Peptide with high anticoagulant activity as well as preparation method and application thereof

Abstract

The invention discloses a high-anticoagulation active peptide and a preparation method and application thereof, and relates to a shrimp paste high-anticoagulation active protein component based on compound enzyme assisted rapid fermentation and a preparation method thereof, and a preparation method of a high-anticoagulation active shrimp paste. The method for rapidly preparing the shrimp paste with high anticoagulation activity and assistance of enzymolysis is obtained by screening enzymolysis-assisted fermentation conditions such as shrimp producing areas, enzyme types, enzymolysis temperature, enzymolysis time, enzymolysis pH and the like. After purification is carried out by combining ultrafiltration, gel chromatography and non-denaturing electrophoresis, the anticoagulant active peptides PSEPSKPVTCKPR and GIAEGCDYPWR are identified by combining proteomics and a virtual screening technology. According to the invention, not only is the preparation method of the high-anticoagulant-activity enzymatic-method-assisted rapid fermentation shrimp paste obtained, but also two novel anticoagulant peptides are innovatively separated from the shrimp paste, and a novel biological agent development basis is provided for treatment of thrombotic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and food processing intersection, and in particular relates to a shrimp paste peptide with high anticoagulant activity based on complex enzyme-assisted rapid fermentation, and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease (CVD) is a class of diseases caused by pathological processes such as atherosclerosis, thrombosis, and vascular inflammation. Its core characteristic is dysfunction of the cardiac and vascular systems. As the leading cause of death worldwide, as of 2019, CVD accounted for 32% of all deaths worldwide, with approximately 18.6 million deaths from CVD each year. Thrombotic diseases rank first among all diseases in terms of morbidity, disability, and mortality. Current antithrombotic drugs, including thrombolytics (such as recombinant tissue prothrombin activator, rt-PA) and direct oral anticoagulants (DOACs) (such as dabigatran and rivaroxaban), increase the risk of intracranial or gastrointestinal bleeding, have low fibrin clot specificity, a short half-life, and other limitations. Therefore, the search for a thrombolytic agent that combines safety, efficacy, high specificity, no side effects such as bleeding, and reasonable price has become an ideal goal.

[0003] Shrimp paste is a traditional fermented condiment rich in various nutrients such as protein, amino acids, vitamins and minerals, and has been shown to contain components with anti-thrombotic activity. As an important part of my country's traditional food culture, it is deeply loved by consumers and is highly suitable for development as a functional food. However, current research on shrimp paste preparation mostly focuses on the control of harmful components such as biogenic amines and the enhancement of fermented flavor, while research on functional active substances in shrimp paste mostly focuses on antioxidant capacity, and there is little research on the screening of anticoagulant proteins. Therefore, in-depth research on the anticoagulant protein components in shrimp paste will not only help promote the functional application of shrimp paste products, but also provide new ideas for the modern application of traditional foods. Summary of the Invention

[0004] In view of this, the present invention provides a high anticoagulant activity peptide and a preparation method and application thereof, which are two shrimp paste high anticoagulant activity peptides based on complex enzyme-assisted rapid fermentation and a preparation method thereof.

[0005] The present invention determines the conditions for producing shrimp paste with high anticoagulant activity by measuring the anticoagulant activity of marine shrimp paste. Based on this, the antithrombotic active components of the shrimp paste are enriched using ammonium sulfate fractional precipitation and purified by ultrafiltration, gel chromatography, and native electrophoresis. Finally, based on proteomics and virtual screening techniques, anticoagulant peptide sequences are identified and synthetically verified. These results provide new insights for the high-value utilization of shrimp paste, a traditional fermented condiment, and the research of novel antithrombotic functional foods.

[0006] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0007] A peptide with high anticoagulant activity, wherein the amino acid sequence of the peptide is as shown in SEQ ID No. 1 (PSEPSKPVTCKPR) or SEQ ID No. 2 (GIAEGCDYPWR), and the peptide with high anticoagulant activity is used in the preparation of antithrombotic drugs.

[0008] The preparation method of the high anticoagulant activity peptide comprises the following steps: preparing shrimp paste using Penaeus vannamei as raw material, performing enzymatic hydrolysis with a biological enzyme, and then fermenting to obtain fermented shrimp paste containing the high anticoagulant activity peptide; and obtaining the high anticoagulant activity peptide through post-processing.

[0009] Selecting biological enzymes for enzymatic hydrolysis and then fermenting to obtain fermented shrimp paste containing high anticoagulant activity peptides, specifically including:

[0010] Chitosanase was used to assist enzymatic hydrolysis of Yantai shrimp at 44-46℃ and pH 6.5-7.5 for 5-7h, followed by natural fermentation for 14-16d to produce fermented shrimp paste containing high anticoagulant active peptides.

[0011] Post-processing specifically includes:

[0012] The supernatant of fermented shrimp paste containing high anticoagulant activity peptides after water extraction was enriched with ammonium sulfate aqueous solution, and then purified by ultrafiltration, gel chromatography and native electrophoresis. Combined with proteomics and virtual screening technology, the structure of the anticoagulant peptides was identified to obtain high anticoagulant activity peptides.

[0013] The supernatant of the fermented shrimp paste containing high anticoagulant activity peptides after water extraction is enriched by an ammonium sulfate aqueous solution, specifically comprising:

[0014] The fermented shrimp paste containing high anticoagulant active peptides is centrifuged with water, the supernatant is collected, and then the anticoagulant active components are enriched using a 30-60% saturation ammonium sulfate aqueous solution.

[0015] The material-liquid ratio of the fermented shrimp paste containing high anticoagulant activity peptides to water is 1g:6-7mL.

[0016] Ultrafiltration was performed using an ultrafiltration tube with a molecular weight cut-off of 10 kDa to obtain M W A mixture of anticoagulant active components >10kDa.

[0017] Gel chromatography was performed using Sephadex G-50 gel chromatography.

[0018] Identify the structure of anticoagulant peptides, including:

[0019] Combining proteomics and virtual screening technology to identify the sequence of anticoagulant peptides.

[0020] The invention provides a method for preparing shrimp paste with high anticoagulant activity. The method comprises the following parameters: fermenting Yantai shrimp for 6 hours with 1% (E / S, w / w) chitosanase at 45°C and pH 7.0, followed by natural fermentation for 15 days to obtain a shrimp paste with the strongest antithrombotic activity.

[0021] Taking anticoagulant activity as an indicator, the enzymatic hydrolysis-assisted fermentation conditions such as raw material origin, enzyme type, enzymatic hydrolysis temperature, enzymatic hydrolysis time, and enzymatic hydrolysis pH were screened and optimized.

[0022] The present invention provides a method for enriching components with high anticoagulant activity in shrimp paste, which comprises the following steps:

[0023] (1) Enrichment of anticoagulant active components of shrimp paste by ammonium sulfate fractionation precipitation method;

[0024] The crude extract was prepared into saturated concentration (30%, 60%, 100%) precipitates by adding ammonium sulfate, and the active components of shrimp paste with strong health-promoting ability were further purified.

[0025] (2) Purification by ultrafiltration, gel chromatography, and native electrophoresis.

[0026] Furthermore, the components in step (1) were separated by ultrafiltration at 10 kDa, and the components with Mw>10 kDa were retained. Three peaks were finally obtained by Sephadex G-50 gel filtration chromatography;

[0027] Furthermore, the peak with the strongest activity was selected for non-denaturing polyacrylamide gel electrophoresis to detect the anticoagulant ability of bands with different molecular weights, and proteomics technology and virtual screening technology were combined to perform peptide scoring and activity prediction.

[0028] The present invention combines proteomics technology with virtual screening technology to identify the structure of anticoagulant peptides, obtains two protein sequences with high anticoagulant activity as described above, and verifies their activity.

[0029] The beneficial effects of the present invention are as follows: 1. The present invention obtains a method for rapidly preparing shrimp paste with high anticoagulant activity by enzymatic hydrolysis assistance through screening of conditions for enzymatic hydrolysis-assisted fermentation, such as the origin of shrimp, the type of enzyme, the enzymatic hydrolysis temperature, the enzymatic hydrolysis time, and the enzymatic hydrolysis pH. The method of composite enzymatic hydrolysis-assisted fermentation is different from similar products reported so far, which lays the foundation for high-value applications of shrimp paste and provides ideas for functional research and optimization of traditional fermented products. 2. Anticoagulant active components are enriched with the assistance of ammonium sulfate precipitation, and after purification by ultrafiltration, gel chromatography, and non-denaturing electrophoresis, anticoagulant peptides are screened using virtual screening technology. The present invention not only obtains a method for preparing shrimp paste with high anticoagulant activity by enzyme-assisted rapid fermentation, but also the two anticoagulant peptides obtained in the present invention have significantly better anticoagulant effects at a concentration of 3 mg / mL than sodium heparin at the same concentration, providing a new biological preparation basis for the treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The effect of different saturation of ammonium sulfate on protein anticoagulant activity and protein content;

[0031] Figure 2 For different M W The anticoagulant capacity of shrimp paste protein components;

[0032] Figure 3 is the anticoagulant activity of chromatographic fractions A1 to A3;

[0033] Figure 4 for electrophoretic band distribution and antithrombotic activity;

[0034] Figure 5 This is the docking diagram of the antithrombotic peptide and thrombin molecules;

[0035] Figure 6 To verify the activity of anticoagulant peptides. DETAILED DESCRIPTION

[0036] The present invention will be further explained below with reference to specific embodiments.

[0037] Example 1

[0038] The amino acid sequences of the peptide with anticoagulant activity described in this embodiment are: PSEPSKPVTCKPR and GIAEGCDYPWR.

[0039] The enzymatic hydrolysis-assisted rapid fermentation method for preparing shrimp paste with high anticoagulant activity of the present invention is implemented by the following steps:

[0040] Thaw 3-5cm Yantai whiteleg shrimp (Penaeus vannamei, Litopenaeus vannamei) at 25°C for 30 minutes, rinse, remove shell fragments and foreign matter, and air-dry for 8 minutes. Add 10% of the shrimp's weight in water to the mixture in a juicer and blend thoroughly to prepare shrimp paste. Weigh the mixture and add 15% NaCl.

[0041] Second, chitosanase 1% (E / S, w / w) was used to enzymatically hydrolyze the shrimp paste at 45°C and pH 7.0 for 6 h, followed by natural fermentation at 25°C for 15 days.

[0042] Table 1. Anticoagulant activity of shrimp paste fermented with enzyme-assisted hydrolysis under different conditions

[0043]

[0044] As shown in Table 1, Table 1 shows the anticoagulant activity of shrimp paste fermented with enzyme-assisted hydrolysis under different conditions. These results prepare for the subsequent experiment of separating the high anticoagulant activity components of shrimp paste.

[0045] This study found that shrimp paste produced by enzymatic hydrolysis of Yantai shrimp with 1% (E / S, w / w) chitosanase at 45°C and pH 7.0 for 6 hours, followed by natural fermentation at 25°C for 15 days, exhibited the strongest anticoagulant activity. The fermented shrimp paste containing highly anticoagulant peptides was centrifuged with water (at a material-liquid ratio of 1g:6.5mL). The supernatant was collected and used as the shrimp paste aqueous extract. The shrimp paste aqueous extract (at a concentration of 12mg / mL) had an anticoagulant activity of 93.40±0.31%. The R value was used to determine the influence of factors on both activities. It was found that the most significant factor influencing both anticoagulant and antithrombotic activities was enzymatic hydrolysis temperature, followed by enzymatic hydrolysis pH, and the least significant factor was enzymatic hydrolysis time.

[0046] Example 2

[0047] In this embodiment, the anticoagulant active components in shrimp paste are enriched by graded precipitation of shrimp paste water extract with ammonium sulfate of different saturations. Figure 1 The effect of different saturation of ammonium sulfate on the anticoagulant activity and protein content of the protein. The anticoagulant activity of the sample enriched with 30-60% saturation of ammonium sulfate (5 mg / mL) of shrimp paste protein was 91.57±1.40% and the total protein content was 33.16±1.08%.

[0048] The shrimp paste protein with 30-60% saturation ammonium sulfate fractionation was separated and purified by ultrafiltration fractionation (filtration was performed using an ultrafiltration tube with a molecular weight cut-off of 10 kDa) combined with gel chromatography and non-denaturing electrophoresis. Figure 2 、 3 、4 are different MW Anticoagulant ability of shrimp paste protein fraction, anticoagulant activity of chromatographic fractions A1 to A3, and electrophoretic band distribution. W The anticoagulant activity of shrimp paste protein >10kDa (5mg / mL) was 99.76±0.22%. Peak A1 obtained by Sephadex G-50 gel chromatography showed the strongest anticoagulant activity at a concentration of 1mg / mL, which was 86.58±2.33%. Band a9 with the strongest anticoagulant activity was identified by native electrophoresis. W It is in the range of 10 to 25 kDa and was identified by proteomics.

[0049] Example 3 Identification of Anticoagulant Activity Protein Sequence

[0050] Proteomics combined with virtual screening technology was used to analyze the resulting electrophoretic band sequences. Peptide Ranker was used to predict the potential antithrombotic activity of peptides. Peptides were scored between 0 and 1, and 12 peptides with a score > 0.5 were considered to have potential bioactivity. Isoelectric point (pI) was predicted using ProtParam, and hydrophobicity was predicted using ExPASy. The results are shown in Table 2.

[0051] Table 2. Peptide bioactivity prediction and physicochemical indicators

[0052]

[0053] The crystal structure of thrombin (PDB ID: 4UD9) was obtained from the PDB database (http: / / www.rcsb.org), and the 2D and 3D structures of the peptides required for docking were constructed using ChemDraw and Chem 3D software. PyMOL (2.6.0) software was used to delete the original thrombin water molecules and co-crystallized compounds, perform hydrogenation, and set thrombin as a rigid receptor. Next, the ligands and receptors were prepared by merging non-polar hydrogens, assigning Gasteiger charges, and eliminating lone pairs of electrons using SailVina (1.0). Torsional rotations of all ligands were allowed. Molecular docking was performed using AutoDock Vina (1.2.5), and the docking parameters were slightly modified. Molecular simulation docking was performed using a genetic algorithm to analyze the docking energy of the enzyme active site. In order to determine the most stable molecular docking structure with the lowest docking energy, the molecular docking results were visualized using PyMOL. The docking energy results are shown in Table 3.

[0054] Table 3. Molecular docking energy of anticoagulant peptides binding to thrombin

[0055]

[0056] Potential antithrombotic peptides were screened by evaluating their docking energy scores. Six peptides with lower scores were selected for visualization. The docking diagram is shown in Figure 5 .

[0057] Peptide conformations were mapped using Python. RDKit was used to generate 3D conformations and perform energy minimization. MOL files containing peptide molecular information were converted to PDB files using OpenBabel. Peptide-protein interactions were analyzed using Discovery Studio (2020). Finally, PyMOL and Discovery Studio were used to graphically present interaction forces and active site results, allowing analysis of the potential mechanisms of the anticoagulant peptide's activity.

[0058] The binding sites of the six peptides docked with thrombin, including hydrophobic forces, hydrogen bonding forces, π-π stacking forces, and salt bridge forces, were integrated. It was found that Trp60 formed a total of 13 hydrophobic forces with each peptide, and additionally formed 5 hydrogen bonding forces with peptides GR-10, PR-13, and VK-19, and 2 π-π stacking forces with peptides AK-10 and CK-11, for a total of 20 forces. Therefore, it was inferred that Trp60 was one of the main sites. Similarly, Glu192 formed hydrophobic and hydrogen bonding forces with peptides AK-10, CK-11, and VK-19, respectively, and generated a total of 21 interactions with GR-10, PR-13, and VK-19. Gly216 generated a total of 13 hydrogen bonding forces with all peptides except VK-19. Tyr60 generated 5 interactions with peptides AK-10, CK-11, and VK-19. Arg221 generated a total of 4 hydrogen bonding forces with peptides AK-10, CK-11, and GR-11. Therefore, we believe that Trp60, Glu192, Gly216, Tyr60, and Arg221 are the main sites for the six shrimp paste antithrombotic peptides to dock with thrombin, generating strong interactions with thrombin, resulting in a strong thrombin inhibitory effect, and thus exerting an anti-thrombotic effect.

[0059] The screened peptides CEGNDFCDWLK, AFADFPNAFK, GIAEGCDYPWR, VISKPGKPEGPLEVSGIHK, PSEPSKPVTCKPR, and GSPYWQTAAR were synthesized by Shanghai Kaijia Pharmaceutical Technology Co., Ltd. (purity above 98%).

[0060] The peptide was dissolved in ultrapure water to prepare a 3 mg / mL solution for in vitro anticoagulant activity verification. The results are shown in Figure 6At a uniform concentration of 3 mg / mL, the anticoagulant activity of the synthetic peptides showed significant differences: the thrombin inhibition rates of PR-13 (76.25±1.85%) and GR-11 (67.51±2.38%) were significantly better than those of the positive control drug heparin sodium.

[0061] The above description is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited to any technician familiar with the technical field within the technical scope disclosed in the present invention. Any equivalent replacement or change based on the technical solution and inventive concept of the present invention should be covered by the protection scope of the present invention.

Claims

1. A peptide with high anticoagulant activity, characterized in that: The amino acid sequence of the high anticoagulant activity peptide is as shown in SEQ ID No. 1 or SEQ ID No.

2.

2. Use of the peptide with high anticoagulant activity as claimed in claim 1 in the preparation of antithrombotic drugs.

3. The method for preparing a peptide with high anticoagulant activity according to claim 1, wherein The steps are as follows: using marine shrimp Penaeus vannamei as raw material to prepare shrimp paste, selecting biological enzymes for enzymatic hydrolysis, and then fermenting to obtain fermented shrimp paste containing high anticoagulant activity peptides, and then post-processing to obtain high anticoagulant activity peptides.

4. The method for preparing a peptide with high anticoagulant activity according to claim 3, wherein: Selecting biological enzymes for enzymatic hydrolysis and then fermenting to obtain fermented shrimp paste containing high anticoagulant activity peptides, specifically including: Chitosanase was used to assist enzymatic hydrolysis of Yantai shrimp at 44-46℃ and pH 6.5-7.5 for 5-7h, followed by natural fermentation for 14-16d to produce fermented shrimp paste containing high anticoagulant active peptides.

5. The method for preparing a peptide with high anticoagulant activity according to claim 3, wherein: Post-processing specifically includes: The supernatant of fermented shrimp paste containing high anticoagulant activity peptides after water extraction was enriched with ammonium sulfate aqueous solution, and then purified by ultrafiltration, gel chromatography and native electrophoresis. Combined with proteomics and virtual screening technology, the structure of the anticoagulant peptides was identified to obtain high anticoagulant activity peptides.

6. The method for preparing a peptide with high anticoagulant activity according to claim 5, wherein: The supernatant of the fermented shrimp paste containing high anticoagulant activity peptides after water extraction is enriched by an ammonium sulfate aqueous solution, specifically comprising: The fermented shrimp paste containing high anticoagulant activity peptides is added with water for centrifugal treatment, the supernatant is collected, and then the anticoagulant activity component is enriched using a 30-60% saturation ammonium sulfate aqueous solution.

7. The method for preparing a peptide with high anticoagulant activity according to claim 6, wherein: The material-liquid ratio of the fermented shrimp paste containing high anticoagulant activity peptides to water is 1g:6-7mL.

8. The method for preparing a peptide with high anticoagulant activity according to claim 5, wherein: Ultrafiltration was performed using an ultrafiltration tube with a molecular weight cut-off of 10 kDa to obtain M W A mixture of anticoagulant active components >10kDa.

9. The method for preparing a peptide with high anticoagulant activity according to claim 5, wherein: Gel chromatography used Sephadex G-50 gel chromatography.

10. The method for preparing a peptide with high anticoagulant activity according to claim 5, wherein: Identify the structure of anticoagulant peptides, including: Combining proteomics and virtual screening technology to identify the sequence of anticoagulant peptides.

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