Preparation method and application of oyster oligopeptide for relieving dss-induced ulcerative colitis

Abstract

This invention belongs to the fields of marine product functional applications, functional foods, and biotechnology. It discloses an oyster oligopeptide for alleviating DSS-induced ulcerative colitis, its preparation method, and its uses. Using a DSS-induced damage model of normal human colonic epithelial cells as a guide, oyster meat was enzymatically hydrolyzed, defatted, and precipitated with alcohol to obtain purified oyster hydrolysate. Two oyster peptides were identified, screened using bioinformatics, and verified for synthesis. Their amino acid sequences are shown in SEQ ID NO. 1-2. The oyster oligopeptides exhibit strong antioxidant and anti-inflammatory activities, effectively alleviating DSS-induced NCM460 cell damage, with better effects than the positive control 5-aminosalicylic acid. They can be used to prepare drugs for alleviating ulcerative colitis, as well as foods and health products for adjuvant treatment of ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the fields of marine product functional applications, functional foods and biotechnology, specifically relating to an oyster oligopeptide for alleviating DSS-induced ulcerative colitis, its preparation method and application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease that primarily affects the rectum and colon, characterized by periodic mucosal inflammation and ulceration, clinically manifesting as diarrhea, abdominal pain, and weight loss. In recent years, the incidence of ulcerative colitis has been steadily increasing, particularly in low- and middle-income countries. Although the etiology of ulcerative colitis is not fully understood, numerous studies have shown that its pathological process is closely related to oxidative stress and inflammatory responses: increased reactive oxygen species at sites of colonic injury lead to tissue oxidative stress, which in turn triggers a cascade of inflammatory responses, further exacerbating tissue damage.

[0003] Currently used treatments include 5-ASA, corticosteroids, and immunosuppressants. While these have significant therapeutic effects, they also come with side effects such as fever, diarrhea, gastric ulcers, and increased nephrotoxicity. Therefore, there is an urgent need to develop safer, more natural, and non-toxic alternative active ingredients to prevent and treat ulcerative colitis.

[0004] Caspase-1 is a component of the NOD-like receptor heat protein domain-associated protein 3 (NLRP3) inflammasome, primarily existing in an inactive precursor form in the cytoplasm. However, its uncontrolled activation upon stimulation can trigger a severe inflammatory cascade, including the conversion of inactive precursors of pro-inflammatory cytokines such as pro-IL-1β and pro-IL-18 into their active forms via proteolytic cleavage. Therefore, inhibiting the expression and activity of Caspase-1 is considered helpful in slowing the development of inflammatory diseases such as ulcerative colitis. In recent years, numerous studies have shown that dietary peptides can alleviate ulcerative colitis through multiple pathways, including reducing oxidative stress, lowering inflammation levels, and enhancing antioxidant activity, indicating that dietary peptides have great potential in developing drugs to alleviate ulcerative colitis or as adjunctive therapies for ulcerative colitis.

[0005] Oysters are widely cultivated bivalve mollusks with rich nutritional and medicinal value. According to the Compendium of Materia Medica, oysters have astringent properties for the large and small intestines and can treat diarrhea. Their soft parts are rich in protein, accounting for approximately 40-50% of their total dry matter, making them an excellent source for preparing bioactive peptides. Oyster peptides have been proven to possess various biological activities, including antihypertensive, antibacterial, antioxidant, and anti-inflammatory activities, showing significant potential for the preparation of pharmaceuticals, functional foods, and novel marine health products. Therefore, extracting bioactive peptides from oysters that can alleviate DSS-induced ulcerative colitis is of great significance for the treatment, prevention, and adjunctive control of DSS-induced ulcerative colitis, reducing oxidative stress and inflammation levels in the body, promoting intestinal mucosal repair, and the high-value utilization of oyster bioactive peptide resources. Summary of the Invention

[0006] The purpose of this invention is to provide an oyster-active oligopeptide that can improve DSS-induced NCM460 cell damage and has a better anti-damage effect than 5-ASA, and has the potential to be developed into a drug for alleviating DSS-induced ulcerative colitis and a food for adjuvant therapy.

[0007] The first objective of this invention is to provide an oyster oligopeptide that alleviates DSS-induced ulcerative colitis, with the amino acid sequence Phe-Ala-Gly-Asp-Asp-Ala-Pro-Arg (FAGDDAPR). or Gly-Phe-Ala-Gly-Asp-Asp-Ala-Pro-Arg (GFAGDDAPR).

[0008] A second objective of this invention is to provide a method for preparing the above-mentioned oyster oligopeptides, comprising the following steps:

[0009] (1) Preparation of crude peptides from oyster enzymatic hydrolysis: Oyster meat was minced into a homogenate, the pH was adjusted to 8, and the temperature was preheated to 37℃. Trypsin was added for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme activity was inactivated by boiling in a water bath for 10 min. The inactivated hydrolysate was centrifuged at 8000 r / min for 30 min at 4℃. After degreasing, the supernatant was collected, ethanol was added, and the mixture was allowed to stand overnight at 4℃. Subsequently, the mixture was centrifuged at 5000 r / min for 15 min at 4℃, and the supernatant was collected, concentrated, and then freeze-dried.

[0010] (2) In vitro activity evaluation of oyster enzymatic hydrolysed crude peptides: The ABTS and DPPH free radical scavenging capacity and the ability to inhibit the thermal denaturation of bovine serum albumin were determined; the effects of the oyster enzymatic hydrolysed crude peptides on DSS-induced NCM460 cell viability, cell migration at cell wound sites, and the content of interleukin-6 (IL-6), nitric oxide (NO), reactive oxygen species (ROS), and superoxide dismutase (SOD) activity were determined;

[0011] (3) Identification of peptides: The peptide sequences in the crude peptides from oyster enzymatic hydrolysis were identified using nanoLC-timsTOF-MS / MS;

[0012] (4) Screening and validation of active peptides: Potential active peptides were screened by combining network pharmacology with molecular docking technology, synthesized and validated, and their effects on the viability of DSS-induced NCM460 cells as well as the levels of IL-6, tumor necrosis factor (TNF-α), ROS and SOD activity were verified. The binding of these peptides to the target protein Caspase-1 was simulated by molecular dynamics simulation at 100 ns.

[0013] Preferably, in step (1), the homogenate-to-liquid ratio is 1:4, the amount of trypsin added is 3000 U / g, the enzymatic hydrolysis time is 4h, the concentration of ethanol is 95%, and the volume ratio of ethanol to the sample solution is 3:1.

[0014] Preferably, in the analysis of crude peptides from oyster enzymatic hydrolysis using the nanoLC-timsTOF-MS / MS method described in step (3), separation is performed using a U3000 ultra-high performance liquid chromatography system with an Ionopticks Aurora C18 column. The elution conditions are as follows: eluent A is an aqueous solution containing 0.1% formic acid, and eluent B is an 80% acetonitrile solution containing 0.1% formic acid; the elution gradient is: 0-45 min: 4-37% B, 45-50 min: 37-80% B, 50-55 min: 80-80% B, 55-55.1 min: 80-1% B, 55.1-60 min: 1% B; the flow rate is 300 nL / min; the mass spectrometer operates in DDA-PASEF mode, using positive ion mode, with an m / z range of 100-1700, a fixed TIMS accumulation time of 100 ms, and a fixed ion mobility separation time of 100 ms.

[0015] Preferably, the molecular docking receptor proteins described in step (4) include STAT3, PTGS2, CASP1, and MMP9.

[0016] A third objective of this invention is to provide the application of the above-mentioned oyster oligopeptide in the preparation of a medicament for relieving colitis.

[0017] Furthermore, it is used in drugs that protect against ulcerative colitis.

[0018] The fourth objective of this invention is to provide the application of the above-mentioned oyster oligopeptides in the preparation of functional nutritional foods and health products that protect the intestinal mucosal barrier.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0020] (1) This invention utilizes network pharmacology screening and molecular docking technology to screen two peptides targeting Caspase-1 from crude peptides hydrolyzed from oysters. These peptides are effective in alleviating DSS-induced ulcerative colitis, achieving efficient preparation and screening of small molecule bioactive peptides. Compared to traditional peptide development methods, this method is simple to operate and reduces research and development costs.

[0021] (2) The oyster oligopeptide provided by the present invention is derived from food protein and is prepared by enzymatic hydrolysis of food protease. It has a clear structure and obvious efficacy.

[0022] (3) This invention realizes the high-value utilization of oysters and provides a reliable basis for the deep processing of oysters. Attached Figure Description

[0023] Figure 1 In vitro activity assessment of crude peptides hydrolyzed from oysters. (A) ABTS free radical scavenging activity. (B) DPPH free radical scavenging activity. (C) Inhibition of bovine serum albumin thermal denaturation activity.

[0024] Figure 2 Cellular activity evaluation of crude peptides hydrolyzed from oysters. (A) Cytotoxicity assessment of crude peptides hydrolyzed from oysters on NCM460 cells. (BH) Effects of crude peptides hydrolyzed from oysters on the activity of NCM460 cells damaged by DSS, cell migration at the wound site, IL-6 content, NO content, ROS content and SOD activity, respectively. # p<0.05, ## p<0.01, ### p<0.001 indicates a comparison with the Control group. * p<0.05, ** p<0.01, *** p<0.001 indicates a comparison with the DSS group.

[0025] Figure 3 This study focuses on the prediction and screening of targets for the anti-ulcerative colitis effects of oyster bioactive peptides based on bioinformatics. (A) Volcano plot of all genes from the three datasets GSE36807, GSE10616, and GSE87473. (B) Venn diagram of predicted targets from oyster enzymatic hydrolysis crude peptides and targets for ulcerative colitis. (C) Protein-protein interaction network of oyster enzymatic hydrolysis crude peptides in alleviating ulcerative colitis. (D) Top 10 key gene targets.

[0026] Figure 4 The molecular docking binding energies of 15 selected oyster oligopeptides with four ulcerative colitis targets (STAT3, CASP1, PTGS2 and MMP9) were determined.

[0027] Figure 5To establish a DSS-induced intestinal epithelial cell injury model, the cell-protective effects, anti-inflammatory and antioxidant activities of various oligopeptides were comprehensively screened and validated. (A) shows the effect of 15 selected oyster oligopeptides on the viability of DSS-induced NCM460 cells. (B)-(E) show the effects of 15 selected oyster oligopeptides on the levels of IL-6, TNF-α, ROS and SOD activity in DSS-induced NCM460 cells, respectively.

[0028] Figure 6 The effect of oyster oligopeptides with amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2 in this application on DSS-induced NCM460 cell viability (extracted from...) Figure 5 A).

[0029] Figure 7 The interaction mechanism and binding stability with the core target Caspase-1 are shown. (A) and (B) are molecular docking results of oyster oligopeptides FADGGAPR and GFADGGAPR with Caspase-1, respectively. (C)-(E) are Rg, SASA, and RMSF of Caspase-1, respectively. (F) and (G) are molecular snapshots of the complexes of FADGGAPR and GFADGGAPR with Caspase-1, respectively. Detailed Implementation

[0030] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention and do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the equipment used are conventional experimental equipment unless otherwise specified.

[0032] The preparation of an oyster oligopeptide for alleviating DSS-induced ulcerative colitis according to the present invention includes enzymatic hydrolysis and purification, sequence identification, bioinformatics screening, and synthetic verification. The oyster used in the embodiments of this application is the Hong Kong giant oyster.

[0033] The specific steps include:

[0034] (1) Preparation of crude peptides from oyster enzymatic hydrolysis: Oyster meat was taken and crushed into a homogenate at a material-to-liquid ratio of 1:4. The pH was adjusted to 8, and the temperature was preheated to 37°C. Trypsin was added for enzymatic hydrolysis at a dosage of 3000 U / g for 4 h. After enzymatic hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min. The inactivated hydrolysate was cooled at 4°C and centrifuged at 8000 r / min for 30 min. After removing the lipids, the supernatant was taken and 3 times the volume of 95% ethanol was added. The mixture was allowed to stand overnight at 4°C and then centrifuged at 5000 r / min for 15 min. The supernatant was collected, concentrated, and freeze-dried to obtain crude peptides from oyster enzymatic hydrolysis.

[0035] (2) In vitro activity evaluation of crude peptides hydrolyzed from oysters

[0036] 1) ABTS radical scavenging ability: 50 μL of the test solution was placed in a 96-well plate, and then 150 μL of ABTS was rapidly transferred using a multichannel pipette. ·+ The working solution was added to a 96-well plate and reacted at room temperature in the dark for 6 min. The absorbance at 734 nm was measured. The ABTS radical scavenging capacity of the sample was calculated using the following formula:

[0037] ABTS free radical scavenging ability (%) = (A 空白 - A 待测 ) / A 空白 × 100%

[0038] A 空白 Blank control group A 734 nm;A 待测 Test solution group A 734 nm.

[0039] 2) DPPH radical scavenging ability: 100 μL of the test solution was mixed with an equal volume of DPPH solution in a 96-well plate and reacted at room temperature in the dark for 30 min. The absorbance at 517 nm was measured. The DPPH radical scavenging ability of the sample was calculated according to the following formula:

[0040] DPPH free radical scavenging ability (%) = 1 - (A 待测 - A 对照 ) / A 空白 × 100%

[0041] A 空白 Blank control group A 517 nm;A 对照 Control group A 517 nm;A 待测 Test solution group A 517 nm.

[0042] 3) Inhibition of BSA thermal denaturation: 990 μL of 0.5% (w / v) BSA solution was mixed with 110 μL of the test solution and incubated at 37°C for 15 min, followed by incubation in a 90°C water bath for 20 min. After cooling to room temperature, the turbidity was measured at 660 nm. The protein denaturation inhibition rate of the sample was calculated using the following formula:

[0043] Protein denaturation inhibition rate (%) = (A 空白 - A 待测 ) / A 空白 × 100%

[0044] A 空白 Blank control group A 660 nm;A 待测 Test solution group A 660 nm.

[0045] The results are as follows Figure 1 As shown, the crude peptides hydrolyzed from oysters exhibit good antioxidant activity and resistance to protein thermal denaturation.

[0046] (4) Evaluation of cell activity of crude peptides hydrolyzed from oysters:

[0047] 1) Cell culture: NCM460 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured at 37°C in a humidified incubator containing 5% CO2.

[0048] 2) Cell viability assay: NCM460 cells were incubated at 5 × 10⁻⁶ cells / mL. 3 Cells were seeded per well in 96-well plates and incubated for 24 h until adhesion occurred. Except for the control group, all other groups were treated with DSS (2.5%, m / v) to induce cell damage. After 24 h, cells were treated for 24 h with different concentrations of oyster enzyme-digested crude peptides (6.25, 12.5, 25, 50, 100, 200 μg / mL), 5-ASA (30 μg / mL), or physiological saline. Cell viability was assessed using the CCK-8 assay according to the kit instructions. Cell viability was calculated using the following formula.

[0049] Cell viability (%) = (A s -A b ) / (A c -A b ) × 100%

[0050] A s Group A (dosing group) 450 nm;A c Model group A 450 nm;A b : A in the blank group 450 nm.

[0051] 3) Wound healing experiment: NCM460 cells were cultured at 5 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well plates and incubated until a monolayer of cells formed. Except for the control group, cells in all other groups were treated with DSS (2.5%, m / v) to induce cell damage. After 24 h, linear scratches were created by uniformly scraping the cell monolayer with the tip of a 10 μL sterile pipette perpendicular to the cell monolayer. Cells were washed with PBS and incubated in serum-free medium. The experimental groups were given 100 μg / mL oyster enzyme-digested crude peptides, while the positive control group was given 30 μg / mL 5-ASA. The degree of wound healing in each well was photographed under a microscope at 0, 12, 24, and 36 h, and the wound area was calculated. Cell migration was analyzed by wound closure rate, and the wound healing rate was calculated using the following formula.

[0052] Wound healing rate (%) = (S0-S t ) / S0× 100%

[0053] S0: Wound area at 0 h; S t : Wound area at different observation times.

[0054] 4) Cytokine detection: NCM460 cells were cultured at 5 × 10⁻⁶ cells / mL. 5Cells were seeded per well in 96-well plates and incubated for 24 h until adhesion occurred. Except for the control group, all other cell groups were cultured in fresh medium containing DSS (2.5%, m / v). After 24 h, the DSS was removed, and fresh medium containing (6.25, 25, 100 μg / mL) oyster enzymatic hydrolysate, 30 μg / mL 5-ASA, or physiological saline was added, and the cells were incubated for 24 h. Cell supernatants were collected, and the levels of IL-6, NO, ROS, and SOD activity were measured according to the kit instructions.

[0055] The results are as follows Figure 2 As shown, the oyster enzymatic hydrolysis crude peptides prepared by this invention significantly alleviated DSS-induced NCM460 cell damage, improved NCM460 cell activity, promoted cell migration at the cell wound site, reduced intracellular oxidative stress and inflammation levels, and enhanced the cell's antioxidant capacity.

[0056] (5) Identification of crude peptide sequences from oyster enzymatic hydrolysis: The crude oyster peptides were desalted and then their structures were identified using high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-MS / MS). The chromatographic column was an Ionopticks Aurora C18 (250 mm × 75 μm, inner diameter 1.6 μm). Mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The gradient elution conditions were as follows: 0-45 min: 4-37% B; 45-50 min: 37-80% B; 50-55 min: 80-80% B; 55-55.1 min: 80-1% B; 55.1-60 min: 1% B. Mass spectrometry conditions: Nano-electrospray ionization source, positive ion scanning mode, DDA-PASEF mode, TIMS accumulation time fixed at 100 ms, and ion mobility separation time fixed at 100 ms. The mobility values ​​range from 0.7 to 1.3 Vs / cm. 2 (1 / K0), mass scan range 100-1700 m / z. A total of 16,564 peptide sequences were obtained.

[0057] (6) Screening of bioactive peptides using network pharmacology methods

[0058] 1) The 16,564 identified peptide sequences were subjected to biological activity and toxicity predictions, resulting in 1,536 potentially non-toxic active peptides. For example... Figure 3 As shown, target prediction was performed on these 1536 peptide sequences, resulting in 207 predicted oyster peptide targets, which were used as the target set for oyster peptides.

[0059] 2) Based on the thresholds p adj.<0.05 and |log2FC|>1, 2516 differentially expressed genes were screened from the GSE87473, GSE36807 and GSE10616 datasets in the GEO database to obtain a disease target set for ulcerative colitis.

[0060] 3) The intersection of the oyster peptide target set and the disease target set of ulcerative colitis is obtained to obtain the potential target set of oyster peptides for alleviating ulcerative colitis.

[0061] 4) The intersection target points were input into relevant software to construct a protein-protein interaction network, and the top 10 core gene targets in the protein-protein interaction network were obtained. The top 10 core gene targets correspond to 612 different peptide sequences, and by predicting the allergenicity of the peptides, 223 potential non-toxic and non-allergenic bioactive oligopeptides were finally obtained.

[0062] (7) Molecular docking

[0063] 1) Semi-flexible molecular docking was performed using software. Crystal structures were obtained from the RSCB PDB database (https: / / www.rcsb.org / ), including STAT3 (PDB ID: 6NJS), PTGS2 (PDB ID: 5F19), CASP1 (PDB ID: 6F6R), and MMP9 (PDB ID: 4WZV). The receptor protein was dehydrated, hydrogenated, and ligands and impurities were removed before being designated as the receptor. Gasteiger charges were added to 223 peptide sequences, and MM2 force field energy was minimized before designating them as ligands. Docking was performed using the original coordination site or active site of the receptor protein's original ligand.

[0064] 2) Based on the docking results, the 20 peptides with the strongest binding affinity to each target protein were screened, and the interactions between the peptides and target proteins were analyzed. The number of peptides binding to the characteristic binding sites of the target proteins was considered, such as... Figure 4 Fifteen preferred peptides were screened (Table 1) and then prepared by a company using solid-phase synthesis.

[0065] Table 1

[0066] (8) Evaluation of the cell activity of bioactive peptides

[0067] 1) Cell culture: NCM460 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured at 37°C in a humidified incubator containing 5% CO2.

[0068] 2) Cell viability assay: NCM460 cells were incubated at 5 × 10⁻⁶ cells / mL.3 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h until adhesion occurred, followed by treatment with 2.5% (w / v) DSS for 24 h. The old medium was removed, and fresh medium containing different concentrations of oligopeptides (1, 5, 25 μg / mL) or 30 μg / mL 5-ASA was added. After 24 h of culture, CCK-8 assays were performed.

[0069] 3) Cytokine detection: 1 × 10⁶ NCM460 cells were... 5 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h until adhesion occurred, followed by treatment with 2.5% (w / v) DSS for 24 h. The old culture medium was removed, and fresh culture medium containing different concentrations of oligopeptides (1, 5, 25 μg / mL) or 30 μg / mL 5-ASA was added. After 24 h of culture, the cell supernatant was collected, and IL-6, TNF-α, ROS levels, and SOD activity were measured according to the manufacturer's instructions.

[0070] The results are as follows Figure 5 and Figure 6 As shown, among the 15 synthetic peptides prepared in this invention, FADGGAPR and GFADGGAPR showed the most significant effects in improving the activity of DSS-damaged NCM460 cells, exhibiting strong antioxidant and anti-inflammatory capabilities within the cells and significantly reducing cellular inflammation and oxidative stress levels.

[0071] (9) Molecular Dynamics Simulation: Based on the molecular docking results, molecular dynamics simulations were performed on the Caspase-1 protein and peptide complex, generating the protein's topological structure. A GAFF force field was added to the peptide, and hydrogen was added while calculating the RESP potential. The TIP3P water model was used for the simulation, and appropriate amounts of Na+ and Cl- were added to neutralize the charge of the simulated system. The steepest descent algorithm was used to minimize energy, and the Particle Mesh Ewald (PME) method was used to calculate long-range electrostatic interactions. For short-range electrostatic and van der Waals interactions, the cutoff distance was set to 1.0 nm. The LINCS algorithm was used to constrain all participating hydrogen bonds. The system underwent NVT and NPT equilibration before the simulation, each lasting 100 ps. Finally, molecular dynamics simulations were performed for 100 ns in isothermal (300 K) and isobaric (1 bar) systems using periodic boundary conditions. The pressure was maintained using a Berendsen pressure regulator, and the temperature was adjusted using the V-rescale temperature coupling method. The integration time step is set to 2 fs, and the motion trajectory is recorded every 10 ps.

[0072] The results are as follows Figure 7As shown, the synthetic peptides FADGGAPR and GFADGGAPR prepared in this invention both bind stably to the target protein Caspase-1. Among them, FADGGAPR forms a more deeply intercalated binding conformation with Caspase-1, and GFADGGAPR has a greater impact on the protein's active loop region.

[0073] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an oyster oligopeptide in the preparation of a product for relieving colitis, characterized in that, The amino acid sequence of the oyster oligopeptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The colitis mentioned is ulcerative colitis.

3. The application according to claim 1, characterized in that, The product is a medicine, functional nutritional food, or health product.

4. The application of an oyster oligopeptide in the preparation of products for protecting the intestinal mucosal barrier, characterized in that, The amino acid sequence of the oyster oligopeptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

5. A method for preparing the product according to claim 1, characterized in that, Includes the following steps: S1. Provides an oyster oligopeptide with the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2; S2. The oyster oligopeptide is formulated with pharmaceutically or food-acceptable excipients to obtain a product for relieving ulcerative colitis.

6. The preparation method according to claim 5, characterized in that, The oyster oligopeptide mentioned in step S1 is obtained by the following steps: (a) Preparation of oyster enzymatic hydrolysed crude peptides: Oyster meat was crushed into a homogenate, pH was adjusted to 8, preheated to 37°C, and trypsin was added for enzymatic hydrolysis; after enzymatic hydrolysis, the enzyme was inactivated, centrifuged to collect the supernatant, ethanol was added to precipitate impurities, centrifuged again, and the supernatant was collected, concentrated, and dried to obtain oyster enzymatic hydrolysed crude peptides. (b) Peptide sequence screening: The peptide sequences in the crude oyster enzymatic hydrolysate were identified by mass spectrometry, and active peptides with amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2 were screened by network pharmacology and molecular docking techniques.

7. The preparation method according to claim 6, characterized in that, In step (a), the material-to-liquid ratio of the homogenate is 1:4, the amount of trypsin added is 3000 U / g, and the enzymatic hydrolysis time is 4 h; the volume concentration of ethanol is 95%, and the volume ratio of ethanol added to supernatant is 3:

1.

8. The preparation method according to claim 6, characterized in that, In step (b), the receptor proteins for molecular docking include STAT3, PTGS2, CASP1, and MMP9.

9. The preparation method according to claim 6, characterized in that, In step (b), the mass spectrometry analysis method is nanoLC-timsTOF-MS / MS, the chromatographic column is Ionopticks Aurora C18; mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an 80% acetonitrile solution containing 0.1% formic acid; the gradient elution program is as follows: 0-45 min, mobile phase B increases from 4% to 37%; 45-50 min, mobile phase B increases from 37% to 80%; 50-55 min, maintain at 80%; 55-55.1 min, decrease from 80% to 1%; 55.1-60 min, maintain at 1%.

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