β-lactoglobulin-derived plasmin inhibitory peptides, their screening methods, and applications

Abstract

This invention belongs to the field of bioactive peptide technology, and relates to a β-lactoglobulin-derived plasmin inhibitory peptide, its screening method, and its application. The amino acid sequence of the plasmin inhibitory peptide is shown in SEQ ID NO.1. The screening method involves selecting β-lactoglobulin from bovine milk as the source of the plasmin inhibitory peptide. After preliminary verification that β-lactoglobulin has the ability to inhibit plasmin, molecular docking is performed on β-lactoglobulin and plasmin. The complex is then subjected to molecular dynamics simulation to equilibrium. Based on the interaction between the active site of plasmin and the β-lactoglobulin sequence, targeted screening is conducted on polypeptide sequences in the β-lactoglobulin sequence that may have inhibitory activity. This method solves the technical defects in the current preparation process of plasmin inhibitory peptides, such as the scarcity of raw materials, complex processes, and difficulty in large-scale production. This plasmin inhibitory peptide increases the stability of sterilized milk and extends its shelf life, showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a β-lactoglobulin-derived plasmin inhibitory peptide, its screening method, and its application. Background Technology

[0002] Plasmin is an alkaline serine protease found in milk. This enzyme is heat-resistant and can survive most ultra-high temperature sterilization processes. Therefore, in direct ultra-high temperature (dUHT) sterilized milk, the surviving plasmin can cause hydrolysis of milk proteins, resulting in undesirable precipitation and aging gels in the product, which adversely affects the shelf life and flavor of the milk.

[0003] Currently, most existing plasmin inhibitory peptides are derived from naturally occurring polypeptides in animal venom, obtained through isolation and purification. The problem lies in the difficulty and high cost of obtaining these inhibitory peptides from readily available raw materials, and the complex preparation process easily leads to loss of peptide activity, resulting in generally low yields, which is detrimental to subsequent applications and research. Furthermore, the plasmin inhibitory peptides obtained by this method typically have sequences longer than 50 amino acids, containing one or more Kunitz or Kazal-type domains with disulfide bonds, making rapid industrial-scale production difficult. Therefore, there is an urgent need for a method to obtain plasmin inhibitory peptides from common raw materials, thereby obtaining plasmin inhibitory peptides that are easy to synthesize rapidly.

[0004] β-lactoglobulin is the most abundant protein in bovine milk whey and a natural byproduct of cheese production. β-lactoglobulin is readily available, and research has shown that it possesses plasmin inhibitory activity. Therefore, this invention utilizes β-lactoglobulin to prepare plasmin inhibitory peptides, which has significant potential and economic value. Summary of the Invention

[0005] To address the shortcomings and problems of existing technologies in the preparation of plasmin inhibitory peptides, such as complex and time-consuming processes, difficult-to-obtain and expensive raw materials, low yields, and complex product structures that are difficult to synthesize, this invention proposes a β-lactoglobulin-derived plasmin inhibitory peptide, its screening method, and its application. Utilizing bioinformatics processing methods based on molecular docking and molecular dynamics simulations, plasmin inhibitory peptides are screened from the amino acid sequence of β-lactoglobulin, and their activity is verified through artificial synthesis. This yields plasmin inhibitory peptides with shorter peptide segments, which can be prepared in large quantities and applied to increase the stability of sterilized milk.

[0006] The technical solution of this invention is:

[0007] This invention protects a β-lactoglobulin-derived plasmin inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] This invention also protects a method for screening β-lactoglobulin-derived plasmin inhibitory peptides, comprising the following steps:

[0009] (1) Obtain the protein structures of plasmin and β-lactoglobulin respectively, complete the molecular docking of plasmin and β-lactoglobulin, and perform a 200 ns molecular dynamics simulation of the β-lactoglobulin and plasmin complex;

[0010] (2) Analyze the interaction interface between β-lactoglobulin and plasmin, and screen for polypeptide sequences in the β-lactoglobulin sequence that bind to the active site of plasmin; among them, the active site of plasmin is a triplet composed of 624HIS, 667ASP and 762SER.

[0011] (3) Based on the characteristic that the sequence of the plasmin inhibitory peptide is mainly lysine and a small amount of arginine at the P1 site, and combined with the amino acid sequence of β-lactoglobulin covering the plasmin active site, a suitable polypeptide is selected as the target peptide segment, and the plasmin inhibitory peptide is synthesized according to the sequence.

[0012] Furthermore, the plasmin inhibitory peptide is chemically synthesized by solid-phase synthesis and has a purity of ≥98%.

[0013] Furthermore, the plasmin inhibitory peptide comprises 8 amino acid residues and has a molecular weight of 905.08 Da. This plasmin inhibitory peptide is a small molecule peptide, soluble in 0.01–0.1 M PBS (pH 5.5–8) and DMSO.

[0014] This invention also protects the use of the β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of plasminase inhibitors. Furthermore, this invention protects the use of the plasmin inhibitory peptide or any combination thereof in the preparation of plasminase inhibitors.

[0015] The application of the β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of food additives.

[0016] Furthermore, the food additive is used in dairy products.

[0017] A food additive whose active ingredient includes the plasmin inhibitory peptide as described in claim 1.

[0018] The application of the β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of hemostatic drugs.

[0019] To screen for and prepare β-lactoglobulin-derived plasmin inhibitory peptides, this invention first demonstrated through enzyme activity mechanics experiments that β-lactoglobulin can inhibit plasmin activity; structural data files of plasmin and β-lactoglobulin were obtained using a protein database; then, molecular docking of β-lactoglobulin and plasmin was performed using Z-DOCK (3.0.2) and Haddock (2.4), and molecular dynamics simulations of the β-lactoglobulin-plasmin complex were conducted using the GROMACS software package for 200 ns; by analyzing the interaction interface between β-lactoglobulin and plasmin, polypeptide sequences in the β-lactoglobulin sequence that bind to the plasmin active site were screened. Based on the characteristics of plasmin inhibitory peptides, suitable polypeptides were selected as candidate plasmin inhibitory peptides. After obtaining the target peptide, the candidate plasmin inhibitory peptide was synthesized using a solid-phase chemical synthesis method, and the activity of the plasmin inhibitory peptide was verified by inhibition activity experiments, yielding the desired plasmin inhibitory peptide QTMKGLDI.

[0020] The synthesized plasmin inhibitory peptide QTMKGLDI was added to fresh direct ultra-high temperature (dUHT) sterilized milk, and the stability of the milk during storage was observed to confirm that the plasmin inhibitory peptide can extend the shelf life of dUHT milk.

[0021] The beneficial effects of this invention are:

[0022] (1) This invention is the first to conduct research on plasmin inhibitory peptides derived from β-lactoglobulin and has prepared and obtained one plasmin inhibitory peptide. This invention uses molecular docking technology and molecular dynamics simulation technology to screen plasmin inhibitory peptides. Based on the characteristics of the binding of polypeptide sequences to the active sites of plasmin, it efficiently targets and screens plasmin inhibitory peptides from β-lactoglobulin sequences, avoiding the randomness of traditional polypeptide separation. The peptides obtained by this method are short and can be prepared in large quantities by solid-phase chemical synthesis, with lower cost.

[0023] (2) This invention provides a novel rapid screening method for plasmin inhibitory peptides derived from β-lactoglobulin. This method selects β-lactoglobulin from bovine milk as the source of plasmin inhibitory peptides. After initially verifying that β-lactoglobulin has the ability to inhibit plasmin, molecular docking is performed on β-lactoglobulin and plasmin, and molecular dynamics simulation is performed on the complex until equilibrium is reached. Based on the interaction between the active site of plasmin and the β-lactoglobulin sequence, targeted screening is performed on polypeptide sequences in the β-lactoglobulin sequence that may have inhibitory activity. This solves the technical defects of the current plasmin inhibitory peptide preparation process, such as the scarcity of raw materials, the complexity of the process, and the difficulty in large-scale production.

[0024] (3) The present invention verified the inhibitory ability of the screened peptide through solid-phase synthesis and successfully obtained a QTMKGLDI plasmin inhibitory peptide. This peptide increases the stability of sterilized milk, avoids the occurrence of layering and aging gel, and can extend its shelf life, thereby extending the shelf life of sterilized milk, and has good application prospects. Attached Figure Description

[0025] Figure 1 The results show the inhibition of plasmin activity by β-lactoglobulin;

[0026] Figure 2 This is a schematic diagram of the molecular docking and interaction interface between β-lactoglobulin and plasmin.

[0027] Figure 3 This is a schematic diagram illustrating the inhibitory effect of peptide YHX-PIP-1 on plasmin.

[0028] Figure 4 The apparent changes of dUHT milk stored at 37°C with and without YHX-PIP-1 for one week;

[0029] Figure 5 The effect of YHX-PIP-1 on the stability of dUHT milk. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0032] Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods that already exist in the prior art.

[0033] Example 1: Inhibition of plasmin activity by β-lactoglobulin

[0034] The effect of β-lactoglobulin on plasmin activity was determined using a substrate-based colorimetric method. β-lactoglobulin was dissolved in 0.01 M phosphate buffer (pH 7.4) to prepare a protein solution with a concentration of 16 mg / mL. 50 μL of this protein 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 was immediately measured at 405 nm using a microplate reader. After shaking for three seconds, measurements were taken every 5 minutes, maintaining the temperature at 37 °C.

[0035] A blank control group was set up. The blank control group samples differed from the β-lactoglobulin group samples in that the protein solution was replaced with phosphate buffer. The absorbance at 405 nm was also measured. The results are as follows: Figure 1 As shown, β-lactoglobulin inhibits the activity of plasmin, indicating that β-lactoglobulin can be used as a raw material for extracting plasmin inhibitory peptides.

[0036] Example 2: Molecular docking and molecular dynamics simulation of β-lactoglobulin and plasmin

[0037] The plasmin protein structure data file (E1B726) and the β-lactoglobulin structure file (5IO6) were obtained from the AlphaFold protein structure database and the RCSB Protein DataBank protein database, respectively. In this invention, the light chain (amino acid sequence 585-812) containing the active site in the plasminogen structure was selected as the template for the interaction between β-lactoglobulin and plasmin. The protein structures of plasmin and β-lactoglobulin were extracted from the files, and rigid docking was performed using Z-DOCK (3.0.2) software.

[0038] Preliminary analysis of binding energy and interacting amino acids in the docking results was performed using PDBePISA. The docking model with the lowest Gibbs free energy (ΔG = -4.7 kcal / mol in this method) was selected and then submitted to Haddock for further flexible docking. The model with the highest score in the docking results was then selected as the object for molecular dynamics simulation. Molecular dynamics simulation aims to bring the β-lactoglobulin and plasmin complex into equilibrium, which is more realistic. A 200 ns molecular dynamics simulation of the β-lactoglobulin and plasmin complex was performed using Gromacs software, employing the amber99sb-ildn force field and selecting the tip3p water model to dissolve the complex in a dodecahedral box. 0.1 mol / L Na₂ was randomly inserted. + and Cl -Ions were used to neutralize the system's charge, followed by energy minimization. A 200 ps NVT and 200 ps NPT-bound pre-equilibration was then performed to bring the equilibrium temperature to 298 K. A Berendsen thermostat and Parrinello-Rahman pressure were used to maintain the temperature and pressure, respectively. All nonbonded interactions, including Coulomb and van der Waals potentials, were represented using... Verlet cutoff mode. Electrostatic interactions are handled using the Particle Mesh Ewald (PME) summation method. Finally, position constraints are removed, and the system is simulated with a trajectory time of 200 ns, yielding the following results: Figure 2 The structure of the β-lactoglobulin and plasmin complex in equilibrium is shown.

[0039] Example 3: Screening for potential plasmin inhibitory peptides based on β-lactoglobulin sequences

[0040] Plasmin, as a serine protease, has an active site consisting of a triplet of 624HIS, 667ASP, and 762SER. For example... Figure 2 As shown, after β-lactoglobulin binds to plasmin, the active site of plasmin is partially covered by the β-lactoglobulin sequence. Analysis of existing plasmin inhibitory peptide sequences revealed that they are predominantly lysine (Lys) and contain a small amount of arginine (Arg) at the P1 site. Combined with the β-lactoglobulin amino acid sequence covering the plasmin active site, an octapeptide sequence was screened as a potential plasmin inhibitory peptide, YHX-PIP-1, with the amino acid sequence QTMKGLDI (SEQ ID NO.1).

[0041] Example 4: Peptide Synthesis and Activity Verification

[0042] The plasmin inhibitory peptide YHX-PIP-1 was synthesized using a solid-phase peptide synthesis technique, and its activity was verified. The activity of the peptide was verified using the substrate colorimetric method described in Example 1, with the peptide concentration set at 1 mg / mL. A blank control group was set up, and the results are as follows. Figure 3 As shown, the peptide QTMKGLDI(YHX-PIP-1) has plasmin inhibitory activity.

[0043] Example 5: Application of plasmin inhibitory peptide in improving the stability of dUHT milk

[0044] Fresh, direct ultra-high temperature (dUHT) sterilized milk was heat-treated at 153°C for 0.25 s. 23 mL of fresh dUHT milk was added to a dedicated transparent glass vial for Turbiscan stability analysis and sterilized before use. Subsequently, 300 μL of plasmin inhibitor YHX-PIP-1 (0.01 M PBS, pH 7.4) at a peptide concentration of 8 mM was added to the vial. A blank control group was also included. The samples were stored at 37°C for one week, and the stability changes were analyzed using a Formulaction multiple light scattering analyzer.

[0045] like Figure 4 As shown, after a week of storage, the dUHT milk in the control group showed visible precipitation and stratification, which was mainly caused by the hydrolysis of casein by plasmin. In contrast, the milk containing the plasmin inhibitory peptide YHX-PIP-1 (i.e., peptide QTMKGLDI) was evenly dispersed and did not show precipitation or stratification.

[0046] like Figure 5 As shown, a higher TSI index indicates poorer stability. After one week of storage, a stability analysis was performed for 0-24 hours. The TSI index of the blank group increased from 15 to 35, while the TSI index of the milk with added plasmin inhibitory peptide YHX-PIP-1 was approximately 5. This indicates that the peptide QTMKGLDI of the present invention can effectively inhibit plasmin activity, thereby extending the shelf life of dUHT milk.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of food additives, characterized in that, The amino acid sequence of the β-lactoglobulin-derived plasmin inhibitor peptide is shown in SEQ ID NO.

1. The food additive is used in direct ultra-high temperature sterilized milk to extend the shelf life of direct ultra-high temperature sterilized milk.

2. The application of a β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of hemostatic drugs, characterized in that, The amino acid sequence of the β-lactoglobulin-derived plasmin inhibitory peptide is shown in SEQ ID NO.1.

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