Method for preparing gold ear polypeptide and application thereof

Abstract

This invention belongs to the field of cosmetic technology. Specifically, this invention discloses a method for preparing auricularia auricularia polypeptide and its application. It includes the following steps: (1) defatting auricularia auricularia powder with petroleum ether to obtain defatted auricularia auricularia powder; (2) adding defatted auricularia auricularia powder and a compound enzyme to water, performing enzymatic hydrolysis with ultrasonic assistance, inactivating the enzyme, centrifuging, and obtaining a supernatant; (3) ultrafiltration of the supernatant with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, collecting the filtrate, drying, and obtaining auricularia auricularia polypeptide. In this invention, auricularia auricularia powder is first defatted, then subjected to enzymatic hydrolysis with a compound enzyme with ultrasonic assistance, and finally ultrafiltration is performed sequentially with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and an ultrafiltration membrane with a molecular weight cutoff of 10000 Da, thereby obtaining auricularia auricularia polypeptide with excellent moisturizing and antioxidant properties, and the yield of the auricularia auricularia polypeptide is high.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a method for preparing auricular peptides and their applications. Background Technology

[0002] Golden ear fungus (Tremella aurantialba) is an edible and medicinal fungus rich in protein. Its polypeptides have antioxidant, moisturizing, and anti-inflammatory bioactivities, and have broad application prospects in the cosmetics and pharmaceutical fields.

[0003] Traditional methods for extracting peptides from *Auricularia auricula-judae* (such as acid hydrolysis and alkaline hydrolysis) suffer from low yields, severe damage to active ingredients, and environmental pollution. Enzymatic hydrolysis has gradually become mainstream 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 hydrolysis efficiency, resulting in insufficient peptide yields (usually <15%); low activity retention, and unstable antioxidant and moisturizing properties; and unoptimized process parameters (such as enzyme type, temperature, and time), leading to high costs and poor reproducibility.

[0004] Therefore, this application is submitted. Summary of the Invention

[0005] This invention provides a method for preparing auricularia auricularia peptide and its application. The auricularia auricularia peptide of this invention has excellent moisturizing properties and antioxidant properties, and the yield of the auricularia auricularia peptide is high.

[0006] The present invention solves its technical problem by adopting the following technical solution: A method for preparing auricularia auricula polypeptide includes the following steps: (1) Degrease the golden ear powder with petroleum ether to obtain defatted golden ear powder; (2) Add defatted golden ear powder and compound enzyme to water, use ultrasound to assist enzymatic hydrolysis, inactivate enzyme, centrifuge, and obtain supernatant; (3) The supernatant was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide; The complex enzyme includes cellulase and papain.

[0007] This invention first defatts the golden ear fungus powder, then performs ultrasonic-assisted enzymatic hydrolysis using a compound enzyme, and then uses an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to collect the filtrate, thereby obtaining golden ear fungus polypeptides with excellent moisturizing and antioxidant properties, and the yield of the golden ear fungus polypeptides is high.

[0008] This invention employs a combination of cellulase and papain to target and decompose polysaccharides and proteins in the cell wall of *Auricularia auricula-judae*, thereby improving enzymatic hydrolysis efficiency. Simultaneous ultrasound application during hydrolysis accelerates substrate disruption and enzyme-substrate contact through cavitation, shortening reaction time. Directional molecular weight control is used: ultrafiltration purification (below 3 kDa) retains highly active small-molecule peptides while avoiding interference from large-molecule impurities. The resulting *Auricularia auricula-judae* peptides exhibit high enzymatic hydrolysis efficiency, high peptide yield, high activity retention, and stable antioxidant and moisturizing properties.

[0009] In a preferred embodiment of the present invention, the particle size of the gold ear powder is 80-100 mesh.

[0010] In a preferred embodiment of the present invention, the solid-liquid ratio of the gold ear powder and petroleum ether is 1g:(2~10)mL.

[0011] In a preferred embodiment of the present invention, the mass ratio of cellulase to papain is 1:(0.4~0.6).

[0012] As a preferred embodiment of the present invention, the ratio of defatted golden ear powder, compound enzyme and water is 1g:(0.005~0.02)g:(4~20)mL.

[0013] In a preferred embodiment of the present invention, the enzymatic hydrolysis temperature is 45~55℃ and the enzymatic hydrolysis time is 2~6h.

[0014] In a preferred embodiment of the present invention, the power of the ultrasound is 200~300W and the frequency is 30~50kHz.

[0015] In a preferred embodiment of the present invention, the enzyme inactivation temperature is 80~90℃ and the time is 5~20min.

[0016] The present invention also provides a golden ear polypeptide, which is prepared by the method described above.

[0017] This invention also provides an application of auricular peptide in the preparation of cosmetics.

[0018] The beneficial effects of the present invention are as follows: (1) The present invention first defatted the golden ear powder, then used a compound enzyme for ultrasonic-assisted enzymatic hydrolysis, and used an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain golden ear polypeptide with excellent moisturizing and antioxidant properties, and the yield of the golden ear polypeptide is high.

[0019] (2) The present invention uses a combination of cellulase and papain to target and decompose polysaccharides and proteins in the cell wall of Auricularia auricula-judae, thereby improving the enzymatic hydrolysis efficiency; ultrasound is applied simultaneously during the enzymatic hydrolysis process to accelerate substrate breakage and enzyme-substrate contact through cavitation effect, thereby shortening the reaction time; directional molecular weight control: ultrafiltration purification (below 3 kDa) retains highly active small molecule peptides and avoids interference from large molecule impurities. The Auricularia auricula-judae peptides have high enzymatic hydrolysis efficiency, high peptide yield, high activity retention rate, and stable antioxidant and moisturizing properties. Attached Figure Description

[0020] Figure 1 A representative image of the fluorescent staining area of ​​zebrafish embryos for photodamage testing (repair).

[0021] Figure 2 This is a graph showing the relative fluorescence area of ​​different samples in the photodamage test (repair).

[0022] Figure 3 A representative image of neutrophils in the tail of a zebrafish embryo used for anti-inflammatory testing.

[0023] Figure 4 The effect of different samples on neutrophil count in anti-inflammatory testing. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0025] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0026] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0027] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0028] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0029] Example 1

[0030] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted auricularia auricula-judae powder and compound enzyme to water, and perform enzymatic hydrolysis at 50°C (ultrasonic power of 200W, frequency of 40kHz) for 4 hours. Inactivate the enzyme at 90°C for 10 minutes, centrifuge, and obtain the supernatant. The mass ratio of cellulase to papain is 1:0.4. The material-liquid ratio of defatted auricularia auricula-judae powder, compound enzyme, and water is 1g:0.01g:10mL.

[0031] (3) The supernatant was ultrafiltered sequentially through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0032] The molecular weight of the golden ear polypeptide prepared in Example 1 was determined as follows: Table 1

[0033] Example 2

[0034] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted auricularia auricula-judae powder and compound enzyme to water, and perform enzymatic hydrolysis at 50°C (ultrasonic power of 200W, frequency of 40kHz) for 4 hours. Inactivate the enzyme at 90°C for 10 minutes, centrifuge, and obtain the supernatant. The mass ratio of cellulase to papain is 1:0.6. The material-to-liquid ratio of defatted auricularia auricula-judae powder, compound enzyme, and water is 1g:0.01g:10mL.

[0035] (3) The supernatant was ultrafiltered sequentially through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0036] Example 3

[0037] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted golden ear powder and compound enzyme to water, and use ultrasound (ultrasound power of 200W, frequency of 40kHz) at 50℃ to assist enzymatic hydrolysis for 4h, inactivate enzyme at 90℃ for 10min, centrifuge to obtain supernatant; the mass ratio of cellulase and papain is 1:1.

[0038] The ratio of defatted golden ear powder, compound enzyme, and water is 1g:0.01g:10mL.

[0039] (3) The supernatant was ultrafiltered sequentially through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0040] Example 4

[0041] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted golden ear powder and compound enzyme to water, and use ultrasound (ultrasound power of 200W, frequency of 40kHz) at 50℃ to assist enzymatic hydrolysis for 4h, inactivate enzyme at 90℃ for 10min, centrifuge to obtain supernatant; the mass ratio of cellulase and papain is 1:1.

[0042] The ratio of defatted golden ear powder, compound enzyme, and water is 1g:0.01g:8mL.

[0043] (3) The supernatant was ultrafiltered sequentially through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0044] Example 5

[0045] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted golden ear powder and compound enzyme to water, and use ultrasound (ultrasound power of 200W, frequency of 40kHz) at 50℃ to assist enzymatic hydrolysis for 4h, inactivate enzyme at 90℃ for 10min, centrifuge to obtain supernatant; the mass ratio of cellulase and papain is 1:1.

[0046] The ratio of defatted golden ear powder, compound enzyme, and water is 1g:0.01g:20mL.

[0047] (3) The supernatant was ultrafiltered sequentially through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0048] Example 6

[0049] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted golden ear powder and compound enzyme to water, and use ultrasound (ultrasound power of 200W, frequency of 40kHz) at 50℃ to assist enzymatic hydrolysis for 4h, inactivate enzyme at 90℃ for 10min, centrifuge to obtain supernatant; the mass ratio of cellulase and papain is 1:1.

[0050] The ratio of defatted golden ear powder, compound enzyme, and water is 1g:0.01g:4mL.

[0051] (3) The supernatant was ultrafiltered sequentially through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0052] Comparative Example 1

[0053] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted auricularia auricula-judae powder and compound enzyme to water, and perform enzymatic hydrolysis at 50°C (ultrasonic power of 200W, frequency of 40kHz) for 4 hours. Inactivate the enzyme at 90°C for 10 minutes, centrifuge, and obtain the supernatant. The mass ratio of cellulase to pectinase is 1:0.4. The material-liquid ratio of defatted auricularia auricula-judae powder, compound enzyme, and water is 1g:0.01g:10mL.

[0054] (3) The supernatant was ultrafiltered sequentially with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and an ultrafiltration membrane with a molecular weight cutoff of 10000 Da. The retentate with a molecular weight cutoff of 3000~10000 Da was collected, dried, and the golden ear polypeptide was obtained.

[0055] Comparative Example 2

[0056] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted auricularia auricula-judae powder and compound enzyme to water, and perform enzymatic hydrolysis at 50°C (ultrasonic power of 200W, frequency of 40kHz) for 4 hours. Inactivate the enzyme at 90°C for 10 minutes, centrifuge, and obtain the supernatant. The mass ratio of pectinase to β-glucanase is 1:0.4. The material-to-liquid ratio of defatted auricularia auricula-judae powder, compound enzyme, and water is 1g:0.01g:10mL.

[0057] (3) The supernatant was ultrafiltered sequentially with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and an ultrafiltration membrane with a molecular weight cutoff of 10000 Da. The retentate with a molecular weight cutoff of 3000~10000 Da was collected, dried, and the golden ear polypeptide was obtained.

[0058] Comparative Example 3

[0059] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted golden ear powder and cellulase to water, and use ultrasound (ultrasound power of 200W and frequency of 40 kHz) at 50℃ to assist enzymatic hydrolysis for 4h. Inactivate enzyme at 90℃ for 10min, centrifuge, and obtain supernatant. The material-liquid ratio of defatted golden ear powder, cellulase and water is 1g:0.01g:10mL.

[0060] (3) The supernatant was ultrafiltered sequentially with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and an ultrafiltration membrane with a molecular weight cutoff of 10000 Da. The retentate with a molecular weight cutoff of 3000~10000 Da was collected, dried, and the golden ear polypeptide was obtained.

[0061] Comparative Example 4

[0062] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted golden ear powder and papain to water, and use ultrasound (ultrasound power of 200W, frequency of 40 kHz) at 50℃ to assist enzymatic hydrolysis for 4h, inactivate enzyme at 90℃ for 10min, centrifuge to obtain supernatant; the ratio of defatted golden ear powder, papain and water is 1g:0.01g:10mL.

[0063] (3) The supernatant was ultrafiltered sequentially with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and an ultrafiltration membrane with a molecular weight cutoff of 10000 Da. The retentate with a molecular weight cutoff of 3000~10000 Da was collected, dried, and the golden ear polypeptide was obtained.

[0064] Comparative Example 5

[0065] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted auricularia auricula-judae powder and compound enzyme to water, and perform enzymatic hydrolysis at 50°C (ultrasonic power of 200W, frequency of 40kHz) for 4 hours. Inactivate the enzyme at 90°C for 10 minutes, centrifuge, and obtain the supernatant. The mass ratio of cellulase to papain is 1:0.4. The material-liquid ratio of defatted auricularia auricula-judae powder, compound enzyme, and water is 1g:0.01g:10mL.

[0066] (3) The supernatant was ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the retentate was collected and dried to obtain the golden ear polypeptide.

[0067] Comparative Example 6

[0068] A method for preparing auricularia auricula polypeptide includes the following steps: (1) Grind the dried golden ear fungus into 100 mesh to obtain golden ear fungus powder; The golden ear fungus powder was extracted and defatted with petroleum ether to obtain defatted golden ear fungus powder; the solid-liquid ratio of the golden ear fungus powder to petroleum ether was 1g:5mL. (2) Add defatted auricularia auricula-judae powder and compound enzyme to water, and perform enzymatic hydrolysis at 50°C (ultrasonic power of 200W, frequency of 40kHz) for 4 hours. Inactivate the enzyme at 90°C for 10 minutes, centrifuge, and obtain the supernatant. The mass ratio of cellulase to papain is 1:0.4. The material-liquid ratio of defatted auricularia auricula-judae powder, compound enzyme, and water is 1g:0.01g:10mL.

[0069] (3) The supernatant was ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide.

[0070] Test case

[0071] 1. Yield of golden ear peptides (%) = (mass of extracted peptides / mass of raw materials) × 100%.

[0072] Folin-phenol method (Lowry method): 1.1 Preparation of Standard Curve: Prepare a 0.1 mg / mL bovine serum albumin (BSA) standard solution, and perform serial dilutions (0, 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL). Take 1 mL of each standard solution, add 5 mL of Lowry's reagent (containing basic copper reagent and Folin-phenol reagent), mix well, and let stand at room temperature for 10 minutes. Measure the absorbance (A) at 750 nm and plot the standard curve.

[0073] 1.2 Sample determination: Take 1 mL of the supernatant to be tested, process it in the same way and measure the absorbance. Calculate the peptide concentration (C, mg / mL) according to the standard curve.

[0074] 1.3 Calculation of total peptide mass: Peptide mass .

[0075]

[0076] 2. DPPH free radical scavenging rate: Tested according to T / SHRH006-2018 "Cosmetics - Experimental Method for Free Radical (DPPH) Scavenging".

[0077] 3. Moisturizing properties: 3.1 Reagents and Instruments Reagents: Auricularia auricula-judae polypeptide powder, anhydrous ammonium sulfate (simulating high humidity), and anhydrous magnesium chloride (simulating low humidity).

[0078] Instruments: constant temperature and humidity chamber, analytical balance (accuracy 0.1mg), desiccator, weighing bottle.

[0079] 3.2 Moisture retention test (low humidity environment) Pre-moisture absorption treatment: The sample was saturated by absorbing moisture at 81% humidity for 24 hours, and its weight was recorded as W1.

[0080] Moisture absorption experiment: After absorbing moisture, the sample was transferred to a desiccator containing a saturated magnesium chloride solution (humidity 33%) and placed at a constant temperature of 25°C. The sample was weighed at regular intervals (1, 3, 6, 12, and 24 hours) and the weight W2 after drying was recorded.

[0081] 3.3 Calculation of Moisturizing Rate: .

[0082] Table 2

[0083] As can be seen from Table 1, the golden ear polypeptide of the present invention has excellent moisturizing properties and antioxidant properties, and the yield of the golden ear polypeptide is high.

[0084] 4. Security Testing Human patch test (cosmetic application) 4.1. Test Method Test basis International Standards: ISO 10993-10 (Biological Evaluation of Medical Devices) or Appendix 7 of the "Cosmetic Safety Technical Specifications" (2022 Edition).

[0085] Ethical requirements: Compliance with the Declaration of Helsinki, approval by the ethics committee, and informed consent from the participants.

[0086] 4.2. Subject Selection Number of participants: 30 healthy adults (half male and half female, aged 18-60).

[0087] Exclusion criteria: history of skin diseases (such as eczema, psoriasis), allergic constitution.

[0088] There is a wound or inflammation at the test site (back or inner forearm).

[0089] Use antihistamines or immunosuppressants within 3 days prior to the test.

[0090] 4.3. Sample Preparation Test group: 5% auricularia auricula-judae polypeptide aqueous solution.

[0091] Negative control: physiological saline.

[0092] Positive control: 1% sodium dodecyl sulfate (SDS, known as a mild irritant).

[0093] Application material: FinnChambers® (8mm in diameter) or similar spot treatment device.

[0094] 4.4. Test Procedure Application: Inject 50 μL of sample, negative control, and positive control into the patch applicator and apply it to the healthy skin on the back of the subject. The patch is left in place for 48 hours.

[0095] Initial observation: 30 minutes after removing the spot tester (D0), record the immediate skin reaction (erythema, edema, etc.).

[0096] Follow-up observation: Skin reaction was assessed at 24 hours (D1), 48 hours (D2), and 72 hours (D3) after removal.

[0097] 4.5. Evaluation Criteria Skin reaction grading (International Contact Dermatitis Study Group, ICDRG criteria): Table 3

[0098] Results Interpretation: Irritation: The reaction rate in the test group was significantly higher than that in the negative control group (p<0.05).

[0099] Sensitization: Delayed erythema (appears on days 2-3) with a reaction grade ≥1.

[0100] 4.6. Data analysis: SPSS software was used to perform chi-square test (χ²) or Fisher's exact test, with a significance level of α=0.05.

[0101] 4.7. Test Results 4.7.1. Skin irritation Table 4

[0102] Conclusion: There was no significant difference between the test group and the negative control group (p>0.05), and there was no skin irritation.

[0103] Skin sensitization Table 5

[0104] Conclusion: The golden ear polypeptide described in this invention is mild and non-irritating, has good safety, no delayed erythema reaction, and is non-sensitizing.

[0105] 5. Photodamage Repair Test Detection Principle: Repair refers to helping maintain the skin in a normal state, keeping it hydrated, and reducing the irritation and damage caused by external factors. Damaged skin barriers lead to increased sensitivity to external stimuli, a major cause of erythema, dryness, peeling, and itching. In daily life, various factors such as sunlight and chemical irritants can damage the skin barrier, leading to increased skin permeability. Therefore, reducing light damage to the skin and stabilizing skin barrier function are important ways to maintain normal skin condition. Using non-specific fluorescent staining reagents, changes in zebrafish skin barrier permeability can be efficiently reflected, showing a positive correlation between the fluorescent area of ​​zebrafish embryonic skin and skin barrier permeability. Therefore, this experiment aims to construct a zebrafish embryo photodamage model, use fluorescent staining reagents to label skin permeability, and evaluate the activity of the test sample in inhibiting skin permeability by testing the red fluorescent area of ​​zebrafish skin, thereby reflecting the photodamage repair efficacy of the test sample.

[0106] 5.1 Laboratory Animals and Environment 5.1.1 Rearing conditions for zebrafish: In the rearing of adult wild-type zebrafish and in the dedicated aquaculture system, the pH range is 7.0-8.0, the conductivity range is 500-800 μS / cm, the room temperature range is (26 ℃ ± 1 ℃), the water temperature range is (28℃ ± 1 ℃), and the photoperiod is 14 h light and 10 h darkness.

[0107] 5.1.2 Embryo Preparation: On the afternoon of the day before egg collection, 3-18 month old male and female zebrafish that had already started feeding were transferred to the mating tank, and a partition was inserted. The next morning between 9:00 and 9:30, the partition in the middle of the mating tank was removed, and the embryos were collected 15 minutes later. The embryos were placed in a constant temperature incubator (28±0.5 ℃) and embryo buffer (see Table 6 for embryo buffer preparation method) was added. When the embryos reached 48 hpf, normally developing embryos were selected for the experiment.

[0108] Table 6

[0109] Add the ingredients to the measuring cups according to the table, and bring the volume to 4.5 L with ultrapure water. Filter using a vacuum filter (0.2 μM pore size), sterilize using an autoclave, and store at room temperature.

[0110] 5.2 Test Methods 5.2.1 Experimental Groups: This experiment consists of 1+1+1+1 groups, namely blank group, model group, positive group, and sample group*1.

[0111] 5.2.2 Group design: Use 6-well cell culture plates, with 1 well per group and 20 embryos per well. See Table 7.

[0112] Table 7

[0113] 5.3 Experimental Procedure: 1) Collect wild-type zebrafish embryos at 48 hpf and group them according to the group design in 4.2, with 20 embryos per group. Add 6 mL of embryo buffer to the blank group and model group, and add 6 mL of the corresponding concentration solution to the positive group and drug-treated group. Incubate in a constant temperature incubator at 28 ℃±1 ℃ for 4 h before drug administration.

[0114] 2) Except for the blank group, the rest were placed in a UV irradiation chamber and irradiated for 2.5 h.

[0115] 3) After treatment, staining was performed, and after washing, the fluorescent staining area of ​​each group of zebrafish was observed under an upright fluorescence microscope and photographed and recorded.

[0116] 4) Statistical results and data analysis.

[0117] 5.4 Data Analysis and Processing ImageJ image analysis software was used to statistically analyze the fluorescent staining area of ​​zebrafish tails. The statistical results were imported into Excel for data processing, and the mean and standard error (SEM) of each group were calculated. All results are expressed as mean ± standard error (Mean ± SEM). GraphPad Prism 8 statistical analysis software was used to analyze the data, and differences between different groups were analyzed using a t-test. Generally, P < 0.05 (*) was considered statistically significant.

[0118] 5.5 Test Results The efficacy of zebrafish samples in resisting light damage is shown in the following figures. Figure 1 , Figure 2 See Table 8.

[0119] Table 8

[0120] Note: Comparison between the sample and the model group: **** (P<0.0001) shows a highly significant difference; *** (P<0.001) shows a significant difference; ** (P<0.01) shows a significant difference; * (P<0.05) shows a significant difference; Comparison between the blank and the model group: #### (P<0.0001) shows a highly significant difference; ### (P<0.001) shows a significant difference; ## (P<0.01) shows a significant difference; # (P<0.05) shows a significant difference.

[0121] 5.6 Experimental Conclusions Based on laboratory standards, the results of this experiment show that, under the experimental conditions, a concentration of 2 g / L has an inhibitory effect on photodamage in zebrafish compared to the model group, indicating that the sample has a certain improvement effect on the damage caused by UV light.

[0122] In summary, the golden ear polypeptide sample of the present invention has the effect of resisting light damage.

[0123] 6. Anti-inflammatory experiment of zebrafish Detection Principle: Neutrophils in zebrafish embryos are highly similar to human neutrophils in morphology, biochemistry, and physiological function. Neutrophils are the first white blood cells to appear at sites of injury or pathogen invasion, playing a role in clearing infection or harmful substances. A model of neutrophil aggregation induced by copper sulfate-induced damage to neurothalamic cells in the lateral line region of zebrafish embryos was used for testing. The changes in the number of neutrophils in the lateral line region of the embryos in the test substance treatment group and the model control group were compared, and the neutrophil count was calculated to evaluate the anti-inflammatory efficacy of the raw material, formulation, or product.

[0124] 6.1 Laboratory Animals and Environment 6.1.1 Rearing conditions for zebrafish: In the rearing of adult wild zebrafish and in a dedicated aquaculture system, the pH range is 7.0-8.0, the conductivity range is 500-800 μS / cm, the room temperature range is 26 ℃ ± 1 ℃, the water temperature range is 28 ℃ ± 1 ℃, and the photoperiod is 14 h light and 10 h darkness.

[0125] 6.2 Embryo Preparation: On the afternoon of the day before egg collection, feed-ready 3-18 month old male and female zebrafish were transferred to the mating tank, and a partition was inserted. The next morning between 9:00 and 9:30, the partition in the middle of the mating tank was removed, and the embryos were collected 15 minutes later. The embryos were placed in a constant temperature incubator (28±0.5 ℃) and embryo buffer (see Table 9 for embryo buffer preparation method) was added. When the embryos reached 72 hpf, normally developing embryos were selected for the experiment.

[0126] Table 9

[0127] Add the ingredients to the measuring cups according to the table, and bring the volume to 4.5 L with ultrapure water. Filter using a vacuum filter (0.2 μM pore size), sterilize using an autoclave, and store at room temperature.

[0128] 6.3 Experimental Methods: This experiment consisted of 1+1+1+3 groups, namely, blank group, model group, positive group, and different sample groups*3.

[0129] 6.4 Group design: Use 6-well cell culture plates, with 1 well per group and 20 embryos per well. See Table 10.

[0130] Table 10

[0131] 6.5 Experimental Procedure: 1) Collect wild-type zebrafish embryos at 72 hpf and group them according to the group design in 4.2, with 20 embryos per group. Add 6 mL of embryo buffer to the blank group and the model group, and add 6 mL of the corresponding concentration solution to the positive group and the drug treatment group. Incubate them in a constant temperature incubator at 28 ℃±1 ℃ for 2 h after drug treatment, and then switch to copper sulfate for 2 h for modeling.

[0132] 2) Fish embryo fixation and staining procedure.

[0133] 3) After processing, observe the neutrophils of each group of zebrafish using an upright fluorescence microscope and take photos for recording.

[0134] 4) Statistical results and data analysis.

[0135] 6.6 Data Analysis and Processing ImageJ image analysis software was used to count the number of neutrophils in the caudal fin of zebrafish. The statistical results were imported into Excel for data processing, and the mean and standard error (SEM) of each group were calculated. All results are expressed as mean ± standard error (Mean ± SEM). GraphPad Prism 8 statistical analysis software was used to analyze the data, and differences between different groups were analyzed using a t-test. Generally, P < 0.05 (*) was considered statistically significant.

[0136] 6.7 Test Results The anti-inflammatory efficacy of the zebrafish samples can be found in the following figures. Figure 3 , Figure 4 See Table 11.

[0137] Table 11

[0138] Note: Compared with the model group, **** (P<0.0001) showed a highly significant difference; *** (P<0.001) showed a significant difference; ** (P<0.01) showed a significant difference; * (P<0.05) showed a significant difference; Compared with the model group, #### (P<0.0001) showed a highly significant difference; ### (P<0.001) showed a significant difference; ## (P<0.01) showed a significant difference; # (P<0.05) showed a significant difference.

[0139] 6.8 Experimental Conclusions In this experiment, based on laboratory standards, the results showed that at concentrations of 2 g / L, 4 g / L, and 8 g / L, the sample inhibited neutrophil migration in zebrafish compared to the model group, indicating that the sample had a certain ameliorative effect on the inflammatory response induced by copper sulfate. In conclusion, the *Gynostemma pentaphyllum* polypeptide described in this invention has anti-inflammatory effects.

[0140] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing auricularia auricula-judae polypeptide, characterized in that, Includes the following steps: (1) Degrease the golden ear powder with petroleum ether to obtain defatted golden ear powder; (2) Add defatted golden ear powder and compound enzyme to water, use ultrasound to assist enzymatic hydrolysis, inactivate enzyme, centrifuge, and obtain supernatant; (3) The supernatant was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, the filtrate was collected and dried to obtain the golden ear polypeptide; The complex enzyme includes cellulase and papain.

2. The method for preparing auricularia auricula-judae polypeptide according to claim 1, characterized in that, The particle size of the golden ear powder is 80-100 mesh.

3. The method for preparing auricularia auricula-judae polypeptide according to claim 1, characterized in that, The solid-liquid ratio of the auricularia auricula powder and petroleum ether is 1g:(2~10)mL.

4. The method for preparing auricularia auricularia polypeptide according to claim 1, characterized in that, The mass ratio of cellulase to papain is 1:(0.4~0.6).

5. The method for preparing auricularia auricula-judae polypeptide according to claim 1, characterized in that, The ratio of defatted golden ear powder, compound enzyme, and water is 1g:(0.005~0.02)g:(4~20)mL.

6. The method for preparing auricularia auricularia polypeptide according to claim 1, characterized in that, The enzymatic hydrolysis temperature is 45~55℃, and the enzymatic hydrolysis time is 2~6h.

7. The method for preparing auricularia auricula-judae polypeptide according to claim 1, characterized in that, The ultrasonic power is 200~300W and the frequency is 30~50kHz.

8. The method for preparing auricularia auricularia polypeptide according to claim 1, characterized in that, The enzyme inactivation temperature is 80~90℃, and the time is 5~20min.

9. A golden ear polypeptide, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

10. The application of the auricular polypeptide according to claim 9 in the preparation of cosmetics.

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