A five-black-chicken precise active peptide with improved uterine health and a preparation method and functional product thereof

Abstract

The application relates to the technical field of five black chicken active peptide preparation, in particular to five black chicken precise active peptide with the function of improving uterine health, a preparation method thereof and a functional product. The preparation method is as follows: taking five black chickens as raw materials, the five black chickens are subjected to medium-temperature pretreatment, step-by-step enzymolysis by using alkaline protease 37071 and papain, decolorization and deodorization, centrifugal filtration and drying process, and five black chicken precise active peptide is prepared. The five black chicken precise active peptide prepared by the application can realize multi-target synergistic effect, improve irregular menstruation and repair endometrial receptivity, and also has the functions of efficiently removing free radicals, reducing the content of inflammation-related factors and promoting the expression of anti-apoptosis genes. The 9 peptide segments in the active peptide combination screened by the application are all reported for the first time, the non-toxic active peptide segments have the functions of precisely and efficiently improving uterine health. In addition, the application scheme is suitable for large-scale production, the active ingredients prepared have high natural safety, and can be applied to functional products as functional factors.

Description

Technical Field

[0001] This application relates to the field of Wuhai chicken active peptide preparation technology, and in particular to a Wuhai chicken precise active peptide that improves uterine health, its preparation method, and functional products. Background Technology

[0002] With economic development, women's fast-paced lifestyles, unbalanced diets, and environmental influences have exacerbated uterine health problems, leading to decreased endometrial receptivity, inflammation, and damage. The uterus is crucial for women's reproductive health; its dysfunction not only affects fertility but also contributes to menstrual disorders and gynecological inflammation. Currently, uterine health has become a significant factor hindering the improvement of women's reproductive health and quality of life. Wuhai chicken (scientific name: Black-feathered Silkie chicken, *Gallus gallus domesticus*) is a traditional Chinese medicinal and edible poultry breed, rich in high-quality small-molecule active peptides and other nutrients. Precise active peptides from Wuhai chicken, extracted through targeted enzymatic hydrolysis, can improve uterine health from multiple targets, specifically:

[0003] 1) Optimizes endometrial receptivity: Increases uterine LIF content, upregulates HOXA10 and ITGβ3 expression, improves endometrial morphology, enhances embryo adhesion, and creates a favorable environment for conception; 2) Regulates immune and anti-inflammatory effects: Inhibits the expression of pro-inflammatory factors such as TNF-α and IL-1β, upregulates the content of the anti-inflammatory factor IL-10, and maintains endometrial immune tolerance; 3) Regulates cell apoptosis and repair: Reduces Bax levels, increases Bcl-2 content, inhibits excessive cell apoptosis, and enhances endometrial repair capacity. Wuhai chicken active peptides, through multi-target synergy, regulate uterine physiological function and improve reproductive endocrine function, providing a natural intervention solution for women's uterine health.

[0004] Patent application CN103589772A discloses a method for extracting Silkie chicken polypeptides using ultrasound. The core technology employs ultrasound-assisted enzymatic hydrolysis, optimizing ultrasound parameters and enzymatic hydrolysis conditions to achieve efficient degradation of Silkie chicken egg white. This method offers advantages such as short production cycle, complete preservation of active ingredients, and high purity; however, it lacks functional validation and targeted research. Existing Silkie chicken polypeptide products for improving uterine health suffer from weak component targeting and a single mechanism of action, necessitating the development of efficient, safe, and multi-target active peptide preparation processes based on Silkie chicken, along with functional validation and targeted research. Summary of the Invention

[0005] To address the shortcomings of the prior art mentioned in the background section, this invention provides a precise active peptide from Wuhai chicken that improves uterine health, its preparation method, and a functional product thereof. The technical solution for preparing this precise active peptide from Wuhai chicken that improves uterine health is as follows: This application provides a method for preparing precise active peptides from Wuhai chicken, which includes the following steps: S1. Pretreatment: After removing the internal organs, mince the black-boned chicken into a paste, add water and mix, and heat-treat at 54-56℃ for 0.5-1.5 hours to obtain a pre-hydrolyzed solution. S2. First hydrolysis: Adjust the pH of the pre-hydrolysate, add alkaline protease, and hydrolyze at 50-60℃ for 1-3 hours. After inactivation of the enzyme, the first hydrolysate is obtained. The alkaline protease is alkaline protease 37071, and its addition amount is 0.5% to 1.5% of the weight of the Wuhai chicken. S3. Second hydrolysis: Adjust the pH of the first hydrolysate, add papain, and hydrolyze at 45-65℃ for 1.5-2.5 hours. After enzyme inactivation, the second hydrolysate is obtained. The amount of papain added is 0.25-0.75% of the weight of the Wuhai chicken. S4, Post-processing: After the second hydrolysate is decolorized and deodorized, and solid-liquid separated, the supernatant is taken and subjected to filtration, ultrafiltration, nanofiltration desalination and concentration and spray drying to obtain powdered Wuhai chicken precise active peptides. Among them, the molecular weight cutoff of ultrafiltration is <5000Da, and the mass content of the concentrated Wuhai chicken precise active peptides is 10-25%.

[0006] In some embodiments, the alkaline protease 37071 has an enzyme activity of 0.91 to 2.73 million U / g based on the unit mass of protein in Wuhai chicken; and the papain has an enzyme activity of 0.23 to 0.69 million U / g.

[0007] In some embodiments, in step S2, the pH of the pre-hydrolysate is adjusted to 8.0–9.0, alkaline protease 37071 is added, and the mixture is enzymatically hydrolyzed at 50–60°C for 1–3 hours. After enzyme inactivation, the first hydrolysate is obtained.

[0008] In some embodiments, in step S3, the pH of the first hydrolysate is adjusted to 6.0–7.0, papain is added, and the mixture is hydrolyzed at 45–65°C for 1.5–2.5 hours. After enzyme inactivation, a second hydrolysate is obtained.

[0009] In some embodiments, in step S2, the enzyme is inactivated at 84–86°C for 15–20 min to obtain a first hydrolysate. In some embodiments, in step S3, the enzyme is inactivated at 84–86°C for 15–20 min to obtain a second hydrolysate. In some embodiments, in step S1, fresh Wuhai chicken is gutted, minced into a paste, mixed with water, and heat-treated at 54–56°C for 0.5–1.5 h to obtain a pre-hydrolysate; wherein the mass ratio of the minced meat to water is 1:3 to 1:5.

[0010] In some embodiments, in step S4, after the second hydrolysate is decolorized and deodorized by activated carbon and separated by centrifugation, the supernatant is taken and treated by diatomaceous earth decolorization, filtration, ultrafiltration, nanofiltration desalination and concentration and spray drying to obtain powdered Wuhai chicken precise active peptides.

[0011] In some embodiments, in step S4, the second hydrolysate is cooled to 50-60°C, activated carbon is added and treated for 0.5-1.5 hours, centrifuged, and the supernatant is decolorized with diatomaceous earth and filtered through a 0.45μm aqueous membrane; wherein the amount of activated carbon added is 5.0-7.0% of the mass of the black-boned chicken.

[0012] In some embodiments, the centrifugation conditions are 5000 rpm for 5 to 10 minutes.

[0013] This application also provides a precise active peptide for black-boned chicken, which is prepared by the method described above.

[0014] In some embodiments, the Wuhai chicken precise active peptide is a composition comprising active peptide segments with a relative molecular weight <5000 Da; it includes at least one active peptide segment with the amino acid sequence Leu-Pro-Gln-Pro-Pro-Gln (LPQPPQ), Ile-Pro-Phe (IPF), Leu-Pro-Phe (LPF), Leu-Gly-Pro-Leu (LGPL), Leu-Gly-Pro-Ile (LGPI), Ile-Gly-Pro-Leu (IGPL), Ala-Val-Phe (AVF), Phe-Pro-Pro-Asp-Val-Ala (FPPDVA), and Met-Pro-Phe (MPF).

[0015] This application also provides a functional product whose active ingredients include the Wuhai chicken precise active peptides as described above.

[0016] In some embodiments, the functional product has at least one of the following functions: (1) Anti-inflammatory; (2) Improves menstrual irregularities; (3) Repairs endometrial receptivity.

[0017] Based on the above, compared with the prior art, the present invention has the following beneficial effects: The precise active peptides from Wuhai chicken obtained in this application have significant effects on improving uterine health. They work by significantly scavenging DPPH and ABTS free radicals, increasing / restoring LIF content in human endometrial stromal cells, decreasing TNF-α secretion in human endometrial stromal cells, promoting IL-10 secretion in human endometrial stromal cells, promoting the relative mRNA expression levels of LIF, ITGβ3, HOXA10, and Bcl-2, and inhibiting the relative mRNA expression levels of TNF-α, IL-1β, and Bax, thus collectively improving uterine health. This peptide can be used as a functional factor in functional products that improve uterine health. Furthermore, the method used in this application employs stepwise enzymatic hydrolysis and conventional separation techniques, which have advantages such as low equipment requirements, simple and easy operation, and high extraction efficiency, making it suitable for industrial production. This application method has excellent application and economic prospects.

[0018] In summary, the Wuhai chicken precise active peptides obtained in this application exhibit multi-target synergistic effects, improving menstrual irregularities and repairing endometrial receptivity. They also possess highly efficient free radical scavenging capabilities, reduce the content of inflammation-related factors, and promote the expression of anti-apoptotic genes. Of the nine peptide segments screened in this application, five are previously unreported. These non-toxic active peptides demonstrate precise and efficient improvement in uterine health. Furthermore, the process described in this application is simple and easy to control, requires minimal equipment, and is suitable for large-scale production. The resulting active ingredients are derived from natural sources, ensuring high safety. They can be used as functional factors in functional products, providing a safe and diverse intervention solution for endometrial receptivity and related inflammatory issues. Attached Figure Description

[0019] Figure 1 Graph showing the effect of different concentrations of Wuhai chicken precise active peptides on the viability of human endometrial stromal cells in the examples and comparative examples; Figure 2 A graph showing the LIF content data of human endometrial stromal cells induced by H2O2-induced damage from the precise active peptides of Wuhai chicken. Figure 3 A graph showing the effect of precise active peptides from Wuhai chicken on TNF-α content in human endometrial stromal cells damaged by H2O2. Figure 4 A graph showing the effect of precise active peptides from Wuhai chicken on IL-10 levels in human endometrial stromal cells damaged by H2O2. Figure 5 A graph showing the effect of Wuhai chicken's precise active peptides on the relative expression level of LIF mRNA in H2O2-induced damaged human endometrial stromal cells; Figure 6 A graph showing the effect of Wuhai chicken's precise active peptides on the relative expression level of ITGβ3 mRNA in H2O2-induced damaged human endometrial stromal cells; Figure 7 A graph showing the effect of Wuhai chicken's precise active peptides on the relative expression level of HOXA10 mRNA in human endometrial stromal cells induced by H2O2 damage; Figure 8A graph showing the effect of Wuhai chicken's precise active peptides on the relative expression level of Bax mRNA in H2O2-induced damaged human endometrial stromal cells; Figure 9 A graph showing the effect of Wuhai chicken's precise active peptides on the relative expression level of Bcl-2 mRNA in human endometrial stromal cells induced by H2O2 damage; Figure 10 The graph shows the effect of Wuhai chicken's precise active peptides on the relative expression level of TNF-α mRNA in human endometrial stromal cells induced by H2O2. Figure 11 A graph showing the effect of Wuhai chicken's precise active peptides on the relative expression level of IL-1β mRNA in H2O2-induced damaged human endometrial stromal cells; Figure 12 , Figure 15 , Figure 18 , Figure 21 , Figure 24 , Figure 27 , Figure 30 , Figure 33 , Figure 36 3D diagrams showing the docking results of LPQPPQ, IPF, LPF, LGPL, LGPI, IGPL, AVF, FPPDVA, MPF, and LIF receptor proteins (ID: 3E0G), respectively. Figure 13 , Figure 16 , Figure 19 , Figure 22 , Figure 25 , Figure 28 , Figure 31 , Figure 34 , Figure 37 3D diagrams showing the docking results of LPQPPQ, IPF, LPF, LGPL, LGPI, IGPL, AVF, FPPDVA, MPF, and ITGβ3 receptor protein (ID: 6NAJ). Figure 14 , Figure 17 , Figure 20 , Figure 23 , Figure 26 , Figure 29 , Figure 32 , Figure 35 , Figure 38 3D diagrams showing the docking results of LPQPPQ, IPF, LPF, LGPL, LGPI, IGPL, AVF, FPPDVA, MPF, and TNF-α receptor protein (ID: 2AZ5), respectively. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to represent selected embodiments of this application.

[0021] Example 1 1. Take 500g of black-boned chicken (protein content is about 22%, i.e., 110g; the protein content of black-boned chicken is measured by the Kjeldahl method, the same below), remove the internal organs, mince the meat, mix it evenly with 2000g of water, and then heat it at a medium temperature of 54-56℃ for 1.0h to obtain a pre-hydrolyzed solution.

[0022] The mass ratio of the black chicken to water is 1:4. 2. Adjust the pH of the pre-hydrolysate system to 8.5, add 5.0g of alkaline protease (37071), hydrolyze at 55℃ for 2h, and then inactivate the enzyme at 85℃ for 15min to obtain the first hydrolysate.

[0023] The alkaline protease (37071) added was 1.0% of the weight of the Wuhai chicken.

[0024] 3. Adjust the pH of the first hydrolysate system to 6.5, add 2.5g of papain, hydrolyze at 55℃ for 2h, and then inactivate the enzyme at 85℃ for 15min to obtain the second hydrolysate; The amount of papain added was 0.5% of the weight of the Wuhai chicken. 4. Add 30.0g of activated carbon to the second hydrolysate and decolorize and deodorize at 55℃ for 1h to obtain a decolorized and deodorized hydrolysate; The activated carbon added is 6.0% of the weight of the black-boned chicken.

[0025] 5. After centrifugation to remove residue from the decolorized and deodorized hydrolysate, take the supernatant, decolorize it with fine diatomaceous earth and filter it through a 0.45μm aqueous membrane to obtain a clear hydrolysate; The centrifugation process involves a centrifugation speed of 5000 rpm and a centrifugation time of 5 min.

[0026] 6. The clarified hydrolysate (containing Wuhai chicken precise active peptides and amino acids) is subjected to ultrafiltration; the clarified hydrolysate is subjected to ultrafiltration to filter out Wuhai chicken precise active peptide solution with a molecular weight of less than 5000 Da; the Wuhai chicken precise active peptide solution is desalted and concentrated by nanofiltration to a mass content of 10-25% Wuhai chicken precise active peptide.

[0027] 7. Spray dry the concentrated liquid to make it into powder, thus obtaining the powdered Five Black Chicken Precision Active Peptide.

[0028] Example 2 1. Take 500g of black-boned chicken (protein content approximately 22%, or 110g), remove the internal organs, mince it into a paste, mix it evenly with 1500g of water, and then subject it to medium-temperature heat treatment at 54-56℃ for 0.5h to obtain a pre-hydrolyzed solution. The mass ratio of black-boned chicken to water is 1:3.

[0029] 2. Adjust the pH of the pre-hydrolysate system to 8.0, add 2.5g of alkaline protease (37071), hydrolyze at 50℃ for 1h, and then inactivate the enzyme at 84℃ for 15min to obtain the first hydrolysate.

[0030] The alkaline protease (37071) was added at a rate of 0.5% of the weight of the black-boned chicken.

[0031] 3. Adjust the pH of the first hydrolysate system to 6.0, add 1.25g of papain, hydrolyze at 45℃ for 1.5h, and then inactivate the enzyme at 84℃ for 15min to obtain the second hydrolysate; The amount of papain added was 0.25% of the weight of the Wuhai chicken. 4. Add 25.0g of activated carbon to the second hydrolysate and decolorize and deodorize at 55℃ for 0.5h to obtain a decolorized and deodorized hydrolysate; The amount of activated carbon added is 5.0% of the weight of the black-boned chicken.

[0032] 5. After centrifugation to remove residue from the decolorized and deodorized hydrolysate, take the supernatant, decolorize it with fine diatomaceous earth and filter it through a 0.45μm aqueous membrane to obtain a clear hydrolysate; The centrifugation process involves a centrifugation speed of 5000 rpm and a centrifugation time of 5 min.

[0033] 6. The clarified hydrolysate (containing Wuhai chicken precise active peptides and amino acids) is subjected to ultrafiltration; the clarified hydrolysate is subjected to ultrafiltration to filter out Wuhai chicken precise active peptide solution with a molecular weight of less than 5000 Da; the Wuhai chicken precise active peptide solution is desalted and concentrated by nanofiltration to a mass content of 10-25% Wuhai chicken precise active peptide.

[0034] 7. Spray dry the concentrated liquid to make it into powder, thus obtaining the powdered Five Black Chicken Precision Active Peptide.

[0035] Example 3 1. Take 500g of black-boned chicken (protein content of about 22%, i.e., 110g), remove the internal organs and mince it into meat paste. Mix it evenly with 2500g of water, and then heat it at a medium temperature of 54-56℃ for 1.5h to obtain a pre-hydrolyzed solution.

[0036] The mass ratio of the black-boned chicken to water is 1:5; 2. Adjust the pH of the pre-hydrolysate system to 9.0, add 7.5g of alkaline protease (37071), hydrolyze at 60℃ for 3h, and then inactivate the enzyme at 86℃ for 20min to obtain the first hydrolysate.

[0037] The alkaline protease (37071) was added at a rate of 1.5% of the weight of the black-boned chicken.

[0038] 3. Adjust the pH of the first hydrolysate system to 7.0, add 3.75g of papain, hydrolyze at 65℃ for 2.5h, and then inactivate the enzyme at 86℃ for 20min to obtain the second hydrolysate; The amount of papain added is 0.75% of the weight of the Wuhai chicken.

[0039] 4. Add 35.0g of activated carbon to the second hydrolysate and decolorize and deodorize at 55℃ for 0.5h to obtain a decolorized and deodorized hydrolysate; The amount of activated carbon added is 7.0% of the weight of the black-boned chicken.

[0040] 5. After centrifugation to remove residue from the decolorized and deodorized hydrolysate, take the supernatant, decolorize it with fine diatomaceous earth and filter it through a 0.45μm aqueous membrane to obtain a clear hydrolysate; The centrifugation process involves a centrifugation speed of 5000 rpm and a centrifugation time of 5 min.

[0041] 6. The clarified hydrolysate (containing Wuhai chicken precise active peptides and amino acids) is subjected to ultrafiltration; the clarified hydrolysate is subjected to ultrafiltration to filter out Wuhai chicken precise active peptide solution with a molecular weight of less than 5000 Da; the Wuhai chicken precise active peptide solution is desalted and concentrated by nanofiltration to a mass content of 10-25% Wuhai chicken precise active peptide.

[0042] 7. Spray dry the concentrated liquid to make it into powder, thus obtaining the powdered Five Black Chicken Precision Active Peptide.

[0043] Comparative Example 1 In Comparative Example 1, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. Alkaline protease (37071) and neutral protease were used in the hydrolysis process. The specific operating steps were largely the same as in Example 1, except that in step 3, the 0.5% papain was replaced with 1.28% neutral protease, and the pH value was set to the optimal pH value for the selected enzyme (1.28% neutral protease). The remaining operations and processes were the same as in Example 1.

[0044] It should be noted that, based on the protein content of Wuhai chicken, the total amount of alkaline protease (37071) and neutral protease used in Comparative Example 1 is equal to the total amount of alkaline protease (37071) and papain used in Example 1. The specific calculation process is as follows: The amount of black-boned chicken added is 500g, with a protein content of 22%, which is 110g.

[0045] In Example 1, the amount of alkaline protease (37071) added was 5.0g, with an enzyme activity of 400,000 U / g. Converted to the enzyme activity per unit weight of Wuhai chicken, this is 400,000 U / g × 5.0g / 110g = 18,200 U / g. Similarly, the amount of papain added was 2.5g, with an enzyme activity of 150,000 U / g. Converted to the enzyme activity per unit weight of Wuhai chicken egg white, this is 150,000 U / g × 2.5g / 110g = 4,600 U / g. Therefore, in Example 1, based on the protein content of Wuhai chicken, the total amount of alkaline protease (37071) and papain used was 22,800 U / g.

[0046] In Comparative Example 1, the amount of alkaline protease (37071) added was 5.0g. Based on the protein content of Wuhai chicken, the amount of alkaline protease (37071) used was 400,000 U / g × 5.0g / 110g = 18,200 U / g. The amount of neutral protease added was 6.4g, with an enzyme activity of 80,000 U / g. Based on the protein content of Wuhai chicken, the amount of neutral protease used was 80,000 U / g × 6.4g / 110g = 4,600 U / g. Therefore, in Comparative Example 1, based on the protein content of Wuhai chicken, the total amount of alkaline protease (37071) and neutral protease used was 22,800 U / g, which is the same as the total amount of alkaline protease (37071) and acidic protease used in Example 1.

[0047] Comparative Example 2 In Comparative Example 2, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. Neutral protease and papain were used in the hydrolysis process. The specific operating steps were largely the same as in Example 1, except that in step 2, the 1.0% alkaline protease (37071) was replaced with 1.3% neutral protease, and in step 3, the 0.5% papain was replaced with 2.0% papain. The pH value was set to the optimal pH value for the selected enzymes. The remaining operations and processes were the same as in Example 1.

[0048] In Comparative Example 2, the amount of neutral protease added was 6.5g, with an enzyme activity of 80,000 U / g. Based on the protein content of Wuhai chicken, the amount of neutral protease used was 80,000 U / g × 6.5g / 110g = 4,700 U / g. Similarly, the amount of papain added was 10g, with an enzyme activity of 200,000 U / g. Based on the protein content of Wuhai chicken, the amount of papain used was 200,000 U / g × 10g / 110g = 18,200 U / g. Therefore, in Comparative Example 2, based on the protein content of Wuhai chicken, the total amount of neutral protease and papain used was 22,900 U / g.

[0049] Comparative Example 3 In Comparative Example 3, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. During hydrolysis, a compound lipase and alkaline protease (37071) were used. The specific operating steps were largely the same as in Example 1, except that in step 2, the 1.0% alkaline protease (37071) was replaced with 2.0% compound lipase, and in step 3, the 0.5% papain was replaced with 1.0% alkaline protease (37071). The pH value was set to the optimal pH value for the selected enzymes. All other operations and processes were the same as in Example 1.

[0050] In Comparative Example 3, the amount of compound lipase added was 10g, with an enzyme activity of 50,000 U / g. Based on the protein content of Wuhai chicken, the amount of neutral protease used was 50,000 U / g × 10g / 110g = 4,600 U / g. Similarly, the amount of alkaline protease (37071) added was 5g, with an enzyme activity of 400,000 U / g. Based on the protein content of Wuhai chicken, the amount of alkaline protease (37071) used was 400,000 U / g × 5g / 110g = 18,200 U / g. Therefore, in Comparative Example 3, based on the protein content of Wuhai chicken, the total amount of neutral protease and papain used was 22,800 U / g.

[0051] Comparative Example 4 In Comparative Example 4, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. Trypsin and pepsin were used in the hydrolysis process. The specific operating steps were largely the same as in Example 1, except that in step 2, the amount of alkaline protease (37071) added was changed from 1.0% to 1.0% trypsin, and in step 3, the amount of papain added was changed from 0.5% to 2.0% pepsin. The pH value was set to the optimal pH value of the selected enzymes. The remaining operations and processes were the same as in Example 1.

[0052] In Comparative Example 4, the amount of trypsin added was 10g, with an enzyme activity of 250,000 U / g. Based on the protein content of Wuhai chicken, the amount of trypsin used was 250,000 U / g × 10g / 110g = 22,700 U / g. Similarly, the amount of pepsin added was 5g, with an enzyme activity of 3,000 U / g. Based on the protein content of Wuhai chicken, the amount of pepsin used was 3,000 U / g × 5g / 110g = 136 U / g. Therefore, in Comparative Example 4, based on the protein content of Wuhai chicken, the total amount of neutral protease and papain used was 22,800 U / g.

[0053] Comparative Example 5 In Comparative Example 5, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. During hydrolysis, alkaline protease (Alcalase 2.4 L) and acidic protease were used. The specific operating steps were largely the same as in Example 1, except that in step 2, the amount of alkaline protease (37071) added was changed from 1.0% to 2.0%; and in step 3, the amount of papain added was changed from 0.5% to 0.7%; the pH value was set to the optimal pH value for the selected enzymes. The remaining operations and processes were the same as in Example 1.

[0054] In Comparative Example 5, the amount of alkaline protease (Alcalase 2.4 L) added was 10 g, with an enzyme activity of 200,000 U / g. Based on the protein content of Wuhai chicken, the amount of alkaline protease used was 200,000 U / g × 10 g / 110 g = 18,200 U / g. Similarly, the amount of acidic protease added was 3.5 g, with an enzyme activity of 150,000 U / g. Based on the protein content of Wuhai chicken, the amount of acidic protease used was 150,000 U / g × 3.5 g / 110 g = 4,700 U / g. Therefore, in Comparative Example 5, based on the protein content of Wuhai chicken, the total amount of neutral protease and papain used was 22,900 U / g.

[0055] Comparative Example 6 In Comparative Example 6, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. Alkaline protease (37071) was used during hydrolysis. The specific operating steps were largely the same as in Example 1, except that in step 2, the amount of alkaline protease (37071) added was changed from 1.0% to 1.26%, the hydrolysis time was changed from 2h to 4h, the third step was omitted, and the pH value was set to the optimal pH value of the selected enzyme. All other operations were the same as in Example 1.

[0056] In Comparative Example 6, the amount of alkaline protease added was 6.3g, and the enzyme activity was 400,000 U / g. Based on the protein content of Wuhai chicken, the amount of alkaline protease used was 40U / g × 6.3g / 110g = 22,900 U / g. Therefore, in Comparative Example 6, based on the protein content of Wuhai chicken, the total amount of alkaline protease 37071 used was 22,900 U / g.

[0057] Comparative Example 7 In Comparative Example 7, the protein content of 500g of Wuhai chicken was approximately 22%, or 110g. Papain was used in the hydrolysis process, and the specific operating steps were largely the same as in Example 1, except that in step 3, the amount of papain added was changed from 0.5% to 2.5%, the hydrolysis time was changed from 2h to 4h, the second step was omitted, and the pH value was set to the optimal pH value of the selected enzyme. The remaining operations and processes were the same as in Example 1.

[0058] In Comparative Example 7, the amount of papain added was 12.5g, and the enzyme activity was 200,000 U / g. Based on the protein content of Wuhai chicken, the amount of papain used was 20U / g × 12.5g / 110g = 22,700 U / g. Therefore, in this comparative example, based on the protein content of Wuhai chicken, the total amount of neutral protease and papain used was 22,700 U / g.

[0059] The enzyme raw materials used in the above embodiments and comparative examples are: Neutral protease (model: 0.8L): enzyme activity 80,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Papain: enzyme activity 200,000 U / g, Nanning Pangbo Bioengineering Co., Ltd.; Trypsin: enzyme activity 250,000 U / g, Nanning Pangbo Bioengineering Co., Ltd.; Complex lipase: enzyme activity 50,000 U / g, Dongheng Huadao Biotechnology Co., Ltd.; Alkaline protease (model: 37071): enzyme activity 400,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Alkaline protease (model: Alcalase 2.4L): enzyme activity 200,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Acidic protease: enzyme activity 150,000 U / g, Shandong Longket Enzyme Preparation Co., Ltd.; Pepsinase: enzyme activity 3,000 U / g, Shanghai Yuanju Biotechnology Co., Ltd.

[0060] U / g refers to the enzyme activity per gram of enzyme (used to characterize the enzyme's ability to catalyze a specific chemical reaction). This differs in meaning from the unit U / g used to calculate the enzyme addition amount per gram of black-boned chicken protein content (%). The enzymes with the same name used in the above examples and comparative examples are from the same commercially available company and brand; that is, the enzyme activity of the enzymes with the same name used in each example and comparative example is consistent.

[0061] Test Results The performance of the Wuhai chicken precise active peptide products prepared in the above examples and comparative examples was tested: 1. In vitro antioxidant activity assay 1. Determination of the DPPH and ABTS free radical scavenging rate of Wuhai chicken's precise active peptides Under normal conditions, DPPH and ABTS free radicals participate in the redox regulation of uterine tissue, playing a crucial role in maintaining physiological functions such as uterine muscle contraction and endometrial cycle repair. When the uterus faces oxidative stress, chronic inflammation, or endometrial damage, it produces a large number of free radicals. Excessive free radicals attack the biomembrane and DNA of uterine tissue, causing lipid oxidation of muscle cell membranes, metabolic disorders of endometrial cells, and DNA damage, which in turn leads to uterine dysfunction, aggravated inflammation, and problems such as menstrual irregularities and infertility.

[0062] Wuhai chicken's precise active peptides contain specific functional groups that can effectively scavenge DPPH and ABTS free radicals inside and outside uterine tissue cells. It can rapidly bind to excess free radicals and reduce them to stable products, lowering free radical concentration and reducing oxidative damage to uterine tissues. This helps maintain normal uterine physiological function, resist damage caused by oxidative stress and related pathological factors, and is beneficial for maintaining uterine health. The determination of DPPH and ABTS follows the "Antioxidant Determination of Wuhai Chicken's Precise Active Peptides" standard (GB / T39100-2020), with the specific steps as follows.

[0063] (1) Determination of DPPH free radical scavenging rate Experimental methods: Add 3 mL of DPPH solution and 1 mL of Wuhai chicken precise active peptide solution to tube As (experimental group); add 3 mL of anhydrous ethanol solution and 1 mL of Wuhai chicken precise active peptide solution to tube Ac (control group); add 3 mL of DPPH solution and 1 mL of sample solvent (water) solution to tube Ab (blank group); mix thoroughly with the Wuhai chicken precise active peptide solution to be tested (tube As), and react at room temperature in the dark for 30 min. Calculate the DPPH scavenging rate using a UV spectrophotometer at a wavelength of 517 nm (calibrated with distilled water) (or measure 200 μL in a 96-well microplate using a microplate reader). If precipitation occurs in the Wuhai chicken precise active peptide sample after the reaction, centrifuge and collect the supernatant for analysis.

[0064] The DPPH radical scavenging rate is calculated using the following formula [1]: [1]; In the formula, Y: DPPH free radical scavenging rate; As: absorbance of the experimental group with added Wuhai chicken precise active peptide solution; Ac: absorbance of the control group with added Wuhai chicken precise active peptide solution; Ab: absorbance of the empty group with added sample solvent (water) solution.

[0065] (2) Determination of ABTS free radical scavenging rate Experimental methods: Add 3.6 mL of ABTS solution and 0.4 mL of sample or Wuhai chicken precise active peptide solution to tube As (experimental group); add 3.6 mL of 95% ethanol and 0.4 mL of sample or Wuhai chicken precise active peptide solution to tube Ac (control group); add 3.6 mL of ABTS and 0.4 mL of sample solvent (water) solution to tube Ab (experimental blank group); add 3.6 mL of 95% ethanol and 0.4 mL of sample solvent (water) solution to tube A0 (control blank group); mix thoroughly with the Wuhai chicken precise active peptide solution to be tested (tube As), react at room temperature in the dark for 5 min, and measure the absorbance value using a UV spectrophotometer at a wavelength of 734 nm (zeroed with distilled water).

[0066] The ABTS radical scavenging rate is calculated using the following formula [2]: [2]; In the formula, Y: ABTS free radical scavenging rate; As: absorbance of the experimental group with added Wuhai chicken precise active peptide solution; Ac: absorbance of the control group with added Wuhai chicken precise active peptide solution; Ab: absorbance of the blank group with added sample solvent (water) solution; A0: absorbance of the control blank group with added sample solvent (water) solution. Specific detection results are shown in Table 1 below: Table 1. DPPH free radical scavenging rate of precisely active peptides from Wuhai chicken processed with different techniques

[0067] Table 2. Scavenging rate of ABTS free radicals by precise active peptides from Wuhai chicken processed with different techniques

[0068] As shown in Table 1, under different conditions in the embodiments, the DPPH removal rate can reach over 60%, exhibiting a relatively high removal level. This indicates that the embodiments are effective in removing DPPH to a certain extent, and the differences in effectiveness between the embodiments are small. The comparative examples show relatively small variations in DPPH removal rates, with Comparative Example 4 having the highest removal rate at 45.40% and Comparative Example 5 having the lowest at 22.46%, both lower than the removal rates of the embodiments. As shown in Table 2, under different conditions in the embodiments, the ABTS removal rate can reach over 58%, exhibiting a relatively high removal level. This indicates that the embodiments are effective in removing ABTS to a certain extent, and the differences in effectiveness between the embodiments are small. The comparative examples show significant variations in ABTS removal rates, with Comparative Example 6 having the highest removal rate at 95.05% and Comparative Example 4 having the lowest at 8.80%. Comparative Examples 1, 2, 3, and 6 all have higher ABTS removal rates than the embodiments, but Comparative Examples 4, 5, and 7 have lower rates than the embodiments.

[0069] In summary, among the comparative examples, Comparative Example 4 showed the highest DPPH scavenging rate at 45.40%, but the lowest ABTS scavenging rate at 8.80%. Therefore, the scavenging activity against free radicals cannot be judged by a single indicator and requires comprehensive consideration. Based on the DPPH scavenging activity and ABTS scavenging activity, Examples 1, 1, 2, 3, and 6 were selected for cell experiments.

[0070] 2. Proliferative activity of human endometrial stromal cells, construction of an oxidative damage model, and damage protection. Human endometrial stromal cells originate from the stromal layer of the human endometrium and possess the typical morphology and functional characteristics of uterine stromal cells. They participate in key processes such as the cyclical proliferation, secretion, and decidualization of the endometrium, making them an ideal cell model for studying uterine physiology and the pathogenesis of related diseases. Common uterine health problems such as endometrial injury, thin endometrium, and intrauterine adhesions are closely related to abnormal proliferation and differentiation, decreased repair capacity, and local microenvironment disturbances of these cells. Many studies have used these cells to establish models of uterine injury or endometrial dysfunction to explore the mechanisms of proliferation disorders, inflammation, and fibrosis. Endometrial stromal cell-related experiments are often used to assess the effects of exogenous factors (such as growth factors and active peptide components) on their viability, proliferation, differentiation, and function, thereby determining their potential to improve uterine health, promote endometrial repair, or inhibit fibrosis. In the presence of an electron carrier, WST-8 in the CCK-8 reagent can be reduced by dehydrogenases in endometrial stromal cells to produce a water-soluble orange-yellow formazan product, the yield of which is directly proportional to the number of viable cells. By detecting the absorbance value at a wavelength of 450 nm, the activity of human endometrial stromal cells and their damage response after being affected by compounds can be effectively reflected.

[0071] (1) Effect of Wuhai Chicken Precision Active Peptide on the Proliferative Activity of Human Endometrial Stromal Cells The effect of Wuhai chicken's precise bioactive peptides on the proliferation activity of human endometrial stromal cells was determined using the CCK-8 assay. Human endometrial stromal cells in the logarithmic growth phase were collected, digested with trypsin, and 100 μL of a 1×10⁻⁶ ppm solution was added. 4 Human endometrial stromal cells (HMS cells / mL) were seeded into 96-well plates and cultured in HESCs medium (containing 10% fetal serum, 1% penicillin-drug antibiotics, and 1% factor). The plates were incubated at 37°C for 24 hours to allow the HMS cells to adhere. The plates were then removed, the culture medium aspirated, and the plates were washed twice with PBS, which was then discarded. Experimental groups were prepared by adding culture media containing the Wuhai chicken precise active peptide solution from Example 1 at concentrations of 0 (control group), 0.25%, 0.5%, 1.0%, and 2.0 mg / mL, respectively. These were incubated at 37°C for 24 hours. The culture medium was then aspirated, and 10 μL of CCK-8 solution (protected from light) was added to each well. To ensure thorough mixing of the reagent with the HMS cells, the plate was gently shaken on a shaker for 1-2 minutes at low speed, taking care to avoid air bubbles. The 96-well plates containing the CCK-8 reagent were then returned to the incubator for another 2 hours. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader.

[0072] Calculate cell viability using the following formula [3]: [3]; In the formula, Y represents cell viability; A1 represents the absorbance of the experimental group treated with Wuhai chicken precise active peptide solution; A2 represents the absorbance of the control group treated without Wuhai chicken precise active peptide solution; and A0 represents the absorbance of the blank group without cell inoculation. Specific detection results are provided by [the relevant authority / organization]. Figure 1 As shown in Table 3: Table 3. Effects of different concentrations of Wuhai chicken precise active peptides in the examples and comparative examples on the viability of human endometrial stromal cells.

[0073] In the experiment on the effect of different concentrations of Wuhai chicken precise active peptides on the viability of human endometrial stromal cells, the effects of Wuhai chicken precise active peptide solutions at concentrations of 0.25–2.0 mg / mL in Examples 1, 1, 2, 3, and 6 on the viability of human endometrial stromal cells were all higher than those in the control group (100%), indicating that Wuhai chicken precise active peptides in this concentration range have no toxicity to human endometrial stromal cells. The viability of human endometrial stromal cells showed a trend of first increasing and then decreasing. The highest viability of human endometrial stromal cells was observed at a concentration of 0.5 mg / mL for all Wuhai chicken precise active peptides. Further increases in concentration corresponded to a decrease in viability, but all levels remained higher than the control group. Subsequent experiments will be conducted at this concentration.

[0074] (2) H2O2-induced oxidative damage model of human endometrial stromal cells Hydrogen peroxide (H2O2) is used as a modeling agent. The core principle is to disrupt the balance of the oxidation-antioxidant system in human endometrial stromal cells by directly entering them, generating excessive reactive oxygen species (ROS) and attacking intracellular biomolecules such as proteins, lipids, and nucleic acids. This specifically induces oxidative stress-related phenotypes in damaged cells (such as decreased cell viability, increased apoptosis rate, and increased release of inflammatory factors), thereby constructing a human endometrial stromal cell oxidative damage model. This experiment investigated the effect of H2O2 on the activity of human endometrial stromal cells. The cell culture method was the same as in 2(1) above. H2O2 was added at concentrations of 0 (control group), 25, 50, 100, 300, 600, and 900 μmol / L, respectively. The culture medium was replaced with fresh culture medium as the control group. After incubation at 37°C for 24 h in a carbon dioxide incubator, the culture medium was aspirated, and 10 μL of CCK-8 solution (protected from light) was added to each well. The 96-well plate with the added CCK-8 reagent was returned to the incubator for further incubation for 2 h. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the same method as in formula [3]. The specific detection results are shown in Table 4: Table 4. Effects of different concentrations of H2O2 on the viability of human endometrial stromal cells

[0075] In the experiment investigating the effect of different concentrations of H2O2 on the viability of human endometrial stromal cells, the viability of human endometrial stromal cells gradually decreased with increasing H2O2 concentration. Specifically, at a concentration of 900 μmol / L, the viability of human endometrial stromal cells decreased to 56.00%, reaching the moderate damage range of 50-80%, indicating clear oxidative damage while retaining sufficient surviving cells for viability verification. This concentration will be used as the cell modeling condition in subsequent experiments.

[0076] (3) Protective effect of Wuhai chicken precise active peptides on H2O2-induced damage to human endometrial stromal cells This experiment investigated the protective effect of Wuhai chicken precise active peptide on H2O2-induced damage to human endometrial stromal cells. The cell culture method was the same as in 2(1) above. Wuhai chicken precise active peptide at a concentration of 0.5 mg / mL was added according to different processes for Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 6. The culture medium with fresh culture medium was used as the control group. After incubation at 37°C for 24 h in a carbon dioxide incubator, the culture medium was aspirated, and 10 μL of CCK-8 solution (protected from light) was added to each well. The 96-well plate with CCK8 reagent was put back into the incubator and incubated for another 2 h. After incubation, the absorbance of each well was measured at 450 nm using an ELISA reader. The cell viability was calculated in the same way as in [3].

[0077] The specific test results are shown in Table 5: Table 5. Protective effect of precise active peptides from Wuhai chicken processed with different techniques on H2O2-damaged human endometrial stromal cells.

[0078] Compared with the control group, the viability of human endometrial stromal cells in the model group decreased to 50.30%, indicating that the modeling was successful. Compared with Example 1, the viability of human endometrial stromal cells in the model group increased from 50.30% to 66.01%, proving that the precise active peptides of Wuhai chicken can protect human endometrial stromal cells from oxidative damage induced by H2O2. Compared with the comparative example, there was no significant difference in the viability of human endometrial stromal cells in Example 1, indicating that both Example 1 and the comparative example can protect human endometrial stromal cells from oxidative damage induced by H2O2.

[0079] 3. Effects of Wuhai Chicken Precision Active Peptides on LIF, TNF-α, and IL-10 Content in H2O2-Induced Damage to Human Endometrial Stromal Cells H2O2 induces oxidative stress in damaged human endometrial stromal cells, activating inflammatory signaling pathways and increasing the level of the pro-inflammatory factor TNF-α. Simultaneously, it inhibits the secretion of the anti-inflammatory factor IL-10 and the synthesis of endometrial function-related factor LIF, disrupting cytokine balance and exacerbating cellular inflammatory damage and functional disorders. Furthermore, the interaction between oxidative stress and cytokine imbalance further aggravates the damage to human endometrial stromal cells. Specifically, the precise active peptides from Wuhai chicken can alleviate H2O2-induced damage to human endometrial stromal cells by simultaneously increasing LIF and IL-10 levels and decreasing TNF-α levels.

[0080] This experiment investigated the effects of Wuhai chicken's precise active peptides on LIF, TNF-α, and IL-10 levels in H2O2-induced damaged human endometrial stromal cells. The specific steps are as follows: 1) Cell seeding: Human endometrial stromal cells in good growth condition were seeded at 1×10⁻⁶ cells per cell line. 41) Cells were seeded at a density of 1 cell / well in 96-well plates and cultured at 37℃ in a 5% CO2 incubator for 24 hours to allow for cell adhesion; 2) Experimental groups: control group, model group, and experimental group; 3) After cell adhesion, the upper culture medium was discarded. The control group was treated with HESCs medium only, the model group was treated with HESCs medium only, and the experimental group was treated with 0.5 mg / mL of different processed Wuhai chicken precise active peptides and HESCs medium for 24 hours; 4) After 24 hours of intervention, the control group was treated with HESCs medium for 2 hours, while the model group and experimental group were treated with 900 μmol / L H2O2 for 2 hours; 5) After the intervention, LIF, TNF-α, and IL-10 were measured according to the test instructions.

[0081] (1) Effect of Wuhai chicken precise active peptides on LIF content in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 2 As shown in Table 6: Table 6. Effects of precise active peptides from Wuhai chicken processed with different techniques on LIF content in human endometrial stromal cells damaged by H2O2.

[0082] The control group showed a significant difference from the model group. Under H2O2-induced damage, the LIF content in human endometrial stromal cells of the model group was significantly lower than that of the control group, indicating that the low receptivity model was successfully established. The experimental groups also showed significant differences from the model group. Both the examples and comparative examples effectively increased the LIF content in human endometrial stromal cells damaged by H2O2. Example 1 showed the best effect in increasing LIF content. The experimental groups also showed significant differences from the control group. The examples and comparative examples were insufficient to restore LIF content to normal levels, indicating that the Wuhai chicken precise active peptide has the ability to repair LIF content, but the repair ability is slightly weak.

[0083] (2) Effect of Wuhai chicken precise active peptides on TNF-α content in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 3 And as shown in Table 7: Table 7. Effects of precise active peptides from Wuhai chicken processed with different techniques on TNF-α content in human endometrial stromal cells damaged by H2O2.

[0084] The control group showed significant differences compared to the model group. Under H2O2-induced damage, the TNF-α secretion level in human endometrial stromal cells of the model group was significantly higher than that of the control group, indicating that the inflammation model was successfully established. Both the experimental and comparative groups showed significant differences compared to the model group. Both the examples and comparative examples effectively reduced the TNF-α secretion level in human endometrial stromal cells damaged by H2O2. Among them, Example 1 showed the best effect in reducing TNF-α secretion.

[0085] Examples 1, 1, 3, and 6 showed no significant differences compared to the control group, indicating that they could reduce TNF-α secretion to control levels and effectively reduce inflammatory factors in human endometrial stromal cells. Comparative Example 2 showed significant differences compared to both the control and model groups, indicating that it could reduce the secretion of inflammatory factors in human endometrial stromal cells, but not to normal levels.

[0086] (3) Effect of Wuhai chicken precise active peptides on IL-10 content in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 4 As shown in Table 8: Table 8. Effects of precise active peptides from Wuhai chicken processed with different techniques on IL-10 content in human endometrial stromal cells damaged by H2O2.

[0087] The control group showed a significant difference from the model group. Under H2O2-induced damage, the IL-10 content in the human endometrial stromal cells of the model group was significantly lower than that of the control group, indicating that the inflammation model was successfully established. There was no significant difference between the experimental group and the model group. However, the IL-10 content in Example 1 was higher than that in the model group, as shown in the figure, indicating that Example 1 had the best ability to repair the secretion of the anti-inflammatory factor IL-10. Both the experimental group and the control group showed significant differences, indicating that the experimental group could repair the secretion of the anti-inflammatory factor IL-10, but the ability was weak and could not be restored to normal levels.

[0088] 4. Effects of Wuhai Chicken Precision Active Peptides on the Relative Expression Levels of LIF, ITGβ3, HOXA10, TNF-α, IL-1β, Bax, and Bcl-2 mRNA in H2O2-induced Damaged Human Endometrial Stromal Cells LIF (Lipoprotein Fertilizer) is a key cytokine regulating the proliferation and repair of human endometrial stromal cells, maintaining endometrial receptivity, and promoting embryo implantation, directly participating in the homeostatic regulation of endometrial physiological functions. Oxidative damage induced by H2O2 significantly inhibits the relative expression level of LIF mRNA in human endometrial stromal cells, leading to decreased cell proliferation and repair capacity, reduced endometrial receptivity, and consequently affecting the normal physiological state of the endometrium. ITGβ3 is an important adhesion molecule located on the surface of human endometrial stromal cells, responsible for mediating cell-extracellular matrix adhesion and intercellular signal transduction. It is crucial for maintaining the morphological integrity of human endometrial stromal cells, promoting cell migration, and enhancing endometrial receptivity. Oxidative damage induced by H2O2 leads to a significant downregulation of the relative expression level of ITGβ3 mRNA, disrupting the adhesion and migration functions of human endometrial stromal cells and affecting endometrial development and maturation.

[0089] HOXA10 is a core transcription factor regulating endometrial development and maturation and maintaining endometrial receptivity. It directly participates in the differentiation of human endometrial stromal cells and the expression regulation of key genes related to embryo implantation, and its expression level is closely related to endometrial physiological function. Oxidative damage induced by H2O2 significantly inhibits the relative expression of HOXA10 mRNA, leading to abnormal endometrial development and decreased receptivity, thus affecting the embryo implantation process. TNF-α is a key pro-inflammatory cytokine mediating oxidative stress damage to human endometrial stromal cells. While it does not directly regulate basic endometrial physiological function, H2O2-induced oxidative damage activates inflammatory signaling pathways, resulting in a significant increase in the relative expression of TNF-α mRNA. High expression of TNF-α further aggravates damage to human endometrial stromal cells, inhibits cell proliferation and repair, and disrupts the local microenvironment homeostasis of the endometrium, forming a vicious cycle of "oxidative stress → inflammation activation → more severe cell damage."

[0090] IL-1β is a key pro-inflammatory cytokine involved in oxidative stress-induced inflammation of human endometrial stromal cells. It primarily exacerbates oxidative damage by activating downstream inflammatory cascades, disrupting the balance of the local immune microenvironment in the endometrium. H2O2-induced oxidative damage significantly upregulates the relative expression of IL-1β mRNA in human endometrial stromal cells, intensifying the inflammatory response and leading to cellular dysfunction and endometrial microenvironment imbalance. Bax is a core pro-apoptotic gene regulating apoptosis in human endometrial stromal cells, responsible for initiating apoptosis signaling pathways and promoting apoptosis under oxidative stress or injury. H2O2-induced oxidative damage significantly upregulates the relative expression of Bax mRNA, activating the apoptosis program in human endometrial stromal cells, resulting in reduced cell number and impaired function, thereby affecting the normal physiological function of the endometrium.

[0091] Bcl-2 is a key anti-apoptotic gene in human endometrial stromal cells. It prevents abnormal cell apoptosis by inhibiting the activity of pro-apoptotic proteins and maintaining mitochondrial functional stability, thus playing a crucial role in maintaining the survival and normal physiological function of human endometrial stromal cells. Oxidative damage induced by H2O2 leads to a significant downregulation of the relative expression level of Bcl-2 mRNA, weakening the anti-apoptotic ability of cells and exacerbating apoptotic damage in human endometrial stromal cells.

[0092] This experiment investigated the effects of Wuhai chicken's precise bioactive peptides on the relative mRNA expression levels of LIF, ITGβ3, HOXA10, TNF-α, IL-1β, Bax, and Bcl-2 in human endometrial stromal cells induced by H2O2 damage. The expression of related genes in human endometrial stromal cells oxidatively damaged by H2O2 was measured using RT-qPCR. Specifically, human endometrial stromal cells in the logarithmic growth phase were collected, digested with trypsin, and 100 μL of a 1×10⁻⁶ m³ / dose was added. 4 Human endometrial stromal cells (HMS cells / mL) were seeded in 96-well plates and cultured in HESCs medium (containing 10% fetal serum, 1% penicillin-streptomycin, and 1% stem cell factor) and incubated at 37°C for 24 h in a CO2 incubator to allow the HMS cells to adhere. A 0.5 mg / mL concentration of the Wuhai chicken precise active peptide solution (used in the examples and comparatives) was added and incubated at 37°C for 24 h. After culture, the medium was discarded, and the model and experimental groups were further treated with 900 μmol / L H2O2 for 20 min. After culture, the medium was discarded, and the HMS cells were washed three times with PBS to remove residual medium and drugs. Following the instructions of the RNA extraction kit, an appropriate amount of lysis buffer was added to each culture dish to fully lyse the HMS cells, and total RNA was extracted. The concentration and purity of RNA were measured using a micro-nucleic acid analyzer to ensure that the RNA quality met the requirements for subsequent experiments, such as OD200. 260 / OD 280 The ratio should be between 1.8 and 2.0. Take an appropriate amount of total RNA and reverse transcribe it into cDNA following the instructions of the reverse transcription kit.

[0093] The reverse transcription reaction conditions are typically: incubation at 42℃ for 60-90 minutes, followed by heating at 70℃ for 10-15 minutes to terminate the reaction. The resulting cDNA can be stored at -20℃ for later use. Prepare the PCR reaction system, including SYBR Green fluorescent dye, upstream and downstream primers, cDNA template, PCR buffer, dNTPs, and Taq enzyme. Add the reaction system to a 96-well or 384-well plate, with three replicates per sample. The PCR reaction program is generally: pre-denaturation at 95℃ for 3-5 minutes; then annealing at 95℃ for 15-30 seconds, annealing at 60℃ for 30-60 seconds, and extension at 72℃ for 30-60 seconds, for a total of 40-50 cycles; finally, perform melting curve analysis to determine the specificity of the amplified product. Using GAPDH as an internal reference gene as a control, the relative expression level of the target gene is calculated by comparing the Ct value (cycle threshold). - Data analysis was performed using the ΔΔCt method. Primer information for genes related to human endometrial stromal cells is shown in Table 9.

[0094] Table 9. Primers for genes related to human endometrial stromal cells

[0095] (1) Effect of Wuhai chicken precise active peptides on the relative expression level of LIF mRNA in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 5 As shown in Table 10: Table 10. Relative mRNA expression levels of LIF in H2O2-induced damaged human endometrial stromal cells by Wuhai chicken's precise active peptides.

[0096] Example 1 showed no significant difference compared to the control group and the model group. However, under H2O2-induced damage, the relative expression level of LIF mRNA in human endometrial stromal cells of the model group was lower than that of the control group. The relative expression level of LIF mRNA in Example 1 was higher than that of the model group and the control group, as shown in the figure. This indicates that the Wuhai chicken precise active peptide can increase the relative expression level of LIF mRNA and is comparable to that of LIF in the control group, thus restoring it to the normal level.

[0097] The control group showed significant differences compared to the control group. The relative expression level of LIF mRNA in the control group was higher than that in the control group. However, the excessively high relative expression level of LIF mRNA can enhance the adhesion of ectopic endometrial tissue and promote its survival and proliferation in ectopic sites, leading to endometriosis, endometrial hyperplasia and chronic endometritis.

[0098] (2) Effect of Wuhai chicken precise active peptides on the relative expression level of ITGβ3 mRNA in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 6 As shown in Table 11: Table 11. Relative mRNA expression levels of ITGβ3 in H2O2-induced damaged human endometrial stromal cells by the precise active peptides of Wuhai chicken.

[0099] There was no significant difference between the control group and the model group. However, under H2O2-induced damage, the relative expression level of ITGβ3 mRNA in human endometrial stromal cells of the model group was lower than that of the control group. Example 1 showed a significant difference compared to the model group; the relative expression level of ITGβ3 mRNA in human endometrial stromal cells of Example 1 under H2O2-induced damage was significantly higher than that of the model group. Example 1 also showed a significant difference compared to the control group, effectively increasing the relative expression level of ITGβ3 mRNA in human endometrial stromal cells under H2O2-induced damage significantly higher than that of the control group. All comparative examples showed significant differences compared to the control group. Comparative examples 1, 2, and 3 showed significantly higher relative expression levels of ITGβ3 mRNA in human endometrial stromal cells under H2O2-induced damage than the control group. However, comparative example 6 showed significantly lower relative expression levels of ITGβ3 mRNA in human endometrial stromal cells under H2O2-induced damage than the control group. When the relative expression level of ITGβ3 mRNA is too high, it can easily lead to endometriosis; however, when the relative expression level of ITGβ3 mRNA is too low, endometrial receptivity is severely reduced.

[0100] (3) Effect of Wuhai chicken precise active peptides on the relative expression level of HOXA10 mRNA in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 7 As shown in Table 12: Table 12. Relative expression levels of HOXA10 mRNA in H2O2-induced damaged human endometrial stromal cells by the precise active peptides of Wuhai chicken.

[0101] Compared with the model group, the control group showed a significant difference; under H2O2-induced damage, the relative expression level of HOXA10 mRNA in human endometrial stromal cells of the model group was lower than that of the control group. Example 1 showed a significant difference compared with the model group; under H2O2-induced damage, the relative expression level of HOXA10 mRNA in human endometrial stromal cells of Example 1 was significantly higher than that of the model group. Example 1 showed no significant difference compared with the control group; Example 1 effectively restored the relative expression level of HOXA10 mRNA in human endometrial stromal cells under H2O2-induced damage, restoring it to a level comparable to the control group. Comparative Example 2 showed no significant difference compared with the control group; Comparative Example 2 and Example 1 effectively restored the relative expression level of HOXA10 mRNA in human endometrial stromal cells under H2O2-induced damage, restoring it to a level comparable to the control group. Comparative Examples 1, 3, and 6 also restored the relative expression level of HOXA10 mRNA in human endometrial stromal cells under H2O2-induced damage, but failed to restore it to a level comparable to the control group.

[0102] (4) Effect of Wuhai chicken precise active peptides on the relative expression level of Bax mRNA in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 8 As shown in Table 13: Table 13. Relative mRNA expression levels of Bax in H2O2-induced damaged human endometrial stromal cells by the precise active peptides of Wuhai chicken.

[0103] The control group showed a significant difference from the model group; under H2O2-induced damage, the relative expression level of Bax mRNA in human endometrial stromal cells of the model group was higher than that of the control group. Example 1 showed a significant difference from the model group; under H2O2-induced damage, the relative expression level of Bax mRNA in human endometrial stromal cells of Example 1 was significantly lower than that of the model group. Example 1 showed no significant difference from the control group; Example 1 effectively reduced the relative expression level of Bax mRNA in human endometrial stromal cells under H2O2-induced damage, reducing it to a level comparable to the control group. Comparative Examples 2 and 6 showed no significant difference from the control group; Comparative Examples 2, 6, and Example 1 effectively restored the relative expression level of Bax mRNA in human endometrial stromal cells under H2O2-induced damage, achieving levels comparable to the control group. Comparative Examples 1 and 3 also reduced the relative expression level of Bax mRNA in human endometrial stromal cells under H2O2-induced damage, but failed to reduce it to a level comparable to the control group.

[0104] (5) Effect of Wuhai chicken precise active peptides on the relative expression level of Bcl-2 mRNA in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 9 And as shown in Table 14: Table 14. Relative mRNA expression levels of Bcl-2 in H2O2-induced damaged human endometrial stromal cells induced by the precise active peptides of Wuhai chicken.

[0105] Compared with the model group, the control group showed a significant difference; under H2O2-induced damage, the relative expression level of Bcl-2 mRNA in human endometrial stromal cells of the model group was lower than that of the control group. Example 1 showed a significant difference compared with the model group; under H2O2-induced damage, the relative expression level of Bcl-2 mRNA in human endometrial stromal cells of Example 1 was significantly higher than that of the model group. Example 1 also showed a significant difference compared with the control group, effectively increasing the relative expression level of Bcl-2 mRNA in human endometrial stromal cells under H2O2-induced damage, and significantly higher than that of the control group. Comparative Example 3 showed no significant difference compared with the control group, effectively increasing the relative expression level of Bcl-2 mRNA in human endometrial stromal cells under H2O2-induced damage, comparable to the control group; Comparative Examples 1, 2, and 6 showed no significant difference compared with the model group, failing to effectively increase the relative expression level of Bcl-2 mRNA in human endometrial stromal cells under H2O2-induced damage.

[0106] (6) Effect of Wuhai Chicken Precision Active Peptide on the Relative Expression Level of TNF-α mRNA in H2O2-Induced Damage to Human Endometrial Stromal Cells The specific test results are provided by Figure 10 As shown in Table 15: Table 15. Relative mRNA expression levels of TNF-α in H2O2-induced damaged human endometrial stromal cells induced by the precise active peptides of Wuhai chicken.

[0107] The control group showed a significant difference from the model group; under H2O2-induced damage, the relative expression level of TNF-α mRNA in human endometrial stromal cells of the model group was higher than that of the control group. Example 1 showed a significant difference from the model group; under H2O2-induced damage, the relative expression level of TNF-α mRNA in human endometrial stromal cells of Example 1 was significantly lower than that of the model group. Example 1 showed no significant difference from the control group; Example 1 effectively reduced the relative expression level of TNF-α mRNA in human endometrial stromal cells under H2O2-induced damage, and the level was comparable to that of the control group. Comparative Examples 2, 3, and 6 showed no significant difference from the control group; Comparative Examples 2, 3, 6, and Example 1 effectively reduced the relative expression level of TNF-α mRNA in human endometrial stromal cells under H2O2-induced damage, and the level was comparable to that of the control group; Comparative Example 1 also reduced the relative expression level of TNF-α mRNA in human endometrial stromal cells under H2O2-induced damage, but it did not reduce it to a level comparable to that of the control group.

[0108] (7) Effect of Wuhai chicken precise active peptides on the relative expression level of IL-1β mRNA in H2O2-induced damaged human endometrial stromal cells The specific test results are provided by Figure 11 And as shown in Table 16: Table 16. Relative mRNA expression levels of IL-1β in H2O2-induced damaged human endometrial stromal cells induced by the precise active peptides of Wuhai chicken.

[0109] The control group showed a significant difference from the model group; under H2O2-induced damage, the relative expression level of IL-1β mRNA in human endometrial stromal cells of the model group was higher than that of the control group. Example 1 showed a significant difference from the model group; under H2O2-induced damage, the relative expression level of IL-1β mRNA in human endometrial stromal cells of Example 1 was significantly lower than that of the model group. Example 1 showed no significant difference from the control group; Example 1 effectively reduced the relative expression level of IL-1β mRNA in human endometrial stromal cells under H2O2-induced damage, and the level was comparable to that of the control group. Comparative Examples 1, 2, and 6 showed no significant difference from the control group; Comparative Examples 1, 2, 6, and Example 1 effectively reduced the relative expression level of IL-1β mRNA in human endometrial stromal cells under H2O2-induced damage, and the level was comparable to that of the control group; Comparative Example 3 also reduced the relative expression level of IL-1β mRNA in human endometrial stromal cells under H2O2-induced damage, but it did not reduce it to a level comparable to that of the control group.

[0110] In summary, the precise active peptides from Wuhai chicken exert cytoprotective effects by regulating the mRNA expression of relevant genes in H2O2-induced damaged human endometrial stromal cells through multiple pathways. Specifically, by resisting H2O2 oxidative damage, they upregulate the mRNA expression of LIF, ITGβ3, and HOXA10, repairing the proliferation, repair, adhesion, and migration functions of human endometrial stromal cells, and promoting endometrial development and receptivity. They also reduce inflammatory damage to human endometrial stromal cells and maintain endometrial microenvironment stability by inhibiting inflammatory pathways and downregulating the overexpression of TNF-α and IL-1β mRNA. Furthermore, they regulate the apoptosis balance of human endometrial stromal cells, reduce cell death, and protect cell survival and endometrial tissue function by inhibiting the mRNA expression of the apoptosis factor Bax and upregulating the mRNA expression of the pro- and anti-apoptotic factor Bcl-2.

[0111] 4. Molecular docking experiment 1. Preprocessing method 1.1 Dissolution of Wuhai Chicken Precision Active Peptide Samples 1) Accurately weigh approximately 1 mg of sample into a centrifuge tube, and accurately transfer 100 µL of PBS into the sample to dissolve it; 1.2 Sample Ultrafiltration 1) Shake thoroughly to mix, take 100 μL of sample and add it to different 10kDa ultrafiltration tubes for ultrafiltration, centrifuge at 12000 rpm for 10 min; 2) Add 100 μL of water, centrifuge at 12000 rpm for 10 min, repeat the washing process 3 times, and take the fraction less than 10 kDa and the fraction greater than 10 kDa. 1.3 Concentration measurement using nanodrop 1) Rinse the detection head twice with 2 μL of pure water; 2) Load the blank control solution with a sample volume of 2 μL, measure the data at 205 nm, and deduct the result after measurement; 3) Test the sample, loading 2 μL, and read the data after testing; 4) Rinse the detection head twice with 2 μL of pure water.

[0112] 1.4 Reductive Alkylation 1) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, and reduce it in a 56℃ water bath for 1 h; 2) Accurately pipette 2 μL of 1M IAM solution into the sample to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min. 3) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, in order to neutralize unreacted IAM.

[0113] 1.5 C18 desalination 1) Desalted using C18 stage-tip and dried under vacuum at 45°C.

[0114] 2. Computer Lab The processed Wuhai chicken precise active peptides were detected by liquid chromatography-mass spectrometry (LC-MS) under the following conditions: 2.1 Liquid Chromatography Conditions 1) Pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm; 2) Mobile phase A: 0.1% FA (formic acid); 3) Mobile phase B: 0.1% FA, 80% ACN (acetonitrile); 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min.

[0115] Specific chromatographic test conditions are shown in Table 17: Table 17: Specific Chromatographic Conditions

[0116] 2.2 Mass Spectrometry Conditions The first-order mass spectrometry parameters are as follows: Resolution: 70,000; AGC target: 3e6; Maximum IT: 100 ms; Scan range: 100 to 1500 m / z; The secondary mass spectrometry parameters are as follows: Resolution: 17,500; AGC target: 1e5; Maximum IT: 50ms; TopN: 20; NCE / steppedNCE: 28. The raw data were obtained after mass spectrometry acquisition.

[0117] 2.3 Search Criteria The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: 1) Fixed modifications: Carbamidomethyl (C). 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term). 3) Enzyme: Non-specific. 4) Database: uniprotkb_proteome_UP000000539_2025_12_17.fasta 5) Peptide Mass Tolerance (PMT): 20 ppm; 6) Fragment Mass Tolerance (FMT): 0.03 Da.

[0118] The raw files acquired by mass spectrometry contained 923 peptides ranging from 3 to 17. First, peptides with a relative content >1.0% were selected based on peak area, with 11 peptides containing 3 or more peptides. Activity scoring was performed on the PeptideRanker website (http: / / distilldeep.ucd.ie / PeptideRanker / ), selecting 9 peptides with a result >0.50 and containing 3 or more peptides. Further searching of these 9 peptides on the ToxinPred toxicity prediction website (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) showed that they were all non-toxic. Precise bioactive peptides from Wuhai chicken were searched in the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php), and 5 of these peptides were novel peptides not previously reported. The molecular docking and mass spectrometry analysis results yielded a list of peptides, as shown below. The table below lists nine peptide sequences obtained in Example 1 after screening based on peptide sequence, amino acid count, activity score, relative molecular mass, and damage experiment, as detailed in Table 18. Table 18. Precise Active Peptide Sequences and Information Scores of Wuhai Chicken

[0119] The three-dimensional structures of the above nine peptide sequences with LIF (ID: 3E0G), ITGβ3 (ID: 6NAJ), and TNF-α (ID: 2AZ5) were downloaded from the PDB database (http: / / www.rcsb.org / ) using molecular docking software. The docking binding energies of the nine peptide sequences with different receptors are shown in Table 19. Table 19. MoE (Measure of Efficacy) Analysis of Precise Active Peptide Molecular Docking in Wuhai Chicken

[0120] CDOCKERENERGY is an energy value calculated during CDOCKER docking. It primarily reflects the overall binding energy between the ligand and acceptor during docking. This energy comprehensively considers various interactions between the ligand and acceptor, including van der Waals forces and electrostatic interactions. Physically speaking, a lower CDOCKERENERGY value indicates a tighter and more stable binding between the ligand and acceptor.

[0121] like Figure 12 As shown: LPQPPQ interacts with LIF receptor proteins through van der Waals forces, salt bridges, attractive charges, conventional hydrogen bonds, C-H bonds, unfavorable negative-negative charge interactions, and π-alkyl-based interactions; Figure 13 As shown: LPQPPQ interacts with the ITGβ3 receptor protein through van der Waals forces, salt bridges, attractive charges, conventional hydrogen bonds, C-H bonds, and alkyl-based interactions; such as... Figure 14 As shown: LPQPPQ interacts with TNF-α receptor proteins primarily through van der Waals forces, attractive charges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups; Figure 15 As shown: There are van der Waals forces, salt bridges, and predominantly negative charge-negative charge interactions between IPF and LIF receptor proteins; such as... Figure 16 As shown: There are van der Waals forces, salt bridges, conventional hydrogen bonds, and unfavorable donor-donor-dominant interactions between IPF and the ITGβ3 receptor protein; such as... Figure 17 As shown: There are van der Waals forces, conventional hydrogen bonds, π-π stacking, and π-alkyl-based interactions between IPF and the TNF-α receptor protein; for example... Figure 18 As shown: There are interactions between LPF and LIF receptor proteins primarily involving van der Waals forces, attractive charges, conventional hydrogen bonds, unfavorable negative-negative charge pairs, and amide-π stacking; such as... Figure 19 As shown: LPF interacts with the ITGβ3 receptor protein primarily through van der Waals forces, salt bridges, and conventional hydrogen bonds; Figure 20 As shown: LPF interacts with TNF-α receptor proteins primarily through van der Waals forces, conventional hydrogen bonds, π-anions, π-π T-forms, and π-alkyl groups; Figure 21 As shown: LGPL and LIF receptor proteins exhibit van der Waals forces, salt bridges, C-H bonds, and predominantly negative-charge-negative-charge interactions; for example... Figure 22 As shown: LGPL and the ITGβ3 receptor protein interact primarily through van der Waals forces, salt bridges, conventional hydrogen bonds, and C-H bonds; Figure 23As shown: LGPL interacts with TNF-α receptor protein primarily through van der Waals forces, salt bridges, alkyl groups, and π-alkyl groups; Figure 24 As shown: LGPI and LIF receptor proteins exhibit van der Waals forces, salt bridges, C-H bonds, unfavorable negative-negative charge interactions, and alkyl-based interactions; for example... Figure 25 As shown: LGPI and the ITGβ3 receptor protein exhibit van der Waals forces, salt bridges, conventional hydrogen bonds, carbon-hydrogen bonds, and predominantly negative-charge-negative-charge interactions; such as... Figure 26 As shown: LGPI interacts with TNF-α receptor proteins primarily through van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, and π-cation interactions; Figure 27 As shown: There are van der Waals forces, attractive charges, conventional hydrogen bonds, C-H bonds, and alkyl-based interactions between IGPL and the LIF receptor protein; such as... Figure 28 As shown: There are van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, and alkyl-based interactions between IGPL and the ITGβ3 receptor protein; for example... Figure 29 As shown: IGPL interacts with TNF-α receptor proteins primarily through van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-cations, and π-anions; Figure 30 As shown: AVF and LIF receptor proteins exhibit van der Waals forces, salt bridges, C-H bonds, unfavorable negative-negative charge interactions, and π-alkyl-based interactions; for example... Figure 31 The interaction between AVF and the ITGβ3 receptor protein is mainly characterized by van der Waals forces, salt bridges, attractive charges, conventional hydrogen bonds, π-cations, and π-alkyl groups; as shown. Figure 32 As shown: AVF interacts with the TNF-α receptor protein through van der Waals forces, conventional hydrogen bonds, C-H bonds, and primarily alkyl groups; Figure 33 As shown: FPPDVA and LIF receptor proteins exhibit van der Waals forces, attractive charges, conventional hydrogen bonds, C-H bonds, unfavorable negative-negative charge interactions, and alkyl-based interactions; such as... Figure 34 As shown: FPPDVA interacts with the ITGβ3 receptor protein through van der Waals forces, salt bridges, attractive charges, conventional hydrogen bonds, C-H bonds, and alkyl-based interactions; such as... Figure 35 As shown: FPPDVA interacts with the TNF-α receptor protein primarily through van der Waals forces, attractive charges, conventional hydrogen bonds, C-H bonds, unfavorable negative-negative charge interactions, π-anionic interactions, and π-alkyl interactions; Figure 36 As shown: There is a predominantly van der Waals force, attractive charge, and C-H bond interaction between the MPF and LIF receptor protein; such as Figure 37 As shown, there are interactions between MPF ​​and the ITGβ3 receptor protein, mainly consisting of van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, π-cations, and π-alkyl groups. like Figure 38 As shown: There are interactions between MPF ​​and TNF-α receptor protein mainly consisting of van der Waals forces, conventional hydrogen bonds, unfavorable negative-negative interactions, π-anions, π-sulfur atoms, and π-alkyl groups. Leukemia suppressor factor (LIF, ID: 3E0G) belongs to the interleukin-6 family and is mainly expressed in endometrial epithelial cells, glandular cells, and decidual cells. It is a core molecule regulating endometrial receptivity and embryo implantation. It activates the JAK-STAT3 pathway by binding to the receptor LIFR / gp130, initiating the transcription of receptivity-related genes and constructing a suitable microenvironment for embryo implantation. The physiological functions of LIF include: ① inducing damaged endometrium to enter the implantation window and upregulating receptivity markers; ② promoting trophoblast cell adhesion and invasion, ensuring embryo implantation; ③ maintaining maternal-fetal immune tolerance and preventing embryo rejection. Insufficient LIF expression reduces endometrial receptivity, directly leading to recurrent implantation failure and unexplained infertility. Wuhai chicken's precise active peptides can upregulate endometrial LIF expression, activate downstream signaling pathways, improve receptivity, and increase the success rate of embryo implantation.

[0122] Integrin β3 (ITGβ3, ID: 6NAJ) belongs to the integrin family of transmembrane adhesion molecules. It often forms heterodimers with the αV subunit and is highly expressed on the surface of endometrial epithelial cells, trophoblastic cells, and decidual cells, mediating embryo adhesion and placental development. It mediates the adhesion of trophoblastic cells to endometrial epithelial cells by recognizing extracellular matrix ligands, initiating embryonic invasion. The physiological significance of ITGβ3 includes: ① serving as a core marker of endometrial receptivity, with peak expression coinciding with the implantation window; ② participating in placental vascular remodeling to ensure blood supply; ③ regulating trophoblastic cell proliferation and differentiation to maintain pregnancy stability. Decreased ITGβ3 expression leads to insufficient embryo adhesion, increasing the risk of recurrent miscarriage and preterm birth. Wuhai chicken precise active peptides can enhance ITGβ3 expression, strengthen embryo-endometrial adhesion, promote placental development, and stabilize pregnancy progress.

[0123] Tumor necrosis factor-α (TNF-α, ID: 2AZ5) is a key pro-inflammatory cytokine, mainly expressed in endometrial immune cells, epithelial cells, and placental trophoblast cells, playing a dual role in uterine immune regulation and pregnancy homeostasis. Under physiological conditions, it participates in the periodic repair of the endometrium and regulates the immune balance at the maternal-fetal interface; under pathological conditions, abnormally elevated levels break immune tolerance and induce pregnancy complications. The abnormal mechanisms of TNF-α include: ① mediating excessive inflammation at the maternal-fetal interface, activating neutrophils and the complement system, and damaging the placental vascular endothelium; ② inducing impaired remodeling of damaged uterine spiral arteries, leading to insufficient placental perfusion; ③ increasing the TNF-α / IL-10 ratio, exacerbating immune rejection, and increasing the risk of adverse pregnancy outcomes. Wuhai chicken's precise active peptides can inhibit excessive TNF-α secretion, downregulate downstream inflammatory pathways, restore maternal-fetal immune balance, reduce placental damage, and lower the risk of adverse pregnancy outcomes.

[0124] Upregulating LIF improves endometrial receptivity, laying the foundation for embryo implantation; increasing ITGβ3 enhances embryo adhesion and placental development, ensuring the onset of pregnancy; and inhibiting abnormal TNF-α secretion maintains immune homeostasis and avoids inflammatory damage. At the maternal-fetal interface, the receptive microenvironment constructed by LIF provides conditions for ITGβ3-mediated adhesion, while moderate regulation of TNF-α balances the inflammatory response. These three elements form a dynamic synergistic network that precisely regulates embryo implantation, placental development, and pregnancy maintenance. By synergistically maintaining uterine health from three dimensions—"receptivity construction," "implantation protection," and "immune homeostasis"—it prevents infertility, recurrent miscarriage, and other problems. Because the five black chicken precise active peptides prepared in this application example have nine peptide segments—Leu-Pro-Gln-Pro-Pro-Gln (LPQPPQ), Ile-Pro-Phe (IPF), Leu-Pro-Phe (LPF), Leu-Gly-Pro-Leu (LGPL), Leu-Gly-Pro-Ile (LGPI), Ile-Gly-Pro-Leu (IGPL), Ala-Val-Phe (AVF), Phe-Pro-Pro-Asp-Val-Ala (FPPDVA), and Met-Pro-Phe (MPF)—successfully docked with three receptors—LIF (ID: 3E0G), ITGβ3 (ID: 6NAJ), and TNF-α (ID: 2AZ5)—and exhibited a certain binding energy.

[0125] Summary of Experimental Results: Through in vitro antioxidant assays and comparative studies using human endometrial stromal cells, a precise active peptide from Wuhai chicken with optimal activity in improving uterine health was screened for use in amino acid sequence and activity studies related to its uterine health-improving effects. The precise active peptide from Wuhai chicken prepared in this application significantly scavenges DPPH and ABTS free radicals, increases / restores LIF content in human endometrial stromal cells, reduces TNF-α secretion in human endometrial stromal cells, promotes IL-10 secretion in human endometrial stromal cells, promotes the relative mRNA expression levels of LIF, ITGβ3, HOXA10, and Bcl-2, and inhibits the relative mRNA expression levels of TNF-α, IL-1β, and Bax, collectively exerting an effect in improving uterine health. This application employs mass spectrometry, virtual screening, and molecular docking to screen for nine amino acid peptides from all identified peptides in the examples. These peptides are Leu-Pro-Gln-Pro-Pro-Gln (LPQPPQ), Ile-Pro-Phe (IPF), Leu-Pro-Phe (LPF), Leu-Gly-Pro-Leu (LGPL), Leu-Gly-Pro-Ile (LGPI), Ile-Gly-Pro-Leu (IGPL), Ala-Val-Phe (AVF), Phe-Pro-Pro-Asp-Val-Ala (FPPDVA), and Met-Pro-Phe (MPF), all of which possess amino acid activity for improving uterine health. These peptides work synergistically to improve uterine health by promoting the expression of LIF and ITGβ3 mRNA and inhibiting the expression of TNF-α mRNA.

[0126] In summary, the method for preparing the precise active peptides of Wuhai chicken provided in this application has at least the following mechanisms of action and technical effects: (1) This application found that Wuhai chicken, after being minced into meat paste, homogenized, pretreated with medium-temperature heat treatment, and then subjected to a specific enzymatic hydrolysis combination (first enzymatic hydrolysis with alkaline protease (37071) and second enzymatic hydrolysis with papain), has a high level of effect on improving uterine health. (2) The proposed solution uses a specific combination of alkaline protease (37071) and papain to achieve the desired effect; the effect of this application can not be achieved by hydrolyzing the combination of the two enzymes or by any other combination of proteases.

[0127] For the combination of alkaline protease (37071) and papain: Alkaline protease (37071) exhibits optimal activity within an alkaline pH range of 8–11, efficiently adapting to processing requirements in highly alkaline environments. It has high temperature requirements, reaching peak catalytic efficiency within the 50–60°C range. Alkaline protease (37071) is primarily a serine protease, hydrolyzing peptide bonds in proteins through a catalytic triplet mechanism; its active site often contains a key serine residue. Due to its excellent alkali tolerance, it is particularly suitable for applications requiring protein degradation under high pH conditions, such as the detergent and leather industries. Papain has a pH range of slightly acidic to neutral, with an optimal pH concentrated between 5.2 and 5.8. It exhibits strong temperature adaptability, with superior heat resistance compared to most plant proteases, and an optimal operating temperature of 60–65°C, retaining some activity even at short-term temperatures below 70°C. This enzyme belongs to the cysteine ​​protease family. Its active site contains cysteine ​​and histidine residues. It activates peptide bonds through the synergistic action of thiol and imidazole groups, catalyzing protein hydrolysis. It exhibits broad substrate specificity. This characteristic makes it widely applicable in food processing (meat tenderization, beer clarification), pharmaceutical industry (digestive enzyme preparations, anti-inflammatory drug development), cosmetics (exfoliation, hair conditioning), and feed additives—situations requiring protein modification or hydrolysis under mild conditions.

[0128] (3) Based on the effects and functions of the Wuhai chicken precise active peptide powder prepared above, it can be seen that the Wuhai chicken precise active peptide powder prepared in this application can be used as a functional factor in functional products that improve uterine health.

[0129] (4) The method of this application uses whole black-boned chickens as raw materials and adopts a combination of enzymatic hydrolysis and ultrafiltration. First, the cell structure is destroyed by grinding and homogenizing. Then, a specific combination of enzymatic hydrolysis is used to release the precise active peptides of black-boned chickens. Finally, the precise active peptides of black-boned chickens with the target molecular weight are separated by ultrafiltration. The desired precise active peptides of black-boned chickens can be obtained by simply combining mixing, enzymatic hydrolysis, and filtration. It combines the advantages of multiple technologies and has the advantages of low equipment requirements, simple and easy operation, and high extraction efficiency, which is convenient for large-scale industrial production.

[0130] In summary, this application method extracts, separates, and identifies the active peptides from Wuhai chicken to obtain Wuhai chicken active peptide powder that improves uterine health. These active peptides can be used as raw material components in functional products and can be applied to products that improve uterine health. Furthermore, this application obtains Wuhai chicken active peptides through enzymatic hydrolysis of whole Wuhai chickens. These active peptides can significantly scavenge DPPH and ABTS free radicals, increase / restore LIF content in human endometrial stromal cells, reduce TNF-α secretion in human endometrial stromal cells, promote IL-10 secretion in human endometrial stromal cells, promote the relative mRNA expression levels of LIF, ITGβ3, HOXA10, and Bcl-2, and inhibit the relative mRNA expression levels of TNF-α, IL-1β, and Bax, thus collectively improving uterine health. The amino acid sequences of the precise active peptides from Wuhai chicken were identified using LC-MS / MS analysis. Activity prediction tools and molecular docking were used to screen for amino acid sequences of Wuhai chicken precise active peptides that improve uterine health. These peptides were then artificially synthesized to verify their activity in improving uterine health and to further elucidate their mechanism of action. This provides theoretical support for the prediction, screening, and structure-activity relationship studies of Wuhai chicken precise active peptides in improving uterine health. Specifically, mass spectrometry analysis of the synthesized Wuhai chicken precise active peptides was used to determine their amino acid linkage sequence, molecular docking was used to identify their active sites, and the synthesis was verified to confirm their corresponding activities. This provides a scientific theoretical basis for the production and application of Wuhai chicken precise active peptides and has significant theoretical and practical value for the research of Wuhai chicken precise active peptides.

[0131] It should be noted that: In this article, “~” is used to indicate numerical ranges, and this expression includes two endpoints. The term “Da” as used in this article stands for Dalton, a commonly used unit of molecular weight. The terms “ultrafiltration,” “centrifugation,” and “spray drying” used in this article are conventional names for processing steps in the art, and their names accurately describe the processes, so they will not be repeated here. The alkaline protease (37071), papain, neutral protease, trypsin, complex lipase, alkaline protease (Alcalase 2.4L), acidic protease, and pepsinase used are all commercially available enzymes that can be purchased by those skilled in the art. Human endometrial stromal cells were purchased from Zhejiang Nuobo Biotechnology Co., Ltd.

[0132] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of this application, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application. Furthermore, unless otherwise specified, the raw materials used can also be commercially available products in the art, or prepared by conventional methods in the art; that is, the reagents and instruments used in this embodiment, without specifying the manufacturer, are all conventional products that can be purchased on the market. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them; although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing precise active peptides from Wuhai chicken, characterized in that, Includes the following steps: S1. Pretreatment: After removing the internal organs, mince the black-boned chicken into a paste, add water and mix, and heat-treat at 54-56℃ for 0.5-1.5 hours to obtain a pre-hydrolyzed solution. S2. First hydrolysis: Adjust the pH of the pre-hydrolysate, add alkaline protease, and hydrolyze at 50-60℃ for 1-3 hours. After inactivation of the enzyme, the first hydrolysate is obtained. The alkaline protease is alkaline protease 37071, and its addition amount is 0.5% to 1.5% of the weight of the Wuhai chicken. S3. Second hydrolysis: Adjust the pH of the first hydrolysate, add papain, and hydrolyze at 45-65℃ for 1.5-2.5 hours. After enzyme inactivation, the second hydrolysate is obtained. The amount of papain added is 0.25-0.75% of the weight of the Wuhai chicken. S4, Post-processing: After the second hydrolysate is decolorized and deodorized, and solid-liquid separated, the supernatant is taken and subjected to filtration, ultrafiltration, nanofiltration desalination and concentration and spray drying to obtain powdered Wuhai chicken precise active peptides. Among them, the molecular weight cutoff of ultrafiltration is <5000Da, and the mass content of the concentrated Wuhai chicken precise active peptides is 10-25%.

2. The method for preparing the precise active peptides of Wuhai chicken according to claim 1, characterized in that: Based on the unit mass of protein in Wuhai chicken, the enzyme activity of alkaline protease 37071 is 0.91 to 2.73 million U / g. Based on the unit mass of protein in Wuhai chicken, the enzyme activity of the papain is 0.23 to 0.69 million U / g.

3. The method for preparing the precise active peptides of Wuhai chicken according to claim 1, characterized in that: In step S2, the pH of the prehydrolysate is adjusted to 8.0-9.0, alkaline protease 37071 is added, and the mixture is enzymatically hydrolyzed at 50-60°C for 1-3 hours. After enzyme inactivation, the first hydrolysate is obtained. In step S3, the pH of the first hydrolysate is adjusted to 6.0–7.0, papain is added, and the mixture is hydrolyzed at 45–65°C for 1.5–2.5 hours. After enzyme inactivation, the second hydrolysate is obtained.

4. The method for preparing the precise active peptides of Wuhai chicken according to claim 1, characterized in that, In step S2, the enzyme is inactivated at 84–86°C for 15–20 min to obtain the first hydrolysate. In step S3, the enzyme is inactivated at 84–86°C for 15–20 min to obtain the second hydrolysate.

5. The method for preparing the precise active peptides of Wuhai chicken according to claim 1, characterized in that, In step S1, fresh black-boned chicken is gutted, minced into a paste, mixed with water, and heated at 54-56°C for 0.5-1.5 hours to obtain a pre-hydrolyzed solution; wherein the mass ratio of the paste to water is 1:3 to 1:

5. In step S4, after the second hydrolysate is decolorized and deodorized by activated carbon and separated by centrifugation, the supernatant is taken and treated by diatomaceous earth decolorization, filtration, ultrafiltration, nanofiltration desalination and concentration and spray drying to obtain powdered Wuhai chicken precise active peptides.

6. The method for preparing the precise active peptides of Wuhai chicken according to claim 1, characterized in that: In step S4, the second hydrolysate is cooled to 50-60°C, activated carbon is added for treatment for 0.5-1.5 hours, centrifuged, and the supernatant is decolorized with diatomaceous earth and filtered through a 0.45μm aqueous membrane. The activated carbon is added at a rate of 5.0% to 7.0% of the mass of the black-boned chicken.

7. Five-Black Chicken Precision Active Peptides, characterized in that, It is prepared by the preparation method described in any one of claims 1-6.

8. The precise active peptide from Wuhai chicken according to claim 7, characterized in that, The Wuhai chicken precise active peptide is a composition containing active peptide segments with a relative molecular weight of <5000 Da; it includes at least one active peptide segment with an amino acid sequence of LPQPPQ, IPF, LPF, LGPL, LGPI, IGPL, AVF, FPPDVA, and MPF.

9. A functional product, characterized in that, Its active components include the Wuhai chicken precise active peptide as described in any one of claims 7-8.

10. The functional product according to claim 9, characterized in that, The functional product has at least one of the following functions: (1) Anti-inflammatory; (2) Improves menstrual irregularities; (3) Repair endometrial receptivity.

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