Spirulina-derived ace inhibitory peptide, and preparation method and application thereof

Abstract

The present application provides a spirulina-derived ACE inhibitory peptide and a preparation method and application thereof, and belongs to the technical field of marine bioactive peptides. The spirulina-derived ACE inhibitory peptide is prepared from spirulina algal lipochromoprotein, and five small-molecule polypeptides YATY, RYVTY, YVTY, KAYF and SPSWY are screened, wherein the five polypeptides all have good ACE inhibitory activity, and RYVTY has the highest activity. The spirulina-derived polypeptide prepared by the present application has high hydrolysis degree and high ACE inhibitory activity, and can be applied in the fields of food, health products, medicines and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of marine bioactive peptide technology, specifically to spirulina-derived ACE inhibitory peptides, their preparation methods, and applications. Background Technology

[0002] Hypertension is a common chronic disease closely related to non-communicable diseases such as cardiovascular disease, stroke, and chronic kidney disease. The number of people with 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 leading causes of death and disability.

[0003] Angiotensin-I converting enzyme (ACE) is a Zn-containing enzyme widely found in mammalian tissues such as neuroepithelial cells, vascular endothelial cells, and male germ cells. 2+ ACE is a type-dependent dipeptide carboxypeptidase. As a key enzyme in the renin-angiotensin-aldosterone system and the kallikrein-kallikrein system, its activity significantly influences the body's electrolyte balance and blood pressure regulation. In the renin-angiotensin-aldosterone system, ACE converts angiotensin I (which lacks pressor activity) into angiotensin II (which does possess pressor activity), while simultaneously stimulating the adrenal glands to release aldosterone, leading to an increase in sodium levels in the kidneys. + In the bradykinin-kallikrein system, ACE cleaves the Phe-Arg dipeptide fragment at the C-terminus of bradykinin, causing it to lose its antihypertensive activity. Therefore, inhibiting ACE activity has become one of the effective methods for intervening in and treating hypertension. However, commonly used chemically synthesized ACE inhibitors (captopril, enalapril, lisinopril, etc.) have been shown to cause side effects such as dry cough, leukopenia, taste disturbance, rash, angioedema, and renal insufficiency. Therefore, naturally derived ACE inhibitory peptides are receiving increasing attention due to their higher safety profile.

[0004] Spirulina is rich in essential nutrients such as protein, lipids, carbohydrates, vitamins, and minerals. It possesses various physiological functions, including lowering cholesterol, regulating immunity, anti-inflammation, anti-fatigue, and anti-cancer properties, 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." Currently, in the food industry, however, it is only used as a functional food base added to smoothies, energy bars, and other foods, or as a dietary supplement in tablet and powder form. The extremely small amounts of spirulina found in some products indicate that it is merely used as a coloring agent or a commercial gimmick, without fully realizing its nutritional value. Summary of the Invention

[0005] The purpose of this invention is to provide a spirulina-derived ACE inhibitory peptide, along with its preparation method and specific applications, to overcome the shortcomings of existing technologies.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0007] Spirulina-derived ACE inhibitory peptides include YATY, RYVTY, YVTY, KAYF, or SPSWY.

[0008] The specific sequence of YATY is SEQ No. 1: Tyr-Ala-Thr-Tyr;

[0009] The specific sequence of RYVTY is SEQ No. 2: Arg-Tyr-Val-Thr-Tyr;

[0010] The specific sequence of YVTY is SEQ No. 3: Tyr-Val-Thr-Tyr;

[0011] The specific sequence of KAYF is SEQ No. 4: Lys-Ala-Tyr-Phe;

[0012] The specific sequence of SPSWY is SEQ No. 5: Ser-Pro-Ser-Trp-Tyr.

[0013] Preferably, a spirulina-derived ACE inhibitory peptide with high activity is RYVTY.

[0014] The preparation method of the spirulina-derived ACE inhibitory peptide includes the following steps:

[0015] (1) Spirulina was added to ammonium bicarbonate buffer and mixed evenly. The cell walls of Spirulina were broken by repeated freeze-thaw cycles to obtain a Spirulina cell wall-broken suspension. The supernatant and precipitate were separated by freezing and centrifugation. The supernatant was the Spirulina soluble extract. The Spirulina soluble extract was then purified by ammonium sulfate stepwise precipitation method. The extract was then dialyzed and desalted to obtain the phycobiliprotein purified solution.

[0016] (2) Adjust the pH of the solution, add alkaline protease for enzymatic hydrolysis, boil to inactivate the enzyme, and centrifuge after the sample cools to room temperature. The supernatant is a mixture of spirulina-derived ACE inhibitory peptides.

[0017] (3) Solid-liquid separation and vacuum drying are used to obtain spirulina enzyme-hydrolyzed lyophilized powder;

[0018] (4) The spirulina enzyme lyophilized powder obtained in step (3) was reconstituted with distilled water, desalted, and then identified by liquid chromatography-mass spectrometry.

[0019] (5) Use an online platform to predict the ACE inhibitory activity, predict the physicochemical properties and analyze the toxicity of the polypeptide sequences identified in step (4) and screen for potential ACE inhibitory peptides.

[0020] (6) The selected peptides were synthesized in solid phase and the activity of the potential ACE inhibitory peptides obtained in step (5) was verified by in vitro activity evaluation, and finally the five spirulina-derived ACE inhibitory peptides were obtained.

[0021] Furthermore, step (4) involves desalination using a C18 desalination column.

[0022] 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 in 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 the receptor molecule (C domain PDB ID: 1O8A; N domain PDB ID: 2C6F), performing pre-docking pretreatment on the ACE protein receptor and small molecule polypeptide ligand, setting up the docking box, and finally performing molecular docking using Autodock Vina; 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.

[0023] Furthermore, in step (6), hippuryl-histyl-leucine (HHL) was used as a substrate, and high-performance liquid chromatography was employed to evaluate the in vitro ACE inhibitory activity of the solid-phase synthesized peptide, using the half-maximal inhibitory concentration (IC50) as the criterion. 50 ) indicates its activity level, where IC 50 This represents the peptide concentration corresponding to an ACE inhibitory activity of 50%.

[0024] The application of the spirulina-derived ACE inhibitory peptides YATY, RYVTY, YVTY, KAYF, or SPSWY in the preparation of products with antihypertensive effects: the products include drugs, etc.

[0025] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0026] This invention utilizes spirulina phycobiliproteins to prepare spirulina-derived ACE-inhibiting peptides. Five small molecule peptides were screened and obtained: YATY, RYVTY, YVTY, KAYF, and SPSWY. All five peptides exhibited good ACE-inhibiting activity, with RYVTY showing the highest activity. The spirulina-derived peptides prepared by this invention possess both high hydrolysis degree and high ACE-inhibiting activity, making them suitable for applications in food, health products, and pharmaceuticals, and demonstrating broad application prospects. Attached Figure Description

[0027] Figure 1 The figures show the degree of hydrolysis and ACE inhibition rate of different samples in Example 2. The bar chart shows the ACE inhibition rate of different samples, and the line chart shows the corresponding degree of hydrolysis. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. The descriptions herein represent only some, not all, embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] This embodiment provides a method for preparing a mixture of spirulina-derived ACE inhibitory peptides, comprising the following steps:

[0032] (1) Spirulina platensis was added to ammonium bicarbonate buffer (pH 7.0) at a ratio of 1:10 g / mL. After mixing evenly, it was frozen at -20℃ for 12 h. Then it was thawed at room temperature in the dark. The freeze-thaw cycle was repeated 3 times. Then it was centrifuged at 8000 rpm for 20 min. Ammonium sulfate was slowly added to the supernatant until the ammonium sulfate saturation reached 25%. After stirring at 4℃ in the dark for 1 h, it was centrifuged for 20 min. Ammonium sulfate was added to the supernatant again to increase the ammonium sulfate saturation to 50%. After stirring at 4℃ in the dark for 2 h, it was centrifuged for 20 min. The secondary precipitate was reconstituted with as little deionized water as possible. After dialysis and desalting at 4℃, the phycobiliprotein was obtained by freeze drying.

[0033] (2) The phycobiliprotein purified product was reconstituted to 10 mg / mL using 0.1 mol / L phosphate buffer and the pH of the solution was adjusted to 8.0. Alkaline protease was added at an enzyme-to-protein ratio of 2% (w / w). The enzyme was shaken and digested at 50°C for 4 h. The enzyme was then inactivated by boiling. After the sample cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of spirulina-derived ACE inhibitory peptides.

[0034] Comparative Example 1

[0035] The difference between this comparative example and Example 1 is that the protease used is papain, and the enzymatic hydrolysis conditions were appropriately adjusted: the phycobiliprotein purified product was reconstituted to 10 mg / mL using 0.1 mol / L phosphate buffer, and the pH of the solution was adjusted to 7.0. Papain was added at an enzyme-to-protein ratio of 2% (w / w), and the enzyme was hydrolyzed at 50°C with shaking for 4 h. The enzyme was then inactivated by boiling. After the sample cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of spirulina-derived ACE inhibitory peptides.

[0036] Comparative Example 2

[0037] The difference between this comparative example and Example 1 is that the protease used is trypsin, and the enzymatic digestion conditions were appropriately adjusted: the phycobiliprotein purified product was reconstituted to 10 mg / mL using 0.1 mol / L phosphate buffer, and the pH of the solution was adjusted to 8.0. Trypsin was added at an enzyme-to-protein ratio of 2% (w / w), and the enzyme was shaken and digested at 50°C for 4 h. The enzyme was then inactivated by boiling. After the sample cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of spirulina-derived ACE inhibitory peptides.

[0038] Comparative Example 3

[0039] The difference between this comparative example and Example 1 is that the protease used is a neutral protease, and the enzymatic digestion conditions were appropriately adjusted: the phycobiliprotein purified product was reconstituted to 10 mg / mL using 0.1 mol / L phosphate buffer, and the pH of the solution was adjusted to 7.0. Neutral protease was added at an enzyme-to-protein ratio of 2% (w / w), and the enzyme was shaken at 50°C and 120 rpm for 4 h. The enzyme was then inactivated by boiling. After the sample cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of spirulina-derived ACE inhibitory peptides.

[0040] Comparative Example 4

[0041] The difference between this comparative example and Example 1 is that the protease used is a flavor protease, and the enzymatic hydrolysis conditions were appropriately adjusted: the phycobiliprotein purified product was reconstituted to 10 mg / mL using 0.1 mol / L phosphate buffer, and the pH of the solution was adjusted to 7.5. The flavor protease was added at a ratio of 2% (w / w) to the enzyme base, and the enzyme was hydrolyzed at 50°C with shaking for 4 h. The enzyme was then inactivated by boiling. After the sample cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of spirulina-derived ACE inhibitory peptides.

[0042] Comparative Example 5

[0043] The difference between this comparative example and Example 1 is that the protease used is pepsin, and the enzymatic hydrolysis conditions were appropriately adjusted: the phycobiliprotein purified product was reconstituted to 10 mg / mL using 0.1 mol / L phosphate buffer, and the pH of the solution was adjusted to 2.0. Pepsin was added at an enzyme-to-protein ratio of 2% (w / w), and the enzyme was hydrolyzed at 50°C with shaking for 4 h. The enzyme was then inactivated by boiling. After the sample cooled to room temperature, it was centrifuged at 10,000 rpm for 15 min. The supernatant was a mixture of spirulina-derived ACE inhibitory peptides.

[0044] Example 2

[0045] This embodiment uses formaldehyde titration to determine the amino nitrogen content in the mixture of Spirulina-derived ACE inhibitory peptides obtained in Example 1 and Comparative Examples 1-5, and further calculates the degree of hydrolysis of the mixture obtained by enzymatic hydrolysis. The specific steps include:

[0046] (1) Determination of total nitrogen content

[0047] Referring to GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Food", the Kjeldahl method was used to determine the total nitrogen content in the purified phycobiliprotein.

[0048] (2) Determination of amino nitrogen content

[0049] The amino nitrogen content in the enzymatic hydrolysate was determined using 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 a faint pink color was observed; this was recorded as neutral formaldehyde solution. The pH of all enzymatic hydrolysates was adjusted to 8.0. 400 μL of enzymatic hydrolysate (recorded as V2), 7.6 mL of distilled water, and 2 drops of phenolphthalein indicator were added to an Erlenmeyer flask. 2 mL of neutral formaldehyde solution was added to the mixture, and after mixing, titration was performed with 0.01 M NaOH standard solution. The volume of NaOH consumed in each group was recorded as V1. Distilled water was used as a blank control instead of the enzymatic hydrolysate, and the volume of NaOH consumed was recorded as V0. The formula for calculating the amino nitrogen (AN) content in the enzymatic hydrolysate is as follows:

[0050] In the formula, is the molar concentration of NaOH.

[0051] (3) Calculation of degree of hydrolysis

[0052] The formula for calculating the degree of hydrolysis (DH) of the enzymatic hydrolysate is as follows:

[0053] In the formula, N represents the total nitrogen content in the pre-enzymatic hydrolysate phycobiliprotein; AN represents the amino nitrogen content in the hydrolysate.

[0054] This embodiment uses FAPGG as a substrate and employs an enzyme-linked immunosorbent assay (ELISA) reader to determine the inhibitory activity of the mixture of Spirulina-derived ACE inhibitory peptides obtained in Example 1 and Comparative Examples 1-5. The specific steps include:

[0055] (1) Reagent preparation

[0056] 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, dissolve them separately in deionized water by heating, and then bring the volume to 100 mL. Mix the two solutions until the pH of the solution is 8.3. Take 100 mL of the mixture and add 1.7532 g sodium chloride to it, dissolve and mix well.

[0057] 0.1 U / mL ACE enzyme solution: Dissolve 0.1 U ACE enzyme in 1 mL of the above borate buffer.

[0058] 1 mmol / L N-[3-(2-furanyl)acryloyl]-L-phenylpropionamide-glycine-glycine (FAPGG) solution: Accurately weigh 3.994 mg FAPGG powder, dissolve it in the above borate buffer and bring the volume to 10 mL.

[0059] 3 mg / mL sample solution: Accurately weigh 3 mg of solid sample and dissolve the sample in 1 mL of the above borate solution;

[0060] (2) Experimental methods

[0061] 40 μL of 3 mg / mL sample solution was mixed with 80 μL of 1 mmol / L FAPGG solution in a 96-well microplate. After preheating at 37°C for 10 min, 30 μL of 0.1 U / mL ACE enzyme solution preheated at 37°C was added to the mixture. The absorbance at 340 nm was measured, and the mixture was then incubated at 37°C with shaking for 30 min. The absorbance of the mixture at 340 nm was measured again after the reaction. 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L sodium chloride) was used as a blank control.

[0062] (3) Calculation formula

[0063] The formula for calculating the ACE inhibition rate is as follows:

[0064]

[0065] In the formula: ΔA0 and ΔA1 are the changes in absorbance at 340 nm for the blank group and the sample group before and after the reaction, respectively.

[0066] The results of the determination of the degree of hydrolysis and ACE inhibition rate of the spirulina-derived ACE inhibitory peptide mixture are shown in Table 1 and 2. Figure 1 As shown. The mixture prepared by alkaline protease hydrolysis of phycobiliprotein purification in Example 1 has higher ACE inhibitory activity and degree of hydrolysis than the products obtained by hydrolysis with other proteases in Comparative Examples 1-5. This is related to the nonspecificity of alkaline protease, whose extensive cleavage sites allow it to randomly cleave more peptide bonds, thereby releasing shorter peptides. Shorter peptides often have higher ACE inhibitory activity. Therefore, alkaline protease is more conducive to preparing highly active spirulina-derived ACE inhibitory peptides.

[0067] The spirulina-derived ACE-inhibiting peptide prepared by this invention has been shown to have good ACE-inhibiting activity in in vitro experiments. It can be used as an important raw material to assist in the development of functional foods, health products and even food-derived drugs for lowering blood pressure, further improving the comprehensive utilization rate and economic added value of spirulina resources. In addition, the enzymatic hydrolysis method used in this 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.

[0068] Table 1. Degree of hydrolysis and ACE inhibition rate of the Spirulina-derived ACE-inhibiting peptide mixtures in the examples and comparative examples.

[0069] Note: Different letters indicate significant differences between the same indicators (p < 0.05).

[0070] Example 3

[0071] This embodiment describes the LC-MS / MS identification of the mixture of Spirulina-derived ACE inhibitory peptides obtained by alkaline protease digestion in Example 1. The specific steps include:

[0072] (1) Sample pretreatment

[0073] Acetonitrile was added to the sample solution at a ratio of 1:3 (v / v), mixed well, and incubated at 4°C in the dark for 30 min. The mixture was then centrifuged at 10,000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm filter and dried under vacuum. The dried sample was reconstituted with 0.1% trifluoroacetic acid, desalted using a C18 microchromatographic column, and then dried under vacuum. The dried sample was then reconstituted with 0.1% formic acid aqueous solution, centrifuged at 12,000 rpm for 20 min, and the supernatant was transferred to a sample vial for analysis.

[0074] (2) UPLC-Q-Orbitrap-MS analysis

[0075] Peptide sequences were separated and identified using an Ultimate 3000 UPLC system paired 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 a binary gradient elution system was used as the mobile phase. Phase A consisted of ultrapure water containing 0.1% formic acid, and phase B consisted of acetonitrile containing 0.1% formic acid. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion mode, mass spectrometry scan range of m / z 100 ~ m / z 1500, data acquisition mode of data-dependent scan (Full MS-ddMS2, DDA), MS scan resolution of 70000, MS / MS scan resolution of 17500, and collision energy of 28%.

[0076] (3) Data retrieval and processing

[0077] The Proteome Discoverer software was used to search the Spirulina platensis database from NCBI to identify peptides composed of six or more amino acids. The dipeptides to pentapeptides were sequenced using database search combined with de novo analysis.

[0078] In Example 3, 209 polypeptide sequences were identified from the alkaline protease hydrolysis products. Among them, long peptides with more than 12 amino acid residues accounted for only 5.74%, while pentapeptides and shorter small peptides accounted for as much as 27.75%, further confirming the outstanding advantages of alkaline protease in Example 1 in the preparation of spirulina-derived ACE inhibitory peptides.

[0079] Example 4

[0080] This embodiment uses bioinformatics methods to screen for novel ACE inhibitory peptides with high activity from the 209 polypeptide sequences identified in Example 3, specifically including the following steps:

[0081] (1) Calculation of binding energy between polypeptide and ACE

[0082] The crystal structures of the sACE domains were obtained from the RCSB PDB protein database as receptor molecules (C domain PDB ID: 1O8A; N domain PDB ID: 2C6F). AutoDock Tools were used to pre-dock the two domain receptors and all ligands. AutoDock Vina was used to calculate the binding energies between the peptide ligands and the sACE domain receptors in the highly active fraction. A batch algorithm was used to repeat the docking 27 times, and the three lowest binding energies were used for statistical analysis.

[0083] (2) Evaluation of the ACE inhibitory activity of the peptide

[0084] The AHTpin online platform was used to predict whether peptide sequences in highly active components have ACE inhibitory activity. The threshold for support vector machine (SVM) was set to 0, meaning that sequences with an SVM score ≥ 0 were considered to have antihypertensive activity.

[0085] (3) Prediction of the toxicity and physicochemical properties of polypeptides

[0086] The toxicity and physicochemical properties of the sequence were evaluated using the ToxinPred online platform, including steric hindrance, isoelectric point, hydrophobicity, and hydrophilicity.

[0087] After screening in steps (1) and (2), five potential highly active ACE inhibitory peptides were finally obtained in this embodiment, with the sequences: YATY, RYVTY, YVTY, KAYF, and SPSWY. The binding energy calculation results of the five peptides to the two ACE domains and the AHTpin activity score results are shown in Table 2.

[0088] Table 2. Binding energies of the five potential sequences to the ACE domain and their AHTpin activity scores.

[0089] The toxicity and physicochemical properties of the five potential ACE-inhibiting peptides were further evaluated in step (3), and the results are shown in Table 3. None of the five peptides showed physiological toxicity and have potential for practical application.

[0090] Table 3. Predicted results of toxicity and physicochemical properties of the five sequences.

[0091] Example 5

[0092] This embodiment synthesizes the five peptides screened in Example 4 using solid-phase chromatography and employs high-performance liquid chromatography (HPLC) to determine the amount of hippuric acid (HA) produced in different samples, thereby characterizing the activity of the five ACE-inhibiting peptides. The specific steps include:

[0093] (1) Reagent preparation

[0094] HHL powder and synthetic peptide powder were dissolved in 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L sodium chloride) as described in Example 2 to obtain 5 mmol / L HHL solution and peptide solutions of different concentrations from 15.625 to 500 μg / mL.

[0095] The 0.1 U / mL ACE solution was prepared in the same manner as in Example 2.

[0096] (2) Sample processing

[0097] Mix 50 μL of ACE inhibitory peptide solutions of different concentrations with 5 mmol / L HCl solution and incubate at 37°C for 10 min. Add 50 μL of 0.1 U / mL ACE solution preheated at 37°C to the mixture, mix well, and incubate at 37°C for 30 min. After the reaction is complete, add 100 μL of 1 M HCl to terminate the reaction. Filter the reaction system through a 0.22 μm organic filter membrane.

[0098] (3) Chromatographic conditions

[0099] An Aglient Zorbax SB C18 (4.6 mm × 100 mm, 5 μm) column was used. 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℃, and the detection wavelength was set to 228 nm.

[0100] (4) Calculation formula

[0101] The formulas for calculating the ACE inhibition rate of ACE-inhibiting peptides at different concentrations are as follows:

[0102]

[0103] In the formula, A0 and A1 are the peak areas of HA formation in the blank group and the sample group, respectively. The half-maximal inhibitory concentration (IC50) is... 50 () represents the peptide concentration corresponding to an ACE inhibitory activity of 50%.

[0104] As verified in this embodiment, all five peptides exhibit good ACE inhibitory activity, and their corresponding IC50 values ​​are [missing information]. 50 The values ​​are shown in Table 4. Among them, the pentapeptide RYVTY has the strongest ACE inhibitory activity, which has surpassed most reported food-derived ACE inhibitory peptides, and has broad application prospects in the development of food-derived drugs.

[0105] Table 4 IC50 of five ACE inhibitory peptides 50 value

[0106] Traditional screening processes typically involve further separating and purifying the enzymatically digested peptide mixtures using one or more techniques such as ultrafiltration, gel filtration chromatography, ion exchange chromatography, high-performance liquid chromatography, and affinity chromatography. The highly active components are then sequenced to identify one or more highly active ACE inhibitory peptides. This process suffers from drawbacks such as long research cycles, high workload, low efficiency, and high costs. This invention proposes a high-throughput screening process based on proteomics and bioinformatics. The ACE inhibitory peptides screened using this process exhibit high biological activity and favorable physicochemical properties, significantly improving the screening efficiency for ACE inhibitory peptides.

[0107] The spirulina-derived ACE-inhibiting peptides and their mixtures prepared in the above embodiments exhibit good antihypertensive activity and have broad application prospects in the pharmaceutical field. Specifically, they can be used to develop food-derived drugs with fewer side effects and higher safety, in dosage forms including capsules, tablets, granules, and oral liquids.

[0108] This invention provides a highly efficient method for preparing spirulina polypeptides by optimizing process conditions such as protein raw materials, protease type, and enzymatic hydrolysis time. The polypeptides prepared using this method exhibit excellent ACE inhibitory activity. The preferred protein raw material in this invention is purified spirulina phycobiliprotein, which is rich in hydrophobic amino acids. These hydrophobic amino acids contribute to higher ACE inhibitory activity in the polypeptides, and their high protein purity effectively avoids interference from non-protein components such as polysaccharides during the enzymatic hydrolysis process. The preferred protease is an alkaline protease, which has a wide range of cleavage sites, allowing it to randomly cleave more peptide bonds and release shorter peptides. Shorter peptides are more likely to enter the cavity of ACE proteins, subsequently interacting with the active sites and inhibiting their catalytic activity. The preferred hydrolysis time is 2 hours, which efficiently produces low-molecular-weight, highly active enzymatic hydrolysates in a short time. This method has a simple process flow, is relatively easy to operate, and has a short preparation time.

[0109] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that various modifications or variations can be made within the scope of the claims, which do not affect the substantive content of the present invention.

[0110] Finally, although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for 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 embodiments that can be understood by those skilled in the art.

Claims

1. The application of spirulina-derived ACE inhibitory peptides YATY, KAYF, or SPSWY in the preparation of drugs with antihypertensive effects; in, 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 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.

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