CN105821102B - Preparation method of angiotensin-converting enzyme inhibitory peptide of Sinonovacula constricta - Google Patents
Preparation method of angiotensin-converting enzyme inhibitory peptide of Sinonovacula constricta Download PDFInfo
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- CN105821102B CN105821102B CN201511011196.5A CN201511011196A CN105821102B CN 105821102 B CN105821102 B CN 105821102B CN 201511011196 A CN201511011196 A CN 201511011196A CN 105821102 B CN105821102 B CN 105821102B
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Abstract
The invention discloses a method for preparing angiotensin converting enzyme inhibitory peptide of Sinonovacula constricta, which comprises the following steps: (1) pretreatment of sinonovacula constricta; (2) carrying out enzymolysis; (3) performing ultrafiltration separation; (4) separating and purifying by a rapid protein purification system; (5) and (5) separating and purifying by using a high performance liquid chromatography system. According to the invention, sinonovacula constricta is taken as a raw material, the sinonovacula constricta is subjected to enzymolysis through neutral protease, and an enzymolysis solution is separated and purified by using an ultrafiltration technology, a rapid protein purification system and a high performance liquid chromatography system to obtain a novel angiotensin-converting enzyme inhibitory peptide (ACE inhibitory peptide), the extraction and separation process is reasonable, the operability is strong, and an experimental basis is provided for preparing the high-activity ACE inhibitory peptide from the sinonovacula constricta.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a preparation method of angiotensin-converting enzyme inhibitory peptide of sinonovacula constricta.
Background
Hypertension is a cardiovascular disease which afflicts today and can cause serious complications, with great harm to human health. Angiotensin-converting enzyme (ACE) plays an important role in the regulation of blood pressure of a body, on one hand, the ACE converts inactive Angiotensin I into Angiotensin II which can strongly contract systemic arterioles and increase blood pressure; on the other hand, ACE can promote the decomposition of bradykinin with the function of reducing blood pressure, and causes the increase of blood pressure.
Nowadays, synthetic ACE inhibitors such as captopril, enalapril and lisinopril have strong blood pressure lowering effect, but have side effects such as dizziness, nausea, severe cough, abnormal taste and renal function damage after long-term administration. Many studies show that the short peptide derived from food has the efficacy of reducing blood pressure, and the skin of Pacific cod such as Dai-Hung Ngo and the like is taken as a raw material, is subjected to enzymolysis by papain, and is separated and purified to obtain the antihypertensive peptide: Thr-Cys-Ser-Pro; hasan F and the like carry out enzymolysis on bonito flesh by using pepsin, and high-activity antihypertensive peptide (IKYGD) is obtained after separation and purification; the bioactive peptide derived from food has the characteristics of safety, no toxic or side effect, easy absorption by organisms and the like. Therefore, the preparation and isolation of highly active ACE inhibiting peptides from food sources is still a focus of research.
Sinonovacula constricta (Sinonovacula) is a common shellfish in China coastal areas and belongs to a high-quality protein source. The Sinonovacula constricta Lamarck extract has effects of resisting oxidation, resisting tumor, and improving immunity. However, no research report related to the sinonovacula constricta antihypertensive peptide is found at present.
Disclosure of Invention
The invention aims to provide a preparation method of the angiotensin-converting enzyme inhibitory peptide of sinonovacula constricta with reasonable extraction and separation process, strong operability, good product stability and high purity.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing angiotensin-converting enzyme inhibitory peptide of Sinonovacula constricta comprises the following steps:
(1) pretreatment of sinonovacula constricta: removing shells and stripping meat of the sinonovacula constricta, cleaning the sinonovacula constricta with clear water, homogenizing the sinonovacula constricta with a high-speed tissue pulper, soaking the sinonovacula constricta meat with isopropanol for 3-5 h, filtering, soaking and washing filter residues with purified water until no isopropanol odor exists, and dehydrating to obtain sinonovacula constricta meat residues for later use. The pretreatment of the Sinonovacula constricta Lamarck is mainly to remove hetero-protein and remove lipid.
(2) Enzymolysis: adding neutral protease into the sinonovacula constricta meat residues in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 3-4, adding enzyme in an amount of 1500-2000U/g, adjusting the pH value to 6.5-7.5, carrying out enzymolysis at 50-60 ℃ for 9-12 h, inactivating with a boiling water bath after enzymolysis, precooling, and centrifuging to obtain a supernatant.
(3) And (3) ultrafiltration separation: and (3) filtering the supernatant through a 0.45-micron filter membrane, and performing ultrafiltration through an ultrafiltration membrane with the molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: and (4) separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system to obtain two active components, respectively sampling and determining the angiotensin converting enzyme inhibitory activity of the two active components, and taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain three active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the three active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: Pro-Trp-Gly-Met-Phe (PWGMF).
Preferably, in the step (1), the mass ratio of the sinonovacula constricta to the isopropanol is 1: 3 to 5.
Preferably, in step (2), stirring is continued during the enzymatic hydrolysis.
Preferably, in the step (2), the temperature is precooled to 4-6 ℃.
Preferably, in the step (2), the centrifugal process conditions are as follows: the rotating speed is 5000-10000 rpm/min, and the centrifugation time is 10-30 min.
Preferably, in the step (4), the specific method for separating and purifying the rapid protein purification system comprises the following steps: loading gel into a chromatographic column by using a DEAE-Sepharose FF ion exchange column (the grain diameter of a filler is 10 mu m and is 10 multiplied by 300mm), balancing Tris-HCl (pH is 7.4), preparing ultrafiltrate into a solution of 200mg/mL, loading the solution with the amount of 3mL each time, and respectively carrying out stage elution by using NaCl solutions of Tris-HCl (pH is 7.4), 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L and 1mol/L at the elution speed of 2.2 mL/min; the detection wavelength is 280 nm; the peaks were collected.
Preferably, in the step (5), the specific method for separating and purifying by the high performance liquid chromatography system is as follows: preparing the component B into a solution of 200mg/ml by using a Sephadex G-25 chromatographic column (size: 2.6X 80 cm; column material: Sephadex G-25), and loading: 2.0 mL; mobile phase: ultrapure water; eluent flow rate: 1.0 mL/min; detection wavelength: 280 nm; automatic collection speed: 4.0 min/tube; the peaks were collected.
Preferably, in the step (5), the purity detection method of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using isocratic elution: 80% A, 20% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm.
Therefore, the invention has the following beneficial effects: the sinonovacula constricta is used as a raw material, the sinonovacula constricta is subjected to enzymolysis through neutral protease, an enzymolysis solution is separated and purified by using an ultrafiltration technology, a rapid protein purification system and a high performance liquid chromatography system, and then a novel angiotensin converting enzyme inhibitory peptide (ACE inhibitory peptide) is obtained.
Detailed Description
The invention is further described below by means of specific embodiments.
Example 1
(1) Pretreatment of sinonovacula constricta: removing shells and peeling the sinonovacula constricta lamarck, cleaning the sinonovacula constricta lamarck meat with clear water, homogenizing by a high-speed tissue pulper, and soaking the sinonovacula constricta lamarck meat with isopropanol for 5 hours, wherein the mass ratio of the sinonovacula constricta lamarck to the isopropanol is 1: and 5, soaking and washing filter residues after filtration by using purified water until no isopropanol peculiar smell exists, and dehydrating to obtain the sinonovacula constricta meat residues for later use.
(2) Enzymolysis: adding neutral protease into the sinonovacula constricta meat residues in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 4, adding 2000U/g of enzyme, controlling the pH value to be 7.5, carrying out enzymolysis at the temperature of 60 ℃, carrying out enzymolysis for 12 hours, continuously stirring during the enzymolysis, inactivating the mixture by using a boiling water bath after the enzymolysis is finished, precooling the mixture to 6 ℃, and centrifuging the mixture for 30 minutes at the rotation speed of 10000rpm/min to obtain supernatant.
(3) And (3) ultrafiltration separation: and (3) filtering the supernatant through a 0.45-micron filter membrane, and performing ultrafiltration through an ultrafiltration membrane with the molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system (Apurifier UPC-100 type rapid protein purifier, GE Life sciences company) to obtain two active components, respectively sampling and measuring angiotensin converting enzyme inhibitory activity of the two active components, and taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A, wherein the specific method for separating and purifying the rapid protein purification system comprises the following steps: loading gel into a chromatographic column by using a DEAE-Sepharose FF ion exchange column (the grain diameter of a filler is 10 mu m and is 10 multiplied by 300mm), balancing Tris-HCl (pH is 7.4), preparing ultrafiltrate into a solution of 200mg/mL, loading the solution with the amount of 3mL each time, and respectively carrying out stage elution by using NaCl solutions of Tris-HCl (pH is 7.4), 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L and 1mol/L at the elution speed of 2.2 mL/min; the detection wavelength is 280 nm; the peaks were collected.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain three active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the three active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: the specific method for separating and purifying the angiotensin converting enzyme inhibitory peptide of Pro-Trp-Gly-Met-Phe (PWGMF) by a high performance liquid chromatography system comprises the following steps: preparing the component B into a solution of 200mg/ml by using a Sephadex G-25 chromatographic column (size: 2.6X 80 cm; column material: Sephadex G-25), and loading: 2.0 mL; mobile phase: ultrapure water; eluent flow rate: 1.0 mL/min; detection wavelength: 280 nm; automatic collection speed: 4.0 min/tube; collecting each peak; the method for detecting the purity of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using isocratic elution: 80% A, 20% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm.
Example 2
(1) Pretreatment of sinonovacula constricta: removing shells and peeling the sinonovacula constricta lamarck, cleaning the sinonovacula constricta lamarck meat with clear water, homogenizing by a high-speed tissue pulper, and soaking the sinonovacula constricta lamarck meat with isopropanol for 3 hours, wherein the mass ratio of the sinonovacula constricta lamarck to the isopropanol is 1: and 3, soaking and washing filter residues after filtration by using purified water until no isopropanol peculiar smell exists, and dehydrating to obtain the sinonovacula constricta meat residues for later use.
(2) Enzymolysis: adding neutral protease into the sinonovacula constricta meat residues in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 3, adding 1500U/g of enzyme, adjusting the pH value to 6.5, adjusting the enzymolysis temperature to 50 ℃, adjusting the enzymolysis time to 9h, continuously stirring during the enzymolysis, inactivating with boiling water bath after the enzymolysis is finished, pre-cooling to 4 ℃, and centrifuging at the rotating speed of 5000rpm/min for 10min to obtain the supernatant.
(3) And (3) ultrafiltration separation: and (3) filtering the supernatant through a 0.45-micron filter membrane, and performing ultrafiltration through an ultrafiltration membrane with the molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system to obtain two active components, respectively sampling and determining the angiotensin converting enzyme inhibitory activity of the two active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A, and the specific method for separating and purifying by the rapid protein purification system comprises the following steps: loading gel into a chromatographic column by using a DEAE-Sepharose FF ion exchange column (the grain diameter of a filler is 10 mu m and is 10 multiplied by 300mm), balancing Tris-HCl (pH is 7.4), preparing ultrafiltrate into a solution of 200mg/mL, loading the solution with the amount of 3mL each time, and respectively carrying out stage elution by using NaCl solutions of Tris-HCl (pH is 7.4), 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L and 1mol/L at the elution speed of 2.2 mL/min; the detection wavelength is 280 nm; the peaks were collected.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain three active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the three active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: the specific method for separating and purifying the angiotensin converting enzyme inhibitory peptide of Pro-Trp-Gly-Met-Phe (PWGMF) by a high performance liquid chromatography system comprises the following steps: preparing the component B into a solution of 200mg/ml by using a Sephadex G-25 chromatographic column (size: 2.6X 80 cm; column material: Sephadex G-25), and loading: 2.0 mL; mobile phase: ultrapure water; eluent flow rate: 1.0 mL/min; detection wavelength: 280 nm; automatic collection speed: 4.0 min/tube; collecting each peak; the method for detecting the purity of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using isocratic elution: 80% A, 20% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm.
Example 3
(1) Pretreatment of sinonovacula constricta: removing shells and peeling the sinonovacula constricta lamarck, cleaning the sinonovacula constricta lamarck meat with clear water, homogenizing by a high-speed tissue pulper, and soaking the sinonovacula constricta lamarck meat with isopropanol for 4 hours, wherein the mass ratio of the sinonovacula constricta lamarck to the isopropanol is 1: 4, soaking and washing the filter residue after filtration with purified water until no isopropanol odor exists, and dehydrating to obtain the sinonovacula constricta meat residue for later use.
(2) Enzymolysis: adding neutral protease into the sinonovacula constricta meat residues in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 3.5, adding 1800U/g of enzyme, controlling the pH value to be 7, controlling the enzymolysis temperature to be 55 ℃, controlling the enzymolysis time to be 10 hours, continuously stirring during the enzymolysis, inactivating by using boiling water bath after the enzymolysis is finished, precooling to 5 ℃, and centrifuging for 20 minutes at 8000rpm/min to obtain supernatant.
(3) And (3) ultrafiltration separation: and (3) filtering the supernatant through a 0.45-micron filter membrane, and performing ultrafiltration through an ultrafiltration membrane with the molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system to obtain two active components, respectively sampling and determining the angiotensin converting enzyme inhibitory activity of the two active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A, and the specific method for separating and purifying by the rapid protein purification system comprises the following steps: loading gel into a chromatographic column by using a DEAE-Sepharose FF ion exchange column (the grain diameter of a filler is 10 mu m and is 10 multiplied by 300mm), balancing Tris-HCl (pH is 7.4), preparing ultrafiltrate into a solution of 200mg/mL, loading the solution with the amount of 3mL each time, and respectively carrying out stage elution by using NaCl solutions of Tris-HCl (pH is 7.4), 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L and 1mol/L at the elution speed of 2.2 mL/min; the detection wavelength is 280 nm; the peaks were collected.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain three active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the three active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: the specific method for separating and purifying the angiotensin converting enzyme inhibitory peptide of Pro-Trp-Gly-Met-Phe (PWGMF) by a high performance liquid chromatography system comprises the following steps: preparing the component B into a solution of 200mg/ml by using a Sephadex G-25 chromatographic column (size: 2.6X 80 cm; column material: Sephadex G-25), and loading: 2.0 mL; mobile phase: ultrapure water; eluent flow rate: 1.0 mL/min; detection wavelength: 280 nm; automatic collection speed: 4.0 min/tube; collecting each peak; the method for detecting the purity of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAXSB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using isocratic elution: 80% A, 20% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm.
The extraction and separation process is reasonable and strong in operability, sinonovacula constricta is used as a raw material, the enzymolysis liquid is separated and purified by using an ultrafiltration technology, a rapid protein purification system and a high performance liquid chromatography system after the sinonovacula constricta is subjected to enzymolysis by neutral protease to obtain a new angiotensin-converting enzyme inhibitory peptide (ACE inhibitory peptide, the purity of which is more than 99 percent), the N-end degradation sequencing is carried out by an amino acid sequencer to obtain pentapeptide, and the amino acid sequence of the pentapeptide is as follows: Pro-Trp-Gly-Met-Phe (PWGMF) with a relative molecular mass of 636.78Da, IC of PWGMF50Is 0.15mg/ml, has higher ACE inhibitory activity (the ACE inhibitory rate is more than 96.1 percent), and provides experimental basis for preparing high-activity ACE inhibitory peptide from sinonovacula constricta.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Sequence listing
<110> Zhejiang ocean academy
<120> preparation method of angiotensin converting enzyme inhibitory peptide of Sinonovacula constricta
<140>2015110111965
<141>2015-12-30
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>5
<212>PRT
<213> Sinonovacula (Sinonovacula)
<400>1
Pro Trp Gly Met Phe
1 5
Claims (1)
1. The angiotensin converting enzyme inhibitory peptide of the Sinonovacula constricta is characterized in that the amino acid sequence of the angiotensin converting enzyme inhibitory peptide of the Sinonovacula constricta is as follows: Pro-Trp-Gly-Met-Phe.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101845080A (en) * | 2010-01-08 | 2010-09-29 | 宁波大学 | Angiotensin converting enzyme inhibitory peptide and preparation method thereof |
| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN103242430A (en) * | 2013-05-31 | 2013-08-14 | 南京中医药大学 | Angiotensin-converting enzyme inhibitory peptide, and preparation method and application thereof |
| CN104829689A (en) * | 2015-04-30 | 2015-08-12 | 上海市奉贤区中心医院 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101845080A (en) * | 2010-01-08 | 2010-09-29 | 宁波大学 | Angiotensin converting enzyme inhibitory peptide and preparation method thereof |
| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN103242430A (en) * | 2013-05-31 | 2013-08-14 | 南京中医药大学 | Angiotensin-converting enzyme inhibitory peptide, and preparation method and application thereof |
| CN104829689A (en) * | 2015-04-30 | 2015-08-12 | 上海市奉贤区中心医院 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
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