CN1660888A - Peptide of decreasing blood pressure of protein of acetes chinensis, preparing method and application - Google Patents
Peptide of decreasing blood pressure of protein of acetes chinensis, preparing method and application Download PDFInfo
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- CN1660888A CN1660888A CN 200410075836 CN200410075836A CN1660888A CN 1660888 A CN1660888 A CN 1660888A CN 200410075836 CN200410075836 CN 200410075836 CN 200410075836 A CN200410075836 A CN 200410075836A CN 1660888 A CN1660888 A CN 1660888A
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Abstract
A depressor peptide with higher suppression activity to angiotensin-converting enzyme (ACE) and having 6 amino acids is prepared from the protein of Chinese shrimp through enzymolyzing, separation and purifying. Its short peptide with 12 sequences is chemically synthesized. It can be used to prepare antihypertensive medicine.
Description
(1) technical field
The present invention relates to step-down peptide and preparation method thereof and application that from the enzymolysis product of Chinese shrimp (Accedes chinensis) separation and purification has new aminoacid sequence, belong to the marine biotechnology field.
(2) background technology
Small peptide by food proteins process part enzymolysis gained more and more causes food scholar's attention.These small peptides have various activity, such as: improve immunizing power, improve the dietetic alimentation rate, antihypertensive active.Have these active albumen from Enzymatic Hydrolysate of Food Protein purifying come out, these peptides are not have actively in protein sequence, have only to discharge from albumen by the processing of enzymolysis or food just to have activity.
Angiotensin-converting enzyme (ACE) is a kind of carboxypeptidase that can the catalysis hypertensin 1 changes into hypertensin 2, can cause elevation of blood pressure, therefore suppress the ACE activity, can cause the concentration of the hypertensin 2 in the blood to reduce, thereby cause blood pressure to reduce.Though the ACE inhibitor of synthetic has tangible blood pressure lowering effect, side effect is too big, therefore must find now safer, novel, treat hypertensive medicine efficiently.Now from different albumen, carry out enzymolysis by different enzymes, found that in its zymolyte many ACE suppress small peptide, these protein resources comprise milk proteins, soybean protein, egg albumen, chicken protein, marine fishes albumen, algae etc., having ACE in existing till now more than 200 suppresses active enzymolysis activity peptide and is in the news, but the small peptide of the ACE of report inhibition now is mainly derived from the proteic zymolyte in land, the report that derives from the marine protein zymolyte is less, therefore, from now on, the marine organisms protein zymolyte will become the important source of screening new ace inhibitory peptide from now on.
Shrimp is a kind of low value-added marine protein resource in waters, China Bohai Sea Gulf, and annual amount of fishing reaches 300,000 tons, and up to the present, not effectively development and use are just dried and made dried small shrimp or make shrimp paste, and value-added content of product is lower.Because protein content that shrimp is higher and lower lipid content, therefore, shrimp is the good protein resource that is used for enzymolysis, and form with regard to the amino acid of shrimp, being rich in and having branched-chain amino acid and the die aromatischen Aminosaeuren relevant with the blood pressure lowering peptide structure, is the good raw material that enzymolysis separates the antihypertensive peptide.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of Chinese shrimp protein antihypertensive peptide and preparation method thereof and application are provided.
Separation and purification makes new advances from the proteolysis product of Chinese shrimp has six amino acid whose step-down peptides in the present invention, and its sequence is respectively Phe-Cys-Val-Leu-Arg-Pro, Ile-Phe-Val-Pro-Ala-Phe or Lys-Pro-Pro-Glu-Thr-Val.Have higher angiotensin-converting enzyme (ACE) and suppress active.
Consider that above-mentioned three kind of six peptide might be by further enzymolysis in digestive tube, so action site according to the body endoenzyme, the possible enzymolysis product small peptide of above-mentioned three kind of six peptide has been carried out chemosynthesis, through the ACE inhibition test, screening obtains Cys-Val-Leu-Arg-Pro, Val-Leu-Arg-Pro, Phe-Cys-Val-Leu, Cys-Val-Leu, Arg-Pro, Val-Leu, Phe-Cys, Ile-Phe-Val-Pro-Ala, Phe-Val-Pro-Ala-Phe, Val-Pro-Ala-Phe, the small peptide of 12 kinds of sequences such as Val-Pro-Ala and Ile-Phe have good ACE and suppress active.
On vertical, Chinese shrimp protein antihypertensive peptide of the present invention is one of small peptide or the combination with following sequence:
Phe-Cys-Val-Leu-Arg-Pro peptide 1,
IIe-Phe-Val-Pro-Ala-Phe peptide 2,
Lys-Pro-Pro-Glu-Thr-Val peptide 3,
Cys-Val-Leu-Arg-Pro peptide 4,
Val-Leu-Arg-Pro peptide 5,
Phe-Cys-Val-Leu peptide 6,
Cys-Val-Leu peptide 7,
Arg-Pro peptide 8,
Val-Leu peptide 9,
Phe-Cys peptide 10,
Ile-Phe-Val-Pro-Ala peptide 11,
Phe-Val-Pro-Ala-Phe peptide 12,
Val-Pro-Ala-Phe peptide 13,
Val-Pro-Ala peptide 14 or
Ile-Phe peptide 15.
The preparation method of the present invention China shrimp protein antihypertensive peptide comprises that separation and purification has the step-down peptide (ace inhibitory peptide) of new aminoacid sequence and further utilizes enzymolysis solution separation and purification ace inhibitory peptide from the proteolysis product of Chinese shrimp.Comprise that specifically the enzymolysis shrimp prepares enzymolysis solution, the solid phase synthesis of separation and purification step-down peptide, sequencing and step-down peptide from enzymolysis solution.
Above-mentioned enzymolysis shrimp prepares enzymolysis solution can use prior art, and method provided by the invention is (1) and (2) in the following concrete steps.
Describe each operation steps of the present invention below in detail, but be not limited thereto:
(1) preparation of protease preparation
Substratum: 2～3 parts in cake powder, 2～3 parts of Semen Maydis powder, 1～1.5 part in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts of～0.04 parts, water are weight part, down with.Sterilization, cooling, 5～10 parts of the bacteria suspensions of the eggplant bottle bacterial classification of inoculation subtilis SM98011 (Bacillus.Subtilis SM98011) ventilate and stir, and cultivate 15～18 hours, and are stand-by as liquid seeds.
Liquid submerged fermentation prepares protease preparation:
Substratum: 3～3.5 parts of soybean cake powder, 3.5～5.0 parts of Semen Maydis powder, 2.0～2.5 parts in wheat bran, Na
2HPO
40.4～0.5 part, KH
3PO
40.03 100 parts of～0.04 parts, water are weight part, down with.Sterilization, the liquid seeds of inoculation above-mentioned steps, inoculum size is 5～7% of a substratum weight, ventilates to stirring, temperature control fermented 37～40 hours, got protease preparation.Enzyme is lived and is 4000U/ml.
(2) the enzymolysis shrimp prepares enzymolysis solution
Basic raw material: Chinese shrimp, take by weighing 45～50 parts of raw materials of shrimp by fresh weight, pull an oar, add the above-mentioned protease preparation of 45～50 parts of liquid weights then, pH value to 6.8～7.3,45～50 ℃ of temperature stirred enzymolysis 5 hours.Enzymolysis finishes, the centrifuging and taking supernatant.
(3) separation and purification step-down peptide (ace inhibitory peptide) from enzymolysis solution
With the ultra-filtration membrane of 5000Da with the enzymolysis solution ultrafiltration, ultrafiltration is crossed PharmadexLH20 post (2.5 * 100cm less than the ultrafiltrated of 5000Da, medium Pharmadex), use the protein nucleic acid detector, detect, collect respectively according to separating the chromatographic peak that obtains from chromatographic column at the 214nm place, after the elutriant freeze-drying of collecting is weighed, measure ACE and suppress active, repeatedly collect, further separate then with the anti-phase C18 of high pressure liquid chromatography with suppressing active high component.It is 220nm that the PDA detector detects wavelength, according to the spectrogram of the peptide of gained the peptide peak is collected concentratedly then respectively, measures ACE and suppresses active, obtains having the peptide that high ACE suppresses vigor.
(4) sequencing
The 3rd step is separated the pure peptide that high ACE suppresses vigor that has that obtains, measure amino acid with the amino acid automatic sequencer and form, analyze the molecular weight of 3 peptides and the distribution of peptide section by liquid matter logotype instrument again, analyze the aminoacid sequence of 3 peptides.Sequence is respectively Phe-Cys-Val-Leu-Arg-Pro, Ile-Phe-Val-Pro-Ala-Phe or Lys-Pro-Pro-Glu-Thr-Val.
(5) solid phase synthesis of step-down peptide
According to the restriction enzyme site of human body endoproteinase (trypsinase, Chymotrypsin and stomach en-) and the peptide of above-mentioned 3 separation and purification, design 12 peptides (part of former peptide sequence) Cys-Val-Leu-Arg-Pro, Val-Leu-Arg-Pro, Phe-Cys-Val-Leu, Cys-Val-Leu, Arg-Pro, Val-Leu, Phe-Cys, Ile-Phe-Val-Pro-Ala, Phe-Val-Pro-Ala-Phe, Val-Pro-Ala-Phe, Val-Pro-Ala and Ile-Phe.By the prior art solid phase synthesis, the ACE that measures each bar peptide then suppresses active.The result proves that 12 peptides of solid phase synthesis have higher ACE and suppress active.
Add soya-bean oil 0.3～0.35 weight part in the substratum of above-mentioned steps (1) and make defoamer.
The ACE that measures peptide in the above-mentioned steps (3) suppresses activity methods and can as spectrophotometry or high pressure lipuid chromatography (HPLC), the invention provides following ACE and suppress activity determination method-capillary electrophoresis with existing known technology:
10 μ l substrate Hip-His-Leu (1mM), the angiotensin-converting enzyme of 0.8mU (ACE) are mixed into some groups of samples with the Chinese shrimp enzymolysis solution of 0.03～40mg/ml of some 10 μ l respectively.Described reagent all uses the borate buffer of 100mM (to comprise 0.3M NaCl, pH8.3) preparation.Reaction solution is used 0.1% trifluoroacetic acid TFA (10 μ l) stopped reaction then at 37 ℃ of reaction 30min.Angiotensin-converting enzyme inhibited reaction product directly detects on capillary electrophoresis apparatus.Capillary electrophoresis apparatus is Beckman Coulter P/ACE MDQ (Fullerton, CA) device is equipped with PDA (photodiode array) detector, data gathering, analysis, system's control P/ACE MDQ software, this software from Beckman Coulter (Fullerton, CA).Blend sample after the stopped reaction directly detects with capillary electrophoresis, and last sample pressure is 1psi, and the time is 5 seconds, electrophoretic voltage 20kV, and uv-absorbing is 228nm, electrophoresis time 5 minutes, electrophoretic buffer are the borate buffer (pH9.18) of 20mM.Washed kapillary 1 minute with damping fluid after sample of every mensuration.Substrate and product urobenzoic acid went out the peak at 2.5 minutes and 3.5 minutes respectively, according to the peak area of above-mentioned computed in software urobenzoic acid.
ACE suppresses active calculating (IC
50The calculating of value):
Urobenzoic acid is mixed with different concns 0.2,0.4,0.6,0.8,1.0,1.2,2,4, and 6mM measures the linear relationship of its peak area and urobenzoic acid concentration by capillary electrophoresis.
Peak area=K * Chip+N
ACE vigor (umol/min)=V * Chip ÷ t
V: reaction volume, t: reaction times, K: according to the curve constant that peak area and urobenzoic acid concentration are obtained, Chip: urobenzoic acid concentration, N: the intersection point of urobenzoic acid concentration curve and Y-axis.
By the angiotensin-converting enzyme effect, do not contain any inhibiting peptide, the amount of the urobenzoic acid HA that discharges from substrate Hip-His-Leu is defined as the 100%ACE vigor.The amount of the marine protein enzymolysis product of the ACE vigor of inhibition 50% is defined as the inhibition vigor of enzymolysis product.IC
50The linear-logarithmic method is adopted in the calculating of value, and to Log (hydrolysate concentration mg/ml) mapping, the value that the gained straight line is corresponding with the intersection point of X-axis is M with Log (ACE vigor/(1-ACE vigor)), and 10
MThe IC that suppresses the ACE vigor for enzymolysis product
50Value.
The used high pressure liquid chromatography post of above-mentioned steps (3) is anti-phase C18 post.
Above-mentioned steps (4) amino acid analysis be with peptide with the HCl of 6N 115 ℃ of all-hydrolytics 22 hours, measure by the amino acid automatic sequencer.
Chinese shrimp protein antihypertensive peptide product of the present invention is white powder, and is soluble in water, can be a kind of or its combination in the peptide 1-peptide 15.
Chinese shrimp protein antihypertensive peptide of the present invention is used to prepare antihypertensive drugs.
Excellent results of the present invention is land microbial enzyme engineering is applied to the deep level development utilization of shrimp.Be embodied in 1, with the ocean shrimp as enzymolysis raw material 2, have the peptide 3 of antihypertensive function, react with ACE and detect the antihypertensive activity 4 that respectively goes on foot product and by the chromatographic process separation and purification to the part of the peptide that obtains with new sequence from enzymolysis solution, it is synthetic to carry out sequence, and the little peptide that obtains has higher ACE and suppresses vigor.
(4) description of drawings
Fig. 1 is the shrimp enzymolysis solution process PharmadexLH20 post isolating color atlas of embodiment 1 step (3) through the membrane ultrafiltration of 3000Da.
Fig. 2, Fig. 3 are respectively that II, III, IV sample separate the chromatographic peak that obtains on the anti-phase C18 post of high-pressure liquid phase with Fig. 4.
Fig. 5, Fig. 6 and Fig. 7 are respectively the IC of II, III, IV sample
50The linear-logarithmic figure of value proves that II, III, IV sample are to have the pure peptide that high ACE suppresses vigor, and they are respectively 3 peptides (peptide 1, peptide 2 and peptide 3) of separation and purification.
(5) embodiment
Embodiment 1.
(1) preparation of protease preparation
Seed culture medium: with 0.3 part of weight part extractum carnis, 1 part of peptone, 2 parts in agar, NaCl0.5 part is substratum, and 98 parts in water, pH are 7.1, sterilization, line connects subtilis SM98011 (Bacillus.SubtilisSM98011) bacterial classification, and 30 ℃ of cultivations were cultivated 20 hours.
Fermention medium: 100 parts of 3 parts of soybean cake powder, 2 parts of Semen Maydis powder, 1 part in wheat bran, 0.4 part of Na2HPO4, KH2PO40.04 part, water add soya-bean oil and make defoamer for 0.3 part.120 ℃ of sterilizations 30 minutes after the cooling, connect 10 parts of the bacteria suspensions of seed, in 150 liters of fermentor tanks, and 70 liters of sample-loading amounts, under 30 ℃, ventilation (V
Wind/ V
LiquidTime) 1: 0.6, stir 330 rev/mins, cultivated 36 hours.This moment pH7.0.
(2) preparation of enzymolysis small peptide
At first take by weighing the Chinese shrimp of 50 parts (fresh weights), making beating.The protease preparation that adds 50 parts (liquid weights) then, adjust pH to 7.2,50 ℃, stirred enzymolysis 5 hours, enzymolysis finishes, and 5000rpm is centrifugal, and supernatant is an enzymolysis small peptide liquid.
(3) separation and purification of step-down peptide
With the membrane ultrafiltration of supernatant liquor,, separate obtaining 9 peaks then with Pharmadex LH20 post on the ultrafiltrated with 5000Da, collect respectively, measure ACE and suppress vigor, obtain II, III, the IV peak has higher ACE and suppresses the vigor (see figure 1), collects repeatedly, last high pressure liquid chromatography, with II, III, the IV sample further separates, and obtains 19 chromatographic peak (see figure 2)s, wherein has 3 peaks to have higher ACE and suppresses vigor, the peptide of these 3 peak correspondences is a peptide 1, peptide 2 and peptide 3.
(4) sequencing of step-down peptide
The higher ACE that has that step (3) is obtained suppresses the HCl all-hydrolytic of 3 pure peptides of vigor with 6N, measuring amino acid with the amino acid automatic sequencer forms, analyze the molecular weight of 3 peptides and the distribution of peptide section with liquid matter logotype instrument then, analyze the aminoacid sequence of 3 peptides.Phe-Cys-Val-Leu-Arg-Pro，Ile-Phe-Val-Pro-Ala-Phe，Lys-Pro-Pro-Glu-Thr-Val。Capillary electrophoresis is measured IC
50Value is respectively 12.3 μ m, 19.8 μ m and 24.1 μ M.As Fig. 5, Fig. 6 and shown in Figure 7.The IC that has the enzymolysis ace inhibitory peptide according to prior art
50Value is 0.2 μ M-600 μ M, IC
50Be worth more for a short time, illustrate the restraining effect of ACE by force more, blood pressure lowering effect is good more.Prove that thus the peptide 1 of separation and purification from Chinese shrimp zymolyte, peptide 2 and peptide 3 have higher ACE and suppress active.
(5) solid phase synthesis of peptide
According to body endoproteinase (trypsinase, Chymotrypsin, stomach en-) restriction enzyme site, 12 peptides have been designed, be respectively the part of 3 former peptide sequences of separation and purification: Cys-Val-Leu-Arg-Pro (peptide 4), Val-Leu-Arg-Pro (peptide 5), Phe-Cys-Val-Leu (peptide 6), Cys-Val-Leu (peptide 7), Arg-Pro (peptide 8), Val-Leu (peptide 9), Phe-Cys (peptide 10), Ile-Phe-Val-Pro-Ala (peptide 11), Phe-Val-Pro-Ala-Phe (peptide 12), Val-Pro-Ala-Phe (peptide 13), Val-Pro-Ala (peptide 14) and Ile-Phe (peptide 15).By the prior art solid phase synthesis, the ACE that the synthetic peptide has been measured them with capillary electrophoresis suppresses active, IC
50Value is followed successively by 141.3,22.3, and 19.8,17.6,28.1,34.5,478.6,23.9,24.5,575.4,34.4,26.7 μ M.Illustrate that the synthetic peptide is strong to the restraining effect of ACE.
The small peptide product drying of present embodiment gained is handled the back and is white powder, and is soluble in water.
Embodiment 2: as described in embodiment 1, the consumption of different is raw material shrimp is different, the shrimp of 45 parts (fresh weights), making beating.The protease preparation that adds 50 parts (liquid weights) then.
Small peptide of the present invention has higher ACE and suppresses active, even enter digestive tube behind enzymolysis, its enzymolysis product still has higher antihypertensive activity.Characteristics of the present invention are: integrate enzyme engineering technology and peptide purification authenticate technology, the marine protein resource that this amount of shrimp is not used effectively greatly and so far deep development and use have been carried out, found the ace inhibitory peptide of forming by new aminoacid sequence and guaranteed in vivo functional, for the exploitation of antihypertensive drugs is later on laid a good foundation.
Sequence table
＜110〉Shandong University
＜120〉Chinese shrimp protein antihypertensive peptide and preparation method thereof and application
<160>11
<170>Patent?In3.1
<210>1
<211>6
<212>PRT
＜213〉Chinese shrimp (Accedes chinensis)
<400>1
Phe?Cys?Val?Leu?Arg?Pro
<210>2
<211>6
<212>PRT
<400>2
Ile?Phe?Val?Pro?Ala?Phe
<210>3
<211>6
<212>PRT
<400>3
Lys?Pro?Pro?Glu?Thr?Val
<210>4
<211>5
<212>PRT
<400>4
Cys?Val?Leu?Arg?Pro
<210>5
<211>4
<212>PRT
<400>5
Val?Leu?Arg?Pro
<210>6
<211>4
<212>PRT
<400>6
Phe?Cys?Val?Leu
<210>7
<211>3
<212>PRT
<400>7
Cys?Val?Leu
<210>8
<211>2
<212>PRT
<400>8
Arg?Pro
<210>9
<211>2
<212>PRT
<400>9
Val?Leu
<210>10
<211>2
<212>PRT
<400>10
Phe?Cys
<210>11
<211>5
<212>PRT
<400>11
Ile?Phe?Val?Pro?Ala
<210>12
<211>5
<212>PRT
<400>12
Phe?Val?Pro?Ala?Phe
<210>13
<211>4
<212>PRT
<400>13
Val?Pro?Ala?Phe
<210>14
<211>3
<212>PRT
<400>14
Val?Pro?Ala
<210>15
<211>2
<212>PRT
<400>15
Ile?Phe
Claims (7)
1. Chinese shrimp protein antihypertensive peptide is characterized in that, is one of small peptide or the combination with following sequence:
Phe-Cys-Val-Leu-Arg-Pro peptide 1,
Ile-Phe-Val-Pro-Ala-Phe peptide 2,
Lys-Pro-Pro-Glu-Thr-Val peptide 3,
Cys-Val-Leu-Arg-Pro peptide 4,
Val-Leu-Arg-Pro peptide 5,
Phe-Cys-Val-Leu peptide 6,
Cys-Val-Leu peptide 7,
Arg-Pro peptide 8,
Val-Leu peptide 9,
Phe-Cys peptide 10,
Ile-Phe-Val-Pro-Ala peptide 11,
Phe-Val-Pro-Ala-Phe peptide 12,
Val-Pro-Ala-Phe peptide 13,
Val-Pro-Ala peptide 14 or
Ile-Phe peptide 15.
2. the preparation method of the described Chinese shrimp protein antihypertensive peptide of claim 1, comprise that the enzymolysis shrimp prepares enzymolysis solution, the solid phase synthesis of separation and purification step-down peptide, determined amino acid sequence and peptide from enzymolysis solution, it is characterized in that six the amino acid whose step-down peptides that have that separation and purification makes new advances from the proteolysis product of Chinese shrimp, its sequence is respectively Phe-Cys-Val-Leu-Arg-Pro, Ile-Phe-Val-Pro-Ala-Phe or Lys-Pro-Pro-Glu-Thr-Val; Action site according to the body endoenzyme, the possible enzymolysis product small peptide of above-mentioned three kind of six peptide has been carried out chemical solid phase synthesis, menses angiotensin saccharase inhibition test, screening obtains Cys-Val-Leu-Arg-Pro, Val-Leu-Arg-Pro, Phe-Cys-Val-Leu, Cys-Val-Leu, Arg-Pro, Val-Leu, Phe-Cys, Ile-Phe-Val-Pro-Ala, Phe-Val-Pro-Ala-Phe, Val-Pro-Ala-Phe, the small peptide of Val-Pro-Ala and 12 kinds of sequences of Ile-Phe has good angiotensin converting enzyme inhibition activity.
3. the preparation method of Chinese shrimp protein antihypertensive peptide as claimed in claim 2 is characterized in that described enzymolysis shrimp prepares enzymolysis solution, and concrete steps are as follows:
(1) preparation of protease preparation
Substratum: 2～3 parts in cake powder, 2～3 parts of Semen Maydis powder, 1～1.5 part in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts in～0.04 part and water, sterilization, cooling, 5～10 parts of the bacteria suspensions of the eggplant bottle bacterial classification of inoculation subtilis SM98011 ventilate and stir, and cultivate 15～18 hours, and are stand-by as liquid seeds, are weight part; Liquid submerged fermentation prepares protease preparation then, and is specific as follows:
Substratum: 3～3.5 parts of soybean cake powder, 3.5～5.0 parts of Semen Maydis powder, 2.0～2.5 parts in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts in～0.04 part and water are weight part, above-mentioned liquid seeds inoculate in sterilization, and inoculum size is 5～7% of a substratum weight, ventilates and stirs, and temperature control fermented 37～40 hours, got protease preparation;
(2) the enzymolysis shrimp prepares enzymolysis solution
Basic raw material: Chinese shrimp, take by weighing 45～50 parts of raw materials of shrimp by fresh weight, pull an oar, add the above-mentioned protease preparation of 45～50 parts of liquid weights then, adjust pH to 6.8～7.3,45～50 ℃ of temperature stirred enzymolysis 5 hours, and enzymolysis finishes, the centrifuging and taking supernatant.
4. the preparation method of Chinese shrimp protein antihypertensive peptide as claimed in claim 2, it is characterized in that described from enzymolysis solution separation and purification step-down peptide specific as follows:
With the ultra-filtration membrane of 5000Da with the enzymolysis solution ultrafiltration, ultrafiltration is crossed the PharmadexLH20 post less than the ultrafiltrated of 5000Da, 2.5 * 100cm, use the protein nucleic acid detector, detect at the 214nm place, collect respectively according to separating the chromatographic peak that obtains from chromatographic column, after the elutriant freeze-drying of collecting is weighed, measure angiotensin converting enzyme inhibition activity, repeatedly collect suppressing active high component, further separate with the high pressure liquid chromatography post then, it is 220nm that the PDA detector detects wavelength, according to the spectrogram of the peptide of gained the peptide peak is collected concentratedly then respectively, measured angiotensin converting enzyme inhibition activity.
5. the preparation method of Chinese shrimp protein antihypertensive peptide as claimed in claim 4 is characterized in that described upward high pressure liquid chromatography post is anti-phase C18 post.
6. the preparation method of Chinese shrimp protein antihypertensive peptide as claimed in claim 2, it is characterized in that described determined amino acid sequence be with peptide with the HCl of 6N 115 ℃ of all-hydrolytics 22 hours, measure by the amino acid automatic sequencer.
7. Chinese shrimp protein antihypertensive peptide is used to prepare antihypertensive drugs.
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| CNB2006101082979A Division CN100503633C (en) | 2004-12-29 | 2004-12-29 | Acetes chinensis protein antigypertensive peptide and preparation method and application thereof |
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| CN102276690A (en) * | 2011-08-05 | 2011-12-14 | 成都中医药大学 | Blood pressure lowering functional polypeptide and application thereof |
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| CN102276690A (en) * | 2011-08-05 | 2011-12-14 | 成都中医药大学 | Blood pressure lowering functional polypeptide and application thereof |
| CN102276690B (en) * | 2011-08-05 | 2013-03-20 | 成都中医药大学 | Blood pressure lowering functional polypeptide and application thereof |
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| CN104159912B (en) * | 2012-03-09 | 2016-11-09 | 森永乳业株式会社 | DPP IV inhibitor |
| JPWO2013133031A1 (en) * | 2012-03-09 | 2015-07-30 | 森永乳業株式会社 | Dipeptidyl peptidase-IV inhibitor |
| US20150232510A1 (en) * | 2012-03-09 | 2015-08-20 | Morinaga Milk Industry Co., Ltd | Dipeptidyl peptidase-iv inhibitor |
| EP2824110A4 (en) * | 2012-03-09 | 2015-12-30 | Morinaga Milk Industry Co Ltd | Dipeptidyl peptidase-iv inhibitor |
| CN105833256A (en) * | 2012-03-09 | 2016-08-10 | 森永乳业株式会社 | Dipeptidyl peptidase-IV inhibitor |
| WO2013133031A1 (en) * | 2012-03-09 | 2013-09-12 | 森永乳業株式会社 | Dipeptidyl peptidase-iv inhibitor |
| CN104159912A (en) * | 2012-03-09 | 2014-11-19 | 森永乳业株式会社 | Dipeptidyl peptidase-iv inhibitor |
| US9617300B2 (en) * | 2012-03-09 | 2017-04-11 | Morinaga Milk Industry Co., Ltd. | Dipeptidyl peptidase-IV inhibitor |
| CN111323606A (en) * | 2020-03-16 | 2020-06-23 | 中南大学 | Method for detecting antioxidant effect of antioxidant by using oxidative damage of chromoprotein |
| CN111323606B (en) * | 2020-03-16 | 2021-08-10 | 中南大学 | Method for detecting antioxidant effect of antioxidant by using oxidative damage of chromoprotein |
| CN114921516A (en) * | 2022-05-26 | 2022-08-19 | 上海海洋大学 | Macrobrachium nipponense immunoactive peptide and preparation method and application thereof |
| CN114921516B (en) * | 2022-05-26 | 2024-03-15 | 上海海洋大学 | Chinese lobster immunocompetent peptide, and preparation method and application thereof |
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