Egg white protein-derived peptides having an immunocompromised improving effect and use thereof

Abstract

The application discloses an egg white protein source peptide with the function of improving immunocompromise and application thereof, and belongs to the technical field of small molecule peptides. The egg white protein is subjected to enzymolysis by using a protease, and an albumin peptide is prepared by intercepting an enzymolysis product with a molecular weight less than 1000 Da, wherein the albumin peptide contains a tetrapeptide MSPF with the function of improving immunocompromise. Animal model function verification shows that the tetrapeptide MSPF and the albumin peptide containing the peptide segment can significantly improve immunocompromise, the number of macrophages is increased by 62.94%, the number of neutrophils is increased by 62.69%, and the content of interferon gamma is increased by 76.11%, thereby enhancing the immunity of the organism. The food-derived bioactive peptide has high biological safety. Therefore, the tetrapeptide MSPF or the albumin peptide containing the peptide segment can be applied to the preparation of medicines for improving immunocompromise or health care food for enhancing immunity, and has good market prospect and application potential.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to egg white protein-derived peptides that have the effect of improving immunodeficiency and their applications. Background Technology

[0002] Immunodeficiency refers to a pathological state in which the body's immune system is insufficient to fight pathogens, clear metabolic waste, and maintain homeostasis. The immune system, composed of immune organs, immune cells, and immune factors, is a vital system for self-defense. Impaired function can increase the risk of bacterial and viral infections and affect the body's ability to clear abnormal cells. In recent years, the incidence of immunodeficiency diseases induced by anti-tumor drugs and life stress has been rising annually. Therefore, improving the immunity of immunocompromised patients is of great significance for disease prevention.

[0003] Bioactive peptides are a class of peptide compounds derived from proteins that are beneficial or physiologically active in the life processes of organisms. They vary in size, being molecular polymers intermediate between amino acids and proteins, ranging from a single amino acid to dozens linked by peptide bonds. Bioactive peptides are widely available, exhibit diverse activities including antioxidant, antibacterial, and immunomodulatory effects, and are highly safe for consumption, possessing significant potential application value in the food and pharmaceutical industries.

[0004] For the development of bioactive peptides with immunomodulatory functions, common immunomodulators include thymosin. Clinically used thymosin is a small molecule polypeptide with non-specific immune effects, discovered and purified from calf thymus. It can induce T cell differentiation and maturation, enhance cytokine production, and enhance antibody responses in B cells. Bestim (γ-Glu-Trp) is a dipeptide molecule with immunomodulatory activity that can inhibit the activity of adenylate cyclase (Kolobov AA) in mouse macrophage and thymocyte membranes. et al Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes. Bioorg Khim. 2008,34(1):43-49.). Chen Dongliang et al. studied the physicochemical properties and regulatory effects on immune function of peptides obtained by protease hydrolysis of chicken ovalbumin. The results showed that ovalbumin peptides had an immune-enhancing effect on experimental mice (Immune-enhancing effect of ovalbumin peptides on mice. Food Science and Technology, 2007: 219-223), but this study did not analyze the specific active ingredients.

[0005] Eggs are rich in high-quality protein. The proteins in egg white are mainly ovalbumin and ovoglobulin, containing all eight essential amino acids. The composition of egg white protein is very similar to that of human protein, and the human body can absorb up to 98% of egg protein. The products of ovalbumin hydrolysis by specific proteases possess various physicochemical properties and biological activities, such as enhancing immunity, anti-oxidation, and inhibiting angiotensin-converting enzyme activity. Studies have shown that small peptides have better mobility and absorption rates from the stomach to the small intestine than amino acids of the same composition. Bioactive small peptides can be absorbed through the intestine and can regulate bodily functions. Therefore, it is necessary to conduct in-depth research on the immune-enhancing components of egg white protein peptides to provide theoretical support for the development of products using egg white protease hydrolysates to improve immune function. Summary of the Invention

[0006] The purpose of this invention is to provide a natural small molecule bioactive peptide that can improve immunodeficiency, and to apply it to the development of drugs for improving immunodeficiency or health foods for enhancing immunity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention utilizes alkaline protease, papain, and neutral protease to sequentially enzymatically hydrolyze egg white protein powder, extracting the enzymatic hydrolysate with a molecular weight less than 1000 Da to obtain the egg white protease hydrolysate. The peptide sequences in the egg white protease hydrolysate are analyzed using LC-MS / MS peptide mapping, and the interaction between the peptide and the TNF-α receptor is investigated using molecular docking technology. A candidate peptide is screened, and its amino acid sequence is identified as Met-Ser-Pro-Phe (MSPF) by mass spectrometry, with a molecular weight of 480.243 Da. Further functional verification through the artificial synthesis of the tetrapeptide MSPF reveals that this peptide has the effect of improving immunodeficiency.

[0009] Therefore, this invention provides the application of tetrapeptide MSPF in the preparation of drugs that improve immunodeficiency or health foods that enhance immunity.

[0010] Furthermore, the manifestations of the impaired immune function include a significant decrease in at least one of the following indicators: macrophage count, neutrophil count, and interferon-γ content.

[0011] This invention demonstrates that in an immunocompromised animal model, intervention with tetrapeptide MSPF can significantly improve symptoms of immunocompromise and increase the number of macrophages, neutrophils, and interferon-γ.

[0012] Furthermore, the tetrapeptide MSPF can be prepared by solid-phase synthesis or by enzymatic hydrolysis of egg white protein. The enzymatic hydrolysis involves sequentially hydrolyzing egg white protein with alkaline protease, papain, and neutral protease, and then separating the tetrapeptide MSPF from the hydrolysis products.

[0013] Another object of the present invention is to provide an albumin peptide that enhances immunity, wherein the albumin peptide contains MSPF, a tetrapeptide with the amino acid sequence Met-Ser-Pro-Phe.

[0014] The method for preparing the albumin peptide includes the following steps:

[0015] (1) Mix egg white protein powder with water at a mass ratio of 1:15-20, and then cut to obtain an egg white protein powder solution;

[0016] (2) Heat the egg white protein powder solution to 50℃, adjust the pH value to 8.0±0.2, add alkaline protease at 0.5%-1.0% of the weight of egg white protein powder, and hydrolyze for 2-3 hours; then add papain at 0.4%-0.8% of the weight of egg white protein powder, and continue hydrolyzing for 4-6 hours; then add neutral protease at 0.05%-0.1% of the weight of egg white protein powder, and continue hydrolyzing for 2-3 hours to obtain the enzymatic hydrolysate;

[0017] (3) After centrifuging the enzymatic hydrolysate at a speed of 4000-6000 r / min, the supernatant is collected and filtered through a membrane with a molecular weight cutoff of 1000 Da. The filtrate is then concentrated, sterilized, and dried to obtain the albumin peptide.

[0018] Further, in step (2), 0.8% of alkaline protease by weight of egg white protein powder is added and enzymatically hydrolyzed for 2 hours; then 0.6% of papain by weight of egg white protein powder is added and enzymatically hydrolyzed for 4 hours; then 0.1% of neutral protease by weight of egg white protein powder is added and enzymatically hydrolyzed for 2 hours to obtain the enzymatic hydrolysate.

[0019] This invention demonstrates that the albumin peptides prepared by the above method have the effect of improving immunodeficiency. In immunodeficiency animal models, intervention with the albumin peptides significantly increased the number of macrophages, neutrophils, and interferon-γ levels.

[0020] Therefore, the present invention provides the application of the albumin peptide in the preparation of drugs for improving immunodeficiency or health foods for enhancing immunity.

[0021] Specifically, the present invention provides a pharmaceutical composition for improving immunodeficiency, the pharmaceutical composition comprising an effective dose of a tetrapeptide MSPF with the amino acid sequence Met-Ser-Pro-Phe or an albumin peptide containing a tetrapeptide MSPF, and a pharmaceutically acceptable carrier.

[0022] In this invention, the tetrapeptide MSPF or albumin peptide containing tetrapeptide MSPF can be used as the sole active ingredient to improve immunodeficiency, or it can be combined with other active ingredients that enhance immunity.

[0023] In this invention, the pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of this invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject.

[0024] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

[0025] The present invention also provides a health food for enhancing immunity, the health food comprising a tetrapeptide MSPF with the amino acid sequence Met-Ser-Pro-Phe as an active ingredient or an albumin peptide containing a tetrapeptide MSPF, and food-grade excipients.

[0026] In this invention, the food-grade excipients that are acceptable in food science are those capable of delivering an effective dose of the active substance of this invention without interfering with the bioactivity of the active substance.

[0027] Furthermore, the dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

[0028] The beneficial effects of this invention are as follows:

[0029] This invention focuses on the immune-enhancing components of albumin peptides, providing a bioactive peptide with the effect of improving immunodeficiency: tetrapeptide MSPF and albumin peptides containing this peptide. Animal model functional validation shows that tetrapeptide MSPF and albumin peptides containing this peptide can significantly improve immunodeficiency, increase the number of macrophages, neutrophils, and interferon-γ levels, thereby enhancing the body's immunity. The food-derived bioactive peptide exhibits high biosafety. Therefore, tetrapeptide MSPF or albumin peptides containing this peptide can be used to prepare drugs for improving immunodeficiency or health foods for enhancing immunity, showing promising market prospects and application potential. Attached Figure Description

[0030] Figure 1The effect of different concentrations of cyclophosphamide on macrophages after 24 h was shown in the figure. ns indicates no significant difference compared to the control group (CON); * indicates a significant difference compared to the control group (CON); ** indicates... P <0.01; **** indicates P <0.0001.

[0031] Figure 2 The effect of different concentrations of cyclophosphamide on macrophages after 48 h of modeling is shown in the figure. ns indicates no significant difference compared to the control group (CON); * indicates a significant difference compared to the control group (CON); **** indicates... P <0.0001.

[0032] Figure 3 The effect of different concentrations of cyclophosphamide on neutrophils after 24 h is shown in the figure. * indicates a significant difference compared to the control group (CON), and ** indicates a statistically significant difference. P <0.01; *** indicates P <0.001; **** indicates P <0.0001.

[0033] Figure 4 The effect of different concentrations of cyclophosphamide on neutrophils after 48 h of modeling was shown in the figure. There was no significant difference in ns between the control group (CON) and the control group (*). * indicates a significant difference compared to the control group (CON), and **** indicates a significant difference compared to the control group (CON). P <0.0001.

[0034] Figure 5 The figure shows the effect of albumin peptides on zebrafish head macrophages. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P <0.0001; * indicates a significant difference compared to the model group (MOD), ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001.

[0035] Figure 6 The figure shows the effect of albumin peptide on zebrafish neutrophils. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P <0.0001; * indicates a significant difference compared to the model group (MOD), ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001; ns indicates no significant difference compared with the model group (MOD).

[0036] Figure 7 The figure shows the effect of albumin peptide on interferon-γ in zebrafish. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P <0.0001; * indicates a significant difference compared to the model group (MOD), **** indicates P <0.0001.

[0037] Figure 8 It is a major target for albumin peptides to enhance immunity.

[0038] Figure 9 This is the primary mass spectrum of the tetrapeptide MSPF.

[0039] Figure 10 This is a secondary mass spectrum of the tetrapeptide MSPF. In the spectrum, b2-NH3 represents the second deamination fragment ion generated by the N-terminal cleavage of the peptide; b2 represents the second fragment ion generated by the N-terminal cleavage of the peptide; and y2 represents the second fragment ion generated by the C-terminal cleavage of the peptide.

[0040] Figure 11 This is a schematic diagram illustrating the binding of the tetrapeptide MSPF to the TNF-α receptor.

[0041] Figure 12 The figure shows the ameliorative effect of tetrapeptide MSPF on macrophages. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P <0.0001; * indicates a significant difference compared to the model group (MOD), * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001; ns indicates no significant difference compared with the model group (MOD).

[0042] Figure 13 The figure shows the effect of the tetrapeptide MSPF on neutrophils. The # symbol indicates a significant difference compared to the control group (CON), and ### indicates... P <0.001; * indicates a significant difference compared to the model group (MOD), ** indicates P <0.01; ns indicates no significant difference compared with the model group (MOD).

[0043] Figure 14 The figure shows the effect of the tetrapeptide MSPF on interferon. The # symbol indicates a significant difference compared to the control group (CON), and #### indicates... P <0.0001; * indicates a significant difference compared to the model group (MOD), * indicates P<0.05; ns indicates no significant difference compared with the model group (MOD). Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0046] The egg white protein powder used in the following examples was purchased from Zhejiang Aige Egg Products Co., Ltd.; the alkaline protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Biological Products Co., Ltd.; the papain was purchased from Guangxi Dongheng Huadao Biological Products Co., Ltd.; and the neutral protease (derived from Aspergillus oryzae) was purchased from Nanning Pangbo Biological Products Co., Ltd.

[0047] Example 1: Preparation of albumin peptides

[0048] This embodiment provides a method for sequentially enzymatically hydrolyzing egg white protein powder using alkaline protease, papain, and neutral protease. The specific steps are as follows:

[0049] (1) Add 1500 kg of water to the reactor, and add 100 kg of egg white protein powder while stirring. Stir and mix evenly, and turn on the reactor to shear at 10000 r / min for 30 minutes.

[0050] (2) After shearing, the temperature of the liquid is raised to 50°C. After the temperature is reached, the pH of the material is adjusted to 8.0 with alkaline solution. 800 g of alkaline protease (from Bacillus subtilis) is added to the egg white protein powder solution and enzymatically hydrolyzed for 2 hours. Then 600 g of papain is added and enzymatically hydrolyzed for another 4 hours. Then 100 g of neutral protease (from Aspergillus oryzae) is added and enzymatically hydrolyzed for another 2 hours to obtain the enzymatic hydrolysate.

[0051] (3) The enzymatic hydrolysate was separated using a disc separator (6000 r / min) to obtain the supernatant. The supernatant was then passed through a membrane with a molecular weight cutoff of 1000 Da, and the filtrate was collected.

[0052] (4) Then the liquid is evaporated and concentrated in a vacuum low-temperature thin film evaporator until the sugar content of the liquid is 25° to obtain a concentrated liquid;

[0053] (5) The concentrated liquid was sterilized at high temperature and spray-dried to obtain the protein hydrolysate (albumin peptide).

[0054] The methods for determining the moisture content of albumin peptides refer to the national standard GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food", the methods for determining the ash content refer to the national standard GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Food", the methods for determining the protein content refer to the national standard GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food", the methods for determining the degree of hydrolysis refer to the o-phthalaldehyde method in Nielsen et al., and the methods for determining the relative molecular weight distribution refer to Appendix A of the national standard GB / T 22729-2008 "Marine Fish Oligopeptide Powder".

[0055] The test results are as follows:

[0056] Table 1. Physicochemical detection results of albumin peptides

[0057]

[0058] Table 1 shows that the albumin peptides prepared in this embodiment have a high protein content, and peptides with a relative molecular weight of less than 1000 Da account for as much as 90.73%. This indicates that the enzymatic hydrolysis process in this embodiment can effectively hydrolyze egg white protein powder into bioactive small molecule peptides, providing high-quality raw materials for subsequent research and applications. Furthermore, the physicochemical properties of the hydrolysate are stable, providing a reliable guarantee for its application in the preparation of drugs to improve immunodeficiency or health foods to enhance immunity.

[0059] Example 2: Confirmation of the conditions for establishing a zebrafish model of immunodeficiency

[0060] 1. Experimental Principle

[0061] Macrophages are immune cells with multiple functions. They are white blood cells located in tissues and participate in non-specific defense (innate immunity) and specific defense (cellular immunity) in vertebrates. They activate lymphocytes or other immune cells to respond to pathogens.

[0062] Neutrophils can shape adaptive immunity by influencing T cell development. In chronic inflammatory diseases, neutrophil elastase (NE) promotes dendritic cell (DC)-driven T cell differentiation and development.

[0063] Cyclophosphamide (CTX) is an immunosuppressant. In this embodiment, a zebrafish model of immunodeficiency induced by CTX was used. Specifically, the effect on macrophages was studied using zebrafish (AB). CTX reduces the number of macrophages, and immunity was assessed by counting the number of macrophages. The effect on neutrophils was studied using neutrophil fluorescent expression zebrafish (LYZ). After CTX treatment, the fluorescence intensity was reduced, and immunity was assessed by counting the fluorescence intensity of neutrophils.

[0064] 2. Experimental Methods

[0065] Wild-type zebrafish (AB) and neutrophil fluorescent expression zebrafish (LYZ) were housed on the zebrafish platform of Zhejiang University School of Medicine. This experiment was approved by the Center for Experimental Animal Ethics of Zhejiang University (ethics number ZJU20250649).

[0066] Wild-caught AB strain zebrafish (48 hpf) were placed in six-well plates containing 0.5% dimethyl sulfoxide (DMSO) system water, with 10-15 zebrafish per well. The plates were incubated at 28.5°C. Cyclophosphamide was added at concentration gradients of 50, 100, 125, 150, 175, 200, 250, 300, 500, 800, and 1000 μg / mL, allowing for co-treatment for 24 and 48 hours. Neutral red dye and phenylthiourea (PTU) were added to each well, and the plates were stained in the dark for 6 hours. The embryos were washed with system water in the dark, anesthetized, and fixed with 6% methylcellulose. The embryos were photographed under a stereomicroscope, and the number of head macrophages was counted. Data analysis was performed using GraphPad Prism software. The number of head macrophages in the control group (without cyclophosphamide) and the model group (with cyclophosphamide) was counted.

[0067] The system water formula is as follows: dissolve 35 grams of sodium chloride (NaCl), 2 grams of sodium bicarbonate (NaHCO3), 1 gram of calcium chloride (CaCl2), and 0.5 grams of potassium chloride (KCl) in 1 liter of pure water.

[0068] The same method was used to establish a neutrophil-fluoresced zebrafish model (LYZ). Embryos were washed, anesthetized, and photographed under a stereofluorescence microscope. Fluorescence intensity from the cloaca to the tail tip was recorded, and data analysis was performed using GraphPad Prism software. The fluorescence intensity from the cloaca to the tail tip was statistically analyzed in both the control group and the model group.

[0069] The modeling time and concentration of cyclophosphamide were determined by statistically analyzing the number of macrophages and the fluorescence intensity of neutrophils after cyclophosphamide modeling.

[0070] 3. Results Analysis

[0071] Figure 1 and Figure 2The modeling results showed that at 24 and 48 h, cyclophosphamide concentrations approached a stable state at 200 μg / mL, with 200 μg / mL showing the best modeling effect, achieving nearly half of the damage values. Furthermore, at 48 h, 200 μg / mL showed significant differences compared to 100, 125, and 150 μg / mL. Therefore, 200 μg / mL at 48 h was selected as the macrophage modeling concentration.

[0072] Figure 3 and Figure 4 The results showed that cyclophosphamide concentrations of 175, 200, and 500 μg / mL were optimal for modeling at 24 hours using the unit area fluorescence method, while concentrations of 800 and 1000 μg / mL were optimal at 48 hours, followed by 175 and 200 μg / mL. The 24-hour modeling area was determined by human intervention, thus introducing a human element; therefore, the 24-hour modeling was discarded. Furthermore, high concentrations of 800 and 1000 μg / mL at 48 hours could cause irreversible damage. Therefore, the more stable 200 μg / mL concentration between 175 and 200 μg / mL was selected as the modeling condition for zebrafish neutrophils at 48 hours.

[0073] comprehensive Figures 1-4 Based on the data, the modeling conditions for macrophages and neutrophils were both 48 h and cyclophosphamide at a concentration of 200 μg / mL.

[0074] Example 3: Effects of albumin peptide intervention on zebrafish macrophages

[0075] Based on the modeling conditions of Example 2, the effect of the albumin peptide prepared in Example 1 on improving cyclophosphamide-induced immunosuppression in zebrafish was evaluated. The zebrafish rearing conditions were the same as in Example 2.

[0076] 1. Experimental Methods

[0077] Wild-caught AB strain zebrafish embryos (48 hpf) were demembranous and placed in six-well plates, with two parallel wells per group and 10-15 embryos per well. After 48 h of treatment with the drug, 2.5 μg / mL neutral red dye and PTU were added to each well, and the plates were stained in the dark for 6 h. The embryos were washed with systemic water in the dark, anesthetized, and fixed with 6% methylcellulose. The embryos were photographed under a stereomicroscope, and the number of macrophages in the head was counted. Data were analyzed using GraphPad Prism software.

[0078] The groups are as follows:

[0079] (1) Blank control group (CON): 0.5% DMSO + system water + PTU;

[0080] (2) Model group (MOD): 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%;

[0081] (3) Albumin peptide group (BDB): albumin peptide 50, 100, 125, 150 μg / mL + 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%.

[0082] 2. Experimental Results

[0083] from Figure 5 The results showed that albumin peptides could significantly alleviate the immune decline in zebrafish caused by cyclophosphamide modeling, with 100 μg / mL albumin peptides showing the best effect. Compared with the model group (MOD), the number of macrophages in the head of zebrafish increased by 62.94%.

[0084] Example 4: Effects of albumin peptide intervention on zebrafish neutrophils

[0085] Based on the modeling conditions of Example 2, the effect of the albumin peptide prepared in Example 1 on improving cyclophosphamide-induced immunosuppression in zebrafish was evaluated. The zebrafish rearing conditions were the same as in Example 2.

[0086] 1. Experimental Methods

[0087] 48 hpf transgenic Tg (Lyz:DsRed) zebrafish embryos were demembranously placed in six-well plates, with two parallel wells per group and 15-20 embryos per well. After 48 h of drug treatment, the embryos were washed, anesthetized, and photographed under a stereofluorescence microscope. The fluorescence intensity from the cloaca to the tail tip was counted, and the data were analyzed using GraphPad Prism software. The groups are as follows:

[0088] (1) Blank control group (CON): 0.5% DMSO + system water + PTU;

[0089] (2) Model group (MOD): 200 μg / m³ LTX (dissolved in DMSO) + system water + PTU, DMSO volume fraction was 0.5%;

[0090] (3) Albumin peptide group (BDB): albumin peptide 50, 100, 125, 150, 175, 200, 250 μg / mL + 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%.

[0091] 2. Experimental Results

[0092] from Figure 6The results showed that albumin peptides could significantly alleviate the decrease in neutrophils in zebrafish induced by cyclophosphamide modeling, with 100 μg / mL albumin peptides showing the best effect. Compared with the model group (MOD), the fluorescence intensity of zebrafish neutrophils increased by 62.69%.

[0093] Example 5: Effects of albumin peptide intervention on interferon-gamma in zebrafish

[0094] Based on the modeling conditions of Example 2, the effect of the albumin peptide prepared in Example 1 on improving cyclophosphamide-induced immunosuppression in zebrafish was evaluated. The zebrafish rearing conditions were the same as in Example 2.

[0095] 1. Experimental Methods

[0096] 48 hpf AB strain zebrafish embryos were demembranous and placed in 6-well plates, with 3 parallel wells per group and 30 embryos per well. After 48 h of drug treatment, the 30 embryos from each well were placed into 1.5 mL centrifuge tubes. The embryos were washed with PBS buffer equal to the total liquid volume of each well. After removing the residual PBS buffer, 200 μL of PBS buffer was added to every 30 samples. The samples were homogenized using a high-speed tissue homogenizer for 90 s and centrifuged (4 ℃, 5000 r / min, 10 min). The supernatant was collected, and the IFN-γ content of each sample was calculated using enzyme-linked immunosorbent assay (ELISA) based on the standard curve. Data analysis was performed using GraphPad Prism 8.0 software.

[0097] The groups are as follows:

[0098] (1) Blank control group (CON): 0.5% DMSO + system water + PTU;

[0099] (2) Model group (MOD): 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%;

[0100] (3) Albumin peptide group (BDB): 50, 100 μg / mL albumin peptide + 200 μg / mL CTX (dissolved in DMSO) + system water + PTU, with DMSO volume fraction of 0.5%.

[0101] 2. Experimental Results

[0102] from Figure 7 The results showed that albumin peptides could significantly alleviate the decrease in interferon-γ content in zebrafish caused by cyclophosphamide modeling. Among them, albumin peptides with a concentration of 100 μg / mL had the best effect, and the content of interferon-γ in zebrafish increased by 76.11% compared with the model group (MOD).

[0103] Example 6: Screening of active ingredients in albumin peptides

[0104] In this embodiment, the albumin peptides prepared in Example 1 were analyzed by LC-MS / MS, and then functional peptides were screened using molecular docking technology. Details are as follows:

[0105] 1. LC-MS / MS analysis

[0106] Specifically, the peptide profiling analysis and detection were commissioned to the National Analytical and Testing Platform of Zhejiang University.

[0107] 2. Molecular docking

[0108] Through network pharmacology software analysis of albumin peptides' immune targets, and after the above-mentioned screening process, nine targets with the strongest correlation to the albumin peptide-immune relationship were finally identified, such as... Figure 8 As shown in the figure. This technique uses the TNF-α receptor as the docking target for molecular docking.

[0109] Peptides with PeptdieRanker scores (≥0.95) and relative peak areas (≥10⁶) were molecularly docked with the TNF-α receptor. Sequences were screened and identified based on docking scores (≤-7.0), number of hydrogen bonds (≥4), number of amino acid residue dockings, and docking amino acids associated with bone growth. Finally, the tetrapeptide MSPF was determined to have potential immune-enhancing capabilities, as shown in Table 2.

[0110] Table 2. Potentially bioactive peptides that bind to TNF-α receptors

[0111]

[0112] The primary and secondary structures of the tetrapeptide MSPF were analyzed by mass spectrometry. Figure 9 , Figure 10 It can be seen that the [M+H]+ ion signal of the tetrapeptide MSPF in the primary structure is 481.214 m / z, which is basically consistent with the molecular weight of the tetrapeptide MSPF, 480.243 Da, and the amino acid sequence of the tetrapeptide MSPF in the secondary structure is Met-Ser-Pro-Phe.

[0113] Molecular docking analysis results ( Figure 11 The results showed that the interaction between the tetrapeptide MSPF and TNF-α involves amino acid residues GLU102, SER99, and PRO100, and the binding is achieved through 5 hydrogen bonds. This multi-point hydrogen bond interaction mode enhances the binding stability of MSPF to the TNF-α receptor, suggesting that it may exert an immunomodulatory effect by blocking the TNF-α-mediated inflammatory signaling pathway.

[0114] Example 7: Synthesis of the active peptide tetrapeptide MSPF

[0115] This embodiment utilizes artificially synthesized MSPF peptides. Specifically, the synthesis was commissioned to Shenzhen Borunsida Biotechnology Co., Ltd., with a purity ≥98%.

[0116] S1. Weigh Fmoc-Phe-Wang Resin and place it in a glass reaction column. Add DCM to swell the solution for 30 min, then remove the DCM under reduced pressure.

[0117] S2. Wash the resin three times with DMF, add 20% piperidine / DMF solution and react for 20 min to remove the protecting group fmoc, remove the solution under reduced pressure, and wash with DMF six times.

[0118] S3. Weigh out the second amino acid Fmoc-Pro-OH and TBTU respectively and add them to the resin. Dissolve them in DMF and add DIEA. React for 30 minutes. Take the resin to perform a color reaction and observe the color of the solution and the color of the resin. If the solution is bright yellow and the resin is yellow, it means that the reaction is complete. Remove the solvent under reduced pressure.

[0119] S4. Repeat steps S2 and S3 until the last amino acid Fmoc-Met(otbu)-oh is precipitated. Then wash three times each with DMF, DCM and methanol, and dry the resin.

[0120] S5. Add lysis buffer to remove resin and amino acid side chain protecting groups, filter with sand core, add diethyl ether to the filtrate to precipitate, centrifuge and wash the solid 3 times, dry and detect by MS to obtain tetrapeptide MSPF.

[0121] Example 8: Efficacy Verification of Active Peptides

[0122] Following the operational requirements of Examples 3-5, the effects of tetrapeptide MSPF on macrophages, neutrophils, and interferon-γ in zebrafish modeled with cyclophosphamide were verified at different concentrations (0.5, 1, 5, 10, 25, 50 μg / mL).

[0123] from Figures 12-14 The results showed that, compared with the model group (MOD), tetrapeptide MSPF could significantly alleviate the immune decline in zebrafish induced by cyclophosphamide modeling, with 10 μg / mL tetrapeptide MSPF having the best effect on the number of macrophages in the head of zebrafish. p <0.001), 1 μg / mL of tetrapeptide MSPF was most effective against zebrafish neutrophils. p <0.01); 1 μg / mL tetrapeptide MSPF showed the best effect against zebrafish interferon-gamma ( p <0.05).

[0124] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of tetrapeptide MSPF in the preparation of drugs for improving immunodeficiency or health foods for enhancing immunity, characterized in that, The amino acid sequence of the tetrapeptide MSPF is Met-Ser-Pro-Phe.

2. The application as described in claim 1, characterized in that, The manifestations of the weakened immune function include a significant decrease in at least one of the following indicators: macrophage count, neutrophil count, and interferon-γ content.

3. The application as described in claim 1, characterized in that, The tetrapeptide MSPF is prepared by solid-phase synthesis or by enzymatic hydrolysis of egg white protein.

4. An albumin peptide with immune-enhancing effects, characterized in that, The albumin peptide contains a tetrapeptide MSPF with the amino acid sequence Met-Ser-Pro-Phe; the preparation method of the albumin peptide includes the following steps: (1) Mix egg white protein powder with water at a mass ratio of 1:15-20, and then cut to obtain an egg white protein powder solution; (2) Heat the egg white protein powder solution to 50℃, adjust the pH value to 8.0±0.2, add alkaline protease at 0.5%-1.0% of the weight of egg white protein powder, and hydrolyze for 2-3 hours; then add papain at 0.4%-0.8% of the weight of egg white protein powder, and continue hydrolyzing for 4-6 hours; then add neutral protease at 0.05%-0.1% of the weight of egg white protein powder, and continue hydrolyzing for 2-3 hours to obtain the enzymatic hydrolysate; (3) After centrifuging the enzymatic hydrolysate at a speed of 4000-6000 r / min, the supernatant is collected and filtered through a membrane with a molecular weight cutoff of 1000 Da. The filtrate is then concentrated, sterilized, and dried to obtain the albumin peptide.

5. The use of the albumin peptide as described in claim 4 in the preparation of drugs for improving immunodeficiency or health foods for enhancing immunity.

6. A pharmaceutical composition for improving immunodeficiency, characterized in that, The pharmaceutical composition comprises an effective dose of the albumin peptide as described in claim 4, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

8. A health food product for enhancing immunity, characterized in that, The health food includes albumin peptide as an active ingredient as described in claim 4, and food-grade excipients.

9. The health food product as described in claim 8, characterized in that, The dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

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