Bird's nest peptide with ACE inhibitory activity as well as preparation method and application of bird's nest peptide

Abstract

The invention discloses a cubilose peptide with ACE inhibitory activity and a preparation method and application thereof, the cubilose peptide with the ACE inhibitory activity is prepared through enzymolysis, meanwhile, LC-MS / MS analysis is performed on peptides in enzymatic hydrolysate, a peptide fragment with potential ACE inhibitory activity is screened out in combination with a bioinformatics means, and the obtained peptide fragment has high ACE inhibitory activity through synthesis verification. The bird's nest ACE inhibitory peptide disclosed by the invention has a good application prospect in functional foods or health-care products and food-borne medicines.

Description

Technical Field

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

[0002] Hypertension is a common cardiovascular disease in human life, which is manifested as the diastolic or systolic blood pressure of the human body exceeding the normal range. At present, the number of people with hypertension is increasing year by year worldwide, and the age of people with hypertension is getting younger. Hypertension has seriously affected people's quality of life, and the solution to this major disease problem is imminent. Angiotensin converting enzyme (ACE) is a dipeptidyl carboxypeptidase that plays a role in regulating the balance of human physiological processes by the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). It increases blood pressure by converting to angiotensin II and hydrolyzing to bradykinin. Therefore, inhibiting the activity of ACE has become an important method for treating hypertension. At present, ACE inhibitors on the market are mainly drugs, such as captopril, enalapril, ramipril, lisinopril, etc. Although some drug-based ACE inhibitors have significant therapeutic effects, there are problems such as blood pressure rebound after discontinuation of medication, dizziness, cough, rash and other side effects after long-term use. Compared with drug-based ACE inhibitors, food-derived ACE inhibitory peptides have the advantages of a wide range of raw material sources, high safety, and strong specificity. The research on the separation and extraction of ACE inhibitory peptides from suitable food raw materials has attracted much attention. At present, studies have reported that ACE inhibitory peptides have been extracted from different raw materials such as bovine casein, soy protein, and egg white protein through fermentation, enzymatic hydrolysis, and solid-phase synthesis, and have achieved certain results.

[0003] The nutrients in bird's nest are mainly protein and sialic acid, which have high nutritional value. Traditional Chinese medicine believes that bird's nest has the effects of nourishing lung and yin, relieving cough and reducing phlegm, nourishing the face and stomach, etc. Modern research has proved that bird's nest has multiple functional activities such as anti-oxidation, anti-virus, anti-aging, promoting brain development and bone growth, whitening, and anti-inflammatory. Bird's nest peptides are an important source of substances that produce these functional activities. At present, the application of bird's nest peptides in functional foods, health products, and cosmetics is mostly based on its antioxidant, whitening, anti-aging and anti-inflammatory activities, while there are few studies on the ACE inhibitory activity of bird's nest peptides. The methods for extracting bird's nest peptides from natural bird's nests mainly include extraction, enzymatic hydrolysis, and fermentation. Among them, enzymatic hydrolysis is widely used in the extraction of different types of bioactive peptides due to its mild conditions, safety, environmental protection, and strong specificity. However, bird's nest protein has poor water solubility. Enzymatic extraction under conventional conditions is prone to problems such as low protein dissolution and low degree of hydrolysis. If other extraction methods are used as auxiliary, the process flow will be complicated, the extraction cycle will be extended, and the production cost will increase. Therefore, optimizing the enzymatic hydrolysis process conditions and finding a simple, safe and efficient enzymatic extraction method for bird's nest peptides with ACE inhibitory activity are of great significance for broadening the application of bird's nest peptides in food-borne antihypertensive drugs. Summary of the invention

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

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

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

[0007] In order to solve the above technical problems, the present invention 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] Alternatively, the amino acid sequence of the bird's nest peptide is APFDGIL, i.e. Ala-Pro-Phe-Asp-Gly-Ile-Leu.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide a bird's nest peptide with ACE inhibitory activity for use in functional foods or health products, and food-derived medicines.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a bird's nest peptide having ACE inhibitory activity, characterized in that: comprising:

[0011] Mix the bird's nest silk that has been cleaned and air-dried with pure water and soak it;

[0012] Boil the soaked bird's nest for 30 minutes and then cool to room temperature;

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

[0014] Add protease to the bird's nest homogenate for enzymatic hydrolysis, then inactivate the enzyme, centrifuge at 10000×g for 10 min, and obtain the enzymatic hydrolysis supernatant;

[0015] Ultrafiltration and freeze drying were performed to obtain bird's nest peptide powder <3 kDa;

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

[0017] Screening of peptides with potential to inhibit ACE activity;

[0018] The screened peptides were verified using molecular docking technology;

[0019] The screened peptides were synthesized chemically in solid phase and their ACE inhibitory activity was evaluated in vitro.

[0020] As a preferred embodiment of the preparation method of the present invention, the bird's nest filaments are mixed with pure water and foamed, wherein the mass ratio of the bird's nest filaments to the pure water is 1:20 to 1:40.

[0021] As a preferred embodiment of the preparation method of the present invention, the bird's nest after being soaked is stewed for 30 minutes, wherein the stewing temperature is 90°C to 120°C.

[0022] As a preferred embodiment of the preparation method of the present invention, the stewed bird's nest liquid is shear dispersed, wherein the shear rate is 8000 rpm and the shear time is 5 min.

[0023] As a preferred embodiment of the preparation method of the present invention, wherein: a protease is added to the bird's nest homogenate for enzymatic hydrolysis, wherein the protease is at least one of trypsin, papain, alkaline protease, neutral protease, and flavor protease.

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

[0025] As a preferred embodiment of the preparation method of the present invention, the screening of peptides with potential to inhibit ACE activity includes: using the PeptideRanker tool to score the biological activity of the peptide sequences identified by LC-MS / MS, and selecting peptides with a score ≥0.7; using the MultiPep tool to predict the ACE inhibitory activity of the selected peptides, and selecting peptides with an ACEinhibitor score ≥0.9; using the ToxinPred tool, AllerTOP tool, Proteomics tool and admetSAR tool to predict the toxicity, sensitization, water solubility and HIA, BBB properties of the peptides with an abundance of ≥1×10 7 , and the above-mentioned physicochemical properties generally show good peptides.

[0026] As a preferred embodiment of the preparation method of the present invention, the peptide screened out is verified by using molecular docking technology, including obtaining 1O8A crystal as the receptor ACE structure from the PDB database, deleting its ligand and water molecules using Pymol software, drawing the molecular structure of the ACE inhibitory peptide using Chemdraw 22.2.0 software, and minimizing the energy of the molecular structure, and then using AutoDockTools-1.5.7 to process the ACE receptor and the small molecule peptide ligand respectively, setting the docking box, and finally using AutoDockVina to perform molecular docking.

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention provides a method for preparing bird's nest peptides with ACE inhibitory activity by enzymatic hydrolysis, and simultaneously performing LC-MS / MS analysis on the peptides in the enzymatic hydrolyzate, and screening out peptides with potential ACE inhibitory activity by combining bioinformatics means, and the obtained peptides are synthesized and verified to have high ACE inhibitory activity. The bird's nest ACE inhibitory peptides in the present invention have good application prospects in functional foods or health products, and food-borne drugs.

[0029] (2) The present invention stews the bird's nest before enzymatic hydrolysis. Preliminary experiments have shown that after high-temperature heat treatment, the conformation of the bird's nest protein changes, the internal structure becomes loose, and the hydrophobic groups are exposed, thereby increasing the opportunity for the protease to interact with the bird's nest protein, so that the protein extraction degree of the stewed bird's nest homogenate is higher than that of the dry bird's nest raw material, and the degree of hydrolysis is deeper.

[0030] (3) The present invention has studied a method for efficiently preparing bird's nest peptides with ACE inhibitory activity by screening proteases and optimizing the enzymatic hydrolysis process. The preferred protease in the present invention is a combination of alkaline protease and flavor protease. Alkaline protease specifically hydrolyzes the ends of hydrophobic amino acids and is widely used in the preparation of ACE inhibitory peptides. The amino acid composition of bird's nest protein is mainly hydrophobic amino acids, which can be well hydrolyzed by alkaline protease. Flavor protease has the characteristics of both endo- and exo-enzymes, with more enzyme cleavage sites, and can quickly hydrolyze large molecular weight peptides into short peptides. The process flow of this method is simple and easy to operate. The bird's nest peptide prepared by this method not only has a high protein extraction rate and a high degree of hydrolysis, but also has good ACE inhibitory activity.

[0031] (4) The traditional bioactive peptide screening method first performs a series of separation and purification operations such as ion exchange, gel filtration, and reversed-phase high-performance liquid chromatography on the peptide, and then identifies the purified peptide to obtain the target bioactive peptide. The operation process is cumbersome, time-consuming, and labor-intensive, and it is difficult to apply to large-scale industrial production. This method first performs LC-MS / MS identification on the bird's nest peptide, and then virtually screens out the target bioactive peptide through a bioinformatics method. The obtained target bioactive peptide not only has high ACE inhibitory activity, but also has good physical and chemical properties, and can be better absorbed and utilized by the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

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

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

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

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

[0037] Figure 5 This is the mass spectrum of YGRY.

[0038] Figure 6 This is the mass spectrum of APFDGIL.

[0039] Figure 7is the ACE activity inhibition rate of peptides YGRY and APFDGIL.

[0040] Figure 8 It is the ACE activity inhibition rate of peptides YGRY and APFDGIL after simulated digestion in vitro. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0042] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

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

[0045] Bird’s nest: Xiamen Yanzhiwu Sinong Food Co., Ltd.; alkaline protease, flavor protease, papain, neutral protease: Shanghai Bomeifeike Chemical Technology Co., Ltd.; trypsin: Sigma-Aldrich, USA; BCA protein concentration determination kit: Shanghai Biyuntian Biotechnology Co., Ltd.; sodium hydroxide, formaldehyde: Shanghai Titan Technology Co., Ltd.; ACE: Nanjing Dulai Biotechnology Co., Ltd.; acetonitrile: Honeywell, USA.

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

[0047] HH-2 digital constant temperature water bath: Changzhou Ronghua Instrument Manufacturing Co., Ltd.; YXQ-LS-50A vertical pressure steam sterilizer: Wuxi Capex Technology Co., Ltd.; AvantiJXN-26 intelligent high-efficiency centrifuge, BeckmanCoulter, USA; KDN-16C nitrogen analyzer: Shanghai Fiber Inspection Instrument Co., Ltd.; M2 microplate reader: Molecular Devices, USA; XX80EL230 tangential flow ultrafiltration system: MilliPore, Germany; UltiMate 3000 high performance liquid chromatograph, Orbitrap Eclipse mass spectrometer: Thermo Fisher, USA.

[0048] Example 1

[0049] This embodiment provides a method for preparing a bird's nest enzymatic hydrolysis supernatant, comprising the following steps:

[0050] (1) Mix the cleaned bird's nest silk with purified water in a ratio of raw material to water = 1:25 (w / w), and soak for 1 hour.

[0051] (2) Stew the soaked bird's nest at 120°C for 30 minutes and cool to room temperature after stewing.

[0052] (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.

[0053] (4) Add composite protease (alkaline protease: flavor protease = 2:1) to the bird's nest homogenate, the total enzyme amount is 7000U / g (enzyme activity / bird's nest raw material mass), control the pH to 8, enzymolyze at 50°C for 2.5h, inactivate the enzyme in a boiling water bath for 10min, cool to room temperature, and centrifuge at 10000×g for 10min to obtain the bird's nest enzymolysis supernatant.

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

[0055] Protein extraction rate determination method: The protein content in the bird's nest raw material was determined by the Kjeldahl method (GB5009.5-2016), and the protein content in the bird's nest enzymatic supernatant was determined by the BCA method. The determination process was carried out according to the instructions of the BCA protein concentration determination kit, and the protein extraction rate was calculated according to the following formula:

[0056]

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

[0058]

[0059] ACE inhibitory activity determination method: freeze-dry the bird's nest enzymatic hydrolysis supernatant and redissolve it to a sample solution with a concentration of 2 mg / ml, and prepare the solution with 80 mmol / L HEPES buffer (pH = 8.2, containing 0.3 mol / L NaCl). Mix 40 μl of sample solution with 50 μl FAPGG solution (1 mmol / L), incubate at 37°C for 10 minutes, and then add 10 μl ACE enzyme solution (0.1 U / ml). Measure the initial absorbance value (A0 and B0) at 340 nm, and then measure the end point absorbance value (A1 and B1) after reacting at 37°C in the dark for 30 minutes. The ACE activity inhibition rate is calculated as follows:

[0060]

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

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

[0063]

[0064] Example 2

[0065] The difference between this embodiment and embodiment 1 is that the enzymolysis conditions are different, and the following steps are included:

[0066] (1) Mix the cleaned bird's nest silk with purified water in a ratio of raw material to water = 1:25 (w / w), and soak for 1 hour.

[0067] (2) Stew the soaked bird's nest at 120°C for 30 minutes and cool to room temperature after stewing.

[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) Add composite protease (trypsin: flavor protease = 2:1) to the bird's nest homogenate, the total enzyme amount is 7000U / g (enzyme activity / bird's nest raw material mass), control the pH to 7.5, enzymolyze at 45°C for 2.5h, inactivate the enzyme in a boiling water bath for 10min, cool to room temperature, and centrifuge at 10000×g for 10min to obtain the bird's nest enzymolysis supernatant.

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

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

[0072]

[0073] Comparative Example 1

[0074] The difference between this comparative example and Example 1 is that the bird's nest silk after being soaked is directly enzymatically hydrolyzed, eliminating the process of stewing and homogenizing the bird's nest, specifically:

[0075] (1) Mix the cleaned bird's nest silk with purified water in a ratio of raw material to water = 1:25 (w / w), and soak for 1 hour.

[0076] (2) Adding a composite protease (alkaline protease: flavor protease = 2:1) to the soaked bird's nest liquid, the total enzyme amount is 7000U / g (enzyme activity / bird's nest raw material mass), controlling the pH to 8, enzymolysis at 50°C for 2.5h, inactivating the enzyme in a boiling water bath for 10min, cooling to room temperature, and centrifuging at 10000×g for 10min to obtain the bird's nest enzymolysis supernatant.

[0077] The protein extraction rate, protein hydrolysis degree and ACE inhibitory activity of the enzymatic hydrolyzate obtained in Comparative Example 1 are shown in Table 3. The results show that the protein extraction rate, protein hydrolysis degree and ACE inhibitory activity of the enzymatic hydrolyzate prepared by this method are significantly reduced compared with those in Example 1.

[0078] Table 3 Protein extraction rate, hydrolysis degree and ACE inhibitory activity of bird's nest enzymatic 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 composite protease is adjusted to 1:1 neutral protease and papain, specifically:

[0082] (1) Mix the cleaned bird's nest silk with purified water in a ratio of raw material to water = 1:25 (w / w), and soak for 1 hour.

[0083] (2) Stew the soaked bird's nest at 120°C for 30 minutes and cool to room temperature after stewing.

[0084] (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.

[0085] (4) Add composite protease (neutral protease: papain = 1:1) to the bird's nest homogenate, the total enzyme amount is 7000U / g (enzyme activity / bird's nest raw material mass), control the pH to 7, enzymolyze at 55°C for 2.5h, inactivate the enzyme in a boiling water bath for 10min, cool to room temperature, and centrifuge at 10000×g for 10min to obtain the bird's nest enzymolysis supernatant.

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

[0087] Table 4 Protein extraction rate, hydrolysis degree and ACE inhibitory activity of bird's nest enzymatic hydrolysate in comparative example 2

[0088]

[0089] Comparative Example 3

[0090] The difference between this comparative example and Example 1 is that the enzymolysis time is extended to 4 hours, specifically:

[0091] (1) Mix the cleaned bird's nest silk with purified water in a ratio of raw material to water = 1:25 (w / w), and soak for 1 hour.

[0092] (2) Stew the soaked bird's nest at 120°C for 30 minutes and cool to room temperature after stewing.

[0093] (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.

[0094] (4) Adding composite protease (alkaline protease: flavor protease = 2:1) to the bird's nest homogenate, the total enzyme amount is 7000U / g (enzyme activity / bird's nest raw material mass), controlling the pH to 8, enzymolysis at 50°C for 4h, inactivating the enzyme in a boiling water bath for 10min, cooling to room temperature and centrifuging at 10000×g for 10min to obtain the bird's nest enzymolysis supernatant.

[0095] The protein extraction rate, protein hydrolysis degree and ACE inhibitory activity of the enzymatic hydrolyzate obtained in Comparative Example 3 are shown in Table 5. The results show that the protein extraction rate of the enzymatic hydrolyzate prepared by this method is slightly increased, the hydrolysis degree is slightly decreased, and the ACE inhibitory activity is significantly decreased compared with Example 1.

[0096] Table 5 Protein extraction rate, hydrolysis degree and ACE inhibitory activity of bird's nest enzymatic hydrolysate in comparative example 3

[0097]

[0098]

[0099] Example 3

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

[0101] (1) The bird's nest hydrolysate was ultrafiltered using a tangential flow ultrafiltration system, and the bird's nest hydrolysate supernatant was passed through ultrafiltration membranes with a molecular weight cutoff of 10,000 Da and a molecular weight cutoff of 3,000 Da in sequence, and the final permeate was collected.

[0102] (2) freeze-drying the permeate to obtain bird's nest peptide powder with a molecular weight of less than 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 peptide content in the material composition of the obtained bird's nest peptide powder is high, reaching 83.34%, and its ACE inhibitory activity is significantly higher than that of the bird's nest enzymatic hydrolysate, 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 perform LC-MS / MS identification on the bird's nest oligopeptides with a molecular weight of <3000Da obtained in Example 3, and screen out peptides with strong ACE inhibitory activity, good physicochemical properties, and easy digestion, absorption and utilization by the human body from the identified peptides using a bioinformatics method, and finally perform molecular docking on the screened peptides to verify the reliability of the screening results, specifically comprising the following steps:

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

[0109] The chromatographic conditions were as follows: the sample loading volume was 0.1 μL, the analytical column was Acclaim Pep Map C18, the mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile content 80%), the mobile gradient was: starting with 2.2% B, linearly increasing to 44% within 51 min, then increasing to 90% within 3 min, and maintaining for 6 min. The flow rate was 400 nL / min, and the column temperature was 40°C.

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

[0111] (2) Data processing: The tandem mass spectra were analyzed by PEAKS Studio version 10.6 and analyzed by combining Denovo (de novo sequencing algorithm) with database matching. The database was set to Uniprot-Apodidae, and the enzyme digestion was set to none. The search parameters included the fragment ion mass tolerance of 0.02 Da, the parent ion mass tolerance of 10 ppm, the protein card value of at least 1 unique peptide, and the peptide card value of -101 GP ≥ 20. Peptides matched by the database or with a Denovo result confidence of ≥ 85% were retained.

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

[0113] (4) The peptides selected in step (3) were used to predict ACE inhibitory activity using the MultiPep tool, and peptides with an ACEinhibitor score ≥ 0.9 were selected.

[0114] (5) The peptides selected in step (4) were used to predict the toxicity, sensitization, water solubility, HIA and BBB properties of the peptides using the ToxinPred tool, AllerTOP tool, Proteomics tool and admetSAR tool, respectively. 7 , and the above-mentioned physicochemical properties generally show good peptides.

[0115] (6) Molecular docking of the peptide selected in step (5): 1O8A crystal was obtained from the PDB database as the receptor ACE structure, and its ligand and water molecules were deleted using Pymol software. The molecular structure of the ACE inhibitory peptide was drawn using Chemdraw22.2.0 software, and the molecular structure was energy minimized. Then, AutoDockTools-1.5.7 was used to hydrogenate the ACE receptor and the small molecule peptide ligand, calculate the charge, etc., and set the docking box with coordinates of center_x=39.995, center_y=30.621, and center_z=43.626. Finally, AutoDockVina was used for molecular docking.

[0116] After virtual screening in steps (3), (4), and (5), two potential peptides with high ACE activity scores, non-toxicity, non-sensitization, and good HIA properties were finally obtained. The peptide sequences are: YGRY and APFDGIL. The mass spectra of peptides YGRY and APFDGIL are shown in Figure 2. Figure 5 , Figure 6 shown.

[0117] The peptides YGRY and APFDGIL were subjected to molecular docking, and the docking results were as follows: Figure 1 and Figure 3 As shown, the interactions between peptides YGRY and APFDGIL and ACE on the two-dimensional plane are shown in Figure 2 and Figure 4 As shown, peptides YGRY and APFDGIL bind to multiple active sites in ACE, such as Gln281, His353, Tyr520, and Lys511, through hydrogen bonds and hydrophobic interactions, thereby inhibiting ACE activity. The binding energy of their molecular docking is shown below:

[0118] Table 7 Molecular docking binding energy of peptide 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 synthetic peptides. The determination method was as follows: a 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), incubated at 37°C for 10 min, and then 10 μL of ACE enzyme solution (0.1 U / ml) was added. The initial absorbance was determined at 340 nm, and then the end point absorbance was determined after 30 min of reaction at 37°C in the dark. The ACE activity inhibition rate was calculated as follows:

[0122]

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

[0124] Example 6

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

[0126] Gastric digestion stage: weigh an appropriate amount of YGRY or APFDGIL into a centrifuge tube, add ultrapure water to make the peptide concentration reach 1% (w / v), then prepare simulated gastric digestion fluid (containing 2000U / ml pepsin, 47.2mM NaCl, 6.9mM KCl, 0.9mM KH2PO4, 25mM NaHCO3, 0.12mM MgCl2, 0.5mM (NH4)2CO3, 0.15mM CaCl2, pH = 3), mix the peptide solution with simulated gastric fluid at a ratio of substrate protein: pepsin = 10:1 (w / w), adjust the system pH to 2 with 1M HCl, digest in a water bath 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 fluid (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 gastric digestion stage with the simulated intestinal fluid in a ratio of 1:1 (v / v), adjust the pH of the system to 7 with 1 M NaOH, digest in a water bath at 37°C for 2 h, inactivate the enzyme in a boiling water bath, and end the intestinal digestion stage.

[0128] The ACE inhibitory activities of the digested peptides YGRY and APFDGIL were as follows Figure 8 The results showed that the ACE activity inhibition rates of peptides YGRY and APFDGIL after in vitro simulated digestion at each concentration were close to those before digestion, and their IC 50 The values ​​were 146.85 μg / mL and 220.69 μg / mL, respectively, which were consistent with the IC 50 The values ​​differed by no more than 11%, indicating that peptides YGRY and APFDGIL could well retain their ACE inhibitory activity after digestion, and peptides YGRY and APFDGIL had anti-digestion properties.

[0129] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention 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 invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. 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; Alternatively, the amino acid sequence of the bird's nest peptide is APFDGIL, i.e. Ala-Pro-Phe-Asp-Gly-Ile-Leu.

2. Use of the bird's nest peptide as claimed in claim 1 in functional foods or health products, and food-derived medicines.

3. The method for preparing a bird's nest peptide having ACE inhibitory activity according to claim 1, characterized in that: include, Mix the bird's nest silk that has been cleaned and air-dried with pure water and soak it; Boil the soaked bird's nest for 30 minutes and then cool to room temperature; The stewed bird's nest liquid is sheared and dispersed to obtain a bird's nest homogenate; Add protease to the bird's nest homogenate for enzymatic hydrolysis, then inactivate the enzyme, and centrifuge at 10000×g for 10 min to obtain an enzymatic hydrolysis supernatant; Ultrafiltration and freeze drying were performed to obtain bird's nest peptide powder <3 kDa; LC-MS / MS peptide identification of bird's nest peptide powder <3kDa; Screening of peptides with potential to inhibit ACE activity; The screened peptides were verified using molecular docking technology; The screened peptides were synthesized chemically in solid phase and their ACE inhibitory activity was evaluated in vitro.

4. The preparation method according to claim 3, characterized in that: The bird's nest filaments are mixed with pure water and foamed, wherein the mass ratio of the bird's nest filaments to the pure water is 1:20 to 1:

40.

5. The preparation method according to claim 3, characterized in that: The soaked bird's nest is stewed for 30 minutes, wherein the stewing temperature is 90° C. to 120° C.

6. The preparation method according to claim 3, characterized in that: The stewed bird's nest liquid is sheared and dispersed, wherein the shear rate is 8000rpm and the shear time is 5min.

7. The preparation method according to claim 3, characterized in that: The protease is added to the bird's nest homogenate for enzymolysis, wherein the protease is at least one of trypsin, papain, alkaline protease, neutral protease and flavor protease.

8. The preparation method according to claim 7, characterized in that: The amount of enzyme used is 4000-9000 U / g, the enzymolysis temperature is 40-55° C., the enzymolysis time is 1.5-3.5 hours, and the enzymolysis pH is 6.5-9.

5.

9. The preparation method according to claim 3, characterized in that: The screening of peptides with potential ACE inhibitory activity includes: using the PeptideRanker tool to score the biological activity of the peptide sequences identified by LC-MS / MS, and selecting peptides with a score of ≥0.7; using the MultiPep tool to predict the ACE inhibitory activity of the selected peptides, and selecting peptides with an ACE inhibitor score of ≥0.9; using the ToxinPred tool, the AllerTOP tool, the Proteomics tool, and the admetSAR tool to predict the toxicity, sensitization, water solubility, and HIA and BBB properties of the peptides with an ACE inhibitor score of ≥0.9, and selecting peptides with an abundance of ≥1×10 7 , and the above-mentioned physicochemical properties generally show good peptides.

10. The preparation method according to claim 3, characterized in that: The screened peptides were verified using molecular docking technology, including obtaining 1O8A crystals as the receptor ACE structure from the PDB database, deleting its ligands and water molecules using Pymol software, drawing the molecular structure of the ACE inhibitory peptide using Chemdraw22.2.0 software, and minimizing the energy of the molecular structure, then using AutoDockTools-1.5.7 to process the ACE receptor and the small molecule peptide ligand respectively, setting the docking box, and finally using AutoDockVina for molecular docking.

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