A mytilus mucin polypeptide or composition thereof, preparation method and application

Abstract

The application discloses a mussel mucin polypeptide or a composition thereof, a preparation method and application. The amino acid sequences of the mussel mucin polypeptide are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 25, SEQ ID NO. 27 or SEQ ID NO. 31. The polypeptide component prepared by the application can be used for preparing a medicine or food for improving inflammatory bowel disease. The mussel mucin polypeptide can effectively improve the inflammatory bowel disease, is safe, non-toxic and free of side effects, simple in steps and remarkable in effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a mytilus edulis mucin polypeptide or a composition thereof, a preparation method and application. BACKGROUND

[0002] Mytilus spp. is a kind of marine bivalve mollusk widely distributed in intertidal zone to shallow sea area, belonging to Mollusca, Bivalvia and Mytilidae, and is one of important aquaculture economic species. Its adapted marine high-osmotic pressure, high salinity and complex microecological environment endow its metabolic products with unique structure and significant biological function. In recent years, studies have shown that Mytilus is rich in proteins, polysaccharides, active peptides and various trace elements. Mytilus-derived mucin exhibits wide application potential in the fields of biological medicine and material science due to its excellent underwater adhesion performance and unique biocompatibility, such as biological adhesive, drug delivery carrier, tissue engineering scaffold, marine antifouling coating and the like. These biological effects provide great application prospect of Mytilus edulis mucin in surgical operation, wound repair, tissue regeneration and functional material development. Traditional researches are mostly focused on extraction, purification and macroscopic adhesion performance evaluation of Mytilus edulis mucin. However, with the development of protein chemistry and biomimetic material science, researchers gradually realize that the biological function of Mytilus edulis mucin is closely related to its amino acid sequence composition, special modification (such as dihydroxyphenylalanine Dopa), repeated motif arrangement and three-dimensional spatial conformation.

[0003] However, at present, Mytilus edulis mucin is only widely applied in materials and medical devices due to its physical properties, and the research on its improvement of inflammation is limited. Patent (CN114375298A, CN110139662A) mentions that the repeated peptide segment AKPSYPPTYK of Mytilus edulis mucin Mefp-I and its derivative polypeptide have the effect of improving inflammation, but there is no report that other polypeptides of Mytilus edulis mucin Mefp-I have the function of improving inflammation.

[0004] Inflammatory bowel disease (IBD) is a kind of chronic and recurrent intestinal inflammatory disease induced by immunity, which can be divided into Crohn's disease and ulcerative colitis according to the characteristics and site of the disease, and its characteristic is that the structure and function of intestinal epithelial barrier are destroyed. How to reduce the high incidence of IBD has become one of the difficult problems to be solved. Therefore, it is of great significance to seek an effective drug or functional food for improving inflammatory bowel disease. SUMMARY

[0005] The purpose of the application is that after depleting the intestinal flora of mice, it is found that the anti-inflammatory activity of Mytilus edulis mucin Mefp-I disappears when it is given to the rectum, and the anti-inflammatory activity recovers after colonization Akkermansia muciniphila and Bifidobacterium bifidum , which indicates that Mefp-I plays a key role in the intestinal flora of mice.Akkermansia muciniphila and Bifidobacterium bifidum The degraded polypeptide component has an effect of improving inflammatory bowel disease, and therefore, the technical problem to be solved by the present application is to provide a mussel mucin polypeptide or a composition thereof having an effect of improving inflammatory bowel disease.

[0006] The technical problem to be solved by the present application is also to provide a preparation method of the mussel mucin polypeptide or the composition thereof.

[0007] The technical problem to be solved by the present application is also to provide a preparation method of the mussel mucin polypeptide or the composition thereof.

[0008] Technical solution: In order to solve the above technical problem, the present application provides a mussel mucin polypeptide or a composition thereof, wherein the mussel mucin polypeptide or the composition thereof is selected from one or more of the following polypeptides: the amino acid sequences of the polypeptides are respectively shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 25, SEQ ID NO. 27 or SEQ ID NO. 31.

[0009] The content of the present application also includes a preparation method of the mussel mucin polypeptide or the composition thereof, comprising the following steps: adding Akkermansia muciniphila BNCC341917 or Bifidobacterium bifidum After the BNCC186304 bacterial solution is added to the specific culture medium containing mussel mucin as a carbon source for anaerobic culture, centrifugation, ultrafiltration by an ultrafiltration tube, and collection of the lower solution, freeze-drying is performed to obtain the mussel mucin polypeptide.

[0010] The specific culture medium comprises the following components in 500 mL of water: 1.4 g of Na2HPO4, 0.25 g of (NH4)2SO4, 0.0005 mg of CuCl2·2H2O, 0.015 mg of H3BO3, 0.1 mg of FeSO4·7H2O, 0.5 mg of KH2PO4, 0.0015 mg of MnCl2·4H2O, 0.001 mg of NiCl2·6H2O, 0.25 mg of Na2EDTA, 0.01 mg of CoCl2·6H2O, 0.005 mg of ZnSO4·7H2O, 0.0015 mg of Na2MoO4·2H2O, and 2.5 g of mussel mucin Mefp-1.

[0011] The specific culture medium comprises the following components in 500 mL of water: 1.4 g of Na2HPO4, 0.25 g of (NH4)2SO4, 0.0005 mg of CuCl2·2H2O, 0.015 mg of H3BO3, 0.1 mg of FeSO4·7H2O, 0.5 mg of KH2PO4, 0.0015 mg of MnCl2·4H2O, 0.001 mg of NiCl2·6H2O, 0.25 mg of Na2EDTA, 0.01 mg of CoCl2·6H2O, 0.005 mg of ZnSO4·7H2O, 0.0015 mg of Na2MoO4·2H2O, and 2.5 g of mussel mucin Mefp-1. Akkermansia muciniphilaBNCC341917 and Bifidobacterium bifidum BNCC186304 are activated bacteria liquid obtained after recovery culture in BHI culture medium and MRS culture medium respectively, the composition of the BHI broth culture medium is: 12.5 g of beef brain extract powder, 5.0 g of beef heart extract powder, 10.0 g of tryptone, 2.0 g of glucose, 5.0 g of sodium chloride, 2.5 g of disodium hydrogen phosphate per 1L of water; the composition of the MRS culture medium is: 20.0 g of glucose, 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of sodium acetate, 5.0 g of yeast extract, 2.0 g of potassium phosphate, 2.0 g of ammonium citrate, 1.08 g of Tween 80, 0.2 g of magnesium sulfate and 0.05 g of manganese sulfate per 1L of water.

[0012] The Mefp-I polypeptide or the composition thereof is used for preparing a medicament for preventing or treating inflammatory bowel disease. Akkermansia muciniphila BNCC341917 or Bifidobacterium bifidum The inoculation amount of the recovery culture of BNCC186304 is 1-3%, and the recovery time is 24-48h, and the pH is 6-8.

[0013] The Mefp-I polypeptide or the composition thereof is used for preparing a medicament for preventing or treating inflammatory bowel disease. Akkermansia muciniphila BNCC341917 or Bifidobacterium bifidum The concentration of the BNCC186304 bacteria liquid is OD 600 =0.5-0.6.

[0014] The mass concentration of the mussel adhesive protein in the specific culture medium is 0.2%-1%.

[0015] The anaerobic culture condition is 37 DEG C culture for 24-48h.

[0016] The centrifugal speed is 4000-5000rmp, the centrifugal time is 10-15min, the bacteria are removed by 0.22muM filter membrane, and then the ultrafiltration tube with a pore size of 3KDa is directly used for ultrafiltration.

[0017] The application also includes the use of the mussel adhesive protein polypeptide or the composition thereof in the preparation of a medicament for preventing or treating inflammatory bowel disease, preferably, the inflammatory bowel disease includes colitis.

[0018] The colitis includes but is not limited to ulcerative colitis.

[0019] Beneficial effects: compared with the prior art, the application has the following advantages: the mussel adhesive protein Mefp-I is used as a raw material, and the Mefp-I is subjected to Akkermansia muciniphilaBNCC341917 or Bifidobacterium bifidum The BNCC186304 co-incubation, supernatant extraction, ultrafiltration and freeze-drying process steps are prepared to provide a natural and safe drug or food for improving inflammatory bowel disease. The preparation method of the present application is simple in operation and has a degradation efficiency of 78%, and the polypeptide yield is 60%. Moreover, the prepared mussel mucin polypeptides MP-A and MP-B are mixed polypeptide components less than 3KDa, and it is verified that 11 specific polypeptides among them have anti-inflammatory activity through NCM-460 inflammation model. The mussel mucin polypeptides MP-A and MP-B prepared by the present application can significantly improve the dextran sulfate sodium (DSS) induced ulcerative colitis in mice, and the high dose has the same effect as the positive drug. The present application is prepared by incubating natural marine mussel mucin with intestinal probiotics, and is safe, non-toxic and without side effects. Therefore, the polypeptide components prepared by the present application can be used for preparing a drug or food for improving inflammatory bowel disease. The mussel mucin polypeptide MP of the present application can effectively improve the verified inflammatory bowel disease, is safe, non-toxic and without side effects, has a simple step and remarkable effect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 For Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum The growth curve of BNCC186304;

[0021] Figure 2 For Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum The degradation efficiency of BNCC186304 on mussel mucin;

[0022] Figure 3 The HPLC chromatogram of mussel mucin Mefp-1 under different conditions;

[0023] Figure 4 For Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum The HPLC chromatogram of the degradation sample of Mefp-1 after 48h co-incubation of BNCC186304;

[0024] Figure 5 For Figure 4 The HPLC chromatogram of mussel mucin polypeptides MP-A and MP-B after ultrafiltration of the two fermentation products in through a 3KDa ultrafiltration tube;

[0025] Figure 6 The relative expression amount of inflammatory factor genes in the NCM-460 inflammation model affected by Top15 polypeptides; IL6

[0026] Figure 7 ​Top 15 peptides in inflammatory factor genes in the NCM-460 inflammation model IL1B The effect of relative expression levels;

[0027] Figure 8 The results show the effects of mussel adhesive peptides MP-A and MP-B on the disease activity index of DSS-induced ulcerative colitis mice.

[0028] Figure 9 The results show the effects of mussel adhesive peptides MP-A and MP-B on colon length in mice with DSS-induced ulcerative colitis.

[0029] Figure 10 Representative images showing the effects of mussel adhesive peptides MP-A and MP-B on colon length in mice with DSS-induced ulcerative colitis;

[0030] Figure 11 Figure showing the effects of mussel adhesive peptides MP-A and MP-B on the histopathological characteristics of colon tissue in mice with DSS-induced ulcerative colitis. Detailed Implementation

[0031] Example 1: Growth curve determination of AKK and BIF bacteria

[0032] The BHI broth medium formula (1L) is as follows: 12.5 g bovine brain extract powder, 5.0 g bovine heart extract powder, 10.0 g tryptone, 2.0 g glucose, 5.0 g sodium chloride, and 2.5 g disodium hydrogen phosphate. Prepare the BHI medium according to this formula and autoclave it (115℃, 15 minutes).

[0033] The MRS medium formula (1L) is as follows: 20.0 g glucose, 10.0 g peptone, 10.0 g beef extract, 5.0 g sodium acetate, 5.0 g yeast extract, 2.0 g dipotassium phosphate, 2.0 g ammonium citrate, 1.08 g Tween 80, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate. After preparing the medium, adjust the pH to 6.4 ± 0.2, and then autoclave the medium (121°C, 15 minutes).

[0034] The carbon-free medium, Minimal Salt Medium (MM) (500mL), has the following formula: 1.4g Na2HPO4, 0.25g (NH4)2SO4, 0.0005mg CuCl2·2H2O, 0.015mg H3BO3, 0.1mg FeSO4·7H2O, 0.5mg KH2PO4, 0.0015mg MnCl2·4H2O, 0.001mg NiCl2·6H2O, 0.25mg Na2EDTA, 0.01mg CoCl2·6H2O, 0.005mg ZnSO4·7H2O, and 0.0015mg Na2MoO4·2H2O.

[0035] The specific culture medium formula (BM) (500ml) is as follows: 1.4g Na2HPO4, 0.25g (NH4)2SO4, 0.0005mg CuCl2·2H2O, 0.015mg H3BO3, 0.1mg FeSO4·7H2O, 0.5mg KH2PO4, 0.0015mg MnCl2·4H2O, 0.001mg NiCl2·6H2O, 0.25mg Na2EDTA, 0.01mg CoCl2·6H2O, 0.005mg ZnSO4·7H2O, 0.0015mg Na2MoO4·2H2O, 2.5g mussel adhesive protein (purchased from Jiangyin Beiruisen Biochemical Technology Co., Ltd., naturally extracted Mefp-I subtype). Sterilize at 121℃ for 20min.

[0036] To explore Akkermansia muciniphila BNCC341917 (AKK bacteria) (purchased from Beina Biotechnology) and Bifidobacterium bifidum Can BNCC186304 (BIF bacteria) (purchased from Beina Biotechnology) utilize mussel adhesive protein as a carbon source? We first investigated this under anaerobic conditions at 37°C using BHI broth medium. Akkermansia muciniphila BNCC341917 was pre-cultured using MRS medium. Bifidobacterium bifidum BNCC186304 (BIF bacteria) was pre-cultured. The OD of the bacterial culture was to be measured. 600 When the concentration reached 0.5, the samples were transferred to two different media: carbon-free medium (MM) and BM medium with 0.5% mussel adhesive protein as the sole carbon source. Three biological replicates were set up for each group, and OD was measured every 12 hours. 600 To monitor bacterial growth. See details below. Figure 1The results showed that AKK and BIF bacteria grew well on BHI and MRS media. Compared with MM medium without mucilage, the growth of AKK and BIF bacteria was significantly promoted in BM medium supplemented with mussel mucilage. This demonstrates that AKK and BIF bacteria can effectively utilize mussel mucilage as a carbon source for growth and proliferation.

[0037] Example 2: Determination of the efficiency of AKK and BIF bacteria in degrading mussel adhesive protein

[0038] To detect the degradation efficiency of mussel adhesive protein, 1×10⁻⁶ mcg was used. 8 CFU Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum BNCC186304 was inoculated into BM medium supplemented with 1% mussel mussel adhesive protein as the sole carbon source. The bacteria were anaerobically grown at 37°C for 48 hours. The loss of hexose in the supernatant was assessed using anthrone reagent to evaluate the level of mussel adhesive protein degradation. Detailed results are shown below. Figure 2 The degradation efficiencies of AKK bacteria and BIF bacteria were 78.3% and 63.3%, respectively.

[0039] To optimize the degradation conditions, we used an L9(3³) orthogonal experimental design to investigate three key factors: incubation time (factor A), inoculum size (factor B), and pH (factor C). Each factor was set at three levels. The specific factors and levels are shown in Table 1.

[0040] Nine experiments were conducted using an orthogonal array, with each experiment repeated three times. The average degradation efficiency was taken as the result. The degradation efficiency was calculated by determining hexose loss using the anthrone method. Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidumThe degradation efficiency results of BNCC186304 are shown in Tables 2 and 3. Statistical analysis was performed on the orthogonal experimental results to calculate the average degradation efficiency (k-value) and range (R-value) of each factor at different levels, in order to assess the influence of each factor on the degradation efficiency. The orthogonal experimental results of AKK bacteria are as follows: Factor A (incubation time): k1 = 39.7%, k2 = 69.0%, k3 = 73.5%, R = 29.3; Factor B (inoculum size): k1 = 54.5%, k2 = 54.6%, k3 = 63.1%, R = 8.6; Factor C (pH): k1 = 56.2%, k2 = 63.3%, k3 = 59.6%, R = 7.1. Range (R-value) analysis showed that factor A (incubation time) had the largest range (R=29.3), indicating that incubation time had the most significant impact on degradation efficiency. Factors B (inoculum size) and C (pH) had smaller ranges (8.6 and 7.1, respectively), indicating their relatively weaker effects. The optimal combination of factors was A3 (incubation time 48 hours), B3 (inoculum size 5%), and C2 (pH 7), meaning the optimal conditions were an incubation time of 48 hours, an inoculum size of 5%, and pH 7. Under these optimized conditions, the degradation efficiency reached 78.3%, significantly higher than other experimental groups. The orthogonal experimental results of BIF bacteria are as follows: Factor A (incubation time): k1=54.7%, k2=59.5%, k3=62.0%, range R=7.3; Factor B (inoculum size): k1=58.8%, k2=59.2%, k3=58.8%, range R=0.4; Factor C (pH): k1=59.5%, k2=58.8%, k3=57.8%, range R=1.7. Range (R-value) analysis shows that Factor A (incubation time) has the largest range (R=7.3), indicating that incubation time has the most significant impact on degradation efficiency; Factors B (inoculum size) and Factor C (pH) have smaller ranges (1.7 and 0.4, respectively), indicating that their effects are relatively weak. The optimal combination of levels for each factor is A3 (incubation time 48 hours), B3 (inoculum size 3%), and C2 (pH 6), i.e., the optimal conditions are incubation time 48 hours, inoculum size 3%, and pH 6. Under these optimized conditions, the degradation efficiency reached 63.3%, significantly higher than that of other experimental groups.

[0041] This embodiment clarifies the optimal process parameters for the degradation of mussel adhesive protein through orthogonal experiments, providing a reliable basis for the efficient degradation of mussel adhesive protein, and is applicable to relevant industrial production or applications.

[0042] Table 1 L9(3) 3 Orthogonal Experiment Factors and Levels

[0043]

[0044] Table 2. Results of orthogonal experiments and degradation efficiency (AKK bacteria)

[0045]

[0046] Table 3. Results of orthogonal experiments and degradation efficiency (BIF bacteria)

[0047]

[0048] Example 3 Preparation of mussel adhesive protein polypeptide

[0049] Take it out of the -80℃ freezer Akkermansia muciniphila BNCC341917 (AKK bacteria) Bifidobacterium bifidum BNCC186304 (BIF bacteria) cryovials were rapidly thawed in an anaerobic workstation. 200 µL of the thawed product was then aspirated using a sterile pipette. Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum BNCC186304 (BIF bacteria) (purchased from Beina Biotechnology) bacterial suspension was inoculated into 5 mL of BHI broth and MRS medium, which had been incubated for 30 min under anaerobic conditions at room temperature, at an inoculation volume of 1%, and gently mixed. The culture was then incubated in an anaerobic incubator for 48 h until OD... 600 The value was 0.5. The bacterial pellet was then centrifuged, washed three times with deionized water, and 20 mL of a specific culture medium was added. The mixture was incubated in an anaerobic incubator at 37°C for 48 hours, with OD measured every 12 hours during this period. 600 Growth curves were plotted. After 48 hours, the cells were centrifuged at 10,000 rpm for 15 minutes, the supernatant was collected, filtered through a 0.22 μM filter membrane to remove the bacterial cells, and then ultrafiltered through a 3 kDa ultrafiltration tube. The lower layer liquid was freeze-dried to obtain mussel adhesive protein degradation peptides MP-A (AKK bacteria fermentation peptide product) and MP-B (BIF bacteria fermentation peptide product).

[0050] Example 4: HPLC chromatographic analysis of mussel adhesive peptides

[0051] The degradation of mussel adhesive protein peptides prepared by fermentation with the two probiotics of Example 3, and their 3 kDa ultrafiltration samples MP-A and MP-B, was detected by HPLC. The chromatographic conditions were as follows: Instrument: Shimadzu SPD-M20A high performance liquid chromatograph; Detector: DAD detector; Column: Ultimate HILIC Amphion II column (4.6*250 mm, 5 μM); Mobile phase: A mobile phase was 1‰ ammonium acetate-acetic acid aqueous solution; B mobile phase was 1‰ ammonium acetate-acetic acid acetonitrile solution; Flow rate: 0.4 ml / min; Column temperature: 40 ºC; Injection volume: 20 μl. The HPLC programs for different fermentation products are shown in Tables 4 and 5.

[0052] Table 4. HPLC program for AKK fermentation products

[0053]

[0054] Table 5 HPLC program for BIF fermentation products

[0055]

[0056] Before loading the sample, the chromatographic column needs to be equilibrated. The equilibration solution is mobile phase B, a 1‰ ammonium acetate-acetic acid acetonitrile solution. The equilibration conditions are: 100% equilibration of mobile phase B for 20 min, flow rate: 0.4 ml / min, column temperature: 40 ºC. During the formal analysis, samples containing mussel adhesive protein Mefp-1, co-fermentation products of AKK bacteria and BIF bacteria, as well as mussel adhesive protein peptide products MP-A (fermented by AKK bacteria) and MP-B (fermented by BIF bacteria, <3 kDa) were analyzed.

[0057] Figure 3 The results showed that the characteristic peak of the mussel adhesive protein Mefp-1 appeared at 7.94 min and 10.83 min under different conditions. Figure 4 The disappearance of the characteristic peak (7.94 min) of the mussel adhesive protein Mefp-1 in the left figure indicates that... Akkermansia muciniphila After co-incubating Mefp-1 with BNCC341917 for 48 h, the characteristic peak of Mefp-1 (7.94 min) disappeared, indicating that Mefp-1 was completely degraded. At the same time, three new peaks appeared at 10.600 min, 12.675 min and 15.628 min, indicating that degradation products were present. Figure 4 The disappearance of the characteristic peak (10.83 min) of the mussel adhesive protein Mefp-1 in the right figure indicates that... Bifidobacterium bifidum After co-incubating Mefp-1 with BNCC186304 for 48 h, the characteristic peak of Mefp-1 (10.83 min) disappeared, indicating that Mefp-1 was completely degraded; at the same time, multiple new peaks appeared at 28-33 min and 43-55 min, indicating the presence of degradation products. Figure 5 for Figure 4 HPLC chromatogram of the fermentation sample after ultrafiltration using a 3 kDa ultrafiltration tube. Figure 5 The spectral results on the left show that the product at 10-15 min is a component with a value less than 3 kDa. Figure 5 The spectral results on the right show that the products at 28-33 min and 43-55 min contain components with less than 3 kDa, proving that different mussel adhesive protein peptides were obtained.

[0058] Example 5: Identification of Mussel Adhesive Protein Peptide Omics

[0059] The main steps of peptide proteomics analysis include: peptide desalting, LC-MS / MS analysis, database retrieval, and data analysis. The specific steps are as follows:

[0060] Will Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum Samples of BNCC186304 cultured in carbon-free medium for 48 hours were used as controls (blank). Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum BNCC186304 was incubated for 48 hours in the specific culture medium of Example 1 to obtain mussel adhesive peptides MP-A and MP-B as samples.

[0061] For both groups of samples, mussel adhesive peptides MP-A and MP-B, 1 mL of each was added to an appropriate amount of 0.1% TFA, mixed well, centrifuged at 20000 g for 5 min, and the supernatant was collected and transferred to a 3 kDa ultrafiltration centrifuge tube. The tubes were then centrifuged at 12000 g for 15 min. 200 µL of 0.1% TFA was added, and the tubes were centrifuged at 12000 g for 15 min. This process was repeated twice. The filtrates were collected, desalted using a C18 StageTip (Thermo Fisher Scientific, catalog number: 13-110-055), and then vacuum dried. The dried peptides were reconstituted with 0.1% TFA, and their concentrations were determined for LC-MS analysis.

[0062] After processing, an appropriate amount of peptide was taken from each sample and chromatographically separated using a Nano-flow-rate Easy nLC 1200 chromatography system (Thermo Scientific). Buffer solutions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a mixture of 0.1% formic acid, acetonitrile, and water (acetonitrile comprising 80%). The column was equilibrated with 100% solution A. A sample loading of 0.5 μg was injected into a Trap Column (100µm*20mm, 5µm, C18, Dr. Maisch GmbH) for enrichment and desalting, followed by gradient separation using an analytical column (75µm*150mm, 3µm, C18, Dr. Maisch GmbH) at a flow rate of 300 nL / min.

[0063] The liquid chromatography separation gradients for both columns were as follows: 0-2 min, linear gradient of solution B from 2% to 5%; 2-44 min, linear gradient of solution B from 5% to 28%; 44-51 min, linear gradient of solution B from 28% to 40%; 51-53 min, linear gradient of solution B from 40% to 100%; 53-60 min, solution B maintained at 100%. After peptide separation, data-dependent secondary mass spectrometry (DDA) was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time was 60 min, detection mode: positive ion; precursor ion scan range: 350-1800 m / z; primary mass spectrometry resolution: 60000 @m / z 200; AGC target: 3e6; primary maximum IT: 50 ms. Peptide secondary mass spectrometry analysis was performed using the following method: after each full scan, the secondary mass spectra of the 20 highest intensity precursor ions were acquired (MS2 scan). Secondary mass spectrometry resolution: 15000 @ m / z 200, AGC target: 1e5, secondary maximum IT: 50ms, MS2 activation type: HCD, isolation window: 1.6m / z, normalized collision energy: 28.

[0064] MP-A and MP-B were identified by peptidomics as containing 80 and 128 specific peptides, respectively. The results are shown in Tables 6 and 7, which show the top 20 specific peptides of MP-A and MP-B, respectively. These do not include peptide sequences known to improve inflammatory bowel disease, such as AKPSYPPTYK and its derivative peptides.

[0065] Table 6 MP-A specific peptide sequences (Top 20)

[0066]

[0067] Table 7 MP-B specific peptide sequences (Top 20)

[0068]

[0069] Example 6: AI-Driven Virtual Screening

[0070] This invention identified 26 potential druggable targets through GWAS combined with MR, constructing a virtual target library for inflammatory bowel disease (IBD). Since IBD is a complex chronic disease, drugs screened for a single target may not meet clinical needs; therefore, this invention employs a multi-target, multi-molecule virtual screening approach. The screening process involves constructing 3D target structures and predicted active sites using the protein database PDB and AlphaFold3 software, and performing one-to-one docking with over 200 peptides. The docking process includes initial screening using Schrödinger SP software, followed by screening using the Schrödinger GBSA module, and finally, final screening using the FEP module. After obtaining all docking scores, the peptide molecules are ranked based on personalized scoring, with the top 15 peptides proceeding to further research. The top 15 peptide sequences are NYLPLAKKLSSYKPIKTT, AKKLSSYKPIKTTYNAK, KDMSH(DOPA)IL, NIYNAHGSAYA, YPRTYK, SKTNYLPL, PAGPQGPAGPR, TYKAKPSYSST, LAKKLSSYKPIK, AVFPSIVGRP, FPSIVGRP, GPVYKPVKTS, TYKAKPSY, HGPVYKPVKTS, or IWHHTF.

[0071] Table 8 Top 15 Peptide Molecules

[0072]

[0073] Example 7: NCM-460 Inflammation Model Validates Anti-inflammatory Activity

[0074] The anti-inflammatory activity of peptides was verified by evaluating their inhibitory effect on the expression of inflammatory factors using an NCM-460 inflammation model. The specific procedures were as follows: First, logarithmically growing NCM-460 cells (purchased from BNCC, catalog number: BNCC339288) were digested and seeded at an appropriate density into multi-well plates, and cultured overnight at 37°C in a 5% CO2 incubator to ensure full cell adhesion. The next day, when cell confluence reached over 80%, the medium was replaced with DMEM, and the cells were divided into groups: a blank control group (DMEM medium only), a model group (only an inflammation inducer, such as TNF-α at a final concentration of 50 ng / mL, was added to induce the NCM-460 inflammation model), a positive drug control group (TNF-α and a positive drug, such as 5-ASA (methalazine) were added), and eight peptide experimental groups (peptide final concentration of 1 μM and TNF-α final concentration of 50 ng / mL). Each group was repeated at least three times. After treatment, the multi-well plate was returned to the incubator for a specified incubation time (e.g., 24 hours). After incubation, the culture medium was discarded, and the cells were washed with ice-cold PBS. Then, an appropriate amount of TRIzol was added to lyse the cells, and the lysis buffer was collected. Total RNA was extracted from the cells, and the concentration and purity (A260 / A280) were measured. The total RNA was reverse transcribed to obtain cDNA. The cDNA was used as a template for real-time quantitative PCR (qPCR) targeting inflammatory cytokine genes (e.g.,...). IL1B and IL6 Specific primers were used for amplification. The β-actin gene was used as an internal control. Primer sequence information is shown in Table 9, qRT-PCR reaction system is shown in Table 10, and RT-PCR reaction procedure is shown in Table 11. The relative gene expression levels of inflammatory factors in each treatment group were calculated and compared using the ΔΔCt method.

[0075] Table 9 Primer sequence information

[0076]

[0077] Table 10 qRT-PCR reaction system

[0078]

[0079] Table 11 RT-PCR reaction procedure

[0080]

[0081] like Figure 6 Of the top 15 peptides, only peptides-6,-9,-11, and-12 were inactive; the remaining peptides all exhibited good activity. Peptides-2 and-8 showed the best activity, with highly significant differences between them. Figure 7Of the top 15 peptides, only peptide-6, peptide-9, peptide-11, and peptide-12 were inactive, while the remaining peptides showed good activity, exhibiting highly significant differences. (Where ** and *** represent significant differences from the Model results, the quantified values ​​are: ** indicates P < 0.01, indicating a highly significant difference, and *** indicates P < 0.001, indicating an extremely significant difference).

[0082] Example 8: Effects of mussel adhesive peptides MP-A and MP-B on DSS-induced ulcerative colitis in mice.

[0083] (1) Experimental animals: 40 male C57BL / 6J mice, aged 6-8 weeks and weighing 20±2 g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0084] (2) Animal grouping: After one week of acclimatization, animals were randomly grouped into 5 groups (8 animals / group): control group, model group, 5-ASA treatment group, and low-dose treatment group of two mussel adhesive protein polypeptides prepared in Example 1, namely MP-A (product of mussel adhesive protein co-fermentation by AKK bacteria, <3 KDa) (20 mg / kg) and MP-B (product of mussel adhesive protein co-fermentation by BIF bacteria, <3 KDa) (20 mg / kg).

[0085] (3) Animal modeling and drug administration: The control group mice received standard drinking water, while the drinking water of the other groups was supplemented with 3% DSS to induce acute ulcerative colitis for 7 days. One day after modeling, 100 μL of mussel adhesive peptides MP-A and MP-B (20 mg / kg) were slowly injected into the rectum daily, while the 5-ASA positive control group was slowly injected into the rectum daily with 100 μL of 5-ASA (20 mg / kg).

[0086] (4) DAI score: During the experiment, the three clinical symptoms of UC were assessed daily according to DAI, including the percentage of weight loss, stool viscosity, and rectal bleeding. The DAI score is the sum of the daily weight loss score, stool viscosity score, and rectal bleeding score. DAI score indicators: the percentage of weight loss (0 points: no change in weight, 1 point: 1%-5% decrease, 2 points: 5%-10% decrease, 3 points: 10%-20% decrease, 4 points: >20%), stool consistency (0 points: normal, 2 points: slightly moist but not attached to the perianal area, 4 points: diarrhea), and rectal bleeding (0 points: normal, 2 points: slight bleeding, 4 points: hematochezia) are the sum of the three scores.

[0087] (5) Animal sampling: Eight hours after the last administration, mice were euthanized by cervical dislocation. The peritoneal cavity of the mice was exposed, and the colon and rectum were separated. The length of the colon and rectum was measured and photographed. Subsequently, pre-cooled physiological saline was repeatedly injected into the intestinal lumen to remove the contents. Approximately 1 cm of the distal colon was excised and fixed in 4% paraformaldehyde solution for histopathological examination.

[0088] (6) Experimental results: The specific results of the Disease Activity Index (DAI) score for mouse colitis are shown in [the table below]. Figure 8 Compared to the control group, mice given 3% DSS in their drinking water exhibited acute clinical symptoms, including weight loss, altered fecal characteristics, and severe bloody stools, which were reflected in increased DAI scores. Compared to the model group mice, mussel adhesive peptides MP-A and MP-B significantly improved the clinical symptoms and reduced DAI scores in mice in a dose-dependent manner.

[0089] The specific results of the mouse colon length measurement are shown in [link to relevant documentation]. Figure 9 and Figure 10 Compared with the control group, mice treated with DSS showed significant congestion, extensive mucosal ulceration, and shortened length in the colorectal tissue. Compared with the model group, rectal injection of mussel adhesive peptides MP-A and MP-B significantly reversed intestinal congestion and shortened colorectal length in mice, suggesting that mussel adhesive peptides MP-A and MP-B can improve the severity of ulcerative colitis (UC).

[0090] For detailed results of the histopathological evaluation of mouse colon tissue, please refer to [link / reference]. Figure 11 Compared with the control group, DSS intervention caused damage to the colonic epithelial mucosa, necrosis of crypt structures, and extensive infiltration of inflammatory cells in mice. However, compared with the model group, rectal injection of mussel adhesive peptides MP-A and MP-B significantly alleviated the damage to colonic tissue structure and reduced the infiltration of inflammatory cells, suggesting that mussel adhesive peptides MP-A and MP-B have a protective effect against DSS-induced pathological damage to colonic tissue.

Claims

1. A mussel adhesive protein polypeptide, characterized in that, The amino acid sequence of the mussel adhesive protein polypeptide is shown in SEQ ID NO.

4.

2. A composition of mussel adhesive protein polypeptide, characterized in that, The composition comprises the mussel adhesive protein polypeptide of claim 1.

3. The composition according to claim 2, characterized in that, The composition further includes one or more of the following polypeptides: the amino acid sequences of the polypeptides are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12, SEQ ID NO.14, KDMSH-DOPA-IL, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.27 or SEQ ID NO.31, respectively.

4. A method for preparing a mussel adhesive protein polypeptide or a composition thereof, characterized in that, Includes the following steps: Will Akkermansia muciniphila BNCC341917 bacterial solution or Bifidobacterium bifidum BNCC186304 was added to a specific culture medium containing mussel adhesive protein as a carbon source, anaerobically cultured, centrifuged, and then ultrafiltered. The lower layer solution was collected, lyophilized, and obtained as mussel adhesive protein polypeptide or a combination thereof. The anaerobic culture conditions were: 37℃ for 24-48 h, centrifugation speed of 4000-5000 rpm, centrifugation time of 10-15 min, and ultrafiltration was performed directly using a 3 kDa ultrafiltration tube after sterilization with a 0.22 μM filter membrane.

5. The method for preparing the mussel adhesive protein polypeptide or its composition according to claim 4, characterized in that, The specific culture medium is composed of the following components per 500 mL of water: 1.4 g Na2HPO4, 0.25 g (NH4)2SO4, 0.0005 mg CuCl2·2H2O, 0.015 mg H3BO3, 0.1 mg FeSO4·7H2O, 0.5 mg KH2PO4, 0.0015 mg MnCl2·4H2O, 0.001 mg NiCl2·6H2O, 0.25 mg Na2EDTA, 0.01 mg CoCl2·6H2O, 0.005 mg ZnSO4·7H2O, 0.0015 mg Na2MoO4·2H2O, and 2.5 g Mefp-1.

6. The method for preparing the mussel adhesive protein polypeptide or its composition according to claim 4, characterized in that, The Akkermansia muciniphila BNCC341917 and Bifidobacterium bifidum BNCC186304 was revived and cultured in BHI broth and MRS medium to obtain activated bacterial solutions. The BHI medium consisted of the following components per 1L of water: 12.5 g bovine brain extract, 5.0 g bovine heart extract, 10.0 g tryptone, 2.0 g glucose, 5.0 g sodium chloride, and 2.5 g disodium hydrogen phosphate. The MRS medium consisted of the following components per 1L of water: 20.0 g glucose, 10.0 g peptone, 10.0 g beef extract, 5.0 g sodium acetate, 5.0 g yeast extract, 2.0 g dipotassium phosphate, 2.0 g ammonium citrate, 1.08 g Tween 80, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

7. The method for preparing the mussel adhesive protein polypeptide or its composition according to claim 6, characterized in that, The Akkermansia muciniphila BNCC341917 or Bifidobacterium bifidum The inoculum size for BNCC186304 resuscitation culture is 1-3%, the resuscitation time is 24-48 hours, and the pH is 6-8. Akkermansia muciniphila BNCC341917 or Bifidobacterium bifidum OD of BNCC186304 bacterial culture 600 =0.5~0.

6.

8. The method for preparing the mussel adhesive protein polypeptide or its composition according to claim 4, characterized in that, The mussel adhesive protein has a mass concentration of 0.2% to 1% in the specific culture medium.

9. The use of the mussel adhesive protein polypeptide of claim 1 or the composition of claim 2 or 3 in the preparation of a medicament for the prevention or treatment of inflammatory bowel disease, characterized in that, The inflammatory bowel disease mentioned is colitis.

10. The application according to claim 9, characterized in that, The colitis mentioned includes ulcerative colitis.

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