CN104757561A - Application of mussel protein antihypertensive peptide - Google Patents
Application of mussel protein antihypertensive peptide Download PDFInfo
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- CN104757561A CN104757561A CN201510177317.7A CN201510177317A CN104757561A CN 104757561 A CN104757561 A CN 104757561A CN 201510177317 A CN201510177317 A CN 201510177317A CN 104757561 A CN104757561 A CN 104757561A
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Abstract
The invention discloses an application of a mussel protein antihypertensive peptide. The antihypertensive peptide has antihypertensive activity and is characterized in that an amino acid sequence of the antihypertensive peptide is Val-Ser-Trp-Pro-Cys-Arg-Trp(VSWPCRW); the molecular weight determined by ESI-MS (electrospray ionization mass spectrometry) is 935.07Da. The active peptide Val-Ser-Trp-Pro-Cys-Arg-Trp is obtained by shelling mussels, taking the meat of the mussels, carrying out enzymolysis on the meat, carrying out ultrafiltration on the enzymatic hydrolysate, adsorbing the enzymatic hydrolysate with an agarose magnetic microsphere immobilized ACE (angiotensin converting enzyme) and carrying out purification by RP-HPLC (reversed-phase high-performance liquid chromatography). The active peptide prepared by the invention has obvious ACE inhibitory activity and can be used in hypertension treatment related drugs.
Description
Technical field
The invention belongs to the application purpose of biotechnology, be specifically related to a kind of purposes of mussel protein Antihypertensive Peptides.
Background technology
ACE (ACE) is also called peptidyl-carboxypeptidase or kininaseⅡ, and molecular weight is 150,000 Da, belongs to endothelial cellular membrane desmoenzyme.The angiotensinⅠ of decapeptide can be hydrolyzed into the angiotensinⅡ of octapeptide by ACE, impels blood vessel to shrink further, causes blood pressure to raise.Meanwhile, ACE also promotes the secretion of aldosterone by acting on adrenal cortex.Therefore, ACE is the important component of Re-A-A (RAS).In addition, ACE catalysis can also have the bradykinin hydrolysis of hypotensive effect and makes it lose activity.Therefore, find the material of ACE activity in suitable suppression body, just can reach the object reducing or control blood pressure.
ACEI (ACEI) is that a class develops drug for hypertension rapidly, it is mainly through suppressing ACE active, cause angiotensinⅠ not to be converted to angiotensinⅡ, suppress ACE to the deactivation of bradykinin simultaneously, thus produce pressure reduction effect.Conventional ACEI (ACEI) mostly is chemicals, as captopril (Captopril), alacepril (Alacepril), lisinopril (Lisinopril) and fosinopril (Fosinopril) etc. clinically.But, there is more side effect in this type of chemical synthetic drug process of clinical application, as granulocyte minimizing, cough, fash, heating and parageusia etc., bring certain misery to patient.
At present, ACE inhibitory activity peptide (ACEP) is subject to extensive concern due to its significant physiologically active, compared with the ACEI of chemical synthesis, the advantage of ACEP is mainly reflected in: (1) adopts food grade protease hydrolytic to prepare, security is high, toxic and side effect is little, and multiple ACEP zoopery and clinical test results, do not observe obvious toxic-side effects; (2) edible protein raw material sources are wide, and enzyme-squash techniqued mild condition, is easy to industrialization; (3) ACEP antihypertensive effect is single-minded, and low being easier to of molecular weight is digested and assimilated.Therefore, research and develop out the active peptide food or medicine with hypotensive activity, become an important channel of aquatic products efficiency utilization.
In recent years, magnetic microsphere causes extensive attention as the research of fixed enzyme vector.And adopting " emulsification complex technique " to carry out modification to agarose, the Agarose Magnetic Microsphere of preparation has following remarkable advantage: (1) has magnetic, can be separated rapidly under magnetic fields; (2) good biocompatibility; (3) surface is containing a large amount of functional group, high adsorption capacity; (4) dispersed and good stability.Adopt Agarose Magnetic Microsphere do carrier, the magnetic microsphere immobilized ACE of preparation, in the solution can with ACEP specific adsorption, can be separated with enzymolysis liquid rapidly again under magnetic fields.But researcher finds not yet there is the research utilizing this technology to prepare Antihypertensive Peptides from mussel zymolyte at present.
Based on this, the present invention adopts zymolysis technique degraded mussel protein, and utilize ultrafiltration, magnetic microsphere immobilized ACE adsorbs and high-efficient liquid phase chromatogram technology prepares Antihypertensive Peptides, the higher value application for mussel provides a new approach.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of purposes of mussel active peptide in hypertension treatments include with ACE inhibitory activity.
The present invention for solving the problems of the technologies described above taked technical scheme is: a kind of purposes of mussel protein Antihypertensive Peptides: this mussel protein Antihypertensive Peptides Val-Ser-Trp-Pro-Cys-Arg-Trp(VSWPCRW) to ACE, there is significant inhibitory action, half inhibiting rate (IC
50) be 37.53 ± 1.09 μMs, can be applicable to treat the relevant medicine of hypertension or health food.
The fast preparation method of this mussel protein Antihypertensive Peptides is:
1) preparation of Agarose Magnetic Microsphere and activation:according to w/v 1 ~ 1.5g: 30 mL, agarose is added to the water, jointly boils and dissolve completely to agarose, add 1 ml Fe
3o
4magnetic fluid, vortex mixer fully mixes; Under agitation mixed liquor is added dropwise in the organic phase (Span-80: atoleine=1g:25mL) of 80 DEG C, and reacts 15 ~ 20 min at 80 DEG C; In 250 rpm rotating speed mechanical agitation 10 ~ 15 min, microballoon is shaped after organic phase is cooled to 40 DEG C, and is separated in magnetic field, obtained microballoon uses benzinum and washed with de-ionized water 3 times respectively, to remove remaining organic phase, obtains Agarose Magnetic Microsphere; By NaBH
4join in NaOH solution (0.8 ~ 1.0 mol/L) according to w/v 2 ~ 3mg: 1mL, then epoxychloropropane is joined in NaOH solution (0.8 ~ 1.0 mol/L) according to volume ratio 0.5 ~ 0.7: 1 again, finally according to w/v 2 ~ 3 g: 1 mL, Agarose Magnetic Microsphere is joined in NaOH solution (0.8 ~ 1.0 mol/L), at 40 DEG C, stir 3 ~ 5 h, obtain activated agarose magnetic microsphere.
) ACE fixing:according to mass ratio 1 ~ 1.5: 10, the Agarose Magnetic Microsphere of ACE (ACE) and activation is added in borate buffer solution (0.1 mol/L), after 40 ~ 50 DEG C of vibration 2 ~ 3 h, magnetic field put into by mixed liquor, Agarose Magnetic Microsphere is separated, and fully wash with distilled water, until washed out without zymoprotein, vacuum freeze drying obtains Agarose Magnetic Microsphere Immobilized ACE.
) enzymolysis of mussel protein and ultrafiltration:mussel is shelled, mussel meat mixes according to the w/v of 1g:3 ~ 5mL with glycine-NaOH buffer, homogenate, regulate pH value to 9.5 ~ 10, obtain mixed liquor; Mixed liquor temperature regulating to 45 ~ 55 DEG C, add alkali protease (2.0 × 10 according to 0.5 ~ 0.8% of mussel meat quality
5u/g), enzymolysis 4 ~ 6 h; Enzymolysis liquid is warming up to 90 ~ 95 DEG C, and after this temperature keeps 10 ~ 15min; Temperature regulating to 35 ~ 40 DEG C, adjust pH to 8.5 ~ 9.0 with 1 mol/LNaOH solution, add trypsase (1.9 × 10 according to 0.8 ~ 1.0% of mussel meat quality
4u/g), after enzymolysis 4 ~ 6 h, adopted by enzymolysis liquid 1 kDa milipore filter to carry out hyperfiltration treatment, collect molecular weight and be less than 1 kDa part, freeze-drying, be ultrafiltration zymolyte.
) preparation of mussel protein Antihypertensive Peptides:by ultrafiltration zymolyte successively through the absorption of Agarose Magnetic Microsphere Immobilized ACE and RPLC (RP-HPLC) purifying, obtain Antihypertensive Peptides.
As preferably, the mussel in described step 3) be Trachyostracous mussel (
mytilus coruscus).
As improvement, the Agarose Magnetic Microsphere Immobilized ACE absorption in described step 4) and RP-HPLC purifying are:
The absorption of Agarose Magnetic Microsphere Immobilized ACE: to borate buffer solution (0.1 mol/L, pH 7.8) to add NaCl to concentration be 0.2 mol/L, then adding ultrafiltration zymolyte to concentration is 5 ~ 8 mg/mL, finally add Agarose Magnetic Microsphere Immobilized ACE according to 30 ~ 50 times of zymolyte weight in solution, after adsorbing 45 min, magnetic field is utilized to be separated with solution by magnetic microsphere, with borate buffer solution cyclic washing microballoon to cleaning solution 220 nm and 280 nm without after UV absorption, by magnetic microsphere desorption in the NaCl solution of 1.0 mol/L, magnetic field is utilized to be separated with solution by magnetic microsphere, solution freeze-drying, obtain Antihypertensive Peptides mixture.
RP-HPLC purifying: Antihypertensive Peptides mixture is made into 45 ~ 55 μ g/mL solution, utilizes RP-HPLC to carry out purifying, according to the inhibit activities to ACE Antihypertensive Peptides Val-Ser-Trp-Pro-Cys-Arg-Trp(VSWPCRW).
Preferably, described RP-HPLC condition is: sample size is 19 ~ 21 μ L; Chromatographic column is Zorbax C18(250 × 4.6mm, 5 μm); Mobile phase is 40% acetonitrile; Elution speed is 1.0 mL/min; UV detect wavelength is 220 nm.
Enzymolysis mussel protein of the present invention prepares the method for Antihypertensive Peptides, and tool has the following advantages:
What the present invention adopted is a kind of biological enzyme, easily by the monitoring to enzymolysis process, ACEP is farthest discharged, and improves the utilization rate of raw material.Prepared ACEP is that mussel meat obtains through enzyme hydrolysis, and safely, have no side effect, ACE inhibitory activity is remarkable, has hypotensive effect to hyperpietic.
Product of the present invention can as medicine and health food etc., and craft science is reasonable, simple to operate, has stronger industrial implementation.Than existing preparation method, enzymolysis, ultrafiltration, the absorption of magnetic microsphere immobilized enzyme and RP-HPLC multiple means have been merged in the present invention simultaneously, and method is more perfect, and the Antihypertensive Peptides obtained has higher activity.
Accompanying drawing explanation
Fig. 1 is embodiment of the present invention Zorbax C18(250 × 4.6mm, 5 μm) chromatogram that obtains when carrying out chromatography of post.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment:
A fast preparation method for mussel protein Antihypertensive Peptides, preparation flow is as follows: absorption " RP-HPLC purifying " Antihypertensive Peptides of mussel meat " historrhexis " enzymolysis " ultrafiltration " Agarose Magnetic Microsphere Immobilized ACE.
) preparation of Agarose Magnetic Microsphere and activation:according to w/v 1.5g: 30 mL, agarose is added to the water, jointly boils and dissolve completely to agarose, add 1 ml Fe
3o
4magnetic fluid, vortex mixer fully mixes; Under agitation mixed liquor is added dropwise in the organic phase (Span-80: atoleine=1g:25mL) of 80 DEG C, and reacts 20 min at 80 DEG C; Organic phase is cooled to 40 DEG C makes microballoon be shaped in 250 rpm rotating speed mechanical agitation 15 min, and is separated in magnetic field, and obtained microballoon uses benzinum and washed with de-ionized water 3 times respectively, to remove remaining organic phase, obtains Agarose Magnetic Microsphere; By NaBH
4join in NaOH solution (0.8 mol/L) according to w/v 2mg: 1mL, then epoxychloropropane is joined in NaOH solution (0.8 mol/L) according to volume ratio 0.5: 1 again, finally according to w/v 3 g: 1 mL, Agarose Magnetic Microsphere is joined in NaOH solution (0.8 mol/L), at 40 DEG C, stir 3 h, obtain activated agarose magnetic microsphere.
) ACE fixing:according to mass ratio 1.5: 10, the Agarose Magnetic Microsphere of ACE (ACE) and activation is added in borate buffer solution (0.1 mol/L), after 50 DEG C of vibration 3 h, magnetic field put into by mixed liquor, be separated Agarose Magnetic Microsphere, and fully wash with distilled water, until washed out without zymoprotein, vacuum freeze drying obtains Agarose Magnetic Microsphere Immobilized ACE.
) enzymolysis of mussel protein and ultrafiltration:by Trachyostracous mussel (
mytilus coruscus) shell, mussel meat mixes according to the w/v of 1g:4 mL with glycine-NaOH buffer, homogenate, regulates pH value to 9. 5, obtains mixed liquor; Mixed liquor temperature regulating to 50 DEG C, then adds alkali protease (2.0 × 10 according to 0.8% of mussel meat quality
5u/g), enzymolysis 5 h; Enzymolysis liquid is warming up to 95 DEG C, and after this temperature keeps 10 min; Temperature regulating to 37 DEG C, adjusts pH to 8.6 by 1 mol/L NaOH solution, adds trypsase (1.9 × 10 according to 1.0% of mussel meat quality
4u/g), after enzymolysis 6 h, adopted by enzymolysis liquid 1 kDa milipore filter to carry out hyperfiltration treatment, collect molecular weight and be less than 1 kDa part, freeze-drying, be ultrafiltration zymolyte.
) preparation of mussel protein Antihypertensive Peptides:by ultrafiltration zymolyte successively through the absorption of Agarose Magnetic Microsphere Immobilized ACE and RPLC system (RP-HPLC) purifying, obtain Antihypertensive Peptides.
1. the absorption of Agarose Magnetic Microsphere Immobilized ACE: to borate buffer solution (0.1 mol/L, pH 7.8) to add NaCl to concentration be 0.2 mol/L, then adding ultrafiltration zymolyte to concentration is 6 mg/mL, finally add Agarose Magnetic Microsphere Immobilized ACE according to 40 times of zymolyte weight in solution, after adsorbing 45 min, magnetic field is utilized to be separated with solution by magnetic microsphere, with borate buffer solution cyclic washing microballoon to cleaning solution 220 nm and 280 nm without after UV absorption, then by magnetic microsphere desorption in the NaCl solution of 1.0 mol/L, magnetic field is utilized to be separated with solution by magnetic microsphere, solution freeze-drying, obtain Antihypertensive Peptides mixture.
2. RP-HPLC purifying: Antihypertensive Peptides mixture is made into 45 μ g/mL solution, utilizes RP-HPLC(condition: sample size is 20 μ L; Chromatographic column is Zorbax C18(250 × 4.6mm, 5 μm); Mobile phase is 40% acetonitrile; Elution speed is 1.0 mL/min; UV detect wavelength is 220 nm) carry out purifying, according to the inhibit activities of ACE being obtained to a high activity Antihypertensive Peptides (see figure 1).
3. structure detection: collecting the highest active Antihypertensive Peptides is simple spike after testing, utilizing protein/polypeptide sequenator to measure amino acid sequence is Val-Ser-Trp-Pro-Cys-Arg-Trp(VSWPCRW), ESI/MS detection molecules amount is 935.07 Da.
By above-mentioned obtained mussel protein Antihypertensive Peptides Val-Ser-Trp-Pro-Cys-Arg-Trp(VSWPCRW) carry out ACE inhibitory activity experiment.Experimental result shows: the half inhibiting rate (IC of this polypeptide
50) be 37.53 ± 1.09 μMs.Thus, this polypeptide can be used for preparing relevant cardiovascular health, hypotensive, treat hypertensive health food or Medicines.
Finally, it is also to be noted that what enumerate above is only a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
SEQUENCE LISTING
<110> Oceanography Institute Of Zhejiang
The purposes of a <120> mussel protein Antihypertensive Peptides
<130> zjou-wb-201504-4
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7
<212> PRT
<213> Prof. Du Yucang
<400> 1
Val Ser Trp Pro Cys Arg Trp
1 5
Claims (2)
1. a purposes for mussel protein Antihypertensive Peptides, is characterized in that this Antihypertensive Peptides is for the preparation of care food for reducing blood pressure, prevention or treat hypertensive health food, and the purposes in cardiovascular health food.
2. the purposes of a kind of mussel protein Antihypertensive Peptides according to claim 1, is characterized in that the amino acid sequence of described Antihypertensive Peptides be Val-Ser-Trp-Pro-Cys-Arg-Trp, ESI-MS determining molecular weight is 935.07 Da.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510177317.7A CN104757561A (en) | 2015-04-15 | 2015-04-15 | Application of mussel protein antihypertensive peptide |
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| CN201510177317.7A CN104757561A (en) | 2015-04-15 | 2015-04-15 | Application of mussel protein antihypertensive peptide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106852501A (en) * | 2016-10-26 | 2017-06-16 | 浙江海洋大学 | A kind of purposes of clam extract |
| CN112501230A (en) * | 2020-12-17 | 2021-03-16 | 浙江海洋大学 | Preparation method and application of urechis unicinctus ACE inhibitory peptide |
| CN114044807A (en) * | 2021-11-19 | 2022-02-15 | 浙江海洋大学 | Mussel blood fat reducing oligopeptide for treating hyperlipidemia |
-
2015
- 2015-04-15 CN CN201510177317.7A patent/CN104757561A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 毋瑾超 等: "贻贝蛋白酶解降血压肽的降压活性及相对分子质量与氨基酸组成", 《水产学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106852501A (en) * | 2016-10-26 | 2017-06-16 | 浙江海洋大学 | A kind of purposes of clam extract |
| CN112501230A (en) * | 2020-12-17 | 2021-03-16 | 浙江海洋大学 | Preparation method and application of urechis unicinctus ACE inhibitory peptide |
| CN114044807A (en) * | 2021-11-19 | 2022-02-15 | 浙江海洋大学 | Mussel blood fat reducing oligopeptide for treating hyperlipidemia |
| CN114044807B (en) * | 2021-11-19 | 2023-08-22 | 浙江海洋大学 | Mussel hypolipidemic oligopeptide for treating hyperlipidemia |
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