CN118561957A - Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide VKGVF, FGGAL, WLDAN and application thereof - Google Patents

Abstract

The present invention provides Antarctic krill ACE inhibitory peptides VKGVF, FGGAL, WLDAN and their applications, belonging to the technical field of bioactive peptides. The present invention uses defatted Antarctic krill powder to prepare Antarctic krill ACE inhibitory peptides, and the obtained polypeptides have excellent ACE inhibitory activity. Through mass spectrometry identification, a total of 13 small molecule polypeptides were screened, and these 13 polypeptides have different degrees of ACE inhibitory activity. The Antarctic krill ACE inhibitory peptide prepared by the present invention has an ACE inhibitory ability that is dozens or even hundreds of times that of the currently confirmed Antarctic krill ACE inhibitory peptide, and has an excellent ACE inhibitory effect. Antarctic krill ACE inhibitory peptide can be applied to food, health products, medicines and other fields. The present invention makes high-value and precise use of Antarctic krill protein resources, which can effectively improve resource utilization and industrial value, and also provides new ways and methods for preventing and treating hypertension.

Description

Antarctic krill ACE inhibitory peptides VKGVF, FGGAL, WLDAN and their applications

This application is a divisional application of 2023115869254 "Antarctic krill ACE inhibitory peptide and its preparation method and application", and the original application date is November 24, 2023.

Technical Field

The present invention relates to the technical field of bioactive peptides, and in particular to Antarctic krill ACE inhibitory peptides VKGVF, FGGAL, WLDAN and applications thereof.

Background Art

Hypertension is one of the common chronic diseases that have affected the health of Chinese residents in recent years. Its main clinical feature is the continuous increase in arterial blood pressure, which is an important risk factor for cardiovascular and cerebrovascular diseases such as coronary heart disease, stroke, and heart failure. If the upper limb blood pressure is measured three times on different days without taking antihypertensive drugs, and the systolic blood pressure is ≥140mmHg or the diastolic blood pressure is ≥90mmHg, it is considered hypertension.

Angiotensin-I converting enzyme (ACE, EC 3.2.1.41) plays an important role in regulating blood pressure balance. It causes blood pressure to rise by converting angiotensin I into angiotensin II (vasoconstrictor), inactivating bradykinin, which has a vasodilating effect, and promoting aldosterone secretion. The effect of treating hypertension can be achieved by inhibiting ACE activity. Currently, clinically used antihypertensive drugs such as chemically synthesized ACE inhibitors such as captopril and enalapril have obvious therapeutic effects, but long-term use can easily cause adverse reactions such as kidney damage, angioedema, and hyperkalemia. It is particularly urgent to find natural, safe and reliable ACE inhibitors.

Bioactive peptides have multiple physiological functions and can be divided into antioxidant peptides, immune active peptides, antihypertensive peptides, uric acid-lowering peptides, antimicrobial peptides, etc. Compared with chemically synthesized ACE inhibitors, food-derived polypeptide ACE inhibitors have the advantages of high safety, easy absorption, and few side effects, and can provide a new way to prevent and treat hypertension.

Antarctic krill (Euphausia superba) resources are abundant, and vigorously developing the Antarctic krill industry is an important choice for building my country's second offshore fishery. The protein content of Antarctic krill can reach 65% of dry weight. It is rich in multiple essential amino acids needed by the human body and is a high-quality marine protein with great development value. However, the industry's current high-value utilization of Antarctic krill is still focused on the development of lipid products such as Antarctic krill oil, and the utilization of its protein resources is obviously insufficient. Defatted Antarctic krill meal is a by-product of Antarctic krill meal after processing and extracting Antarctic krill oil. It has the characteristics of high protein and low fat. It is a good raw material for preparing bioactive peptides, but it is currently usually used as aquaculture feed for low value or directly discarded, resulting in a waste of resources. Scientifically utilizing the high-quality protein in defatted Antarctic krill meal to develop polypeptide ACE inhibitors can provide important support for the development of Antarctic krill protein functional products and food-borne polypeptide antihypertensive drugs.

Summary of the invention

One of the purposes of the present invention is to provide Antarctic krill ACE inhibitory peptides VKGVF, FGGAL, WLDAN, and another purpose is to provide applications thereof to make up for the deficiencies of the prior art.

To achieve the above objectives, the technical solution provided by the present invention is as follows:

Antarctic krill ACE inhibitory peptides VKGVF, FGGAL, and WLDAN, wherein the specific sequence of VKGVF is SEQ No. 3: Val-Lys-Gly-Val-Phe; the specific sequence of FGGAL is SEQ No. 8: Phe-Gly-Gly-Ala-Leu; and the specific sequence of WLDAN is SEQ No. 9: Trp-Leu-Asp-Ala-Asn.

A method for preparing Antarctic krill ACE inhibitory peptide comprises the following steps:

(1) Raw material pretreatment;

(2) Enzymatic hydrolysis;

(3) High temperature inactivation of enzymes;

(4) solid-liquid separation to obtain Antarctic krill enzymatic hydrolysis supernatant;

(5) desalting the Antarctic krill enzymatic hydrolysis supernatant obtained in step (4), spray drying or freeze drying the treated solution to obtain Antarctic krill enzymatic hydrolysis lyophilized powder, i.e., Antarctic krill ACE inhibitory peptide mixture 1;

(6) The Antarctic krill ACE inhibitory peptide mixture 1 obtained in step (5) is reconstituted with deionized water and then purified, and the retentate is collected by ultrafiltration, and spray-dried or freeze-dried to obtain a preliminarily purified Antarctic krill ACE inhibitory peptide mixture 2;

(7) The Antarctic krill ACE inhibitory peptide mixture 2 obtained in step (6) is re-dissolved in deionized water and purified again, gel filtration chromatography is used to collect the chromatographic separation components, and spray drying or freeze drying is performed to obtain the Antarctic krill ACE inhibitory peptide mixture 3;

(8) The Antarctic krill ACE inhibitory peptide mixture 3 obtained in step (7) is identified by liquid chromatography-mass spectrometry technology to finally obtain the 13 Antarctic krill ACE inhibitory peptides.

Preferably, in the step (1), the defatted Antarctic krill powder is added to the buffer solution at a material-liquid ratio of 1:4 to 1:8 g/mL and mixed, and the defatted Antarctic krill powder is fully crushed by a wet ultrafine grinding method, and the crushing time is 1 to 10 minutes; preferably, the material-liquid ratio is 1:6 and the crushing time is 2 minutes.

Preferably, the step (2) is performed by enzymolysis, the pH of the buffer solution is adjusted to 2.0-12.0, 200-8000U/g of protease is added according to the mass of the substrate to start the enzymolysis reaction, the enzymolysis time is 1-6h, and the enzymolysis temperature is 25-75°C; the protease is selected from one or more of alkaline protease, trypsin, pepsin, neutral protease, flavor protease, and papain; alkaline protease is preferred, the addition amount is 4000U/g, the enzymolysis pH is 7.5, the enzymolysis time is 3.5h, and the enzymolysis temperature is 55°C.

Preferably, the enzyme inactivation temperature in step (3) is 90-100°C, and the enzyme inactivation time is 10-20 min; preferably, the enzyme inactivation temperature is 100°C, and the enzyme inactivation time is 20 min.

Preferably, the centrifugal speed in step (4) is 3000-8000 r/min, and the centrifugal time is 10-30 min; preferably, the centrifugal speed is 7500 r/min, and the centrifugal time is 20 min.

Preferably, the desalination method in step (5) is: desalting the Antarctic krill enzymatic hydrolysis supernatant using a nanofiltration membrane with a molecular weight cutoff of 200Da, with a treatment pressure of 0.6 to 1.4MPa and a cycle treatment of 1 to 5 times; preferably, the treatment pressure is 1.2MPa and the cycle treatment is 3 times.

Preferably, the ultrafiltration conditions in step (6) are: the Antarctic krill ACE inhibitory peptide mixture 1 is redissolved with deionized water to a solution with a mass concentration of 0.5 to 10 g/L, and ultrafiltration treatment is performed using an ultrafiltration membrane with a molecular weight cutoff of 1 KDa, 3 KDa or 5 KDa, with a treatment pressure of 0.5 to 1.5 MPa and a treatment volume of 0.5 to 10 L/h; preferably, the solution mass concentration is 5 g/L, the ultrafiltration membrane molecular weight cutoff is 1 KDa, the treatment pressure is 1.0 MPa, and the treatment volume is 3 L/h.

Preferably, the gel filtration chromatography conditions in step (7) are as follows: the Antarctic krill ACE inhibitory peptide mixture 2 is reconstituted with deionized water to a solution with a mass concentration of 10 to 100 mg/mL, and purified using an AKTA protein separation and purification system through a dextran gel Sephadex G-15, with a sample volume of 1 to 10 mL, an eluent of deionized water, and an elution flow rate of 0.2 to 1.0 mL/min; preferably, the solution mass concentration is 30 mg/mL, the sample volume is 5 mL, and the elution flow rate is 0.5 mL/min.

The Antarctic krill ACE inhibitory peptide is used in the preparation of products inhibiting angiotensin converting enzyme (ACE); the products include health products, medicines, etc.

The application of the Antarctic krill ACE inhibitory peptide in the preparation of products with blood pressure lowering efficacy; the products include food, health products, medicines, etc.

Application of the Antarctic krill ACE inhibitory peptides VKGVF, FGGAL and WLDAN in the preparation of blood pressure lowering drugs or blood pressure auxiliary foods.

Advantages and beneficial effects of the present invention:

The present invention uses defatted Antarctic krill powder to prepare Antarctic krill ACE inhibitory peptides, and the obtained polypeptides have excellent ACE inhibitory activity. Through mass spectrometry identification, a total of 13 small molecule polypeptides were screened: GLGIF, APGR, VKGVF, LFAGA, LGGIF, FGAGGL, LSGAY, FGGAL, WLDAN, RDWPEGR, DWPEGR, YLGGAL and LGGLNQ. These 13 polypeptides have different degrees of ACE inhibitory activity.

The Antarctic krill ACE inhibitory peptide prepared by the present invention has an ACE inhibitory ability that is dozens or even hundreds of times that of the currently confirmed Antarctic krill ACE inhibitory peptide, and has an excellent ACE inhibitory effect. Antarctic krill ACE inhibitory peptide can be applied to the fields of food, health care products, and medicines. The present invention makes high-value and precise use of Antarctic krill protein resources, which can effectively improve resource utilization and industrial value, and also provides new ways and methods for preventing and treating hypertension.

Various terms and phrases used herein have the general meanings that are well known to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Gel filtration chromatography separation profile of Example 12.

Figure 2 is a liquid-mass total ion current diagram of the Antarctic krill ACE inhibitory peptide in Example 13.

Figure 3 shows the secondary mass spectrum of the ACE inhibitory peptide of GLGIF with amino acid sequence.

FIG4 is a secondary mass spectrum of the ACE inhibitory peptide of APGR with an amino acid sequence.

Figure 5 is the secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of VKGVF.

Figure 6 is the secondary mass spectrum of the ACE inhibitory peptide whose amino acid sequence is LFAGA.

Figure 7 shows the secondary mass spectrum of the ACE inhibitory peptide of LGGIF with an amino acid sequence.

FIG8 is a secondary mass spectrum of an ACE inhibitory peptide with the amino acid sequence of FGAGGL.

FIG. 9 is a secondary mass spectrum of the ACE inhibitory peptide whose amino acid sequence is LSGAY.

FIG10 is a secondary mass spectrum of the ACE inhibitory peptide of FGGAL, whose amino acid sequence is shown in FIG.

FIG11 shows the amino acid sequence of the ACE inhibitory peptide of WLDAN.

FIG. 12 is a secondary mass spectrum of an ACE inhibitory peptide whose amino acid sequence is RDWPEGR.

FIG. 13 is a secondary mass spectrum of an ACE inhibitory peptide whose amino acid sequence is DWPEGR.

FIG. 14 is a secondary mass spectrum of an ACE inhibitory peptide whose amino acid sequence is YLGGAL.

FIG. 15 is a secondary mass spectrum of the ACE inhibitory peptide whose amino acid sequence is LGGLNQ.

Figure 16 Flow chart of the preparation process of Antarctic krill ACE inhibitory peptide.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with embodiments. The specific examples described herein are only for explaining the present invention and are not limited thereto.

In the following examples, the experimental method for determining the ACE inhibition rate of each sample is as follows:

(1) Solution preparation

0.1mol/L borate buffer (pH 8.3, containing 0.3mol/L sodium chloride): weigh 1.730g of boric acid, heat to dissolve, and then dilute to 100mL for use; weigh 1.907g of borax ( _Na2B4O7 · _10H2O ), heat to dissolve _, and then dilute to _100mL for use; measure 32.5mL of the above boric acid solution and 17.5mL of the above borax solution, mix well, adjust the pH to 8.3, and then add 1.753g of sodium chloride and dilute to 100mL.

0.125U/mL ACE enzyme solution: Take 0.5U ACE enzyme and dilute to 4mL with deionized water.

0.25mmol/L N-[3-(2-furyl)acryloyl]-L-phenylalanamide-glycine-glycine solution (FAPGG): weigh 5.0mg FAPGG powder, dissolve it with the above 0.1mol/L borate buffer (pH 8.3, containing 0.3mol/L sodium chloride) and make up to 50mL.

(2) Experimental methods

Pipette 550 μL of sample solution, add 275 μL of the above FAPGG solution, mix, then add 275 μL of the above ACE enzyme solution, measure the absorbance at 340 nm immediately after mixing, then incubate at 37°C for 30 min, and measure the absorbance at 340 nm again after the reaction is completed; use deionized water instead of the sample solution as a control.

(3) Calculation formula

The ACE inhibitory activity was calculated as follows:

Wherein: _A1 represents the initial absorbance value of the sample group; _A2 represents the absorbance value of the sample group after 30 minutes of reaction; _A3 represents the initial absorbance value of the control group; _A4 represents the absorbance value of the control group after 30 minutes of reaction.

Embodiment 1:

A method for preparing Antarctic krill ACE inhibitory peptide, the preparation method comprising the following steps:

(1) Raw material pretreatment: add appropriate amount of defatted Antarctic krill powder to phosphate buffer at a material-liquid ratio of 1:6, and wet ultrafine grind for 2 min;

(2) Enzymatic hydrolysis: adjust the pH to 7.5, add alkaline protease to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U/g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

(3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

(4) Solid-liquid separation: centrifugation at 7500 r/min for 20 min to obtain the Antarctic krill enzymatic hydrolysis supernatant;

(5) Desalting: The Antarctic krill enzymatic hydrolysis supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle of treatment for 3 times to obtain an Antarctic krill ACE inhibitory peptide mixture.

Embodiment 2:

A method for preparing Antarctic krill ACE inhibitory peptide, the preparation method comprising the following steps:

(1) Raw material pretreatment: add appropriate amount of defatted Antarctic krill powder to phosphate buffer at a material-liquid ratio of 1:6, and wet ultrafine grind for 2 min;

(2) Enzymatic hydrolysis: adjust the pH to 8.0, add trypsin to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U/g, the enzymatic hydrolysis temperature is 37°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

(3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

(4) Solid-liquid separation: centrifugation at 7500 r/min for 20 min to obtain the Antarctic krill enzymatic hydrolysis supernatant;

(5) Desalting: The Antarctic krill enzymatic hydrolysis supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle of treatment for 3 times to obtain an Antarctic krill ACE inhibitory peptide mixture.

Embodiment 3:

A method for preparing Antarctic krill ACE inhibitory peptide, the preparation method comprising the following steps:

(1) Raw material pretreatment: add appropriate amount of defatted Antarctic krill powder to phosphate buffer at a material-liquid ratio of 1:6, and wet ultrafine grind for 2 min;

(2) Enzymatic hydrolysis: adjust the pH to 2.5, add pepsin to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000 U/g, the enzymatic hydrolysis temperature is 40°C, and the enzymatic hydrolysis reaction is carried out for 3.5 h;

(3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

(4) Solid-liquid separation: centrifugation at 7500 r/min for 20 min to obtain the Antarctic krill enzymatic hydrolysis supernatant;

(5) Desalting: The Antarctic krill enzymatic hydrolysis supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle of treatment for 3 times to obtain an Antarctic krill ACE inhibitory peptide mixture.

Embodiment 4:

A method for preparing Antarctic krill ACE inhibitory peptide, the preparation method comprising the following steps:

(1) Raw material pretreatment: add appropriate amount of defatted Antarctic krill powder to phosphate buffer at a material-liquid ratio of 1:6, and wet ultrafine grind for 2 min;

(2) Enzymatic hydrolysis: adjust the pH to 7.0, add neutral protease to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U/g, the enzymatic hydrolysis temperature is 45°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

(3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

(4) Solid-liquid separation: centrifugation at 7500 r/min for 20 min to obtain the Antarctic krill enzymatic hydrolysis supernatant;

(5) Desalting: The Antarctic krill enzymatic hydrolysis supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle of treatment for 3 times to obtain an Antarctic krill ACE inhibitory peptide mixture.

Embodiment 5:

A method for preparing Antarctic krill ACE inhibitory peptide, the preparation method comprising the following steps:

(1) Raw material pretreatment: add appropriate amount of defatted Antarctic krill powder to phosphate buffer at a material-liquid ratio of 1:6, and wet ultrafine grind for 2 min;

(2) Enzymatic hydrolysis: adjust the pH to 7.0, add flavor protease to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U/g, the enzymatic hydrolysis temperature is 50°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

(3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

(4) Solid-liquid separation: centrifugation at 7500 r/min for 20 min to obtain the Antarctic krill enzymatic hydrolysis supernatant;

(5) Desalting: The Antarctic krill enzymatic hydrolysis supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle of treatment for 3 times to obtain an Antarctic krill ACE inhibitory peptide mixture.

Embodiment 6:

A method for preparing Antarctic krill ACE inhibitory peptide, the preparation method comprising the following steps:

(1) Raw material pretreatment: add appropriate amount of defatted Antarctic krill powder to phosphate buffer at a material-liquid ratio of 1:6, and wet ultrafine grind for 2 min;

(2) Enzymatic hydrolysis: adjust the pH to 6.5, add papain to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U/g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

(3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

(4) Solid-liquid separation: centrifugation at 7500 r/min for 20 min to obtain the Antarctic krill enzymatic hydrolysis supernatant;

(5) Desalting: The Antarctic krill enzymatic hydrolysis supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle of treatment for 3 times to obtain an Antarctic krill ACE inhibitory peptide mixture.

Embodiment 7:

The present invention uses a spectrophotometer to measure the activity of the Antarctic krill ACE inhibitory peptide mixture obtained in Examples 1 to 6, and the results are shown in Table 1. The measurement results show that the product obtained by enzymatic hydrolysis using alkaline protease in Example 1 has significantly higher ACE inhibitory activity than the enzymatic hydrolysis products obtained under other conditions in Examples 2 to 6; enzymatic degreasing of Antarctic krill powder using alkaline protease is more conducive to the preparation of highly active Antarctic krill ACE inhibitory peptides.

The Antarctic krill ACE inhibitory peptide prepared by the method of the present invention has excellent active effects, can be used as a common health food, health food or pharmaceutical raw material for preventing and treating hypertension, effectively improves the utilization rate and added value of defatted Antarctic krill powder, and uses a protease enzymatic hydrolysis method to obtain active peptide powder, which is economical, environmentally friendly, highly safe and has good application prospects.

Table 1 ACE inhibition rate of the mixture of Antarctic krill ACE inhibitory peptides obtained in Examples 1 to 6

Embodiment 8:

A method for preparing Antarctic krill ACE inhibitory peptides, the preparation method is to further purify the Antarctic krill ACE inhibitory peptide mixture in Example 1, comprising the following steps:

(1) The Antarctic krill ACE inhibitory peptide mixture obtained in Example 1 was redissolved in deionized water to a solution with a mass concentration of 5 g/L;

(2) The Antarctic krill ACE inhibitory peptide mixture solution was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1 KDa, with a treatment pressure of 1.0 MPa and a treatment volume of 3 L/h to obtain retentates less than 1 KDa and greater than 1 KDa, respectively. The retentates less than 1 KDa were collected and spray-dried or freeze-dried to obtain the preliminarily purified Antarctic krill ACE inhibitory peptide mixture.

Embodiment 9:

A method for preparing Antarctic krill ACE inhibitory peptides, the preparation method is to further purify the Antarctic krill ACE inhibitory peptide mixture in Example 1, comprising the following steps:

(1) The Antarctic krill ACE inhibitory peptide mixture obtained in Example 1 was redissolved in deionized water to a solution with a mass concentration of 5 g/L;

(2) The Antarctic krill ACE inhibitory peptide mixture solution was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 KDa, with a treatment pressure of 1.0 MPa and a treatment volume of 3 L/h to obtain retentates less than 3 KDa and greater than 3 KDa, respectively. The retentates less than 3 KDa were collected and spray-dried or freeze-dried to obtain the preliminarily purified Antarctic krill ACE inhibitory peptide mixture.

Embodiment 10:

A method for preparing Antarctic krill ACE inhibitory peptides, the preparation method is to further purify the Antarctic krill ACE inhibitory peptide mixture in Example 1, comprising the following steps:

(1) The Antarctic krill ACE inhibitory peptide mixture obtained in Example 1 was redissolved in deionized water to a solution with a mass concentration of 5 g/L;

(2) The Antarctic krill ACE inhibitory peptide mixture solution was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 KDa, with a treatment pressure of 1.0 MPa and a treatment volume of 3 L/h to obtain retentates less than 5 KDa and greater than 5 KDa, respectively. The retentates less than 5 KDa were collected and spray-dried or freeze-dried to obtain the preliminarily purified Antarctic krill ACE inhibitory peptide mixture.

Embodiment 11:

The present invention uses a spectrophotometer to measure the activity of the Antarctic krill ACE inhibitory peptide mixture obtained in Example 1 and Examples 8, 9, and 10, and the results are shown in Table 2. The measurement results show that the product obtained by first enzymatic hydrolysis with alkaline protease and further ultrafiltration purification with an ultrafiltration membrane with a molecular weight cutoff of 1KDa in Example 8 has a significantly higher ACE inhibitory activity than the products obtained in Example 1 and Examples 9 and 10; enzymatic degreasing of Antarctic krill powder using alkaline protease and ultrafiltration separation and purification of the enzymatic hydrolysis product to obtain a component with a relative molecular mass of less than 1KDa is more conducive to the preparation of highly active Antarctic krill ACE inhibitory peptides.

The Antarctic krill ACE inhibitory peptide prepared by the method of the present invention has excellent active effects, can be used as a common health food, health food or pharmaceutical raw material for preventing and treating hypertension, effectively improves the utilization rate and added value of defatted Antarctic krill powder, and uses a protease enzymatic hydrolysis method to obtain active peptide powder, which is economical, environmentally friendly, highly safe and has good application prospects.

Table 2 ACE inhibition rate of the Antarctic krill ACE inhibitory peptide mixture obtained in Examples 1, 8, 9, and 10

Embodiment 12:

A method for preparing Antarctic krill ACE inhibitory peptides, the preparation method is to further purify the Antarctic krill ACE inhibitory peptide mixture in Example 8, comprising the following steps:

(1) The Antarctic krill ACE inhibitory peptide mixture obtained in Example 8 was redissolved in deionized water to a solution with a mass concentration of 30 mg/mL;

(2) 5 mL of the Antarctic krill ACE inhibitory peptide mixture solution was loaded onto the AKTA protein separation and purification system, and separated and purified by Sephadex G-15 chromatography. The eluent was deionized water, and the elution flow rate was 0.5 mL/min, and four components A, B, C, and D were obtained respectively (Figure 1); the activities of components A, B, C, and D were determined by spectrophotometry. The results are shown in Table 3. The target component C with high ACE inhibitory activity was collected and spray-dried or freeze-dried to obtain the Antarctic krill ACE inhibitory peptide mixture.

Table 3 ACE inhibition rate of each component obtained by gel filtration chromatography separation in Example 8 and Example 12

The measurement results show that the product C obtained in Example 12 by first using alkaline protease enzymatic hydrolysis, then using ultrafiltration treatment to separate and recover components with a relative molecular mass of less than 1 KDa, and further using Sephadex G-15 chromatography separation, has an ACE inhibitory activity significantly higher than the product obtained in Example 8; using alkaline protease to enzymatically degrease Antarctic krill powder, and then ultrafiltration and gel filtration chromatography purification of the enzymatic hydrolysis product in sequence is more conducive to the preparation of highly active Antarctic krill ACE inhibitory peptides.

Embodiment 13:

The identification of ACE inhibitory peptides from Antarctic krill using liquid chromatography-mass spectrometry technology includes the following steps:

(1) Sample pretreatment: The Antarctic krill ACE inhibitory peptide obtained in Example 12 was desalted using a Waters SEP-PAK C18 solid phase extraction column, vacuum freeze-dried, re-dissolved in 0.1% (v/v) formic acid solution, and tested after filtration through a 0.45 μm microporous filter membrane.

(2) Instrument detection conditions: the sample was injected into a C18 capillary capture column (100 μm × 20 mm, 5 μm, Dr. Maisch GmbH) and then passed through a C18 separation column (75 μm × 150 mm, 3 μm, Dr. Maisch GmbH) for gradient separation; the mobile phase A was 0.1% (v/v) formic acid solution, and the mobile phase B was 80% (v/v) acetonitrile solution (containing 0.1% formic acid); the chromatographic column was fully equilibrated with 92% mobile phase A and gradient elution was performed, with the volume ratio of A to B being 92:8 at 0 min, 76:28 at 98 min, 63:37 at 113 min, 0:100 at 117 min, 0:100 at 120 min, and 0:100 at 125 min. The volume ratio of A to B was 92:8, and the elution program ended after 130 min; the flow rate was 300 nL/min; the injection volume was 1 μL; and the detection mode was positive ion mode.

The mass-to-charge ratios of the peptides and their fragments were collected according to the following method: 20 fragment spectra (MS2 scan) were collected after each full scan; scanning range: 400-1800 m/z; primary resolution: 60000@m/z200, secondary resolution: 150000@m/z200; collision energy CE28 eV.

(3) The software Proteome Discoverer 2.5 was used to search and analyze the Uniprot protein database. The parameter settings are shown in Table 4, and finally the peptide identification results were obtained.

Table 4 Proteome Discoverer analysis parameter settings

The total ion current of the Antarctic krill ACE inhibitory peptide is shown in Figure 2. After the mass spectrometry data was retrieved, FDR≤0.01 was used as the screening standard for credible protein sequences, and a total of 733 peptides and 65 protein sequences were identified, of which 13 peptide sequences with high credibility were GLGIF, APGR, VKGVF, LFAGA, LGGIF, FGAGGL, LSGAY, FGGAL, WLDAN, RDWPEGR, DWPEGR, YLGGAL and LGGLNQ; the secondary mass spectra of the 13 peptide chains are shown in Figures 3 to 15.

Embodiment 14:

The 13 polypeptides obtained in Example 13 were subjected to solid phase synthesis and activity verification, comprising the following steps:

(1) The peptides GLGIF, APGR, VKGVF, LFAGA, LGGIF, FGAGGL, LSGAY, FGGAL, WLDAN, RDWPEGR, DWPEGR, YLGGAL, and LGGLNQ were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method.

(2) The synthetic peptides were prepared into solutions with a mass concentration of 0.035-0.1 mg/mL, and the in vitro ACE inhibitory activity of the synthetic peptides was determined by spectrophotometry, and the results are shown in Table 5. The results showed that the peptides GLGIF, APGR, VKGVF, LFAGA and LGGIF had higher ACE inhibitory activity at 0.1 mg/mL or lower concentrations of 0.04 mg/mL or 0.035 mg/mL.

Table 5 Peptide information and ACE inhibition rate of 13 polypeptides in Example 14

Further, the above 5 polypeptides, GLGIF, APGR, VKGVF, LFAGA and LGGIF, were prepared into solutions with a mass concentration of 0.01-1 mg/mL, and the in vitro ACE inhibition rate of the synthetic polypeptides at different concentrations was determined by spectrophotometry, and the IC ₅₀ values were calculated. The results are shown in Table 6. The measurement results show that the 5 polypeptides, GLGIF, APGR, VKGVF, LFAGA and LGGIF, all have excellent ACE inhibitory activity, among which the peptide segment with the amino acid sequence of GLGIF exhibits the highest ACE inhibitory effect. It is worth noting that the Antarctic krill ACE inhibitory peptide mixture prepared in Example 1 has an IC ₅₀ value of 0.336 mg/mL, that is, the polypeptide prepared by the present invention can increase the ACE inhibitory activity by 28 times, the technical improvement effect is obvious, and the product has good application prospects.

Table 6 Peptide information and ACE inhibitory activity _IC50 values of 5 polypeptides in Example 14

The overall preparation method flow of the above-mentioned embodiments 1-14 is shown in FIG16 .

Embodiment 15:

The application of the Antarctic krill ACE inhibitory peptide obtained in Examples 1 to 14 specifically includes:

The Antarctic krill ACE inhibitory peptides obtained in Examples 1 to 14 can be applied to the fields of ordinary health foods, health foods, medicines, etc. for preventing and treating hypertension. The product preparations can be in the form of solid beverages, capsules, compressed candies, gel candies, liquid drinks, etc.

The embodiments described above are only preferred implementations of the present invention and do not constitute limitations on the scope of the claims. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are within the protection scope of the present invention.

Claims (2)

1. The euphausia superba ACE inhibitory peptide VKGVF, FGGAL, WLDAN is characterized in that the specific sequence of the ACE inhibitory peptide VKGVF is SEQ No.3: val-Lys-Gly-Val-Phe; the specific sequence of the ACE inhibitory peptide FGGAL is SEQ No.8: phe-Gly-Gly-Ala-Leu; the specific sequence of the ACE inhibitory peptide WLDAN is SEQ No.9: trp-Leu-Asp-Ala-Asn.

2. Use of the antarctic krill ACE inhibiting peptide VKGVF, FGGAL, WLDAN of claim 1 in the preparation of a antihypertensive drug or an ancillary antihypertensive food.