Cubilose antihypertensive peptide, polypeptide mixture and preparation method and application thereof

Abstract

This invention discloses a bird's nest antihypertensive peptide with the amino acid sequence RPWLY. A method for preparing a polypeptide mixture containing the above-mentioned bird's nest antihypertensive peptide includes the following steps: Bird's nest is pulverized and dried, mixed with 30 times its volume of water, and ultrasonically crushed to obtain bird's nest stock solution. The ultrasonic power is 25 kW, and the time is 25 s. The pH of the bird's nest stock solution is adjusted to 7.5-8.5, trypsin is added, and enzymatic hydrolysis is performed for 4 hours. After inactivating the enzyme with boiling water, a trypsin hydrolysate is obtained. The pH of the trypsin hydrolysate is adjusted to 1.5-2.5, pepsin is added, and enzymatic hydrolysis is performed for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The pepsin hydrolysate is centrifuged, the supernatant is collected, and ultrafiltration is performed to obtain a polypeptide mixture. The bird's nest antihypertensive peptide of this invention has the advantages of strong angiotensin-converting enzyme inhibition, safety, non-toxicity, and no side effects, and can be used as a blood pressure lowering product to broaden the added value of bird's nest.

Description

Technical Field

[0001] This invention belongs to the field of bird's nest processing technology, and specifically relates to a bird's nest antihypertensive peptide, a polypeptide mixture, its preparation method and application. Background Technology

[0002] Bird's nest, made from the saliva and down feathers of swiftlets (Apodidae and other genus species), originates from Central Java Province, Indonesia, and is a prized food for medicinal purposes. Its main nutrients include water-soluble protein, carbohydrates, trace elements, and various essential amino acids, possessing antioxidant and immune-regulating effects. Although bird's nest has high nutritional and medicinal value, its deep processing research is still in its early stages. Currently, it is mainly processed by steaming or boiling, which makes its large protein molecules difficult for the intestines to digest and absorb. Therefore, further research is needed on its deep processing methods, utilizing biotechnology to extract nutrients and prepare natural products.

[0003] In recent years, bird's nest peptides have gradually attracted attention, and researchers have discovered many methods for preparing them. For example, Chinese patent application CN202211554326.X discloses a method for preparing bird's nest peptides with high antioxidant activity and their applications. This method involves adding bird's nest fragments to a mixed solution of SDS and urea at a certain concentration, which completely dissolves the bird's nest protein, allowing fine feathers and other impurities to be effectively separated from the protein. This avoids the need for manual feather removal and achieves 100% utilization of the fragments, resulting in a high yield of small-molecule bird's nest peptides with strong antioxidant activity. Another example is Chinese patent application CN202410156762.4, which discloses bird's nest peptide II with skin elasticity protection and anti-inflammatory effects and its applications. The amino acid sequence of bird's nest peptide II is DPFYGGEYLK. This bird's nest peptide II has a high elastase inhibition rate, which can inhibit the degradation of elastin in the skin and delay skin aging. Bird's nest peptide II also has the effect of improving inflammation. This invention is of great significance for improving the economic and utilization value of bird's nest.

[0004] Hypertension, as a chronic disease, poses a serious threat to human health. In the human physiological mechanism, blood pressure fluctuations are closely related to angiotensin-converting enzyme (ACE). ACE has a dual function: on the one hand, it can inactivate bradykinin in the body; on the other hand, it promotes the conversion of angiotensin I to angiotensin II. This series of changes ultimately leads to an increase in blood pressure. In recent years, ACE-inhibiting peptides screened from various natural products have gradually emerged as promising candidates. Compared with traditional chemical drugs, these ACE-inhibiting peptides are safer and more effective, and have been widely used in drug development, demonstrating a very broad and promising research prospect. They are expected to open up new and effective avenues for the treatment of hypertension and promote further development in related medical fields. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a bird's nest antihypertensive peptide, a polypeptide mixture, its preparation method, and its application.

[0006] The technical solution of the present invention is as follows:

[0007] One of the objectives of this invention is to provide a bird's nest antihypertensive peptide, the amino acid sequence of which is RPWLY.

[0008] A second objective of this invention is to provide a polypeptide mixture comprising the aforementioned bird's nest antihypertensive peptide.

[0009] A third objective of this invention is to provide a method for preparing the above-mentioned polypeptide mixture, comprising the following steps:

[0010] S1: After pulverizing and drying the bird's nest, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kW and the time is 25s.

[0011] S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 7.5-8.5, add trypsin, and enzymatically hydrolyze at 40-50℃ for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 1-2% of the mass of the bird's nest.

[0012] S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 1.5-2.5, add pepsin, and enzymatically hydrolyze at 35-40℃ for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, the pepsin hydrolysate is obtained. The amount of pepsin added is 1-2% of the weight of the bird's nest.

[0013] S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and ultrafilter out the components with a molecular weight of less than 3 kDa from the supernatant to obtain the polypeptide mixture.

[0014] Furthermore, in step S2, the trypsin has an enzyme activity ≥2500 U / mg.

[0015] Furthermore, in step S3, the pepsin has an enzyme activity ≥3000 U / mg.

[0016] The fourth objective of this invention is to provide a method for preparing the above-mentioned bird's nest antihypertensive peptide. The method involves performing chromatographic treatment on the above-mentioned polypeptide mixture. The chromatographic treatment process is as follows: using a Superdex™ peptide 10 / 300GL gel chromatography column equipped with an AKTA system to perform column chromatography on the pepsin hydrolysate after ultrafiltration, a total of 4 elution peaks are obtained. The eluents corresponding to the 4 elution peaks are collected respectively, and after freeze-drying, 4 polypeptides are obtained. The ACE inhibition rate is used as the evaluation index, and the bird's nest antihypertensive peptide with the strongest ACE inhibition rate is screened through in vitro ACE inhibition experiments.

[0017] The loading concentration was 20 mg / mL, the loading volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected for every 200 μL. The absorbance was detected at 220 nm and a curve was plotted.

[0018] The fifth objective of this invention is to provide an application of the above-mentioned bird's nest antihypertensive peptide in the preparation of antihypertensive products.

[0019] The sixth objective of this invention is to provide an application of the above-mentioned polypeptide mixture in the preparation of antihypertensive products.

[0020] The seventh objective of this invention is to provide an application of the polypeptide mixture prepared according to the above preparation method in the preparation of antihypertensive products.

[0021] The eighth objective of this invention is to provide an application of the bird's nest antihypertensive peptide prepared according to the above preparation method in the preparation of antihypertensive products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention provides a bird's nest antihypertensive peptide, which has the advantages of strong angiotensin-converting enzyme inhibition and safety without toxic side effects, and can be used as a blood pressure lowering product to broaden the added value of bird's nest.

[0024] 2. The present invention provides a method for preparing a polypeptide mixture, which combines two-step enzymatic hydrolysis, ultrafiltration and gel chromatography. The resulting polypeptide component not only has higher activity, but also has low impurity content and high purity after multiple separation and purification steps. Furthermore, the amino acid sequence of the chromatographic active component was identified by LC-MS / MS mass spectrometry, and RPWLY ​​was determined to be the peptide with the strongest ACE inhibitory activity by Autodock molecular docking simulation screening. Attached Figure Description

[0025] Figure 1 This is a bar chart showing the angiotensin-converting enzyme inhibitory activity of polypeptides 1-1, 1-2, 1-3, and 1-4 in Example 1 of this invention.

[0026] Figure 2 This is the absorbance curve of the component obtained by gel chromatography in Example 1 of this invention under a 220 nm ultraviolet wavelength detector;

[0027] Figure 3 This is a secondary mass spectrum of the bird's nest antihypertensive peptide obtained in Example 1 of this invention;

[0028] Figure 4 This invention provides the conformation of the bird's nest antihypertensive peptide obtained in Example 1 in the ACE pocket, its binding mode in the ACE active site, and its interaction planar diagram.

[0029] Figure 5 This is a bar chart showing the angiotensin-converting enzyme inhibitory activity of EBNP-1 in Example 1, EBNP-2 in Comparative Example 1, and EBNP-3 in Comparative Example 2 in this invention.

[0030] Figure 6 This is the result of the polypeptide mixture prepared in Example 1 of this invention on the cytotoxicity of LO2 cells. Detailed Implementation

[0031] The following describes a preferred embodiment, with reference to the appendix. Figure 1-6 To further illustrate the present invention, the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values; for numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein; the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified; the experimental methods in the following embodiments are conventional methods unless otherwise specified.

[0032] This invention provides a bird's nest antihypertensive peptide, the amino acid sequence of which is: RPWLY ​​(SEQ ID NO:1).

[0033] This invention obtains a polypeptide mixture by ultrafiltration and chromatography of the enzymatic hydrolysate of dried bird's nest powder.

[0034] The bird's nest antihypertensive peptide and polypeptide mixture provided by this invention can also be used to prepare antihypertensive products.

[0035] The following specific embodiments further illustrate the bird's nest antihypertensive peptide of the present invention and its applications:

[0036] The experimental indicators and methods involved in the following embodiments are as follows:

[0037] (1) Gel column chromatography

[0038] The gel chromatography column used was a Superdex™ peptide 10 / 300 GL gel chromatography column. The chromatography method was as follows: the sample concentration was 20 mg / mL, the sample volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected every 200 μL. The absorbance curves of each chromatographic component were detected under a 220 nm wavelength UV detector and plotted.

[0039] (2) In vitro ACE inhibition experiment:

[0040] The peptide was dissolved in borate buffer (0.1 M boric acid and 0.3 M sodium chloride mixed, pH adjusted to 8.3 with NaOH) to obtain the sample. 50 μL of the sample was mixed with 100 μL of 5 mM HCl and incubated at 37°C for 10 min. Then, 50 μL of 0.1 U / mL ACE solution was added and mixed, then incubated at 37°C for 10 min. Finally, 150 μL of 1 M hydrochloric acid was added to terminate the reaction, followed by 1.5 mL of ethyl acetate, and the mixture was shaken to mix. After centrifugation (4000 r / min, 10 min), 0.8 mL of the ethyl acetate layer was collected in a test tube, dried in a 95°C oven, and 4 mL of deionized water was added and mixed. The absorbance at 228 nm was then measured.

[0041] Control group: No peptides were added; all other procedures were the same as for the sample group.

[0042] Blank group: Add 150 μL of 1 M hydrochloric acid to terminate the reaction before the reaction, and the rest of the operation is the same as the sample group;

[0043] Positive control group: Captopril was used as the positive control group.

[0044] ACE inhibition rate (%) = [(A1 – A0) / (A1 – A2)] × 100%

[0045] In the formula: A0 is the sample group; A1 is the control group; A2 is the blank group.

[0046] Example 1

[0047] This embodiment provides a bird's nest antihypertensive peptide, the preparation method of which includes the following steps:

[0048] 1) A method for preparing a polypeptide mixture, comprising the following steps:

[0049] S1: After pulverizing and drying the bird's nest, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kW and the time is 25s.

[0050] S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 8.5, add trypsin, and enzymatically hydrolyze at 50°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 1% of the mass of the bird's nest.

[0051] S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 2.0, add pepsin, and enzymatically hydrolyze at 37°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 1% of the weight of the bird's nest.

[0052] S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and ultrafilter out the components with a molecular weight of less than 3 kDa from the supernatant to obtain the polypeptide mixture, denoted as EBNP-1.

[0053] In this embodiment, in step S2, the enzyme activity of the trypsin is ≥2500 U / mg.

[0054] In this embodiment, in step S3, the enzyme activity of the pepsin is ≥3000 U / mg.

[0055] 2) Preparation method of bird's nest antihypertensive peptides:

[0056] The above-mentioned peptide mixture was subjected to chromatographic treatment. The chromatographic treatment process was as follows: the ultrafiltration-treated pepsin hydrolysate was subjected to column chromatography using a Superdex™ peptide 10 / 300 GL gel chromatography column equipped with an AKTA system. Four elution peaks were obtained. The eluents corresponding to the four elution peaks were collected, freeze-dried, and the peptide mixture was obtained and labeled as 1-1, 1-2, 1-3, and 1-4, respectively. The ACE inhibition rate was used as the evaluation index. The peptide with the strongest ACE inhibition rate in the peptide mixture was screened through in vitro ACE inhibition experiments and identified as the bird's nest antihypertensive peptide. Figure 1 This is a bar chart showing the angiotensin-converting enzyme inhibitory activity of peptides 1-1, 1-2, 1-3, and 1-4 in this embodiment. Figure 1 As shown, in vitro ACE inhibition experiments revealed that components 1-4, with the smallest molecular weight, exhibited the strongest ACE inhibitory activity.

[0057] The loading concentration was 20 mg / mL, the loading volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected for every 200 μL. The absorbance was detected at 220 nm and a curve was plotted.

[0058] Figure 2 This is an absorbance curve of the component obtained by gel chromatography in this embodiment under a 220 nm ultraviolet wavelength detector.

[0059] 3) Amino acid sequence identification

[0060] The peptides 1-1, 1-2, 1-3, and 1-4 selected above were measured by LC-MS / MS, and the results were analyzed using PEAKS Studio 11 mass spectrometry software.

[0061] In this embodiment, the LC-MS / MS measurement conditions are as follows:

[0062] In the liquid chromatography method: the column was a NanoViper C18 1.9 μm, 100A, with phase A being 0.1% FA and phase B being 0.1% FA and 80% ACN. The flow rate was 50 nL / min, with a chromatographic gradient of 70 min. The specific elution gradient was as follows: 0-1 min, phase B increased uniformly from 8% to 12%; 1-9 min, phase B increased uniformly from 12% to 15%; 9-31 min, phase B increased uniformly from 15% to 25%; 31-38 min, phase B increased uniformly from 25% to 36%; 38-45 min, phase B increased uniformly from 36% to 60%; 45-53 min, phase B increased uniformly from 60% to 95%; 53-60 min, the mobile phase remained unchanged; 60-70 min, phase B decreased uniformly from 95% to 8%.

[0063] In the mass spectrometry method: First-order mass spectrometry parameters: Resolution: 120000; AGC target: Custorm; Maximum IT: Custorm; Scan range: 100 to 1000 m / z. Second-order mass spectrometry parameters: Resolution: 15000; AGC target: Custorm; Maximum IT: Custorm; Cycle time: 3 s; NCE / stepped NCE: 30.

[0064] Search criteria: The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Peptide N-term); Enzyme: Non-specific; Database: Apodiformes; Peptide Mass Tolerance (PMT): 20 ppm; Fragment Mass Tolerance (FMT): 0.02 Da.

[0065] The amino acid sequence of the bird's nest antihypertensive peptide in the polypeptide mixture prepared in this embodiment is RPWLY, and its secondary mass spectrum is shown below. Figure 3 As shown.

[0066] 4) Molecular docking screening

[0067] The activity of several peptides obtained from the PEAKS Studio 11 mass spectrometry analysis software was evaluated using the PeptideRanker tool, with a score greater than 0.9 (indicating a high probability of biological activity). Furthermore, the ToxinPred tool was used to predict the potential toxicity of the peptides to exclude those with potential application risks; all results showed no toxicity. Simultaneously, the CPPpred tool was used to screen for peptides with a score greater than 0.5 (indicating good cell penetration). New peptides were then screened using the Biopep database.

[0068] Based on molecular docking, the software Autodock (version 1.5.7) was used to screen new peptides obtained from mass spectrometry identification and simulation screening for bioactivity, identifying peptide sequences with strong protein binding ability. The spatial structure of the peptides was drawn using ChemDraw 22 software, and energy minimization was performed. Protonation states and hydrogen orientations were optimized in Autodock, and the results were saved as pdbqt files as ligand files.

[0069] The 3D structure of Alpha-glucosidase (PDB ID: 3WY1) was obtained from the PDB database. Repeating sequences and water molecules were removed, and polar hydrogen atoms were added. A gridbox was set within the predicted active pocket of Alpha-glucosidase, with active sites at X: 1.098, Y: -4.174, and Z: -7.923; the grid size was 21.75 Å × 25.5 Å × 18.75 Å.

[0070] Figure 4 The diagram shows the conformation of the bird's nest antihypertensive peptide RPWLY ​​obtained in this embodiment in the ACE pocket, its binding mode in the ACE active site, and its interaction plane. As can be seen from the diagram, the bird's nest antihypertensive peptide with the amino acid sequence RPWLY ​​of this invention has a strong binding ability to ACE, which is -10.6 kcal / mol. The active peptide enters the active site of ACE, occupies the hydrophobic pocket, and forms bonds with the surrounding amino acid residues. The binding is relatively tight, and the ligand molecules that interact with these residues can reduce the activity of ACE enzymes through competitive inhibition.

[0071] Example 2

[0072] This embodiment provides a polypeptide mixture containing the bird's nest antihypertensive peptide of Example 1.

[0073] The method for preparing the polypeptide mixture includes the following steps:

[0074] S1: After pulverizing and drying the bird's nest, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kW and the time is 25s.

[0075] S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 7.5, add trypsin, and enzymatically hydrolyze at 40°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, obtain the trypsin hydrolysate. The amount of trypsin added is 2% of the mass of the bird's nest.

[0076] S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 1.5, add pepsin, and enzymatically hydrolyze at 35°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 2% of the weight of the bird's nest.

[0077] S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and ultrafilter out the components with a molecular weight of less than 3 kDa from the supernatant to obtain the polypeptide mixture.

[0078] In this embodiment, in step S2, the enzyme activity of the trypsin is ≥2500 U / mg.

[0079] In this embodiment, in step S3, the enzyme activity of the pepsin is ≥3000 U / mg.

[0080] In this embodiment, in step S4, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to separate and collect components with a molecular weight less than 3 kDa; the chromatography process is as follows: using a Superdex™ peptide 10 / 300 GL gel chromatography column equipped with an AKTA system to perform column chromatography on the ultrafiltration-treated pepsin hydrolysate, obtaining a total of 4 elution peaks, collecting the eluents corresponding to the 4 elution peaks respectively, freeze-drying to obtain a peptide mixture, and using the ACE inhibition rate as the evaluation index, screening the peptide with the strongest ACE inhibition rate in the peptide mixture through in vitro ACE inhibition experiments;

[0081] The loading concentration was 20 mg / mL, the loading volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected for every 200 μL. The absorbance was detected at 220 nm and a curve was plotted.

[0082] Example 3

[0083] This embodiment provides a polypeptide mixture containing the bird's nest antihypertensive peptide of Example 1.

[0084] The method for preparing the polypeptide mixture includes the following steps:

[0085] S1: After pulverizing and drying the bird's nest, add 30 times the amount of water and mix evenly. After ultrasonic crushing, bird's nest liquid is obtained. The ultrasonic power is 25kW and the time is 25s.

[0086] S2: Adjust the pH of the bird's nest liquid obtained in step S1 to 8.0, add trypsin, and enzymatically hydrolyze at 45°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a trypsin hydrolysate is obtained; wherein, the amount of trypsin added is 1.5% of the mass of the bird's nest.

[0087] S3: Adjust the pH of the trypsin hydrolysate obtained in step S2 to 2.5, add pepsin, and enzymatically hydrolyze at 40°C for 4 hours. After inactivating the enzyme with boiling water for 10 minutes, a pepsin hydrolysate is obtained. The amount of pepsin added is 1.5% of the weight of the bird's nest.

[0088] S4: Centrifuge the pepsin hydrolysate obtained in step S3, take the supernatant, and ultrafilter out the components with a molecular weight of less than 3 kDa from the supernatant to obtain the polypeptide mixture.

[0089] In this embodiment, in step S2, the enzyme activity of the trypsin is ≥2500 U / mg.

[0090] In this embodiment, in step S3, the enzyme activity of the pepsin is ≥3000 U / mg.

[0091] In this embodiment, in step S4, the ultrafiltration process is as follows: using an ultrafiltration tube with a molecular weight cutoff of 3 kDa to collect components with a molecular weight less than 3 kDa.

[0092] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro ACE inhibition test in this embodiment are similar to those in Example 2.

[0093] Comparative Example 1

[0094] This comparative example provides a polypeptide mixture, the preparation method of which differs from that of Example 1 in that:

[0095] In step S4 of this comparative example, ultrafiltration tubes with molecular weight cutoffs of 3 kDa and 10 kDa are used to collect components with molecular weights between 3 kDa and 10 kDa to obtain the polypeptide mixture, denoted as EBNP-2.

[0096] Comparative Example 2

[0097] This comparative example provides a polypeptide mixture, the preparation method of which differs from that of Example 1 in that:

[0098] In step S4 of this comparative example, an ultrafiltration tube with a molecular weight cutoff of 10 kDa was used to collect components with a molecular weight greater than 10 kDa to obtain the polypeptide mixture, denoted as EBNP-3.

[0099] Implementation effect evaluation

[0100] The following specific experiments on the polypeptide mixtures obtained in Example 1 and Comparative Examples 1-2 further illustrate the excellent effects achieved by the present invention:

[0101] 1) In vitro ACE inhibition assay:

[0102] EBNP-1 from Example 1, EBNP-2 from Comparative Example 1, and EBNP-3 from Comparative Example 2 were selected for in vitro ACE inhibition assays. Figure 5 This is a bar chart showing the angiotensin-converting enzyme inhibitory activities of EBNP-1, EBNP-2, and EBNP-3 according to the present invention. Figure 5 As can be seen from the above, the polypeptide mixture of the present invention has a good inhibitory effect on angiotensin-converting enzyme.

[0103] 2) Cytotoxicity assay:

[0104] LO2 cells in logarithmic growth phase were prepared into a homogeneous single-cell suspension, counted, and diluted with culture medium to 10,000 cells / 100 μL. 100 μL of each cell was seeded into 96-well plates and cultured for 12–16 h. The old culture medium was discarded, and different concentrations of peptide-containing culture medium (medium:peptide solution = 99:1) were added, and the cells were cultured for another 24 h. Finally, under light-protected conditions, 100 μL of fresh culture medium and 15 μL of 5 mg / mL thiazolyl blue solution were added to each well, and the cells were incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well, which was then shaken for 10 min in the dark. The absorbance was measured at 490 nm. Six parallel wells were set up for each sample group, and a PBS control group was included.

[0105] The formula for calculating cell viability is as follows:

[0106] Cell viability (%) = [(A1 – A0) / (A2 – A0)] × 100%

[0107] In the formula: A0 is the blank group; A1 is the sample group; A2 is the control group.

[0108] Experimental results are as follows Figure 6 As shown, from Figure 6 As can be seen, the bird's nest antihypertensive peptide prepared in Example 1 of the present invention has a growth-promoting effect on LO2 cells under the conditions of 0.1-10 μg / mL concentration, and is non-toxic to cells at a concentration of 100 μg / mL, with a cell survival rate between 95-110%.

[0109] In summary, the bird's nest antihypertensive peptide of the present invention has the characteristic of strong angiotensin-converting enzyme inhibition, and its molecular weight is 733.3911 Da. This polypeptide also has the advantages of being safe and free of toxic side effects, and can be used as a product to lower blood pressure, thus expanding the added value of bird's nest.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A bird's nest antihypertensive peptide, characterized in that, The amino acid sequence of the bird's nest antihypertensive peptide is SEQ ID NO:

1.

2. A mixture of polypeptides, characterized in that, The polypeptide mixture comprises the bird's nest antihypertensive peptide of claim 1.

3. A method for preparing a mixture of polypeptides, for preparing a mixture of polypeptides according to claim 2, characterized in that, The method comprises the following steps: S1: crushing and drying the bird's nest, adding 30 times the amount of water and mixing uniformly, and preparing a bird's nest stock solution by ultrasonic crushing, wherein the ultrasonic power is 25 kW and the time is 25 s; S2: adjusting the pH of the bird's nest stock solution obtained in step S1 to 7.5-8.5, and adding trypsin for enzymatic treatment at 40-50℃ for 4 h, and then boiling for 10 min to inactivate the enzyme to obtain a trypsin enzymatic hydrolysate; wherein the addition amount of trypsin is 1-2% of the mass of the bird's nest; S3: adjusting the pH of the trypsin enzymatic hydrolysate obtained in step S2 to 1.5-2.5, and adding pepsin for enzymatic treatment at 35-40℃ for 4 h, and then boiling for 10 min to inactivate the enzyme to obtain a pepsin enzymatic hydrolysate; wherein the addition amount of pepsin is 1-2% of the mass of the bird's nest; S4: centrifuging the pepsin enzymatic hydrolysate obtained in step S3, taking the supernatant, and ultrafiltering a component with a molecular weight less than 3 kDa from the supernatant to obtain the polypeptide mixture.

4. The production method according to claim 3, characterized by, In step S2, the enzyme activity of the trypsin is ≥2500 U / mg.

5. The production method according to claim 3, characterized by, In step S3, the enzyme activity of the pepsin is ≥3000 U / mg.

6. Use of the bird's nest antihypertensive peptide of claim 1 in the preparation of an antihypertensive drug.

7. Use of the polypeptide mixture of claim 2 in the preparation of an antihypertensive drug.

8. Use of the polypeptide mixture prepared by the method of any one of claims 3-5 in the preparation of an antihypertensive drug.

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