A nonapeptide with immune-boosting effect and preparation and application thereof

Abstract

This invention discloses a nonapeptide with immune-enhancing effects, its preparation, and its applications, belonging to the field of biotechnology. This invention screened and identified a bioactive peptide EK9 with immunomodulatory effects from whey protein hydrolysates, with the amino acid sequence Glu-Asp-Lys-Leu-Asp-Leu-Asp-His-Lys. Functional verification showed that this peptide significantly improved immunodeficiency. Specifically, in an immunodeficiency model, treatment with bioactive peptide EK9 significantly increased the number of immune cells such as macrophages and neutrophils, and significantly increased the content of immune factors such as IFN-γ. Furthermore, the food-derived peptide exhibits high biosafety; therefore, it can be used to prepare immunomodulatory products. This invention provides a new method and theoretical basis for enhancing immunity, and has good market prospects and application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a whey nonapeptide with immune-enhancing effects, its preparation method, and its applications. Background Technology

[0002] Immune dysfunction refers to a pathological state in which the body's immune system is insufficient in its ability to fight pathogens, clear metabolic waste, and maintain homeostasis. The immune system is composed of immune organs, immune cells, and immune factors. Impaired function can increase the risk of bacterial and viral infections and affect the body's ability to clear abnormal cells. Therefore, improving immune function has become a focus of public attention.

[0003] Whey protein is a major protein component of dairy products and is widely used in sports nutrition and dietary supplements. Besides providing nutrition and aiding muscle recovery, research has also found a close relationship between whey protein and the immune system, potentially offering benefits for the prevention and treatment of immune diseases. However, a weakened immune system can actually increase the risk of food protein allergies.

[0004] Food-derived bioactive peptides refer to functional peptide compounds released during the hydrolysis of food proteins or specific processing. Their molecular structure lies between that of amino acids and proteins. These compounds possess advantages such as small relative molecular weight, weak immunogenicity, and high biological activity, positively impacting the life activities of organisms. For example, patent document CN114903176B discloses the preparation of collagen peptides, soybean peptides, and whey peptides through various protein decomposition processes. These peptides are then formulated into a polypeptide nutritional powder. This powder does not contain allergenic proteins, contains various essential and non-essential amino acids, and can enhance immune cell activity, humoral immunity, and cellular immunity, fundamentally improving human immunity. Patent document CN101518295B discloses the use of trypsin to enzymatically hydrolyze whey, achieving a hydrolysis degree of 20-21%, to obtain whey peptides rich in 1000 Dalton molecular weight peptides, which have functions such as anti-protein allergy and enhanced immunity.

[0005] Whey peptides are produced from whey protein through processes such as enzymatic hydrolysis. Current research reports on the immune-enhancing effects of whey peptides are mostly at the mixture level, failing to isolate and identify the key active peptides, making precise quality control and targeted efficacy optimization difficult. The functional properties of bioactive peptides are closely related to their amino acid composition and sequence, making the rational design and synthesis of bioactive peptides an effective strategy for improving immunity. Therefore, peptide profiling of whey peptides to screen for highly efficient peptides related to immune enhancement will provide a foundation for the development of novel immunomodulators. Summary of the Invention

[0006] The purpose of this invention is to screen and identify natural small molecule bioactive peptides with immunomodulatory effects from whey protein hydrolysates, and to apply them to the development of related products that enhance immunity.

[0007] To achieve the above objectives, this invention utilizes alkaline protease, trypsin, and flavor protease to perform stepwise enzymatic hydrolysis of whey protein. LC-MS / MS peptide mapping analysis is used to analyze the polypeptide sequences in the whey protein hydrolysates. Molecular docking technology is then used to investigate the interaction between the peptides and aryl hydrocarbon receptors (AHRs), resulting in the nonapeptide EDKLDLDHK (EK9), with the amino acid sequence Glu-Asp-Lys-Leu-Asp-Leu-Asp-His-Lys. Further artificial synthesis of the nonapeptide EK9 and functional verification revealed that this peptide has the effect of enhancing immunity.

[0008] Therefore, the present invention provides a bioactive peptide EK9, wherein the amino acid sequence of the bioactive peptide EK9 is Glu-Asp-Lys-Leu-Asp-Leu-Asp-His-Lys.

[0009] This invention also provides a method for preparing the bioactive peptide EK9. As a specific embodiment of this invention, the bioactive peptide EK9 can be prepared by solid-phase synthesis. Specifically, an Fmoc solid-phase synthesis strategy is adopted, using Fmoc-protected amino acids as raw materials and Wang resin as a solid-phase carrier. Lysine, histidine, aspartic acid, leucine, aspartic acid, leucine, lysine, aspartic acid, and glutamic acid residues are introduced sequentially to extend the peptide chain from the C-terminus to the N-terminus, thereby synthesizing the nonapeptide EK9 in the solid phase.

[0010] In another specific embodiment of the present invention, the bioactive peptide EK9 can be prepared by genetic engineering.

[0011] In another specific embodiment of the present invention, the bioactive peptide EK9 can be obtained by targeted enzymatic hydrolysis of whey protein. Specifically, the targeted enzymatic hydrolysis method includes: mixing water and whey protein at a mass ratio of 10:1, adjusting the pH to 8.0±0.2, adding 1.0% (by weight of total whey protein) of alkaline protease, and hydrolyzing for 30 min; subsequently adding 1.0% (by weight of total whey protein) of trypsin, and continuing enzymatic hydrolysis for 60 min; finally adding 0.5% (by weight of total whey protein) of flavor protease, and hydrolyzing for 30 min; after enzymatic hydrolysis, heating to inactivate the enzyme, centrifuging at 6000 r / min for 20 min, collecting the supernatant, and obtaining an enzymatic hydrolysis product containing nonapeptide EK9. The alkaline protease activity is ≥150 U / mg; the trypsin activity is ≥20 U / mg; and the flavor protease activity is ≥60 U / mg.

[0012] This invention also provides the application of the bioactive peptide EK9 in the preparation of immunomodulatory products. The immunomodulation includes at least one of increasing the proliferation of immune cells and increasing the levels of immune factors.

[0013] This invention demonstrates that the bioactive peptide EK9 possesses immunomodulatory properties. In a chloramphenicol-induced immunodeficiency model, intervention with nonapeptide EK9 significantly improved the immunodeficiency state, restoring the number of immune cells such as macrophages and neutrophils, and the levels of immune factors such as interferon-γ, to near-normal levels. Mechanistic studies suggest that nonapeptide EK9 exerts its immunomodulatory effects by binding to and activating aryl hydrocarbon receptors. Therefore, nonapeptide EK9 can be applied to the preparation of products that enhance immunity, including but not limited to pharmaceuticals and health foods.

[0014] In one specific embodiment of the present invention, the product is a drug for the prevention or treatment of immunodeficiency. The drug can be used to treat immunodeficiency diseases, which can refer to diseases accompanied by immunodeficiency that require the administration of immune enhancers or whose symptoms are more effectively relieved by the administration of immune enhancers.

[0015] Furthermore, the manifestations of the weakened immune function include a decrease in the number of immune cells and a decrease in the level of immune factors. Specifically, the immune cells include macrophages and neutrophils; the immune factors include interferon-γ.

[0016] In another specific embodiment of the present invention, the product is a health food that helps enhance immunity. This health food can be used to improve a sub-healthy state characterized by weakened immune function.

[0017] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of immunodeficiency, said pharmaceutical composition comprising an effective dose of the bioactive peptide EK9 and a pharmaceutically acceptable carrier.

[0018] This invention uses the bioactive peptide EK9 as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a formulation according to pharmaceutically described methods. The pharmaceutical composition provided by this invention can use the bioactive peptide EK9 as the sole active ingredient to enhance immunity, or it can be combined with other active ingredients that have immunomodulatory effects.

[0019] The pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of the present invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject.

[0020] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

[0021] Furthermore, the pharmaceutical composition may be in the form of, but is not limited to, an oral formulation. Specifically, the formulation may be, but is not limited to, an oral liquid, capsule, microcapsule powder, tablet, granules, or emulsion.

[0022] The specific dosage of the pharmaceutical composition is adjusted according to the type of disease, the severity of the disease, the age, and the purpose of administration.

[0023] The present invention also provides a health food that helps to enhance immunity, the health food comprising bioactive peptide EK9 as an active ingredient and food science acceptable excipients.

[0024] The food-grade excipients that are acceptable in food science are those that can deliver an effective dose of the active substance of the present invention without interfering with the bioactivity of the active substance.

[0025] Furthermore, the dosage form of the health food can be, but is not limited to, tablets, capsules, oral liquids, granules, beverages, and powders.

[0026] The beneficial effects of this invention are as follows: This invention provides a novel bioactive peptide, EK9, with immune-enhancing effects. Functional verification shows that this peptide significantly improves immunodeficiency. Specifically, in an immunodeficiency model, treatment with bioactive peptide EK9 significantly increases the number of immune cells such as macrophages and neutrophils, and significantly increases the content of immune factors such as IFN-γ. Furthermore, the food-derived peptide exhibits high biosafety, thus it can be used to prepare immunomodulatory products. This invention provides a new method and theoretical basis for enhancing immunity, and has good market prospects and application potential. Attached Figure Description

[0027] Figure 1 This is the secondary mass spectrum of nonapeptide EK9.

[0028] Figure 2 This is a schematic diagram of the chemical structure of nonapeptide EK9.

[0029] Figure 3 This is a schematic diagram illustrating the binding interaction between nonapeptide EK9 and AHR.

[0030] Figure 4 Images of head macrophages observed under a stereomicroscope after zebrafish models were treated with different concentrations of nonapeptide EK9.

[0031] Figure 5 for Figure 4A statistical chart showing the number of macrophages in the mid-head region. * indicates a significant difference between groups. p <0.05; ** indicates p <0.01; **** indicates p <0.0001.

[0032] Figure 6 Photographs of neutrophil fluorescence from the cloaca to the tail end were observed under a stereomicroscope after zebrafish models were treated with different concentrations of nonapeptide EK9.

[0033] Figure 7 for Figure 6 A statistical graph of neutrophil fluorescence intensity. * indicates a significant difference between groups, ** indicates a significant difference. p <0.01; *** indicates p <0.001.

[0034] Figure 8 The effect of different concentrations of nonapeptide EK9 on the immune factor IFN-γ in a zebrafish model. * In the figure, * indicates significant differences between groups. p <0.05; ** indicates p <0.01.

[0035] Figure 9 To compare the effects of nonapeptide EK9 and decapeptide KV10 on a zebrafish model, photographs of head macrophages were observed under a stereomicroscope.

[0036] Figure 10 for Figure 9 A chart showing the number of macrophages in the mid-head region. * indicates a significant difference between groups, and ** indicates a significant difference between groups. p <0.01; *** indicates p <0.001; **** indicates p <0.0001.

[0037] Figure 11 To compare the fluorescence of neutrophils from the cloaca to the tail end in a zebrafish model after treatment with nonapeptide EK9 and decapeptide KV10.

[0038] Figure 12 for Figure 11 The graph shows the fluorescence intensity of neutrophils. * indicates a significant difference between groups. p <0.05; ** indicates p <0.01; *** indicates p <0.001.

[0039] Figure 13To compare the effects of nonapeptide EK9 and decapeptide KV10 on the immune factor IFN-γ in a zebrafish model. * In the figure, * indicates significant differences between groups. p <0.05; ** indicates p <0.01. Detailed Implementation

[0040] 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.

[0041] 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.

[0042] Whey protein was purchased from Hilmar Cheese Company; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme Preparations (Yichang) Co., Ltd., with an enzyme activity ≥150 U / mg; trypsin was purchased from Nanning Pangbo Biotechnology Co., Ltd., with an enzyme activity ≥20 U / mg; flavor protease was purchased from Angel Enzyme Preparations (Yichang) Co., Ltd., with an enzyme activity ≥60 U / mg.

[0043] Example 1: Screening of active peptides 1. Preparation of whey peptides Water and whey protein were added to the reactor at a ratio of 10:1. After feeding, the pH of the solution was adjusted to 8.0 ± 0.2, and alkaline protease (1.0% of the total whey protein weight) was added for enzymatic hydrolysis for 30 min. The pH of the solution was maintained at ≥7.5 throughout the hydrolysis process. After hydrolysis, trypsin (1.0% of the total whey protein weight) was added for enzymatic hydrolysis for 60 min. After hydrolysis, flavor protease (0.5% of the total whey protein weight) was used for 30 min, followed by enzyme inactivation at 100 ℃ for 30 min, centrifugation at 6000 r / min for 20 min, and collection of the supernatant. The sugar content of the solution was then concentrated to 25°Bx and spray-dried to obtain whey peptide powder.

[0044] 2. Screening for active peptides Whey peptides were analyzed by LC-MS / MS peptide profiling. The peptide fragments were then organized according to screening criteria, and further molecular docking was used to determine the final theoretically effective peptides based on their scores. The specific analytical process is as follows: (a) Identification of whey peptide sequences LC-MS / MS was used to analyze the peptide sequences of whey peptide samples. A C18 reversed-phase column with a pore size of 300 Å, a particle size of 100 mm × 2.1 mm, and a particle size of 1.7 μm was used. Separation was performed at a flow rate of 0.3 mL / min at a column temperature of 40 °C. Mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) formic acid. A Thermo Fisher Q Exactive high-resolution tandem mass spectrometer was used for full scan and MS2 scans in positive ionization mode. Full scan mode mass spectrometry conditions: scan range: 200 m / z ~ 2000 m / z, resolution: 70000; MS2 mode mass spectrometry conditions: collision voltage: 30 V, resolution: 17500. Raw mass spectrometry data were obtained and then compared with protein databases to identify and analyze the whey peptide sequences.

[0045] (b) Screening of peptides with potential immunomodulatory activity Aromatic hydrocarbon receptors (AHRs) play a crucial role in the immune system. As ligands that activate transcription factors, AHRs mediate immune responses to dietary sources, microbial metabolites, and environmental chemicals, regulating the Th17 / Treg balance, inflammatory cytokine secretion, and intestinal barrier function. In this embodiment, whey peptides were molecularly docked with AHRs to screen for peptides with potential immunomodulatory functions.

[0046] The peptides were molecularly docked with the aryl hydrocarbon receptor (AHR). First, the crystal structure of AHR (5NJ8) was downloaded from the PDB protein database. Using Discovery Studio software, water molecules were removed from the receptor target, and hydrogen atoms were added to define its active site. The structures of the selected whey peptides were constructed using Discovery Studio, and their energies were minimized using the CHARMm force field to define these peptides as ligands. The constructed whey peptides were then docked with AHR using CDOCKER to simulate the binding modes, sites, and active amino acid residues with the lowest and highest binding energies. The peptides were then screened based on binding energy and the number of hydrogen bonds. Finally, one nonapeptide (EDKLDLDHK) and one decapeptide (KFPKAEFVEV) were identified, as shown in Table 1.

[0047] Table 1. Peptides in whey peptides with potential immunomodulatory functions The secondary mass spectrum of the nonapeptide EDKLDLDHK is shown below. Figure 1As shown, peptide fragment ions include N-terminal fragment ions (types a, b, and c) and C-terminal fragment ions (types x, y, and z). a, y, and z-type ions break their side chains to form d, v, and w-type ions, respectively. In addition, there are internal ions formed by breakage at both ends, with b and y series ions being the most common. The primary structure of a peptide can be deduced from its b or y series fragment ions. 556.79 m / z is the [M+H]+ ion signal of the nonapeptide, with a charge number (z) of 2 and a molecular weight of 1112.57 Da. Secondary mass spectrometry analysis of the nonapeptide using source collision-induced dissociation technology confirmed its primary structure as glutamic acid-aspartic acid-lysine-leucine-aspartic acid-leucine-aspartic acid-histidine-lysine (Glu-Asp-Lys-Leu-Asp-Leu-Asp-His-Lys). The chemical structure of the nonapeptide EDKLDLDHK is shown below. Figure 2 As shown.

[0048] Molecular docking 2D and 3D diagrams of the nonapeptide EDKLDLDHK with AHR are shown below. Figure 3 As shown. Analysis of the chemical bonds revealed that EDKLDLDHK primarily binds to AHR through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), electrostatic interactions (salt bridges and attracted charges), and hydrophobic interactions (alkyl and π-alkyl groups), with a docking energy of -171.37 kcal / mol. EDKLDLDHK forms 9 van der Waals forces with amino acid residues PHE117, SER153, SER157, GLU116, ASP161, TYR76, GLY109, and TYR154; 10 hydrogen bonds with TYR311, GLN162, GLN113, GLY152, LYS155, THR160, and LEU142; 2 electrostatic interactions with LYS313 and ARG143; and 3 electrostatic interactions with LEU112. Based on the molecular docking results, it is inferred that the nonapeptide EK9 can bind to AHR, thereby activating AHR and exerting immunomodulatory effects.

[0049] 3. Artificially synthesized peptides The peptides EDKLDLDHK and KFPKAEFVEV with a purity of ≥98% were synthesized by Shenzhen Borunsida Biotechnology Co., Ltd. for subsequent functional verification.

[0050] Example 2: The Proliferative Effect of Nonapeptide (EDKLDLDHK) on Macrophages To assess the immunomodulatory function of the target peptide, this study used a zebrafish in vivo model for characterization. As a model organism, zebrafish share approximately 87% genome homology with humans, and their immune system is highly similar in composition, particularly in innate immunity, where their response mechanisms effectively reflect the physiological characteristics of mammals. Zebrafish possess a complete innate and adaptive immune system: macrophages and neutrophils, as the core cell groups of innate immunity, develop in a timeline consistent with mammals, appearing in the embryo within 30 hours after fertilization; while T and B lymphocytes begin development from 4 days post-fertilization (dpf), and the adaptive immune system only matures 4-6 weeks post-fertilization, indicating that zebrafish embryos primarily rely on innate immunity for defense. At the molecular level, various immune molecules such as TLRs, TNF, and ILs participate in the immune response during embryonic development. Among them, the TLR family with MyD88 as the linker can activate downstream signaling cascades such as MAPK and NF-κB by recognizing pathogen-associated model molecules. Accordingly, exogenous immunomodulatory substances can intervene in the embryonic innate immune system through the aforementioned molecular pathways. Fluctuations in the number of macrophages and neutrophils, as well as changes in the levels of cytokines such as IFN-γ, can all serve as effective parameters for evaluating the immunomodulatory activity of substances.

[0051] Macrophages, originating from bone marrow progenitor cells and tissue colonization precursors, are key effector cells of the innate immune system. They possess the functions of phagocytizing and clearing pathogens and abnormal cells, and can secrete various cytokines. Under microenvironmental stimulation, macrophages can polarize into two phenotypes: pro-inflammatory (M1) and anti-inflammatory (M2). The M1 type is responsible for releasing pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, as well as bactericidal effector molecules such as NO and reactive oxygen species. The M2 type secretes anti-inflammatory factors and participates in tissue repair, angiogenesis, and fibrosis. Therefore, changes in macrophage numbers can serve as one of the core indicators for measuring the effects of immune regulation.

[0052] In this embodiment, an immunodeficiency model was created in wild AB strain zebrafish by using chloramphenicol to reduce the number of macrophages. The nonapeptide EDKLDLDHK was then used for intervention to characterize the effect of the nonapeptide on the number of macrophages, an important immune cell.

[0053] Wild-caught AB line zebrafish embryos were treated with 10 μL / mL phenylthiourea stock solution (PTU) at 24 hpf and demetabolized at 48 hpf. The demetabolized embryos were then placed in six-well plates, with two parallel wells per group and 10 embryos per well. The groups were as follows: (1) Blank control group: 0.5% (v / v) DMSO + system water (components: 1% sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate, 0.00003% methylene blue, pH=7.00) + PTU with a final concentration of 30 μg / mL; (2) Model control group: Chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (3) Nonapeptide intervention group (EK9-10, EK9-5, EK9-1): Add nonapeptide EDKLDLDHK (EK9) with a final concentration of 10 μg / mL, 5 μg / mL or 1 μg / mL + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%.

[0054] Twenty-four hours after drug exposure, 2.5 μg / mL neutral red staining solution and PTU were added to each well, and the embryos were incubated in the dark for 6 hours. After staining, the embryos were washed with systemic water in the dark, anesthetized, and fixed with 6% methylcellulose. Images were acquired under a stereomicroscope, and the number of macrophages in the head was counted. Data were processed using GraphPad Prism 8.0 with the model group as a reference. Comparisons among multiple groups were performed using one-way ANOVA and Tukey's post-hoc test.

[0055] The results are as follows Figure 4 and Figure 5 As shown, compared with the blank control group, the number of macrophages in the model group was significantly reduced ( p <0.0001), indicating that the immunodeficiency model was successfully induced after using chloramphenicol (150 μg / mL).

[0056] When the nonapeptide EDKLDLDHK (EK9) was used to treat immunocompromised zebrafish, the intervention concentrations of 10 μg / mL and 5 μg / mL significantly promoted macrophage proliferation compared with the model control group. p <0.0001, p <0.01), with no significant difference compared to the blank control group ( p >0.05). At a concentration of 1 μg / mL, there was no statistically significant difference compared to the model control group ( p >0.05), but the difference compared to the blank control group ( p <0.05) is less than the difference between the model control group and the blank control group ( p The result was <0.0001, indicating a certain positive trend at this concentration. The combined significance results at 10 μg / mL and 5 μg / mL demonstrate that the nonapeptide EDKLDLDHK has a significant immune-enhancing effect within the effective concentration range.

[0057] Example 3: Effect of nonapeptide (EDKLDLDHK) on neutrophil proliferation Neutrophils, a subset of white blood cells derived from bone marrow hematopoietic stem cells, constitute the largest proportion of circulating blood cells and are the body's first line of defense against pathogen invasion. This cell group not only performs phagocytic and bactericidal functions by releasing cytotoxic granules and proteases, but also participates in monocyte recruitment and the repair of damaged tissues, playing an irreplaceable role in the innate immune response.

[0058] During zebrafish embryonic development, neutrophils originate from myeloid precursor cells in the embryonic liver or yolk sac. These precursor cells differentiate at the anterior end of the hematopoietic zone and then enter the circulatory system, distributing throughout the body via the bloodstream. Based on this developmental characteristic, immunological studies often use neutrophil-specific fluorescent transgenic strain Tg(Lyz:DsRed) zebrafish larvae as observation vectors, directly quantifying the fluorescence signal of neutrophils in the region from the cloaca to the tail tip using fluorescence microscopy.

[0059] In this embodiment, Tg(Lyz:DsRed) fluorescent transgenic zebrafish larvae were used as a model. Chloramphenicol was used to induce a model of immunodeficiency by reducing the number of neutrophils. The nonapeptide EDKLDLDHK was used for intervention to characterize the effect of the nonapeptide on the number of neutrophils, an important immune cell.

[0060] Tg(Lyz:DsRed) zebrafish embryos were treated with 10 μL / mL phenylthiourea stock solution (PTU) at 24 hpf, and demetabolized at 48 hpf. Embryos expressing fluorescent genes were screened under a stereofluorescence microscope. The demetabolized embryos with fluorescent genes were placed in six-well plates, with two parallel wells per group and 10 embryos per well. The groupings are as follows: (1) Blank control group: 0.5% DMSO + system water + PTU; (2) Model control group: Chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (3) Nonapeptide intervention group (EK9-10, EK9-5, EK9-1): Add nonapeptide EDKLDLDHK (EK9) with a final concentration of 10 μg / mL, 5 μg / mL or 1 μg / mL + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%.

[0061] After 24 hours of combined treatment, the embryos were cleaned, anesthetized, and photographed under a stereofluorescence microscope. The fluorescence intensity from the cloaca to the tail tip was counted, and the model group was compared with other groups using GraphPad Prism 8.0 software for significance testing.

[0062] The results are as follows Figure 6 and Figure 7 As shown, compared with the blank control group, the number of neutrophils in the model group was significantly reduced ( p <0.001), indicating that the immunodeficiency model was successfully induced after using chloramphenicol (150 μg / mL).

[0063] When the nonapeptide EDKLDLDHK (EK9) was used to treat immunocompromised zebrafish, both the intervention concentrations of 10 μg / mL and 5 μg / mL significantly promoted the proliferation of neutrophils. p <0.001, p <0.01), with no significant difference compared to the blank control group ( p >0.05); when the intervention concentration was 1 μg / mL, there was no significant difference compared with the blank control group ( p >0.05). This indicates that, from the perspective of neutrophils, an important immune cell, the nonapeptide EDKLDLDHK has an immune-enhancing effect.

[0064] Example 4: Upregulation of the immune factor IFN-γ by nonapeptide (EDKLDLDHK) Interferons (IFNs), a class of glycoproteins with broad biological activity, are classified into type I and type II based on their structure and function. Type I mainly includes IFN-β and IFN-α, while type II contains only IFN-γ. IFN-γ is primarily secreted by T lymphocytes, natural killer (NK) cells, macrophages, and neutrophils, and is considered a key macrophage activator in mammals. Unlike type I interferons, IFN-γ belongs to the immunomodulatory interferon family, and its immunomodulatory efficacy is far higher than that of antiviral interferons, thus it is regarded as an important biomarker for assessing the body's immune status.

[0065] In this embodiment, a model of immunodeficiency was created in wild AB strain zebrafish using chloramphenicol to reduce IFN-γ levels. The nonapeptide EDKLDLDHK was then used for intervention to characterize the effect of the nonapeptide on the level of the important immune factor IFN-γ.

[0066] Wild-caught AB line zebrafish embryos were treated with 10 μL / mL phenylthiourea stock solution (PTU) at 24 hpf and demetabolized at 48 hpf. The demetabolized embryos were then placed in six-well plates, with two parallel wells per group and 10 embryos per well. The groups were as follows: (1) Blank control group: 0.5% DMSO + system water + PTU; (2) Model control group: Chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (3) Nonapeptide intervention group (EK9-10, EK9-5, EK9-1): Add nonapeptide EDKLDLDHK (EK9) with a final concentration of 10 μg / mL, 5 μg / mL or 1 μg / mL + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%.

[0067] After incubation at 28.5℃ for 24 hours, embryos (120 per well) were collected from each group and placed in 1.5 mL centrifuge tubes. The tubes were washed twice with an equal volume of PBS buffer, and residual liquid was aspirated. The embryos were then homogenized at a ratio of 50 μL PBS per 10 mg of tissue, and centrifuged at 4℃, 5000 r / min for 5 minutes using a high-speed tissue homogenizer. The supernatant was collected. The IFN-γ ELISA kit (Wuhan GeneMe Biotechnology Co., Ltd.) was used to determine the IFN-γ content in the supernatant, strictly following the instructions. Data were statistically analyzed using GraphPad Prism 8.0, and one-way ANOVA combined with Tukey's multiple test was used for comparisons among multiple groups.

[0068] Experimental results are as follows Figure 8 As shown, after treatment with 150 μg / mL chloramphenicol, the IFN-γ level in the model control group was significantly decreased compared to the blank control group. p <0.01), indicating that the immunodeficiency model was successfully constructed.

[0069] Regarding EK9 intervention, both 10 μg / mL and 5 μg / mL concentrations of EK9 significantly increased IFN-γ levels, with statistically significant differences compared to the model control group. p <0.05), and recovered to a level that was not significantly different from the blank control group ( p >0.05). It is noteworthy that even though statistical significance was not achieved between the 1 μg / mL low-dose group and the model group ( p >0.05), but its IFN-γ level was not significantly different from that of the normal group ( p >0.05), indicating that the nonapeptide has a positive regulatory trend on IFN-γ over a wide concentration range.

[0070] In summary, the nonapeptide EDKLDLDHK also exhibits definite immune-enhancing activity at the level of immune factors.

[0071] Example 5: Comparison of the effects of nonapeptide EDKLDLDHK (EK9) and decapeptide KFPKAEFVEV (KV10) on macrophage proliferation. An immunodeficiency model was created in wild AB strain zebrafish by using chloramphenicol to reduce the number of macrophages. The effects of nonapeptide EDKLDLDHK (EK9) and decapeptide KFPKAEFVEV (KV10) on the number of macrophages, an important immune cell, were compared.

[0072] Wild-caught AB line zebrafish embryos were treated with 10 μL / mL phenylthiourea stock solution (PTU) at 24 hpf and demetabolized at 48 hpf. The demetabolized embryos were then placed in six-well plates, with two parallel wells per group and 10 embryos per well. The groups were as follows: (1) Blank control group: 0.5% DMSO + system water + PTU; (2) Model control group: Chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (3) Nonapeptide intervention group (EK9): EDKLDLDHK (EK9) with a final concentration of 5 μg / mL was added + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL was added + system water + PTU, with DMSO volume fraction of 0.5%; (4) Decapeptide intervention group (KV10): KFPKAEFVEV (KV10) with a final concentration of 5 μg / mL was added + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%.

[0073] Twenty-four hours after drug administration, 2.5 μg / mL of neutral red dye and PTU were added to each well, and staining was performed for 6 hours in the dark. Embryos were washed with systemic water in the dark, anesthetized, and fixed with 6% methylcellulose. Images were taken under a stereomicroscope, and the number of macrophages in the head was counted. The model group served as a control. Data analysis was performed using GraphPad Prism 8.0 software, and Tukey's test in One-way ANOVA was used to compare differences between multiple groups.

[0074] Experimental results are as follows Figure 9 and Figure 10 As shown, an immunodeficiency model was successfully induced using chloramphenicol (150 μg / mL), with the number of macrophages significantly lower than that in the blank control group. p <0.0001). When the nonapeptide EDKLDLDHK (EK9) was used to intervene in immunocompromised zebrafish, it significantly promoted the proliferation of macrophages compared with the model control group. p <0.01); however, after intervention with KFPKAEFVEV (KV10), which also has a high docking energy with AHR molecules, there was no significant difference compared with the model control group ( p<0.05). This indicates that, from the perspective of important immune cells such as macrophages, EK9 has a stronger immune-enhancing effect than KV10.

[0075] Example 6: Comparison of the effects of nonapeptide EDKLDLDHK (EK9) and decapeptide KFPKAEFVEV (KV10) on neutrophil proliferation. Using Tg(Lyz:DsRed) fluorescent transgenic zebrafish larvae as a model, chloramphenicol was used to induce a decrease in the number of neutrophils, resulting in an immunodeficiency model. EDKLDLDHK and KFPKAEFVEV were then used for intervention, and the effects of EK9 and KV10 on the number of neutrophils, an important immune cell, were compared.

[0076] Tg(Lyz:DsRed) zebrafish embryos were treated with 10 μL / mL phenylthiourea stock solution (PTU) at 24 hpf, and demetabolized at 48 hpf. Embryos expressing fluorescent genes were screened under a stereofluorescence microscope. The demetabolized embryos with fluorescent genes were placed in six-well plates, with two parallel wells per group and 10 embryos per well. The groupings are as follows: (1) Blank control group: 0.5% DMSO + system water + PTU; (2) Model control group: Chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (3) EK9 intervention group (EK9): EDKLDLDHK (EK9) with a final concentration of 5 μg / mL was added + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL was added + system water + PTU, with DMSO volume fraction of 0.5%; (4) KV10 intervention group (KV10): KFPKAEFVEV (KV10) with a final concentration of 5 μg / mL was added + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with a DMSO volume fraction of 0.5%.

[0077] After 24 hours of combined treatment, the embryos were cleaned, anesthetized, and photographed under a stereofluorescence microscope. The fluorescence intensity from the cloaca to the tail tip was counted, and the model group was compared with other groups using GraphPad Prism 8.0 software for significance testing.

[0078] The results are as follows Figure 11 and Figure 12 As shown, an immunodeficiency model was successfully induced using chloramphenicol (150 μg / mL), with the number of neutrophils significantly lower than that in the blank control group. p<0.001). When 5 μg / mL of the nonapeptide EDKLDLDHK (EK9) was used to treat immunocompromised zebrafish, it significantly promoted neutrophil proliferation compared with the model control group. p <0.01); however, at the same intervention concentration (5 μg / mL), KFPKAEFVEV (KV10) showed no significant difference compared to the model control group ( p >0.05). This indicates that, from the perspective of neutrophils, an important immune cell, EDKLDLDHK has a stronger immune-enhancing effect than KFPKAEFVEV.

[0079] Example 7: Comparison of the effects of nonapeptide EDKLDLDHK (EK9) and decapeptide KFPKAEFVEV (KV10) on the upregulation of the immune factor IFN-γ An immunodeficiency model was created in wild AB strain zebrafish by using chloramphenicol to reduce IFN-γ levels. EDKLDLDHK and KFPKAEFVEV were then used for intervention, and the effects of EK9 and KV10 on the levels of the important immune factor IFN-γ were compared.

[0080] Wild-caught AB line zebrafish embryos were treated with 10 μL / mL phenylthiourea stock solution (PTU) at 24 hpf and demetabolized at 48 hpf. The demetabolized embryos were then placed in six-well plates, with two parallel wells per group and 10 embryos per well. The groups were as follows: (1) Blank control group: 0.5% DMSO + system water + PTU; (2) Model control group: Chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (3) EK9 intervention group (EK9): EDKLDLDHK (EK9) with a final concentration of 5 μg / mL + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%; (4) KV10 intervention group (KV10): KFPKAEFVEV (KV10) with a final concentration of 5 μg / mL + chloramphenicol (dissolved in DMSO) with a final concentration of 150 μg / mL + system water + PTU, with DMSO volume fraction of 0.5%.

[0081] After culturing at 28.5℃ for 24 h, 120 embryos from each group were placed into 1.5 mL centrifuge tubes. The embryos were washed with PBS buffer equal to the total liquid volume in each well. After removing the residual PBS buffer, PBS buffer was added at a ratio of 50 μL of liquid per 10 mg of sample. The mixture was homogenized using a high-speed tissue homogenizer for 90 s and then centrifuged (4 ℃, 5000 r / min, 5 min). The supernatant was collected, and the IFN-γ content in the supernatant was determined using an IFN-γ ELISA kit (Wuhan GeneMe Biotechnology Co., Ltd.) according to the kit instructions. Data analysis was performed using GraphPad Prism 8.0 software, and Tukey's test in One-way ANOVA was used to compare differences among multiple groups.

[0082] The results are as follows Figure 13 As shown, an immunodeficiency model was successfully induced using chloramphenicol (150 μg / mL), with IFN-γ levels significantly lower than those in the normal control group. p <0.01). When 5 μg / mL of the nonapeptide EDKLDLDHK (EK9) was used to intervene in immunocompromised zebrafish, it significantly upregulated IFN-γ levels compared with the model control. p <0.05); however, at the same intervention concentration, KFPKAEFVEV (KV10) showed no significant difference compared to the model group ( p >0.05). This indicates that, from the perspective of the level of the important immune factor IFN-γ, EDKLDLDHK has a stronger immune-enhancing effect than KFPKAEFVEV.

[0083] 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. A biologically active peptide EK9, characterized in that, The amino acid sequence of the bioactive peptide EK9 is Glu-Asp-Lys-Leu-Asp-Leu-Asp-His-Lys.

2. The method for preparing the bioactive peptide EK9 as described in claim 1, characterized in that, The bioactive peptide EK9 is prepared by solid-phase synthesis or genetic engineering, or by targeted enzymatic hydrolysis of whey protein.

3. The application of the bioactive peptide EK9 as described in claim 1 in the preparation of immune-modulating products.

4. Use according to claim 3, wherein the compound is ###0002### The product is a drug for the prevention or treatment of immunodeficiency.

5. The use according to claim 4, wherein the compound is ###0002### The manifestations of weakened immune function include a decrease in the number of immune cells and a decrease in the level of immune factors.

6. Use according to claim 5, wherein The immune cells include macrophages and neutrophils; the immune factors include interferon-γ.

7. The use according to claim 3, wherein the compound is ###0002### The product is a health food that helps boost immunity.

8. A pharmaceutical composition for preventing or treating immunocompromise, characterized by, The pharmaceutical composition comprises an effective dose of the bioactive peptide EK9 as described in claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is in the form of an oral formulation.

10. A health food product that helps boost immunity, characterized in that, The health food includes the bioactive peptide EK9 as described in claim 1 as an active ingredient and food-grade excipients.

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