CN118307633B - Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide LGGIF, YLGGAL, LGGLNQ and application thereof - Google Patents

Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide LGGIF, YLGGAL, LGGLNQ and application thereof Download PDF

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CN118307633B
CN118307633B CN202410592398.6A CN202410592398A CN118307633B CN 118307633 B CN118307633 B CN 118307633B CN 202410592398 A CN202410592398 A CN 202410592398A CN 118307633 B CN118307633 B CN 118307633B
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euphausia superba
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刘小芳
李爽
冷凯良
王西西
于源
苏东
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

The invention provides antarctic krill ACE inhibitory peptide LGGIF, YLGGAL, LGGLNQ and application thereof, and belongs to the technical field of bioactive peptides. According to the invention, the euphausia superba ACE inhibitory peptide is prepared from defatted euphausia superba powder, and the obtained polypeptide has excellent ACE inhibitory activity. Through mass spectrum identification, 13 small molecule polypeptides are obtained through co-screening, and the 13 polypeptides have ACE inhibitory activities with different degrees. The ACE inhibitory capacity of the euphausia superba ACE inhibitory peptide prepared by the method is tens or hundreds of times that of the euphausia superba ACE inhibitory peptide confirmed at present, and the euphausia superba ACE inhibitory peptide has excellent ACE inhibitory effect. The antarctic krill ACE inhibitory peptide can be applied to the fields of foods, health products, medicines and the like. The invention carries out high-value accurate utilization on the euphausia superba protein resource, can effectively improve the resource utilization rate and the industrial value, and simultaneously provides a new way and a new method for preventing and treating hypertension.

Description

Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide LGGIF, YLGGAL, LGGLNQ and application thereof
The application relates to 2023115869254 'antarctic krill ACE inhibitory peptide, a preparation method and application thereof', and the original application date is 2023, 11 and 24.
Technical Field
The invention relates to the technical field of bioactive peptides, in particular to antarctic krill ACE inhibitory peptide LGGIF, YLGGAL, LGGLNQ and application thereof.
Background
Hypertension is one of the common chronic diseases affecting the health of residents in China in recent years, and is an important risk factor for cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral apoplexy and heart failure by taking continuous rising of arterial blood pressure as a main clinical characteristic. Under the condition that no antihypertensive drug is used, the upper limb blood pressure is measured for 3 times on the same day, and the systolic pressure is more than or equal to 140mmHg or the diastolic pressure is more than or equal to 90mmHg, namely the hypertension is considered.
Angiotensin converting enzyme (Angiotensin-I converting enzyme, ACE, EC 3.2.1.41) plays an important role in regulating blood pressure balance, and it can achieve the effect of treating hypertension by inhibiting ACE activity by converting Angiotensin I into Angiotensin II (vasoconstrictor), inactivating bradykinin having vasodilatory effect, and promoting secretion of aldosterone to cause elevation of blood pressure. At present, the chemically synthesized ACE inhibitors of blood pressure lowering drugs such as captopril, enalapril and the like which are clinically used have obvious treatment effects, but the adverse reactions such as kidney damage, vascular edema, hyperkalemia and the like are easy to cause after long-term administration, so that the search of the ACE inhibitors which are of natural sources and safe and reliable is particularly urgent.
The bioactive peptide has various physiological functions, and can be classified into antioxidant peptide, immunocompetent peptide, antihypertensive peptide, uric acid reducing peptide, antibacterial peptide, etc. Compared with chemical synthesis of ACE inhibitors, the food-borne polypeptide ACE inhibitors have the advantages of high safety, easy absorption, small side effect and the like, and can provide a new way for preventing and treating hypertension.
The euphausia superba (Euphausia superba) has rich resource reserves, and the important choice for creating the second ocean fishery in China is to develop the euphausia superba industry greatly. The euphausia superba protein content can reach 65% of dry weight, is rich in various essential amino acids required by human body, and is ocean high-quality protein with great development value. However, the industry is still focused on the development of lipid products such as euphausia superba oil for high-value utilization of euphausia superba, and the utilization of protein resources of the euphausia superba oil is obviously insufficient. The defatted euphausia superba powder is a byproduct of the euphausia superba powder after being processed and extracted to obtain euphausia superba oil, has the characteristics of high protein and low fat, is a good raw material for preparing bioactive peptide, and is usually used as a breeding feed for low-valued use or is directly discarded at present, so that resource waste is caused. The polypeptide ACE inhibitor is developed by scientifically utilizing high-quality protein in the defatted euphausia superba powder, and important support can be provided for the development of euphausia superba protein functional products and food-borne polypeptide antihypertensive drugs.
Disclosure of Invention
One of the objects of the present invention is to provide antarctic krill ACE inhibiting peptide LGGIF, YLGGAL, LGGLNQ, and another object is to provide its use, to remedy the deficiencies of the prior art.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
an antarctic krill ACE inhibiting peptide LGGIF, YLGGAL, LGGLNQ, wherein:
LGGIF has a specific sequence of SEQ No.5, leu-Gly-Gly-Ile-Phe;
YLGGAL the specific sequence is SEQ No.12, tyr-Leu-Gly-Gly-Ala-Leu;
LGGLNQ the specific sequence is SEQ No.13 Leu-Gly-Gly-Leu-Asn-Gln.
The preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Pretreating raw materials;
(2) Performing enzymolysis;
(3) High-temperature enzyme deactivation;
(4) Solid-liquid separation is carried out to obtain euphausia superba enzymolysis supernatant;
(5) Desalting the euphausia superba enzymolysis supernatant obtained in the step (4), and performing spray drying or freeze drying on the treatment liquid to obtain euphausia superba enzyme thawing dry powder, namely euphausia superba ACE inhibitory peptide mixture 1;
(6) Purifying the euphausia superba ACE inhibitory peptide mixture 1 obtained in the step (5) after redissolving by using deionized water, collecting trapped fluid by using an ultrafiltration method, and obtaining a preliminarily purified euphausia superba ACE inhibitory peptide mixture 2 by spray drying or freeze drying;
(7) Re-dissolving the euphausia superba ACE inhibitory peptide mixture 2 obtained in the step (6) by using deionized water, purifying again, collecting chromatographic separation components by using a gel filtration chromatography method, and performing spray drying or freeze drying to obtain the euphausia superba ACE inhibitory peptide mixture 3;
(8) And (3) identifying the euphausia superba ACE inhibitory peptide mixture 3 obtained in the step (7) by a liquid chromatography-mass spectrometry technology, and finally obtaining the 13 euphausia superba ACE inhibitory peptides.
Preferably, in the step (1), the defatted antarctic krill powder is added into a buffer solution according to a feed liquid ratio of 1:4-1:8 g/mL and uniformly mixed, and the defatted antarctic krill powder is fully crushed by a wet superfine grinding method for 1-10 min, preferably a feed liquid ratio of 1:6 and a crushing time of 2min.
Preferably, the step (2) is carried out enzymolysis, the pH of a buffer solution is regulated to 2.0-12.0, 200-8000U/g protease is added according to the mass of a substrate to start the enzymolysis reaction, the enzymolysis time is 1-6 h, the enzymolysis temperature is 25-75 ℃, one or more of alkaline protease, trypsin, pepsin, neutral protease, flavourzyme and papain are selected as protease, preferably alkaline protease is added in 4000U/g, the enzymolysis pH is 7.5, the enzymolysis time is 3.5h, and the enzymolysis temperature is 55 ℃.
Preferably, the enzyme deactivation temperature in the step (3) is 90-100 ℃, the enzyme deactivation time is 10-20 min, and the enzyme deactivation temperature is preferably 100 ℃ and the enzyme deactivation time is 20min.
Preferably, the centrifugation speed in the step (4) is 3000-8000 r/min, the centrifugation time is 10-30 min, and preferably the centrifugation speed is 7500r/min and the centrifugation time is 20min.
Preferably, the desalting method in the step (5) comprises the steps of desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with a molecular weight cut-off of 200Da, and performing cyclic treatment for 1-5 times under the treatment pressure of 0.6-1.4 MPa, preferably 1.2MPa, and performing cyclic treatment for 3 times.
Preferably, the ultrafiltration condition in the step (6) is that the euphausia superba ACE inhibitory peptide mixture 1 is redissolved to a solution with the mass concentration of 0.5-10 g/L by using deionized water, ultrafiltration treatment is carried out by adopting an ultrafiltration membrane with the interception molecular weight of 1kDa or 3kDa or 5kDa, the treatment pressure is 0.5-1.5 MPa, the treatment capacity is 0.5-10L/h, the mass concentration of the solution is preferably 5g/L, the interception molecular weight of the ultrafiltration membrane is 1kDa, the treatment pressure is 1.0MPa, and the treatment capacity is 3L/h.
Preferably, gel filtration chromatography conditions in the step (7) are that the antarctic krill ACE inhibitory peptide mixture 2 is redissolved to a solution with the mass concentration of 10-100 mg/mL by using deionized water, an AKTA protein separation and purification system is adopted to purify by Sephadex G-15, the loading amount is 1-10 mL, the eluent is deionized water, the elution flow rate is 0.2-1.0 mL/min, the preferred solution mass concentration is 30mg/mL, the loading amount is 5mL, and the elution flow rate is 0.5mL/min.
The application of the euphausia superba ACE inhibitory peptide in preparing an Angiotensin Converting Enzyme (ACE) inhibitory product, wherein the product comprises medicines and the like.
The application of the euphausia superba ACE inhibitory peptide in preparing antihypertensive effect products comprises medicines, auxiliary antihypertensive foods and the like.
The application of the euphausia superba ACE inhibitory peptide LGGIF, YLGGAL, LGGLNQ in preparing antihypertensive drugs or auxiliary antihypertensive foods.
The invention has the advantages and beneficial effects that:
according to the invention, the euphausia superba ACE inhibitory peptide is prepared from defatted euphausia superba powder, and the obtained polypeptide has excellent ACE inhibitory activity. Through mass spectrum identification, 13 small molecule polypeptides, GLGIF, APGR, VKGVF, LFAGA, LGGIF, FGAGGL, LSGAY, FGGAL, WLDAN, RDWPEGR, DWPEGR, YLGGAL and LGGLNQ, are obtained through co-screening. These 13 polypeptides have varying degrees of ACE inhibitory activity.
The ACE inhibitory capacity of the euphausia superba ACE inhibitory peptide prepared by the method is tens or hundreds of times that of the euphausia superba ACE inhibitory peptide confirmed at present, and the euphausia superba ACE inhibitory peptide has excellent ACE inhibitory effect. The antarctic krill ACE inhibitory peptide can be applied to the fields of medicines and the like. The invention carries out high-value accurate utilization on the euphausia superba protein resource, can effectively improve the resource utilization rate and the industrial value, and simultaneously provides a new way and a new method for preventing and treating hypertension.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
FIG. 1 example 12 gel filtration chromatographic separation profile.
Figure 2a liquid total ion flow diagram of the ACE inhibiting peptides of krill s in example 13.
FIG. 3 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence GLGIF.
FIG. 4 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence APGR.
FIG. 5 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence VKGVF.
FIG. 6 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence LFAGA.
FIG. 7 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence LGGIF.
FIG. 8 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence FGAGGL.
FIG. 9 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence LSGAY.
FIG. 10 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence FGGAL.
FIG. 11 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence WLDAN.
FIG. 12 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence RDWPEGR.
FIG. 13 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence DWPEGR.
FIG. 14 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence YLGGAL.
FIG. 15 is a secondary mass spectrum of ACE inhibitory peptide with the amino acid sequence LGGLNQ.
Fig. 16 is a flow chart of a process for preparing euphausia superba ACE inhibiting peptides.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are provided to illustrate the invention and not to limit the invention.
In the following examples, the test methods for measuring ACE inhibition rates of the respective samples were as follows:
(1) Solution preparation
A borate buffer solution (pH 8.3, containing 0.3mol/L sodium chloride) of 0.1mol/L is prepared through weighing 1.730g boric acid, heating for dissolving, constant volume to 100mL for later use, weighing 1.907g borax (Na 2B4O7·10H2 O), heating for dissolving, constant volume to 100mL for later use, weighing 32.5mL boric acid solution and 17.5mL borax solution, mixing, regulating pH to 8.3, and adding 1.753g sodium chloride, constant volume to 100 mL.
0.125U/mL ACE enzyme solution 0.5U ACE enzyme was taken and deionized water was used to determine the volume to 4mL.
0.25Mmol/L N- [3- (2-furyl) acryloyl ] -L-phenylpropionamide-glycine solution (FAPGG) 5.0. 5.0mg FAPGG powder was weighed, dissolved in the above 0.1mol/L borate buffer (pH 8.3, containing 0.3mol/L sodium chloride) and sized to 50mL.
(2) Experimental method
Transferring 550. Mu.L of sample solution, adding 275. Mu.L of FAPGG solution, mixing, adding 275. Mu.L of ACE enzyme solution, mixing, immediately measuring the absorbance at 340nm, incubating at 37deg.C for 30min, measuring the absorbance at 340nm again after the reaction, and using deionized water instead of sample solution as control.
(3) Calculation formula
The ACE inhibitory activity was calculated as follows:
Wherein A 1 represents the initial absorbance of the sample group, A 2 represents the absorbance of the sample group when the sample group reacts for 30min, A 3 represents the initial absorbance of the control group, and A 4 represents the absorbance of the control group when the sample group reacts for 30 min.
Example 1:
the preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Raw material pretreatment, namely adding a proper amount of defatted euphausia superba powder into phosphoric acid buffer solution according to a feed-liquid ratio of 1:6, and carrying out wet superfine grinding for 2min;
(2) Enzymolysis, namely adjusting the pH value to 7.5, adding alkaline protease to start enzymolysis reaction, wherein the addition amount of the enzyme is 4000U/g, the enzymolysis temperature is 55 ℃, and the enzymolysis is carried out for 3.5 hours;
(3) High temperature enzyme deactivation, namely heating and deactivating enzyme of the enzymolysis liquid in a boiling water bath at 100 ℃ for 20min after the enzymolysis reaction is finished;
(4) Separating solid and liquid, namely, 7500r/min centrifuging for 20min to obtain euphausia superba enzymolysis supernatant;
(5) Desalting, namely desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with the molecular weight cut-off of 200Da, wherein the treatment pressure is 1.2MPa, and circularly treating for 3 times to obtain the euphausia superba ACE inhibitory peptide mixture.
Example 2:
the preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Raw material pretreatment, namely adding a proper amount of defatted euphausia superba powder into phosphoric acid buffer solution according to a feed-liquid ratio of 1:6, and carrying out wet superfine grinding for 2min;
(2) Enzymolysis, namely adjusting the pH value to 8.0, adding trypsin to start enzymolysis reaction, wherein the adding amount of the enzyme is 4000U/g, the enzymolysis temperature is 37 ℃, and the enzymolysis is carried out for 3.5 hours;
(3) High temperature enzyme deactivation, namely heating and deactivating enzyme of the enzymolysis liquid in a boiling water bath at 100 ℃ for 20min after the enzymolysis reaction is finished;
(4) Separating solid and liquid, namely, 7500r/min centrifuging for 20min to obtain euphausia superba enzymolysis supernatant;
(5) Desalting, namely desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with the molecular weight cut-off of 200Da, wherein the treatment pressure is 1.2MPa, and circularly treating for 3 times to obtain the euphausia superba ACE inhibitory peptide mixture.
Example 3:
the preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Raw material pretreatment, namely adding a proper amount of defatted euphausia superba powder into phosphoric acid buffer solution according to a feed-liquid ratio of 1:6, and carrying out wet superfine grinding for 2min;
(2) Enzymolysis, namely adjusting the pH value to 2.5, adding pepsin to start an enzymolysis reaction, wherein the addition amount of the enzyme is 4000U/g, the enzymolysis temperature is 40 ℃, and the enzymolysis is carried out for 3.5 hours;
(3) High temperature enzyme deactivation, namely heating and deactivating enzyme of the enzymolysis liquid in a boiling water bath at 100 ℃ for 20min after the enzymolysis reaction is finished;
(4) Separating solid and liquid, namely, 7500r/min centrifuging for 20min to obtain euphausia superba enzymolysis supernatant;
(5) Desalting, namely desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with the molecular weight cut-off of 200Da, wherein the treatment pressure is 1.2MPa, and circularly treating for 3 times to obtain the euphausia superba ACE inhibitory peptide mixture.
Example 4:
the preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Raw material pretreatment, namely adding a proper amount of defatted euphausia superba powder into phosphoric acid buffer solution according to a feed-liquid ratio of 1:6, and carrying out wet superfine grinding for 2min;
(2) Enzymolysis, namely adjusting the pH value to 7.0, adding neutral protease to start enzymolysis reaction, wherein the addition amount of the enzyme is 4000U/g, the enzymolysis temperature is 45 ℃, and the enzymolysis is carried out for 3.5 hours;
(3) High temperature enzyme deactivation, namely heating and deactivating enzyme of the enzymolysis liquid in a boiling water bath at 100 ℃ for 20min after the enzymolysis reaction is finished;
(4) Separating solid and liquid, namely, 7500r/min centrifuging for 20min to obtain euphausia superba enzymolysis supernatant;
(5) Desalting, namely desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with the molecular weight cut-off of 200Da, wherein the treatment pressure is 1.2MPa, and circularly treating for 3 times to obtain the euphausia superba ACE inhibitory peptide mixture.
Example 5:
the preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Raw material pretreatment, namely adding a proper amount of defatted euphausia superba powder into phosphoric acid buffer solution according to a feed-liquid ratio of 1:6, and carrying out wet superfine grinding for 2min;
(2) Enzymolysis, namely adjusting the pH value to 7.0, adding flavourzyme to start enzymolysis reaction, wherein the addition amount of the enzyme is 4000U/g, the enzymolysis temperature is 50 ℃, and the enzymolysis is carried out for 3.5 hours;
(3) High temperature enzyme deactivation, namely heating and deactivating enzyme of the enzymolysis liquid in a boiling water bath at 100 ℃ for 20min after the enzymolysis reaction is finished;
(4) Separating solid and liquid, namely, 7500r/min centrifuging for 20min to obtain euphausia superba enzymolysis supernatant;
(5) Desalting, namely desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with the molecular weight cut-off of 200Da, wherein the treatment pressure is 1.2MPa, and circularly treating for 3 times to obtain the euphausia superba ACE inhibitory peptide mixture.
Example 6:
the preparation method of the antarctic krill ACE inhibitory peptide comprises the following steps:
(1) Raw material pretreatment, namely adding a proper amount of defatted euphausia superba powder into phosphoric acid buffer solution according to a feed-liquid ratio of 1:6, and carrying out wet superfine grinding for 2min;
(2) Enzymolysis, namely adjusting the pH value to 6.5, adding papain to start an enzymolysis reaction, wherein the addition amount of the papain is 4000U/g, the enzymolysis temperature is 55 ℃, and the enzymolysis is carried out for 3.5 hours;
(3) High temperature enzyme deactivation, namely heating and deactivating enzyme of the enzymolysis liquid in a boiling water bath at 100 ℃ for 20min after the enzymolysis reaction is finished;
(4) Separating solid and liquid, namely, 7500r/min centrifuging for 20min to obtain euphausia superba enzymolysis supernatant;
(5) Desalting, namely desalting the euphausia superba enzymolysis supernatant by using a nanofiltration membrane with the molecular weight cut-off of 200Da, wherein the treatment pressure is 1.2MPa, and circularly treating for 3 times to obtain the euphausia superba ACE inhibitory peptide mixture.
Example 7:
The activity of the euphausia superba ACE inhibitory peptide mixtures obtained in examples 1-6 was measured by spectrophotometry, and the results are shown in Table 1. The measurement result shows that the ACE inhibition activity of the product obtained by enzymolysis of the alkaline protease in the embodiment 1 is obviously higher than that of the enzymolysis product obtained by other conditions in the embodiments 2-6, and the use of the alkaline protease for enzymolysis of the defatted euphausia superba powder is more beneficial to preparing and obtaining the euphausia superba ACE inhibition peptide with high activity.
The euphausia superba ACE inhibitory peptide prepared by the method has excellent activity effect, can be used as a common medicine raw material for preventing and treating hypertension, effectively improves the utilization rate and the additional value of defatted euphausia superba powder, and obtains active peptide powder by using a protease enzymolysis method, thereby being economical and environment-friendly, high in safety and good in application prospect.
Table 1 ACE inhibition ratios of ACE inhibition peptide mixtures of euphausia superba obtained in examples 1 to 6
Example 8:
A process for preparing antarctic krill ACE inhibiting peptides by continuing to purify the mixture of antarctic krill ACE inhibiting peptides of example 1, comprising the steps of:
(1) The euphausia superba ACE inhibitory peptide mixture obtained in example 1 was reconstituted to a mass concentration of 5g/L with deionized water;
(2) And (3) carrying out ultrafiltration treatment on the euphausia superba ACE inhibitory peptide mixture solution by adopting an ultrafiltration membrane with a molecular weight cutoff of 1kDa, wherein the treatment pressure is 1.0MPa, the treatment capacity is 3L/h, the trapped fluid smaller than 1kDa and the trapped fluid larger than 1kDa are respectively obtained, the trapped fluid smaller than 1kDa is collected, and the initially purified euphausia superba ACE inhibitory peptide mixture is obtained through spray drying or freeze drying.
Example 9:
A process for preparing antarctic krill ACE inhibiting peptides by continuing to purify the mixture of antarctic krill ACE inhibiting peptides of example 1, comprising the steps of:
(1) The euphausia superba ACE inhibitory peptide mixture obtained in example 1 was reconstituted to a mass concentration of 5g/L with deionized water;
(2) And (3) carrying out ultrafiltration treatment on the euphausia superba ACE inhibitory peptide mixture solution by adopting an ultrafiltration membrane with a cut-off molecular weight of 3kDa, wherein the treatment pressure is 1.0MPa, the treatment capacity is 3L/h, cut-off solutions smaller than 3kDa and larger than 3kDa are respectively obtained, the cut-off solutions smaller than 3kDa are collected, and the initially purified euphausia superba ACE inhibitory peptide mixture is obtained through spray drying or freeze drying.
Example 10:
A process for preparing antarctic krill ACE inhibiting peptides by continuing to purify the mixture of antarctic krill ACE inhibiting peptides of example 1, comprising the steps of:
(1) The euphausia superba ACE inhibitory peptide mixture obtained in example 1 was reconstituted to a mass concentration of 5g/L with deionized water;
(2) And (3) carrying out ultrafiltration treatment on the euphausia superba ACE inhibitory peptide mixture solution by adopting an ultrafiltration membrane with a molecular weight cutoff of 5kDa, wherein the treatment pressure is 1.0MPa, the treatment capacity is 3L/h, the trapped fluid smaller than 5kDa and the trapped fluid larger than 5kDa are respectively obtained, the trapped fluid smaller than 5kDa is collected, and the initially purified euphausia superba ACE inhibitory peptide mixture is obtained through spray drying or freeze drying.
Example 11:
The activity of the euphausia superba ACE inhibitory peptide mixtures obtained in example 1 and examples 8, 9, 10 was measured spectrophotometrically, and the results are shown in table 2. The measurement results show that the ACE inhibition activity of the product obtained by carrying out enzymolysis by using alkaline protease and further carrying out ultrafiltration purification by using an ultrafiltration membrane with a molecular weight cutoff of 1kDa in the example 8 is obviously higher than that of the products obtained in the example 1 and the examples 9 and 10, and the components with the relative molecular weight of less than 1kDa are obtained by carrying out enzymolysis by using alkaline protease to degrease euphausia superba powder and carrying out ultrafiltration separation and purification on the enzymolysis product, thereby being more beneficial to preparing and obtaining the high-activity euphausia superba ACE inhibition peptide.
The euphausia superba ACE inhibitory peptide prepared by the method has excellent activity effect, can be used as a medicine raw material for preventing and treating hypertension or an auxiliary blood pressure-reducing food raw material, effectively improves the utilization rate and the additional value of defatted euphausia superba powder, and obtains the active peptide powder by using a protease enzymolysis method, thereby being economic and environment-friendly, having high safety and good application prospect.
Table 2 ACE inhibition ratios of the euphausia superba ACE inhibition peptide mixtures obtained in examples 1, 8, 9, 10
Example 12:
A process for preparing antarctic krill ACE inhibiting peptides by continuing to purify the antarctic krill ACE inhibiting peptide mixture of example 8, comprising the steps of:
(1) The euphausia superba ACE inhibitory peptide mixture obtained in example 8 was reconstituted to a mass concentration of 30mg/mL using deionized water;
(2) 5mL of the euphausia superba ACE inhibitory peptide mixture solution is loaded to an AKTA protein separation and purification system, sephadex G-15 chromatography separation and purification are carried out, deionized water is adopted as eluent, the elution flow rate is 0.5mL/min, A, B, C, D components (figure 1) are respectively obtained, the activity of the component A, B, C, D is measured by a spectrophotometry method, the result is shown in the table 3, the target ACE inhibitory active component C is collected, and the euphausia superba ACE inhibitory peptide mixture is obtained through spray drying or freeze drying.
TABLE 3 ACE inhibition of the fractions isolated by gel filtration chromatography in example 8 and example 12
The measurement result shows that the ACE inhibition activity of the product C obtained by adopting alkaline protease to carry out enzymolysis, then adopting ultrafiltration treatment to separate and recycle components with the relative molecular mass smaller than 1kDa and further adopting Sephadex G-15 chromatographic separation is obviously higher than that of the product obtained in the example 8 in the example 12, and the preparation of the high-activity antarctic krill ACE inhibition peptide is more facilitated by adopting alkaline protease to carry out enzymolysis on defatted antarctic krill powder, and sequentially carrying out ultrafiltration and gel filtration chromatographic purification on the enzymolysis product.
Example 13:
the method for identifying the antarctic krill ACE inhibitory peptide by adopting a liquid chromatography-mass spectrometry technology comprises the following steps of:
(1) Sample pretreatment, namely desalting the euphausia superba ACE inhibitory peptide obtained in example 12 by using WATERS SEP-PAK C18 solid phase extraction column, re-dissolving by using 0.1% (v/v) formic acid solution after vacuum freeze drying, and filtering by using a 0.45 mu m microporous filter membrane for detection.
(2) The instrument detection conditions comprise that a sample is introduced into a C18 capillary capture column (100 mu m multiplied by 20mm,5 mu m, dr. Maisch GmbH) and then subjected to gradient separation through a C18 separation column (75 mu m multiplied by 150mm,3 mu m, dr. Maisch GmbH), a mobile phase A is 0.1% (v/v) formic acid solution, a mobile phase B is 80% (v/v) acetonitrile solution (containing 0.1% formic acid), the chromatographic column is subjected to gradient elution after being fully balanced by 92% of the mobile phase A, the volume ratio of 0min A to B is 92:8, the volume ratio of 98min A to B is 76:28, the volume ratio of 113min A to B is 63:37, the volume ratio of 117min A to B is 0:100, the volume ratio of 120min A to B is 0:100, the volume ratio of 125min A to B is 92:8,130min is over, the flow rate is 300nL/min, the sample introduction amount is 1 mu L, and the detection mode is positive ion mode.
The mass-charge ratio of the polypeptide and fragments of the polypeptide is acquired by acquiring 20 fragment patterns (MS 2 scan) after each Full scan (Full scan), wherein the scanning range is 400-1800 m/z, the primary resolution is 60000@m/z200, the secondary resolution is 150000@m/z200, and the collision energy CE28eV.
(3) And (4) carrying out search analysis on the Uniprot protein database by using software Proteome Discoverer 2.5.5, setting parameters as shown in Table 4, and finally obtaining a polypeptide identification result.
Table 4Proteome Discoverer analysis parameter set
The liquid total ion flow diagram of antarctic krill ACE inhibiting peptide is shown in figure 2. After mass spectrum data retrieval, adopting FDR less than or equal to 0.01 as a screening standard of a trusted protein sequence, and carrying out total identification to obtain 733 peptide fragments and 65 protein sequences, wherein 13 peptide fragments with high credibility, specifically GLGIF, APGR, VKGVF, LFAGA, LGGIF, FGAGGL, LSGAY, FGGAL, WLDAN, RDWPEGR, DWPEGR, YLGGAL and LGGLNQ, are obtained, and the secondary mass spectrum of the 13 peptide chains is shown in figures 3-15.
Example 14:
the 13 polypeptides obtained in example 13 were subjected to solid-phase synthesis and activity verification, comprising the steps of:
(1) Peptide fragments GLGIF, APGR, VKGVF, LFAGA, LGGIF, FGAGGL, LSGAY, FGGAL, WLDAN, RDWPEGR, DWPEGR, YLGGAL and LGGLNQ were both synthesized by Fmoc solid phase synthesis from Nanje peptide Biotechnology Inc.
(2) The synthesized polypeptide is prepared into a solution with the mass concentration of 0.035-0.1 mg/mL, and the in vitro ACE inhibition activity of the synthesized polypeptide is measured by a spectrophotometry method, and the result is shown in the table 5. The measurement results show that the polypeptides GLGIF, APGR, VKGVF, LFAGA and LGGIF have higher ACE inhibition activity at 0.1mg/mL or lower concentration of 0.04mg/mL or 0.035 mg/mL.
TABLE 5 peptide fragment information and ACE inhibition ratio of 13 polypeptides in example 14
Further, 5 polypeptides such as GLGIF, APGR, VKGVF, LFAGA and LGGIF are prepared into a solution with the mass concentration of 0.01-1 mg/mL, the in vitro ACE inhibition rate of the synthesized polypeptides at different concentrations is measured by a spectrophotometry, the IC 50 value of the synthesized polypeptides is calculated, and the result is shown in Table 6. The determination result shows that all 5 polypeptides such as GLGIF, APGR, VKGVF, LFAGA and LGGIF have excellent ACE inhibitory activity, wherein the peptide segment with the amino acid sequence of GLGIF shows the highest ACE inhibitory effect. Notably, the IC 50 value of the euphausia superba ACE inhibitory peptide mixture prepared in the embodiment 1 is 0.336mg/mL, namely the polypeptide prepared by the invention can improve the ACE inhibitory activity by 28 times, the technical improvement effect is obvious, and the product application prospect is good.
TABLE 6 peptide fragment information for 5 polypeptides in example 14 ACE inhibitory Activity IC 50 values
Peptide fragment Molecular weight (Da) IC50(mg/mL)
GLGIF 505.60 0.012
APGR 399.43 0.020
VKGVF 548.67 0.060
LFAGA 477.53 0.073
LGGIF 505.60 0.106
The overall process flow for the preparation of examples 1-14 above is shown in FIG. 16.
Example 15:
the application of the antarctic krill ACE inhibitory peptide obtained in examples 1-14 specifically comprises:
The euphausia superba ACE inhibitory peptide obtained in examples 1-14 can be applied to the fields of medicines for preventing and treating hypertension, auxiliary antihypertensive foods and the like, and the product preparation can be in the forms of solid beverages, capsules, tablet candies, gel candies, liquid drinks and the like.
The above examples are only preferred embodiments of the present invention and do not limit the scope of the claims, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the scope of the invention.

Claims (2)

1.一种南极磷虾ACE抑制肽LGGIF、YLGGAL、LGGLNQ，其特征在于，所述ACE抑制肽LGGIF具体序列为SEQ No.5：Leu-Gly-Gly-Ile-Phe；所述ACE抑制肽YLGGAL具体序列为SEQNo.12：Tyr-Leu-Gly-Gly-Ala-Leu；所述ACE抑制肽LGGLNQ具体序列为SEQ No.13：Leu-Gly-Gly-Leu-Asn-Gln。1. An Antarctic krill ACE inhibitory peptide LGGIF, YLGGAL, LGGLNQ, characterized in that the specific sequence of the ACE inhibitory peptide LGGIF is SEQ No.5: Leu-Gly-Gly-Ile-Phe; the specific sequence of the ACE inhibitory peptide YLGGAL is SEQ No.12: Tyr-Leu-Gly-Gly-Ala-Leu; the specific sequence of the ACE inhibitory peptide LGGLNQ is SEQ No.13: Leu-Gly-Gly-Leu-Asn-Gln. 2.权利要求1所述南极磷虾ACE抑制肽LGGIF、YLGGAL、LGGLNQ在制备降血压药品或辅助降血压食品中的应用。2. Use of the Antarctic krill ACE inhibitory peptides LGGIF, YLGGAL, and LGGLNQ according to claim 1 in the preparation of blood pressure lowering drugs or auxiliary blood pressure lowering foods.
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