A plasmin inhibitory peptide YHX-PIP-4 derived from milk protein and its application
By screening out the plasmin inhibitory peptide YHX-PIP-4 from β-lactoglobulin, the gelation problem caused by plasmin activity in ultra-high temperature sterilized dairy milk was solved, achieving the effects of extending the shelf life and reducing costs.
Patent Information
- Application Number
- CN202311406887.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-27
- Publication Date
- 2025-10-03
- Estimated Expiration
- 2043-10-27
AI Technical Summary
In the prior art, the activity of plasmin in ultra-high temperature sterilized dairy milk leads to gelation, which shortens the shelf life. In addition, traditional plasmin inhibitors are expensive and difficult to obtain.
An octapeptide plasmin inhibitory peptide YHX-PIP-4 was screened from the β-lactoglobulin sequence, synthesized through bioinformatics and molecular dynamics simulation, and added to ultra-high temperature sterilized dairy milk to inhibit plasmin activity and avoid gelation.
It effectively inhibits the activity of plasmin, avoids gelation of the milk system, extends the shelf life of ultra-high temperature sterilized milk, and reduces R&D and production costs.
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Figure CN117430694B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a plasmin inhibitory peptide YHX-PIP-4 derived from bovine milk protein and its application. Background Art
[0002] Plasmin is a fibrinolytic enzyme that is secreted from plasma into milk through the mammary gland and is the main endogenous protease in milk. The plasmin system consists of plasmin, plasminogen, plasminogen activator, plasminogen activator inhibitor and plasmin inhibitor, and its activity depends largely on the processing and storage conditions of milk.
[0003] Studies have found that plasmin is heat-stable and can survive ultra-high temperature sterilization. In ultra-high temperature sterilized dairy milk, plasmin still has relatively good activity. In ultra-high temperature sterilized dairy milk, the hydrolysis of plasmin will cause the milk system to gel. Gelation is a quality defect of ultra-high temperature sterilized milk that forms curd-like substances and reduces fluidity during storage. The occurrence of gelation seriously reduces the shelf life of milk and shortens the shelf life of ultra-high temperature sterilized milk.
[0004] Currently, to suppress the activity of plasmin in ultra-high temperature sterilized milk, plasmin inhibitors are typically added to the milk. Common plasmin inhibitors are mostly natural plasmin inhibitors; however, natural plasmin inhibitors are mostly proteins and peptides, often found in grain proteins or animal venoms. Not only are raw materials challenging to obtain, but their primary use in clinical treatments makes them prohibitively expensive to obtain.
[0005] In this regard, the inventors believe that how to develop a new plasmin inhibitor to address the problem of excessive plasmin activity in sterilized milk leading to gelation and shortening of shelf life is a technical problem that technicians in this field urgently need to solve. This plasmin inhibitor should have good plasmin inhibitory activity and low production cost.
[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention
[0007] In response to the above technical problems, the embodiments of the present invention provide a plasmin inhibitory peptide YHX-PIP-4 derived from bovine milk protein and its application to solve the problems raised in the above background technology.
[0008] The present invention provides the following technical solutions:
[0009] A plasmin inhibitory peptide YHX-PIP-4, the amino acid sequence of which is shown in SEQ ID NO.1.
[0010] Specifically, the plasmin-inhibiting peptide YHX-PIP-4 is a peptide segment with plasmin-inhibiting ability screened from the amino acid sequence of β-lactoglobulin. This peptide segment is an 8-peptide sequence with the amino acid sequence of SEQ ID NO.1: CLVRTPEV, and is named plasmin-inhibiting peptide YHX-PIP-4.
[0011] Preferably, the plasmin inhibitory peptide YHX-PIP-4 is a water-soluble polypeptide; specifically, the plasmin inhibitory peptide YHX-PIP-4 is not only soluble in water, but also soluble in 0.01-0.1 M PBS (pH 5.5-8) and DMSO.
[0012] Preferably, the plasmin inhibitory peptide YHX-PIP-4 has a molecular weight of 916.10 Da.
[0013] Preferably, the plasmin inhibitory peptide YHX-PIP-4 is prepared by solid phase synthesis.
[0014] A use of the plasmin inhibitory peptide YHX-PIP-4 as described above in the preparation of a plasmin inhibitor.
[0015] Preferably, the plasmin inhibitory peptide YHX-PIP-4 is used to prepare a plasmin inhibitor.
[0016] A use of the plasmin inhibitory peptide YHX-PIP-4 as described above in food additives.
[0017] Preferably, the plasmin inhibitory peptide YHX-PIP-4 is used as a milk additive. Adding the plasmin inhibitory peptide YHX-PIP-4 to ultra-high temperature sterilized milk can inhibit the activity of plasmin and avoid gelation of the milk system.
[0018] Specifically, ultra-high temperature sterilized milk refers to milk that has been sterilized at ultra-high temperature. The ultra-high temperature sterilization method refers to the instantaneous sterilization of milk by ultra-high temperature instantaneous sterilization (135℃ to 150℃, 4 to 15 seconds), which completely destroys the microorganisms and spores that can grow in it. It is a relatively common sterilization method at present.
[0019] Preferably, the plasmin inhibitory peptide YHX-PIP-4 is used to increase the shelf life of ultra-high temperature sterilized dairy milk.
[0020] A use of the plasmin inhibitory peptide YHX-PIP-4 as described above in the preparation of hemostatic drugs.
[0021] The present invention provides a plasmin-inhibiting peptide YHX-PIP-4 derived from bovine milk protein and its application, which has the following beneficial effects:
[0022] 1. This study used bioinformatics techniques, molecular docking, and molecular dynamics simulation targeting to screen a plasmin-inhibiting peptide from the β-lactoglobulin sequence. This peptide was named plasmin-inhibiting peptide YHX-PIP-4.
[0023] 2. Studies have shown that the plasmin inhibitory peptide YHX-PIP-4 can inhibit the activity of plasmin. Adding it to ultra-high temperature milk can prevent the milk from precipitating and aging and gelling during storage, thereby extending its shelf life.
[0024] 3. Compared with traditional peptide development methods, the present invention's screening method for the plasmin-inhibiting peptide YHX-PIP-4 avoids the tedious steps of traditional peptide development, simplifies R&D costs, and the screened plasmin-inhibiting peptide YHX-PIP-4 still has strong plasmin inhibition ability in a real, complex milk system.
[0025] 4. The plasmin inhibitory peptide YHX-PIP-4 screened by the present invention is derived from β-lactoglobulin and has good safety. This peptide segment contains only 8 amino acids, has a short synthetic sequence, is easy to synthesize, and has a clear mechanism of action. It can be synthesized quickly, in large quantities and at low cost, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Schematic diagram of microthermophoresis (MST) of the binding of β-lactoglobulin and plasmin;
[0027] Figure 2 The interaction interface of the molecular docking complex after 200 ns molecular dynamics simulation equilibrium;
[0028] Figure 3 The inhibitory effect of peptide YHX-PIP-4 on plasmin;
[0029] Figure 4 The apparent changes of dUHT milk with or without YHX-PIP-4 after storage at 37°C for one week;
[0030] Figure 5 This is the effect of YHX-PIP-4 addition on the TSI value of dUHT milk stability. DETAILED DESCRIPTION
[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.
[0032] 1. Design principle of the present invention
[0033] 1. Research has shown that heating β-lactoglobulin can reduce the activity of plasmin. Further research has revealed that β-lactoglobulin, whether undenatured or heat-denatured, can act as a direct inhibitor of plasmin. This suggests that a natural plasmin inhibitor sequence exists within the β-lactoglobulin structure.
[0034] With the development and application of bioinformatics technology, it has become possible to study the interaction between proteins and enzymes at the amino acid level, which provides a basis for the targeted screening of peptides with plasmin inhibitory ability from the β-lactoglobulin sequence.
[0035] 2. In order to achieve the above object, the present invention adopts the following technical solutions:
[0036] (1) The binding of β-lactoglobulin to plasmin was verified using a microcalorimeter;
[0037] (2) The structural data files of plasmin and β-lactoglobulin were downloaded from the existing protein data;
[0038] (3) Molecular docking of β-lactoglobulin and plasmin was completed with the help of Z-DOCK (3.0.2) and Haddock (2.4), and the complex was subjected to 200 ns analytical dynamics simulation using Gromacs software to achieve equilibrium;
[0039] (4) synthesizing a candidate plasmin inhibitory peptide by a solid-phase chemical synthesis method, and verifying the candidate plasmin inhibitory peptide by an inhibitory activity experiment to obtain the novel plasmin inhibitory peptide;
[0040] (5) The polypeptide of the present invention was added to fresh sterilized milk to observe its effect on the shelf life of milk during storage, and the effect of the polypeptide on improving the storage stability of ultra-high temperature sterilized milk was verified using a stability analyzer.
[0041] 2. The present invention will be further described in detail below with reference to specific embodiments.
[0042] Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods in the prior art.
[0043] Example 1 Analysis of the Binding Ability of β-lactoglobulin and Plasmin Activity
[0044] Take 90μL of plasmin and mix it with 10μL of RED-NHS labeling-dye mixture, incubate it in the dark at room temperature for 30 minutes (enzyme concentration 2.7μM, dye concentration 10μM). Take 100μL of the labeled enzyme mixture and filter it through a gel column. After collecting, the enzyme is about 450μL. After diluting the plasmin, take 15μL and aspirate it with a capillary and place it on the Nanotemper instrument for detection. The instrument quality inspection shows that the fluorescence intensity is greater than 200 and there is no adsorption and aggregation. The maximum concentration of β-lactoglobulin is 200μM, and then a 2-fold concentration gradient dilution is performed in 16 tubes. After dilution, the solution in each tube is 10μL. Finally, 10μL of the diluted protein solution is added and mixed. Aspirate it with a capillary and place it on the Nanotemper instrument for detection (the protein concentration on the machine is 100nM). The results are as follows Figure 1 The equilibrium dissociation constant of β-lactoglobulin and plasmin is Kd(M):3.69×10 -5 .
[0045] Example 2 Molecular Docking and Molecular Dynamics Simulation of β-lactoglobulin and Plasmin
[0046] The structural files of plasmin and β-lactoglobulin were obtained from the AlphaFoldprotein protein structure database (E1B726) and the RCSBProteinDataBank protein database (5IO6). Before molecular docking, the amino acid 585-812 portion of the plasminogen sequence was selected from the plasminogen structure and used as a template for the interaction between plasmin and β-lactoglobulin.
[0047] Plasmin and β-lactoglobulin were rigidly docked using Z-DOCK (3.0.2) software. PDBePISA (https: / / www.ebi.ac.uk / pdbe / pisa / ) was used to preliminarily analyze the binding energy and interacting amino acids in the docking results. The docking model with the lowest Gibbs free energy was selected and then submitted to Haddock for further flexible docking. The complex structure with the highest score was selected. Molecular dynamics simulations of the complex were performed for 200 ns at 300 K using Gromacs software to ensure that the β-lactoglobulin and plasmin complex reached a realistic equilibrium state.
[0048] Example 3 Screening of potential plasmin inhibitory peptides based on β-lactoglobulin sequence
[0049] The β-lactoglobulin and plasmin complex at equilibrium Figure 2As shown, the plasmin active site is a triplet consisting of 624HIS, 667ASP, and 762SER in the amino acid sequence. In the equilibrium β-lactoglobulin-plasmin complex, the plasmin active site is partially covered by the β-lactoglobulin amino acid sequence, suggesting that this peptide segment may play a role in competitively inhibiting plasmin activity.
[0050] The conservative sequence distribution characteristics of existing plasmin inhibitory peptides were analyzed by sequence alignment. It was concluded that the amino acid sequence of plasmin inhibitors at the P1 site is mainly lysine (Lys) and a small part of arginine (Arg). Combined with the amino acid sequence of β-lactoglobulin covering the active site of plasmin, an octapeptide sequence was screened as a potential plasmin inhibitory peptide CLVRTPEV.
[0051] Example 4 Peptide Synthesis and Activity Verification
[0052] The screened peptide CLVRTPEV was synthesized using peptide solid-phase synthesis technology. After testing, the peptide had good water solubility. The effect of the peptide CLVRTPEV on the activity of plasmin was measured using the substrate colorimetric method. 0.01M phosphate buffer (pH 7.4) was configured into a peptide solution with a concentration of 1 mg / mL. 50 μL of the peptide solution was mixed with 10 μL of 0.1 U / mL plasmin, followed by the addition of 140 μL of 0.1 mM D-Val-Leu-Lys p-nitroaniline dihydrochloride. The absorbance at 405 nm was immediately measured using an enzyme reader. After shaking for three seconds, the absorbance was measured every 5 minutes, and the temperature was maintained at 37°C. In the control group sample, the peptide solution was replaced with phosphate buffer and the absorbance at 405 nm was measured.
[0053] like Figure 3 As shown, the addition of polypeptide YHX-PIP-4 can reduce the ability of plasmin to hydrolyze the substrate, which indicates that the polypeptide CLVRTPEV extracted from β-lactoglobulin has the function of inhibiting the activity of plasmin.
[0054] Example 5 Application of plasmin inhibitory peptide in improving storage stability of dUHT milk
[0055] Fresh direct ultra-high temperature (dUHT) sterilized milk was heat treated at 153°C for 0.25 seconds. Fresh dUHT milk was added with 300 μL of 8 mM peptide YHX-PIP-4 and stored at 37°C in the dark to simulate the accelerated storage process. Blank milk was also used as a control. After one week of storage, Figure 4The milk system with YHX-PIP-4 showed no significant changes in appearance, while the control group showed significant precipitation and stratification. This indicates that YHX-PIP-4 still has strong plasminogen activator inhibitory ability in the milk system, thereby increasing the storage stability of sterilized milk and extending its shelf life.
[0056] The stability of the samples was further analyzed using the Formulaaction-Turbiscan Tower stability analyzer. Figure 5 As shown in the figure, after one week of storage, the TSI index of the dUHT milk in the control group increased from 15 to 35, while the TSI index of the milk added with the peptide YHX-PIP-4 slowly increased from 5 to 10 within 24 hours. The larger the TSI, the more unstable the system. The above results indicate that the peptide YHX-PIP-4 of the present invention can effectively inhibit the activity of plasmin in dUHT milk and extend the shelf life of dUHT milk.
[0057] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.
Claims
1. A use of a plasmin inhibitory peptide YHX-PIP-4 in a food additive, characterized in that: The plasmin inhibitory peptide YHX-PIP-4 is used as a milk additive. Adding the plasmin inhibitory peptide YHX-PIP-4 to ultra-high temperature sterilized milk can inhibit the activity of plasmin and prevent the milk system from gelling. The amino acid sequence of the plasmin inhibitory peptide YHX-PIP-4 is shown in SEQ ID NO.
1.
2. The use of the plasmin inhibitory peptide YHX-PIP-4 in food additives according to claim 1, characterized in that: The plasmin inhibitory peptide YHX-PIP-4 is used for improving the shelf life of ultra-high temperature sterilized dairy milk.
3. Use of a plasmin inhibitory peptide YHX-PIP-4 in the preparation of a hemostatic drug, characterized in that: The amino acid sequence of the plasmin inhibitory peptide YHX-PIP-4 is shown in SEQ ID NO.1.