A method for preparing auricularia auricula-judae polypeptide and its application in skin care products.

Abstract

This invention belongs to the field of natural bioactive peptide technology, specifically relating to a method for preparing *Auricularia auricula-judae* polypeptides and their application in skincare products. The preparation method includes the following steps: defatting *Auricularia auricula-judae* powder, fermentation, alkaline protein extraction, isoelectric point precipitation, targeted enzymatic hydrolysis with alkaline protease, impurity removal, and secondary ultrafiltration purification to obtain *Auricularia auricula-judae* small molecule polypeptides <1000 Da, containing two specific sequences, ATFRYAL and MMLKLLR. The *Auricularia auricula-judae* polypeptides obtained by this invention can scavenge UVB-induced ROS, restore mitochondrial membrane potential, and inhibit MMP-1 and hyaluronidase, exhibiting excellent anti-photoaging, anti-aging, and repairing effects. An addition concentration of 50-150 μg / mL can be used in skincare products such as serums, creams, and repair masks. This invention features a stable process, high protein utilization, good transdermal absorption, and high safety, making it suitable for industrial production and high-end skincare product applications.

Description

Technical Field

[0001] This invention belongs to the field of natural bioactive peptide technology, specifically relating to a method for preparing auricularia auricula-judae polypeptide and its application in skin care products. Background Technology

[0002] Golden ear fungus is a traditional Chinese medicinal and edible fungus, rich in active ingredients such as polysaccharides, proteins, and amino acids. Current research focuses only on the antioxidant, immunomodulatory, anti-inflammatory, and hypoglycemic activities of golden ear fungus polysaccharides. The bioactive peptides prepared from its protein components through enzymatic hydrolysis have not yet been developed and utilized, resulting in a serious waste of golden ear fungus protein resources and failure to achieve high-value utilization.

[0003] Photoaging of the skin is a core type of aging induced by UVB radiation. Excessive reactive oxygen species (ROS) cause mitochondrial damage, degradation of the extracellular matrix (collagen, hyaluronic acid), cell senescence and apoptosis, ultimately leading to sagging skin, wrinkles, and dullness. Among existing anti-aging ingredients, chemically synthesized antioxidants pose safety risks such as skin irritation and cytotoxicity; natural plant / fungal-derived peptides, due to their safety, high efficacy, and easy transdermal absorption, have become core ingredients in cosmetics and functional foods.

[0004] Traditional methods for extracting peptides from *Auricularia auricula-judae*, such as acid hydrolysis and alkaline hydrolysis, suffer from problems such as low yield, severe damage to active ingredients, and environmental pollution. Enzymatic hydrolysis has gradually become the mainstream method due to its advantages of mild conditions, high selectivity, and environmental friendliness. However, there is currently limited research on enzymatic hydrolysis processes for *Auricularia auricula-judae* peptides, and existing methods generally suffer from the following problems: low enzymatic hydrolysis efficiency, insufficient peptide yield; low activity retention rate; unstable antioxidant and moisturizing properties, resulting in high costs and poor reproducibility. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing auricularia auricularia peptide with high protein utilization, strong activity, and high purity, so as to obtain auricularia auricularia peptide that can be absorbed through the skin, targets photoaging, and repairs mitochondria, and can be applied to anti-aging skin care products.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing auricularia auricula polypeptide includes the following steps: (1) Degreasing treatment: Fresh golden ear fungus is dried at low temperature, crushed, degreased, filtered and dried to obtain degreased golden ear fungus powder; (2) Mix defatted golden ear powder with water, sterilize, and obtain fermentation material; add bacterial strain to fermentation material, and sterilize after fermentation to obtain golden ear powder fermentation liquid; (3) Alkaline extraction of crude protein: Adjust the pH of the fermentation broth of auricularia auricula-judae powder to alkaline, heat to extract, cool and centrifuge, and collect the supernatant; (4) Precipitation of protein at isoelectric point: Adjust the pH of the supernatant to acidic, let it stand, and centrifuge to collect the crude protein precipitate from the golden ear fungus; (5) Alkaline protease-directed enzymatic hydrolysis: The crude protein precipitate of Auricularia auricula-judae was reconstituted with buffer solution, and alkaline protease was added for enzymatic hydrolysis at constant temperature; (6) Enzyme inactivation treatment: Inactivate enzymes, cool and centrifuge, and collect the supernatant of enzyme hydrolysis; (7) Secondary ultrafiltration purification: The enzymatic hydrolysis supernatant is first passed through an ultrafiltration membrane to remove macromolecular impurities, and then enriched by an ultrafiltration membrane. The filtrate with a value of <1000 Da is collected, freeze-dried, and the golden ear polypeptide is obtained.

[0007] This invention combines fermentation with alkaline extraction. First, fermentation is performed to gently degrade the cell wall of *Auricularia auricula-judae* using a complex enzyme system secreted by microorganisms, thereby disrupting the cross-linking structure of proteins and polysaccharides and allowing the bound proteins to be fully released. Then, alkaline extraction is performed to further dissolve residual proteins and inactivate microorganisms. The synergistic effect of the two methods avoids the severe damage to proteins caused by alkaline extraction alone and overcomes the low efficiency of simple fermentation. Furthermore, the antioxidant activity of the resulting peptides is significantly enhanced.

[0008] Preferred strains include Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus.

[0009] Preferably, the ratio of viable counts of Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus in the bacterial strain is 1:(0.4-0.7):(1.2-1.5).

[0010] Preferably, the fermentation temperature is 35-40℃ and the anaerobic fermentation time is 46-52h.

[0011] This invention uses a specific ratio of Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus for synergistic fermentation over a specific time. Bifidobacterium and lactic acid bacteria co-produce acid, lowering the pH value, activating endogenous proteases, and inhibiting other bacteria. Leuconostoc mesenteroides produces glucan, improving the rheological properties of the fermentation system. The specific fermentation time ensures sufficient cell growth and enzyme secretion while avoiding over-fermentation that would consume proteins into free amino acids. This further facilitates subsequent enzymatic hydrolysis and targeted enrichment of active peptides with a molecular weight <1000 Da, ultimately identifying two specific anti-photoaging sequences, ATFRYAL and MMLKLLR.

[0012] Preferably, the pH of the auricularia auricula-judae fermentation broth is adjusted to 10.0-10.5, and extracted in a water bath at 55-60℃ for 1-2 hours.

[0013] Preferably, the molecular weight of the golden ear polypeptide is <1000 Da, including polypeptides with the amino acid sequences ATFRYAL and MMLKLLR.

[0014] Note: The amino acid sequence uses standard single-letter codes, where A represents alanine; T represents threonine; F represents phenylalanine; R represents arginine; Y represents tyrosine; L represents leucine; M represents methionine; and K represents lysine.

[0015] This method combines microbial fermentation, alkaline extraction, and targeted enzymatic hydrolysis with alkaline protease, resulting in a good synergistic effect. Fermentation first disrupts the dense cell wall and fibrous structure of *Auricularia auricula-judae*, releasing the protein from its binding and making it easier for the protein to dissolve during alkaline extraction. The crude protein prepared has a higher content of effective components, and the alkaline protease can further efficiently cleave the protein peptide chains, ultimately producing *Auricularia auricula-judae* polypeptides with a molecular weight of less than 1000 Da. The product contains the characteristic amino acid sequences ATFRYAL and MMLKLLR. The process combination is reasonable, resulting in better polypeptide yield and quality.

[0016] This invention provides the application of the auricularia auricula-judae polypeptide prepared by the above-described preparation method in the preparation of skin care products that repair UVB photodamage, delay skin aging, repair mitochondria, soothe inflammation, and moisturize and repair.

[0017] This invention provides the application of the golden ear polypeptide prepared by the above-described method in the preparation of functional foods or health foods with antioxidant and anti-aging properties.

[0018] A skincare product comprising the auricularia peptide prepared by the method described above, and cosmetically acceptable excipients.

[0019] The present invention provides that the concentration of the golden ear polypeptide added is 50~150μg / mL.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention develops protein peptides from *Auricularia auricula-judae*, breaking through the technical bottleneck of traditional methods that only utilize *Auricularia auricula-judae* polysaccharides, and achieving high-value utilization of resources. In the extraction process, alkaline protein extraction combined with isoelectric precipitation is used to improve protein utilization. Further targeted enzymatic hydrolysis using alkaline protease is significantly more efficient than papain, cellulase, and pectinase. A two-stage ultrafiltration system is used during purification to target and enrich small peptides with molecular weights below 1000 Da, improving transdermal permeability. The final product yields two specific peptide sequences—ATFRYAL and MMLKLLR—with clearly defined activities and excellent stability.

[0021] 2. The golden ear peptide of this invention has multi-target anti-photoaging effects, capable of scavenging reactive oxygen species, repairing mitochondrial function, and inhibiting matrix metalloproteinase degradation, while also possessing anti-inflammatory, moisturizing, and barrier repair effects. In terms of safety, verification using HaCaT cell and zebrafish embryo models has shown that it is non-toxic, non-teratogenic, and non-lethal, meeting cosmetic and food safety standards. Furthermore, the small molecule peptide is easily transdermal, has good water solubility, and excellent formulation compatibility, making it widely applicable in skincare products, functional foods, and health foods.

[0022] 3. This invention combines fermentation with alkaline extraction. Fermentation is used first, and the two work synergistically. This avoids the severe damage to proteins caused by alkaline extraction alone, and overcomes the low efficiency of simple fermentation. In addition, the antioxidant activity of the obtained peptides is significantly enhanced.

[0023] 4. This invention uses a specific ratio of Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus for synergistic fermentation for a specific time, which ensures sufficient cell growth and enzyme secretion while avoiding over-fermentation that would consume proteins into free amino acids. This further facilitates the subsequent enzymatic hydrolysis and targeted enrichment of active peptides with a molecular weight of <1000 Da, ultimately identifying two specific anti-photoaging sequences, ATFRYAL and MMLKLLR. Attached Figure Description

[0024] Figure 1 The graph shows the DPPH / ABTS free radical scavenging capacity of different *Auricularia auricula-judae* protein hydrolysates. A represents the DPPH free radical scavenging rate; B represents the ABTS free radical scavenging rate.

[0025] Figure 2 The effects of TAH-A peptide on zebrafish embryonic development, oxidative stress, and apoptosis are shown in the figure. (A) Hatching rate; (B) Mortality rate; (C) Representative fluorescence images: reactive oxygen species (ROS) (ascending, green, DCFH-DA staining) and acridine orange (descending, red, AO staining). Scale bar = 200 μm; (D) ROS; (E) Quantitative analysis of acridine orange fluorescence intensity. Data are mean ± SD (n=3). *p<0.05, **p<0.01 vs. control group.

[0026] Figure 3 This diagram illustrates the docking results of the core polypeptide ATFRYAL from *Eurydon fuciformis* with anti-aging target protein molecules; A represents the binding of hyaluronidase (HAase, PDB number: 2CBI) to polypeptide ATFRYAL; B represents the binding of matrix metalloproteinase-1 (MMP-1, PDB number: 966C) to polypeptide ATFRYAL; G represents the binding of type I collagen (Col-Ⅰ, PDB number: 7CWK) to polypeptide ATFRYAL; and H represents the binding of elastase (Elastase, PDB number: 1ELC) to polypeptide ATFRYAL.

[0027] Figure 4 The diagram shows the docking results of the core polypeptide MMLKLLR from *Eurydon fuciformis* with anti-aging target protein molecules; E represents the binding of hyaluronidase (HAase, PDB number: 2CBI) to polypeptide MMLKLLR; F represents the binding of matrix metalloproteinase-1 (MMP-1, PDB number: 966C) to polypeptide MMLKLLR; K represents the binding of type I collagen (Col-Ⅰ, PDB number: 7CWK) to polypeptide MMLKLLR; and L represents the binding of elastase (Elastase, PDB number: 1ELC) to polypeptide MMLKLLR. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] All raw materials used in the following embodiments of the present invention are commercially available products.

[0030] Example 1 This embodiment provides a method for preparing auricular peptide, including the following steps: (1) Degreasing treatment: Fresh golden ear fungus dried at low temperature is pulverized to 100 mesh, and degreased by stirring at 25℃ for 30 minutes with 1g:12mL petroleum ether. The filtrate is filtered until it is colorless and dried at 50℃ to obtain degreased golden ear fungus powder. (2) Mix defatted auricularia auricula-judae powder and water at a mass ratio of 1:9, and sterilize at 121℃ for 15 min to obtain fermentation material; add bacterial strains to the fermentation material, and the total viable count of the bacterial strains after inoculation is 3×10 8 Fermentation was carried out at CFU / mL of the fermentation material at 37℃ for 48 hours under anaerobic conditions. After fermentation, the mixture was pasteurized at 65℃ for 30 minutes to obtain the fermentation broth for *Eurydonium majus* powder. The bacterial strains included *Bifidobacterium adolescentis*, *Leuconostoc mesenteroides*, and *Lactobacillus rhamnosus*, with a viable count ratio of 1:0.5:1.4. The strain number of *Bifidobacterium adolescentis* was CICC6175; the strain number of *Lactobacillus rhamnosus* was CICC6137; and the strain number of *Leuconostoc mesenteroides* was CICC22264. The strain was obtained from the China Industrial Microbial Culture Collection Center.

[0031] (3) Alkaline extraction of crude protein: The pH of the fermentation broth of Auricularia auricula-judae powder was adjusted to 10.0 with 1 mol / L NaOH, and extracted in a water bath at 60℃ for 1 h. After cooling, it was centrifuged at 9000 rpm and 4℃ for 30 min, and the supernatant was collected. (4) Precipitation of protein at isoelectric point: The supernatant was adjusted to pH=4.0 with 1mol / L HCl, allowed to stand at 4℃ for 30min, and then centrifuged to collect the crude protein precipitate from the golden ear fungus; (5) Alkaline protease-directed enzymatic hydrolysis: The crude protein precipitate of Auricularia auricula-judae was reconstituted with 0.01mol / LPBS (pH 7.4) at a material-to-liquid ratio of 1g:50mL, and alkaline protease was added. The enzyme addition amount was 16000U / g crude protein precipitate of Auricularia auricula-judae. The pH was adjusted to 10.0, and the enzymatic hydrolysis was carried out at 45℃ for 2h. (6) Enzyme inactivation treatment: Inactivate enzymes at 90℃ for 15 min, cool and centrifuge, and collect the supernatant of enzyme hydrolysis; (7) Secondary ultrafiltration purification: The enzymatic supernatant is first passed through a 3000 Da ultrafiltration membrane to remove macromolecular impurities, and then enriched through a 1000 Da ultrafiltration membrane. The filtrate with a density of <1000 Da is collected, freeze-dried, and the golden ear polypeptide is obtained.

[0032] Example 2 This embodiment provides a method for preparing auricular peptide, including the following steps: (1) Degreasing treatment: Fresh golden ear fungus dried at low temperature is pulverized to 100 mesh, and degreased by stirring at 25℃ for 30 minutes with 1g:12mL petroleum ether. The filtrate is filtered until it is colorless and dried at 50℃ to obtain degreased golden ear fungus powder. (2) Mix defatted auricularia auricula-judae powder and water at a mass ratio of 1:9, and sterilize at 121℃ for 15 min to obtain fermentation material; add bacterial strains to the fermentation material, and the total viable count of the bacterial strains after inoculation is 3×10 8 Fermentation was carried out at CFU / mL of the fermentation material at 37℃ for 48 hours under anaerobic conditions. After fermentation, the mixture was pasteurized at 65℃ for 30 minutes to obtain the fermentation broth for *Eurydonium majus* powder. The bacterial strains included *Bifidobacterium adolescentis*, *Leuconostoc mesenteroides*, and *Lactobacillus rhamnosus*, with a viable count ratio of 1:0.4:1.2. The strain number of *Bifidobacterium adolescentis* was CICC6175; the strain number of *Lactobacillus rhamnosus* was CICC6137; and the strain number of *Leuconostoc mesenteroides* was CICC22264. The strain was obtained from the China Industrial Microbial Culture Collection Center.

[0033] (3) Alkaline extraction of crude protein: The pH of the fermentation broth of Auricularia auricula-judae powder was adjusted to 10.0 with 1 mol / L NaOH, and extracted in a water bath at 60℃ for 1 h. After cooling, it was centrifuged at 9000 rpm and 4℃ for 30 min, and the supernatant was collected. (4) Precipitation of protein at isoelectric point: The supernatant was adjusted to pH=4.0 with 1mol / L HCl, allowed to stand at 4℃ for 30min, and then centrifuged to collect the crude protein precipitate from the golden ear fungus; (5) Alkaline protease-directed enzymatic hydrolysis: The crude protein precipitate of Auricularia auricula-judae was reconstituted with 0.01mol / LPBS (pH 7.4) at a material-to-liquid ratio of 1g:50mL, and alkaline protease was added. The enzyme addition amount was 16000U / g crude protein precipitate of Auricularia auricula-judae. The pH was adjusted to 10.0, and the enzymatic hydrolysis was carried out at 45℃ for 2h. (6) Enzyme inactivation treatment: Inactivate enzymes at 90℃ for 15 min, cool and centrifuge, and collect the supernatant of enzyme hydrolysis; (7) Secondary ultrafiltration purification: The enzymatic supernatant is first passed through a 3000 Da ultrafiltration membrane to remove macromolecular impurities, and then enriched through a 1000 Da ultrafiltration membrane. The filtrate with a density of <1000 Da is collected, freeze-dried, and the golden ear polypeptide is obtained.

[0034] Comparative Example 1 The difference between this comparative example and Example 1 is that the alkaline protease was replaced with an equal amount of papain.

[0035] Comparative Example 2 The difference between this comparative example and Example 1 is that the alkaline protease is replaced with an equal amount of cellulase.

[0036] Comparative Example 3 The difference between this comparative example and Example 1 is that the alkaline protease was replaced with an equal amount of pectinase.

[0037] Comparative Example 4 The difference between this comparative example and Example 1 is as follows: the bacterial strains include *Bifidobacterium adolescentis* and *Lactobacillus rhamnosus*, with a viable count ratio of 1:1.4; the strain number of *Bifidobacterium adolescentis* is CICC6175; and the strain number of *Lactobacillus rhamnosus* is CICC6137. (China Industrial Microbial Culture Collection Center) Comparative Example 5 The difference between this comparative example and Example 1 is as follows: the bacterial strains include Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus, with a live bacteria ratio of 1:1:1; the strain number of Bifidobacterium adolescentis is CICC6175; the strain number of Lactobacillus rhamnosus is CICC6137; the strain number of Leuconostoc mesenteroides is CICC22264; and the strains are from the China Industrial Microbial Culture Collection Center.

[0038] Comparative Example 6 The difference between this comparative example and Example 1 is that *Leuconostoc mesenteroides* was replaced with an equal amount of *Lactobacillus plantarum*. The strain number of *Lactobacillus plantarum* is CICC25125, from the China Industrial Microbial Culture Collection Center.

[0039] Comparative Example 7 The difference between this comparative example and Example 1 is that the fermentation temperature is 37°C and the anaerobic fermentation time is 40 hours.

[0040] Comparative Example 8 The difference between this comparative example and Example 1 is that the fermentation temperature is 37°C and the anaerobic fermentation time is 60 hours.

[0041] Application Example 1 An anti-photoaging essence containing golden ear polypeptide, the specific formula is shown in Table 1.

[0042] Table 1 Serum Formula Golden Ear Peptide 0.1 Sodium hyaluronate 0.1 glycerin 5.0 Butylene glycol 3.0 Panthenol 0.5 preservative 0.1 Deionized water margin The preparation process of the serum involves dissolving the ingredients in the formula by stirring at room temperature, filtering for sterilization, and aseptic filling.

[0043] Application Example 2 A functional solid beverage containing golden ear polypeptide, the specific formula of which is shown in Table 2.

[0044] Table 2 Solid Beverage Formulations Golden Ear Peptide 0.1 maltodextrin 90 Citrus fruit powder 9.9 The preparation process is as follows: mixing, granulating, drying, and filling the solid beverage formulation components.

[0045] Performance testing I. In vitro antioxidant activity detection (1) DPPH free radical scavenging rate Sample preparation: The auricularia auricula-judae peptides of Examples 1-2 and Comparative Examples 1-8 were prepared with deionized water to a concentration of 800 μg / mL, with 3 replicates.

[0046] Weigh an appropriate amount of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), dissolve it in anhydrous ethanol and prepare a 0.1 mmol / L working solution. Store in the dark and prepare fresh before use.

[0047] Add the following sequentially to the 96-well plate: Sample set: 100 μL LPPH working solution + 100 μL sample solutions of different concentrations; Control group: 100 μL LPPH working solution + 100 μL deionized water; Blank group: 100 μL anhydrous ethanol + 100 μL sample solution; Gently shake to mix, and incubate at room temperature in the dark for 30 minutes. Measure the absorbance at 517 nm using a microplate reader (denoted as A). 样品 A 对照 A 空白 ).

[0048] DPPH free radical scavenging rate = [1-(A)] 样品 -A 空白 )÷A 对照 ]×100%.

[0049] (2) ABTS free radical scavenging rate The samples were the golden ear polypeptides from Examples 1-2 and Comparative Examples 1-8.

[0050] An equal volume of 7 mmol / L LABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) aqueous solution was mixed with 2.45 mmol / L potassium persulfate aqueous solution and reacted at room temperature in the dark for 12 h to obtain the ABTS⁺ stock solution. Before use, the solution was diluted with PBS (pH 7.4) to an absorbance of 0.70 at 734 nm to obtain the ABTS⁺ working solution. Following the DPPH method, each sample was prepared to a concentration of 800 μg / mL.

[0051] Add the following sequentially to the 96-well plate: Sample set: 20 μL sample solution + 200 μL LABTS⁺ working solution; Control group: 20 μL deionized water + 200 μL LABTS⁺ working solution; Blank group: 20 μL sample solution + 200 μL PBS; The reaction was carried out at room temperature in the dark for 6 minutes. The absorbance at 734 nm was measured using a microplate reader (denoted as A). 样品 A 对照 A 空白 ).

[0052] ABTS radical scavenging rate = [1-(A 样品 -A 空白 )÷A 对照 ]×100%.

[0053] The results were plotted using TAH-A (Example 1, golden ear polypeptide), TAH-Pa (Comparative Example 1, papain hydrolysate), TAH-C (Comparative Example 2, cellulose hydrolysate), and TAH-P (Comparative Example 3, pectin hydrolysate). See Table 3 for the results. Figure 1 .

[0054] Table 3 Free radical scavenging rate Example 1 142.70 112.70 Example 2 140.53 110.84 Comparative Example 1 13.21 102.45 Comparative Example 2 52.59 25.31 Comparative Example 3 50.09 61.27 Comparative Example 4 77.34 68.18 Comparative Example 5 95.97 81.92 Comparative Example 6 84.65 73.03 Comparative Example 7 113.16 89.67 Comparative Example 8 125.48 100.46 As shown in Table 1, the DPPH free radical scavenging rates at 800 μg / mL were TAH-A = 142.7%, TAH-Pa = 13.21%, TAH-C = 52.59%, and TAH-P = 50.09%, with TAH-A significantly higher than the other groups (P < 0.05). Similarly, the ABTS free radical scavenging rates at 800 μg / mL were TAH-A = 112.7%, TAH-Pa = 102.45%, TAH-C = 25.31%, and TAH-P = 61.27%, with TAH-A significantly higher than the other groups (P < 0.05).

[0055] II. Wild-type AB lineage zebrafish were reared in a standard recirculating aquaculture system (28.5℃, light / dark cycle 14h / 10h) and fed brine shrimp daily. Healthy, sexually mature zebrafish were paired at a 1:1 female-to-male ratio. After light stimulation, fertilized eggs were collected, washed with fish tank water (0.2g / L sea salt, prepared with deionized water), and placed in a 28.5℃ constant temperature incubator for later use.

[0056] Zebrafish embryos that had developed to 6 hpf (after fertilization at hour 1 hour) were randomly divided into the following 6 groups, with 30 embryos in each group: Control Group: Fish Farm Water Treatment; Model group: UVB irradiation model (300mJ / cm²); TAH-A group: 50, 100, 150 μg / mL of auricularia auricula-judae peptide (prepared in Example 1) + UVB irradiation; TAH-A was dissolved in fish tank water and administered via immersion starting at 6 hpf, with the solution changed daily for a continuous treatment up to 72 hpf. Both the model group and the drug-treated group underwent UVB irradiation at a specific time point (48 hpf) after drug administration.

[0057] Zebrafish embryos were transferred to 6-well plates containing a small amount of aquarium water. Most of the liquid was removed, leaving only a thin film of water on the fish's surface. The embryos were then irradiated with a UVB lamp at a predetermined distance, with the irradiation intensity calibrated to 300 mJ / cm² using a UVB radiometer. The aquarium water was immediately replaced with fresh water after irradiation, and the embryos were cultured until the predetermined time point.

[0058] (1) Developmental indicators (hatching rate and mortality rate) Hatching rate: The hatching status of embryos in each well was observed under a stereomicroscope at 48 hpf and 72 hpf, and the number of hatched individuals was counted. Hatching rate (%) = (number of hatched embryos / total number of embryos) × 100%.

[0059] Mortality rate: Dead embryos (cardiac arrest, tail non-responsiveness, yolk saturation) were removed at the same time point. Mortality rate (%) = (number of dead embryos / total number of embryos) × 100%. Results are as follows: Figure 2 As shown in A and 2B.

[0060] (2) Detection of reactive oxygen species (ROS) levels After the 72 hpf treatment, 10 live zebrafish juveniles from each experimental group were transferred to 24-well plates. DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) (dissolved in fish tank water) was added to a final concentration of 25 μg / mL, and the plates were incubated at 28.5℃ in the dark for 1 h. The plates were then rinsed three times with fish tank water for 5 min each time to remove unbound fluorescent dye. The juveniles were anesthetized with tricaine (0.016%), observed, and photographed under a fluorescence stereomicroscope with excitation wavelength of 488 nm and emission wavelength of 525 nm, and exposure parameters were fixed. Figure 2 C (upwards, green). The average fluorescence intensity of the trunk region of juvenile fish was quantitatively analyzed using ImageJ software. Figure 2 D), the average fluorescence intensity of at least 5 juvenile fish in each experimental group was taken.

[0061] (3) Apoptosis detection (acidine orange staining) Principle: Acridine orange (AO) can pass through the membranes of dead or apoptotic cells, bind to DNA, and emit red fluorescence, which is used to label apoptotic cells.

[0062] Ten zebrafish juveniles from the same batch were taken, washed with fish tank water, and then stained with acridine orange solution to a final concentration of 5 μg / mL at 28.5℃ in the dark for 20 min. They were then quickly washed three times with fish tank water (2 min each time), and immediately after anesthesia, fluorescence observation was performed (excitation wavelength 488 nm, emission wavelength 600 nm). Red fluorescence represented apoptotic cells. Figure 2 C down, red). Similarly, ImageJ was used to quantify fluorescence intensity (C down, red). Figure 2 E). All experiments were repeated 3 times.

[0063] The results show that Figure 2 A: TAH-A50-150μg / mL dose-dependently increased the hatching rate of zebrafish embryos after UVB irradiation, reaching close to the Control group at 150μg / mL. Figure 2 B: There was no significant increase in mortality in any of the treatment groups, and the 72-hour survival rate was ≥95%, indicating that TAH-A has no embryotoxicity. Figure 2 C, 2D: TAH-A significantly reduced UVB-induced ROS green fluorescence (DCFH-DA) in a dose-dependent manner. Figure 2 E: TAH-A also reduced apoptosis-associated red fluorescence (AO) in a dose-dependent manner, and the 150 μg / mL group recovered to the control level.

[0064] Safety: 50–150 μg / mL TAH-A had no lethal or teratogenic toxicity to zebrafish embryos, with a 72-hour survival rate ≥95%, and dose-dependently improved embryo hatching rate; Anti-photoaging: After UVB (300 mJ / cm²) irradiation, TAH-A dose-dependently reduced ROS levels and apoptosis in zebrafish larvae, with the 150 μg / mL group returning to the levels of the control group.

[0065] (4) Detection method for anti-photoaging activity of HaCaT cells Human immortalized keratinocytes (HaCaT) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. When the cells reached 80%–90% confluence, they were passaged by digestion with 0.25% trypsin-EDTA.

[0066] HaCaT cells in logarithmic growth phase were used at a rate of 1×10 5 Cells were seeded in 6-well plates and cultured for 24 hours, then replaced with serum-free medium and starved for 12 hours. Experimental groups: Control group: No irradiation, routine culture; Model group: UVB irradiation (20mJ / cm²); TAH-A group: 50, 100, 150 μg / mL TAH-A + UVB irradiation; Administration method: Before irradiation, cells were pre-protected with different concentrations of TAH-A for 2 hours, then the drug-containing culture medium was discarded, the cells were washed once with PBS, and covered with a layer of PBS for UVB irradiation (the UVB lamp was about 15 cm away from the cell layer, and the intensity was calibrated to 20 mJ / cm² using a UVB radiometer). Immediately after irradiation, the culture medium was replaced with fresh drug-containing medium, and the cells were cultured for another 24 hours before various indicators were measured.

[0067] Collect the treated HaCaT cells, dilute DCFH-DA with serum-free DMEM to a final concentration of 10 μmol / L, and incubate at 37°C in the dark for 30 min.

[0068] Cells were washed twice with PBS, resuspended, and fluorescence intensity of the FL1 channel (525 nm) was detected using a flow cytometer (BDFACSCantoII). 10,000 cells were counted per sample. Simultaneously, images were taken using a fluorescence microscope. Results are expressed as relative fold increases in fluorescence intensity.

[0069] Mitochondrial membrane potential (ΔΨm) was detected using the JC-1 fluorescent probe method: Cells were collected, and JC-1 working solution (Beyotime, C2006) was added and incubated at 37°C for 20 min. JC-1 forms a polymer in normal mitochondria emitting red fluorescence (excitation 525 nm, emission 590 nm); when the membrane potential decreases, it emits green fluorescence as a monomer (excitation 488 nm, emission 525 nm). Flow cytometry was used to detect the red / green fluorescence ratio, which reflects the membrane potential level.

[0070] Lipid peroxidation (MDA) detection: Cell supernatant or cell lysate was collected, and the MDA content was determined by the thiobarbituric acid (TBA) method according to the instructions of the lipid peroxidation malondialdehyde (MDA) detection kit. The results are expressed as nmol / mg protein.

[0071] Fe²⁺ overload detection: The ferroazine colorimetric method was used: After cell lysis, ferroazine chromogenic reagent was added, and the absorbance at 562 nm was measured. The Fe²⁺ concentration was calculated according to the standard curve and expressed as μmol / mg protein.

[0072] As above, using one-way ANOVA and Tukey's test, P<0.05 was considered statistically significant.

[0073] The results showed that after UVB (20 mJ / cm²) irradiation of HaCaT cells, TAH-A (50, 100, 150 μg / mL) dose-dependently reduced intracellular ROS, restored mitochondrial membrane potential, and inhibited lipid peroxidation and Fe²⁺ overload; the 150 μg / mL group recovered to normal cell status.

[0074] III. Identification of Polypeptide Sequences The *Gynostemma pentaphyllum* peptides prepared in Example 1 were analyzed by UPLC-Q-Orbitrap HRMS, revealing the core active peptides as ATFRYAL and MMLKLLR. The core active peptides ATFRYAL and MMLKLLR were then docked with COL-I, MMP-1, HAase, and elastase. The CDOCKER interaction energies are shown in Table 4. Figure 3-4 .

[0075] Table 4. Docking binding energies of each receptor to the two polypeptides (unit: kcal / mol) Hyaluronidase (PDB:2CBI) -102.789 -120.723 Matrix metalloproteinase-1 (PDB:966C) -131.264 -126.255 Type I collagen (PDB:7CWK) -16.6762 -24.546 Elastase (PDB:1ELC) -53.7332 -34.5034 The core active peptides ATFRYAL and MMLKLLR were docked with COL-I, MMP-1, HAase, and elastase. The CDOCKER interaction energy was < -7 kcal / mol, and they were stably bound to the target proteins, which could inhibit the degradation of the extracellular matrix.

[0076] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing auricularia auricula-judae polypeptide, characterized in that, Includes the following steps: (1) Degreasing treatment: Fresh golden ear fungus is dried at low temperature, crushed, degreased, filtered and dried to obtain degreased golden ear fungus powder; (2) Mix defatted golden ear powder with water, sterilize, and obtain fermentation material; add bacterial strain to fermentation material, and sterilize after fermentation to obtain golden ear powder fermentation liquid; (3) Alkaline extraction of crude protein: Adjust the pH of the fermentation broth of auricularia auricula-judae powder to alkaline, heat to extract, cool and centrifuge, and collect the supernatant; (4) Precipitation of protein at isoelectric point: Adjust the pH of the supernatant to acidic, let it stand, and centrifuge to collect the crude protein precipitate from the golden ear fungus; (5) Alkaline protease-directed enzymatic hydrolysis: The crude protein precipitate of Auricularia auricula-judae was reconstituted with buffer solution, and alkaline protease was added for enzymatic hydrolysis at constant temperature; (6) Enzyme inactivation treatment: Inactivate enzymes, cool and centrifuge, and collect the supernatant of enzyme hydrolysis; (7) Secondary ultrafiltration purification: The enzymatic hydrolysis supernatant is first passed through an ultrafiltration membrane to remove macromolecular impurities, and then enriched by an ultrafiltration membrane. The filtrate with a value of <1000 Da is collected, freeze-dried, and the golden ear polypeptide is obtained.

2. The method for preparing the auricularia auricularia polypeptide according to claim 1, characterized in that, The bacterial strains include Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus.

3. The method for preparing the golden ear polypeptide according to claim 2, characterized in that, The ratio of viable counts of Bifidobacterium adolescentis, Leuconostoc mesenteroides, and Lactobacillus rhamnosus in the bacterial strain was 1:(0.4-0.7):(1.2-1.5).

4. The method for preparing the golden ear polypeptide according to claim 2, characterized in that, The fermentation temperature is 35-40℃, and the anaerobic fermentation time is 46-52 hours.

5. The method for preparing the auricularia auricularia polypeptide according to claim 1, characterized in that, Adjust the pH of the auricularia auricula-judae fermentation broth to 10.0-10.5, and extract it in a water bath at 55-60℃ for 1-2 hours.

6. The method for preparing the auricularia auricularia polypeptide according to claim 1, characterized in that, The molecular weight of the golden ear polypeptide is <1000 Da, including polypeptides with the amino acid sequences ATFRYAL and MMLKLLR.

7. The use of the auricularia auricula-judae polypeptide prepared by the preparation method according to any one of claims 1-6 in the preparation of skin care products that repair UVB photodamage, delay skin aging, repair mitochondria, soothe inflammation, and moisturize and repair.

8. The application of the auricularia auricula polypeptide prepared by the preparation method according to any one of claims 1-6 in the preparation of functional foods or health foods with antioxidant and anti-aging properties.

9. A skincare product, characterized in that, It comprises the auricularia polypeptide prepared by the preparation method according to any one of claims 1-6, and cosmetically acceptable excipients.

10. The skincare product according to claim 9, characterized in that, The concentration of the golden ear polypeptide added is 50~150μg / mL.

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