Microscopic nannochloropsis ace inhibiting peptide, preparation method and application thereof

Abstract

The present application provides a kind of micro-nanqiqiao ACE inhibitory peptide, preparation method and application, belong to the technical field of bioactive peptide.The present application mixes micro-nanqiqiao algae powder and water ultrasonically, high pressure treatment, cellulase enzymolysis adjusts pH value ultrasonic water bath extraction centrifugation;The supernatant obtained by repeatedly extracting the precipitate after centrifugation and water is combined, and the pH value is adjusted, and the precipitate is collected by centrifugation, then water is added to adjust the pH value, and the micro-nanqiqiao crude protein powder is obtained by freeze-drying;The micro-nanqiqiao crude protein powder is mixed with water, the pH value is adjusted, and the supernatant is collected by centrifugation after trypsin enzymolysis, concentrated and freeze-dried to obtain nanqiqiao crude polypeptide powder;Nanqiqiao crude polypeptide powder is dissolved in water and separated by ultrafiltration to obtain micro-nanqiqiao ACE inhibitory peptide extract with molecular weight less than 3kDa.The micro-nanqiqiao ACE inhibitory peptide extract prepared by the present application and the polypeptides PGPAIF, GPGPFTVF and WDPLGF screened and separated have good ACE inhibitory activity.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and particularly relates to an ACE inhibitory peptide of *Microcystis aeruginosa*, its preparation method, and its application. Background Technology

[0002] Hypertension is a prevalent cardiovascular disease affecting the health of approximately one billion adults worldwide. Long-term hypertension increases the risk of diseases such as myocardial infarction, kidney failure, and stroke. In recent years, more than 9 million people die annually from hypertension-related diseases. The renin-angiotensin system (RAS) and the kinin-nitric oxide system (KNOS) are crucial for regulating blood pressure in the human body.

[0003] Angiotensin-converting enzyme (ACE) inhibitory peptides, also known as ACE inhibitory peptides, act simultaneously on both the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) for vasodilation, and are often used as targets for the clinical treatment of hypertension. Currently, most drugs used clinically to treat hypertension are developed targeting the ACE inhibitor. For example, captopril, enalapril, and lisinopril are all ACE inhibitors. However, most of these ACE inhibitors are synthetic drugs, and their use often results in various toxic side effects, such as cough, skin itching, taste disturbances, or hypotension.

[0004] Bioactive peptides are composed of short amino acid sequences, possessing simple structures, good bio-penetration, and easy absorption by cells. They are also less likely to bind with other drug molecules, thus reducing the risk of side effects. Algae, rich in unsaturated fatty acids, functional polysaccharides, and polypeptides, have a long history of application in food and medicine. In recent years, algal protein has gained popularity as a novel source of plant protein. Algal protein can be consumed directly or hydrolyzed to prepare functional peptides. Algal protein bioactive peptides exhibit antioxidant, antihypertensive, and anticancer activities.

[0005] *Nannochloropsis*, also known as *Nannochloropsis* var. *microphylla*, is a single-celled green algae. Rich in polyunsaturated fatty acids, proteins, and polysaccharides, *Nannochloropsis* has broad application prospects in aquaculture, food, healthcare, and cosmetics industries. The oil content of *Nannochloropsis* can reach over 60% of its cell biomass, with eicosapentaenoic acid (EPA) content reaching up to 30%. EPA is an ω-3 long-chain polyunsaturated fatty acid that has functions such as lowering cholesterol, reducing blood viscosity, and improving blood circulation. Due to its rapid growth, high biomass, and strong environmental tolerance, *Nannochloropsis* var. *microphylla* is considered the most promising oil-producing microalga for large-scale cultivation. In recent years, increasing research has found that peptides derived from algae possess antioxidant, antihypertensive, and anticancer biological activities. However, research on the application of proteins and peptides from *Nannochloropsis* var. *microphylla* is relatively limited. While research on the preparation, separation, purification, and functional screening of algal-derived biopeptides has increased in recent years, there are few reports of isolating ACE inhibitory peptides from *Nannochloropsis* var. *microphylla*. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a microalgae ACE inhibitory peptide and a method for preparing the same.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an ACE inhibitory peptide from *Microcystis aeruginosa*, wherein the sequence of the ACE inhibitory peptide is Pro-Gly-Pro-Ala-IIe-Phe, Gly-Pro-Gly-Pro-Phe-Thr-Val-Phe, or Trp-Asp-Pro-Leu-Gly-Phe.

[0008] The present invention also provides an ACE inhibitory peptide extract of *Microcystis aeruginosa*, comprising ACE inhibitory peptides with the sequences Pro-Gly-Pro-Ala-IIe-Phe, Gly-Pro-Gly-Pro-Phe-Thr-Val-Phe, and Trp-Asp-Pro-Leu-Gly-Phe.

[0009] This invention also provides a method for preparing an ACE inhibitory peptide extract from *Microcystis aeruginosa*, comprising the following steps: 1) Mix the microalgae powder and water, sonicate, let stand, treat under high pressure at 0.1-0.3 MPa for 20-40 min, adjust the pH to 5-7, add cellulase to hydrolyze and obtain the enzymatic hydrolysate; 2) After adjusting the pH of the enzyme hydrolysate to 10-14, extract using an ultrasonic water bath, centrifuge, and collect the supernatant and precipitate; 3) Mix the precipitate obtained after centrifugation with water and repeat the extraction 1-3 times as in step 2), and collect the supernatant; 4) Combine the supernatants obtained in steps 2) and 3) and remove impurities. Adjust the pH to 2-5 for the first time, let stand, centrifuge, collect the precipitate, add water and adjust the pH to 8-12 for the second time. Pre-freeze at -60~-100℃ and then freeze-dry to obtain crude protein powder of Microcystis thunbergii. 5) Mix the crude protein powder of Microcystis aeruginosa with water and stir, adjust the pH value to 7-9, add trypsin for enzymatic hydrolysis, centrifuge after enzymatic hydrolysis, collect the supernatant, remove impurities, concentrate and freeze dry to obtain crude polypeptide powder of Microcystis aeruginosa. 6) Dissolve the crude polypeptide powder of *Chlorella vulgaris* in water to obtain a crude polypeptide solution of *Chlorella vulgaris*. Then, use ultrafiltration membranes of 10 kDa and 3 kDa to separate the polypeptides and obtain *Chlorella vulgaris* ACE inhibitory peptide extract with a molecular weight of <3 kDa.

[0010] Preferably, the mixing ratio of the microalgae powder and water is (60-100) g: (600-1000) mL; The ultrasonic power in step 1) is 500 W-1000 W, the ultrasonic time is 10-30 min, the settling time is 8-12 h, and the high-pressure treatment temperature is 100-150℃. The cellulase has an enzyme activity of 30-70 U / mg, and the amount of cellulase used is 1.5-3g; The enzymatic hydrolysis temperature is 30-50℃, and the enzymatic hydrolysis time is 1.5-3h.

[0011] Preferably, the ultrasound time in step 2) is 20-40 min; the water bath extraction temperature is 30-50℃; the water bath extraction time is 4-8 h; and stirring is performed during the water bath extraction process at a speed of 100-300 rpm.

[0012] Preferably, the temperature for standing in step 4) is 2-6°C, and the standing time is 20-30h; the pressure for freeze drying is 10-30Pa, and the freeze drying time is 48h-72h.

[0013] In the preferred step 5), the mixing ratio of the crude protein powder of *Microcystis aeruginosa* to water is (15-25) g: (300-500) mL; the enzyme activity of the trypsin is 2000-3000 U / mg, and the amount of trypsin used is 0.1-0.3 g; the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 4-8 h.

[0014] Preferably, the concentration pressure in step 5) is 0.070-0.095 MPa, the concentration temperature is 50-70°C, the freeze-drying pressure is 10-30 Pa, and the freeze-drying time is 48-72 h.

[0015] Preferably, the centrifugation speed in steps 2), 4) and 5) is 6000-8000 rpm, and the centrifugation time is 20-40 min.

[0016] This invention also provides the application of *Microcystis chinensis* ACE inhibitory peptide, *Microcystis chinensis* ACE inhibitory peptide extract, or *Microcystis chinensis* ACE inhibitory peptide extract prepared by the preparation method of *Microcystis chinensis* ACE inhibitory peptide extract in the preparation of drugs for treating hypertension.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The peptides prepared in this invention can effectively inhibit ACE activity, and all of these peptides are small molecule peptides (less than 10 amino acids), making them easily absorbed and utilized. This invention employs three methods—ultrasound-assisted treatment, high-pressure steam treatment, and cellulase hydrolysis—to pretreat microalgae, improving the crude protein dissolution rate and performing preliminary hydrolysis of large protein molecules. Simultaneously, high-pressure steam treatment eliminates the influence of endogenous enzymes on subsequent exogenous enzyme hydrolysis. The peptides PGPAIF, GGPPFTVF, and WDPLGF prepared in this invention exhibit excellent ACE inhibitory activity and good digestibility, making them suitable for use as adjunctive therapies for lowering hypertension or for developing products targeting hypertensive patients. Attached Figure Description

[0018] Figure 1 Inhibitory activity of peptide components (YP and YP-3) on ACE before and after ultrafiltration; Figure 2 Screening peptides for ACE inhibitory activity; Figure 3 PGPAIF, GGPPFTVF, WDPLGF mass spectra; Figure 4 PGPAIF, GGPPFTVF, and WDPLGF exhibit in vitro ACE inhibitory activity; Figure 5 PGPAIF, GGPPFTVF, and WDPLGF simulate gastrointestinal digestive stability. Detailed Implementation

[0019] This invention provides an ACE inhibitory peptide from *Microcystis aeruginosa*, wherein the sequence of the ACE inhibitory peptide is Pro-Gly-Pro-Ala-IIe-Phe, Gly-Pro-Gly-Pro-Phe-Thr-Val-Phe, or Trp-Asp-Pro-Leu-Gly-Phe.

[0020] The present invention also provides an ACE inhibitory peptide extract of *Microcystis aeruginosa*, comprising ACE inhibitory peptides with the sequences Pro-Gly-Pro-Ala-IIe-Phe, Gly-Pro-Gly-Pro-Phe-Thr-Val-Phe, and Trp-Asp-Pro-Leu-Gly-Phe.

[0021] This invention also provides a method for preparing an ACE inhibitory peptide extract from *Microcystis aeruginosa*, comprising the following steps: 1) Mix the microalgae powder and water, sonicate, let stand, treat under high pressure at 0.1-0.3 MPa for 20-40 min, adjust the pH to 5-7, add cellulase to hydrolyze and obtain the enzymatic hydrolysate; 2) After adjusting the pH of the enzyme hydrolysate to 10-14, extract using an ultrasonic water bath, centrifuge, and collect the supernatant and precipitate; 3) Mix the precipitate obtained after centrifugation with water and repeat the extraction 1-3 times as in step 2), and collect the supernatant; 4) Combine the supernatants obtained in steps 2) and 3) and remove impurities. Adjust the pH to 2-5 for the first time, let stand, centrifuge, collect the precipitate, add water and adjust the pH to 8-12 for the second time. Pre-freeze at -60~-100℃ and then freeze-dry to obtain crude protein powder of Microcystis thunbergii. 5) Mix the crude protein powder of Microcystis aeruginosa with water and stir, adjust the pH value to 7-9, add trypsin for enzymatic hydrolysis, centrifuge after enzymatic hydrolysis, collect the supernatant, remove impurities, concentrate and freeze dry to obtain crude polypeptide powder of Microcystis aeruginosa. 6) Dissolve the crude polypeptide powder of *Chlorella vulgaris* in water to obtain a crude polypeptide solution of *Chlorella vulgaris*. Then, use ultrafiltration membranes of 10 kDa and 3 kDa to separate the polypeptides and obtain *Chlorella vulgaris* ACE inhibitory peptide extract with a molecular weight of <3 kDa.

[0022] In this invention, *Microcystis aeruginosa* powder and water are mixed and sonicated, allowed to stand, and then subjected to high pressure treatment at 0.1-0.3 MPa for 20-40 minutes. The pH value is adjusted to 5-7, and cellulase is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The preferred mixing ratio of *Microcystis aeruginosa* powder and water is (60-100) g:(600-1000) mL, more preferably (70-90) g:(700-9000) mL. The sonication is performed under water bath conditions, and the preferred power of the sonication is 500 W-1000 W. The ultrasonic wave (UW) is preferably 700W-900W; the ultrasonic treatment time is preferably 10-30 min, more preferably 15-25 min; the settling time is preferably 8-12 h, more preferably 9-11 h; the high-pressure treatment time is preferably 25-35 min; the high-pressure treatment temperature is preferably 100-150℃, more preferably 110-130℃; the pH is naturally cooled to room temperature before adjustment; the solution for pH adjustment is preferably sodium hydroxide solution, and the pH is further preferably adjusted to 5.5-6.5; the cellulase activity is preferably 30-70. The concentration of cellulase is preferably 40-60 U / mg, more preferably 1.5-3g, more preferably 1.8-2.6g; the enzymatic hydrolysis is carried out under water bath conditions, and the temperature of the enzymatic hydrolysis is preferably 30-50℃, more preferably 35-45℃; the time of the enzymatic hydrolysis is preferably 1.5-3h, more preferably 1.8-2.8h; the enzymatic hydrolysis is carried out by stirring, and the stirring speed is preferably 100-300rpm, more preferably 150-250rpm.

[0023] In this invention, the pH of the enzymatic hydrolysate is adjusted to 10-14, followed by ultrasonic water bath extraction, centrifugation, and collection of the supernatant and precipitate. The solution used to adjust the pH of the enzymatic hydrolysate is preferably a sodium hydroxide solution. The pH of the enzymatic hydrolysate is further adjusted to 11-13. The ultrasonic extraction time is preferably 20-40 min, more preferably 25-35 min. The water bath extraction temperature is preferably 30-50℃, more preferably 35-45℃. The water bath extraction time is preferably 4-8 h, more preferably 5-7 h. During the water bath extraction, stirring is performed. The stirring speed is preferably 100-300 rpm, more preferably 150-250 rpm. The centrifugation speed is preferably 6000-8000 rpm, more preferably 6500-7500 rpm. The centrifugation time is preferably 20-40 min, more preferably 25-35 min.

[0024] In this invention, the precipitate obtained after centrifugation is mixed with water and extracted repeatedly 1-3 times according to step 2), and the supernatant is collected; the number of extractions is preferably 2 times.

[0025] In this invention, the obtained supernatant is combined and impurities are removed. The pH is adjusted to 2-5 for the first time, allowed to stand, centrifuged, and the precipitate is collected. Water is added, and the pH is adjusted to 8-12 for the second time. The mixture is pre-frozen at -60 to -100°C, and then freeze-dried to obtain crude protein powder of *Microcystis aeruginosa*. The preferred method for impurity removal is vacuum filtration, with a pressure of 0.07-0.09. The filter membrane used for the reduced pressure filtration is preferably 0.45 μm; the solution used for the first pH adjustment is preferably hydrochloric acid solution, and the first pH adjustment is further adjusted to 2.5-3.5; the settling temperature is preferably 2-6℃, more preferably 3-5℃; the settling time is preferably 20-30h, more preferably 22-26h; the centrifugation speed is preferably 6000-8000rpm, more preferably 6500-7500rpm; the centrifugation time is preferably 20-40min, more preferably 25-35min; the solution used for the second pH adjustment is preferably sodium hydroxide solution, and the second pH adjustment is further adjusted to 9-11; the pre-freezing temperature is further preferably -70~-90℃; the freeze-drying pressure is preferably 10-30Pa, more preferably 15-25Pa; the freeze-drying time is preferably 48h-72h, more preferably 54-66h.

[0026] In this invention, crude protein powder of *Microcystis aeruginosa* is mixed with water and stirred, then the pH is adjusted to 7-9. Trypsin is added for enzymatic hydrolysis. After hydrolysis, the mixture is centrifuged, the supernatant is collected, impurities are removed, and the solution is concentrated and freeze-dried to obtain crude polypeptide powder of *Microcystis aeruginosa*. The preferred mixing ratio of the crude protein powder of *Microcystis aeruginosa* to water is (15-25) g:(300-500) mL, more preferably (18-22) g:(350-450) mL. The preferred enzyme activity of the trypsin is 2000-3000 U / mg, more preferably 2200-2700 U / mg. The preferred amount of trypsin is 0.1-0.3 g. The preferred hydrolysis temperature is 40-60℃, more preferably 45-55℃. The preferred hydrolysis time is 4-8 h, more preferably 5-7 h. Stirring is performed during the hydrolysis process, and the preferred stirring speed is 100-300 rpm. The enzymatic hydrolysis is performed at 150-250 rpm, more preferably 150-250 rpm; after the enzymatic hydrolysis is completed, the enzyme is heated in a boiling water bath for 10-15 min to inactivate it, with the heating time more preferably 12-14 min; the centrifugation speed is preferably 6000-8000 rpm, more preferably 6500-7500 rpm; the centrifugation time is preferably 20-40 min, more preferably 25-35 min; the impurity removal method is preferably vacuum filtration, with a pressure of 0.07-0.09 rpm. The filter membrane used for vacuum filtration is preferably 0.45 μm; the concentration pressure is preferably 0.070-0.095 MPa, more preferably 0.080-0.090 MPa, and even more preferably 0.085 MPa; the concentration temperature is preferably 50-70℃, more preferably 55-65℃; the concentration is stopped when the volume of the supernatant is 1 / 4 to 1 / 3 of the original volume; the pre-freezing is performed before freeze-drying, and the pre-freezing temperature is preferably -60 to -100℃, more preferably -70 to -90℃; the freeze-drying pressure is preferably 10-30 Pa, more preferably 15-25 Pa; the freeze-drying time is preferably 48 h-72 h, more preferably 54-66 h.

[0027] In this invention, crude peptide powder of *Chlorella vulgaris* is dissolved in water to obtain a crude peptide solution of *Chlorella vulgaris*, which is then separated by ultrafiltration using 10 kDa and 3 kDa ultrafiltration membranes to obtain *Chlorella vulgaris* ACE inhibitory peptide extract with a molecular weight <3 kDa.

[0028] This invention also provides the application of *Microcystis chinensis* ACE inhibitory peptide, *Microcystis chinensis* ACE inhibitory peptide extract, or *Microcystis chinensis* ACE inhibitory peptide extract prepared by the preparation method of *Microcystis chinensis* ACE inhibitory peptide extract in the preparation of drugs for treating hypertension.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] Preparation of ACE inhibitory peptides (YP and YP-3): Take 80 g of *Microcystis aeruginosa* powder, add 800 mL of deionized water and stir well. Sonicate in a water bath for 20 min (800 W), then let stand for 10 h. Next, treat the mixture under high pressure (0.20 MPa, 120℃) for 30 min. After naturally cooling to room temperature, adjust the pH to 6.0 with sodium hydroxide, add 2.4 g of cellulase (50 U / mg), and enzymatically hydrolyze in a water bath at 37℃ for 2 h, stirring continuously at 200 rpm. Adjust the pH of the hydrolyzed solution to 12.0 with saturated sodium hydroxide, sonicate in a water bath for 30 min, then extract in a water bath for 6 h (37℃, 200 rpm stirring). Centrifuge the extract (7000 rpm for 30 min) and collect the supernatant. Add an appropriate amount of deionized water to the precipitate and repeat the extraction once, collecting the supernatant. Combine the supernatants from both extractions and remove impurities by vacuum filtration (0.45 μm, 0.08 MPa). The obtained filtrate was adjusted to pH 3.0 with hydrochloric acid and allowed to stand at 4°C for 24 h. The extract was centrifuged (7000 rpm for 30 min) and the precipitate was collected. The precipitate was dissolved in an appropriate amount of deionized water, and the pH was adjusted to 10.0 with saturated sodium hydroxide. The resulting solution was pre-frozen at -80°C and then freeze-dried (20 Pa) for 60 h to obtain crude protein powder from *Microcystis aeruginosa*. 20 g of the obtained crude protein powder was added to 400 ml of deionized water and stirred until homogeneous, yielding a crude protein solution of 0.05 g / ml. The pH was adjusted to 8.0, and 0.2 g of trypsin (enzyme activity 2500 U / mg) was added. The solution was enzymatically hydrolyzed in a water bath at 50°C for 6 h, with low-speed stirring (200 rpm) during hydrolysis. After hydrolysis, the solution was heated in a boiling water bath for 12 min to inactivate the enzyme. The resulting solution was cooled to room temperature and centrifuged at 7000 rpm for 30 min, and the supernatant was collected. Impurities were removed from the supernatant by vacuum filtration (0.45 μm, 0.08 MPa). The supernatant was concentrated at 60 °C and 0.085 MPa until its volume was reduced to 1 / 4 to 1 / 3 of its original volume. The resulting concentrated solution was pre-frozen at -80 °C and then freeze-dried (20 Pa) for 60 h to obtain crude polypeptide powder of *Microcystis aeruginosa* (i.e., YP). The obtained crude polypeptide powder was prepared into a 5 g / L solution and subjected to ultrafiltration using 10 kDa and 3 kDa ultrafiltration membranes sequentially to obtain the fraction with a molecular weight <3 kDa (YP-3).

[0032] Example 2

[0033] ACE inhibitory activity of *Microcystis aeruginosa* hydrolysates (YP and YP-3): 10 μL of each of the YP and YP-3 solutions prepared in Example 1 were mixed with 30 μL of substrate (hippuryl-histyl-leucine, HHL, 2.5 mM) and preheated in a 37°C water bath for 5 min. Then, 20 μL of ACE solution (0.1 U / mL) was added, and the mixture was reacted in a 37°C water bath for 60 min. Finally, 80 μL of hydrochloric acid solution (1 M) was added to terminate the reaction. The control and blank groups were replaced with an equal volume of borate buffer (0.1 M, pH=8.3) instead of the samples. For the blank group, 80 μL of 1 M hydrochloric acid was added before adding the ACE solution. All solutions used borate buffer (0.1 M, pH=8.3) as the solvent.

[0034] The reaction solution was analyzed using high-performance liquid chromatography (HPLC). The detection conditions were as follows: Chromatographic column: C18 column (4.6) 150 mm, 5 μm); Flow rate: 0.5 mL / min; Column temperature: 25 ℃; Injection volume: 10 μL; Detection wavelength: 228 nm; Mobile phase A: Ultrapure water containing 0.05% trifluoroacetic acid (TFA); Mobile phase B: Acetonitrile; Elution method: Isocratic elution of 78% mobile phase A and 22% mobile phase B for 12 min.

[0035]

[0036] Wherein, A control – the peak area of ​​hippuric acid in the control group; A sample – the peak area of ​​hippuric acid in the sample group; A blank – the peak area of ​​hippuric acid in the blank group.

[0037] Experimental results: such as Figure 1 As shown in the figure, at a concentration of 33.3 μg / mL, the inhibition rates of YP and YP-3 on ACE were (7.11±0.07)% and (30.90±1.82)%, respectively. It is evident that the activity of YP-3 was significantly higher than that of YP. The inhibition rate of both peptides on ACE increased with increasing concentration, and at low concentrations, the inhibition rate of YP-3 on ACE was significantly higher than that of YP. With increasing peptide concentration, the inhibitory effects of both components on ACE tended to saturate. The half-maximal inhibitory concentrations (IC50) of YP and YP-3 on ACE activity were obtained using DoseResponse fitting. 50 The values ​​were 301.0 ± 17.2 μg / mL and 75.8 ± 1.8 μg / mL, respectively. IC50 50 A lower value indicates a better suppression effect, based on IC. 50The comparison of values ​​shows that after ultrafiltration separation, the inhibitory activity of YP-3 on ACE increased by 3 times compared with that before ultrafiltration.

[0038] Example 3

[0039] YP-3 polypeptide sequence identification and screening of ACE-inhibiting oligopeptides: The amino acid sequence of the YP-3 peptide was identified by LC-MS / MS. The sample was dissolved in ultrapure water containing 0.1% formic acid, and 2 μL of the sample solution was loaded onto a nanoViper C18 (Acclaim PepMap 100, 75 μm × 2 cm) column. Online chromatographic separation was performed on an Easy nLC 1200 system, using 20 μL of 100% mobile phase A (ultrapure water containing 0.1% formic acid) for capture and desalting. Then, gradient elution was performed using an analytical column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm 100 Å), with mobile phase B (80% acetonitrile, 0.1% formic acid): 5%–38%, for 30 min. Analysis was performed using a ThermoFisher Q Exactive mass spectrometer combined with a nano-spray ion source at a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 275 °C. The primary mass spectrometry scan resolution was 70,000, the AGC target was 3 × 10⁶, the scan range was 100–1500 m / z, and the maximum injection time was 100 ms. For the secondary mass spectrometry scan, only the spectra with charge states 1–3 were selected. Fragmentation was performed through high-energy collisions with a normalized collision energy of 28 eV, the AGC target was 8,000, the maximum injection time was 50 ms, and the dynamic exclusion was set to 6 s. The raw mass spectrometry files were analyzed using PEAKS Studio 8.5.

[0040] A total of 3233 polypeptide sequences were identified, and 1151 oligopeptides with a sequence ≤9 were selected as the research subjects. Polypeptides with high potential biological activity and good water solubility were selected sequentially using PeptideRanker (≥0.85) and hydrophilicity (Gravy≤1.5). These polypeptides were then molecularly docked with ACE enzyme (1O8A). The top 8 polypeptides with the highest abundance (p<0.001) were selected for chemical synthesis and subsequent activity verification. The polypeptide sequences are shown in Table 1.

[0041] Table 1 Physicochemical properties of ACE inhibitory peptides screened by computer simulation

[0042] Example 4

[0043] Synthesis and activity verification of the 8 selected peptides: Eight potential ACE-inhibiting peptides obtained from the screening were synthesized using a solid-phase method. Solid-phase synthesis: The eight peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method, achieving a purity ≥95%. The synthesis mainly consisted of three cycles: deprotection, activation and cross-linking, elution, and deprotection.

[0044] Activity verification results: Figure 2 As shown, all eight peptides screened exhibited varying degrees of ACE inhibition. At a peptide concentration of 166.7 μg / mL, the inhibition rates of ACE activity by FLSQPF, WDPLGF, GGPPFTVF, FTPGTF, GSPLFDPR, FGPLG, FGPIG, and PGPAIF were (23.70±1.70)%, (64.52±0.65)%, (90.20±0.52)%, (39.04±2.26)%, (60.14±5.81)%, (5.44±0.70)%, (32.83±0.64)%, and (78.73±0.26)%, respectively. Among these, the three peptides with the best ACE inhibition effects were PGPAIF, GGPPFTVF, and WDPLGF.

[0045] Example 5

[0046] The inhibitory effects of the three peptides with the best activity, PGPAIF, GGPPFTVF, and WDPLGF, on ACE at different concentrations were further investigated.

[0047] Experimental results: The mass spectra of the three polypeptides are as follows Figure 3 As shown, its inhibitory activity against ACE is shown in [reference needed]. Figure 4As the concentration increased, the inhibitory activity of each peptide against ACE gradually increased. When the peptide concentration was 333.3 μg / mL, the inhibition rates of ACE by PGPAIF, GGPPFTVF, and WDPLGF were 84.36±1.33%, 97.19±2.49%, and 73.33±1.54%, respectively. Furthermore, the half-maximal inhibitory concentrations (IC50) of these three peptides against ACE were 31.26±1.32 μg / mL, 32.65±1.16 μg / mL, and 79.22±3.55 μg / mL, respectively. Since the molecular weights of PGPAIF, GGPPFTVF, and WDPLGF are 600.20, 820.15, and 733.20, respectively, their half-maximal inhibitory concentrations (WMCs) for ACE, converted to μmol / L, are 52.03±2.19 μmol / L, 39.77±1.42 μmol / L, and 107.96±4.84 μmol / L, respectively. A lower WMC indicates better inhibitory activity. Therefore, GGPPFTVF exhibits the best inhibitory activity against ACE, followed by PGPAIF, and then WDPLGF.

[0048] Example 6

[0049] Three bioactive peptides, PGPAIF, GGPPFTVF, and WDPLGF, were subjected to in vitro simulated gastrointestinal digestion to investigate their stability against digestive enzymes. Solutions of PGPAIF, GGPPFTVF, and WDPLGF at 1 mg / mL were prepared, and then an equal volume of simulated gastric juice was added. The mixture was incubated at 37°C for 90 min to simulate gastric digestion, followed by an equal volume of simulated intestinal juice for 180 min. Samples were taken every 30 min to measure the ACE inhibition rate.

[0050] Experimental results: such as Figure 5 As shown. By Figure 5 As shown in A, after digestion by gastric and intestinal juices, the ACE inhibitory activity of PGPAIF does not change significantly, fluctuating within a range of less than 3%, indicating relative stability. Figure 5 As shown in Figure B, after 30 minutes of digestion by gastric juice, the ACE inhibitory activity of GGPPFTVF increased by approximately 15%, and then stabilized over time. After digestion by intestinal juice, the ACE inhibitory activity decreased. After digestion by gastrointestinal juice, the ACE inhibitory activity decreased to 84.69%. Figure 5 As shown in C, after 30 minutes of digestion by gastric juice, the ACE inhibitory activity of WDPLGF increased by about 15%, and its ACE inhibitory activity tended to stabilize as time went on. After further digestion by intestinal juice, its ACE inhibitory activity increased after 60 minutes, and as time went on, its ACE inhibitory activity increased to 117.50%, and tended to stabilize, with a fluctuation range of less than 20%.

[0051] After gastrointestinal digestion, PGPAIF maintained stable ACE inhibitory activity, while WDPLGF showed an increase in ACE inhibition rate after gastrointestinal digestion. Although GGPPFTVF had slightly poor digestibility, its ACE inhibitory activity decreased by 15.31% after gastrointestinal digestion, but the decrease was acceptable.

[0052] As can be seen from the above embodiments, the *Microcystis aeruginosa* ACE inhibitory peptide extract obtained by the preparation method of the present invention and the polypeptides PGPAIF, GGPPFTVF, and WDPLGF obtained by separation and screening have good inhibitory effects on ACE, and have good digestibility. They can be used to make adjunctive treatments to lower hypertension or to develop products for patients with hypertension.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microalgae-like ACE-inhibiting peptide, characterized in that, The ACE inhibitory peptide sequence of *Microcystis globulus* is Gly-Pro-Gly-Pro-Phe-Thr-Val-Phe.

2. A microalgae ACE-inhibiting peptide extract, characterized in that, This includes an ACE inhibitory peptide with the sequence Gly-Pro-Gly-Pro-Phe-Thr-Val-Phe.

3. The method for preparing the *Microcystis globulus* ACE inhibitory peptide extract according to claim 2, characterized in that, Includes the following steps: 1) Mix the microalgae powder and water, sonicate, let stand, treat under high pressure at 0.1-0.3 MPa for 20-40 min, adjust the pH to 5-7, add cellulase to hydrolyze and obtain the enzymatic hydrolysate; 2) After adjusting the pH of the enzyme hydrolysate to 10-14, extract using an ultrasonic water bath, centrifuge, and collect the supernatant and precipitate; 3) Mix the precipitate obtained after centrifugation with water and repeat the extraction 1-3 times as in step 2), and collect the supernatant; 4) Combine the supernatants obtained in steps 2) and 3) and remove impurities. Adjust the pH to 2-5 for the first time, let stand, centrifuge, collect the precipitate, add water and adjust the pH to 8-12 for the second time. Pre-freeze at -60 to -100℃ and then freeze-dry to obtain crude protein powder of *Microcystis aeruginosa*. 5) Mix the crude protein powder of Microcystis aeruginosa with water and stir, adjust the pH value to 7-9, add trypsin for enzymatic hydrolysis, centrifuge after enzymatic hydrolysis, collect the supernatant, remove impurities, concentrate and freeze dry to obtain crude polypeptide powder of Microcystis aeruginosa. 6) Dissolve the crude polypeptide powder of *Chlorella vulgaris* in water to obtain a crude polypeptide solution of *Chlorella vulgaris*. Then, use ultrafiltration membranes with 10 kDa and 3 kDa to separate the polypeptides and obtain *Chlorella vulgaris* ACE inhibitory peptide extract with a molecular weight <3 kDa.

4. The method for preparing the *Microcystis aeruginosa* ACE inhibitory peptide extract according to claim 3, characterized in that, The mixing ratio of the Microcystis aeruginosa algal powder and water is (60-100)g:(600-1000)mL; The ultrasonic power in step 1) is 500W-1000W, the ultrasonic time is 10-30min; the settling time is 8-12h; and the high-pressure treatment temperature is 100-150℃. The cellulase has an enzyme activity of 30-70 U / mg, and the amount of cellulase used is 1.5-3g; The enzymatic hydrolysis temperature is 30-50℃, and the enzymatic hydrolysis time is 1.5-3h.

5. The method for preparing the *Microcystis aeruginosa* ACE inhibitory peptide extract according to claim 3, characterized in that, The ultrasound time in step 2) is 20-40 min; the water bath extraction temperature is 30-50℃; the water bath extraction time is 4-8 h; and stirring is performed during the water bath extraction process at a speed of 100-300 rpm.

6. The method for preparing the *Microcystis aeruginosa* ACE inhibitory peptide extract according to claim 3, characterized in that, The settling temperature in step 4) is 2-6℃, and the settling time is 20-30h; the freeze-drying pressure is 10-30Pa, and the freeze-drying time is 48h-72h.

7. The method for preparing the *Microcystis aeruginosa* ACE inhibitory peptide extract according to claim 3, characterized in that, In step 5), the mixing ratio of the crude protein powder of *Microcystis aeruginosa* to water is (15-25) g: (300-500) mL; the enzyme activity of the trypsin is 2000-3000 U / mg, and the amount of trypsin used is 0.1-0.3 g; the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 4-8 h.

8. The method for preparing the *Microcystis aeruginosa* ACE inhibitory peptide extract according to claim 3, characterized in that, In step 5), the concentration pressure is 0.070-0.095 MPa and the concentration temperature is 50-70°C; the freeze-drying pressure is 10-30 Pa and the freeze-drying time is 48-72 h.

9. The method for preparing the *Microcystis aeruginosa* ACE inhibitory peptide extract according to claim 3, characterized in that, The centrifugation speed in steps 2), 4), and 5) is 6000-8000 rpm, and the centrifugation time is 20-40 min.

10. The use of the *Microcystis cereus* ACE inhibitory peptide according to claim 1, the *Microcystis cereus* ACE inhibitory peptide extract according to claim 2, or the *Microcystis cereus* ACE inhibitory peptide extract prepared by any one of claims 3 to 9 in the preparation of drugs for treating hypertension.

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