Cordyceps militaris peptide having ACE inhibitory activity, and preparation method and application thereof

Abstract

The application discloses a bird's nest peptide with ACE inhibitory activity and a preparation method and application thereof, and the bird's nest peptide with ACE inhibitory activity is prepared through enzymolysis, and LC-MS / MS analysis is conducted on the peptides in the enzymolysis solution, potential ACE inhibitory activity of the peptide segments is screened by combining bioinformatics means, and the obtained peptide segments are verified by synthesis, and the peptide segments have high ACE inhibitory activity. The bird's nest ACE inhibitory peptide has a good application prospect in functional food or health products and food-derived drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioactive peptides, and particularly relates to a bird's nest peptide with ACE inhibitory activity, a preparation method and application thereof. BACKGROUND

[0002] Hypertension is a common cardiovascular disease in human life, which is specifically manifested as that the diastolic pressure or systolic pressure of the human body exceeds the normal range. At present, the incidence of hypertension is increasing year by year in the global range, and the age of the hypertension population is becoming younger. Hypertension disease has seriously affected the quality of life of the people, and the solution to this major disease problem is imminent. Angiotensin converting enzyme (ACE) is a dipeptidyl carboxypeptidase, which plays a role in the regulation of the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) in the balance of human physiological processes, and makes the blood pressure rise by converting angiotensin II and hydrolyzing bradykinin. Therefore, inhibiting the activity of ACE has become an important method for treating hypertension. At present, the ACE inhibitors on the market are mainly drugs, such as captopril, enalapril, ramipril, lisinopril, etc. Some of the drug ACE inhibitors have significant therapeutic effects, but there are problems of rebound of blood pressure after drug withdrawal, dizziness, cough, skin rash and other side effects after long-term use. Compared with drug ACE inhibitors, food-derived ACE inhibitory peptides have the advantages of wide source of raw materials, high safety and strong specificity, and the related research on the separation and extraction of ACE inhibitory peptides from suitable food raw materials has attracted much attention. At present, it has been reported that ACE inhibitory peptides are extracted from different raw materials such as bovine casein, soybean protein and egg white protein by fermentation, enzymatic hydrolysis and solid-phase synthesis, and certain achievements have been made.

[0003] The nutritional components in bird's nest are mainly protein and sialic acid, and bird's nest has high nutritional value. According to traditional Chinese medicine, bird's nest has the effects of tonifying lung and yin, relieving cough and reducing phlegm, beautifying the skin and nourishing the stomach. Modern research has proved that bird's nest has multiple functional activities such as antioxidant, antiviral, anti-aging, promoting brain development and bone growth, whitening, anti-inflammatory, and so on. Bird's nest peptide is an important source of these functional activities. At present, the application of bird's nest peptide in functional food, health products and cosmetics is mainly based on its antioxidant, whitening, anti-aging and anti-inflammatory activities, and the research on the ACE inhibitory activity of bird's nest peptide is less. The methods for extracting bird's nest peptide from natural bird's nest mainly include extraction method, enzymatic hydrolysis method and fermentation method, among which the enzymatic hydrolysis method is widely used in the extraction of different types of bioactive peptides due to its mild conditions, safety, environmental protection and strong specificity. However, the water solubility of bird's nest protein is poor, and under conventional conditions, the use of enzyme extraction is prone to problems such as low protein dissolution amount and low hydrolysis degree. If other extraction methods are used, the process will be complicated, the extraction cycle will be prolonged, and the production cost will be increased. Therefore, it is of great significance to optimize the enzymatic hydrolysis process conditions and find a simple, safe and efficient enzymatic extraction method of bird's nest peptide with ACE inhibitory activity for widening the application of bird's nest peptide in food-based antihypertensive drugs. SUMMARY

[0004] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification of the present application in order to avoid obscuring the purpose of this section, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0005] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0006] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a bird's nest peptide with ACE inhibitory activity.

[0007] To solve the above technical problems, the present application provides the following technical solutions: a bird's nest peptide with ACE inhibitory activity, characterized in that the amino acid sequence of the bird's nest peptide is YGRY, i.e. Tyr-Gly-Arg-Tyr.

[0008] Or, the amino acid sequence of the bird's nest peptide is APFDGIL, i.e. Ala-Pro-Phe-Asp-Gly-Ile-Leu.

[0009] Another purpose of the present application is to overcome the deficiencies in the prior art and provide an application of the bird's nest peptide with ACE inhibitory activity in functional food or health products and food-based drugs.

[0010] Still another object of the present application is to provide a preparation method of a bird's nest peptide with ACE inhibitory activity, which overcomes the deficiencies in the prior art and is characterized in that it comprises,

[0011] mixing the bird's nest silk after impurity removal and air drying with purified water for soaking;

[0012] stewing the soaked bird's nest for 30 min and then cooling to room temperature;

[0013] shearing and dispersing the stewed bird's nest liquid to obtain a bird's nest homogenate;

[0014] adding a protease to the bird's nest homogenate for enzymolysis, then inactivating the enzyme, and centrifuging at 10,000 x g for 10 min to obtain an enzyme hydrolysis supernatant;

[0015] ultrafiltration and freeze-drying to obtain a <3 kDa bird's nest peptide powder;

[0016] LC-MS / MS peptide segment identification on the <3 kDa bird's nest peptide powder;

[0017] screening of peptides with potential ACE inhibitory activity;

[0018] verification of the screened peptides by molecular docking technology;

[0019] chemical solid-phase synthesis of the screened peptides and in vitro ACE inhibitory activity evaluation.

[0020] As a preferred scheme of the preparation method of the present application, the bird's nest silk is soaked with purified water, and the mass ratio of the bird's nest silk to the purified water is 1:20-1:40.

[0021] As a preferred scheme of the preparation method of the present application, the soaked bird's nest is stewed for 30 min, and the stewing temperature is 90-120°C.

[0022] As a preferred scheme of the preparation method of the present application, the stewed bird's nest liquid is sheared and dispersed, and the shearing rate is 8000 rpm and the shearing time is 5 min.

[0023] As a preferred scheme of the preparation method of the present application, the protease added to the bird's nest homogenate for enzymolysis is at least one of trypsin, papain, alkaline protease, neutral protease and flavor protease.

[0024] As a preferred scheme of the preparation method of the present application, the enzyme amount is 4000-9000 U / g, the enzymolysis temperature is 40-55°C, the enzymolysis time is 1.5-3.5 h, and the enzymolysis pH is 6.5-9.5.

[0025] As a preferred scheme of the preparation method, wherein: the screening of the peptide with potential ACE inhibitory activity comprises: using the PeptideRanker tool to perform biological activity scoring on the peptide sequence identified by LC-MS / MS, and selecting the peptide with a score greater than or equal to 0.7; using the MultiPep tool to perform ACE inhibitory activity prediction on the selected peptide, and selecting the peptide with an ACE inhibitor score greater than or equal to 0.9; using the ToxinPred tool, the AllerTOP tool, the Proteomics tool and the admetSAR tool to predict the toxicity, allergenicity, water solubility and HIA, BBB properties of the peptide with an ACE inhibitor score greater than or equal to 0.9, respectively, and selecting the peptide with an abundance greater than or equal to 1x10 7 , and the peptide with good overall physicochemical properties.

[0026] As a preferred scheme of the preparation method, wherein: the verification of the screened peptide by using the molecular docking technology comprises: obtaining 1O8A crystal as the receptor ACE structure from the PDB database, deleting the ligand and water molecules thereof by using the Pymol software, drawing the molecular structure of the ACE inhibitory peptide by using the Chemdraw 22.2.0 software, and performing energy minimization on the molecular structure, then processing the ACE receptor and the small molecule peptide ligand by using the AutoDockTools-1.5.7, setting the docking box, and finally performing molecular docking by using the AutoDockVina.

[0027] The present application has the following beneficial effects:

[0028] (1) The present application provides a method for preparing ACE inhibitory activity edible bird's nest peptide by enzymatic hydrolysis, and the peptides in the enzymatic hydrolysate are analyzed by LC-MS / MS, and the peptides with potential ACE inhibitory activity are screened by bioinformatics methods, and the obtained peptides are verified by synthesis, and have high ACE inhibitory activity. The edible bird's nest ACE inhibitory peptide in the present application has good application prospect in functional food or health products, and food-derived drugs.

[0029] (2) The present application first stews the edible bird's nest before enzymatic hydrolysis. The pre-experiment shows that the conformation of the edible bird's nest protein changes after high-temperature heat treatment, the internal structure becomes loose, and the hydrophobic group is exposed, thereby increasing the action opportunity of the protease and the edible bird's nest protein, so that the protein extraction degree of the enzyme hydrolysis of the homogenate of the stewed edible bird's nest is higher than that of the direct enzyme hydrolysis of the dry edible bird's nest raw material, and the degree of hydrolysis is deeper.

[0030] (3)The application discloses a method for efficiently preparing a bird's nest peptide with ACE inhibitory activity by screening a protease and optimizing an enzymolysis process. The preferred protease in the application is a combination of alkaline protease and flavor protease. The alkaline protease specifically hydrolyzes the end of a hydrophobic amino acid and is widely applied to the preparation of ACE inhibitory peptides. The amino acid composition in the bird's nest protein is mainly hydrophobic amino acids, which can be well hydrolyzed by the alkaline protease. The flavor protease has the characteristics of endo-enzyme and exo-enzyme, has more enzyme cutting sites, and can quickly hydrolyze large molecular weight peptides into short peptides. The method has a simple process flow and convenient operation. The bird's nest peptide prepared by the method has high protein extraction rate, high hydrolysis degree, and good ACE inhibitory activity.

[0031] (4)The traditional method for screening a bioactive peptide comprises a series of separation and purification operations such as ion exchange, gel filtration and reverse phase high performance liquid chromatography, and then the purified peptide is identified to obtain a target bioactive peptide. The operation process is complicated, time-consuming and laborious, and is difficult to be applied to large-scale industrial production. The method comprises the steps of identifying the bird's nest peptide by LC-MS / MS and virtually screening a target bioactive peptide by bioinformatics. The target bioactive peptide has high ACE inhibitory activity and good physical and chemical properties, and can be well absorbed and utilized by the human body. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0033] Figure 1 It is a molecular docking diagram of the peptide YGRY and ACE.

[0034] Figure 2 It is a molecular interaction diagram of the peptide YGRY and ACE.

[0035] Figure 3 It is a molecular docking diagram of the peptide APFDGIL and ACE.

[0036] Figure 4 It is a molecular interaction diagram of the peptide APFDGIL and ACE.

[0037] Figure 5 It is a mass spectrum diagram of YGRY.

[0038] Figure 6 It is a mass spectrum diagram of APFDGIL.

[0039] Figure 7The ACE activity inhibition rate of the peptide YGRY, APFDGIL.

[0040] Figure 8 The ACE activity inhibition rate of the peptide YGRY, APFDGIL after in-vitro simulated digestion. DETAILED DESCRIPTION

[0041] In order to make the above objectives, features and advantages of the present application more apparent, the specific embodiments of the present application will be described in detail below with reference to the embodiments of the present application.

[0042] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein, and those skilled in the art can make similar generalizations without departing from the concept of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term “one embodiment” or “an embodiment” as used herein refers to a specific feature, structure or characteristic that can be included in at least one implementation of the present application. The term “in one embodiment” appearing in different places in the specification does not refer to the same embodiment, nor is it an embodiment that is independent of or mutually exclusive of other embodiments.

[0044] Experimental materials used in the embodiments of the present application:

[0045] Bird's nest: Xiamen Yanzhiwu Silk Concentrated Food Co., Ltd.; Alkaline protease, flavor protease, papain, neutral protease: Shanghai Bomeifuke Chemical Technology Co., Ltd.; Trypsin: Sigma-Aldrich Company, USA; BCA protein concentration determination kit: Shanghai Biyun Tian Biological Technology Co., Ltd.; Sodium hydroxide, formaldehyde: Shanghai Titan Science and Technology Co., Ltd.; ACE: Nanjungtai Biological Technology Co., Ltd.; Acetonitrile: Honeywell Company, USA.

[0046] Experimental instruments used in the embodiments of the present application:

[0047] HH-2 digital constant-temperature water bath: Changzhou Ronghua Instrument Manufacturing Co., Ltd.; YXQ-LS-50A type vertical pressure steam sterilizer: Wuxi Kaipaike Science and Technology Co., Ltd.; Avanti JXN-26 intelligent high-efficiency centrifuge, Beckman Coulter Company, USA; KDN-16C nitrogen determination instrument: Shanghai Fiber Instrument Co., Ltd.; M2 enzyme label instrument: Molecular Devices Company, USA; XX80EL230 tangential flow ultrafiltration system: MilliPore Company, Germany; UltiMate 3000 high-performance liquid chromatograph, Orbitrap Eclipse mass spectrometer: Thermo Fisher Company, USA.

[0048] Example 1

[0049] The present example provides a preparation method of an enzyme hydrolysis supernatant of bird's nest, comprising the following steps:

[0050] (1) The dry bird's nest silk after impurity removal is mixed with pure water at a ratio of raw material: water = 1:25 (w / w) and soaked for 1 h.

[0051] (2) The soaked bird's nest is stewed at 120℃ for 30 min, and then cooled to room temperature.

[0052] (3) The stewed bird's nest is sheared at 8000 rpm for 5 min with a high-speed dispersion machine to prepare a bird's nest homogenate.

[0053] (4) Compound protease (alkaline protease: flavor protease = 2:1) is added to the bird's nest homogenate, the total amount of enzyme is 7000 U / g (enzyme activity / bird's nest raw material mass), the pH is controlled at 8, the enzyme hydrolysis is carried out at 50℃ for 2.5 h, the enzyme is inactivated in a boiling water bath for 10 min, and then cooled to room temperature and centrifuged at 10000 x g for 10 min to obtain the bird's nest enzyme hydrolysis supernatant.

[0054] The protein extraction rate, protein hydrolysis degree and ACE inhibition activity of the enzyme hydrolysis liquid obtained in Example 1 are shown in Table 1. The bird's nest enzyme hydrolysis liquid prepared by this method has good protein extraction rate, high hydrolysis degree, and good ACE inhibition activity.

[0055] Protein extraction rate determination method: The protein content in the bird's nest raw material is determined by Kjeldahl nitrogen determination method (GB 5009.5-2016), and the protein content in the bird's nest enzyme hydrolysis supernatant is determined by BCA method. The determination process is carried out according to the BCA protein concentration determination kit instruction manual. The protein extraction rate is calculated according to the following formula:

[0056]

[0057] Protein hydrolysis degree determination method: The amino acid nitrogen in the enzyme hydrolysis liquid and the bird's nest raw material is determined according to the neutral formaldehyde titration method of GB 5009.235-2016, and the hydrolysis degree (DH) is calculated according to the following formula:

[0058]

[0059] ACE inhibitory activity assay: The enzyme hydrolyzate supernatant of bird's nest was lyophilized and then reconstituted into a sample solution with a concentration of 2 mg / ml. The solution was prepared using 80 mmol / L HEPES buffer (pH = 8.2, containing 0.3 mol / L NaCl). 40 μl of the sample solution was mixed with 50 μl of FAPGG solution (1 mmol / L), and after incubation at 37°C for 10 min, 10 μl of ACE enzyme solution (0.1 U / ml) was added. The initial absorbance (A0 and B0) was measured at 340 nm, and then the end-point absorbance (A1 and B1) was measured after incubation at 37°C for 30 min in the dark. The ACE activity inhibition rate was calculated according to the following formula:

[0060]

[0061] In the formula: AI: ACE activity inhibition rate; A0: initial absorbance of the sample; A1: absorbance of the sample after reaction for 30 min; B0: initial absorbance of the buffer instead of the sample; B1: absorbance of the buffer instead of the sample after reaction for 30 min.

[0062] Table 1 Protein extraction rate, degree of hydrolysis and ACE inhibitory activity of the enzyme hydrolyzate of bird's nest in Example 1

[0063]

[0064] Example 2

[0065] The difference between this example and Example 1 is in the enzyme hydrolysis conditions, including the following steps:

[0066] (1) The dry bird's nest silk after impurity removal was mixed with purified water at a ratio of raw material: water = 1:25 (w / w), and soaked for 1 h.

[0067] (2) The soaked bird's nest was stewed at 120°C for 30 min, and then cooled to room temperature.

[0068] (3) The stewed bird's nest was sheared at 8000 rpm for 5 min using a high-speed disperser to prepare a bird's nest homogenate.

[0069] (4) Compound protease (trypsin: flavour protease = 2:1) was added to the bird's nest homogenate, and the total amount of enzyme was 7000 U / g (enzyme activity / bird's nest raw material mass), and the pH was controlled at 7.5. The enzyme hydrolysis was carried out at 45°C for 2.5 h, and then the enzyme was inactivated in a boiling water bath for 10 min. After cooling to room temperature, centrifugation was performed at 10000 x g for 10 min to obtain the enzyme hydrolyzate supernatant of bird's nest.

[0070] This example replaces the alkaline protease with trypsin compared to Example 1. The protein extraction rate, degree of hydrolysis and ACE inhibitory activity of the enzyme hydrolysate obtained in Example 2 are shown in Table 2, and the results show that the protein extraction rate of the enzyme hydrolysate prepared by this method is increased by 2.13% compared with Example 1, the degree of hydrolysis is increased by 1.79%, and the ACE inhibitory activity is decreased by 5.55%.

[0071] Table 2 Protein extraction rate, degree of hydrolysis and ACE inhibitory activity of the bird's nest enzyme hydrolysate in Example 2

[0072]

[0073] Comparative Example 1

[0074] The difference between this comparative example and Example 1 is that the enzyme hydrolysis is directly performed on the soaked bird's nest silk, and the process of stewing and homogenizing the bird's nest is omitted. Specifically:

[0075] (1) The dry bird's nest silk after impurity removal was mixed with purified water at a ratio of raw material: water = 1:25 (w / w), and soaked for 1 h.

[0076] (2) Compound protease (alkaline protease: flavor protease = 2:1) was added to the soaked bird's nest liquid, the total amount of enzyme was 7000 U / g (enzyme activity / quality of bird's nest raw material), the pH was controlled at 8, and the enzyme hydrolysis was carried out at 50°C for 2.5 h, the enzyme was inactivated in boiling water for 10 min, and after cooling to room temperature, centrifugation was performed at 10000 x g for 10 min to obtain the bird's nest enzyme hydrolysate supernatant.

[0077] The protein extraction rate, degree of hydrolysis and ACE inhibitory activity of the enzyme hydrolysate obtained in Comparative Example 1 are shown in Table 3, and the results show that the protein extraction rate, degree of hydrolysis and ACE inhibitory activity of the enzyme hydrolysate prepared by this method are all greatly decreased compared with Example 1.

[0078] Table 3 Protein extraction rate, degree of hydrolysis and ACE inhibitory activity of the bird's nest enzyme hydrolysate in Comparative Example 1

[0079]

[0080] Comparative Example 2

[0081] The difference between this comparative example and Example 1 is that the composition of the compound protease is adjusted to 1:1 of neutral protease and papain. Specifically:

[0082] (1) The dry bird's nest silk after impurity removal was mixed with purified water at a ratio of raw material: water = 1:25 (w / w), and soaked for 1 h.

[0083] (2) The soaked bird's nest was stewed at 120°C for 30 min, and after stewing, it was cooled to room temperature.

[0084] (3) The stewed bird's nest was sheared at 8000 rpm for 5 min by a high-speed dispersion machine to prepare a bird's nest homogenate.

[0085] (4) Compound protease (neutral protease: papain = 1:1) was added to the bird's nest homogenate, the total amount of enzyme was 7000 U / g (enzyme activity / bird's nest raw material mass), pH was controlled at 7, and the enzyme was hydrolyzed at 55°C for 2.5 h. The enzyme was inactivated in a boiling water bath for 10 min. After cooling to room temperature, centrifugation was performed at 10000 x g for 10 min to obtain a bird's nest enzyme hydrolysate supernatant.

[0086] The protein extraction rate, protein hydrolysis degree and ACE inhibition activity of the enzyme hydrolysate obtained in Comparative Example 2 are shown in Table 4. The results show that the protein extraction rate and the hydrolysis degree of the enzyme hydrolysate prepared by this method are slightly lower than those of Example 1, and the ACE inhibition activity is significantly decreased.

[0087] Table 4 Protein extraction rate, hydrolysis degree and ACE inhibition activity of the bird's nest enzyme hydrolysate in Comparative Example 2

[0088]

[0089] Comparative Example 3

[0090] The difference between this comparative example and Example 1 is that the enzyme hydrolysis time is extended to 4 h. Specifically:

[0091] (1) The dry bird's nest silk after impurity removal was mixed with pure water at a raw material: water ratio of 1:25 (w / w) and soaked for 1 h.

[0092] (2) The soaked bird's nest was stewed at 120°C for 30 min, and then cooled to room temperature.

[0093] (3) The stewed bird's nest was sheared at 8000 rpm for 5 min by a high-speed dispersion machine to prepare a bird's nest homogenate.

[0094] (4) Compound protease (alkaline protease: flavor protease = 2:1) was added to the bird's nest homogenate, the total amount of enzyme was 7000 U / g (enzyme activity / bird's nest raw material mass), pH was controlled at 8, and the enzyme was hydrolyzed at 50°C for 4 h. The enzyme was inactivated in a boiling water bath for 10 min. After cooling to room temperature, centrifugation was performed at 10000 x g for 10 min to obtain a bird's nest enzyme hydrolysate supernatant.

[0095] The protein extraction rate, protein hydrolysis degree and ACE inhibition activity of the enzyme hydrolysate obtained in Comparative Example 3 are shown in Table 5. The results show that the protein extraction rate of the enzyme hydrolysate prepared by this method is slightly higher than that of Example 1, the hydrolysis degree is slightly decreased, and the ACE inhibition activity is significantly decreased.

[0096] Table 5 Protein extraction rate, hydrolysis degree and ACE inhibition activity of the bird's nest enzyme hydrolysate in Comparative Example 3

[0097]

[0098]

[0099] Example 3

[0100] This example is to purify the enzyme hydrolysate of bird's nest obtained in Example 1 to obtain low molecular weight oligopeptides, including the following steps:

[0101] (1) The enzyme hydrolysate of bird's nest was treated by tangential flow ultrafiltration system, and the supernatant of the enzyme hydrolysate was sequentially passed through ultrafiltration membrane bags with a molecular weight cut-off of 10000 Da and 3000 Da, and the last permeate was collected.

[0102] (2) The permeate was freeze-dried to obtain bird's nest peptide powder with a molecular weight of <3000 Da.

[0103] The peptide content and ACE inhibitory activity of the bird's nest peptide powder obtained by this method are shown in Table 6. The obtained bird's nest peptide powder has a high proportion of peptides in the material composition, reaching 83.34%, and its ACE inhibitory activity is significantly higher than that of the enzyme hydrolysate of bird's nest, reaching 74.62%.

[0104] Table 6 Peptide content and ACE inhibitory activity of bird's nest peptide powder in Example 3

[0105]

[0106] Example 4

[0107] This example is to identify the bird's nest oligopeptide with a molecular weight of <3000 Da obtained in Example 3 by LC-MS / MS, and to screen out peptide segments with strong ACE inhibitory activity, good physicochemical properties and easy digestion and absorption by the human body from the identified peptide segments using bioinformatics methods. Finally, molecular docking is performed on the screened peptide segments to verify the reliability of the screening results, which specifically includes the following steps:

[0108] (1) The bird's nest peptide powder was reconstituted into a 2 mg / ml solution with ultrapure water, desalted by a C18 solid phase extraction column, and then analyzed by LC-MS / MS using an Orbitrap Eclipse mass spectrometer connected to an EASY-nanoLC1200.

[0109] Among them, the chromatographic conditions are as follows: the sample loading amount is 0.1 μL, the analysis column is Acclaim PepMap C18, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile content 80%), and the flow gradient is as follows: 2.2% B starts, linearly increases to 44% within 51 min, then increases to 90% within 3 min, and maintains for 6 min. The flow rate is 400 nL / min, and the column temperature is 40℃.

[0110] Mass spectrometry conditions: (a) MS: scan range (m / z) 200-1200, resolution 120000, AGC target 4e5, maximum injection time 50 ms; (b) HCD-MS / MS: resolution 30000, AGC target 5e4, maximum injection time 54 ms, collision energy 30%, dynamic exclusion time 30 s.

[0111] (2) Data processing: tandem mass spectrum was analyzed by PEAKS Studio version 10.6 using Denovo (de novo sequencing algorithm) and database matching combination, the database was set to Uniprot-Apodidae, none enzyme was set, the search library parameters were as follows: fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, protein card value was at least containing 1 unique peptide, peptide card value was -10 lgP≥20, and the peptides matched by the database or the Denovo results with a confidence of≥85% were reserved.

[0112] (3) The peptides identified by LC-MS / MS were first scored by the PeptideRanker tool for biological activity, and the peptides with a score of≥0.7 were selected.

[0113] (4) The peptides selected in step (3) were subjected to ACE inhibitor activity prediction by the MultiPep tool, and the peptides with an ACE inhibitor score of≥0.9 were selected.

[0114] (5) The peptides selected in step (4) were subjected to toxicity, allergenicity, water solubility and HIA, BBB properties prediction by the ToxinPred tool, the AllerTOP tool, the Proteomics tool and the admetSAR tool respectively, and the peptides with an abundance of≥1×10 7 and good overall performance of the above physicochemical properties were selected.

[0115] (6) The peptides selected in step (5) were subjected to molecular docking: 1O8A crystal was obtained from PDB database as the receptor ACE structure, the ligand and water molecules were removed using Pymol software, the molecular structure of the ACE inhibitor peptide was drawn by Chemdraw22.2.0 software, and the molecular structure was subjected to energy minimization, then the ACE receptor and small molecule peptide ligand were subjected to hydrogenation and charge calculation respectively using AutoDockTools-1.5.7, the docking box was set, the coordinates were center_x=39.995, center_y=30.621, center_z=43.626, and finally AutoDockVina was used for molecular docking.

[0116] After virtual screening of steps (3), (4), (5), two potential ACE active peptides with high score and good HIA property without toxicity and allergenicity were obtained, and the peptide sequences were YGRY and APFDGIL. The mass spectra of the peptides YGRY and APFDGIL were shown in Figure 5 、 Figure 6 .

[0117] The peptides YGRY and APFDGIL were subjected to molecular docking, and the docking results were shown in Figure 1 and Figure 3 , the interaction of the peptides YGRY and APFDGIL with ACE in the two-dimensional plane was shown in Figure 2 and Figure 4 , the peptides YGRY and APFDGIL combined with Gln281, His353, Tyr520, Lys511 and other active sites in ACE through hydrogen bond and hydrophobic interaction, thereby inhibiting the activity of ACE, and the binding energy of the molecular docking was shown as follows:

[0118] Table 7 Binding energy of the molecular docking of the peptides YGRY and APFDGIL

[0119]

[0120] Example 5

[0121] In this example, the two peptides screened in Example 4 were subjected to solid-phase synthesis, and FAPGG was used as a substrate to determine the in vitro ACE inhibitory activity of the two synthesized peptides. The determination method was as follows: a solution was prepared with 80 mmol / L HEPES buffer (pH = 8.2, containing 0.3 mol / L NaCl). 40 μL of sample solution was mixed with 50 μL of FAPGG solution (1 mmol / L), and then 10 μL of ACE enzyme solution (0.1 U / ml) was added after incubation at 37°C for 10 min. The initial absorbance value was determined at 340 nm, and then the end-point absorbance value was determined after incubation at 37°C for 30 min in the dark. The ACE activity inhibition rate was calculated according to the following formula:

[0122]

[0123] The results showed that the peptides YGRY and APFDGIL both had good ACE inhibitory activity, and the ACE inhibitory activity was shown in Figure 7 , and the IC 50 values were 133.46 μg / mL and 211.14 μg / mL, respectively.

[0124] Example 6

[0125] This example is to carry out in vitro simulated digestion of the two synthetic peptides in Example 5, and to determine their ACE inhibitory activities after digestion. Among them, in vitro simulated digestion is divided into two stages of simulated gastric digestion and simulated intestinal digestion, and the experimental method is as follows:

[0126] Gastric digestion stage: weigh an appropriate amount of YGRY or APFDGIL in a centrifuge tube, add ultrapure water to make the peptide concentration reach 1% (w / v), then prepare simulated gastric juice (containing 2000 U / ml pepsin, 47.2 mM NaCl, 6.9 mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3, 0.12 mM MgCl2, 0.5 mM (NH4)2CO3, 0.15 mM CaCl2, pH = 3), mix the peptide solution with the simulated gastric juice at a substrate protein: pepsin ratio of 10:1 (w / w), adjust the system pH to 2 with 1M HCl, and digest at 37°C for 2h, adjust the system pH to 8 with 1M NaOH, and end the gastric digestion stage.

[0127] Intestinal digestion stage: prepare simulated intestinal digestion solution (containing 2 mg / mL trypsin, 38.4 mM NaCl, 6.8 mM KCl, 0.8 mM KH2PO4, 85 mM NaHCO3, 0.33 mM MgCl2, 0.6 mM CaCl2, pH = 7), mix the solution after the end of the gastric digestion stage with the simulated intestinal solution at a ratio of 1:1 (v / v), adjust the system pH to 7 with 1M NaOH, and digest at 37°C for 2h, and then boil in water bath to inactivate the enzyme, and end the intestinal digestion stage.

[0128] The ACE inhibitory activities of the digested peptides YGRY and APFDGIL are shown in Table 1. Figure 8 The results show that the ACE activity inhibition rates of the peptides YGRY and APFDGIL after in vitro simulated digestion at each concentration are close to those before digestion, and their IC 50 values are 146.85 μg / mL and 220.69 μg / mL, respectively, which differ by no more than 11% from the IC 50 values before digestion, indicating that the peptides YGRY and APFDGIL can well retain their ACE inhibitory activities after digestion, and the peptides YGRY and APFDGIL have anti-digestion properties.

[0129] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the present application.

Claims

1. A bird's nest peptide having ACE inhibitory activity, characterized in that: The amino acid sequence of the bird's nest peptide is YGRY, i.e. Tyr-Gly-Arg-Tyr; Or, the amino acid sequence of the bird's nest peptide is APFDGIL, i.e. Ala-Pro-Phe-Asp-Gly-Ile-Leu.

2. The use of the bird's nest peptide according to claim 1 in the preparation of a functional food or a health care product.

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