CN107082807A - Suppress the Yak Bone Protein peptide and preparation method and application of function with ACE - Google Patents
Suppress the Yak Bone Protein peptide and preparation method and application of function with ACE Download PDFInfo
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- CN107082807A CN107082807A CN201710345158.6A CN201710345158A CN107082807A CN 107082807 A CN107082807 A CN 107082807A CN 201710345158 A CN201710345158 A CN 201710345158A CN 107082807 A CN107082807 A CN 107082807A
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- yak bone
- bone protein
- yak
- protein peptide
- protease
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
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- 241000276707 Tilapia Species 0.000 description 3
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 3
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- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 1
- 239000005541 ACE inhibitor Substances 0.000 description 1
- 102000005862 Angiotensin II Human genes 0.000 description 1
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- MVORZMQFXBLMHM-QWRGUYRKSA-N Gly-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 MVORZMQFXBLMHM-QWRGUYRKSA-N 0.000 description 1
- UCDHVOALNXENLC-KBPBESRZSA-N Leu-Gly-Tyr Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 UCDHVOALNXENLC-KBPBESRZSA-N 0.000 description 1
- QMKFDEUJGYNFMC-AVGNSLFASA-N Leu-Pro-Arg Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QMKFDEUJGYNFMC-AVGNSLFASA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000276701 Oreochromis mossambicus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
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- 150000007516 brønsted-lowry acids Chemical class 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kind of Yak Bone Protein peptide and preparation method and application for suppressing function with ACE.The Yak Bone Protein peptide is that Yak Bone Protein is made by the zymolyte of compound protease stepwise discretization；The compound protease is alkali protease, papain, trypsase and flavor protease.There is Yak Bone Protein peptide of the present invention excellent ACE to suppress function, and ACE inhibitory activity (IC50 values) is less than 0.32mg/mL, can be applied to special procure in food and nutraceutical as Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe.The Yak Bone Protein peptide preparation method of the present invention is simple, does not have addition acid or alkali to carry out pH adjustment in whole process, and product keeps preferable functional characteristic, is easier to realize industrialization production.
Description
Technical field
The present invention relates to animal foodstuff manufacture field, more particularly, to a kind of Yak Bone egg for suppressing function with ACE
White peptide and preparation method and application.
Background technology
Hypertension is that, to a kind of great disease of human health risk, incubation period is long.The early stage of hypertension, does not have typically
Have significant discomfort disease, until occur clinical indication heart attack, rupture of blood vessel in brain and cause death.
Angiotensin converting enzyme (Angiotensin I converting enzyme, ACE) is renin-angiotensin
The key enzyme of prime system system.Proangiotensin is converted into angiotensin I in the presence of feritin, and angiotensin I is ACE's
Angiotensin II is converted under effect, vessel retraction is stimulated, causes blood pressure to rise.Suppress ACE activity has to reducing blood pressure
Positive effect, thus the effective Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe of exploitation causes the very big concern of people.Although food-based ACE suppressions
Peptide processed is weaker than artificial synthesized Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe effect, but it has the advantages that its is unique, will not produce the problem of decompression is excessive,
It is safe, have no side effect, in addition to buck functionality, often there is the functions such as Immune enhancement, absorption easy to digest simultaneously.Due to medicine
The exploitation that therapy has the blood pressure-lowering functional factor in side effect, natural food will turn into hypertension non-drug therapy from now on
Pith.
There are the method for preparing ace inhibitory peptide using the stock of fish or clam class, such as Chinese patent in the prior art
CN106399435A discloses a kind of method that utilization fish scale prepares ace inhibitory peptide, and this method includes fish scale washing decontamination
Afterwards, soaked 1-2 days with NaCl solution, removing foreign protein and fish silver powder use HCl solution, solid-liquid ratio 1 again after fish scale is pulled out:10-
1:20,1-2h is soaked, to remove the inorganic salts such as calcium salt；Fish scale is swelled with distilled water immersion, then digested with alkali protease,
It is then centrifuged for, supernatant is enzymolysis liquid, enzymolysis liquid freeze-drying is obtained into ace inhibitory peptide；Resulting product ACE inhibiting rates compared with
Height, compared with the ace inhibitory peptide of chemical synthesis, the ace inhibitory peptide safety non-toxic of preparation, be easy to digestion；Such as Chinese patent
CN104593466A discloses a kind of method that utilization endogenous enzymes prepare blue clam albumen source ACE inhibitor peptides, and this method utilizes blue clam
Internal endogenous protease is digested, and is obtained by self-dissolving in natural A CE peptide for inhibiting, preparation process and is added without any external source
Protease, whole preparation process include new vivid blue clam pretreatment, homogenate, endogenous enzyme activition, enzyme strive, it is the enzyme that goes out, deodorant, centrifugation, cold
It is lyophilized dry；Also have been reported that and use tilapia fishskin and vegetable protein to produce ace inhibitory peptide, such as Chinese patent for raw material
CN101240312A discloses a kind of preparation method for the ace inhibitory peptide for coming from fish-skin, and this method realizes Tilapia mossambica discarded object
Comprehensive utilization, specifically using tilapia fishskin as raw material, the fish-skin albumen in water extraction tilapia fishskin enters water-filling using protease
Solution, fish-skin protein enzymatic hydrolyzate (TSP-1) is obtained through the process such as the enzyme that goes out, de- bitter, decolouring, de- taste, filtering, ion exchange and concentration,
After TSP-1 hyperfiltration treatments, then through gel layer collecting high-activity component, the ace inhibitory peptide product of high activity is made in freeze-drying；
Chinese patent CN104131055A discloses a kind of preparation method of phycoerythrin ace inhibitory peptide, and this method includes phycoerythrin
Extraction step, add pepsin enzymolysis adds trypsin digestion；Freeze-dried powder after enzymolysis is dissolved in pure water, is splined on
SephadexG-15 gel columns, it is phycoerythrin ACE constituents for suppressing to collect active top.The component is continued to be splined on height
Effect liquid phase chromatogram ZORBAX300SB-C18 posts are separated, to detect that in the component prepared by phycoerythrin ace inhibitory peptide, the hair
Bright method can avoid active peptide from being degraded and inactivate by pipe intestinal digesting liquid after being taken in through human body, while reducing cost.
But pH generally is adjusted using by adding acid, alkali in these preparation methods, the quality of product is influenceed, while to albumen
The main composition of peptide is indefinite.China's Yak Bone aboundresources, has no in patent utilize Yak Bone Protein resource at present both at home and abroad
Prepare the patent literature of inhibiting peptide of tonin.
The content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of there is ACE to suppress function
Yak Bone Protein peptide and its preparation method and application.
In order to realize the purpose of the present invention, one aspect of the present invention provides a kind of Yak Bone egg for suppressing function with ACE
White peptide, the Yak Bone Protein peptide is that Yak Bone Protein is made by the zymolyte of compound protease stepwise discretization；Wherein, it is combined egg
White enzyme is alkali protease, papain, trypsase and flavor protease.
Yak Bone Protein is distributed using specific compound protease of the invention and digested, there is obtained peptide ACE to suppress work
Property, reasonably and efficiently utilizes Yak Bone Protein.
In order to obtain purer Yak Bone Protein peptide, following step is preferably included using the distribution enzymolysis of above-mentioned compound protease
Suddenly：
Adjust Yak Bone Protein liquid in protein content be 8-10wt%, according to protein by weight 0.4 in Yak Bone Protein liquid~
1.0wt% percentage by weight adds compound protease and carries out stepwise discretization.
In order to improve the inhibitory activity of obtained yak protein peptides, in a preferred embodiment of the invention, using upper
State compound protease distribution enzymolysis and specifically include following steps：
First step enzyme digestion reaction：0.2~0.5wt% first is added into protein by weight 8-10wt% Yak Bone Protein liquid
Compound protease, carries out 0.5~1.5h of enzyme digestion reaction under the conditions of 50~60 DEG C, wherein, the first compound protease is alkaline egg
White enzyme and papain；
Second step enzyme digestion reaction：The compound proteases of 0.2~0.5wt% second are added into first step enzyme digestion reaction product,
1.0~2.0h of enzyme digestion reaction is carried out under the conditions of 45~55 DEG C, wherein, the second compound protease is trypsase and local flavor albumen
Enzyme.
In a preferred embodiment of the invention, the matter of the first compound protease neutral and alkali protease and papain
Amount is than being 1~2:1.
In a preferred embodiment of the invention, the quality of trypsase and flavor protease in the second compound protease
Than for 1:1~2.
In order to further improve the inhibitory activity of obtained yak protein peptides, in a preferred embodiment of the invention,
The preparation method of Yak Bone Protein peptide comprises the following steps：
1) preparation of Yak Bone Protein liquid；
2) by step 1) made from Yak Bone Protein liquid pass through compound protease stepwise discretization；
3) by step 2) enzymolysis after product removal of impurities, UF membrane, gel separation and reversed-phase HPLC separation, concentrate, freezing
Produced after drying.
Without any bronsted lowry acids and bases bronsted lowry in the preparation method.
In a preferred embodiment of the invention, step 1) in also include obtained Yak Bone Protein liquid in 75-85
At DEG C, handled 15-25 minutes for 80-100kH ultrasonic echography through frequency.Under this condition, yak can effectively be changed
The institutional framework of bone collagen, can using the Yak Bone Protein liquid of specific frequency ultrasonic wave technical finesse at such a temperature
To be obviously improved sensitiveness of the Yak Bone Protein to enzyme, the usage amount of enzyme is effectively reduced.
In step 3) in, impurity-removing method general in this area can be used, for the active preferably polypeptide that is inhibited,
In a preferred embodiment of the invention, step 3) in removal of impurities be specially：
10~20 minutes are incubated at 90~95 DEG C, room temperature is cooled to, enzymolysis liquid weight 0.2-0.6wt% activity is added
Charcoal is separated in enzymolysis liquid by diatomite, collects separating liquid.
In the preferred embodiment, by using activated carbon removal of impurities so that protein peptides have good local flavor and color and luster,
It can be widely used in special procuring in food, medicine and nutraceutical.
In order that obtained Yak Bone Protein liquid inhibition more preferably, step 3) in UF membrane can be specially：Step
3) UF membrane is specifically as follows in：
Product after removal of impurities is handled by two step hyperfiltration process, successively using aperture for 5000 dalton film and
Aperture carries out ultrafiltration for the film of 3000 dalton.
It is further preferred that the step of UF membrane is：
Obtained separating liquid is handled by two step hyperfiltration process, surpassed using aperture for the ceramic membrane of 5000 dalton
Filter, first comes out albumen and peptide separation that molecular weight is less than 5000 dalton, then will be divided for the film of 3000 dalton with aperture
Son amount is separated less than the protein peptides of 3000 dalton.
In a preferred embodiment of the invention, step 3) in gel separation and reversed-phase HPLC can be：
Product after UF membrane is separated by Sephadex G-25 gels, eluent is deionized water, and eluting peak exists
Detected under 280nm, collect the 2nd eluting peak；By concentration, freeze-drying, Yak Bone Protein peptide crude product is obtained；Use again
RP-HPLC RPLCs carry out 1 separation, collect by 16-18 in the separation of RP-HPLC RPLCs
Peptide eluent in point.
Resulting peptide eluent is concentrated, freeze-drying, you can obtain most preferably Yak Bone Protein peptide of the invention.
The ACE inhibitory activity (IC50 values) of the Yak Bone Protein peptide of the present invention is less than 0.32g/mL, when the concentration of protein peptides
During for 1.0mg/mL, ACE inhibiting rates are up to more than 80%, preferably more than 90%.
The present invention extracts the ratio of obtained peptide of the Yak Bone Protein peptide middle-molecular-weihydroxyethyl less than 1000 from Yak Bone Protein
For more than 90%.
In a preferred embodiment of the invention, the Yak Bone polypeptide prepared using the above method includes having such as
The polypeptide of lower amino acid sequence：
Glu-Ile-Arg-Met-Leu (GIAML, as shown in SEQ ID NO.1).
In a preferred embodiment of the invention, the Yak Bone polypeptide prepared using the above method includes having such as
The polypeptide of lower amino acid sequence：
Thr-Val-Ser-Leu-Pro-Arg (TVSLPA, as shown in SEQ ID NO.2)
In a preferred embodiment of the invention, the Yak Bone polypeptide prepared using the above method includes having such as
The polypeptide of lower amino acid sequence：Ser-Pro-Ile-Leu-Gly-Tyr-Trp (SPILGTT, as shown in SEQ ID NO.3).
Preferable ACE is respectively provided with containing Yak Bone Protein peptide any in above-mentioned 3 kinds of polypeptides and suppresses function, and ACE suppresses
Active (IC50 values) is less than 0.32g/mL.
In a preferred embodiment of the invention, active component includes above-mentioned 3 kinds of polypeptides in Yak Bone Protein peptide, enters one
Preferably, active component is the combination of above-mentioned 3 kinds of polypeptides to step, it is further preferred that the content of the combination of above-mentioned 3 kinds of peptides is 50%
More than.
The Yak Bone Protein peptide of the present invention can be powdered.
In order that whole process it is simpler and without addition acid or alkali carry out pH adjustment, step 1) in yak
Bone protein liquid is, using Yak Bone as raw material, to be obtained through thermophilic digestion and degreasing.
I.e. present invention also offers the preparation method of above-mentioned Yak Bone Protein peptide, comprise the following steps：
1) using Yak Bone as raw material, Yak Bone Protein liquid is obtained through thermophilic digestion and degreasing；
2) by step 1) made from Yak Bone Protein liquid pass through compound protease stepwise discretization；
3) by step 2) enzymolysis after product removal of impurities, UF membrane, gel separation and reversed-phase HPLC separation, concentrate, freezing
Produced after drying.
Wherein, step 2) and step 3) further preferred scheme with reference to the above.
In a preferred embodiment of the invention, step 1) in the preparation of Yak Bone Protein liquid be specifically as follows：
From fresh Yak Bone is freezed, cleaned with the clean water for meeting sanitary standard for drinking water, by yak after Yak Bone cleaning
Bone breaks into skeletal grain, and the water for adding 1.0~3.0 times of yak osseous granules total amount handles 2-5 hours under the conditions of 125-135 DEG C, then
Obtain boning the Yak Bone thermophilic digestion liquid of slag by centrifugal filtration, is cooled to 15-30 DEG C, by a point flow container degreasing, obtains yak
Ox bone protein liquid.
In order to keep preferable functional characteristic, it is easier to realize industrialization production, in a preferred embodiment of the invention,
The preparation method of Yak Bone Protein peptide is specifically as follows：
1) from fresh Yak Bone is freezed, cleaned with the clean water for meeting sanitary standard for drinking water, ox is used after Yak Bone cleaning
Bone is crushed breaks into skeletal grain by Yak Bone, and the water for adding 1.0~3.0 times of yak osseous granules total amount is handled under the conditions of 125-135 DEG C
2-5 hours, the Yak Bone thermophilic digestion liquid for the slag that then obtains boning by centrifugal filtration was cooled to 15-30 DEG C, passes through a point flow container
Degreasing, obtains Yak Bone Protein liquid；
By the Yak Bone Protein liquid 1) obtained, temperature is adjusted to 75-85 DEG C, using supersonic generator through ultrasonic wave (frequency
Rate is 80-100kH) handle 15-25 minutes, change the institutional framework of Yak Bone collagen.
2) protein content is 8-10wt% in regulation Yak Bone Protein liquid, according to protein by weight 0.4 in Yak Bone Protein liquid
~1.0wt% percentage by weight adds compound protease and carries out stepwise discretization：
The first step is constituted using 0.2~0.5wt% compound proteases one (alkali protease and papain), they it
Between mass ratio be 1~2：1) 0.5~1.5h of enzyme digestion reaction, is carried out under the conditions of 50~60 DEG C；
Then carry out second step using 0.2~0.5% compound protease two (trypsase and flavor protease composition, it
Between mass ratio be 1：1~2), 1.0~2.0h of enzyme digestion reaction is carried out under the conditions of 45~55 DEG C；
3) removal of impurities：10~20 minutes are incubated at 90~95 DEG C, room temperature is cooled to, enzymolysis liquid weight 0.2- is added
0.6wt% activated carbon is separated in enzymolysis liquid by diatomite, collects separating liquid；
UF membrane：Separating liquid obtained by step (3) is handled by two step hyperfiltration process, is 5000 roads using aperture
You ceramic membrane ultrafitration, first by molecular weight be less than 5000 dalton albumen and peptide separation come out, then with aperture be 3000
The film of dalton separates the protein peptides that molecular weight is less than 3000 dalton.
Gel is separated and reversed-phase HPLC separation：It is the protein peptides liquid less than 3000 to take molecular weight, then by Sephadex
G-25 gels are separated, and eluent is deionized water, and eluting peak is detected under 280nm, collect the 2nd eluting peak；By dense
Contracting, freeze-drying, obtain Yak Bone Protein peptide；1 separation is carried out with RP-HPLC RPLCs again, collects and passes through
Peptide eluent in the separation of RP-HPLC RPLCs in 16-18 points；
By obtained peptide solution by concentration, freeze-drying, Yak Bone Protein Gly-His-Lys are obtained.
The ratio for obtaining peptide of the Yak Bone Protein peptide molecular weight less than 1000 by above-mentioned preparation method is more than 90%.
Prevention is being prepared present invention also offers above-mentioned Yak Bone Protein peptide and the above-mentioned method for preparing ox bone protein peptides
Or the application in the medicine or food of the disease of ACE suppression is benefited from treatment.
The yak protein peptides of the present invention can be as medicine, or dietary supplements, or is added to as foodstuff base material general
In logical food such as beverage, dairy products, medicine of the invention or food can be used for treatment hypertension etc. to be controlled with Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe
The other diseases for the treatment of.Those skilled in the art can according to the actual conditions of higher or lower IC50 value combined treatment objects,
Carry out the dosage that Reasonable adjustment is used.
There is the Yak Bone Protein peptide that the present invention is extracted from Yak Bone Protein excellent ACE to suppress function, and ACE suppresses to live
Property (IC50 values) be less than 0.32mg/mL；The Yak Bone Protein peptide preparation method that the present invention is developed is simple, does not have in whole process
There are addition acid or alkali to carry out pH adjustment, product keeps preferable functional characteristic, is easier to realize industrialization production；Present invention exploitation
Product safety, the present invention is using numerous food level specific compound protease (alkali protease, papain, tryptose
Enzyme and flavor protease), through in a mild condition, the Yak Bone Protein of the peptide composition of specified molecular weight being obtained by appropriateness enzymolysis
Peptide, is not added with any additive, is 100% Yak Bone Protein peptide.
Embodiment
With reference to embodiment, the embodiment to the present invention is described in further detail.Following examples are used for
Illustrate the present invention, but be not limited to the scope of the present invention.
Unless otherwise specified, the routine techniques hand that technological means used in embodiment is well known to those skilled in the art
Section.Unless otherwise specified, reagent used in embodiment is commercially available.
Embodiment 1 has the preparation method of the Yak Bone Protein peptide of ACE suppression functions
(1) from fresh 100 grams of Yak Bone is freezed, cleaned with the clean water for meeting sanitary standard for drinking water, Yak Bone cleaning
Yak Bone is broken into skeletal grain with ox bone pulverizer afterwards, adding the water of 2 times of yak osseous granules total amount, that 4 are handled under the conditions of 125 DEG C is small
When, the Yak Bone cooking liquor for the slag that obtains boning by centrifugal filtration is cooled to 20 DEG C, by a point flow container degreasing, obtains Yak Bone
Protein liquid.
(2) the Yak Bone Protein liquid for obtaining (1), temperature is adjusted to 80 DEG C, using supersonic generator through ultrasonic wave (frequency
Rate is 85kH) handle 15 minutes.
(3) protein content is 8% in regulation Yak Bone Protein liquid, according to protein by weight 0.8% in Yak Bone Protein liquid
Percentage by weight adds compound protease and carries out stepwise discretization, the first step using 0.4% compound protease one (alkali protease and
The mass ratio of papain is 1：1) enzyme digestion reaction 1.5h, is carried out under the conditions of 55 DEG C；Then carry out second step and utilize 0.4%
(mass ratio of trypsase and flavor protease is 1 to compound protease two：2) enzyme digestion reaction 1.0h, is carried out under the conditions of 50 DEG C；
10 minutes are incubated at 95 DEG C, room temperature is cooled to, the activated carbon of enzymolysis liquid weight 0.2% is added in enzymolysis liquid, passes through diatom
Soil is separated, and collects separating liquid；
(4) it is first that molecular weight is small by the separating liquid obtained by step (3) using the ceramic membrane ultrafitration that aperture is 5000 dalton
Albumen and peptide separation in 5000 come out, then the egg with the film that aperture is 3000 dalton by molecular weight less than 3000 dalton
White peptide is separated.
(5) it is the protein peptides liquid less than 3000 to take molecular weight, then by the separation of Sephadex G-25 gels, eluent is
Deionized water, eluting peak is detected under 280nm, collects the 2nd eluting peak；By concentration, freeze-drying, obtain by solidifying
The Yak Bone Protein peptide of glue separation；1 separation is carried out with RP-HPLC RPLCs again, the 16-18 points of peptides collected are taken
Solution；
(6) peptide solution for obtaining step (5) obtains suppressing with ACE the ossein of function by concentration, freeze-drying
Protein peptide powder.The main component of bone collagen peptide is measured by LC-MS/MS, its amino acid sequence is Glu-Ile-
The content of Arg-Met-Leu, Thr-Val-Ser-Leu-Pro-Arg, Ser-Pro-Ile-Leu-Gly-Tyr-Trp peptide is
50.9%.
Embodiment 2 has the preparation method of the Yak Bone Protein peptide of ACE suppression functions
(1) from fresh 500 grams of Yak Bone is freezed, cleaned with the clean water for meeting sanitary standard for drinking water, Yak Bone cleaning
Yak Bone is broken into skeletal grain with ox bone pulverizer afterwards, the water for adding 1.5 times of yak osseous granules total amount handles 3 under the conditions of 130 DEG C
Hour, the Yak Bone cooking liquor for the slag that obtains boning by centrifugal filtration is cooled to 25 DEG C, by a point flow container degreasing, obtains yak
Bone protein liquid.
(2) the Yak Bone Protein liquid for obtaining (1), temperature is adjusted to 82 DEG C, using supersonic generator through ultrasonic wave (frequency
Rate is 90kH) handle 20 minutes.
(3) protein content is 9% in regulation Yak Bone Protein liquid, according to protein by weight 1.0% in Yak Bone Protein liquid
Percentage by weight adds compound protease and carries out stepwise discretization, the first step using 0.4% compound protease one (alkali protease and
The mass ratio of papain is 1：1) enzyme digestion reaction 1.5h, is carried out under the conditions of 50 DEG C；Then carry out second step and utilize 0.6%
(mass ratio of trypsase and flavor protease is 1 to compound protease two：1) enzyme digestion reaction 1.0h, is carried out under the conditions of 50 DEG C；
10 minutes are incubated at 95 DEG C, room temperature is cooled to, the activated carbon of enzymolysis liquid weight 0.4% is added in enzymolysis liquid, passes through diatom
Soil is separated, and collects separating liquid；
(4) it is first that molecular weight is small by the separating liquid obtained by step (3) using the ceramic membrane ultrafitration that aperture is 5000 dalton
Albumen and peptide separation in 5000 come out, then the egg with the film that aperture is 3000 dalton by molecular weight less than 3000 dalton
White peptide is separated.
(5) it is the protein peptides liquid less than 3000 to take molecular weight, then by the separation of Sephadex G-25 gels, eluent is
Deionized water, eluting peak is detected under 280nm, collects the 2nd eluting peak；By concentration, freeze-drying, obtain by solidifying
The Yak Bone Protein peptide of glue separation；1 separation is carried out with RP-HPLC RPLCs again, the 16-18 points of peptides collected are taken
Solution；
(6) peptide solution for obtaining step (5) obtains suppressing with ACE the ossein of function by concentration, freeze-drying
Protein peptide powder.The main component of bone collagen peptide is measured by LC-MS/MS, its amino acid sequence is Glu-Ile-
The content of Arg-Met-Leu, Thr-Val-Ser-Leu-Pro-Arg, Ser-Pro-Ile-Leu-Gly-Tyr-Trp peptide is
51.8%.
Embodiment 3 has the preparation method of the Yak Bone Protein peptide of ACE suppression functions
(1) from fresh 1000 grams of Yak Bone is freezed, cleaned with the clean water for meeting sanitary standard for drinking water, Yak Bone cleaning
Yak Bone is broken into skeletal grain with ox bone pulverizer afterwards, the water for adding 3.0 times of yak osseous granules total amount handles 3 under the conditions of 132 DEG C
Hour, the Yak Bone thermophilic digestion liquid for the slag that then obtains boning by centrifugal filtration is cooled to 25 DEG C, by a point flow container degreasing,
Obtain Yak Bone Protein liquid.
(2) the Yak Bone Protein liquid for obtaining (1), temperature is adjusted to 85 DEG C, using supersonic generator through ultrasonic wave (frequency
Rate is 90kH) handle 25 minutes.
(3) protein content is 9% in regulation Yak Bone Protein liquid, according to protein by weight 0.6% in Yak Bone Protein liquid
Percentage by weight adds compound protease and carries out stepwise discretization, the first step using 0.3% compound protease one (alkali protease and
Papain is constituted, and the mass ratio between them is 2：1) enzyme digestion reaction 1.0h, is carried out under the conditions of 60 DEG C；Then is carried out
Using 0.3% compound protease two, (trypsase and flavor protease composition, the mass ratio between them is 1 to two steps：1), exist
Enzyme digestion reaction 2.0h is carried out under the conditions of 50 DEG C；20 minutes are incubated at 90 DEG C, room temperature is cooled to, enzymolysis liquid weight 0.6% is added
Activated carbon in enzymolysis liquid, separated by diatomite, collect separating liquid；
(4) separating liquid obtained by step (3) is handled by two step hyperfiltration process, is 5000 dalton using aperture
Ceramic membrane ultrafitration, first by molecular weight be less than 5000 albumen and peptide separation come out, then with aperture for 3000 dalton film
The protein peptides that molecular weight is less than 3000 dalton are separated.
(5) it is the protein peptides liquid less than 3000 to take molecular weight, then by the separation of Sephadex G-25 gels, eluent is
Deionized water, eluting peak is detected under 280nm, collects the 2nd eluting peak；By concentration, freeze-drying, obtain by solidifying
The Yak Bone Protein peptide of glue separation；1 separation is carried out with RP-HPLC RPLCs again, the 16-18 points of peptides collected are taken
Solution；
(6) peptide solution for obtaining step (5) obtains suppressing with ACE the ossein of function by concentration, freeze-drying
Protein peptide powder.The main component of bone collagen peptide is measured by LC-MS/MS, its amino acid sequence is Glu-Ile-
The content of Arg-Met-Leu, Thr-Val-Ser-Leu-Pro-Arg, Ser-Pro-Ile-Leu-Gly-Tyr-Trp peptide is
52.6%.
The determination test of embodiment 4 Yak Bone Protein peptide angiotonin invertase (ACE) rejection ability
Test specimen：Yak Bone Protein peptide and sample 4 prepared by embodiment 1, embodiment 2, embodiment 3 is embodiment 1
Yak Bone Protein liquid passes through the Yak Bone Protein powder that freeze-drying reaches.ACE rejection abilities are carried out as follows：
The method of ACE inhibiting rates.The Hip amounts discharged can be quantitatively detected under 228nm with high performance liquid chromatography, so that
Calculate the ACE inhibiting rates of polypeptide.
1st, the configuration of reagent
PH8.3 phosphate buffer solution：Prepared with ultra-pure water, wherein phosphate-containing 50mmol/L, NaCl 300mmol/
L, adjusts pH to 8.3；
ACE enzyme liquids：In the ACE that 2mL phosphate buffer is added to 1U, its concentration is set to be changed into 0.5U/mL.
HHL solution：HHL is dissolved using phosphate buffer, makes its final concentration of 5mmol/L.
Sample solution：Appropriate amount of sample is weighed, with the solution of concentration needed for phosphate buffer.
2nd, the chromatographic condition that ACE inhibiting rates are determined
Detection wavelength：228nm；Flow velocity:1mL/min；Mobile phase A:Ultra-pure water (contains 0.1% trifluoroacetic acid), Mobile phase B:
Methanol (contains 0.1% trifluoroacetic acid)；Sample size:10 μ L, hand sampling；
3rd, the method for determining ACE inhibitory activity
120 μ L HHL substrate solutions are taken, the sample for adding 20 μ L is well mixed, and 10min is incubated in 37 DEG C of waters bath with thermostatic control.
Then 10 μ L ACE enzyme liquids are added and react 30min in 37 DEG C of waters bath with thermostatic control, the HCl for adding 150 μ L 1mol/L is terminated instead
Should, obtain reaction solution.The reaction solution is analyzed using HPLC, while setting blank control group.ACE inhibitory activity calculation formula
It is as follows：
ACE inhibitory activity %=(M-N)/M × 100,
In formula, M is the peak area of hippuric acid in control group, and N is the peak area of hippuric acid in the sample sets added.
4th, the measure of 503nhibiting concentration
Its inhibitory activity is determined according to ace inhibitory peptide external detection method, using concentration as abscissa, ACE inhibiting rates are vertical
Coordinate plots round and smooth curve, and IC50 values are calculated from curve.It the results are shown in Table 1.
5th, the measure of Yak Bone Protein peptide middle-molecular-weihydroxyethyl distribution：Determined with reference to GB/T22729-2008 methods.
As shown in Table 1, the Yak Bone Protein peptide that prepared by the present invention has good ACE inhibitory activity, and its IC50 value is less than
0.32mg/mL, Yak Bone Protein peptide middle-molecular-weihydroxyethyl reaches more than 90% less than 1000.
The ACE inhibitory activity experimental result of the Yak Bone Protein peptide of table 1
Finally, method of the invention is only preferably embodiment, is not intended to limit the scope of the present invention.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Sequence table
<110>Anhui Guo Tai bio tech ltd
<120>Suppress the Yak Bone Protein peptide and preparation method and application of function with ACE
<130> KHP171112765.1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213>Yak Bone
<400> 1
Glu Ile Arg Met Leu
1 5
<210> 2
<211> 6
<212> PRT
<213>Yak Bone
<400> 2
Thr Val Ser Leu Pro Arg
1 5
<210> 3
<211> 7
<212> PRT
<213>Yak Bone
<400> 3
Ser Pro Ile Leu Gly Tyr Trp
1 5
Claims (10)
1. a kind of Yak Bone Protein peptide for suppressing function with ACE, it is characterised in that the Yak Bone Protein peptide is Yak Bone egg
The white zymolyte by compound protease stepwise discretization is made；The compound protease is alkali protease, papain, pancreas
Protease and flavor protease.
2. Yak Bone Protein peptide according to claim 1, it is characterised in that the preparation method bag of the Yak Bone Protein peptide
Include following steps：
1) preparation of Yak Bone Protein liquid；
2) by step 1) made from Yak Bone Protein liquid pass through compound protease stepwise discretization；
3) by step 2) enzymolysis after product removal of impurities, UF membrane, gel separation and reversed-phase HPLC separation, concentrate, freeze-drying
After produce.
3. Yak Bone Protein peptide according to claim 1 or 2, it is characterised in that the compound protease stepwise discretization bag
Include following steps：
First step enzyme digestion reaction：0.2~0.5wt% first is added into protein by weight 8-10wt% Yak Bone Protein liquid to be combined
Protease, carries out 0.5~1.5h of enzyme digestion reaction under the conditions of 50~60 DEG C, wherein, the first compound protease is alkali protease
And papain；
Second step enzyme digestion reaction：The compound proteases of 0.2~0.5wt% second are added into first step enzyme digestion reaction product, 45
1.0~2.0h of enzyme digestion reaction is carried out under the conditions of~55 DEG C, wherein, the second compound protease is trypsase and flavor protease.
4. Yak Bone Protein peptide according to claim 2, it is characterised in that the step 1) in also include obtained yak
Ox bone protein liquid is handled 15-25 minutes at 75-85 DEG C through frequency for 80-100kH ultrasonic echography.
5. Yak Bone Protein peptide according to claim 2, it is characterised in that the step 3) in removal of impurities be specially：
Be incubated 10~20 minutes at 90~95 DEG C, be cooled to room temperature, add enzymolysis liquid weight 0.2-0.6wt% activated carbon in
In enzymolysis liquid, separated by diatomite, collect separating liquid.
6. Yak Bone Protein peptide according to claim 2, it is characterised in that the step 3) in UF membrane be specially：
Product after removal of impurities is handled by two step hyperfiltration process, successively the film using aperture for 5000 dalton and aperture
Ultrafiltration is carried out for the film of 3000 dalton.
7. Yak Bone Protein peptide according to claim 2, it is characterised in that the step 3) in gel separation and anti-phase
HPLC is specially：
Product after UF membrane is separated by Sephadex G-25 gels, eluent is deionized water, and eluting peak is in 280nm
It is lower to be detected, collect the 2nd eluting peak；By concentration, freeze-drying, Yak Bone Protein peptide crude product is obtained；RP-HPLC is used again
RPLC carries out 1 separation, collects by the separation of RP-HPLC RPLCs 16-18 points
Peptide eluent.
8. the Yak Bone Protein peptide according to any one of claim 1-7, it is characterised in that including with following amino acid
The polypeptide of sequence：
Glu-Ile-Arg-Met-Leu,
Thr-Val-Ser-Leu-Pro-Arg,
And/or Ser-Pro-Ile-Leu-Gly-Tyr-Trp.
9. preparing the method for the Yak Bone Protein peptide any one of claim 1-8, comprise the following steps：
1) using Yak Bone as raw material, Yak Bone Protein liquid is obtained through thermophilic digestion and degreasing；
2) by step 1) made from Yak Bone Protein liquid pass through compound protease stepwise discretization；
3) by step 2) enzymolysis after product removal of impurities, UF membrane, gel separation and reversed-phase HPLC separation, concentrate, freeze-drying
After produce.
10. the method described in Yak Bone Protein peptide or claim 9 any one of claim 1-8 prepare prevention or
The application in the medicine or food of the disease of ACE suppression is benefited from treatment.
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| CN108342441A (en) * | 2018-02-27 | 2018-07-31 | 国肽生物工程（常德）有限公司 | One kind relieves fatigue and oxidation resistant Yak Bone Protein peptide and preparation method |
| CN112574294A (en) * | 2020-12-30 | 2021-03-30 | 青海瑞肽生物科技有限公司 | Collagen peptide and preparation method and application thereof |
| CN113072621A (en) * | 2021-04-07 | 2021-07-06 | 安徽国肽生物科技有限公司 | Yak bone antihypertensive peptide and preparation method and application thereof |
| CN113072621B (en) * | 2021-04-07 | 2022-02-18 | 安徽国肽生物科技有限公司 | Yak bone antihypertensive peptide and preparation method and application thereof |
| CN113201065A (en) * | 2021-04-19 | 2021-08-03 | 国肽生物工程（常德）有限公司 | Bovine bone collagen peptide with functions of relieving fatigue and improving bone density and preparation method thereof |
| CN113201065B (en) * | 2021-04-19 | 2022-05-20 | 国肽生物工程（常德）有限公司 | Bovine bone collagen peptide with functions of relieving fatigue and improving bone density and preparation method thereof |
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