gradient elution with 0.2-0.5 mol / L NaCl solution to collect the target eluted fraction and obtain purified Tremella polysaccharide; (4) Controllable degradation: Prepare a 2% aqueous solution of the refined tremella polysaccharide obtained in step (3), add 30% hydrogen peroxide to a final concentration of 1.5%-2.0%, and degrade at 50-55℃ for 2.5-3.0 hours to obtain the degradation solution; (5) Post-processing: After dialysis of the degradation solution obtained in step (4) through a dialysis bag, the solution is freeze-dried under vacuum to obtain Tremella polysaccharide.

[0009] As a preferred technical solution: In step (2), the amount of propylene carbonate added is 1.5% of its volume ratio in the crude extract.

[0010] In step (2), the amount of polycrylene potassium added is 0.3% of its mass-volume ratio in the crude extract.

[0011] In step (2), the optimal conditions for the enzymatic hydrolysis treatment are: papain concentration of 0.3% (w / v), hydrolysis pH of 6.5, hydrolysis temperature of 50℃, and hydrolysis time of 2 hours.

[0012] In step (3), the alcohol precipitation conditions are: add 4 times the volume of anhydrous ethanol and precipitate at 4°C for 12 hours.

[0013] In step (3), the elution method of the ion exchange chromatography is as follows: gradient elution is performed sequentially with distilled water, 0.2 mol / L NaCl, and 0.5 mol / L NaCl, and the elution fraction of 0.5 mol / L NaCl is collected.

[0014] In step (4), the optimal conditions for the controlled degradation of hydrogen peroxide are: 30% H2O2 final concentration 1.8%, degradation at 53°C for 2.5 hours.

[0015] In step (5), the molecular weight cutoff of the dialysis bag is 100-500 Da.

[0016] On the other hand, the present invention also provides the application of Tremella fuciformis polysaccharide obtained by the aforementioned preparation method in the preparation of cosmetics. Furthermore, it provides the application of Tremella fuciformis polysaccharide obtained by the aforementioned preparation method in the preparation of cosmetics with moisturizing, skin barrier repair, anti-inflammatory, and / or antioxidant effects.

[0017] Compared with the prior art, the technical advantages of the present invention are as follows: 1. High extraction efficiency and high polysaccharide yield: This invention optimizes key process parameters in the hot water cooking and shearing process, and innovatively uses propylene carbonate and potassium polycrylene as enzymatic hydrolysis promoters in combination in the enzymatic hydrolysis step. This effectively destroys the cell wall structure of Tremella fuciformis, reduces the steric hindrance of glycoproteins, and significantly improves the extraction rate and yield of Tremella fuciformis polysaccharides.

[0018] 2. Thorough protein removal and low polysaccharide loss: This invention adopts a protein removal strategy of "enzymatic hydrolysis followed by isoelectric point method", combined with the promoting effect of propylene carbonate and potassium polycrylene, so that the protein removal rate is at a high level, while the polysaccharide loss rate is controlled within a low range. The overall performance is significantly better than that of the single isoelectric point method or enzymatic hydrolysis method.

[0019] 3. Good bioactivity and clear skin care effects: Through in vitro cell experiments and in vivo animal models, the Tremella fuciformis polysaccharide prepared in this invention can promote the migration and proliferation of keratinocytes and accelerate the healing of skin wounds; significantly inhibit the secretion of inflammatory factors and exert anti-inflammatory effects; in animal photodamage models, it can reduce transepidermal water loss, increase the water content of the stratum corneum, repair the skin barrier function, and improve the pathological damage of skin photoaging induced by ultraviolet rays. Attached Figure Description

[0020] Figure 1 Curve showing the change in yield of tremella polysaccharide with the ratio of solid to liquid.

[0021] Figure 2 Curve showing the change in the yield of tremella polysaccharides with the time of crushing.

[0022] Figure 3 Curve showing the change in yield of Tremella polysaccharide with extraction temperature.

[0023] Figure 4 : Surface plot of the effect of material-liquid ratio on the yield of Tremella polysaccharide.

[0024] Figure 5 : Surface plot of the effect of crushing time on the yield of Tremella polysaccharides.

[0025] Figure 6 : Surface plot of the effect of extraction temperature on the yield of Tremella polysaccharides.

[0026] Figure 7Contour plot of the yield of Tremella polysaccharide based on the ratio of solid to liquid.

[0027] Figure 8 Contour plot of the effect of crushing time on the yield of Tremella polysaccharides.

[0028] Figure 9 Contour plot of the effect of extraction temperature on the yield of Tremella polysaccharides.

[0029] Figure 10 Effect of different particle size on the yield of Tremella polysaccharide.

[0030] Figure 11 The effect of different pH values ​​on protein removal rate and polysaccharide loss rate.

[0031] Figure 12 The effect of enzyme concentration on protein removal rate.

[0032] Figure 13 The effect of temperature on protein removal rate.

[0033] Figure 14 The effect of pH on protein removal rate.

[0034] Figure 15 The effect of time on protein removal rate.

[0035] Figure 16 : DEAE-52 ion exchange chromatography elution curve.

[0036] Figure 17 The effect of Tremella polysaccharide on the secretion of multicellular inflammatory factors.

[0037] Figure 18 H&E staining results of skin tissue pathology in the skin photodamage model group (A) and the tremella polysaccharide intervention group (B). Detailed Implementation

[0038] To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0039] 1. Research on Tremella fruiting body cell disruption technology 1.2 Optimization of parameters such as material-liquid ratio, crushing time, and extraction temperature. 1.2.1 Single-factor investigation (1) Single-factor investigation of liquid-to-material ratio Under fixed conditions: crushing time 17.5 min (intermediate level), extraction temperature 80℃ (intermediate level), the yield of Tremella polysaccharide was investigated at material-to-liquid ratios of 10, 15, 20, 25, 30, 35, and 40 g:mL. The results are shown in Table 1. Table 1 Effect of material-to-liquid ratio on the yield of Tremella polysaccharide Table 1 and Figure 1 It is evident that the yield is higher when the material-to-liquid ratio is in the range of 20-35 g:mL. When the ratio is below 20 g:mL, the raw material extraction is insufficient, and when it is above 35 g:mL, the excessive solvent leads to increased costs for subsequent concentration. Therefore, the response surface optimization range is determined to be 20-35 g:mL.

[0040] (2) Single factor analysis of crushing time With a fixed liquid-to-solid ratio of 27.5 g:mL (intermediate level) and extraction temperature of 80℃ (intermediate level), the yield of Tremella fuciformis polysaccharide was investigated at crushing times of 5, 10, 15, 20, 25, 30, and 35 min. The results are shown in Table 2. Table 2 Effect of crushing time on the yield of Tremella polysaccharides Table 2 and Figure 2 The results show that the yield is relatively high when the crushing time is 10-25 min. When the time is less than 10 min, the cell wall of Tremella fuciformis is not completely crushed, and when the time is more than 25 min, excessive crushing leads to increased dissolution of impurities. Therefore, the optimal range for response surface methodology is determined to be 10-25 min.

[0041] (3) Single-factor investigation of temperature The liquid-to-solid ratio was fixed at 27.5 g:mL (intermediate level), and the crushing time was 17.5 min (intermediate level). The yield of Tremella polysaccharide was investigated at extraction temperatures of 50, 60, 70, 80, 90, 100, and 110 ℃. The results are shown in Table 3.

[0042] Table 3 Effect of different extraction temperatures on the yield of Tremella polysaccharides Table 3 and Figure 3 The results show that the yield is higher when the extraction temperature is 70-90 ℃, the polysaccharide dissolution rate is slow when the temperature is below 70 ℃, and the polysaccharide may be degraded when the temperature is above 90 ℃. Therefore, the response surface optimization range is determined to be 70-90 ℃.

[0043] 1.2.2 Optimization of Box-Behnken Experimental Design Response surface methodology was used to systematically optimize the three core parameters. Using the yield of Tremella fuciformis polysaccharides as the response value, 17 experimental groups were designed, and the factor levels were determined as follows: liquid-to-solid ratio 20-35 (g:mL), crushing time 10-25 minutes, and extraction temperature 70-90℃. The formulation was optimized using a 3-factor, 3-level Box-Behnken experimental design; the factors and levels are shown in Table 4.

[0044] Table 4. Factor Levels in Box-Behnken Experimental Design Table 5 Optimization results of the Box-Behnken experiment Table 6. Results of the analysis of variance for the Box-Behnken experimental model. The effect surface plot of the material-to-liquid ratio (A), crushing time (B), and extraction temperature (C) on the yield of Tremella polysaccharides is shown below. Figures 4-6 Contour plots showing the effects of material-to-liquid ratio (A), crushing time (B), and extraction temperature (C) on the yield of Tremella fuciformis polysaccharides. The effect surface plot and contour plots clearly demonstrate the influence of these three factors on the yield of Tremella fuciformis polysaccharides.

[0045] To further enhance its adaptability to industrialization, the project also studied the impact of the particle size of the Tremella fuciformis raw material (passing through 20-mesh, 40-mesh, 60-mesh, and 80-mesh sieves) on the crushing efficiency. Figure 10 The method determined that grinding the raw materials to 20-40 mesh would meet the requirements, avoiding excessive grinding that would increase energy consumption. The precise control of this parameter provides an important basis for industrialization.

[0046] 2. Research on enzymatic and isoelectric point-based purification techniques 2.1 Comparative Study of Isoelectric Point Method, Papain Method, and Enzyme-Isoelectric Point Binding Method To address the technical challenge of high protein content (15%-20%, mainly glycoproteins and free proteins) in crude extracts of Tremella polysaccharides, the project team systematically compared the efficiency of three protein removal processes.

[0047] 2.1.1 Isoelectric point precipitation method The pH of the crude extract of Tremella fuciformis polysaccharides (concentration 2.0 mg / mL) was adjusted to 2.1-2.9, and the solutions were allowed to stand at 4℃ for 12 hours before centrifugation. The protein and polysaccharide contents were measured before and after pH adjustment, and the protein removal rate and polysaccharide loss rate were calculated. Protein content was determined using the Coomassie Brilliant Blue method, and the protein removal rate was calculated using Formula 1. Polysaccharide content was determined using the phenol-sulfuric acid method, and the polysaccharide loss rate was calculated using Formula 2.

[0048] Protein removal rate (%) = Formula 1 Polysaccharide loss rate (%) = Formula 2 Where w1 represents the protein content after pH adjustment; w0 represents the protein content before pH adjustment; m1 represents the polysaccharide content after pH adjustment; and m0 represents the polysaccharide content before pH adjustment. The effects of different pH values ​​on protein removal rate and polysaccharide loss rate are shown in [reference needed]. Figure 11 .

[0049] The results showed that the protein removal rate reached a peak of 78.3% when pH=2.5, but the polysaccharide loss rate was as high as 18.7%. The main reason was that the strong acid environment caused partial hydrolysis of acidic polysaccharides, and the precipitate was gel-like, making centrifugal separation difficult and industrialization feasible.

[0050] 2.1.2 Papain hydrolysis method Papain (800 U / mg) was added to the crude extract, and the parameter space was investigated for enzyme concentrations of 0.1%-0.5%, temperatures of 40-60℃, pH of 6.0-7.0, and time of 1-4 hours. Single-factor experiments were conducted to examine the effects of different treatments on protein removal rate, and the results are as follows: Figures 12-15 As shown, the optimal combination of individual factors was enzyme concentration 0.3%, temperature 50℃, pH 6.5, and time 2 hours, at which the protein removal rate was 72.1% and the polysaccharide loss rate was 15.4%. This method is mild, but it is difficult to completely remove glycoproteins tightly bound to polysaccharides when used alone, and high-temperature inactivation is required after enzymatic hydrolysis, resulting in high energy consumption.

[0051] 2.1.3 Enzyme-Isoelectric Point Binding Method Combining Propylene Carbonate and Polykrylene Potassium This invention proposes a combined strategy of "enzymatic hydrolysis with propylene carbonate and potassium polycrylene followed by isoelectric point precipitation." The crude extract is first hydrolyzed with papain under optimal conditions to break the glycoprotein covalent bonds and release the encapsulated polysaccharides. After cooling to room temperature, the hydrolysate is immediately adjusted to pH 2.5 with 10% hydrochloric acid and allowed to stand overnight at 4°C, allowing the enzymatically cleaved protein fragments to precipitate efficiently at the isoelectric point. This combined process achieves synergistic effects: protein removal rate is increased to 92.3%, and polysaccharide loss rate is reduced to 8.7%, with overall performance significantly better than single methods. This is because enzymatic hydrolysis cleaves large glycoprotein molecules into smaller peptides, reducing molecular resistance to isoelectric point precipitation. Simultaneously, rapid acidification after enzymatic hydrolysis (pH adjustment completed within <30 minutes) minimizes the degradation of the polysaccharide backbone under acidic conditions.

[0052] The comparative study concluded that the combination of propylene carbonate and polycrylene potassium with enzyme-isoelectric point binding method showed the best performance in terms of protein removal rate, polysaccharide loss rate and process cost, and was identified as the core process for industrialization.

[0053] 3. Study on the combined enzyme-isoelectric point binding process of propylene carbonate and polycrylene potassium. During the extraction of polysaccharides from Tremella fuciformis, the crude extract contains a large number of glycoproteins bound to the polysaccharides and cell wall fragments. Conventional enzymatic hydrolysis is insufficient to fully expose the polysaccharide molecules, resulting in low hydrolysis efficiency. To address this issue, this invention innovatively introduces propylene carbonate and potassium polycrylene as enzymatic hydrolysis promoters in the enzymatic hydrolysis step. Propylene carbonate can disrupt the lipid bilayer structure of the cell membrane, increasing cell wall permeability; potassium polycrylene neutralizes the negative charge on the surface of glycoproteins through electrostatic interactions, reducing steric hindrance. The combined effect of these two agents significantly improves the contact efficiency between the protease and the substrate.

[0054] 3.1 Effect of propylene carbonate addition on enzymatic hydrolysis efficiency The enzymatic hydrolysis conditions were fixed as follows: papain concentration 0.3%, polykrylamide potassium addition 0.3 w / v%, pH 6.5, temperature 50℃, and time 2 h. Propylene carbonate was added to the crude extract at volume ratios of 0, 0.5%, 1.0%, 1.5%, 2.0%, and 2.5%, respectively, to investigate its effect on protein removal rate and polysaccharide loss rate. The results are shown in Table 7.

[0055] Table 7. Effect of propylene carbonate addition on enzymatic hydrolysis efficiency As shown in Table 7, the protein removal rate gradually increased with the increase of propylene carbonate addition. When the addition reached 1.5%, the protein removal rate was significantly higher than that of the group without addition. Further increases above 2.0% resulted in a slower increase in the rate of increase, while the polysaccharide loss rate began to increase significantly. Considering all factors, an addition of 1.5% is the preferred amount.

[0056] 3.2 Effect of Porcolin Potassium Addition on Enzymatic Hydrolysis Efficiency The enzymatic hydrolysis conditions were fixed as follows: 1.5% propylene carbonate, 0.3% papain, pH 6.5, temperature 50℃, and time 2 h. Polykrylamide potassium was added to the crude extract at mass-volume ratios of 0, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, respectively, and its effect on the enzymatic hydrolysis effect was investigated. The results are shown in Table 8.

[0057] Table 8 Effect of polykrylamide potassium addition on enzymatic hydrolysis efficiency The results showed that when the amount of potassium polykrylamide added was in the range of 0.1% to 0.5%, the protein removal rate increased with the increase of the amount added, while the polysaccharide loss rate showed a decreasing trend.

[0058] 3.3 Verification of the combined effect of propylene carbonate and polycrylene potassium To clarify the combined effect of propylene carbonate and potassium polycrylene in the enzymatic hydrolysis of Tremella fuciformis polysaccharides, a comparative experiment was designed in this invention. Under fixed enzymatic hydrolysis conditions (papain concentration 0.3%, pH 6.5, temperature 50℃, time 2 h), the following four groups were set up for comparison: Control group: No accelerators added; Propylene carbonate group: 1.5% propylene carbonate by volume is added alone, without adding polycrylene potassium; Polycrylene potassium group: Polycrylene potassium is added alone at a mass-volume ratio of 0.3%, without propylene carbonate; Combined group: with the addition of 1.5% propylene carbonate and 0.3% polycrylene potassium.

[0059] The experimental results are shown in Table 9.

[0060] Table 9 Comparison of the combined effects of propylene carbonate and polycrylene potassium carbonate Table 9 shows that adding propylene carbonate or potassium polycrylene alone can improve the protein removal rate to some extent, but the polysaccharide loss rate is still relatively high. The combined group, however, significantly improved the protein removal rate to 92.3%, while reducing the polysaccharide loss rate to 8.7%, demonstrating a clear synergistic effect. Propylene carbonate effectively disrupts the lipid bilayer structure of the *Tremella fuciformis* cell membrane, enhancing cell wall permeability and promoting the contact between protease and substrate; potassium polycrylene, by neutralizing the negative charge on the glycoprotein surface and reducing steric hindrance, further enhances the hydrolysis efficiency of the protease. The combined use of the two achieves a complementary effect of physical and chemical promotion, significantly improving the enzymatic hydrolysis effect.

[0061] 4. DEAE-52 ion exchange chromatography purification process After removing over 90% of the protein using the enzyme-isoelectric point method, a small amount of free protein, pigments, and small molecule salts still remain in the extract. The project employs DEAE-52 cellulose anion exchange chromatography for further purification; this resin has selective adsorption capacity for acidic polysaccharides.

[0062] 4.1 Pretreatment of chromatography column After the resin swells by soaking in distilled water for 1 hour, it is treated sequentially with 0.5 mol / L HCl for 2 hours, washed with deionized water until neutral, and then treated with 0.5 mol / L NaOH for 2 hours and washed with water until neutral to remove production residues and activate ion exchange groups. After column packing, the column is equilibrated with distilled water for 6 hours to ensure column bed stability.

[0063] 4.2 Sample loading and elution The enzyme-isoelectric point treated polysaccharide solution (concentration 10 mg / mL) was filtered through a 0.45 μm filter membrane and loaded at a flow rate of 10 mL / min, with the loading volume controlled at 5% of the column volume. Stepwise elution was performed: ① Elute with distilled water for 2 column volumes to remove neutral sugars and oligosaccharides; ② Elute with 0.2 mol / L NaCl for 3 column volumes to remove pigments and some impurities; ③ Elute with 0.5 mol / L NaCl for 4 column volumes to collect the main component, Tremella fuciformis polysaccharide; ④ Elute with 1.0 mol / L NaCl for 2 column volumes to regenerate the column. The eluent was collected fractionally, and the polysaccharide content was monitored using the phenol-sulfuric acid method. Elution curves were plotted. Figure 16 The main peak is concentrated in the 0.5 mol / L NaCl component.

[0064] 5. Protein removal rate and polysaccharide loss rate 5.1 Protein removal rate Statistical analysis of 10 batches of data showed that the average protein content of the extract after pretreatment was 18.7 mg / mL, which was reduced to 1.45 mg / mL after enzyme-isoelectric point binding method treatment, with an average removal rate of 92.3%, ranging from 91.8% to 93.1%, and RSD=0.48%.

[0065] Table 10 Statistical Table of Protein Removal Rate 5.2 Polysaccharide loss rate The average polysaccharide content of the extract was 156.2 mg / mL, which decreased to 142.5 mg / mL after purification. The average polysaccharide loss rate was 8.7%, ranging from 7.9% to 9.4%, with an RSD of 5.2%. The loss mainly occurred during the isoelectric point precipitation stage, where a small amount of polysaccharide encapsulated in the protein precipitate was lost with the centrifuged liquid. By optimizing the centrifugation speed (increasing from 5000 rpm to 8000 rpm) and centrifugation time (extending from 15 minutes to 20 minutes), the polysaccharide loss rate was reduced from the initial 10.2% to 8.7% while maintaining the protein removal rate.

[0066] Table 11 Process Data Table of Polysaccharide Loss Rate This invention employs a three-step method: "enzymatic pretreatment with propylene carbonate and polycrylene potassium, isoelectric point precipitation, and ion exchange purification." This method ensures a protein removal rate of >92% while controlling the polysaccharide loss rate to <9%, achieving the optimized goal of "high-efficiency impurity removal and low-loss preservation."

[0067] 6. Skin Repair Evaluation: Establishment of In Vivo and In Vitro Models and Validation of Efficacy 6.1 In vitro evaluation Cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin and cultured in an incubator at 37°C and 5% CO2. When the cells reached the logarithmic growth phase and the confluence reached 70%-80%, an ultraviolet damage model was established: HaCaT cells were irradiated with UVB at a dose of 10 J / cm² to construct a cell damage model. After irradiation, the cells were randomly divided into a damage control group and treatment groups with different concentrations of Tremella fuciformis polysaccharide. The treatment groups were given Tremella fuciformis polysaccharide solutions with final concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. A hyaluronic acid treatment group was set up as a positive control. After each group was cultured in an incubator at 37°C and 5% CO2 for a period of time, the cell viability was detected by the CCK-8 assay to evaluate the damage repair effect of Tremella fuciformis polysaccharide.

[0068] Table 12 Effects of Tremella polysaccharide on cell viability The results showed that the cell survival rate in the damaged control group was only 58.3%, while the cell survival rate in the 100 μg / mL Tremella polysaccharide treatment group was significantly restored to 89.7%. Statistical analysis showed that the survival rate was significantly different from that in the damaged control group (p<0.001), and its repair effect was significantly better than that in the hyaluronic acid treatment group (p<0.05). Other concentrations of Tremella polysaccharide treatment groups also showed varying degrees of survival rate improvement.

[0069] The culture system used was a high-glucose DMEM medium (4.5 g / L glucose, containing L-glutamine) containing 10% fetal bovine serum (FBS, which required inactivation at 56℃ for 30 min to remove complement components) and 1% penicillin-streptomycin mixture (final penicillin concentration 100 U / mL, final streptomycin concentration 100 μg / mL). The culture environment was set as a constant temperature incubator at 37℃ with 5% CO2 volume fraction, ensuring that the pH of the medium was kept stable at 7.2-7.4. 2 × 10⁶ cells / well were inoculated per well of a 6-well plate. 5Cells were seeded and allowed to adhere to the plate for 24 hours. The culture medium was changed every 2-3 days during subsequent culture. After the cells reached 100% confluence in the 6-well plate, a sterile 200 μL pipette tip was used to make a straight line with uniform width in the center of the cell monolayer in each well [20,21]. The cells were gently washed 3 times with phosphate-buffered saline (PBS) to remove the cells detached from the scratch. Then, serum-free DMEM medium containing different concentrations of Tremella fuciformis polysaccharide was added according to the group (100 μg / mL concentration with the best repair effect was selected for the experiment, and a damage control group without drug was set up). The well plates were placed in a 37℃, 5% CO2 incubator. Images were taken in the same field of view using an inverted microscope at 0 h and 24 h after scratching. The scratch width was measured and the healing rate was calculated using ImageJ software.

[0070] Table 13 Results of cell scratch assay The results showed that the 24-hour scratch healing rate in the tremella polysaccharide treatment group reached 76.4%, while that in the damage control group was only 41.2%, confirming that it can significantly accelerate cell migration.

[0071] 6.2 Measurement of inflammatory factors To further explore its anti-inflammatory and repair mechanism, an ELISA inflammatory factor detection experiment was carried out simultaneously. Cell culture supernatant that had been treated with 100 μg / mL Tremella polysaccharide for 24 h after UVB damage was taken. The TNF-α and IL-6 ELISA detection kits were operated according to the instructions. Standards, samples and detection antibodies were added in sequence. After incubation, washing and color development, the absorbance value was measured at 450 nm and the secretion of factors was calculated.

[0072] Figure 17 The results showed that the TNF-α secretion level in the damaged control group was as high as 1285 pg / mL and the IL-6 level was 895 pg / mL, while the TNF-α secretion level in the tremella polysaccharide treatment group decreased to 425 pg / mL and the IL-6 level decreased to 312 pg / mL. The secretion level of inflammatory factors was significantly downregulated. Combined with the results of the cell scratch assay, this fully demonstrates that tremella polysaccharide has dual anti-inflammatory and repair effects of promoting cell migration and inhibiting inflammatory response.

[0073] 6.3 In vivo evaluation A photodamage model of skin was established in Kunming mice. After hair removal on their backs, mice were irradiated with UVB daily (1.26 J / cm²) for 30 consecutive days, while simultaneously receiving topical application of Tremella fuciformis polysaccharide (10 mg / cm² / day). After 14 days of intervention, the skin water content of the mice recovered from 52.3%±3.8% in the model group to 74.6%±2.9%, approaching the level of the normal group (76.8%±2.1%).

[0074] Six- to eight-week-old SPF-grade Kunming mice (half male and half female, weighing 20-22g) were selected and acclimatized for one week (temperature 22-25℃, humidity 45%-55%, 12-hour light-dark cycle, free access to food and water). After acclimatization, the mice were randomly divided into three groups: a normal control group (no hair removal, no UVB irradiation, topical application of physiological saline), a model control group (hair removal, UVB irradiation, topical application of physiological saline), and a Tremella fuciformis polysaccharide intervention group (hair removal, UVB irradiation, topical application of Tremella fuciformis polysaccharide), with 10 mice in each group.

[0075] On day 1 of the experiment, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). Hair was then shaved from a 3cm x 3cm area on the back using an electric shaver. Depilatory cream (such as sodium sulfide depilatory cream) was then applied, and after 30 seconds, the area was gently wiped clean with sterile saline solution, ensuring the skin in the shaved area remained intact and undamaged. After hair removal, the mice were housed separately to prevent biting and skin damage. Hair removal was repeated weekly to maintain the hair-free state.

[0076] A 312nm UVB ultraviolet irradiation instrument was used, and the irradiation intensity was calibrated before irradiation (ensuring the intensity at the target site was 1.26 J / cm²). Mice were anesthetized and fixed on a dedicated irradiation frame, exposing the hair-removed area on their backs, while non-irradiated areas were tightly covered with aluminum foil. Irradiation was performed once daily from 9:00 AM to 10:00 AM, with the duration of each irradiation calculated based on the irradiation intensity (irradiation time = 1.26 J / cm² ÷ instrument irradiation intensity), for 30 consecutive days.

[0077] The intervention group received topical intervention 30 minutes after each UVB irradiation: a sterile cotton swab was dipped in a 100 mg / mL solution of Tremella fuciformis polysaccharide, and applied evenly to the hair removal area on the back at a dose of 10 mg / cm² (i.e., 0.1 mL / cm²), and gently massaged for 30 seconds to promote absorption; the normal group and the model group were treated with the same method and an equal volume of sterile saline solution was applied once a day, simultaneously with the irradiation, for 30 consecutive days.

[0078] On day 14 of the experiment (14 days after intervention), after anesthetizing the mice, the skin moisture content of the hair-removed area on the back was measured using a skin moisture meter (such as Corneometer CM825). Three test points were selected for each mouse (avoiding skin lesions), and each point was measured three times and the average value was taken. Finally, the mean ± standard deviation of the skin moisture content of each group of mice was calculated.

[0079] Table 14 Raw data and statistical analysis results of mouse skin moisture content on day 14 of the experiment. VISIA skin imaging multispectral analysis showed that the skin texture variance in the tremella polysaccharide group decreased from 42.3 to 18.5 (compared to 16.2 in the normal group), the erythema index (EI) decreased from 285 to 112, and pigmentation was significantly reduced.

[0080] Table 15 Results of VISIA Skin Imaging System Multispectral Analysis of Skin Parameters Skin analysis showed that transepidermal water loss (TEWL) decreased from 48.2 g / h·m² in the model group to 28.5 g / h·m², restoring skin barrier function.

[0081] Table 16 Transdermal Water Loss (TEWL) Detection Data Histopathological H&E staining results showed ( Figure 18 Compared with the normal control group, the skin photodamage model group mice showed significantly increased epidermal thickness (manifested as increased number of epidermal cell layers and disordered structure), obvious defects and breaks in the stratum corneum, loose and disordered collagen fiber arrangement in the dermis, and collagen fiber degradation and breakage in some areas. Simultaneously, the number of hair follicles decreased and their structure atrophied, and the volume and number of sebaceous glands decreased significantly, exhibiting typical pathological characteristics of skin photoaging. However, after intervention with Tremella fuciformis polysaccharide, the morphology of mouse skin tissue was significantly improved: epidermal thickness returned to the level of the normal control group, and epidermal cells... The collagen fibers in the dermis were tightly arranged and clearly layered, with a complete and continuous stratum corneum structure without defects or breaks, effectively repairing the damage to the epidermal barrier function caused by photodamage. The collagen fibers in the dermis were regularly and densely arranged, with clearly visible fiber bundles, and the degree of disorder and degradation was significantly reduced, suggesting that Tremella fuciformis polysaccharide can inhibit collagen fiber damage caused by photoaging. Further quantitative analysis showed that the number of hair follicles and sebaceous glands in the Tremella fuciformis polysaccharide group was significantly increased compared to the model group, and the hair follicle structure was intact and the sebaceous gland secretion function was restored. Statistical analysis showed that this difference was statistically significant (P<0.01). These results confirm that Tremella fuciformis polysaccharide can significantly improve the pathological damage of UVB irradiation-induced photoaging of mouse skin by repairing epidermal structure, protecting the integrity of collagen fibers, and promoting the regeneration of skin appendages (hair follicles and sebaceous glands), providing clear histomorphological evidence for its anti-photoaging effect.

Claims

1. A method for preparing tremella polysaccharide, characterized in that, Includes the following steps: (1) Add water to the fruiting body of Tremella fuciformis at a material-to-liquid ratio of 1:25-1:35 g:mL, steam and soften at 70-90℃, and then perform high-speed shearing and crushing for 10-25 minutes to obtain crude extract; (2) Add 0.5%-2.0% propylene carbonate and 0.1%-0.5% potassium polykrylate to the crude extract, then add papain to a final concentration of 0.1%-0.5%, adjust the pH to 6.0-7.0, enzymatically hydrolyze at 40-60℃ for 1-4 hours, inactivate, adjust the pH to 2.5-3.0, let stand at 4℃ to precipitate, centrifuge to remove protein precipitate, and obtain purified solution; (3) Add 3-5 times the volume of anhydrous ethanol to the purification solution, precipitate at 4℃ for 10-15 hours, and collect the crude polysaccharide by centrifugation; after redissolving the crude polysaccharide, load it onto the sample, elute the DEAE-52 ion exchange chromatography column with a gradient of 0.2-0.5 mol / L NaCl, collect the eluent fraction, and obtain the purified Tremella polysaccharide. (4) Prepare a 2% aqueous solution of refined tremella polysaccharide, add 30% hydrogen peroxide to a final concentration of 1.5%-2.0%, and degrade it at 50-55℃ for 2.5-3.0 hours to obtain the degradation solution; (5) After dialysis of the degradation solution through a dialysis bag, freeze-dry it under vacuum to obtain Tremella polysaccharide.

2. The production method according to claim 1, characterized by, The amount of propylene carbonate added in step (2) is 1.5% of the volume of the crude extract.

3. The preparation method according to claim 1, characterized in that, The amount of polycrylene potassium added in step (2) is 0.3% of the mass of the crude extract.

4. The preparation method according to claim 1, characterized in that, The enzyme used in step (2) for enzymatic hydrolysis is papain, with an enzyme concentration of 0.3% (w / v), a hydrolysis pH of 6.5, a hydrolysis temperature of 50°C, and a hydrolysis time of 2 hours.

5. The preparation method according to claim 1, characterized in that, The alcohol precipitation in step (3) involves adding 4 times the volume of anhydrous ethanol and precipitating at 4°C for 12 hours.

6. The preparation method according to claim 1, characterized in that, The ion exchange chromatography in step (3) uses a DEAE-52 ion exchange chromatography column, and is eluted stepwise with distilled water, 0.2 mol / L NaCl, and 0.5 mol / L NaCl, and the 0.5 mol / L NaCl elution fraction is collected.

7. The preparation method according to claim 1, characterized in that, The conditions for the controlled degradation of hydrogen peroxide in step (4) are as follows: the polysaccharide is prepared into a 2% aqueous solution, 30% H2O2 is added to a final concentration of 1.8%, and it is degraded at 53°C for 2.5 hours.

8. The preparation method according to claim 1, characterized in that, Step (5) After dialysis of the degradation solution through a low molecular weight cutoff dialysis bag, it is freeze-dried under vacuum to obtain Tremella polysaccharide.

9. The application of the Tremella polysaccharide obtained by the preparation method of claim 1 in the preparation of cosmetics.

10. The application of the Tremella polysaccharide obtained by the preparation method of claim 1 in the preparation of cosmetics with moisturizing, skin barrier repair, anti-inflammatory and / or antioxidant effects.

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