A precise albumin peptide for enhancing immunity and promoting wound healing and a preparation method and application thereof

Abstract

This application discloses a precise albumin peptide that enhances immunity and promotes wound healing, as well as its preparation method and applications, belonging to the field of biotechnology. The precise albumin peptide contains at least one of ADHPF, INF, FNP, FPN, YCPL, YCPI, CLLC, or YCP, and possesses functions such as enhancing immunity, promoting wound healing, and promoting albumin synthesis. Furthermore, the preparation method of the precise albumin peptide provided by this invention has significant advantages such as low equipment investment, simple operation, high product yield, and ease of industrial scale-up, providing a practical and feasible technical solution for the large-scale production of functional active peptides.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a precise albumin peptide that enhances immunity and promotes wound healing, as well as its preparation method and application. Background Technology

[0002] Albumin bioactive peptides are small-molecule bioactive peptides prepared from ovalbumin using modern enzymatic hydrolysis technology. In recent years, they have attracted considerable attention due to their unique nutritional properties and multifunctional bioactivities. Ovalbumin, as the main protein component of egg white (accounting for approximately 54% of total protein), possesses a complete amino acid profile and excellent digestibility and absorption characteristics. Studies have shown that egg white peptides obtained through targeted enzymatic hydrolysis not only retain the nutritional value of the original protein but also exhibit a variety of physiological regulatory functions exceeding those of the intact protein. Their molecular weight is typically less than 1000 Da, making them easily absorbed and less allergenic. They can be directly absorbed through the intestines, significantly improving bioavailability. In terms of functional properties, albumin bioactive peptides have been proven to possess various bioactivities, including immunomodulation, antioxidant, antibacterial, and mineral absorption enhancement. In particular, their mechanisms of action in promoting albumin synthesis and improving liver function have become a hot research topic.

[0003] With advancements in proteomics, bioinformatics, and enzyme engineering, researchers are able to more precisely design and screen albumin bioactive peptides with specific functions. Currently, research on albumin bioactive peptides has shifted from basic activity identification to molecular mechanism analysis and clinical application development, showing broad application prospects in areas such as medical foods, sports nutrition, and functional foods. It also provides new technological pathways for the high-value utilization of poultry egg resources.

[0004] Given that albumin bioactive peptides possess multiple physiological activities and high bioavailability, exploring their specific functional peptide segments is of great significance for developing novel bioactive preparations and realizing the high-value utilization of poultry egg resources. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a precise albumin peptide that enhances immunity and promotes wound healing, as well as its preparation method and application.

[0006] According to a first aspect of the present invention, a precise albumin peptide with functions of enhancing immunity, promoting wound healing, and promoting albumin synthesis is provided, wherein the precise albumin peptide comprises at least one of ADHPF, INF, FNP, FPN, YCPL, YCPI, CLLC, or YCP; the specific sequence of ADHPF is shown in SEQ ID NO.1, the specific sequence of YCPL is shown in SEQ ID NO.2, the specific sequence of YCPI is shown in SEQ ID NO.3, and the specific sequence of CLLC is shown in SEQ ID NO.4.

[0007] Furthermore, the precise albumin peptide comprises at least one of INF, FNP, or YCPL.

[0008] According to a second aspect of the present invention, a method for preparing a precise albumin peptide with functions of enhancing immunity, promoting wound healing, and promoting albumin synthesis is provided, comprising the following steps:

[0009] S1. Mix egg white powder with water and then heat treat to obtain a pretreatment solution;

[0010] S2. Add Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase to the pretreatment solution in sequence for three-stage hydrolysis. After each stage of hydrolysis, the enzymes are inactivated to obtain the hydrolysate.

[0011] S3. The hydrolysate is ultrafiltered and spray-dried to obtain the precise albumin peptide;

[0012] The prepared precise albumin peptide contains at least one of ADHPF, INF, FNP, FPN, YCPL, YCPI, CLLC, or YCP; the specific sequence of ADHPF is shown in SEQ ID NO.1, the specific sequence of YCPL is shown in SEQ ID NO.2, the specific sequence of YCPI is shown in SEQ ID NO.3, and the specific sequence of CLLC is shown in SEQ ID NO.4.

[0013] Furthermore, the amount of Bacillus licheniformis alkaline protease added is 1% to 3% of the mass of the egg white powder; the amount of Bacillus subtilis neutral protease added is 1% to 3% of the mass of the egg white powder; and the amount of aminopeptidase added is 1% to 3% of the mass of the egg white powder.

[0014] Furthermore, the alkaline protease activity of the Bacillus licheniformis is 400,000 U / g; the neutral protease activity of the Bacillus subtilis is 80,000 U / g; and the aminopeptidase activity is 100,000 U / g.

[0015] Furthermore, the hydrolysis conditions for the Bacillus licheniformis alkaline protease are: adjusting the system pH to 8.0–8.5 and hydrolyzing at 50°C–55°C for 1–2 hours; the hydrolysis conditions for the Bacillus subtilis neutral protease are: adjusting the system pH to 6.5–7.5 and hydrolyzing at 50°C–55°C for 1–2 hours; and the hydrolysis conditions for the aminopeptidase are: adjusting the system pH to 6.5–7.5 and hydrolyzing at 50°C–55°C for 1–2 hours.

[0016] Furthermore, the mass ratio of egg white powder to water is 1:(15~30), and the heat treatment conditions are 90℃ for 10~30 min.

[0017] Furthermore, the enzyme inactivation treatment is performed at 85℃~90℃ for 15~20 min, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

[0018] Furthermore, the precise albumin peptide comprises at least one of INF, FNP, or YCPL.

[0019] According to a third aspect of the present invention, the application of the precise albumin peptide as described herein in the preparation of functional products that enhance immunity, promote wound healing, or promote albumin synthesis is proposed.

[0020] According to a fourth aspect of the present invention, the application of a precise albumin peptide prepared by the method described herein in the preparation of functional products for enhancing immunity, promoting wound healing, or promoting albumin synthesis is proposed.

[0021] The beneficial effects of this invention are:

[0022] This invention focuses on three aspects: enhancing immunity, improving wound healing, and promoting albumin synthesis. It develops a precise albumin peptide suitable for improving the body's immunity, accelerating wound repair, and stimulating liver albumin synthesis. The precise albumin peptide prepared by this invention has the effects of enhancing immunity, promoting wound healing, and promoting albumin synthesis, and is particularly suitable for postoperative recovery, patients with chronic wounds, and patients with hypoalbuminemia. Furthermore, the precise albumin peptide has no obvious bitter or astringent taste, has a good taste and flavor, and is beneficial to improving the user experience. The solution of this invention uses a simple combination of operations such as mixing, pretreatment, enzymatic hydrolysis, and filtration to obtain the desired precise albumin peptide. It has low equipment requirements, a simple and easy-to-operate process, and is suitable for large-scale industrial production. Attached Figure Description

[0023] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention.

[0024] Figure 1 This is a graph showing the phagocytic rate of neutral red blood cells in macrophages according to a specific embodiment of the present invention;

[0025] Figure 2 This is a graph showing the NO content in macrophages according to a specific embodiment of the present invention;

[0026] Figure 3 This is a graph showing the concentration of TNF-α in macrophages according to a specific embodiment of the present invention;

[0027] Figure 4 This is a graph showing the concentration of IL-6 in macrophages according to a specific embodiment of the present invention;

[0028] Figure 5This is a diagram showing the expression level of the IL-6 gene in macrophages according to a specific embodiment of the present invention.

[0029] Figure 6 This is a diagram showing the expression level of the TNF-α gene in macrophages according to a specific embodiment of the present invention.

[0030] Figure 7 This is a diagram showing the expression level of the IL-1β gene in macrophages according to a specific embodiment of the present invention.

[0031] Figure 8 This is a diagram showing the expression level of the COX-2 gene in macrophages according to a specific embodiment of the present invention.

[0032] Figure 9 This is a diagram showing the hydroxyproline content in fibroblasts according to a specific embodiment of the present invention.

[0033] Figure 10 This is a diagram showing the expression level of the COL-1 gene in fibroblasts according to a specific embodiment of the present invention.

[0034] Figure 11 This is a diagram showing the expression level of the TGF-β gene in fibroblasts according to a specific embodiment of the present invention.

[0035] Figure 12 This is a diagram showing the expression level of the Ang-1 gene in fibroblasts according to a specific embodiment of the present invention.

[0036] Figure 13 This is a diagram showing the expression level of the CTGF gene in fibroblasts according to a specific embodiment of the present invention.

[0037] Figure 14 The graph shows the relative albumin content in HepG2 cells according to a specific embodiment of the present invention.

[0038] Figure 15 This is a diagram showing the expression level of the HNF4α gene in HepG2 cells according to a specific embodiment of the present invention.

[0039] Figure 16 This is a diagram showing the expression level of the SIRT1 gene in HepG2 cells according to a specific embodiment of the present invention.

[0040] Figure 17 This is a graph showing the expression level of the KLF5 gene in HepG2 cells according to a specific embodiment of the present invention.

[0041] Figure 18 This is a diagram showing the expression level of the H-CEBPα gene in HepG2 cells according to a specific embodiment of the present invention.

[0042] Figure 19 This is a 2D diagram of the docking of INF and TLR4 molecules in a specific embodiment of the present invention;

[0043] Figure 20 This is a 2D diagram of the docking of FPN and TLR4 molecules in a specific embodiment of the present invention;

[0044] Figure 21 This is a 2D diagram of the docking of YCPL and TLR4 molecules in a specific embodiment of the present invention;

[0045] Figure 22 This is a 2D diagram of the docking of INF and VEGFR2 molecules in a specific embodiment of the present invention;

[0046] Figure 23 This is a 2D diagram of the docking of FPN and VEGFR2 molecules in a specific embodiment of the present invention;

[0047] Figure 24 This is a 2D diagram of the docking of YCPL and VEGFR2 molecules in a specific embodiment of the present invention;

[0048] Figure 25 This is a 2D diagram of the docking of INF and HNF-4α molecules in a specific embodiment of the present invention;

[0049] Figure 26 This is a 2D diagram of the docking of FPN and HNF-4α molecules in a specific embodiment of the present invention;

[0050] Figure 27 This is a 2D diagram of the docking of YCPL with HNF-4α molecules in a specific embodiment of the present invention;

[0051] Reference numbers: 1-ILE A:450; 2-ASN A:497; 3-ASN A:448; 4-CYS A:1045; 5-VAL A:899; 6-LEU A:889; 7-GLU A:885; 8-MET A:342; 9-VAL A:255; 10-MET A:252; 11-GLN A:345; 12-SER A:256; 13-VAL A:178. Detailed Implementation

[0052] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0053] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0054] Given that albumin bioactive peptides possess multiple physiological activities and high bioavailability, exploring their specific functional peptide segments is of great significance for developing novel bioactive preparations and realizing the high-value utilization of poultry egg resources. Therefore, the first objective of this invention is to propose a precise albumin peptide that enhances immunity, promotes wound healing, and promotes albumin synthesis.

[0055] The second objective of this invention is to propose a method for preparing a precise albumin peptide that enhances immunity, promotes wound healing, and promotes albumin synthesis. The preparation method has low equipment requirements, is simple and easy to operate, and is suitable for large-scale industrial production.

[0056] The third objective of this invention is to propose the application of the precise albumin peptide as described above in the preparation of functional products that enhance immunity, promote wound healing, or promote albumin synthesis.

[0057] A fourth objective of this invention is to propose the application of a precise albumin peptide prepared by the method described above in the preparation of functional products for enhancing immunity, promoting wound healing, or promoting albumin synthesis.

[0058] To achieve the above objectives, this invention provides a precise albumin peptide with functions of enhancing immunity, promoting wound healing, and promoting albumin synthesis. The precise albumin peptide comprises at least one or more of ADHPF, INF, FNP, FPN, YCPL, YCPI, CLLC, or YCP. The specific sequence of ADHPF is shown in SEQ ID NO.1, the specific sequence of YCPL is shown in SEQ ID NO.2, the specific sequence of YCPI is shown in SEQ ID NO.3, and the specific sequence of CLLC is shown in SEQ ID NO.4. The precise albumin peptide comprises at least one or more of INF, FNP, or YCPL.

[0059] This invention also provides a method for preparing precise albumin peptides with functions of enhancing immunity, promoting wound healing, and promoting albumin synthesis. The specific steps are as follows:

[0060] Step 1

[0061] Mix egg white powder and water evenly, with the mass ratio of egg white powder to pure water being 1:(15-30). Heat treat at 90℃ for 10-30 minutes to obtain a pretreated solution.

[0062] Step Two

[0063] (1) Add Bacillus licheniformis alkaline protease (enzyme activity 400,000 U / g) to the pretreatment solution, adjust the pH of the system to 8.0-8.5, hydrolyze at 50℃-55℃ for 1-2 h, and then inactivate the enzyme at 85℃-90℃ for 15-20 min to obtain the first hydrolysate; wherein, the amount of Bacillus licheniformis alkaline protease added is (1-3)% of the mass of egg white powder.

[0064] (2) Add Bacillus subtilis neutral protease (enzyme activity 80,000 U / g) to the first hydrolysate, adjust the pH of the system to 6.5-7.5, hydrolyze at 50℃-55℃ for 1-2 h, and then inactivate the enzyme at 85℃-90℃ for 15-20 min to obtain the second hydrolysate; wherein, the amount of Bacillus subtilis neutral protease added is (1-3)% of the mass of egg white powder.

[0065] (3) Add aminopeptidase (enzyme activity 100,000 U / g) to the second hydrolysate, adjust the pH of the system to 6.5-7.5, hydrolyze at 50℃-55℃ for 1-2 h, and then inactivate the enzyme at 85℃-90℃ for 15-20 min to obtain the third hydrolysate; wherein, the amount of aminopeptidase added is (1-3)% of the mass of egg white powder.

[0066] Step 3

[0067] The third hydrolysate was cooled to room temperature and filtered. After filtration, the filtrate with a molecular weight of less than 5000 Da was obtained by ultrafiltration and then spray-dried to obtain the precise albumin peptide.

[0068] Step Four

[0069] Activity detection and cell experiments on precise albumin peptides revealed that they can enhance immunity, promote wound healing, and promote albumin synthesis.

[0070] It should be noted that the enzymes with the same name used in the following examples and comparative examples are from the same commercially available company and brand, meaning that the enzyme activities of the enzymes with the same name used in each example and comparative example are consistent. The enzyme activities of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, aminopeptidase, alkaline protease 1, alkaline protease 2, and neutral protease 1 are 400,000 U / g, 80,000 U / g, 100,000 U / g, 240,000 U / g, 200,000 U / g, 500,000 U / g, 600,000 U / g, 600,000 U / g, papain, and 300,000 U / g, respectively.

[0071] Example 1

[0072] 1. Take 100g of egg white powder (80% protein content), mix it evenly with 2000g of pure water, and heat-treat at 90℃ for 20 minutes;

[0073] 2. Heat to 55℃, adjust pH to 8.5 with sodium hydroxide, add 1g of Bacillus licheniformis alkaline protease to hydrolyze for 2h, and heat to 85℃ to inactivate enzyme for 20min;

[0074] 3. Cool down to 55℃, adjust the pH to 7.0 with hydrochloric acid, add 2g of Bacillus subtilis neutral protease to hydrolyze for 1 hour, and heat up to 85℃ to inactivate the enzyme for 20 minutes.

[0075] 4. After cooling to 55℃, adjust the pH to 6.5 with hydrochloric acid, add 1g of aminopeptidase to hydrolyze for 1 hour, and then heat to 85℃ to inactivate the enzyme for 20 minutes.

[0076] 5. After filtration, ultrafiltration is performed to obtain a precise albumin peptide solution with a molecular weight of less than 5000 Da;

[0077] 6. Precise albumin peptide powder is obtained through spray drying.

[0078] Example 2

[0079] 1. Take 100g of egg white powder (80% protein content), mix it evenly with 1500g of pure water, and heat-treat at 90℃ for 10 minutes;

[0080] 2. Heat to 50℃, adjust pH to 8.0 with sodium hydroxide, add 3g of Bacillus licheniformis alkaline protease for 1 hour, and heat to 90℃ for 15 minutes to inactivate the enzyme.

[0081] 3. After cooling to 50℃, adjust the pH to 7.5 with hydrochloric acid, add 1g of Bacillus subtilis neutral protease to hydrolyze for 2h, and then heat to 85℃ to inactivate the enzyme for 20min.

[0082] 4. After cooling to 50℃, adjust the pH to 7.5 with sodium hydroxide, add 3g of aminopeptidase to hydrolyze for 1 hour, and then heat to 85℃ to inactivate the enzyme for 20 minutes.

[0083] 5. After filtration, ultrafiltration is performed to obtain a precise albumin peptide solution with a molecular weight of less than 5000 Da;

[0084] 6. Precise albumin peptide powder is obtained through spray drying.

[0085] Example 3

[0086] 1. Take 100g of egg white powder (80% protein content), mix it evenly with 3000g of pure water, and heat-treat at 90℃ for 30min;

[0087] 2. Heat to 55℃, adjust pH to 8.0 with sodium hydroxide, add 2g of Bacillus licheniformis alkaline protease for 1 hour, and heat to 90℃ for 15 minutes to inactivate the enzyme.

[0088] 3. After cooling to 50℃, adjust the pH to 6.5 with hydrochloric acid, add 3g of Bacillus subtilis neutral protease to hydrolyze for 1 hour, and then heat to 85℃ to inactivate the enzyme for 20 minutes.

[0089] 4. After heating to 55℃, adjust the pH to 6.5 with sodium hydroxide, add 1g of aminopeptidase to hydrolyze for 2h, and then heat to 85℃ to inactivate the enzyme for 20min.

[0090] 5. After filtration, ultrafiltration is performed to obtain a precise albumin peptide solution with a molecular weight of less than 5000 Da;

[0091] 6. Precise albumin peptide powder is obtained through spray drying.

[0092] Comparative Example 1

[0093] The hydrolysis process in this comparative example uses Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease. The only difference from Example 1 is that the amount of Bacillus subtilis neutral protease added in step 3 is changed to 3.25g, and step 4 is removed. The remaining operations and processes are the same as in Example 1.

[0094] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0095] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0096] Therefore, in this comparative example, the amount of Bacillus licheniformis alkaline protease added is 1g, which, based on the protein content in the egg white powder, represents an enzyme addition of 5000 U / g; the amount of Bacillus subtilis neutral protease added is 3.25g, which, based on the protein content in the egg white powder, represents an enzyme addition of 3250 U / g; this is the same as the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase used in Example 1.

[0097] Comparative Example 2

[0098] The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and aminopeptidase. The only difference from Example 1 is that the amount of aminopeptidase added in step 4 is changed to 2.6g, and step 3 is removed. The rest of the operation and process are the same as in Example 1.

[0099] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0100] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0101] Therefore, in this comparative example, the amount of Bacillus licheniformis alkaline protease added is 1g, which, based on the protein content in the egg white powder, represents an enzyme addition of 5000 U / g; the amount of aminopeptidase added is 2.6g, which, based on the protein content in the egg white powder, represents an enzyme addition of 3250 U / g; this is the same as the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase used in Example 1.

[0102] Comparative Example 3

[0103] The hydrolysis process in this comparative example uses papain and bromelain. The only difference from Example 1 is that: the alkaline protease of Bacillus licheniformis in step 2 is replaced with papain, pH 7.0, and the amount added is 0.3g; the neutral protease of Bacillus subtilis in step 3 is replaced with bromelain, pH 7.0, and the amount added is 1.4g; step 4 is removed, and the remaining operations and processes are the same as in Example 1.

[0104] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0105] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0106] Therefore, in this comparative example, the amount of papain added was 0.3g, which is 3000 U / g based on the protein content of egg white powder; the amount of bromelain added was 1.4g, which is 5250 U / g based on the protein content of egg white powder; and the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase used in Example 1 was equal to that used in Example 1.

[0107] Comparative Example 4

[0108] The hydrolysis process in this comparative example uses only alkaline protease 1 and proline endonuclease. The difference from Example 1 is that: the Bacillus licheniformis alkaline protease in step 2 of Example 1 is replaced with alkaline protease 1, and the amount added is changed to 1.5g; the Bacillus subtilis neutral protease in step 3 is replaced with proline endonuclease, and the amount added is changed to 2g; step 4 is removed, and the remaining operations and processes are the same as in Example 1.

[0109] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0110] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0111] Therefore, in this comparative example, the amount of alkaline protease 1 added was 1.5g, which is 4500 U / g based on the protein content of egg white powder; the amount of proline endopeptidase added was 2g, which is 3750 U / g based on the protein content of egg white powder; which is the same as the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase used in Example 1.

[0112] Comparative Example 5

[0113] The hydrolysis process in this comparative example uses alkaline protease 2 and neutral protease 1. The difference from Example 1 is that: the Bacillus licheniformis alkaline protease in step 2 of Example 1 is replaced with alkaline protease 2, and the amount added is changed to 1.5g; the Bacillus subtilis neutral protease in step 3 is replaced with neutral protease 1, and the amount added is changed to 0.72g; step 4 is removed, and the remaining operations and processes are the same as in Example 1.

[0114] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0115] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0116] Therefore, in this comparative example, the amount of alkaline protease 2 added was 1.5g, which is 3750 U / g based on the protein content of egg white powder; the amount of neutral protease 1 added was 2g, which is 4500 U / g based on the protein content of egg white powder; which is the same as the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase used in Example 1.

[0117] Comparative Example 6

[0118] The difference from Example 1 is that steps 2 and 3 are swapped, i.e., one step of hydrolysis to Bacillus subtilis neutral protease, two steps of hydrolysis to Bacillus licheniformis alkaline protease, and three steps of hydrolysis to aminopeptidase. The remaining operations and processes are the same as in Example 1.

[0119] Since the amount of enzyme added was not changed, the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase used in this comparative example is the same as that used in Example 1.

[0120] Comparative Example 7

[0121] The difference from Example 1 is that steps 2 and 4 are swapped, i.e., one step of hydrolysis to aminopeptidase, two steps of hydrolysis to Bacillus licheniformis alkaline protease, and three steps of hydrolysis to Bacillus subtilis neutral protease. The remaining operations and processes are the same as in Example 1.

[0122] Since the amount of enzyme added was not changed, the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase used in this comparative example is the same as that used in Example 1.

[0123] Comparative Example 8

[0124] The hydrolysis process in this comparative example uses Bacillus licheniformis alkaline protease and acidic protease. The difference from Example 1 is that: the amount of Bacillus licheniformis alkaline protease added in step 2 is changed to 0.75g; the neutral protease of Bacillus in step 3 is changed to acidic protease, with an added amount of 0.6g, a temperature of 37℃, and a pH of 2.5; step 4 is removed, and the remaining operations and processes are the same as in Example 1.

[0125] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0126] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0127] Therefore, in this comparative example, the amount of Bacillus licheniformis alkaline protease added was 0.75g, which is 3750 U / g based on the protein content of egg white powder; the amount of acidic protease added was 0.6g, which is 4500 U / g based on the protein content of egg white powder; which is the same as the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase used in Example 1.

[0128] Comparative Example 9

[0129] The hydrolysis process in this comparative example uses a single egg white hydrolase. The difference from Example 1 is that the Bacillus licheniformis alkaline protease in step 2 is replaced with egg white hydrolase, the amount added is changed to 1.1g, and the pH is 9.0; steps 3 and 4 are removed, and the remaining operations and processes are the same as in Example 1.

[0130] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 1g. Based on the protein content (80%) in the egg white powder, the enzyme addition amount of Bacillus licheniformis alkaline protease was 5000 U / g. The specific calculation process is as follows:

[0131] The enzyme activity of 1g of Bacillus subtilis neutral protease is 400,000 U / g × 1g = 400,000 U. Converted to the amount of enzyme added per gram of egg white powder, this translates to 400,000 U / (100g × 80%)g = 5000 U / g (the denominator 'g' represents the amount of protein per gram of egg white powder). Similarly, adding 2g of Bacillus subtilis neutral protease, based on the amount of protein in egg white powder, results in an enzyme addition of 2000 U / g; adding 1g of aminopeptidase, based on the amount of protein in egg white powder, results in an enzyme addition of 1250 U / g.

[0132] Therefore, in this comparative example, the amount of egg white hydrolase added was 1.1g, which is 8250U / g based on the protein content in the egg white powder; this is the same as the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase used in Example 1.

[0133] Example 4

[0134] This embodiment describes the activity detection and cell experiments of the precise albumin peptide products obtained in Examples 1-3 and Comparative Examples 1-9.

[0135] 1. Immunomodulatory capabilities of precise albumin peptides

[0136] 1.1 Cytotoxicity assay

[0137] Cell viability was determined using the CCK8 assay (Cell Counting Kit-8). The results were analyzed for significance, and the highest non-toxic concentration of 1 mg / mL was selected for drug administration to the cells.

[0138] 1.2 Phagocytic Activity Test

[0139] The effect of precise albumin peptide on phagocytosis was evaluated using the neutral red uptake assay. Log-phase RAW264.7 macrophages were collected and cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a cell density of 1.0 × 10⁶ cells / mL.5 Cells were seeded at 100 cells / mL in 96-well plates and cultured at 37°C for 24 hours in a CO2 incubator to allow cell adhesion. The 96-well plates were then removed, the culture medium was aspirated, and the following medium was used as experimental groups: 1 mg / mL of precise albumin peptide solution was added; 1 μg / mL of lipopolysaccharide (LSP) solution was added; fresh culture medium was added; and no cells were added to the medium as the blank group. Each group had four replicates. After 24 hours of culture, the supernatant was discarded, and 100 μL of 0.1% neutral red solution was added to each well for staining. After staining, the staining solution was discarded, and the cells were washed three times with PBS. Cells were lysed with 100 μL of lysis buffer (acetic acid: ethanol = 1:1, v / v) at room temperature for 1 hour, and the absorbance was measured at 540 nm. The neutral red phagocytosis rate was calculated according to formula (1):

[0140] Attrition rate = (A1 - A0) / (A2 - A0) ×100% (1)

[0141] In the formula:

[0142] A1: Absorbance values ​​of experimental groups with added precise albumin peptide or lipopolysaccharide (LSP) solution;

[0143] A2: Absorbance of the control group without the addition of precise albumin peptide solution;

[0144] A0: Absorbance of the blank control group without cell access.

[0145] Neutral red is a weakly basic dye that can be taken up by phagocytes into acidic organelles such as lysosomes. In an acidic environment, neutral red aggregates and develops its color. By detecting the intracellular neutral red content, the phagocytic activity of cells can be indirectly reflected. The degree of phagocytosis is typically quantitatively analyzed by measuring absorbance at a specific wavelength using a spectrophotometer.

[0146] like Figure 1 As shown, compared with the control group, the degree of cell phagocytosis in Example 1, Comparative Example 6 and Comparative Example 5 was significantly improved.

[0147] 1.3 NO content test

[0148] Collect macrophages at a rate of 2 × 10 4Cells were seeded at a density of 10 cells / mL in 24-well plates and incubated for 24 h. When the cells reached 70%–75% confluence, the safe concentration of the cell culture medium selected by CCK-8 screening was added to the control and comparative samples. A blank control group and an LPS group (Model group) were also set up, with 3 replicates in each group. After 24 h of incubation, the supernatant was discarded, and 50 μL of culture medium was aspirated from each well into a 96-well plate. The NO release was measured according to the NO detection reagent instructions.

[0149] NO is an important gaseous signaling molecule involved in numerous physiological processes, including vasodilation, neurotransmission, and immune regulation. When immune cells are stimulated by pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS), intracellular signal transduction pathways are activated, leading to upregulation of the iNOS gene and the production of large amounts of NO. Appropriate amounts of NO can enhance the bactericidal and phagocytic functions of macrophages, promote the proliferation and differentiation of T lymphocytes, and regulate the maturation and antigen presentation functions of dendritic cells.

[0150] like Figure 2 As shown, compared with the control group, the NO content of Example 1, Comparative Example 5, Comparative Example 6, and Comparative Example 8 was significantly increased.

[0151] 1.4 Immune factor content test

[0152] Collect macrophages at a rate of 2 × 10 4 Cells were seeded at a density of 10 cells / mL in 24-well plates and incubated for 24 h. When the cells reached 70%–75% confluence, the safe concentration of the cell culture medium selected by CCK-8 screening was added to the control and comparative samples. A blank control group and an LPS group (model group) were also set up, with 3 replicates in each group. After 24 h of incubation, the supernatant was discarded, and 100 μL of culture medium was aspirated from each well into a 96-well plate. The cytokine release was measured according to the ELISA kit (IL-6 and TNF-α) instructions.

[0153] TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6) are cytokines secreted by immune cells, primarily originating from monocytes, macrophages, and T lymphocytes. In the early stages of an immune response, the secretion levels of both TNF-α and IL-6 begin to rise simultaneously. TNF-α mainly enhances the phagocytic and killing abilities of macrophages and the antigen-presenting function of immune cells to initiate the immune response. IL-6 promotes the proliferation and differentiation of B cells and T lymphocytes, leading to antibody secretion.

[0154] like Figure 3 and Figure 4As shown, the TNF-α concentrations in Comparative Example 4, Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 6, Comparative Example 7, and Comparative Example 9 were significantly higher than those in the control group; the IL-6 concentration in Comparative Example 6 was significantly higher than that in the control group.

[0155] 1.5 Immunological factor gene expression level test

[0156] Collect macrophages at a rate of 2 × 10 4 Cells were seeded at a density of [number] cells / mL in 24-well plates and incubated for 24 h. When the cells reached 70%–75% confluence, the safe concentration of the CCK-8 assay and comparative samples was added. A blank control group (control group) and an LPS group (model group) were also included, with three replicates per group. Cells were incubated for another 24 h. Total RNA was then extracted using the Simply P Total RNA Extraction Kit (BioFlux). 2 µL of sterile, enzyme-free water was dropped onto a Nanodrop one plate to determine RNA concentration and purity. A 20 µL qRT-PCR reaction system was established. PCR amplification conditions were: pre-denaturation 95℃, 30 s; denaturation 95℃, 15 s; annealing 54℃, 30 s; extension 72℃, 45 s; 40 cycles. Melting curve conditions were: 95℃, 30 s; 60℃, 1 min; fluorescence signal was collected every 5℃ increase. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., with GAPDH as an internal reference. Primer sequence information is shown in Table 1.

[0157] Table 1 Primer sequence information for Example 4

[0158] ;

[0159] IL-6 is a multifunctional cytokine, primarily secreted by macrophages, playing a dual role in immune regulation. It can promote B cell differentiation and antibody production, enhancing humoral immunity, and also induce the synthesis of acute-phase proteins, participating in inflammatory responses. Moderately increasing IL-6 levels helps enhance the body's resistance to infection, such as... Figure 5 As shown, the expression levels of IL-6 gene were significantly increased in Comparative Examples 5 and 7 compared to the control group.

[0160] TNF-α is a core regulator of the inflammatory response, released by activated macrophages. It enhances the phagocytic function of macrophages, activates the NF-κB pathway, and promotes the secretion of other inflammatory factors, thereby strengthening the innate immune response. Moderate expression of TNF-α helps clear pathogens, such as... Figure 6As shown, compared with the control group, the expression levels of TNF-α gene in Comparative Example 4, Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 6, Comparative Example 7 and Comparative Example 9 were significantly increased.

[0161] IL-1β is a potent pro-inflammatory cytokine produced upon activation of the inflammasome, participating in inflammatory responses such as fever and neutrophil recruitment. It can synergistically amplify the immune response with TNF-α and IL-6, and promote Th17 cell differentiation, enhancing defense against pathogens, such as... Figure 7 As shown, compared with the control group, the expression levels of IL-1β gene in Comparative Example 4, Example 1 and Comparative Example 7 were significantly increased.

[0162] COX-2 is a key enzyme in prostaglandin synthesis, catalyzing the production of inflammatory mediators such as PGE2, which are involved in fever, pain, and vasodilation. It plays a dual role in immune regulation: on the one hand, it promotes the release of inflammatory factors; on the other hand, high concentrations of PGE2 may inhibit excessive immune responses. For example... Figure 8 As shown, compared with the control group, the expression level of COX-2 gene in comparative examples 5 and 7 was significantly increased.

[0163] 2. The effect of precise albumin peptides on wound healing

[0164] Fibroblasts are the core functional cells for wound repair; therefore, fibroblasts were used to measure the effect of precise albumin peptides on wound healing. Cell viability was determined using the CCK8 assay (Cell Counting Kit-8). The results were analyzed for significance, and the highest non-toxic concentration of 1 mg / mL was selected for drug administration to the cells.

[0165] 2.1 Determination of hydroxyproline content

[0166] HSF cells were digested, centrifuged, and resuspended, then injected at a dose of 1×10⁻ 5 After seeding 6-well plates at a density of cells / mL and incubating for 12-16 hours, 1 mg / mL of sample was added and incubated for another 24 hours. The cell supernatant was then collected, centrifuged at 10,000 rpm / min at 4°C for 20 minutes to remove cell debris, and stored at -80°C for later use. The hydroxyproline assay was performed according to the Nanjing Jiancheng Hydroxyproline Reagent Kit method.

[0167] The hydroxyproline content in fibroblasts directly reflects their ability to synthesize collagen, a key structural component of wound repair. Elevated hydroxyproline levels indicate active collagen synthesis, promoting granulation tissue formation and wound contraction, thus accelerating healing; conversely, decreased levels suggest insufficient collagen deposition, potentially leading to delayed healing or chronic wounds (such as diabetic ulcers). Figure 9As shown, the hydroxyproline content in Examples 1, 5, 7 and 8 was higher than that in the control group, indicating that the precise albumin peptides in Examples 1, 5, 7 and 8 can all promote the production of hydroxyproline in fibroblasts.

[0168] 2.2 Determination of expression levels of key genes in fibroblasts

[0169] Collect HSF cells at 2 × 10⁻⁶ 4 Cells were seeded at a density of [number] cells / mL in 24-well plates and incubated for 12-16 h. When the cells reached 70-75% confluence, a safe concentration of the sample selected by CCK-8 screening was added. A blank control group was also included. Each group had three replicates, and the cells were incubated for 24 h. Total RNA was then extracted using the Simply P Total RNA Extraction Kit (BioFlux). 2 µL of sterile, enzyme-free water was dropped onto a Nanodrop one plate to determine RNA concentration and purity. A 20 µL qRT-PCR reaction system was established. The PCR amplification conditions were: pre-denaturation 95℃ for 30 s; denaturation 95℃ for 15 s; annealing 54℃ for 30 s; extension 72℃ for 45 s; 40 cycles. The melting curve conditions were: 95℃ for 30 s, 60℃ for 1 min, with fluorescence signals collected every 5℃ increase. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., with GAPDH as an internal reference. Primer sequence information is shown in Table 2.

[0170] Table 2 Primer sequence information for Example 5

[0171] ;

[0172] COL-1 (type I collagen) is a major component of the extracellular matrix (ECM), synthesized by skin fibroblasts, providing mechanical strength and structural support to the skin, such as... Figure 10 As shown, the expression levels of COL-1 in Examples 1, 5, 7, and 8 were all higher than those in the control group, indicating that the precise albumin peptides of Examples 1, 5, 7, and 8 can all increase the expression of COL-1 in fibroblasts, thereby promoting its synthesis. Among them, the expression level in Example 1 was higher.

[0173] TGF-β (transforming growth factor-β) is a core factor regulating fibroblast activation, promoting COL-1 synthesis and myofibroblast differentiation (through α-SMA expression). Figure 11As shown, the expression levels of TGF-β in Examples 1, 5, 7, and 8 were all increased compared to the control group, indicating that the precise albumin peptides of Examples 1, 5, 7, and 8 can all increase the expression of TGF-β in fibroblasts, thereby promoting COL-1 synthesis and myofibroblast differentiation. Among them, the expression level in Example 1 was higher.

[0174] Ang-1 (angiopoietin-1) optimizes local blood supply to wounds by stabilizing endothelial cell junctions in newly formed blood vessels, reducing vascular leakage, and enhancing microvascular maturation, in synergy with VEGF. Increased expression of Ang-1 during the healing process helps improve tissue perfusion, and is particularly crucial in chronic wounds or ischemic injuries. Figure 12 As shown, the expression levels of Ang-1 in Examples 1, 5, 7 and 8 were all increased compared to the control group, indicating that the precise albumin peptides of Examples 1, 5, 7 and 8 can all increase the expression of Ang-1 in fibroblasts.

[0175] CTGF (connective tissue growth factor) is a downstream effector molecule of TGF-β, amplifying the pro-fibrotic signaling of TGF-β and directly stimulating COL-1 deposition and fibroblast proliferation. Its high expression is closely related to pathological scars (such as keloids) and organ fibrosis. Figure 13 As shown, the expression levels of CTGF in Examples 1, 5, 7 and 8 were all lower than those in the control group, indicating that CTGF was inhibited, which prevented excessive deposition of extracellular matrix (ECM) and reduced the risk of scarring.

[0176] The above results demonstrate that precise albumin peptides can effectively promote the early stages of wound repair (by upregulating COL-1 and TGF-β to enhance ECM synthesis and fibroblast activation, while increasing Ang-1 to improve vascular stability), and at the same time, avoid the risk of excessive fibrosis by selectively inhibiting CTGF. This "repair-fibrosis decoupling" characteristic ensures both the matrix reconstruction and vascular support required for rapid wound closure, and significantly reduces the possibility of pathological scar formation, demonstrating ideal healing potential.

[0177] 3. The effect of precise albumin peptides on albumin synthesis

[0178] 3.1 Effect of precise albumin peptides on albumin content in HepG2 cells

[0179] HepG2 (human liver cancer cell line) cells were collected at a concentration of 5 × 10⁻⁶. 5Cells were seeded at a density of 10000 cells / mL in 12-well plates and incubated for 12-16 hours. When the cells reached 70-75% confluence, a safe concentration of the sample selected by CCK-8 screening was added. A blank control group and a positive control group (dexamethasone: its main effect on HepG2 cells is to promote the differentiation and maintenance of liver-specific functions. As a glucocorticoid, it enhances the expression of liver-specific genes such as albumin and tyrosine aminotransferase (TAT) by activating glucocorticoid receptor (GR) and upregulating liver-related transcription factors (such as HNF4α and C / EBPα)). Each group was divided into 3 replicates. After culturing for 24 hours, the cell supernatant was collected, centrifuged at 10,000 rpm / min for 20 minutes at 4°C to remove cell debris, and stored at -80°C for later use. The assay was performed according to the method of the human albumin protein-linked immunosorbent assay kit (ELISA).

[0180] like Figure 14 As shown, compared with the blank group, the relative content of albumin in HepG2 cells treated with Example 1, Comparative Example 5, Comparative Example 7 and Comparative Example 8 all increased to varying degrees, indicating that the precise albumin peptide can promote the synthesis and secretion of albumin in HepG2 cells.

[0181] 3.2 Determination of expression levels of key genes in HepG2 cells

[0182] HepG2 cells were collected at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in 12-well plates and incubated for 12-16 hours. When the cells reached 70-75% confluence, a safe concentration of the sample selected by CCK-8 screening was added. A blank control group and a positive control group were also included, with three replicates per group. Cells were incubated for 24 hours. Total RNA was then extracted using the Simply P Total RNA Extraction Kit (BioFlux). 2 µL of sterile, enzyme-free water was dropped onto a Nanodrop One plate to determine RNA concentration and purity. A 20 µL qRT-PCR reaction system was established. PCR amplification conditions were: pre-denaturation 95℃ for 30 s; denaturation 95℃ for 15 s; annealing 54℃ for 30 s; extension 72℃ for 45 s; 40 cycles. Melting curve conditions were: 95℃ for 30 s; 60℃ for 1 min; fluorescence signal was collected every 5℃ increase. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., with GAPDH as an internal reference. Primer sequence information is shown in Table 3.

[0183] Table 3 Primer sequence information for Example 6

[0184] ;

[0185] HNF4α (hepatocyte nuclear factor 4α) is a key liver-specific transcription factor in HepG2 cells (human hepatocellular carcinoma cell line) that directly regulates the expression of the albumin (ALB) gene. It activates the transcription of the albumin gene by binding to a specific sequence in the promoter region, thus maintaining the liver's synthetic function. Figure 15 As shown, compared with the control group, the expression level of HNF4α in Example 1 and the other comparative examples was increased to varying degrees, with the expression level in Example 1 being the highest.

[0186] SIRT1 (Silencing Information Regulator 1) indirectly affects albumin synthesis in HepG2 cells by deacetylation of the HNF4α transcription factor. SIRT1 activation enhances HNF4α stability and promotes albumin expression. Figure 16 As shown, compared with the control group, the expression level of SIRT1 in Example 1 and the other comparative examples was increased to varying degrees, with the expression level of Example 1 being the highest.

[0187] KLF5 (Krüppel-like factor 5) is mainly involved in proliferation and metabolic regulation in HepG2 cells, but its direct effect on albumin is relatively weak. Studies have shown that KLF5 may indirectly affect hepatocyte differentiation status by regulating the PPARγ or Wnt / β-catenin pathway, thereby interfering with HNF4α-mediated albumin expression. Figure 17 As shown, compared with the control group, the SIRT1 expression levels in Example 1 and the other comparative examples were reduced to varying degrees. Among them, the expression levels in Example 1, Comparative Example 7 and Comparative Example 8 were almost the same as those in the positive control group, while the expression level in Comparative Example 5 was slightly higher.

[0188] H-CEBPα (CCAAT / enhancer-binding protein α) is closely related to hepatocyte differentiation and metabolic function in HepG2 cells, but its regulatory effect on albumin is weaker than that of HNF4α. H-CEBPα can maintain the expression of hepatocyte-specific genes (including albumin) through synergistic action with HNF4α. Figure 18 As shown, compared with the control group, except for Comparative Example 7, the expression level of H-CEBPα in Example 1 and the other comparative examples was increased to varying degrees, with Example 1 showing the highest expression level.

[0189] 4. Peptide screening

[0190] Peptide screening: Raw files acquired by mass spectrometry were searched in a database to screen for peptides with an abundance greater than 49,000,000. Activity prediction was performed on the fragments, and peptides with a result >0.7 were selected. Simultaneously, abundance ranking was used for further screening, resulting in 8 peptides. Peptide-related information is listed in Table 4.

[0191] Table 4 Peptide Information

[0192] ;

[0193] Note: Peptide<ProteinMetrics Confidential> : Amino acid sequence of the identified peptide; Mass: molecular weight of the peptide; Score: activity prediction score; Lenth: number of amino acids in the peptide; RT: retention time of the peptide, in min; -10 lgP: significance score of the peptide identified by database search or de novo sequencing; Intensity: peptide abundance.

[0194] Molecular docking

[0195] TLR4 (Toll-like receptor 4) is a key receptor of the innate immune system. It primarily recognizes pathogen-associated molecules such as bacterial lipopolysaccharide (LPS), activates the NF-κB and MAPK signaling pathways, and induces the release of pro-inflammatory cytokines (such as TNF-α and IL-6), playing a central role in anti-infection and inflammatory responses. Overactivation of TLR4 may lead to sepsis or autoimmune diseases, while inhibiting TLR4 signaling can reduce inflammatory damage; therefore, TLR4 is an important target for immune regulation.

[0196] VEGFR2 (vascular endothelial growth factor receptor 2) is a key regulator of angiogenesis. By binding to VEGF and activating downstream PI3K-Akt and ERK signaling pathways, it promotes endothelial cell proliferation, migration, and angiogenesis, driving granulation tissue growth and revascularization during wound repair. Inhibition of VEGFR2 can reduce pathological angiogenesis (such as in tumors or scars), while activation of VEGFR2 helps accelerate the healing of chronic wounds (such as diabetic ulcers), making it an important target for tissue repair.

[0197] HNF-4α (hepatocyte nuclear factor 4α) is a liver-specific transcription factor that directly regulates the expression of liver function genes such as albumin (ALB) and maintains plasma colloid osmotic pressure and substance transport. Decreased expression or activity of HNF-4α can lead to hypoalbuminemia (such as cirrhosis or malnutrition), while enhancing its function can promote albumin synthesis and improve liver function. Therefore, HNF-4α is a key regulatory target for liver metabolism and protein synthesis.

[0198] The eight selected fragments were docked with the three targets mentioned above using molecular docking software. The binding energies of the peptides to the receptors are summarized in Tables 5 and 6. The three-dimensional structures of TLR4 (PDB: 2Z63), VEGFR2 (PDB: 3VHE), and HNF-4α (PDB: 1PZL) were downloaded from the database.

[0199] Table 5. Binding energies of peptides to receptors (TLR4 and VEGFR2)

[0200] ;

[0201] Table 6. Binding energy of peptides to receptors (HNF-4α)

[0202] ;

[0203] Peptides 1-5 and 8 can all bind to the TLR4 receptor. Combined with previous experimental results, this indicates that the binding of peptides to TLR4 can inhibit its function and thus enhance immunity. Peptides 2-6 and 8 can all bind to the VEGFR2 receptor. Combined with previous experimental results, this indicates that the binding of peptides to VEGFR2 activates related signaling pathways, thereby promoting wound healing. Peptides 2-5 and 8 can all bind to the HNF-4α receptor. Combined with previous experimental results, this indicates that the binding of peptides to HNF-4α can activate its expression, thereby directly regulating the expression and production of ALB.

[0204] Based on binding energy and peptide abundance, three sequences, INF, FPN, and YCPL, were selected as characteristic peptide sequences in precise albumin peptides that enhance immunity, promote wound healing, and promote albumin synthesis.

[0205] Figure 19 This is a 2D diagram showing the docking of INF with TLR4 molecules. Figure 20 A 2D diagram showing the docking of FPN and TLR4 molecules; Figure 21 A 2D diagram showing the docking of YCPL and TLR4 molecules; Figure 22 This is a 2D diagram showing the docking of INF with VEGFR2 molecules; Figure 23 This is a 2D diagram showing the docking of FPN and VEGFR2 molecules. Figure 24 A 2D diagram showing the docking of YCPL with VEGFR2 molecules; Figure 25 This is a 2D diagram showing the docking of INF with HNF-4α molecules. Figure 26 This is a 2D diagram of the docking of FPN and HNF-4α molecules. Figure 27 This is a 2D diagram showing the docking of YCPL with HNF-4α molecules.

[0206] like Figure 19 As shown, INF and TLR4 interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds; as Figure 20 As shown, FPN and TLR4 interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds; as Figure 21 As shown, YCPL and TLR4 interact through covalent hydrogen bonds, van der Waals forces, salt bridges, and attractive forces.

[0207] like Figure 22 As shown, INF and VEGFR2 interact through carbon-hydrogen bonds, van der Waals forces, and covalent hydrogen bonds; as Figure 23As shown, FPN and VEGFR2 interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds; as Figure 24 As shown, YCPL and VEGFR2 interact through attractive forces, covalent hydrogen bonds, and van der Waals forces.

[0208] like Figure 25 As shown, INF and HNF-4α interact through covalent hydrogen bonds and van der Waals forces; Figure 26 As shown, FPN and HNF-4α mainly interact through van der Waals forces; Figure 27 As shown, YCPL interacts with HNF-4α through covalent hydrogen bonds, van der Waals forces, and carbon-hydrogen bonds.

[0209] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing a precise albumin peptide with functions of enhancing immunity, promoting wound healing, and promoting albumin synthesis, characterized in that, Includes the following steps: S1. Mix egg white powder with water and then heat treat to obtain a pretreatment solution; S2. Add Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase to the pretreatment solution in sequence for three-stage hydrolysis. After each stage of hydrolysis, the enzymes are inactivated to obtain the hydrolysate. S3. The hydrolysate is ultrafiltered and spray-dried to obtain the precise albumin peptide; The amount of Bacillus licheniformis alkaline protease added is 1% to 3% of the mass of the egg white powder; the amount of Bacillus subtilis neutral protease added is 1% to 3% of the mass of the egg white powder; the amount of aminopeptidase added is 1% to 3% of the mass of the egg white powder. The alkaline protease activity of the *Bacillus licheniformis* is 400,000 U / g; the neutral protease activity of the *Bacillus subtilis* is 80,000 U / g; and the aminopeptidase activity is 100,000 U / g. The hydrolysis conditions for the Bacillus licheniformis alkaline protease are: adjusting the system pH to 8.0–8.5 and hydrolyzing at 50–55℃ for 1–2 hours; the hydrolysis conditions for the Bacillus subtilis neutral protease are: adjusting the system pH to 6.5–7.5 and hydrolyzing at 50–55℃ for 1–2 hours; the hydrolysis conditions for the aminopeptidase are: adjusting the system pH to 6.5–7.5 and hydrolyzing at 50–55℃ for 1–2 hours. The mass ratio of egg white powder to water is 1:(15~30), and the heat treatment conditions are 90℃ for 10~30 min; The enzyme inactivation treatment is performed at 85℃~90℃ for 15~20 min, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

2. The preparation method according to claim 1, characterized in that, The precise albumin peptide comprises at least one of ADHPF, INF, FNP, FPN, YCPL, YCPI, CLLC, or YCP; the specific sequence of ADHPF is shown in SEQ ID NO. 1, the specific sequence of YCPL is shown in SEQ ID NO. 2, the specific sequence of YCPI is shown in SEQ ID NO. 3, and the specific sequence of CLLC is shown in SEQ ID NO.

4.

3. The use of a precise albumin peptide prepared by the method described in claim 1 in the preparation of functional products for enhancing immunity, promoting wound healing, or promoting albumin synthesis.

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