Purification of sardine ACE inhibitory peptide and its preparation method and application

Abstract

The application provides a scomber ACE inhibitory peptide as well as a preparation method and application thereof, and relates to the technical field of ACE inhibitory peptides.The scomber ACE inhibitory peptide provided by the application comprises an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.Through papain enzymolysis, ultrafiltration, gel chromatography, liquid chromatography-tandem mass spectrometry sequence identification and QSAR modeling, the application improves the working efficiency of ACE inhibitory peptide screening, prepares and screens scomber ACE inhibitory peptides with high angiotensin converting enzyme inhibitory effect, and the scomber ACE inhibitory peptides can be used for developing new antihypertensive drugs, and have a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of ACE inhibitory peptide, and particularly relates to a scomber ACE inhibitory peptide and a preparation method and application thereof. BACKGROUND

[0002] Hypertension is a global cardiovascular disease with high incidence, is the main risk factor for causing stroke, myocardial infarction, heart disease and other complications, affects the health of about 15% to 20% of adults in the world, causes about 9.4 million patients to die every year, and ranks first in the world with a proportion of 12.8%.

[0003] Angiotensin-I-converting-enzyme (ACE) plays a role in blood pressure regulation through renin-angiotensin-system (RAS) and kallikrein-kinin-system (KKS), and is considered as an important target for treating hypertension. By inhibiting ACE activity, the purpose of treating hypertension can be achieved. Many synthetic ACE inhibitors such as enalapril, captopril and lisinopril are widely used as antihypertensive drugs. However, they have obvious side effects, including allergic reactions, taste disorders, kidney function damage, skin rash and cough, etc. Therefore, food-derived ACE inhibitory peptides with less toxic and side effects have become a research hotspot at present.

[0004] Scomber contains rich protein, fat, niacin, thiamine, riboflavin, calcium, phosphorus, iron and other substances, has high nutritional value, can enhance physical fitness, improve the body's immunity and disease resistance, and is beneficial to the health of human body. However, at present, scomber is mainly processed into canned food, and the utilization rate of high value is low, and has great development space. At present, there are many ACE inhibitory peptides screened from marine fish and by-products thereof, such as Pseudosciaena heterura, Pseudosciaena crocea and Cobia head. However, there is no report on the separation of ACE inhibitory peptides from scomber. SUMMARY

[0005] The present application aims to provide a scomber ACE inhibitory peptide and a preparation method and application thereof. The present application improves the working efficiency of ACE inhibitory peptide screening through papain enzymolysis, ultrafiltration, gel chromatography, liquid chromatography-tandem mass spectrometry sequence identification and QSAR modeling, and prepares and screens ACE inhibitory peptides with amino acid sequences of PLITT (Pro-Leu-Ile-Thr-Thr) and VPLYT (Val-Pro-Leu-Tyr-Thr). The ACE inhibitory peptides have high angiotensin converting enzyme (ACE) inhibitory effect, and can be used for developing new antihypertensive drugs.

[0006] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions.

[0007] The present application provides a scomberomorus ACE inhibitory peptide, which comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0008] The present application also provides a preparation method of the above-mentioned scomberomorus ACE inhibitory peptide, comprising the following steps:

[0009] (1) After pretreatment of scomberomorus meat, papain is used for enzymolysis, and the supernatant of enzymolysis is collected, concentrated and dried to obtain a crude polypeptide powder;

[0010] (2) The crude polypeptide powder is prepared into a solution for ultrafiltration, and a component with a molecular weight less than 3 kDa is collected;

[0011] (3) The component with a molecular weight less than 3 kDa is subjected to chromatography separation, and a polypeptide component with the maximum ACE inhibitory activity is collected for sequence identification;

[0012] (4) The sequence identified in step (3) is subjected to model prediction, and a scomberomorus ACE inhibitory peptide is screened.

[0013] Preferably, the pretreatment method is to boil the minced scomberomorus meat with water; the ratio of scomberomorus meat to water is 1:2-4; and the boiling time is 10-20 min.

[0014] Preferably, the enzyme addition amount of papain is 2000-4000 U / g; the enzymolysis temperature is 60-80℃, the enzymolysis time is 1.5-3.0 h, the enzymolysis pH is 6.5-7.5; and the rotation speed during enzymolysis is 200-500 rpm.

[0015] Preferably, the concentration method is rotary evaporation, the rotary evaporation rotation speed is 100-150 rpm, the rotary evaporation temperature is 60-80℃, the rotary evaporation pressure is 0.090-0.095 MPa, the volume of the supernatant after rotary evaporation is 1 / 10-3 / 10 of the volume of the original supernatant, the drying is freeze drying, the freeze drying temperature is-80--40℃, the freeze drying pressure is less than 30 Pa, and the freeze drying time is 48-60 h.

[0016] Preferably, the concentration of the solution prepared from the crude polypeptide powder is 10-20 g / L.

[0017] Preferably, the chromatography is Sephadex G-15 gel chromatography, the sample loading amount of the gel chromatography is 2-4 mL, the sample loading concentration is 40-60 mg / mL, and the flow rate is 0.5-1.5 mL / min.

[0018] Preferably, the sequence identification is performed by liquid chromatography-tandem mass spectrometry.

[0019] Preferably, the model used in the model prediction is: y = 1.707 + 0.090x1-0.017x2+0.070x3+0.0668x4-0.113x5-0.030x6+0.101x7-0.015x8-0.076x9-0.084x 10 -0.059x 11 +0.049x 12 -0.029x 13 +0.106x 14 +0.054x 15 .

[0020] The application also provides a use of the above-mentioned scomber ACE inhibitory peptide or the scomber ACE inhibitory peptide prepared by the above-mentioned preparation method in the preparation of antihypertensive drugs.

[0021] The application provides a scomber ACE inhibitory peptide, a preparation method and application thereof. The application combines a traditional ACE inhibitory peptide purification method with a bioinformatics method, improves the working efficiency of ACE inhibitory peptide screening through papain enzymolysis, ultrafiltration, gel chromatography, LC-MS / MS analysis and QSAR modeling, and finds new ACE inhibitory peptides PLITT (Pro-Leu-Ile-Thr-Thr) and VPLYT (Val-Pro-Leu-Tyr-Thr) derived from scomber. The IC 50 of the ACE inhibitory peptides in inhibiting angiotensin converting enzyme is 167.42±8.59 μM and 610.12±0.68 μM respectively, and the ACE inhibitory peptides show high angiotensin converting enzyme (ACE) inhibitory effect in vitro. The ACE inhibitory peptides can be used for developing new antihypertensive drugs and have wide application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Table 1 shows the ACE inhibitory rates of the crude polypeptide, the component with a molecular weight of >10 kDa, the component with a molecular weight of 3-10 kDa and the component with a molecular weight of <3 kDa in Example 1, all of which have a concentration of 250 μg / mL.

[0023] Figure 2 Figure 1 shows a Sephadex G-15 gel chromatography chart in Example 1.

[0024] Figure 3 Table 2 shows the ACE inhibitory rates of the six components after chromatography in Example 1, all of which have a concentration of 166.67 μg / mL.

[0025] Figure 4The mass spectrum identification chart of the pout ACE inhibitory peptide PLITT prepared in Example 1 is shown in the following figure:

[0026] Figure 5 The mass spectrum identification chart of the pout ACE inhibitory peptide VPLYT prepared in Example 1 is shown in the following figure:

[0027] Figure 6 The ACE inhibitory rates of PLITT and VPLYT at different concentrations in Example 1. DETAILED DESCRIPTION

[0028] The present application provides a pout ACE inhibitory peptide, which comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0029] In the present application, the amino acid sequence as shown in SEQ ID NO. 1 is Pro-Leu-Ile-Thr-Thr, abbreviated as PLITT. The molecular weight is 543.65 Da.

[0030] In the present application, the amino acid sequence as shown in SEQ ID NO. 2 is Val-Pro-Leu-Tyr-Thr, abbreviated as VPLYT. The molecular weight is 591.69 Da.

[0031] The present application also provides a preparation method of the above-mentioned pout ACE inhibitory peptide, comprising the following steps:

[0032] (1) After pretreatment of pout meat, papain is used for enzymolysis, and the supernatant of enzymolysis is collected, concentrated and dried to obtain a crude polypeptide powder;

[0033] (2) The crude polypeptide powder is prepared into a solution for ultrafiltration, and a component with a molecular weight <3 kDa is collected;

[0034] (3) The component with a molecular weight <3 kDa is subjected to chromatographic separation, and a polypeptide component with the largest ACE inhibitory activity is collected for sequence identification;

[0035] (4) The sequence identified in step (3) is subjected to model prediction, and a pout ACE inhibitory peptide is screened.

[0036] In the present application, the pout meat is pretreated, and then papain is used for enzymolysis, and the supernatant of enzymolysis is collected, concentrated and dried to obtain a crude polypeptide powder.

[0037] In the present application, the pout meat is preferably pout meat after removing the head, internal organs, fish skin and fish bones.

[0038] In the present application, the pretreatment method is preferably boiling the ground pout meat with water.

[0039] In the present application, the water is preferably deionized water.

[0040] In the present application, the ratio of scomber meat to water is preferably 1:2-4, and more preferably 1:3.

[0041] In the present application, the boiling time is preferably 10-20 min, and more preferably 15 min.

[0042] In the present application, the boiled scomber meat is preferably naturally cooled to room temperature (25°C) before the enzymatic hydrolysis using papain.

[0043] In the present application, the enzyme loading of papain is preferably 2000-4000 U / g, and more preferably 3000 U / g.

[0044] In the present application, the temperature of the enzymatic hydrolysis is preferably 60-80°C, and more preferably 70°C.

[0045] In the present application, the time of the enzymatic hydrolysis is preferably 1.5-3.0 h, and more preferably 2 h.

[0046] In the present application, the pH of the enzymatic hydrolysis is preferably 6.5-7.5, and more preferably 7.0.

[0047] In the present application, the rotation speed during the enzymatic hydrolysis is preferably 200-500 rpm, and more preferably 350 rpm.

[0048] In the present application, it is also preferred that the papain is boiled to inactivate after the enzymatic hydrolysis, and the boiling time is preferably 5-15 min, and more preferably 10 min.

[0049] In the present application, the method of collecting the supernatant of the enzymatic hydrolysis is preferably centrifugation.

[0050] In the present application, the rotation speed of the centrifugation is preferably 6000-8000 rpm, and more preferably 7000 rpm.

[0051] In the present application, the temperature of the centrifugation is preferably 1-5°C, and more preferably 4°C.

[0052] In the present application, the time of the centrifugation is preferably 15-25 min, and more preferably 20 min.

[0053] In the present application, it is also preferred that the supernatant is filtered to remove impurities before the concentration.

[0054] In the present application, the process of the filtration is preferably that the liquid is added to the Buchner funnel while the filtration is performed, until the liquid is completely filtered.

[0055] In the present application, the pressure of the suction filtration is preferably 0.090-0.095 MPa, and more preferably 0.095 MPa.

[0056] In the present application, the method of concentration is preferably rotary evaporation.

[0057] In the present application, the rotation speed of the rotary evaporation is preferably 100-150 rpm, and more preferably 120 rpm.

[0058] In the present application, the temperature of the rotary evaporation is preferably 60-80°C, and more preferably 70°C.

[0059] In the present application, the pressure of the rotary evaporation is 0.090-0.095 MPa, and more preferably 0.095 MPa.

[0060] In the present application, the volume of the enzyme hydrolysis supernatant after the rotary evaporation is preferably 1 / 10-3 / 10 of the volume of the original enzyme hydrolysis supernatant, and more preferably 1 / 5 of the volume of the original enzyme hydrolysis supernatant.

[0061] In the present application, the drying is preferably freeze-drying.

[0062] In the present application, the temperature of the freeze-drying is preferably -80 to -40°C, and more preferably -80°C.

[0063] In the present application, the pressure of the freeze-drying is preferably less than 30 Pa, and more preferably 20 Pa.

[0064] In the present application, the time of the freeze-drying is preferably 48-60 h, and more preferably 50 h.

[0065] The obtained crude polypeptide powder is dissolved and subjected to ultrafiltration, and the component with a molecular weight of <3 kDa is collected.

[0066] In the present application, the concentration of the solution of the crude polypeptide powder is preferably 10-20 g / L, and more preferably 15 g / L.

[0067] In the present application, in the process of collecting the component with a molecular weight of <3 kDa, 10 kDa and 3 kDa ultrafiltration membranes are used in sequence for ultrafiltration, and the components are collected. It is determined that the component with a molecular weight of <3 kDa has the highest ACE inhibitory activity, and therefore, the component with a molecular weight of <3 kDa is selected for chromatographic separation.

[0068] The collected component with a molecular weight of <3 kDa is subjected to chromatographic separation, and the polypeptide component with the highest ACE inhibitory activity is collected and subjected to sequence identification.

[0069] In the present application, the chromatography is preferably Sephadex G-15 gel chromatography.

[0070] In the present application, the sample loading amount of the gel chromatography is preferably 2-4 mL, and further preferably 3 mL.

[0071] In the present application, the sample loading concentration is preferably 40-60 mg / mL, and further preferably 50 mg / mL.

[0072] In the present application, the flow rate is preferably 0.5-1.5 mL / min, and further preferably 1 mL / min.

[0073] In the present application, when chromatographic separation is performed, one tube of chromatography is collected every 2 min, and 6 fractions are separated. The ACE inhibitory activity of the 6 fractions is determined, and the fraction with the highest ACE inhibitory activity is selected for sequence identification.

[0074] In the present application, the sequence identification is preferably performed by liquid chromatography-tandem mass spectrometry.

[0075] The identified sequence is subjected to model prediction in the present application, and a scomber ACE inhibitory peptide is screened.

[0076] In the present application, the partial least squares method (PLS) is preferably used to establish a quantitative structure-activity relationship (QSAR) model of ACE inhibitory pentapeptides for model prediction.

[0077] In the present application, the QSAR model is:

[0078] y = 1.707 + 0.090x1-0.017x2+0.070x3+0.0668x4-0.113x5-0.030x6+0.101x7-0.015x8-0.076x9-0.084x 10 -0.059x 11 +0.049x 12 -0.029x 13 +0.106x 14 +0.054x 15 .

[0079] The present application also provides a use of the above-mentioned scomber ACE inhibitory peptide or the scomber ACE inhibitory peptide prepared by the above-mentioned preparation method in the preparation of antihypertensive drugs.

[0080] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0081] Example 1

[0082] The application provides a scomber ACE inhibitory peptide, and a specific preparation process is as follows:

[0083] The scomber is thawed, and then the internal organs, fish skin and fish bones are removed, and the fish meat is washed and ground. 500 g of the ground scomber meat is taken, 1500 mL of deionized water is added, and the mixture is stirred uniformly and boiled for 15 min. After natural cooling, the pH is adjusted to 7.0 by using sodium hydroxide, 7.5 g of papain with an enzyme activity of 200 U / mg is added, and the mixture is subjected to enzymolysis in a water bath at 70 DEG C for 2 h. The mixture is stirred at a rotating speed of 350 rpm during the enzymolysis. After the enzymolysis, the mixture is boiled for 10 min to inactivate the enzyme, and an enzymolysis solution is obtained. The cooled enzymolysis solution is centrifuged at 7000 rpm for 20 min at 4 DEG C, and the supernatant of the enzymolysis solution is collected. The supernatant of the enzymolysis solution is added to a Buchner funnel, and impurities are removed by suction filtration under a pressure of 0.095 MPa until the liquid is completely suctioned. The supernatant of the enzymolysis solution after the removal of impurities by suction filtration is subjected to rotary evaporation under a pressure of 0.095 MPa at 70 DEG C at a rotating speed of 120 rpm, and the rotary evaporation is stopped when the volume of the supernatant of the enzymolysis solution is concentrated to 1 / 5 of the original volume. The concentrated supernatant of the enzymolysis solution is freeze-dried at -80 DEG C under a pressure of 20 Pa for 50 h, and a scomber crude polypeptide powder is obtained. The in-vitro activity of the scomber crude polypeptide powder is determined. Meanwhile, the scomber crude polypeptide powder is configured into a 15 g / L solution, and the solution is subjected to ultrafiltration by using 10 kDa and 3 kDa ultrafiltration membranes in sequence, and components with a molecular weight of >10 kDa, a molecular weight of 3-10 kDa and a molecular weight of <3 kDa are obtained, respectively. The in-vitro activity of the components obtained by ultrafiltration is determined.

[0084] The in-vitro activity is determined by using the following method:

[0085] A blank group, a control group and a sample group are set. 10 μL of the scomber crude polypeptide or the components obtained by ultrafiltration is mixed with 30 μL of a 2.5 mM hippuryl-histidyl-leucine (HHL) substrate solution uniformly in the sample group, and the mixture is preheated in a water bath at 37 DEG C for 5 min; then 20 μL of a 0.1 U / mL ACE solution is added, and the mixture is reacted in a water bath at 37 DEG C for 60 min; after the reaction, 80 μL of a 1 M hydrochloric acid solution is added to terminate the reaction. The control group and the blank group are replaced by a 0.1 M boric acid buffer (containing 0.3 M NaCl, pH=8.3) instead of the sample, and the blank group is added with 80 μL of a 1 M hydrochloric acid before the addition of the ACE solution. All the above solutions are prepared by using a 0.1 M boric acid buffer (containing 0.3 M NaCl, pH=8.3) as a solvent.

[0086] The reaction solution is detected by using a high-performance liquid chromatography method. The detection conditions are as follows:

[0087] Chromatographic column: C18 column (4.6*150 mm, 5 μm); flow rate: 0.5 mL / min; column temperature: 25 °C; 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: 78% mobile phase A and 22% mobile phase B isocratic elution for 12 min.

[0088]

[0089] Wherein, A control - peak area of control group hippurate; A sample - peak area of sample group hippurate; A blank - peak area of blank group hippurate

[0090] The in vitro activity determination results are shown in Table 1. Figure 1 As shown in Table 1, when the concentration is 250 μg / mL, the ACE inhibition rate of the sardine crude polypeptide is 48.62±1.40%, the ACE inhibition rate of the component with molecular weight <3 kDa is 53.39±2.48%, the ACE inhibition rate of the component with molecular weight 3-10 kDa is 44.41±1.27%, and the ACE inhibition rate of the component with molecular weight >10 kDa is 35.40±2.20%. Therefore, the ACE inhibition activity of the component with molecular weight <3 kDa after ultrafiltration is the highest, and the IC 50 is further determined to be 223.91±4.76 μg / mL.

[0091] Sephadex G-15 gel chromatography is used to chromatographically separate the component with molecular weight <3 kDa. The chromatographic conditions are set as follows: sample amount 3 mL, sample concentration 50 mg / mL, flow rate 1 mL / min; 1 tube is collected every 2 mL. Six components are separated, as shown in Table 2, which are F1-F6, respectively. Figure 2 The in vitro activity of the components F1-F6 is determined, and the determination method is the same as that of the sardine crude polypeptide. The results are shown in Table 3. Figure 3 As shown in Table 3, when the concentration is 166.67 μg / mL, the ACE inhibition rate of F1 is 54.23±2.26%, the ACE inhibition rate of F2 is 25.53±1.19%, the ACE inhibition rate of F3 is 45.60±1.72%, the ACE inhibition rate of F4 is 31.14±1.01%, the ACE inhibition rate of F5 is 45.33±0.81%, and the ACE inhibition rate of F6 is 49.12±1.50%. Therefore, the ACE inhibition activity of the component F1 is the highest, and the IC 50 is further determined to be 131.56±3.46 μg / mL.

[0092] The component F1 was subjected to amino acid sequence identification by LC-MS / MS method, and 285 polypeptide sequences with reliability (-10lgP) greater than 20 were identified, all of which contained ≥5 amino acids, and most of which were pentapeptides. 58 ACE inhibitory peptides and their IC 50 values were collected from the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), and a QSAR model of ACE inhibitory pentapeptides was established based on partial least squares (PLS) using Z-scales amino acid descriptors:

[0093] y = 1.707 + 0.090x1-0.017x2+0.070x3+0.0668x4-0.113x5-0.030x6+0.101x7-0.015x8-0.076x9-0.084x 10 -0.059x 11 +0.049x 12 -0.029x 13 +0.106x 14 +0.054x 15 . Two principal components were extracted from the model according to the size of cross-validity index, the model correlation coefficient R 2 = 0.829, the 7-fold cross-validation correlation coefficient Q 2 = 0.523, and the root mean square error RMSEE = 0.240. According to the model, two ACE inhibitory peptides with better activity were screened, which were PLITT (Pro-Leu-Ile-Thr-Thr) and VPLYT (Val-Pro-Leu-Tyr-Thr).

[0094] PLITT and VPLYT were subjected to mass spectrometric identification, and the results are shown in Figure 4 and 5 . PLITT and VPLYT were prepared into solutions with different concentrations for in vitro activity determination. The concentrations of PLITT were 16.67 μg / mL, 33.33 μg / mL, 66.67 μg / mL, 166.67 μg / mL, 333.33 μg / mL, and 833.33 μg / mL, respectively, and the concentrations of VPLYT were 42.67 μg / mL, 106.67 μg / mL, 266.67 μg / mL, 666.67 μg / mL, and 1666.67 μg / mL, respectively. The determination method was the same as that of the rough polypeptide of the blue shark. The results are shown in Figure 6 . It can be seen that with the increase of the concentrations of PLITT and VPLYT, their ACE inhibitory rates also increase, and the IC 50167.42 ± 8.59 μM, 610.12 ± 0.68 μM, respectively.

[0095] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A sardine ACE inhibitory peptide, characterized in that, The amino acid sequence of the sardine ACE inhibitory peptide is shown in SEQ ID NO. 1 or SEQ ID NO.

2.

2. A method for preparing the sardine ACE inhibitory peptide according to claim 1, characterized by, The method comprises the following steps: (1) After pretreatment, the sardine meat is subjected to enzymatic hydrolysis using papain, and the enzyme hydrolysis supernatant is collected, concentrated and dried to obtain a crude polypeptide powder; The pretreatment method is to boil the minced sardine meat with water for 10-20 min; The enzyme addition amount of the papain is 2000-4000 U / g; the enzymatic hydrolysis temperature is 60-80℃, the enzymatic hydrolysis time is 1.5-3.0 h, the enzymatic hydrolysis pH is 6.5-7.5; and the rotation speed during the enzymatic hydrolysis is 200-500 rpm; (2) The crude polypeptide powder is prepared into a solution for ultrafiltration, and a component with a molecular weight of less than 3 kDa is collected; (3) The component with a molecular weight of less than 3 kDa is subjected to chromatographic separation, and a polypeptide component with the largest ACE inhibitory activity is collected for sequence identification; (4) The sequence identified in step (3) is subjected to model prediction to screen a sardine ACE inhibitory peptide.

3. The preparation method according to claim 2, characterized in that, The ratio of the sardine meat to water during the pretreatment is 1:2-4.

4. The production method according to claim 3, characterized by, The concentration method is rotary evaporation, the rotary evaporation rotation speed is 100-150 rpm, the rotary evaporation temperature is 60-80℃, the rotary evaporation pressure is 0.090-0.095 MPa, the volume of the enzyme hydrolysis supernatant after rotary evaporation is 1 / 10-3 / 10 of the original enzyme hydrolysis supernatant volume, and the drying is freeze drying, the freeze drying temperature is -80--40℃, the freeze drying pressure is less than 30 Pa, and the freeze drying time is 48-60 h.

5. The preparation method according to claim 4, characterized in that, The concentration of the solution prepared from the crude polypeptide powder is 10-20 g / L.

6. The production method according to claim 5, wherein The chromatography is Sephadex G-15 gel chromatography, the sample loading amount of the gel chromatography is 2-4 mL, the sample loading concentration is 40-60 mg / mL, and the flow rate is 0.5-1.5 mL / min.

7. The preparation method according to claim 6, characterized in that, The sequence identification is performed by liquid chromatography-tandem mass spectrometry.

8. Use of the sardine ACE inhibitory peptide of claim 1 or the sardine ACE inhibitory peptide prepared by the preparation method of any one of claims 2-7 in the preparation of antihypertensive drugs.

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