Bacterial-source ACE inhibitory peptide and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a bacterial-derived ACE inhibitory peptide and application thereof. The ACE inhibitor is a novel peptide, is named as PVQRFF, has remarkable ACE inhibitory activity, and can effectively reduce the level of angiotensin II so as to achieve the purpose of controlling blood pressure. The PVQRFF has the potential application value in hypertension prevention and treatment by reducing blood pressure, improving blood circulation and protecting the cardiovascular system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a bacterial-derived ACE inhibitory peptide and its application. Background Art

[0002] Hypertension, as a prevalent chronic disease globally, has become one of the important factors affecting public health. Its incidence has been increasing year by year. Due to its potential serious complications such as heart disease, stroke, renal failure, etc., it has led to a significant social and economic burden. Traditional treatment methods include lifestyle changes and drug interventions. Among them, ACE (angiotensin-converting enzyme) inhibitors are commonly used drugs in antihypertensive treatment. These drugs inhibit the activity of ACE, reduce the level of angiotensin II in the blood, and achieve the effects of vasodilation and blood pressure reduction.

[0003] However, existing ACE inhibitors such as enalapril and lisinopril still have many side effects, including but not limited to dry cough, hyperkalemia, dizziness, etc. These side effects have limited their use to a certain extent and increased the compliance problems of patients. Therefore, there is an urgent need to explore new antihypertensive drugs to reduce side effects and improve the quality of life of patients. In recent years, studies have shown that natural-derived peptides exhibit good biological activities in ACE inhibition. These peptides can be extracted from natural resources such as foods (such as dairy products, fish, etc.), plants, and microorganisms. Among them, bacterial-derived peptides have received increasing attention due to their rich variety, strong activity, low cost, etc. By screening and optimizing specific bacterial strains, effective ACE inhibitory peptides can be obtained, thus providing a new treatment option for hypertension.

[0004] The current technological development in this field is still insufficient, especially in the discovery and application of highly efficient, safe and available bacterial-derived ACE inhibitory peptides. Therefore, in response to this need, the present invention aims to develop a new type of bacterial-derived ACE inhibitory peptide. We will extract the peptide components from the selected specific bacterial strains and verify their ACE inhibitory activity through experiments. At the same time, we will study its biosafety and effectiveness, with a view to providing a new, safe and efficient intervention means for the treatment of hypertension and related diseases. This research not only has the potential to provide a theoretical basis for new drug development, but also opens up new directions for research in related fields. It is hoped that with the help of advanced separation and purification technologies and bioactivity analysis methods, new health options can be brought to hypertension patients. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a bacterial-derived ACE inhibitory peptide and its applications, aiming to provide small polypeptide products with strong ACE inhibitory activity for the market, and further lay a certain technical foundation for its research and application in the development of drugs for preventing or treating hypertension, the food industry, the health product industry, and functional supplements.

[0006] On the one hand, the present invention provides an ACE inhibitory peptide named PVQRFF. The amino acid sequence of the ACE inhibitory peptide PVQRFF is Pro-Val-Gln-Arg-Phe-Phe (SEQ ID NO.1: PVQRFF), which is a novel peptide with a novel sequence. Its molecular weight is 705.82 g / mol, and its molecular formula is C 31 H 45 N 7 O 8 。

[0007] In one aspect of the present invention, the nucleic acid sequence of the ACE inhibitory peptide PVQRFF is provided, as shown in SEQ ID NO.2.

[0008] SEQ ID NO.2: CCAGUUCAAAGAUUUUUU.

[0009] On the other hand, the present invention also provides a method for preparing ACE inhibitory peptide by hydrolyzing black bean protein using a strain combined with alkaline protease, which is characterized by including the following steps: (1) Mix and ferment alkaline protease, the bacterial solution of the strain, and black bean protein solution to obtain a fermentation broth; (2) After inactivating the enzyme and adjusting the pH value of the fermentation broth, take the supernatant; (3) After ultrafiltration of the supernatant through an ultrafiltration membrane, take the ultrafiltrate with high ACE inhibitory activity, and perform separation and identification to obtain the ACE inhibitory peptide.

[0010] The present invention also provides an application of the ACE inhibitory peptide PVQRFF in the preparation of antihypertensive drugs.

[0011] Beneficial effects: (1) The present invention prepares ACE inhibitory peptide by combining lactic acid bacteria fermentation and alkaline protease hydrolysis, aiming to shorten the cycle of the fermentation and hydrolysis processes, and at the same time screen out effective ACE inhibitory peptides with high activity. We selected the high-yield potential strain Lactobacillus fermentum FJ-113 screened in the laboratory, and used it together with alkaline protease to construct a co-fermentation hydrolysis process. Further separate and purify the hydrolysis product, and finally identify a novel ACE inhibitory peptide.

[0012] (2) Different from the prior art, the ACE inhibitory peptide PVQRFF provided by the above technical solution is composed of 6 amino acids, with the amino acid sequence of Pro-Val-Gln-Arg-Phe-Phe, a molecular weight of 705.82 g / mol, and the molecular formula of C 31 H 45 N 7 O 8 . It has high ACE inhibitory activity, no cytotoxicity, good digestive stability and cytoprotective effect. The ACE inhibitory peptide PVQRFF provided by the above technical solution is expected to play a positive role in the production and preparation of drugs for preventing or treating hypertension and health foods for assisting in reducing blood pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not constitute an improper limitation to the invention.

[0014] Figure 1 is the inhibition rate of ACE of each component; Figure 2 is the molecular docking diagram of the ACE inhibitory peptide provided by the specific embodiment of the present invention; Figure 3 is the result of the cytotoxicity test of the ACE inhibitory peptide under different concentration conditions in the specific embodiment of the present invention; Figure 4 is the result of the test on the protective effect of the ACE inhibitory peptide under different concentration conditions on AngII-induced damaged HUVEC cells in the specific embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] The embodiments of the present invention will be described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0016] In the following embodiments, the chemical reagents used are all purchased from regular chemical reagent suppliers, and the purity is analytical pure.

[0017] Example 1 Fermented Lactobacillus combined with enzymatic fermentation to hydrolyze black bean protein solution Prepare a 5% (m / v) black bean protein solution using PBS buffer (pH 7.0, 0.05 mo1 / L), add a certain amount of 1 mo1 / L NaOH to pH 7.0, heat in a boiling water bath for 15 min, and after it is fully dissolved, sterilize it by high-pressure steam (115 °C, 15 min) to obtain a 5% (m / v) sterile black bean protein solution.

[0018] Inoculate 5 (v / v)% Lactobacillus fermentum into the sterile black bean protein solution Lactobacillus fermentum bacterial solution FJ-113 (1.0x10 9 CFU / mL), and ferment at 37 °C for 6 h, 10 h, and 14 h respectively, and calculate the degree of hydrolysis and ACE inhibitory rate. The results show that the degree of hydrolysis after 14 h of fermentation is 10.56%, and the ACE inhibitory rate is 55.39%. It indicates that Lactobacillus fermentum bacterial solution FJ-113 has high activity in hydrolyzing black bean protein.

[0019] Add a single protease (alkaline protease, flavor protease, papain, trypsin or neutral protease) to the 5% sterile black bean protein solution at an addition amount of 1 g:200 mL of enzyme to substrate ratio, and then inoculate 5% (v / v) Lactobacillus fermentum FJ-113. After fermenting at 37 °C for 14 h, measure the ACE inhibitory rate, degree of hydrolysis, peptide content and pH of each group. By comprehensively comparing various indexes, determine the best protease for co-fermentation hydrolysis with Lactobacillus fermentum FJ-113. It is found that Lactobacillus fermentum FJ-113 has the best hydrolysis effect when combined with alkaline protease for hydrolysis.

[0020] Through single-factor experiments and orthogonal experiments, determine that the addition amount of alkaline protease is 1 g:200 mL, the inoculation amount of Lactobacillus fermentum FJ-113 is 5% (v / v), the enzymolysis time is 14 h, and the substrate pH is 7.0. Under this condition, the ACE inhibitory rate is increased to 65.24%.

[0021] Example 2 Preparation of mixed polypeptides Collect the fermentation broth with an ACE inhibitory rate of 65.24%, and place it in a water bath at 90 °C for 10 min to inactivate the enzyme. After adjusting the pH to 8.3, directly freeze-dry a part to obtain a freeze-dried sample for standby. Another part is centrifuged at 4 °C and 8000 rpm / min for 15 min, and the supernatant is taken for standby.

[0022] Weigh a certain amount of freeze-dried sample and dissolve it with deionized water to prepare solutions with concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL respectively, and draw the ACE inhibition rate curve of the sample at different concentrations. Ultrafiltration is carried out using 10KDa and 3KDa filter membranes, and the supernatant is separated and purified into 3 different components (i.e., M3>10KDa, 3KDa<M2<10KDa, M1<3KDa). Specifically, take 10 mL of the supernatant and place it in a 10KDa ultrafiltration tube, centrifuge and ultrafilter at 4°C and 3500 rpm / min for 20 min, take the ultrafiltrate with a molecular weight less than 10KDa, and continue centrifugal ultrafiltration with a 3KDa ultrafiltration tube under the same conditions. Ultrafiltrates with molecular weights greater than 10KDa (component M3), less than 10KDa and greater than 3KDa (component M2), and less than 3KDa (component M1) are obtained respectively. Collect the filtrates of each component, rotary evaporate to about 50 ml and then freeze-dry. It is measured that the ACE inhibitory activity of component M1 is the highest, reaching 73.15%, which is significantly lower than that of the crude enzyme solution, component M2 and M3 (P<0.001).

[0023] Component M1 was separated and purified by Sephadex G-15 chromatography. Take a certain amount of Sephadex G-15 powder and put it into five times distilled water, expand it at room temperature for 3 h to fully swell G-15, and pour off the suspended gel particles. After the chromatography column is fixed, slowly pour the pretreated gel into the column along the upper port of the column, and let it naturally precipitate to two-thirds of the column height in the column. Before injection, it is necessary to continuously balance the gel with distilled water until there are no bubbles and no stratification in the gel before injection can be carried out. Sample loading volume: 1 mL, eluent: distilled water, sample loading concentration: 200 mg / mL, flow rate: 1 mL / min, UV detection wavelength of AKTA protein chromatography system: 280 nm. Collect the peak components to obtain three components G1, G2, and G3. After freeze-drying, the ACE inhibition rate of each component is measured. As Figure 1 shown, when the concentration is 1 mg / mL, the ACE inhibition rate of component G2 is the highest, which is 74.10%, and its inhibitory activity is significantly higher than that of other components.

[0024] Component G2 was identified by liquid chromatography, tandem mass spectrometry analysis and de novo sequencing to obtain 3512 peptide segments, among which hexapeptides were the most prominent, with a total of 1231. The amino acid distribution shows that the number of peptide segments containing hydrophobic amino acids in component G2 reaches more than 90%, and the peptide segments with hydrophobic amino acid content in the range of 45-70% are the most abundant, accounting for 45.9% of the total number of peptide segments, and are mostly located at both ends of the polypeptide. Since the content and position of hydrophobic amino acids in ACE inhibitory peptides are closely related to their activity, these polypeptides rich in hydrophobic amino acids significantly enhance the activity of component G2.

[0025] Example 3 Screening of polypeptides inhibiting ACE activity in combination with a database Screen the polypeptides obtained by sequencing from the following six dimensions: Confidence level: From the polypeptide sequences identified by LC-MS / MS, select peptide segments with a confidence level ALC>80% (relatively reliable); Polypeptide content: The peak area of the peptide segment can reflect its content. From the peptide segments with a confidence level>80%, select peptide segments with a peak area Area>10e5; Bioactivity score: Predict the peptide segments with Area>10×e5 on the PeptideRanker website, and select peptide segments with PeptideRanker>0.5; Water solubility prediction: For the peptide segments that may have biological activity (PeptideRanker>0.5) obtained by prediction, perform water solubility prediction in Proteomics tools under the INNOVAGEN website, and select peptide segments with "Good" water solubility; Toxicity prediction: Perform peptide segment toxicity prediction on the peptide segments with good water solubility obtained by screening in the "BatchSubmission" module of the Toixinpred website, and select peptide segments with "Non-Toxin"; Allergy prediction: Perform allergy prediction on the non-toxic peptide segments obtained by screening in the "AllerTOPServerPage", and select peptide segments with "NON-ALLERGEN"; Example 4 Screening of ACE inhibitory peptides by molecular docking Nine peptide segments were screened from the above six dimensions, and further screened for the peptide segment with the strongest potential for binding to ACE by molecular docking. The specific experimental steps are as follows: Pretreatment of macromolecular receptor: Download the three-dimensional structure of the X-ray diffraction protein of the human ACE crystal complex 1O8A (https: / / www.rcsb.org / structure / 1O8A) from the PDB database (http: / / www.rcsb.org), and use Autodocktools 1.5.6 to process the protein structure: remove water, delete the ligand, then add hydrogen, calculate the charge, combine non-polar hydrogen, and further optimize the three-dimensional structure of the protein. After processing, export it in PDB format.

[0026] Pretreatment of small molecule ligand: Import the peptide segments to be docked on the website (https: / / cloud.yinfotek.com / ), process the small molecule structure in Autodocktools 1.5.6, add all hydrogen, automatically distribute the charge, and detect the torsion bond. After processing, export it in PDB format.

[0027] Docking and analysis: Set the position of Zn in the crystal structure of 1O8A as the active site, and set the central coordinates of the active pocket site as (40.586, 39.831, 43.427). The number of grid points in each of the XYZ directions is set to 50×50×50, the grid point spacing is set, the number of docking times is set to 100, and the remaining parameters are set to default values. Select the peptide segments with a binding energy to the 1O8A crystal higher than that of the positive control captopril (ACE inhibitor), and use Pymol 2.3.0 to analyze and visualize the interaction mode between the receptor and the ligand. Two peptide segments with good binding to ACE were screened out from the molecular docking calculation of the binding energy, namely PVQRFF and QPVPFQ, and their docking binding energies to ACE are -12.3 and -10.5 kcal / mol respectively. 2+ The specific visualized docking results are as follows

[0028] as Figure 2 shown. Among them, hydrogen bonds, π-π interactions, etc. are formed between the polypeptide PVQRFF and the residues Arg402, Ser517, and Arg522 at the active site of ACE, and it can bind to the active site of ACE relatively stably, making it unable to bind to the angiotensin precursor and catalyze its conversion into angiotensin, thereby leading to a decrease in ACE activity.

[0029] Example 5 Cytotoxicity study of ACE inhibitory peptide SPFW Entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize the ACE inhibitory peptide PVQRFF by solid-phase chemical synthesis method, and the purity is above 95%.

[0030] The CCK-8 method was used to measure cell viability. HUVECs in good growth state were seeded at 5x10 3 cells, 100 μL per well in a 96-well plate. After growing for 24 h, 10 μL of active peptide with different concentrations was added to the wells, and the cells were continuously cultured in an incubator for 24 h. Then 10 μL of CCK-8 (1 mg / mL) was added to each well and treated for 2 h, and the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay reader. As shown in the results Figure 3 , the test results show that when PVQRFF is 20 ng / mL, 40 ng / mL, and 80 ng / mL, the cell viability has no significant difference from the blank control, indicating that the ACE inhibitory peptide SPFW has no toxicity to HUVEC cells.

[0031] Example 6 Study on the protective effect of ACE inhibitory peptide PVQRFF on AngII-induced damaged human umbilical vein endothelial cells (HUVECs) After treatment with 2 μg / mL captopril (positive control) and different concentrations of SPFW (20 ng / mL, 40 ng / mL, and 80 ng / mL) for 1 h, 1 μM AngII was used to treat for 24 h. The method for cell viability determination was as follows: HUVECs were seeded at 5x10 3 cells, 100 μL per well, in a 96-well plate. After growing for 24 h, 10 μL of 1 mg / mL CCK-8 was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader. As Figure 4 shown by the experimental results of the protective effect of the ACE inhibitory peptide PVQRFF on AngII-induced HUVEC damage cells under different concentration conditions. The experimental results showed that compared with the damaged cell group, different concentrations of PVQRFF significantly increased the cell viability of damaged HUVEC cells, and the effect of increasing cell viability was significantly better than that of the captopril positive control group.

[0032] The present invention provides a novel ACE inhibitory peptide named PVQRFF. This peptide not only exhibits good ACE inhibitory activity but also has the characteristic of being non-toxic, making it have broad application potential in the biomedical field. It has been found that PVQRFF can significantly increase the cell viability of damaged human umbilical vein endothelial cells (HUVECs). This provides strong support for the development of new hypertension treatment methods and also lays a foundation for the research and development of related health products and functional foods. Through the optimized molecular structure, the biological activity of PVQRFF can effectively promote cell repair and growth, demonstrating its prospect as a potential drug ingredient.

Claims

1. An ACE inhibitory peptide, named PVQRFF, whose amino acid sequence is shown in SEQ ID NO.

1.

2. The nucleotide sequence corresponding to the ACE inhibitory peptide according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO.

2.

3. Use of the ACE inhibitory peptide according to claim 1 in the preparation of antihypertensive drugs.

4. A method for preparing the ACE inhibitory peptide according to any one of claims 1 to 3 by using a strain in combination with alkaline protease to hydrolyze black bean protein, characterized in that: The following steps are involved: (1) mixing alkaline protease, bacterial liquid of the strain and black bean protein solution for fermentation to obtain a fermentation liquid; (2) treating the fermentation broth to inactivate the enzyme, adjusting the pH value, and taking out the supernatant; (3) The supernatant is subjected to ultrafiltration using an ultrafiltration membrane, and the ultrafiltrate with high ACE inhibitory activity is taken out for separation and identification to obtain ACE inhibitory peptides.

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