Spirulina-derived ACE inhibitory peptide as well as preparation method and application thereof

Abstract

The invention provides a spirulina-derived ACE (angiotensin converting enzyme) inhibitory peptide as well as a preparation method and application thereof, and belongs to the technical field of marine bioactive peptides. Spirulina phycobiliprotein is used for preparing the ACE inhibitory peptide of the spirulina source, five kinds of small molecule polypeptides including YATY, RYVTY, YVTY, KAYF and SPSWY are obtained through co-screening, the five kinds of polypeptides all have good ACE inhibitory activity, and the RYVTY activity is the highest. The spirulina-derived polypeptide prepared by the preparation method disclosed by the invention has high hydrolysis degree and high ACE inhibitory activity, can be applied to the fields of food, health care products, medicines and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of marine bioactive peptides, and in particular to a spirulina-derived ACE inhibitory peptide and a preparation method and application thereof. Background Art

[0002] Hypertension is a common chronic disease that is closely related to non-communicable diseases such as cardiovascular disease, stroke and chronic kidney disease. The number of people suffering from hypertension is as high as 245 million (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg or long-term use of antihypertensive drugs). Hypertension has become one of the main causes of death and disability.

[0003] Angiotensin-I converting enzyme (ACE) is a Zn-converting enzyme widely present in mammalian tissues such as neuroepithelial cells, vascular endothelial cells and male germ cells. 2+ ACE is a key enzyme in the renin-angiotensin aldosterone system and the kinin-kallikrein system. Its activity greatly affects the body's electrolyte balance and blood pressure regulation process. In the renin-angiotensin aldosterone system, ACE can convert angiotensin I, which has no pressor activity, into angiotensin II, which has pressor activity, and at the same time stimulate the adrenal glands to release aldosterone, causing Na in the kidneys. + In the kinin-kallikrein system, ACE cleaves the dipeptide fragment Phe-Arg at the carboxyl end of bradykinin, causing it to lose its antihypertensive activity. Therefore, inhibiting the activity of ACE has become one of the effective methods for intervening and treating hypertension. However, the commonly used chemically synthesized ACE inhibitors (captopril, enalapril, lisinopril, etc.) have been proven to cause side effects such as dry cough, leukopenia, taste disorders, rash, angioedema and renal insufficiency. Therefore, ACE inhibitory peptides from natural sources are receiving more and more attention due to their high safety.

[0004] Spirulina ( Spirulina ) is rich in essential nutrients such as protein, lipids, carbohydrates, vitamins, minerals, etc. It has multiple physiological effects such as lowering cholesterol, regulating immunity, anti-inflammatory, anti-fatigue and anti-cancer, and has been recommended by the Food and Agriculture Organization of the United Nations as "the most ideal health food for mankind in the 21st century". At present, in the food industry, it is only added to smoothies, energy bars and other foods as a functional food matrix, or used as a dietary supplement in the form of tablets and powders. The very small amount of spirulina in some products shows that it is only used as a colorant or as a commercial gimmick, and its nutritional value has not been fully utilized. Summary of the invention

[0005] The purpose of the present invention is to provide a spirulina-derived ACE inhibitory peptide, and to provide a preparation method and specific application of the ACE inhibitory peptide, so as to make up for the deficiencies of the prior art.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows: The ACE inhibitory peptides derived from Spirulina include YATY, or RYVTY, or YVTY, or KAYF, or SPSWY.

[0007] Among them, the specific sequence of YATY is SEQ No.1: Tyr-Ala-Thr-Tyr; The specific sequence of RYVTY is SEQ No. 2: Arg-Tyr-Val-Thr-Tyr; The specific sequence of YVTY is SEQ No. 3: Tyr-Val-Thr-Tyr; The specific sequence of KAYF is SEQ No. 4: Lys-Ala-Tyr-Phe; The specific sequence of SPSWY is SEQ No. 5: Ser-Pro-Ser-Trp-Tyr.

[0008] Preferably, a Spirulina-derived ACE inhibitory peptide with higher activity is RYVTY.

[0009] The method for preparing the ACE inhibitory peptide derived from Spirulina comprises the following steps: (1) Adding spirulina to ammonium bicarbonate buffer and mixing evenly, repeatedly freezing and thawing to break the cell wall of spirulina to obtain a spirulina suspension; separating the supernatant and precipitate by freeze centrifugation, wherein the supernatant is the spirulina soluble extract; purifying the spirulina soluble extract by ammonium sulfate stepwise precipitation method, and desalting by dialysis to obtain a phycobiliprotein purified solution; (2) adjusting the pH of the solution, adding alkaline protease for enzymatic hydrolysis, boiling to inactivate the enzyme, and centrifuging the sample after cooling to room temperature. The supernatant is a mixture of ACE inhibitory peptides derived from Spirulina. (3) solid-liquid separation and vacuum drying to obtain lyophilized powder of Spirulina; (4) using distilled water to reconstitute the lyophilized powder of Spirulina obtained in step (3), desalting it, and then identifying the polypeptide sequence thereof by liquid chromatography-mass spectrometry; (5) Using an online platform to predict ACE inhibitory activity, physical and chemical properties, and toxicity analysis of the peptide sequence identified in step (4), and screen for potential ACE inhibitory peptides; (6) The screened peptides are subjected to solid phase synthesis, and the activity of the potential ACE inhibitory peptides obtained in step (5) is verified by in vitro activity evaluation, thereby finally obtaining the 5 Spirulina-derived ACE inhibitory peptides.

[0010] Furthermore, the step (4) uses a C18 desalting column for desalting.

[0011] Furthermore, in step (5), the online platform is used to screen potential ACE inhibitory peptides, including using the AHTpin tool to predict the ACE inhibitory activity of peptides identified by LC-MS / MS of the enzymatic hydrolysate, and selecting peptides with an activity score ≥ 0; obtaining the crystal structure of the sACE domain from the RCSB PDB database as a receptor molecule (C domain PDB ID: 1O8A; N domain PDB ID: 2C6F), performing docking pre-processing on the ACE protein receptor and the small molecule polypeptide ligand, setting up a docking box, and finally using Autodock Vina to perform molecular docking; using the ToxinPred tool to predict the physicochemical properties and toxicity analysis of the ACE inhibitory peptides predicted and screened by the AHTpin tool and the molecular docking tool, wherein the predicted physicochemical properties include steric hindrance, isoelectric point, hydrophobicity and hydrophilicity.

[0012] Furthermore, in step (6), the solid phase synthesized polypeptide is evaluated for in vitro ACE inhibitory activity using high performance liquid chromatography using hippuryl-histidyl-leucine (HHL) as a substrate, and the half inhibitory concentration (IC 50 ) indicates its activity level, among which IC 50 It is the peptide concentration corresponding to 50% ACE inhibitory activity.

[0013] The use of the spirulina-derived ACE inhibitory peptide YATY, or RYVTY, or YVTY, or KAYF, or SPSWY in the preparation of products with blood pressure lowering effect: the products include health foods, medicines, etc.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are: The present invention uses Spirulina phycobiliprotein to prepare Spirulina-derived ACE inhibitory peptides, and a total of 5 small molecule polypeptides are screened: YATY, RYVTY, YVTY, KAYF and SPSWY, all of which have good ACE inhibitory activity, among which RYVTY has the highest activity. The Spirulina-derived polypeptide prepared by the present invention has both high hydrolysis degree and high ACE inhibitory activity, can be applied to the fields of food, health products, medicines, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the hydrolysis degree and ACE inhibition rate of different samples in Example 2, wherein the bar graph shows the ACE inhibition rate of different samples, and the line graph shows the corresponding hydrolysis degree. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. What is described here are some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0017] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0018] Example 1

[0019] This embodiment provides a method for preparing a mixture of ACE inhibitory peptides derived from Spirulina, comprising the following steps: (1) Add ammonium bicarbonate buffer (pH 7.0) to Spirulina platensis at a ratio of 1:10 g / mL, mix well, freeze at -20°C for 12 h, then thaw at room temperature in the dark, repeat freeze-thaw cycles 3 times, then centrifuge at 8000 rpm for 20 min, slowly add ammonium sulfate to the supernatant until the ammonium sulfate saturation reaches 25%, stir at 4°C in the dark for 1 h, and centrifuge for 20 min, then add ammonium sulfate to the supernatant again to increase the ammonium sulfate saturation to 50%, stir at 4°C in the dark for 2 h, and centrifuge for 20 min, re-dissolve the secondary precipitate with as little deionized water as possible, dialyze and desalt at 4°C, and lyophilize to obtain the purified phycobiliprotein.

[0020] (2) The purified phycobiliprotein was reconstituted to 10 mg / mL in 0.1 mol / L phosphate buffer and the pH of the solution was adjusted to 8.0. Alkaline protease was added at an enzyme-substrate ratio of 2% (w / w). The mixture was oscillated at 50°C for 4 h and the enzyme was inactivated by boiling. After the sample was cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of ACE inhibitory peptides derived from Spirulina.

[0021] Comparative Example 1 The difference between this comparative example and Example 1 is that the protease used is papain, and the enzymatic hydrolysis conditions are appropriately adjusted: the purified phycobiliprotein is redissolved to 10 mg / mL in 0.1 mol / L phosphate buffer, and the pH of the solution is adjusted to 7.0, papain is added at an enzyme-substrate ratio of 2% (w / w), and the enzymatic hydrolysis is carried out at 50°C for 4 h with shaking, and the enzyme is inactivated by boiling. After the sample is cooled to room temperature, it is centrifuged at 10000 rpm for 15 min, and the supernatant is a mixture of ACE inhibitory peptides from Spirulina.

[0022] Comparative Example 2 The difference between this comparative example and Example 1 is that the protease used is trypsin, and the enzymatic hydrolysis conditions are appropriately adjusted: the purified phycobiliprotein is redissolved to 10 mg / mL in 0.1 mol / L phosphate buffer, and the pH of the solution is adjusted to 8.0, trypsin is added at an enzyme-substrate ratio of 2% (w / w), and the enzymatic hydrolysis is carried out at 50°C for 4 h with shaking, and the enzyme is inactivated by boiling. After the sample is cooled to room temperature, it is centrifuged at 10000 rpm for 15 min, and the supernatant is a mixture of ACE inhibitory peptides from Spirulina.

[0023] Comparative Example 3 The difference between this comparative example and Example 1 is that the protease used is a neutral protease, and the enzymatic hydrolysis conditions are appropriately adjusted: the purified phycobiliprotein is redissolved to 10 mg / mL using 0.1 mol / L phosphate buffer, and the pH of the solution is adjusted to 7.0, neutral protease is added according to an enzyme-substrate ratio of 2% (w / w), and the enzymatic hydrolysis is carried out under shaking at 50°C and 120 rpm for 4 h, and the enzyme is boiled to inactivate the enzyme. After the sample is cooled to room temperature, it is centrifuged at 10,000 rpm for 15 min, and the supernatant is a mixture of ACE inhibitory peptides from Spirulina.

[0024] Comparative Example 4 The difference between this comparative example and Example 1 is that the protease used is flavor protease, and the enzymatic hydrolysis conditions are appropriately adjusted: the purified phycobiliprotein is redissolved to 10 mg / mL in 0.1 mol / L phosphate buffer, and the pH of the solution is adjusted to 7.5, flavor protease is added at an enzyme-substrate ratio of 2% (w / w), and the enzymatic hydrolysis is carried out at 50°C for 4 h with shaking, and the enzyme is inactivated by boiling. After the sample is cooled to room temperature, it is centrifuged at 10000 rpm for 15 min, and the supernatant is a mixture of ACE inhibitory peptides from Spirulina.

[0025] Comparative Example 5 The difference between this comparative example and Example 1 is that the protease used is pepsin, and the enzymatic hydrolysis conditions are appropriately adjusted: the purified phycobiliprotein is redissolved to 10 mg / mL in 0.1 mol / L phosphate buffer, and the pH of the solution is adjusted to 2.0, pepsin is added at an enzyme-substrate ratio of 2% (w / w), and the enzymatic hydrolysis is carried out at 50°C for 4 h with shaking, and the enzyme is inactivated by boiling. After the sample is cooled to room temperature, it is centrifuged at 10000 rpm for 15 min, and the supernatant is a mixture of ACE inhibitory peptides from Spirulina.

[0026] Example 2

[0027] This example uses formaldehyde titration to determine the amino nitrogen content in the Spirulina-derived ACE inhibitory peptide mixture obtained in Example 1 and Comparative Examples 1-5, and further calculates the degree of hydrolysis of the mixture obtained by enzymatic hydrolysis, specifically comprising the following steps: (1) Determination of total nitrogen content With reference to GB 5009.5-2016 “National Food Safety Standard Determination of Protein in Food”, the total nitrogen content in the purified phycobiliprotein was determined by the Kjeldahl method.

[0028] (2) Determination of amino nitrogen content The amino nitrogen content in the enzymatic hydrolysate was determined by formaldehyde titration. 1 mL of 0.5% phenolphthalein ethanol solution was added to 50 mL of formaldehyde aqueous solution and titrated with 0.01 M NaOH standard solution until it turned slightly red, which was recorded as neutral formaldehyde solution. The pH of all enzymatic hydrolysates was adjusted to 8.0, and 400 μL of enzymatic hydrolysate (recorded as V 2 ), 7.6 mL of distilled water and 2 drops of phenolphthalein indicator, add 2 mL of neutral formaldehyde solution to the mixture, mix well and titrate with 0.01 M NaOH standard solution, and record the volume of NaOH consumed by each group. V 1 Distilled water was used instead of the enzymatic solution as a blank control, and the volume of NaOH consumed was recorded as V 0 The calculation formula of amino nitrogen (AN) in the enzymatic hydrolysate is as follows: Where, is the molar concentration of NaOH.

[0029] (3) Calculation of degree of hydrolysis The calculation formula of the degree of hydrolysis (DH) of the enzymatic hydrolyzate is as follows: Wherein, N represents the total nitrogen content in phycobiliprotein before enzymatic hydrolysis; AN represents the amino nitrogen content in the enzymatic hydrolyzate.

[0030] In this example, FAPGG was used as a substrate, and the inhibitory activity of the spirulina-derived ACE inhibitory peptide mixture obtained in Example 1 and Comparative Examples 1-5 was determined using an ELISA instrument, specifically comprising the following steps: (1) Reagent preparation 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L sodium chloride): Accurately weigh 3.8137 g sodium tetraborate (borax) and 0.6183 g boric acid, heat and dissolve them in deionized water, and then dilute to 100 mL for use. Mix the two solutions until the pH of the solution is 8.3. Take 100 mL of the mixed solution, add 1.7532 g sodium chloride, and dissolve and mix.

[0031] 0.1 U / mL ACE enzyme solution: Dissolve 0.1 U ACE enzyme in 1 mL of the above borate buffer.

[0032] 1 mmol / L N-[3-(2-furyl)acryloyl]-L-phenylalanamide-glycine-glycine (FAPGG) solution: Accurately weigh 3.994 mg of FAPGG powder, dissolve it in the above borate buffer and make up to 10 mL.

[0033] 3 mg / mL sample solution: Accurately weigh 3 mg of solid sample and dissolve the sample with 1 mL of the above borate solution; (2) Experimental methods 40 μL of 3 mg / mL sample solution and 80 μL of 1 mmol / L FAPGG solution were mixed in a 96-well ELISA plate, preheated at 37°C for 10 min, and then 30 μL of 0.1 U / mL ACE enzyme solution preheated at 37°C was added to the mixture, mixed and the absorbance at 340 nm was measured, followed by constant temperature oscillation reaction at 37°C for 30 min, and the absorbance of the mixture after the reaction at 340 nm was measured again. 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L sodium chloride) was used as a blank control.

[0034] (3) Calculation formula The calculation formula of ACE inhibition rate is as follows:

[0035] Where: ΔA 0 and ΔA 1 are the changes in absorbance at 340 nm of the blank group and the sample group before and after the reaction, respectively.

[0036] The results of the hydrolysis degree and ACE inhibition rate of the mixture of ACE inhibitory peptides from Spirulina are shown in Tables 1 and Figure 1 As shown. The mixture prepared by enzymatic hydrolysis of purified phycobiliprotein by alkaline protease in Example 1 has higher ACE inhibitory activity and hydrolysis degree than the products obtained by enzymatic hydrolysis by other proteases in Comparative Examples 1-5. This is related to the non-specificity of alkaline protease. Its wide range of cleavage sites enables it to randomly cut more peptide bonds, thereby releasing shorter peptide segments, and shorter peptide segments often have higher ACE inhibitory activity. Therefore, alkaline protease is more conducive to the preparation of highly active ACE inhibitory peptides derived from Spirulina.

[0037] The spirulina-derived ACE inhibitory peptide prepared by the present invention has been confirmed to have good ACE inhibitory activity by in vitro experiments, and can be used as an important raw material for the development of functional foods, health products and even food-borne drugs for assisting in lowering blood pressure, thereby further improving the comprehensive utilization rate and economic added value of spirulina resources. In addition, the enzymatic hydrolysis method adopted by the present invention has mild reaction conditions, simple operation, high preparation efficiency and strong specificity, and has broad application prospects in the field of active peptide preparation.

[0038] Table 1 Hydrolysis degree and ACE inhibition rate of the mixture of ACE inhibitory peptides from Spirulina in the examples and comparative examples

[0039] Note: Different letters indicate significant differences among the same indicators ( p < 0.05).

[0040] Example 3 This example is to perform LC-MS / MS identification on the mixture of ACE inhibitory peptides from Spirulina obtained by alkaline protease hydrolysis in Example 1, which specifically includes the following steps: (1) Sample pretreatment Add acetonitrile to the sample solution at a ratio of 1:3 (v / v), mix well, stand at 4°C in the dark for 30 min, centrifuge at 10,000 rpm for 15 min, filter the supernatant through a 0.22 μm filter, and dry under vacuum. Redissolve the dried sample with 0.1% trifluoroacetic acid, desalt the sample with a C18 microchromatographic column, and dry under vacuum. Redissolve the dried sample with 0.1% formic acid aqueous solution, centrifuge at 12,000 rpm for 20 min, and transfer the supernatant to a sample injection bottle for testing.

[0041] (2) UPLC-Q-Orbitrap-MS analysis The peptide sequences were separated and identified using the Ultimate 3000 UPLC system with a Q Exactive mass spectrometer. UPLC chromatographic conditions: an ACQUITY UPLC Peptide CSH C18 column (1 mm × 150 mm, 1.7 µm, Waters) was used for component separation, the injection volume was 10 μL, the flow rate was set to 0.15 mL / min, and the mobile phase was a binary gradient elution system, in which phase A was ultrapure water containing 0.1% formic acid, and phase B was acetonitrile containing 0.1% formic acid. Mass spectrometry conditions: electrospray ionization source (ESI), positive ion mode, mass spectrometry scanning range of m / z 100 ~ m / z 1500, data acquisition mode of data-dependent scanning (Full MS-ddMS2, DDA), MS scanning resolution of 70000, MS / MS scanning resolution of 17500, and collision energy of 28%.

[0042] (3) Data retrieval and processing Proteome Discoverer software was used to search the Spirulina platensis database from NCBI to identify peptides composed of six or more amino acids, and database search combined with de novo analysis methods were used to identify the sequences of dipeptides to pentapeptides.

[0043] In Example 3, 209 polypeptide sequences were identified from the alkaline protease hydrolysis products, of which long peptides with more than 12 amino acid residues accounted for only 5.74%, while pentapeptides and shorter small peptides accounted for as high as 27.75%, further confirming the outstanding advantages of alkaline protease in Example 1 in the preparation of Spirulina-derived ACE inhibitory peptides.

[0044] Example 4 This example uses bioinformatics to screen for highly active novel ACE inhibitory peptides from the 209 polypeptide sequences identified in Example 3, specifically comprising the following steps: (1) Calculation of the binding energy between peptide and ACE The crystal structure of the sACE domain was obtained from the RCSB PDB protein database as the receptor molecule (C domain PDB ID: 1O8A; N domain PDB ID: 2C6F). AutoDock Tools was used to pre-process the two domain receptors and all ligands. AutoDock Vina was used to calculate the binding energy between the peptide ligand and the sACE domain receptor in the highly active component. The batch algorithm was used to repeat the docking 27 times, and the three lowest binding energies were used for statistical analysis.

[0045] (2) Evaluation of ACE inhibitory activity of peptides The AHTpin online platform was used to predict whether the peptide sequences in the high-activity fraction had ACE inhibitory activity, and the threshold of the support vector machine (SVM) was set to 0, that is, sequences with an SVM score ≥ 0 were considered to have antihypertensive activity.

[0046] (3) Prediction of toxicity and physicochemical properties of peptides The ToxinPred online platform was used to evaluate the toxicity and physicochemical properties of the sequences, including steric hindrance, isoelectric point, hydrophobicity, and hydrophilicity.

[0047] After screening in steps (1) and (2), this example finally obtained five potential highly active ACE inhibitory peptides, whose sequences are: YATY, RYVTY, YVTY, KAYF and SPSWY. The binding energy calculation results of the five peptides with the two domains of ACE and the AHTpin activity score results are shown in Table 2.

[0048] Table 2 Binding energy of five potential sequences to the ACE domain and their AHTpin activity scores

[0049] The toxicity and physicochemical properties of the five potential ACE inhibitory peptides were further evaluated through step (3), and the results are shown in Table 3. All five peptides have no physiological toxicity and have practical application potential.

[0050] Table 3 Prediction results of toxicity and physicochemical properties of five sequences

[0051] Example 5 In this example, the five polypeptides screened in Example 4 were subjected to solid phase synthesis, and HHL was used as a substrate. High performance liquid chromatography was used to determine the amount of hippuric acid (HA) generated by the reaction of different samples, thereby characterizing the activity of the five ACE inhibitory peptides. Specifically, the following steps were included: (1) Reagent preparation The HHL powder and the synthetic peptide powder were dissolved in the 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L sodium chloride) in Example 2 to obtain a 5 mmol / L HHL solution and peptide solutions with different concentrations ranging from 15.625 to 500 μg / mL.

[0052] The preparation of 0.1 U / mL ACE solution was the same as in Example 2.

[0053] (2) Sample processing 50 μL of ACE inhibitory peptide solutions of different concentrations were mixed with 5 mmol / L HHL solution and incubated at 37°C for 10 min. 50 μL of 0.1 U / mL ACE solution preheated at 37°C was added to the mixture, mixed and incubated at 37°C for 30 min. After the reaction was completed, 100 μL of 1 M HCl was added to terminate the reaction, and the reaction system was filtered through a 0.22 μm organic filter membrane.

[0054] (3) Chromatographic conditions An Aglient Zorbax SB C18 (4.6 mm × 100 mm, 5 μm) column was selected, the mobile phase was 25% acetonitrile (containing 0.1% trifluoroacetic acid), the injection volume was set to 10 μL, the flow rate was set to 0.4 mL / min, the column temperature was set to 30 °C, and the detection wavelength was set to 228 nm.

[0055] (4) Calculation formula The calculation formula of ACE inhibition rate of different concentrations of ACE inhibitory peptides is as follows:

[0056] In the formula, A 0 and A 1 are the peak areas of HA generated in the blank group and the sample group, respectively. 50 ) is the peptide concentration corresponding to 50% ACE inhibitory activity.

[0057] As verified in this example, all five peptides have good ACE inhibitory activity, and their corresponding IC 50 The values ​​are shown in Table 4. Among them, the pentapeptide RYVTY has the strongest ACE inhibitory activity, which has exceeded most reported food-borne ACE inhibitory peptides and has broad application prospects in the development of functional foods, health foods and food-borne drugs.

[0058] Table 4 IC values ​​of five ACE inhibitory peptides 50 value

[0059] The traditional screening process generally uses one or more techniques such as ultrafiltration, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography, affinity chromatography, etc. to further separate and purify the enzymatic hydrolysis products, and then sequence the high-activity components to screen out one or several high-activity ACE inhibitory peptides, which has the disadvantages of long research cycle, large workload, low efficiency, high cost, etc. The present invention proposes a high-throughput screening process based on proteomics technology and bioinformatics means. The ACE inhibitory peptides screened out by this process have high biological activity and good physical and chemical properties, which greatly improves the screening efficiency of ACE inhibitory peptides.

[0060] The ACE inhibitory peptides derived from Spirulina and the mixture thereof prepared in the above examples have good antihypertensive activity and have broad application prospects in the fields of food and medicine. Specifically, they include: (1) Used for the development of general health foods, including compressed candies, gel candies, polypeptide freeze-dried powder, etc.; (2) For the development of health foods that have the effect of preventing and treating hypertension. Product types include compressed candies, oral liquids, capsules, etc.; (3) Used to develop food-borne drugs with fewer side effects and higher safety. The dosage forms include capsules, tablets, granules, oral solutions, etc.

[0061] The present invention obtains a method for efficiently preparing spirulina polypeptides by optimizing the process conditions such as protein raw materials, protease types, and enzymolysis time, and the polypeptides prepared by the present method have good ACE inhibitory activity. The preferred protein raw material in the present invention is a purified product of spirulina phycobiliprotein, and the hydrophobic amino acids in the phycobiliprotein are rich, and the hydrophobic amino acids help the polypeptides have higher ACE inhibitory activity, and its higher protein purity effectively avoids the interference of non-protein components such as polysaccharides on the enzymolysis process; the preferred protease is alkaline protease, which has a wide range of enzyme cleavage sites, so that it can randomly cut more peptide bonds and release shorter peptide segments, and short peptides are easier to enter the cavity of ACE protein, and then interact with the active site to inhibit its catalytic activity; the preferred enzymolysis time is 2 h, which can efficiently produce low molecular weight and highly active enzymolysis products in a short time. The process flow of this method is simple, the operation difficulty is relatively small, and the preparation time is relatively short.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that various variations or modifications may be made within the scope of the claims, which do not affect the essential content of the present invention.

[0063] Finally, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Spirulina-derived ACE inhibitory peptide, characterized in that: Including ACE inhibitory peptide YATY, or ACE inhibitory peptide RYVTY, or ACE inhibitory peptide YVTY, or ACE inhibitory peptide KAYF, or ACE inhibitory peptide SPSWY; Among them, the specific sequence of the ACE inhibitory peptide YATY is SEQ No.1: Tyr-Ala-Thr-Tyr; The specific sequence of the ACE inhibitory peptide RYVTY is SEQ No. 2: Arg-Tyr-Val-Thr-Tyr; The specific sequence of the ACE inhibitory peptide YVTY is SEQ No. 3: Tyr-Val-Thr-Tyr; The specific sequence of the ACE inhibitory peptide KAYF is SEQ No. 4: Lys-Ala-Tyr-Phe; The specific sequence of the ACE inhibitory peptide SPSWY is SEQ No. 5: Ser-Pro-Ser-Trp-Tyr.

2. The ACE inhibitory peptide derived from Spirulina according to claim 1, characterized in that The ACE inhibitory peptide is RYVTY.

3. The method for preparing the ACE inhibitory peptide derived from Spirulina according to claim 1, characterized in that: The following steps are involved: (1) repeatedly freezing and thawing the Spirulina suspension to obtain a broken Spirulina suspension; separating the supernatant and the precipitate by freezing centrifugation, wherein the supernatant is the Spirulina soluble extract; purifying the Spirulina soluble extract by precipitation method, and desalting by dialysis to obtain a purified phycobiliprotein solution; (2) adjusting the pH of the solution, adding alkaline protease for enzymatic hydrolysis, boiling to inactivate the enzyme, and centrifuging the sample after cooling to room temperature. The supernatant is a mixture of ACE inhibitory peptides derived from Spirulina. (3) solid-liquid separation and vacuum drying to obtain lyophilized powder of Spirulina; (4) The lyophilized powder of Spirulina obtained in the reconstitution step (3) is desalted and then the polypeptide sequence is identified by liquid chromatography-mass spectrometry; (5) Using an online platform to predict ACE inhibitory activity, physical and chemical properties, and toxicity analysis of the peptide sequence identified in step (4), and screen for potential ACE inhibitory peptides; (6) The screened peptides are subjected to solid phase synthesis, and the activity of the potential ACE inhibitory peptides obtained in step (5) is verified by in vitro activity evaluation, thereby finally obtaining the 5 Spirulina-derived ACE inhibitory peptides.

4. The preparation method according to claim 3, characterized in that: In the step (4), a C18 desalting column is used for desalting.

5. The preparation method according to claim 3, characterized in that: In the step (5), an online platform is used to screen potential ACE inhibitory peptides, including using the AHTpin tool to predict the ACE inhibitory activity of peptides identified by LC-MS / MS of the enzymatic hydrolysate, and selecting peptides with an activity score ≥ 0; obtaining the crystal structure of the sACE domain from the RCSB PDB database as a receptor molecule, performing docking pre-processing on the ACE protein receptor and the small molecule polypeptide ligand, and finally using Autodock Vina for molecular docking; using the ToxinPred tool to predict the physicochemical properties and perform toxicity analysis on the ACE inhibitory peptides predicted and screened by the AHTpin tool and the molecular docking tool.

6. The preparation method according to claim 3, characterized in that: In the step (6), hippuryl-histidyl-leucine is used as a substrate, and high performance liquid chromatography is used to evaluate the in vitro ACE inhibitory activity of the solid phase synthesized polypeptide, and the half inhibition concentration is used to indicate the activity.

7. Use of the spirulina-derived ACE inhibitory peptide YATY, or RYVTY, or YVTY, or KAYF, or SPSWY according to claim 1 in the preparation of health food or medicine with blood pressure lowering effect.

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