CN117964693A

CN117964693A - Chlorella antihypertensive peptide and composition, preparation method and application thereof

Info

Publication number: CN117964693A
Application number: CN202410158834.9A
Authority: CN
Inventors: 岳洋; 索齐善; 张全斌; 吴宁; 王晶; 耿丽华
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2024-02-04
Filing date: 2024-02-04
Publication date: 2024-05-03

Abstract

The invention belongs to the technical field of biology, and relates to a chlorella antihypertensive peptide and a composition, a preparation method and application thereof in foods, health products or medicines. The invention takes chlorella as raw material, uses several specific proteases to carry out enzymolysis on chlorella, and polypeptide after enzymolysis obtains chlorella enzymolysis peptide extract through the technologies of enzyme deactivation, centrifugation, drying and the like, and researches find that the chlorella enzymolysis peptide extract has remarkable Angiotensin Converting Enzyme (ACE) inhibitory activity. Wherein the chlorella enzymatic peptide extract obtained after trypsin enzymatic hydrolysis contains various active oligopeptides, such as chlorella oligopeptides LKKAP and PGLRP. Experiments show that the isolated chlorella oligopeptides have remarkable ACE inhibitory activity, and the minimum IC ₅₀ can reach 36.19 mu M and 44.78 mu M. The invention provides a method for preparing chlorella antihypertensive peptide, and successfully identifies two chlorella oligopeptide with good antihypertensive activity and novel sequence, provides a reference for fully utilizing chlorella protein resources, and has good development and utilization prospects.

Description

Chlorella antihypertensive peptide and composition, preparation method and application thereof

Technical Field

The invention relates to the field of biology, in particular to a chlorella antihypertensive peptide and a composition, a preparation method and application thereof.

Background

Hypertension is a systemic disease characterized mainly by elevated blood pressure. Diagnostic criteria for hypertension: in the case of no use of the drop-off drug, the blood pressure of the consulting room is measured 3 times a day, the systolic pressure (SBP) is not less than 140mmHg and/or the diastolic pressure (DBP) is not less than 90mmHg. The prevalence of hypertension of residents in China is generally rising, wherein the coarse prevalence of hypertension of adults over 18 years old is about 27.9%, and the estimated number of people suffering from hypertension of adults is about 2.45 hundred million. Untreated hypertension can eventually lead to stroke, heart failure, kidney damage, etc., and in severe cases can lead to death. Therefore, the method still has great prospect for developing effective antihypertensive drugs and health-care foods.

The renin-angiotensin-aldosterone system (RAAS) plays a vital role in the regulation of blood pressure. In RAAS, angiotensin I converting enzyme catalyzes the degradation of angiotensin I to angiotensin II on the one hand and bradykinin on the other hand. Angiotensin II causes vasoconstriction and water-sodium reabsorption by binding to angiotensin II type II receptors, which in turn causes an increase in blood pressure. At the same time, the elevation of bradykinin level can also play a role in lowering blood pressure. Thus, ACE inhibitors are always first line drugs for the treatment of hypertension. However, since the existing ACE inhibitors have a series of side effects, such as dry cough, rash, hypokalemia, etc., the development of more efficient and safer ACE inhibitors remains a research hotspot.

Chlorella is a unicellular green algae rich in proteins, lipids, minerals, vitamins, and antioxidant substances (vitamin C, lutein, carotenoids), etc. The chlorella protein content is high, can account for more than 50% of dry weight, and is obviously higher than milk and soybean protein. Studies have shown that taking chlorella has biological activities of lowering blood pressure, anti-inflammatory, lowering blood sugar, lowering blood lipid, and anti-oxidizing. The food protein can release rich bioactive peptide through enzymolysis, but the bioactive peptide prepared and identified from chlorella at present is less, especially ACE inhibitory peptide with blood pressure reducing activity. In 2009, I-Chuan Sheih and the like prepared chlorella antioxidant peptide by pepsin, the amino acid sequence of the chlorella antioxidant peptide is VECYGPNRPQF, various free radicals can be effectively removed, and the chlorella antioxidant peptide has an obvious protection effect on DNA. Domestic researchers in 2014 utilize chlorella protein as a raw material to prepare antioxidant polypeptide through enzymolysis of acid protease. In 2018, researchers successfully prepare a plurality of chlorella oligodipeptides with the blood pressure reducing effect by compounding and hydrolyzing chlorella proteins, can obviously inhibit angiotensin converting enzyme-I, and shows obvious blood pressure reducing effect in vivo. Thus, chlorella ACE inhibitory peptides remain to be further studied.

Disclosure of Invention

The invention aims to provide a chlorella antihypertensive peptide, a composition thereof and a preparation method thereof; the invention also aims to provide the application of the chlorella antihypertensive peptide and the composition thereof in foods, medicines or health care products.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a chlorella enzymatic hydrolysis peptide extract, which is characterized in that: the extract contains one or more peptide sequences of LKKAP, PGLRP, ATKFP, VASPK, KVLP, VAIGQK, IGRKFS, VGAGAH, SAKTRAE, and has an average molecular weight of less than 2000Da and an enzymatic peptide ratio of not less than 80%.

The invention also provides an inhibiting oligopeptide of chlorella Angiotensin Converting Enzyme (ACE), which is characterized in that: the oligopeptide is one or more of LKKAP, PGLRP, ATKFP, VASPK, KVLP, VAIGQK, IGRKFS, VGAGAH.

In addition, the invention also provides a chlorella antihypertensive peptide or a chlorella antihypertensive composition, which is characterized by comprising the chlorella enzymolysis peptide extract, or any one or more oligopeptides of chlorella Angiotensin Converting Enzyme (ACE) inhibitory oligopeptides, or a composition of the chlorella enzymolysis peptide extract and any one or more oligopeptides of chlorella Angiotensin Converting Enzyme (ACE) inhibitory oligopeptides.

Further, the invention also provides a chlorella food, which is characterized in that: the chlorella food contains a chlorella enzymolysis peptide extract or chlorella Angiotensin Converting Enzyme (ACE) inhibitory oligopeptide or any chlorella enzymolysis peptide sequence or composition.

The application of the chlorella antihypertensive peptide is characterized in that: the application of the enzymatic hydrolysis peptide extract or ACE inhibition oligopeptide or the composition thereof in preparing an angiotensin converting enzyme inhibitor or a health-care product or a pharmaceutical preparation related to the prevention or treatment of hypertension and cardiovascular diseases.

The angiotensin converting enzyme inhibitor, or the health-care product or the pharmaceutical preparation related to the prevention or treatment of hypertension and cardiovascular diseases is characterized in that: the extract, or any one or more chlorella antihypertensive peptides, or the composition is prepared by adding any auxiliary materials which meet the production allowance of foods or medicines.

Further, the invention provides a method for preparing chlorella enzymatic hydrolysis peptide extract, which is characterized by comprising the following steps of: taking chlorella as a raw material, breaking the wall of the chlorella, carrying out enzymolysis by protease, inactivating enzyme and centrifuging to obtain chlorella zymolyte supernatant; purifying and/or drying the obtained supernatant of the zymolyte to obtain the chlorella zymolyte peptide extract. The protease is any one of trypsin, alkaline protease, chymotrypsin and neutral protease.

Preferably, the preparation method of the chlorella enzymatic peptide extract comprises the following steps: the ratio of the material to the water (chlorella powder: water, w/w) is 1:10-1:30 (w/w), the ultrasonic cell disruption instrument is used for breaking the wall of the feed liquid with the power of 200-800w, the supernatant is centrifugally taken after 3-5 times of circulation, and protease is added into the supernatant with the enzyme bottom ratio (chlorella powder: enzyme, w/w) of 1% -10%, and the enzymolysis conditions are as follows: pH is 7-10, temperature is 40-65 ℃, and enzymolysis time is 1-12h; and then heating at 95 ℃ for 15min to inactivate enzyme, centrifuging to obtain an enzymatic hydrolysate supernatant, and drying to obtain the chlorella enzymatic peptide extract. Separating and purifying the supernatant of the enzymolysis product by using one or more of ultrafiltration membrane concentration, sephadex G-15 gel column, DEAE anion exchange column chromatography and C18 reverse phase column chromatography, and freeze drying the obtained components to obtain refined chlorella enzymolysis peptide extract.

The antihypertensive peptide LKKAP (SEQ ID NO. 2) is prepared from chlorella pyrenoidosa (Chlorella pyrenoidosa) by enzymolysis and various chromatographic separation and purification. The sequence is Leu-Lys-Lys-Ala-Pro, and the relative molecular weight is 555.78Da. The polypeptide can be applied to preparation of antihypertensive drugs or pharmaceutical compositions, health-care foods or common foods through enzymolysis, separation and purification, microbial fermentation or chemical synthesis.

The antihypertensive peptide PGLRP (SEQ ID NO. 10) is prepared from chlorella pyrenoidosa (Chlorella pyrenoidosa) by enzymolysis and various chromatographic separations and purifications. The sequence is Pro-Gly-Leu-Arg-Pro, and the relative molecular weight is 538.71Da. The polypeptide can be applied to preparation of antihypertensive drugs or pharmaceutical compositions, health-care foods or common foods through enzymolysis, separation and purification, microbial fermentation or chemical synthesis.

The chlorella antihypertensive peptide can obviously inhibit the activity of Angiotensin Converting Enzyme (ACE) under the in vitro experimental condition of taking the maleyl-histidine-leucine as a substrate, and the half inhibition rates (IC ₅₀) of LKKAP and PGLRP are 36.19 mu M and 44.78 mu M respectively.

The invention has the advantages that:

The polypeptide obtained by the invention has excellent in vitro ACE inhibitory activity, and has no obvious cytotoxicity in a cell level experiment. The polypeptide is pentapeptide with molecular weight of 555.78Da and 538.71Da, and the polypeptide has small molecular weight and easy absorption. The polypeptide has good application prospect in the fields of foods, health care products, medicaments and the like with blood pressure regulating activity.

Description of the drawings:

FIG. 1 molecular weight distribution of Chlorella zymolytic peptide extract was determined by gel chromatography.

FIG. 2 ACE inhibitory Activity of Chlorella enzymatic peptide extract

FIG. 3A is a mass spectrum total ion flow chart of Chlorella enzymatic hydrolysis peptide extract

FIG. 4 ACE inhibition of Chlorella oligopeptide at 400. Mu.g/mL

FIG. 5 high performance liquid chromatography method for identifying purity of synthetic Chlorella oligopeptide

FIG. 6 mass spectrum of synthetic Chlorella oligopeptide

FIG. 7 dose curves for LKKAP and PGLRP ACE inhibition

FIG. 8 double reciprocal plot determination LKKAP and PGLRP inhibition patterns on ACE

FIG. 9 pH, temperature and in vitro simulated digestion stability of LKKAP and PGLRP inhibiting ACE

FIG. 10 Mass spectrogram of corresponding parent ion of AP, PG, LRP produced by in vitro simulated digestion LKKAP and PGLRP

The specific embodiment is as follows:

The invention is further illustrated by the following figures and examples. The invention aims to take chlorella as a raw material, isolate and screen the chlorella with a definite sequence through proteolysis, and it is understood that the examples are only used for illustrating the invention and are not used for limiting the scope of the invention. Further, it is understood that various changes and modifications of the invention may be made by those skilled in the art after reading the disclosure of the invention, and such equivalents are intended to fall within the scope of the invention as defined by the claims.

EXAMPLE 1 preparation of Chlorella enzymatic peptide extract

Weighing 15g of chlorella dry powder, freezing in 240mL of deionized water for 4h at minus 20 ℃, thawing at 37 ℃, and crushing chlorella suspension by using an ultrasonic cell crusher, wherein parameters of the ultrasonic cell crusher are set to work for 15s, the interval is 15s, and the power is 300W for 30min. The container is placed on ice during ultrasonic treatment to prevent the liquid from rising temperature due to ultrasonic treatment. Freezing thawing-ultrasonic circulating for 3 times, centrifuging at 4deg.C for 10min at 8000 RCF, and collecting supernatant to obtain Chlorella zymolyte supernatant. The protein concentration in the supernatant was measured and adjusted to 10mg/mL by adding deionized water, and after adjusting the pH shown in Table 1 by adding NaOH solution having a concentration of 0.1M, pepsin, trypsin, snail protease, alpha-chymotrypsin, alkaline protease, flavourzyme and neutral protease were added respectively in an amount of 1% by mass of the protein, and the conditions of the enzymatic hydrolysis pH and temperature were as shown in Table 1. After reacting for 4 hours, heating for 15min at 95 ℃ to inactivate enzyme, centrifuging for 10min at 8000 RCF, taking supernatant and freeze-drying to obtain chlorella enzymatic peptide crude extract.

TABLE 1 temperature and pH conditions for the preparation of Chlorella enzymatic peptide extracts

Enzymes	pH	Temperature (. Degree. C.)
			Alpha-chymotrypsin	8	37
Trypsin, trypsin and its preparation method	8	37
			Pepsin	2	37
Neutral protease	7	50
			Alkaline protease	8	50
Snail enzyme	7	37
			Flavoured protease	7	50

EXAMPLE 2 high Performance liquid gel chromatography determination of molecular weight distribution of Chlorella zymolytic peptides

And measuring the molecular weight distribution of the zymolyte by adopting a high performance liquid chromatography. The liquid chromatography system was fitted with TSKG SWXL (7.8x300 mm) column (TOSOH co., japan). The sample was dissolved in ultrapure water at a concentration of 5mg/mL, filtered through a 0.22 μm membrane, and loaded. The sample eluting mobile phase was: 45% acetonitrile +55% ultra pure water (v/v contains 0.1% TFA), flow rate 0.5mL/min, detection wavelength 214nm. The molecular weights of cytochrome C, insulin, bacitracin, glycine-tyrosine-arginine tetrapeptide and glycine-glycine tripeptide are used for making standard curves for peak time, and the standard curves are as follows:

y＝-0.22x+0.671,R²＝0.9961

The percentage of peptides with molecular weights <1kDa, 1-2 kDa, 2-3 kDa and 3-5 kDa in the samples was calculated based on the established standard curve.

The molecular weight distribution of the chlorella enzymatic peptide is shown in figure 1. According to the molecular weight distribution diagram of fig. 1, the molecular weight distribution of the chlorella enzymatic peptides calculated by adopting a normalization method is shown in table 2, and the ratio of the enzymatic peptides with the average molecular weight lower than 1000 Da in chlorella after enzymolysis by trypsin, alkaline protease, chymotrypsin and neutral protease is 91.5889%, 97.7888%, 85.8229% and 94.474% respectively, which are obviously higher than those obtained by pepsin (71.2799%) and snail protease (71.911%).

Example 3 High Performance Liquid Chromatography (HPLC) determination of in vitro ACE inhibitory Activity of Chlorella enzymatic peptide extract

The experimental method comprises the following steps:

The principle of the method is as follows: the hippocampal-histamine-leucine (Hip-His-Leu, HHI, sigma-Aldrich) can be used as a substrate of Angiotensin Converting Enzyme (ACE) to be decomposed to generate hippuric acid, after different ACE inhibitors are added, the generation amount of the hippuric acid is correspondingly reduced, and the inhibition activity of the inhibitor on the ACE activity can be evaluated by calculating the peak area of the hippuric acid at 228 nm.

Experimental reagent:

ACE (0.1U/mL), maleyl-histidine-leucine (HHI), captopril, 0.1M sodium borate solution (pH 8.3, 0.3M sodium chloride)

ACE was dissolved in sodium borate buffer to a final concentration of 0.1U/mL for the assay. The samples were dissolved in sodium borate buffer to prepare polypeptide solutions of different concentrations. Then, 20. Mu.L of a polypeptide solution of a certain concentration was mixed with 10. Mu.L of ACE solution. The mixture was incubated at 37℃for 5min, and 50. Mu.L of HHT (sodium borate buffer pH8.3, containing 0.3M sodium chloride) at a molar concentration of 5mM was added to the mixture to initiate the reaction. The reaction was maintained at 37℃for 60min, and then 150. Mu.L of HCl having a molar concentration of 1M was added to terminate the reaction. The solution was passed through a 0.22 μm filter to obtain a reaction solution. mu.L of the reaction solution was loaded on RP-HPLC, which was coupled to Eclipse XDB-C18 column (4.6 mm. Times.150 mm. Times.5 μm), and the concentration of Hippuric Acid (HA) was detected by ultraviolet. Hippuric acid was detected as absorbance at 228 nm. All absorbance measurements were repeated three times. ACE inhibition activity was calculated as follows:

ACE inhibitory activity (%) = (AControl-AInhibitor)/AControl x 100)

Wherein AInhibitor is the relative area of the Hippuric Acid (HA) peaks obtained from the reaction of ACE and HIL with the inhibitor. AControl is the relative area of the Hippuric Acid (HA) peak obtained from the reaction of ACE and HHL without inhibitor. IC50 is defined as the concentration of polypeptide that can inhibit half of ACE activity.

Chromatographic conditions:

c18 column (4.6mm x 150mm x 5 μm, agilent), detection wavelength: 228nm; mobile phase: 78% ultrapure water (containing 0.05% TFA) +22% acetonitrile (containing 0.05% TFA); flow rate: 0.8mL/min.

The in vitro ACE inhibitory activity measurement is shown in figure 2, and the result shows that only chlorella enzymatic hydrolysis peptide extract obtained after enzymolysis of trypsin, alkaline protease, chymotrypsin and neutral protease has obvious ACE inhibitory activity, wherein the ACE inhibitory rates of the enzymatic hydrolysis peptide extract obtained by trypsin, alkaline protease and neutral protease are equivalent when the concentration is 400 mug/mL, and are 63.65%, 64.215% and 65.31% respectively; the highest ACE inhibition activity of chymotrypsin zymolyte reaches 76.60 percent.

As a reference example, the enzymatic peptide extracts prepared from pepsin, snail protease and flavourzyme have lower ACE inhibition activity, and the inhibition rate ranges from 2 to 26 percent (figure 2).

Example 4 purification refinement of Chlorella enzymatic peptide extract and ACE inhibitory Activity assay

The chlorella enzymatic hydrolysis peptide extract prepared by trypsin is used as a representative, and is separated and purified to obtain further refined chlorella enzymatic hydrolysis extract, and ACE inhibitory peptide with a definite sequence is identified. Firstly, separating and purifying the chlorella antihypertensive peptide crude extract by using a gel chromatographic column (Sephadex G15, 1.6X100 cm), taking deionized water as a mobile phase, using the flow rate of 1mL/min, separating CT into 5 components according to an elution curve, and freeze-drying the components to obtain chlorella refined enzymatic peptide extracts respectively named as CTF1, CTF2, CTF3 and CTF 4.

The ACE inhibitory activity of 4 components was evaluated according to the calculation method of ACE inhibitory activity in example 3, and the result shows that the ACE inhibitory rate of CTF3 at a concentration of 400. Mu.g/mL is 54.86%.

CTF3 is separated, separated and purified by Sepharose DEAE Fast Flow (1.6X100 cm) anion exchange chromatography, and eluted by pure water until the conductivity is stable after sample loading, then eluted by 0-1mol/L NaCl solution linearly, and the elution curve is obtained by detecting the elution process at 230nm and 280 nm. According to the elution curve, separating CTF3 into 2 refined components of chlorella enzymatic hydrolysis peptide extract, which are respectively a water elution component CTD1 and a salt elution component CTD2. The ACE inhibitory activity of 2 components was evaluated according to the ACE inhibitory activity calculation method in example 3, and CTD1 showed a 62.46% ACE inhibition at a concentration of 400 μg/mL.

The CTD1 fraction obtained by separation was further separated on an Agilent Zorbax SB-Aq C18 column (4.6X250 mm,5 μm) with the following elution procedure: 1-5min:5% acetonitrile (v%); 5-35min:5% -30% acetonitrile (v%); 35-40min:30-100% acetonitrile (v%); the flow rate was 0.8mL/min. The fractions were combined and collected to further obtain 3 refined fractions of chlorella enzymatic peptide extract, CTR1, CTR2 and CTR3, respectively, and ACE inhibitory activities were evaluated according to the method of example 3, with ACE inhibitory rates of 77.18%,73.29% and 52.54% at 50. Mu.g/mL.

Examples 4 and 5 show that the chlorella enzymatic peptide extract prepared by the enzymolysis of specific protease has remarkable ACE inhibitory activity, and the chlorella enzymatic peptide extract with different refining degrees obtained by the purification means has remarkable ACE inhibitory activity.

Example 5 high resolution Mass Spectrometry identification of Chlorella antihypertensive peptide sequences

Chlorella enzymolysis peptide sample is dissolved in a proper amount of ddH2O and added with DTT to make the final concentration 10mmol/L, IAA solution is added to make the final concentration 50mmol/L after water bath at 56 ℃ for 1h, desalting is carried out after 40min of light-proof reaction, and 0.1% formic acid solution is used for LC-MS/MS analysis after vacuum volatilizing solvent.

Nano LC-MS/MS: the column packing was ReproSil-Pur C18-AQ (1.9 μm,) The specification is 150 μm by 150mm. The mobile phases A, B were water and acetonitrile containing 0.1% formic acid, respectively, and 5. Mu.L was applied and then subjected to gradient elution at a flow rate of 600 nL/min. Gradient procedure is as follows:

TABLE 3 elution gradient Table

And performing analysis by Q Exactive Hybrid Quadrupole-Orbitrap-MS/MS (Thermo FISHER SCIRNTIFIC, USA) to obtain secondary mass spectrum data, and performing mass spectrum characteristic comparison in a self-contained database by using Byonic software to obtain polypeptide sequence information. The mass spectrum total ion flow chart is shown in figure 3, and the chlorella peptide sequence obtained by mass spectrum characteristic comparison and identification is marked in the mass spectrum, and the result shows that the chlorella enzymatic hydrolysis peptide extract contains LKKAP, PGLRP, ATKFP, VASPK, KVLP, VAIGQK, IGRKFS, VGAGAH, SAKTRAE and other oligopeptide molecules (SEQ ID NO.1-10, table 4).

Example 6 discovery of the Chlorella angiotensin converting enzyme ACE inhibitory oligopeptide

The first round of screening was performed on the identified sequences based on molecular weight error (Mass error), score (score) and signal strength (intensity) at the time of polypeptide identification. The two-and three-dimensional structures of chlorella oligopeptides were mapped using Chemdraw software. In molecular docking software, chlorella polypeptides were stored after protonation and energy minimization at pH 7.4. The crystal structure (PDBID:1O 86) of ACE is taken as a receptor molecule, and a Glide module is adopted for molecular docking. The butt joint site is centered on Zn ²⁺ of ACE active center, and the radius isIs connected with the ball. The binding energy of the conformation obtained by docking was calculated to characterize the affinity of the polypeptide to the ACE active center and the docking binding energy results are shown in table 4.

9 Polypeptides with binding energy below-30 kcal/mol were selected for synthesis and were tested for ACE inhibitory activity in vitro, the results are shown in FIG. 4. The results show that the selected 9 chlorella oligopeptides all show different degrees of ACE inhibitory activity at the concentration of 400 mug/mL, wherein the inhibition rates of LKKAP and PGLRP on ACE are highest and reach 95.68% and 97.60% respectively.

EXAMPLE 7 purity identification of Chlorella antihypertensive peptide

0.5Mg of the sample was weighed and dissolved in 0.5mL of ultrapure water, and then analyzed by a high performance liquid chromatography system equipped with NanoChrom Chromcore TM C18 (4.6×250mm×5 μm) column. The loading was 40. Mu.L, and the mobile phases were acetonitrile and ultrapure water containing 0.1% trifluoroacetic acid, respectively. The flow rate was 1.0mL/min, and the peak was detected at 214nm, and the chromatogram was shown in FIG. 5.

Analysis of polypeptide chromatographic peaks by a normalization method shows that the purity of the selected 9 chlorella oligopeptides is more than or equal to 95 percent.

Example 8 Mass spectrometric identification of Chlorella antihypertensive peptides

0.1Mg of the sample was dissolved in 0.5mL of ultrapure water, and after passing through a C18 column, the sample was analyzed by a Q-Exactive mass spectrometer with a loading of 1. Mu.L, a carrier gas flow rate of 1.5L/min, and a liquid phase mobile phase of 50% H2O+50% MeOH. The molecular weight of each peptide is shown in Table 4, and the mass spectrum is shown in FIG. 6.

TABLE 4 sequence, molecular weight and molecular docking score of Chlorella oligopeptides

EXAMPLE 9 IC ₅₀ determination of Chlorella antihypertensive peptide

The IC50 values of the polypeptides were calculated by measuring the ACE inhibition of LKKAP and PGLRP at concentrations of 400, 100, 80, 10,1,0.1,0.01,0.001. Mu.g/mL as described in example 2, setting three replicates, taking the average and plotting the log of the concentration versus the inhibition (FIG. 7).

The IC ₅₀ values of chlorella antihypertensive peptides LKKAP, PGLRP were determined to be 36.19 μm and 44.78 μm, respectively (fig. 7).

Example 10 inhibition pattern assay of Chlorella antihypertensive peptides

Polypeptide solutions having final HHT concentrations of 0.5,0.25,0.125,0.0625mM and chlorella antihypertensive peptides concentrations of 80. Mu.g/mL and 40. Mu.g/mL were prepared, and the activities of the polypeptides at different concentrations and HHT solutions at different concentrations (maleyl-histidyl-leucine solutions) were evaluated as described in example 1. The inhibition pattern of ACE by the polypeptides was analyzed by the double reciprocal method, plotted as the reciprocal of the rate of hippuric acid production versus the reciprocal of the substrate concentration. The rate of hippuric acid production is proportional to ACE activity, and the stronger the inhibition activity of ACE inhibitory peptide, the less the corresponding hippuric acid produced, the stronger the antihypertensive activity.

As shown in fig. 8, as the concentration of the polypeptide increases, the slope of the double reciprocal curve increases and the cross-sectional distance is unchanged, i.e., V _max becomes smaller Km, and the curves intersect on the negative half-axis of the horizontal axis, so that the inhibition patterns of LKKAP and PGLRP are considered to be non-competitive inhibition.

Example 11 determination of pH, temperature and in vitro simulated digestion stability of Chlorella antihypertensive peptides

Thermal stability study: synthetic peptides (80. Mu.g/mL) were incubated at various temperatures (0, 20, 40, 60, 80 and 100 ℃) for 2 hours, and then their ACE inhibitory activity was determined.

PH stability study: after incubation for 2 hours at different pH values (2, 4, 6, 8, 10 and 12), the ACE inhibitory activity of the synthetic peptides (80. Mu.g/mL) was determined.

Virtual gastrointestinal digestion of ACE inhibiting peptides: a computer simulated gastrointestinal digestion was performed using ExPASy Peptidecutter (https:// web. Expasy. Org/peptide_ cutter). Synthetic peptides were digested with pepsin (pH 1.3), trypsin and alpha-chymotrypsin (low specificity) cleavage sites. Peptide fragments have been searched on the web to determine their novelty and ACE inhibiting activity.

In vitro gastrointestinal stability studies: the synthetic peptides were incubated in simulated gastric (pH 1.3, 1% (m/v) pepsin in 1mol/L HCl) and intestinal environments (pH 6.8, 6.8g K ₂PO₄ and 1% (m/v) trypsin and alpha-chymotrypsin in 500mL deionized water), respectively, for 2 hours. ACE inhibitory activity of the mixture was measured. The mixture was then subjected to high resolution LC/MS to determine if predicted fragments of each peptide were present.

As shown in fig. 9, the ACE inhibition capacity of LKKAP and PGLRP was not affected by pH, temperature changes, and the in vitro simulated digestion had less effect on its ACE inhibition capacity. However, the results of the virtual enzymolysis show that two peptides can be digested by digestive enzymes to generate AP, PG, LRP and other fragments, and then the results of the LC-MS analysis show that a mass spectrum peak of AP, PG, LRP is detected in the in vitro simulated digestion process (figure 10), which shows that LKKAP and PGLRP can be digested into oligopeptide molecules with smaller molecular weight in the digestive tract in vivo, wherein IC ₅₀ of PG and LRP are 13.93 and 15.20 mu M respectively, which shows that the chlorella ACE inhibitory peptides LKKAP and PGLRP can still play an ACE inhibitory role after being digested by digestive enzymes in vivo.

EXAMPLE 12 practicality of Chlorella antihypertensive peptide in the prevention and treatment of hypertension and cardiovascular diseases

Chlorella is approved as a new resource food for use as early as 2012, has a protein content of up to 60%, contains various essential amino acids, has balanced nutrition, and is an ideal protein source. The chlorella enzymolysis peptide and the chlorella enzymolysis peptide composition are obtained by enzymolysis of chlorella protein, and have good safety as a food raw material; meanwhile, the preparation process is suitable for large-scale industrial production and has strong practicability.

ACE is an important target for the prevention and treatment of hypertension and cardiovascular diseases. ACE inhibitors currently on the market are widely used for the treatment and prevention of diseases such as hypertension and heart failure. The benefits and clinical indications of ACE inhibitors in a variety of cardiovascular diseases have been clarified, including chronic heart failure, asymptomatic left ventricular dysfunction, hypertension and high risk patients for cardiovascular events. Patients with the above diseases would benefit more if they were to have diabetes in combination. The chlorella enzymatic hydrolysis peptide or oligopeptide and the composition thereof provided by the invention have the effect of obviously inhibiting ACE, are beneficial to maintaining the healthy level of blood pressure, protecting target organs such as heart and kidney, can be applied to health products, medicines or pharmaceutical preparations, and have good market development prospect no matter being used as foods, health products or medicines, and have the effect of improving or treating hypertension, chronic cardiovascular diseases and the like.

Claims

1. An enzymatic peptide extract of chlorella, which is characterized in that: the enzymatic peptide with average molecular weight of 0-1000Da accounts for 80% or more in the extract, and contains one or more peptide sequences of LKKAP, PGLRP, ATKFP, VASPK, KVLP, VAIGQK, IGRKFS, VGAGAH, SAKTRAE.

2. A chlorella Angiotensin Converting Enzyme (ACE) inhibitory oligopeptide, characterized in that: the oligopeptide is one or more of LKKAP, PGLRP, ATKFP, VASPK, KVLP, VAIGQK, IGRKFS, VGAGAH.

3. A chlorella antihypertensive peptide or composition, characterized in that: a composition comprising the extract of claim 1 or the oligopeptide of claim 2 or the extract of claim 1 and the oligopeptide of claim 2.

4. A chlorella food, characterized in that: the chlorella food contains one or more of the enzymatic peptide extracts or the chlorella enzymatic peptide sequences or the compositions in the composition consisting of the extract or the oligopeptides of any one or more of claim 2 or the oligopeptides of claim 3.

5. Use of the chlorella antihypertensive peptide or composition of claim 3, characterized in that: use of the enzymatic peptide extract of claim 1, or the ACE inhibiting oligopeptide of claim 2, or the composition of claim 3, as described in claims 1-3, for the preparation of an angiotensin converting enzyme inhibitor, or a health product or pharmaceutical preparation associated with the prevention or treatment of hypertension, cardiovascular diseases.

6. The use according to claim 5, wherein: the extract, or any one or more chlorella antihypertensive peptides, or the composition of the extract is added with any auxiliary materials which meet the production allowance of foods or medicines for preparing angiotensin converting enzyme inhibitors, or health-care products or pharmaceutical preparations related to the prevention or treatment of hypertension and cardiovascular diseases.

7. A method for preparing chlorella enzymatic peptide extract according to claim 1, characterized in that: taking chlorella as a raw material, breaking the wall of the chlorella, carrying out enzymolysis by protease, inactivating enzyme and centrifuging to obtain chlorella zymolyte supernatant; and drying and/or purifying the obtained supernatant of the zymolyte to obtain the chlorella zymolyte peptide extract.

8. The method of manufacturing according to claim 7, wherein: the preparation method of the chlorella enzymatic peptide extract comprises the following steps: the ratio of the material to the water (chlorella powder: water, w/w) is 1:10-1:30 (w/w), the ultrasonic cell disruption instrument is used for breaking the wall of the feed liquid with the power of 200-800w, the supernatant is centrifugally taken after 3-5 times of circulation, and protease is added into the supernatant with the enzyme bottom ratio (chlorella powder: enzyme, w/w) of 1% -10%, and the enzymolysis conditions are as follows: pH is 7-10, temperature is 40-65 ℃, and enzymolysis time is 1-12h; and then heating at 95 ℃ for 15min to inactivate enzyme, centrifuging to obtain an enzymatic hydrolysate supernatant, and freeze-drying to obtain the chlorella enzymatic peptide extract.

9. The process according to claim 8, wherein: the supernatant of the zymolyte obtained by the centrifugation of the reaction is separated and purified by one or a combination of more of ultrafiltration membrane concentration, sephadex G-15 gel column, DEAE anion exchange column chromatography and C18 reverse phase column chromatography.

10. The process according to claim 7, wherein: the protease is any one of trypsin, alkaline protease, chymotrypsin and neutral protease.