Bacterial-derived ace inhibitory peptide and use thereof

Abstract

The application belongs to the field of medicine, and particularly relates to a bacterial source ACE inhibiting peptide and application thereof. The ACE inhibitor is a novel peptide, named PVQRFF, has significant ACE inhibiting activity, can effectively reduce the level of angiotensin II, so as to achieve the purpose of controlling blood pressure. PVQRFF can reduce blood pressure, improve blood circulation, and has a protective effect on the cardiovascular system, and shows its potential application value in the prevention and treatment of hypertension.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a bacterial source ACE inhibitory peptide and application thereof. BACKGROUND

[0002] Hypertension is a chronic disease that exists globally and has become one of the important factors affecting public health. Its incidence is rising year by year, and due to its potential serious complications such as heart disease, stroke, and renal failure, it has caused significant social and economic burden. Traditional treatment methods include lifestyle changes and drug intervention, and ACE (angiotensin-converting enzyme) inhibitors are commonly used drugs in the treatment of hypertension. This kind of drug can reduce the level of angiotensin II in the blood by inhibiting the activity of ACE, so as to achieve the effect of dilating blood vessels and reducing blood pressure.

[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 limit their use to some extent and increase the patient's compliance problem. Therefore, it is urgent to explore new antihypertensive drugs to reduce side effects and improve the quality of life of patients. In recent years, research has shown that natural source peptides exhibit good biological activity in ACE inhibition. These peptides can be extracted from natural resources such as food (such as dairy products, fish, etc.), plants, and microorganisms. Among them, peptides from bacteria are attracting more and more attention due to their rich variety, strong activity, and low cost. By screening and optimizing specific bacterial strains, effective ACE inhibitory peptides can be obtained, thereby providing a new treatment for hypertension.

[0004] Current technological development is still insufficient in this field, especially in the discovery and application of efficient, safe, and usable bacterial source ACE inhibitory peptides. Therefore, in view of this demand, the present application aims to develop a new type of bacterial source ACE inhibitory peptide. We will extract peptide components from specific bacterial strains selected and verify their ACE inhibitory activity through experiments. At the same time, we will study their biological safety and effectiveness in order to provide a new, safe, and efficient intervention for the treatment of hypertension and related diseases. This research is expected to provide a theoretical basis for new drug development and open up new directions for related fields. With the help of advanced separation and purification technology and biological activity analysis methods, we hope to bring new health choices for hypertension patients. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides a bacterial source ACE inhibitory peptide and an application thereof, so as to provide a polypeptide small molecule product with strong ACE inhibitory activity for the market, and further lay a certain technical foundation for the development and application of the polypeptide small molecule product in the prevention or treatment of hypertension, the food industry, the health product industry and the functional supplement industry.

[0006] In one aspect, the present application provides an ACE inhibitory peptide, which is named as 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. The molecular weight of the ACE inhibitory peptide PVQRFF is 705.82 g / mol, the molecular formula is C 31 H 45 N7O8.

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

[0008] SEQ ID NO. 2: CCAGUUCAAAGAUUUUUU.

[0009] In another aspect, the present application further provides a method for preparing an ACE inhibitory peptide by using a strain and alkaline protease to hydrolyze black bean protein, and the method is characterized by comprising the following steps:

[0010] (1) mixing alkaline protease, bacterial liquid of the strain and black bean protein solution to perform fermentation, so as to obtain a fermentation liquid;

[0011] (2) after the fermentation liquid is subjected to enzyme inactivation and pH value adjustment, a supernatant is taken;

[0012] (3) after the supernatant is subjected to ultrafiltration by using an ultrafiltration membrane, an ultrafiltrate with high ACE inhibitory activity is taken, and separation and identification are performed, so as to obtain the ACE inhibitory peptide.

[0013] The present application further provides an application of the ACE inhibitory peptide PVQRFF in preparation of a blood pressure lowering drug.

[0014] Beneficial effects:

[0015] (1) the present application uses the method of combining lactic acid bacteria fermentation and alkaline protease hydrolysis to prepare the ACE inhibitory peptide, aims to shorten the period of fermentation and hydrolysis process, and screen out effective ACE inhibitory peptides with high activity. We selected a high-ACE inhibitory peptide potential strain-fermenting lactobacillus FJ-113 screened in the laboratory, and used the strain and alkaline protease to construct a synergistic fermentation and hydrolysis process. Further separation and purification were performed on the hydrolysis product, and finally a novel ACE inhibitory peptide was identified.

[0016] (2) Different from the prior art, the ACE inhibitory peptide PVQRFF provided by the above technical scheme is composed of 6 amino acids, the amino acid sequence is Pro-Val-Gln-Arg-Phe-Phe, the molecular weight is 705.82 g / mol, the molecular formula is C 31 H 45 N7O8. It has high ACE inhibitory activity, no cytotoxicity, good digestive stability and cell protection effect. The ACE inhibitory peptide PVQRFF provided by the above technical scheme is expected to have a positive effect in the production of drugs for preventing or treating hypertension and health care foods for assisting in reducing blood pressure. BRIEF DESCRIPTION OF DRAWINGS

[0017] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0018] Figure 1 is the inhibition rate of each component ACE;

[0019] Figure 2 is the molecular docking diagram of the ACE inhibitory peptide provided by the specific embodiment of the present application;

[0020] Figure 3 is the cell toxicity test result of the ACE inhibitory peptide of the specific embodiment of the present application under different concentration conditions;

[0021] Figure 4 is the protection effect test result of the ACE inhibitory peptide of the specific embodiment of the present application on AngII-induced HUVEC damaged cells under different concentration conditions. DETAILED DESCRIPTION

[0022] The embodiments of the present application will be described in detail below, and the embodiments are implemented on the premise of the technical scheme of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0023] In the following embodiments, the chemical reagents used are purchased from regular chemical reagent suppliers and have an analytical purity.

[0024] Example 1: Fermentation of Lactobacillus fermentum combined enzyme to hydrolyze black bean protein solution

[0025] A 5% (m / v) black bean protein solution was prepared by using PBS buffer (pH 7.0, 0.05 mol / L), and a certain amount of 1 mol / L NaOH was added dropwise to pH 7.0. After being heated in a boiling water bath for 15 min to fully dissolve, it was sterilized by high-pressure steam (115℃, 15 min) to obtain a 5% (m / v) sterile black bean protein solution.

[0026] Inoculate 5 (v / v) % Lactobacillus fermentum into the sterile black bean protein solution Lactobacillus fermentum FJ-113 bacterial solution (1.0 x 10 9 CFU / mL) and fermented for 6h, 10h and 14h respectively at 37°C, and the degree of hydrolysis and ACE inhibition rate were calculated. The results showed that the degree of hydrolysis after 14h fermentation was 10.56%, and the ACE inhibition rate was 55.39%. This indicated that Lactobacillus fermentum FJ-113 had high activity in hydrolyzing black bean protein.

[0027] A single protease (alkaline protease, flavor protease, papain, trypsin or neutral protease) was added to 5% sterile black bean protein solution at an enzyme-to-substrate ratio of 1g:200mL, and then 5% (v / v) Lactobacillus fermentum FJ-113 was inoculated. After 14h fermentation at 37°C, the ACE inhibition rate, degree of hydrolysis, peptide content and pH were measured. The best protease for combined fermentation and hydrolysis with Lactobacillus fermentum FJ-113 was determined by comprehensive comparison of the indicators. The results showed that Lactobacillus fermentum FJ-113 had the best hydrolysis effect when combined with alkaline protease.

[0028] The single factor experiment and orthogonal experiment were used to determine the optimal conditions for the combined fermentation and hydrolysis of Lactobacillus fermentum FJ-113 and alkaline protease. The results showed that the ACE inhibition rate increased to 65.24% under the following conditions: alkaline protease addition amount of 1g:200mL, Lactobacillus fermentum FJ-113 inoculation amount of 5% (v / v), enzyme hydrolysis time of 14h, and substrate pH of 7.0.

[0029] Example 2 Preparation of mixed polypeptides

[0030] The fermentation broth with an ACE inhibition rate of 65.24% was collected and placed in a 90°C water bath for 10min to inactivate the enzyme. After adjusting the pH to 8.3, a portion was directly freeze-dried to obtain a freeze-dried sample for standby. Another portion was centrifuged at 8000 rpm / min for 15min at 4°C, and the supernatant was collected for standby.

[0031] A certain amount of freeze-dried sample was weighed and dissolved in deionized water to prepare 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL solutions, and the ACE inhibition rate curves of the samples at different concentrations were plotted. Ultrafiltration was performed using 10KDa and 3KDa filter membranes to separate and purify the supernatant into three different fractions (i.e., M3>10KDa, 3KDa, <M2<10KDa,M1<3KDa)。具体的,取10mL上清液于10KDa超滤管中,4℃,3500rpm / min离心超滤20min,取分子量小于10KDa部分超滤液,相同条件下,用3KDa的超滤管继续离心超滤。分别得到分子质量大于10KDa(组分M3)、小于10KDa且大于3KDa(组分M2)、小于3KDa(组分M1)的超滤液。收集各组分滤液旋转蒸发至约50ml后冷冻干燥。测得组分M1的ACE抑制活性最大,达到73.15%,显著低于粗酶解液、组分M2和M3(P<0.001)。

[0032] Component M1 was purified by chromatography on Sephadex G-15. Take a certain amount of Sephadex G-15 powder and put it into five times distilled water. Let it swell for 3 hours at room temperature to fully swell the G-15 and pour out 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, so that it will naturally precipitate in the column to two-thirds of the column height. Before injection, the gel must be continuously balanced with distilled water until there are no bubbles and no stratification before injection. Sample volume: 1 mL, eluent is distilled water, sample concentration: 200 mg / mL, flow rate: 1 mL / min, AKTA protein chromatography system UV detection wavelength: 280 nm. Collect the peak components to obtain three components: G1, G2 and G3. After freeze-drying, the ACE inhibition rate of each component was determined. Figure 1 As shown in the figure, when the concentration was 1 mg / mL, the ACE inhibition rate of component G2 was the highest, which was 74.10%, and its inhibitory activity was significantly higher than that of other components.

[0033] Liquid chromatography, tandem mass spectrometry, and de novo sequencing identified 3,512 peptides in component G2, of which 1,231 were hexapeptides. Amino acid distribution revealed that over 90% of the peptides in component G2 contained hydrophobic amino acids. Peptides with a hydrophobic amino acid content of 45-70% were the most abundant, accounting for 45.9% of the total peptides, and were mostly located at both ends of the peptides. Because the content and location of hydrophobic amino acids in ACE inhibitory peptides are closely related to their activity, these hydrophobic amino acid-rich peptides significantly enhanced the activity of component G2.

[0034] Example 3 Screening of polypeptides inhibiting ACE activity by combining database

[0035] The polypeptides obtained by sequencing were screened from the following six dimensions:

[0036] Confidence: from the polypeptide sequences identified by LC-MS / MS, the peptides with confidence ALC>80% (more reliable) were screened;

[0037] Polypeptide content: the peak area of the peptide can reflect its content, from the peptides with confidence >80%, the peptides with peak area Area>10e5 were screened;

[0038] Bioactivity score: the peptides with Area>10e5 were predicted on the PeptideRanker website, and the peptides with PeptideRanker>0.5 were screened;

[0039] Water solubility prediction: the peptides with possible biological activity (PeptideRanker>0.5) obtained by prediction were subjected to water solubility prediction in Proteomics tools under INNOVAGEN website, and the peptides with water solubility "Good" were screened;

[0040] Toxicity prediction: the peptides with better water solubility obtained by screening were subjected to peptide toxicity prediction in the "Batch Submission" module of Toixinpred website, and the "Non-Toxin" peptides were selected;

[0041] Allergy prediction: the non-toxic peptides obtained by screening were subjected to allergy prediction in "AllerTOP Server Page", and the "NON-ALLERGEN" peptides were selected;

[0042] Example 4 Screening of ACE inhibiting peptides by molecular docking

[0043] Nine peptides were screened from the above six dimensions, and the peptides with the strongest potential binding potential to ACE were further screened by molecular docking. The specific experimental steps were as follows:

[0044] Macromolecular receptor pretreatment: the X-ray protein three-dimensional structure of human ACE crystal complex 1O8A (https: / / www.rcsb.org / structure / 1O8A) was downloaded from PDB database (http: / / www.rcsb.org), and the protein structure was processed by Autodocktools 1.5.6: water removal, ligand deletion, hydrogen addition, charge calculation, merging of non-polar hydrogen, and further optimization of the three-dimensional structure of the protein. After processing, it was exported to PDB format.

[0045] Small molecule ligand preprocessing: The peptide to be docked was imported from the website (https: / / cloud.yinfotek.com / ), and the small molecule structure was processed in Autodocktools 1.5.6, with full hydrogen addition, automatic charge distribution, and torsion bond detection. After processing, the structure was exported to PDB format.

[0046] Docking and analysis: setting the Zn in the 1O8A crystal structure 2+ The active site was located at the active pocket, and the coordinates of the active pocket center were set to (40.586, 39.831, 43.427). The grid number in each X, Y, and Z directions was set to 50×50×50, the grid spacing was set to 100 for the docking number, and all other parameters were left as default. Peptides with higher binding energies to 1O8A crystals than the positive control captopril (ACE inhibitor) were selected, and the receptor-ligand interaction pattern was analyzed and visualized using Pymol 2.3.0. Molecular docking binding energy calculations identified two peptides with good binding to ACE: PVQRFF and QPVPFQ, with docking binding energies of -12.3 and -10.5 kcal / mol, respectively.

[0047] The specific visualization docking results are as follows: Figure 2 As shown, the polypeptide PVQRFF forms hydrogen bonds and π-π interactions with the residues Arg402, Ser517 and Arg522 of the ACE action site, which can bind to the ACE action site more firmly, preventing it from binding to the angiotensin precursor and catalyzing its conversion into angiotensin, thereby leading to a decrease in ACE activity.

[0048] Example 5 Cytotoxicity Study of ACE Inhibitory Peptide SPFW

[0049] We commissioned Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the ACE inhibitory peptide PVQRFF using solid-phase chemical synthesis with a purity of over 95%.

[0050] The cell activity was determined by CCK-8 method. HUVECs in good growth state were plated at 5×10 3 100 μL of cells were seeded into each well of a 96-well plate and grown for 24 hours. 10 μL of active peptides at different concentrations were added to the wells and cultured in an incubator for another 24 hours. 10 μL of CCK-8 (1 mg / mL) was added to each well for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Figure 3 The test results showed that when PVQRFF was 20 ng / mL, 40 ng / mL and 80 ng / mL, there was no significant difference in cell viability compared with the blank control, indicating that the ACE inhibitory peptide SPFW had no toxicity to HUVEC cells.

[0051] Example 6 Study on protective effect of ACE inhibitory peptide PVQRFF on AngII-induced damaged human umbilical vein endothelial cells (HUVECs)

[0052] After 1h treatment with 2ug / mL captopril (positive control) and different concentrations of SPFW (20ng / mL, 40ng / mL and 80ng / mL) respectively, 1uM AngII was used for 24h treatment. Cell viability determination method: HUVECs were seeded in 96-well plates at 5x10 3 3 Figure 4 The test results of protective effect of ACE inhibitory peptide PVQRFF on AngII-induced damaged HUVECs under different concentrations are shown in the table. The results show that different concentrations of PVQRFF significantly improve the cell viability of damaged HUVECs compared with the damaged cell group, and the effect of improving cell viability is significantly better than that of the captopril positive control group.

[0053] The present application provides a novel ACE inhibitory peptide named PVQRFF. This peptide not only exhibits good ACE inhibitory activity, but also has the characteristics of non-toxicity, making it have wide application potential in the field of biomedicine. It is found that PVQRFF can significantly improve 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, showing its potential 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 molecule corresponding to the ACE inhibitory peptide according to claim 1, wherein the sequence of the nucleotide molecule is shown in SEQ ID NO.

2.

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

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