A casein-derived peptide having an anti-allergic activity and a preparation method and use thereof

Abstract

The application discloses a casein-derived peptide with anti-allergy activity and a preparation method and application thereof, and belongs to the technical field of proteins, and particularly relates to a casein-derived peptide with anti-allergy activity, which comprises ALLERVIA NO.1 and ALLERVIA NO.2, the amino acid sequence of the ALLERVIA NO.1 is as shown in SEQ ID NO.1, and the amino acid sequence of the ALLERVIA NO.2 is as shown in SEQ ID NO.2. The casein-derived peptide with anti-allergy activity is prepared by using a method of cold plasma assistance combined with computer-aided virtual screening network pharmacology, molecular docking, MD dynamics simulation and ADMET prediction, and the problems of great blindness, long cycle and low efficiency of target peptide discovery in the development process of traditional food-derived bioactive peptides are solved, and the casein-derived peptide with anti-allergy activity is efficiently screened.

Description

Technical Field

[0001] This invention belongs to the field of protein technology, and particularly relates to a casein-derived peptide with anti-allergic activity, its preparation method, and its application. Background Technology

[0002] Food allergy (FA) refers to a specific immune response triggered by contact with certain foods. It can repeatedly affect multiple organ systems, including the skin, gastrointestinal tract, and respiratory tract, and in severe cases, can lead to anaphylactic shock or even death. Currently, there is no cure. Bovine casein (CN) accounts for approximately 80% of the total protein in milk and is an important source of high-quality dietary protein and bioactive peptides (especially immunomodulatory peptides). However, its high allergenicity has become a key technological bottleneck restricting the in-depth development of related dairy products.

[0003] In recent years, food-derived bioactive peptides have become a research hotspot due to their low allergenicity and easy absorption. Traditional methods such as protein enzymatic hydrolysis and microbial fermentation have limitations, including poor hydrolysis specificity, long development cycles, and high costs. Furthermore, the screening of bioactive peptides relies excessively on cumbersome separation and purification processes, resulting in low efficiency. By leveraging bioinformatics and molecular dynamics simulation techniques, computer-aided high-throughput screening of the dynamic interactions between peptide molecules and allergy receptors can be achieved, providing a new approach to overcome these bottlenecks.

[0004] In existing technologies, the preparation and screening of anti-allergic bioactive peptides mainly rely on protein cleavage and blind screening, which suffers from drawbacks such as lack of specificity in cleavage, high workload, high cost, and low efficiency in target peptide discovery. Crucially, the target and mechanism of action of most bioactive peptides remain unclear, lacking evidence of dynamic binding at the molecular level to core allergy receptor proteins (such as the IgE / FcεRI complex), and lacking multi-dimensional consistency verification from computer theory predictions, biochemical enzymology to cellular immunity levels. This casts doubt on their true anti-allergic efficacy and severely limits the development of related targeted products.

[0005] Cold plasma (CP), as an emerging non-thermal processing technology, can precisely cleave peptide bonds using active particles to generate small peptides, thereby targeting and destroying antigenic epitopes of proteins, providing a novel approach for the preparation of hypoallergenic active peptides. However, there is currently no technical solution that couples CP's efficient targeted degradation technology with computer-aided directed screening strategies, and combines this with in vitro enzymatic and cellular validation to obtain novel functional peptides with clear anti-allergic activity. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a casein-derived peptide with anti-allergic activity, its preparation method, and its applications. The method utilizes cold plasma-assisted combined with computer-aided virtual screening network pharmacology, molecular docking, MD kinetic simulation, and ADMET prediction to prepare casein-derived peptides with anti-allergic activity. This solves the problems of high screening blindness, long cycles, and low target peptide discovery efficiency in the traditional research and development of food-derived bioactive peptides. The aim is to efficiently screen casein-derived peptides with anti-allergic activity by integrating CP processing and computer-aided analysis.

[0007] To achieve the above objectives, the present invention provides a casein-derived peptide with anti-allergic activity, wherein the casein-derived peptide with anti-allergic activity includes ALLERVIA NO.1 and ALLERVIA NO.2, wherein the amino acid sequence of ALLERVIA NO.1 is as shown in SEQ ID NO.1, and the amino acid sequence of ALLERVIA NO.2 is as shown in SEQ ID NO.2.

[0008] The present invention also provides a method for preparing the casein-derived peptide with anti-allergic activity, comprising the following steps: 1) Bovine milk casein was pretreated with cold plasma to obtain CP-pretreated casein small molecule peptides; 2) Collect the CP pretreated casein small molecule peptides obtained in step 1) to construct a database, combine bioinformatics and network pharmacology target prediction for high-throughput virtual screening, and verify the anti-allergic activity by measuring hyaluronidase inhibitory activity and the inhibitory activity on KU812 cell degranulation and inflammatory factor release, and obtain casein-derived peptides with anti-allergic activity.

[0009] Preferably, in step 1), the cold plasma pretreatment of bovine casein uses dielectric barrier discharge, with argon as the working gas, an input power of 75W, and a treatment time of 12min.

[0010] Preferably, step 2) includes the following steps: using bioinformatics to screen peptides in the database to obtain candidate short peptides; using network pharmacology to predict the targets of the obtained candidate short peptides and taking the intersection with food allergy disease targets to obtain core targets; and performing KEGG pathway enrichment analysis on pathways closely related to inflammation and immune response, docking the candidate short peptides with the receptors of the above-mentioned pathways closely related to inflammation and immune response to obtain bindable peptides; measuring the hyaluronidase inhibitory activity of the bindable peptides and their inhibitory activity on KU812 cell degranulation and inflammatory factor release to verify anti-allergic activity, thus obtaining casein-derived peptides with anti-allergic activity.

[0011] Further preferably, the candidate short peptide is a peptide with a length <10, a bioactivity score >0.8, an intestinal absorption potential (HIA) >20%, a blood-brain barrier crossing (BBB) ​​>0.1, and is non-toxic and non-sensitizing.

[0012] Preferably, the core targets are AKT1, NFKB1, IL-1β and STAT3.

[0013] Preferably, the pathways closely related to inflammation and immune response are AGE-RAGE, TNF signaling pathway and Th17 cell differentiation.

[0014] Preferably, the step of docking the candidate short peptide with receptors of the aforementioned pathways closely related to inflammation and immune response to obtain a bindable peptide specifically involves docking the candidate short peptide with AKT1, NFKB1, IL-1β, and STAT3 to obtain a peptide with an AutoDock Vina binding energy of <-5 kcal / mol and a discovery Studio binding energy of <-50 kcal / mol.

[0015] The present invention also provides the application of the casein-derived peptide with anti-allergic activity in the preparation of anti-allergy products.

[0016] Preferably, the anti-allergy products include those for food allergies, allergic dermatitis, allergic asthma, allergic rhinitis, and / or allergic conjunctivitis.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a casein-derived peptide with anti-allergic activity, its preparation method, and its applications. The method utilizes cold plasma-assisted, computer-aided virtual screening network pharmacology, molecular docking, MD kinetic simulation, and ADMET prediction to prepare casein-derived peptides with anti-allergic activity. This solves the problems of high screening blindness, long cycle, and low target peptide discovery efficiency in the traditional research and development of food-derived bioactive peptides. Combined with in vitro enzymatic and cellular verification, a casein-derived peptide with high anti-allergic activity was obtained, providing a core material basis for its application in the development of anti-allergic functional drugs. The casein-derived peptides ALLERVIA NO.1 and ALLERVIA NO.2 described in this invention not only form highly stable dynamic complexes with the core allergy target in molecular dynamics simulations, but also exhibit significant hyaluronidase inhibitory activity in vitro and effectively block basophil degranulation and inflammatory factor release at the in vivo cellular level, achieving a synergistic effect from biochemical inhibition to cellular immune intervention. This provides a new theoretical basis and safe and efficient food-derived intervention substances for the development of anti-allergy products. The present invention describes a method for preparing casein-derived peptides with anti-allergic activity. This method utilizes cold plasma instead of traditional strong acid-base hydrolysis and high-temperature treatment, resulting in milder reaction conditions that maximize the retention of active peptides. Physical cleavage reduces the allergenicity of the casein parent compound, leaving no chemical reagent residues, making it a green and efficient approach. It overcomes the limitations of traditional blind screening by integrating network pharmacology and molecular dynamics simulations to clarify the stable binding conformation of the target peptide to the core target site, significantly shortening the research and development cycle and greatly improving the accuracy of active peptide screening. ADMET prediction and evaluation show that the intestinal absorption potential (HIA) of the ultrashort peptides ALLERVIA NO.1 and ALLERVIA NO.2 obtained by this invention is >20%, effectively solving the technical pain point of low oral bioavailability of traditional large-molecule active peptides. CCK-8 cytotoxicity experiments verify that the target ALLERVIA NO.1 and ALLERVIA NO.2 have no significant toxicity to human immune cells within the effective activity concentration range, exhibiting good cell survival rates and providing reliable safety support for long-term application. The ALLERVIA NO.1 and ALLERVIA NO.2 obtained by this invention can be used as core active ingredients and widely applied in anti-allergy drugs. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1Venn diagram showing the target of candidate short peptides and the disease target of "food allergy"; Figure 2 A network diagram of the core target points; Figure 3 A bar chart showing the core target points; Figure 4 GO enrichment analysis plot; Figure 5 A graph showing KEGG pathway enrichment analysis; Figure 6 Heatmaps of molecular docking results using different docking software programs are shown. A represents the docking result from Discovery Studio, and B represents the docking result from AutoDock Vina. Figure 7 The graph shows the correlation between the inhibitory activities of different concentrations of FRQFY, ALLERVIA NO.2, FRQF, ALLERVIA NO.1, and sodium cromoglycate (SCG) on hyaluronidase. In the graph, A represents the inhibitory activity of different concentrations of FRQFY, ALLERVIA NO.2, FRQF, ALLERVIA NO.1, and sodium cromoglycate (SCG) on hyaluronidase; B represents the inhibition rate of the logarithmic SCG concentration on hyaluronidase; and C represents the inhibition rate of the logarithmic ALLERVIA NO.1 concentration on hyaluronidase. The figures show the ns... and Representative difference significance analysis; Figure 8 The effects of different concentrations of FRQFY, ALLERVIA NO.2, FRQF and ALLERVIA NO.1 on the viability of KU812 cells; Figure 9 The effects of FRQFY, ALLERVIA NO.2, FRQF, and ALLERVIA NO.1 at concentrations of 25 μg / mL on degranulation and release of inflammatory factors in KU812 cells were investigated. In the figure, A represents histamine release level, B represents the release rate of degranulation markers, C represents IL-4 release level, and D represents TNF-α release level. CN represents the casein positive control group, CN+CP represents the casein and plasma treatment group (12 min), CN+NO.2 represents the casein and ALLERVIA NO.2 group, CN+FRQFY represents the casein and FRQFY group, CN+FRQF represents the casein and FRQF group, CN+OLO represents the casein and olopatadine group, CN+NO.1 represents the casein and ALLERVIA NO.1 group, and PBS represents the PBS blank control group. Different lowercase letters indicate significant differences. Figure 10The effects of FRQFY, ALLERVIA NO.2, FRQF, and ALLERVIA NO.1 at concentrations of 25 μg / mL on the expression of STAT3 and AKT1 proteins in KU812 cells were investigated. In the figures, A represents the Western blot images of AKT1, STAT3, p-AKT1, and p-STAT3; B represents the relative expression level of p-STAT3 / STAT3; C represents the relative expression level of p-AKT / AKT; CN represents the casein positive control group; ALLERVIA NO.2 and CN+NO.2 represent the casein and peptide ALLERVIA NO.2 group; CN+OLO represents the casein and drug olopatadine group; ALLERVIA NO.1 and CN+NO.1 represent the casein and peptide ALLERVIA NO.1 group; PBS represents the PBS blank control group; different lowercase letters indicate significance analysis. Figure 11 Statistical charts of the average RMSD of ALLERVIA NO.1-STAT3, ALLERVIA NO.2-STAT3, and STAT3; Figure 12 Statistical charts of root mean square fluctuations (RMSF) for ALLERVIA NO.1-STAT3, ALLERVIA NO.2-STAT3, and STAT3; Figure 13 Statistical chart of solvent accessible surface area (SASA) for ALLERVIA NO.1-STAT3, ALLERVIA NO.2-STAT3, and STAT3; Figure 14 A statistical chart showing the number of hydrogen bonds in ALLERVIA NO.1-STAT3 and ALLERVIA NO.2-STAT3; Figure 15 This is a schematic diagram of the overall experimental process of Embodiments 1, 2, and 3 of the present invention; Figure 16 Total ion chromatogram of CP-pretreated casein small peptide fragments; Figure 17 This is a detailed secondary mass spectrum of ALLERVIA NO.1; Figure 18 This is a detailed secondary mass spectrum of ALLERVIA NO.2. Detailed Implementation

[0020] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementations of the present invention. The specification and embodiments of the present invention are merely exemplary.

[0021] The materials used in this invention were sourced as follows: cold plasma was purchased from Nanjing Suman Plasma Technology Co., Ltd.; bovine casein was purchased from Shanghai Yuanye Company. The overall experimental process of Examples 1, 2, and 3 of this invention is illustrated below. Figure 15 As shown.

[0022] Example 1 Bovine milk casein was pretreated with cold plasma (cracking peptide bonds, destroying antigenic epitopes and reducing sensitization). The cold plasma pretreatment adopted dielectric barrier discharge (DBD), with argon as the working gas, an input power of 75W, and a treatment time of 12min to obtain CP-pretreated casein small molecule peptides.

[0023] Peptide composition was identified by LC-MS / MS, confirming that ALLERVIA NO.1 and ALLERVIA NO.2 were indeed present in the treated product. Figure 16 , Figure 17 and Figure 18 As shown.

[0024] Combining bioinformatics and network pharmacology target prediction, high-throughput virtual screening is performed, specifically as follows: 1) A database of CP-pretreated casein small peptides was constructed. The PeptideRanker tool was used to predict the activity of each peptide, and peptides with a bioactivity score > 0.8 were screened. Furthermore, the potential sensitization of these peptides was examined online using AllerTOPv2.1 and ALLERDET to confirm non-toxicity, and the potential toxicity of the peptides was analyzed online using ToxinPred3.0. The intestinal absorption potential (HIA > 20%), blood-brain barrier crossing (BBB > 0.1), and toxicity (non-toxic) of the peptides were analyzed using admetSAR3.0. Finally, BIOPEP-UWM was used to determine the novelty of the active peptides in immunomodulation, thereby screening peptides in the database to identify candidate peptides. 2) Network pharmacology prediction: SwissTargetPrediction (Probability ≥ 0.1) and SuperPred databases (Probability ≥ 0.5) were used to predict target genes for candidate peptides. GeneCards, OMIM, and DisGeNET databases were searched using the keyword "food allergy" to identify potential disease targets. A "candidate peptide-target-disease" interaction network diagram was constructed using Cytoscape 3.7.2 software and the STRING database. DAVID was used to perform GO and KEGG pathway enrichment analysis on the intersection target genes of candidate peptides acting on allergy relief. The results were visualized using a microbioinformatics platform to determine the targets of candidate peptides acting on allergies. The targets of candidate short peptides predicted by network pharmacology were intersected with the disease targets of "food allergy" (e.g., ...). Figure 1 As shown), the core targets AKT1 (3QKL), IL-1β (5R85), and STAT3 (6NJS) were obtained (as shown). Figure 2 and Figure 3 As shown), GO and KEGG pathway enrichment analysis was performed, and the disease targets were mainly located in the plasma membrane, exhibiting specific serine-type endopeptidase activity and protein hydrolysis function, and significantly enriched in the AGE-RAGE, TNF signaling pathway, and Th17 cell differentiation pathway, thereby jointly mediating the regulation of inflammation and immune response (e.g. Figure 4 and Figure 5 As shown in the figure, the target NFKB1 (8TQD) was found to occupy a core position in the above three pathways. Therefore, AutoDockVina and Discovery Studio were used to dock the candidate short peptides with AKT1 (3QKL), NFKB1 (8TQD), IL-1β (5R85) and STAT3 (6NJS), obtaining peptides with binding energies of AutoDock Vina < -5 kcal / mol and Discovery Studio < -50 kcal / mol: FRQFY (SEQ ID NO.3:FRQFY), ALLERVIA NO.2 (SEQ ID NO.2:AYFY, derived from amino acids 158-161 of bovine α-S1 casein (UniProt number: P02662), FRQF (SEQ ID NO.4:FRQF), and ALLERVIA NO.1 (SEQ ID NO.4:FRQF). NO.1: FYQL, derived from amino acid positions 168-171 of bovine α-S1 casein (UniProt ID: P02662) (e.g. Figure 6 China A and Figure 6 (As shown in B) Hyaluronidase inhibitory activity assay: In an acetate buffer system at pH 5.6, FRQFY, ALLERVIA NO.2, FRQF, or ALLERVIA NO.1 or a positive control were dissolved in 5% DMSO and incubated with 1250 U / mL hyaluronidase and calcium chloride solution at 37°C. Then, 0.5 mL of 0.5 mg / mL sodium hyaluronate substrate was added and incubated at 37°C for 30 min, followed by a 5 min reaction at room temperature. After the reaction was complete, 0.1 mL of 0.4 mol / L sodium hydroxide and 0.5 mL of acetylacetone (3.5 mL of acetylacetone dissolved in 50 mL of 1.0 mol / L sodium carbonate solution) were added sequentially. The mixture was then placed in a boiling water bath at 100 °C for 15 min, followed by an ice bath for 5 min. After cooling to room temperature, 1 mL of Ehrlich's reagent was added (15 mL of concentrated hydrochloric acid (37%) and 5 mL of anhydrous ethanol were mixed thoroughly, and then 0.8 g of p-dimethylaminobenzaldehyde was weighed and dissolved in the above mixed solution). After the color development was performed, each test tube was shaken thoroughly, and 3.0 mL of anhydrous ethanol was added to stop the color development. After standing at room temperature for 20 min, the absorbance was measured at 530 nm to calculate the enzyme inhibition rate.

[0025] The results are as follows Figure 7 China A Figure 7 China B and Figure 7 As shown in Figure C, the peptides ALLERVIA NO.2, FRQF, and ALLERVIA NO.1 screened in this invention exhibit excellent and sequence-specific inhibitory efficacy. ALLERVIA NO.1, at an extremely low concentration (25 μg / mL), demonstrates a potent enzyme-blocking effect exceeding 50%, significantly superior to the positive control. ALLERVIA NO.2, on the other hand, exhibits sustained and stable moderate inhibitory activity over a very wide concentration window (25–400 μg / mL). These results, from the perspective of blocking extracellular matrix degradation, confirm that peptides ALLERVIA NO.1 and ALLERVIA NO.2 possess an excellent pharmacodynamic basis for their anti-allergic and anti-inflammatory effects through a synergistic effect of "potent low-dose blocking" and "wide-window stable regulation."

[0026] Assay for inhibitory activity against KU812 cell degranulation and inflammatory factor release: To investigate the allergic effects of different peptides on allergic reactions, the density of the logarithmic phase KU812 cell suspension was adjusted to 2 × 10⁻⁶. 6Inoculation was performed at a rate of 1 mL / well in a 24-well plate pre-filled with 1 mL of RPMI-1640 basal medium. After mixing and standing, the plate was incubated overnight at 37°C with 5% CO2. The next day, 20 μL of patient serum containing IgE was added to each well (an equal volume of sterile PBS was added to the blank control group), and incubation was continued for 24 hours to induce sensitization. After 24 hours of sensitization, the test sample FRQFY, ALLERVIA NO.2, FRQF, or ALLERVIA NO.1 was added to the system to achieve a final concentration of 25 μg / mL (an equal volume of sterile PBS was added to the blank and positive control groups), and pre-incubated for 30 minutes. Then, casein solution was added to achieve a final concentration of 50 μg / mL (an equal volume of sterile PBS was added to the blank control group), and co-incubation was continued for 4 hours to induce degranulation. The secretion of inflammatory factors (TNF-α, IL-4), histamine, and β-hexosidase (β-hex) in the cells of each group was detected.

[0027] The results are as follows Figure 9 China A~ Figure 9 The results showed that, compared with the positive control group (CN), the histamine release levels in the ALLERVIA NO.1 and ALLERVIA NO.2 treatment groups were significantly reduced (p<0.05), decreasing from 12.73 ng / mL to 4.43 ng / mL and 8.73 ng / mL, respectively (3.21 ng / mL in the blank PBS group and 6.36 ng / mL in the olopatadine (OLO) group). Regarding degranulation markers, the release rate of β-hex also showed a significant decreasing trend (p<0.05), decreasing significantly from 60.48% in the control group to 49.83% (ALLERVIA NO.1 group) and 51.72% (ALLERVIA NO.2 group), respectively. Furthermore, in terms of inflammatory cytokine secretion, ALLERVIA NO.1 significantly inhibited the release levels of IL-4 and TNF-α (p<0.05). While ALLERVIA NO.2 caused a similar significant decrease in TNF-α levels, its inhibitory effect on IL-4 release was not significant, showing no significant difference compared to the positive control group. In summary, both ALLERVIA NO.1 and ALLERVIA NO.2 possess certain anti-allergic activities, but ALLERVIA NO.1 demonstrates more comprehensive performance in inhibiting cell degranulation and the release of inflammatory factors.

[0028] By measuring the release levels of β-aminohexosidase (β-HEX), histamine, IL-4, and TNF-α, ALLERVIA NO.1 and ALLERVIA NO.2 were verified to be anti-allergic synthetic peptides that can effectively block the degranulation reaction of KU812 basophils and significantly downregulate the release levels of β-HEX, histamine, IL-4, and TNF-α, thus being casein-derived peptides with anti-allergic activity.

[0029] Example 2 Casein-derived peptides with anti-allergic activity were prepared and purified using a solid-phase synthesis method. All peptides used in this experiment were custom-synthesized by Shanghai ChuTai Biotechnology Co., Ltd. (Shanghai, China). The specific synthetic steps were as follows: The target peptides were synthesized using the standard Fmoc solid-phase synthesis method with blank Wang resin as the solid-phase carrier. First, the resin was activated and swollen in anhydrous N,N-dimethylformamide (DMF) after being dehydrated by molecular sieves for 30 minutes. Then, 5 equivalents of C-terminal Fmoc-amino acids, DIC, and DMAP were added, and the reaction proceeded at room temperature for 3 hours. Unreacted active sites were blocked using pyridine and acetic anhydride (volume ratio 1:1). Peptide chain elongation was strictly performed according to a "deprotection-condensation" cycle: first, the Fmoc protecting group was removed using a 20% piperidine / DMF solution; then, 3 equivalents of amino acids were added in an HBTU / HOBT / DIEA (molar ratio 3:3:10) activation system for condensation for 40 minutes. The reaction progress was monitored at each step using the ninhydrin assay (Kaiser test). After completing full-sequence ligation and removing terminal Fmoc groups, the peptide chain was cleaved and protected by reacting with a mixed lysis buffer containing 95% TFA, 2% TIS, 2% EDT, and 1% H2O at room temperature for 2 hours. The resulting lysis buffer was concentrated by nitrogen purging, and the crude peptide solid was obtained by repeated precipitation and washing with ice-cold ether. The crude product was finally purified by Agilent 1100 high-performance liquid chromatography (HPLC), freeze-dried to precipitate crystals, and the molecular weight was identified using a Waters ZQ2000 mass spectrometer (MS).

[0030] Example 3 I. Evaluation of KU812 cytotoxicity (CCK-8 assay): Prepare a suspension of KU812 cells at a density of 2 × 10⁻⁶ cells per well. 5 Cells were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Then, CCK-8 was added and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0031] Prepare a suspension of KU812 cells in the logarithmic growth phase, using 2×10⁻⁶ cells per cell. 5Cells were seeded at a rate of 100 μL per well in a 96-well plate; the control group received only 100 μL of RPMI-1640 basal medium. After seeding, the plates were incubated statically at 37°C and 5% CO2 for 24 hours. The peptide samples were dissolved in preheated (37°C) sterile PBS. After 24 hours of incubation, 10 μL of peptide sample solutions of different concentrations were added to each well of the sample groups, resulting in final concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. Simultaneously, 10 μL of sterile PBS was added to each well of the control and control groups. All groups were co-incubated for another 24 hours in a CO2 incubator under the same conditions. After incubation, 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated in the dark for 2 hours. The absorbance at 450 nm was then measured and recorded using a microplate reader.

[0032] The results are as follows Figure 8 As shown, the cell viability of FRQFY, ALLERVIA NO.2, FRQF, or ALLERVIA NO.1 was >80% at concentrations ranging from 0 to 200 μg / mL, indicating that FRQFY, ALLERVIA NO.2, FRQF, or ALLERVIA NO.1 had no significant toxicity to KU812 cells. Furthermore, at a concentration of 25 μg / mL, the cell viability of all four peptides was >90%, and no abnormal proliferation was observed. Therefore, a concentration of 25 μg / mL was selected as the concentration for subsequent degranulation experiments.

[0033] II. Effects of casein-derived peptides with anti-allergic activity on the expression of STAT3 and AKT1 proteins in KU812 cells: KU812 cells were seeded in 24-well plates according to the aforementioned method. After culture and drug intervention, the supernatant was discarded, and the cells were washed three times with pre-cooled PBS buffer. Total protein was extracted from the cells using a freshly prepared composite lysis buffer (RIPA lysis buffer: 100×PMSF: 100×phosphatase inhibitor) at a volume ratio of 100:1:1, and proteins were separated by SDS-PAGE polyacrylamide gel electrophoresis. Subsequently, the target protein bands were transferred to a PVDF membrane using a transfer technique, blocked, and then incubated sequentially with specific primary and secondary antibodies. Finally, the immunoreaction signal was captured using band exposure imaging technology, and the expression level of the target protein was analyzed.

[0034] The STAT3 and AKT signaling pathways play a crucial regulatory role in allergic and immune responses by controlling cell proliferation, survival, and the release of various inflammatory factors. For example... Figure 10 China A~ Figure 10As shown in Figure C, quantitative analysis revealed that treatment with the peptide ALLERVIA NO.1 significantly inhibited the abnormally high phosphorylation level of STAT3 (a signal transduction and transcription activator 3) induced by casein (CN) (p<0.05). Furthermore, combined analysis showed that ALLERVIA NO.1 could also significantly reverse the CN-induced decrease in phosphorylation level of the key downstream signaling molecule protein kinase B (AKT) (p<0.05), indicating that it effectively and comprehensively intervened in the abnormal activation and suppression of the STAT3 / AKT signaling pathway. In contrast, treatment with the peptide ALLERVIA NO.2 mainly showed a significant upregulation of suppressed AKT phosphorylation levels, but had no significant inhibitory effect on STAT3 activation.

[0035] III. Molecular dynamics simulation verification of STAT3 by peptides ALLERVIA NO.1 and ALLERVIA NO.2: The complexes of peptides (ALLERVIA NO.2 and ALLERVIA NO.1) with the core target STAT3 were simulated at 100 ns using GROMACS 2020.6-MODIFIED software. The system employed an AMBER99SB-ILDN protein force field, and the ligand topology was constructed using Multiwfn and Sobtop to allocate GAFF force field parameters. The complexes were placed in a rhombic dodecahedral TIP3P water box with a 1.0 nm margin and Na was added. + and Cl - Neutralizing charge. After the system underwent energy minimization via the steepest descent method and ensemble equilibrium of NVT and NPT under position constraints, unconstrained final product simulations were conducted under isothermal and isobaric conditions. Finally, by extracting the trajectory and calculating the root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), and number of dynamic hydrogen bonds, the results were verified and confirmed that peptides ALLERVIA NO.1 and ALLERVIA NO.2 can form highly stable composite structures with the core target.

[0036] Trajectory analysis showed that the root mean square deviation (RMSD) of the complex system of each peptide and the core target tended to reach equilibrium after 20 ns; such as Figures 11-14 As shown, this invention employs all-atom molecular dynamics simulations to further verify the binding mode between the tested peptides and the core target from thermodynamic and conformational evolution perspectives. Trajectory analysis indicates that the root mean square deviation (RMSD) of each complex system tends to reach dynamic equilibrium after 20 ns. Figures 11-14As shown, the average RMSD of the ALLERVIA NO.1-STAT3 complex (0.20 nm) and the ALLERVIA NO.2-STAT3 complex (0.22 nm) were both lower than those of the pure STAT3 system (0.25 nm). Combined with the stable trends of root mean square fluctuation (RMSF) and solvent accessible surface area (SASA), this confirms that peptide binding has a significant stabilizing effect on the spatial conformation of the target protein, with the conformational binding effect of peptide ALLERVIA NO.1 being more pronounced. Further analysis of the interaction forces revealed that during the simulation, ALLERVIA NO.1 and ALLERVIA NO.2 maintained an average of 3.67 and 1.92 dynamic hydrogen bonds with STAT3, respectively. ALLERVIA NO.1 exhibited a denser hydrogen bond network, which is the core molecular structural basis for its ability to efficiently block the abnormally elevated phosphorylation of STAT3 in in vitro cell experiments. In summary, the polypeptides ALLERVIA NO.1 and ALLERVIA NO.2 described in this invention can form an extremely stable thermodynamic complex structure with the target STAT3. The simulation results strongly support the technical effect of the polypeptides of this invention as intracellular signaling pathway regulators to exert potential anti-allergic activity at the molecular level and in terms of spatial conformation.

[0037] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a casein-derived peptide in the preparation of an anti-allergic drug, characterized in that, The amino acid sequence of the casein-derived peptide is shown in SEQ ID NO.1 or SEQ ID NO.2; the anti-allergy is selected from anti-food allergy, anti-allergic dermatitis, anti-allergic rhinitis and / or anti-allergic conjunctivitis.

2. The use according to claim 1, characterized in that, The method for preparing the casein-derived peptide includes the following steps: 1) Cold plasma pretreatment of bovine milk casein yielded cold plasma pretreated casein small molecule peptides; 2) Collect the cold plasma pretreated casein small molecule peptides obtained in step 1) to construct a database, combine bioinformatics and network pharmacology target prediction for high-throughput virtual screening, and verify the anti-allergic activity by measuring hyaluronidase inhibitory activity and the inhibitory activity on KU812 cell degranulation and inflammatory factor release, and obtain casein-derived peptides with anti-allergic activity. Step 2) includes the following steps: Bioinformatics was used to screen peptides in the database to obtain candidate short peptides. Network pharmacology was used to predict the targets of the candidate short peptides and to obtain the core targets by intersecting with targets of food allergies. KEGG pathway enrichment analysis was performed on pathways closely related to inflammation and immune response. The candidate short peptides were docked with receptors of the above-mentioned pathways closely related to inflammation and immune response to obtain bindable peptides. The hyaluronidase inhibitory activity and the inhibitory activity on KU812 cell degranulation and inflammatory factor release of the bindable peptides were measured to verify the anti-allergic activity, thus obtaining casein-derived peptides with anti-allergic activity.

3. The use according to claim 2, characterized in that, In step 1), the cold plasma pretreatment of bovine casein is performed using dielectric barrier discharge, with argon as the working gas, an input power of 75W, and a processing time of 12min.

4. The use according to claim 2, characterized in that, The candidate short peptides are peptides with a length <10, a bioactivity score >0.8, an intestinal absorption potential (HIA) >20%, a blood-brain barrier crossing (BBB) ​​>0.1, and are non-toxic and non-sensitizing.

5. The use according to claim 2, characterized in that, The core targets are AKT1, NFKB1, IL-1β, and STAT3.

6. The use according to claim 2, characterized in that, The pathways closely related to inflammation and immune response are AGE-RAGE, TNF signaling pathway, and Th17 cell differentiation.

7. The use according to claim 2, characterized in that, The process of docking candidate short peptides with receptors of the aforementioned pathways closely related to inflammation and immune responses to obtain bindable peptides specifically involves docking candidate short peptides with AKT1, NFKB1, IL-1β, and STAT3 to obtain peptides with AutoDock Vina binding energies <-5kcal / mol and Discovery Studio binding energies <-50kcal / mol.

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