CN106146644A - Antithrombotic peptide and directional enzymatic preparation method thereof - Google Patents
Antithrombotic peptide and directional enzymatic preparation method thereof Download PDFInfo
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- CN106146644A CN106146644A CN201610808452.1A CN201610808452A CN106146644A CN 106146644 A CN106146644 A CN 106146644A CN 201610808452 A CN201610808452 A CN 201610808452A CN 106146644 A CN106146644 A CN 106146644A
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- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- DKWNMCUOEDMMIN-PKOBYXMFSA-N melagatran Chemical compound C1=CC(C(=N)N)=CC=C1CNC(=O)[C@H]1N(C(=O)[C@H](NCC(O)=O)C2CCCCC2)CC1 DKWNMCUOEDMMIN-PKOBYXMFSA-N 0.000 description 1
- 229960002137 melagatran Drugs 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000005645 nematicide Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 108010043393 protease N Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000012958 reprocessing Methods 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 229940080237 sodium caseinate Drugs 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 108010065972 tick anticoagulant peptide Proteins 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 1
- 229960005080 warfarin Drugs 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 235000021250 α-S2-casein Nutrition 0.000 description 1
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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- 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
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Abstract
Antithrombotic peptide and directional enzymatic preparation method thereof, the invention belongs to food-borne protein deep process technology field, the problem that it is relatively low in order to solve the existing suppression ratio of thrombin peptide for inhibiting prepared with casein for source.Antithrombotic peptide of the present invention comprises multiple polypeptides.Preparation method: during one, casein is dissolved in the aqueous solution that pH is 7.0～8.0, prepare casein solution；Two, regulate the pH to 7.0～8.5 of casein solution system, add trypsin and be hydrolyzed reaction, after enzyme denaturing process, obtain enzymolysis solution；Three, ultra-filtration filters is carried out after centrifugal treating；Four, homogenizing process is carried out；Five, through sterilizing, obtain after drying the molecular weight antithrombotic peptide that peptide masses content is 40%～80% less than 5000.The present invention is hydrolyzed by control and ultra-filtration process, increases the content of antithrombotic peptide small molecular amount polypeptide, makes the thrombin suppression ratio of antithrombotic peptide dry powder reach 60%～95%.
Description
Technical field
The invention belongs to food-borne protein deep process technology field, be specifically related to Lac Bovis seu Bubali or casein for raw material system
Get everything ready the preparation method of antithrombotic acitivity polypeptide.
Background technology
Owing to modern people's operating pressure is big, rhythm of life is fast, and do not have enough sleep, excessive diet, addicted to cigarette and excessive drinking etc.
The impact of bad living habit, the dynamic equilibrium of human body intravascular coagulation system is broken so that blood solidifies easily in the blood vessel
Form thrombosis.Thrombotic disease in cardiovascular and cerebrovascular disease has become the number one killer of the mankind, and at present, cardiovascular disease is in Europe
Millions of people's premature death is caused in continent every year, and in China, cardiovascular disease fashion trend is the most notable.According to investigations, China's cardiovascular
Disease prevalence male is 1.78%, and women 1.10%, and prevalence raises with the age and increases.
It is used for treating the drug main heparin to be had of thrombosis, warfarin etc. at present, but these medicines all exist certain secondary work
With, such as thrombocytopenia and causing bleeding.The shortcoming recognizing these antithrombotic reagents, people begin one's study exploitation safer
The anticoagulation of health, antithrombotic reagent.Under this background, biologically active peptide is due to its absorbability is good, safety is high characteristic
Cause the concern of vast researcher.At present, studies have reported that some peptides can significantly inhibit thrombin and live
Property, there is significant antithrombotic acitivity.Non-food-borne peptide and food-borne can be classified as by the source difference of anticoagulant active peptide
The big class of peptide two, the research history that the most non-food-borne anticoagulant peptide is the longest, and the research of food-borne anticoagulant peptide is the most just drawn
Rise and pay close attention to widely.
At present, study hotspot is concentrated mainly on the first two aspect.And from prevention angle for, anticoagulant enzymatic activity
Or factor active anticoagulant is the main point of penetration of exploitation antithrombotic product, the ideal selection being well recognized as.
Hirudin be relatively early studied can anticoagulant enzymatic activity polypeptide, hirudin with on prothrombin molecule surface
The Fibrinogen recognition site of basic amino acid composition combines, and makes thrombin configuration that slight change to occur, so hirudin with
The enzyme active center of thrombin combines, thus the catalysis of enzyme anticoagulant is active, stops thrombin and Fibrinogen phase interaction
With.Tsetse fly thrombin inhibitor TTI is isolated from the salivary gland extract of tsetse fly, compares N terminal amino acid suitable
Sequence finds that itself and own identified serpin and other natural anticoagulant are without homology.Tick anticoagulant peptide TAP
The strand acidity peptide being made up of 60 aminoacid, can be combined with the catalytic site of thrombin, molecular structure and hirudin class
Seemingly, it is by being combined with Xa factor, thus stops the formation of thromboplastin complex, with the activation of proenzyme anticoagulant.Nematicide
Anticoagulant peptide is a series of micromolecule polypeptides, by being combined with coagulation factor activity site, carrys out protoenzyme anticoagulant or thrombin closes
Become.Bivalirudin is the thrombin inhibitor of a kind of synthesis, belongs to the micromolecule polypeptide that hirudin derives.Bivalirudin is with solidifying
Hemase combines and forms 1:1 complex, and direct anticoagulant, enzymatic activity, thus played anticoagulation, but its of enzymatic activity anticoagulant
Persistent period shorter.Melagatran is a kind of fibrin dipeptide analog, and its anticoagulant mechanism is and thrombin activity site
In conjunction with, can competitive directly enzyme anticoagulant.
All kinds of bioactive peptide with anticoagulant functions reported at present, some itself is the effective of animal saliva or venom
Composition, although anticoagulant effect is good, but is likely to have strong side effect simultaneously, there is potential safety hazard；Some anticoagulant peptide
Few at natural biological in-vivo content, separate and purification cost is high, so most of polypeptide is difficult to large-scale production；Also have is anti-
Fvii polypeptide activity cycle is short, can not being administered orally of having.These drawbacks promote researchers to do one's utmost to develop the anticoagulant peptide of food-borne,
And the exploitation of these anticoagulative substances is all to carry out from the angle of clinical medicine.For the early prevention of thrombosis, from function
It is ideal means that the angle of property food component is started with.
All there is bio-active peptide sequence in most food proteins, can be by food proteins sequence by zymolysis
Peptide discharges, and obtains the multiple bioactive peptide with biological function or physiological effect.Food-borne anticoagulant active peptide refers to sky
So animal or plant albumen is precursor substance, extracting directly or carry out the peptide with antithrombotic acitivity that reprocessing obtains.Relevant
Applied basic research and product development highlight significance.
In recent years, find from different food proteins and there is the research of anticoagulant functions protolysate gradually closed
Note.Shimizu etc. utilize papain (Papain) to hydrolyze Carnis Sus domestica, and the peptide composition of isolated shows relatively after being administered rat
Good antithrombotic acitivity.Yang Wangen etc. utilize Alcalase 2.4L and Protease N to hydrolyze albumen, have obtained having anti-
The hydrolyzate of blood coagulation activity.Rajapakse etc. obtain from fish protein zymolyte has anticoagulation and suppression platelet
The strand monomeric protein assembled.Nie Yilei etc. use trypsin hydrolyzing gelatin, find that the anticoagulant effect of gelatin hydrolysied matter is notable.
Rojas Ronquillo etc. utilizes lactic acid bacteria fermentation bovine casein, and research shows that tunning has an anticoagulant active, and
During fermentation 27h to thrombin inhibition the most by force, tunning obtains multiple bioactive peptide after purification through HPLC.Also studies have found that
It is derived from k caseic antithrombotic acitivity peptide and can reduce the platelet aggregation that thrombin causes, and in significant dose-effect relationship.
But the control of these research hydrolytic processes has certain blindness, and mostly rests on laboratory stage, this is that this type of produces
Technical bottleneck in product exploitation.
It is the specific amino acids site utilizing proteolytic enzyme protolysate that enzymatic isolation method prepares active polypeptide, thus obtains having life
The polypeptide of thing activity.The method operation is relatively easy, working condition is gentle, Product Safety is high, is to obtain food-grade organism to live
The common method of property peptide.China's Lac Bovis seu Bubali aboundresources, casein is the good source of Several Active Peptides, therefore by casein
Use orientation enzymatic hydrolysis to prepare thrombin peptide for inhibiting for raw material be just increasingly subject to the attention of Chinese scholars, it opened further
Outbreak is used for preventing thrombotic disease for functional food ingredient, has wide market prospect.
Summary of the invention
The invention aims to solve the suppression of the existing thrombin peptide for inhibiting prepared with casein for source
The problem that rate is relatively low, the concrete aminoacid sequence of active polypeptide component in the clearest and the most definite hydrolyzate, and provide antithrombotic peptide and
Its directional enzymatic preparation method.
Comprising following polypeptide in antithrombotic peptide of the present invention, the aminoacid sequence of every peptide species is respectively His-Gln-
Gly-Leu-Pro-Gln-Glu-Val-Leu-Asn-Glu-Asn-Leu-Leu-Arg、Phe-Phe-Val-Ala-Pro-Phe-
Pro-Glu-Val-Phe-Gly-Lys、Glu-Asp-Val-Pro-Ser-Glu-Arg、Tyr-Leu-Gly-Tyr-Leu-Glu-
Gln-Leu-Leu-Arg、Asn-Met-Ala-Ile-Asn-Pro-Ser-Lys、Asn-Ala-Val-Pro-Ile-Thr-Pro-
Thr-Leu-Asn-Arg、Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys、Val-Leu-Pro-Val-Pro-Gln-Lys、
Ala-Val-Pro-Tyr-Pro-Gln-Arg、Gly-Pro-Phe-Pro-Ile-Ile-Val。
The directional enzymatic preparation method of antithrombotic peptide of the present invention follows these steps to realize:
One, casein is dissolved in the aqueous solution that pH is 7.0～8.0, is heated to 50～90 DEG C and processes 5～10min, cooling
Casein solution is obtained to room temperature；
Two, the pH to 7.0～8.5 of alkaline conditioner regulation casein solution system is used, then at 30～45 DEG C of constant temperature
Under water bath condition, addition E/S 2.0%～5.0% trypsin is hydrolyzed and reacts 0.5～4h, and enzyme denaturing obtains enzymolysis after processing
Solution；
Three, it is centrifuged enzymolysis solution processing, collects supernatant, then use the film of molecular cut off 1K～10KDa
Carry out ultra-filtration filters, the enzymolysis solution after being filtered；
Four, the enzymolysis solution after filtering carries out homogenizing process under conditions of 50～75 DEG C, 15～30MPa, obtains all
Enzymolysis solution after matter；
Five, use the mode of reverse osmosis to carry out desalting processing the enzymolysis solution after homogenizing, through sterilization, divided after drying
The son amount peptide masses percentage composition less than 5000 accounts for the antithrombotic peptide of 40%～80%.
The preparation method of the biologically active peptide with anticoagulant active of the present invention, is former with Lac Bovis seu Bubali or casein
Material, chooses suitable protease, and enzymolysis prepares the casein thick product of antithrombotic peptide under certain condition, then uses membrane technology to enter
Row concentrates, and uses the mode of reverse osmosis to carry out desalination, and uses Vacuum Freezing & Drying Technology or low temperature spray drying technology,
Prepare antithrombotic peptide eventually.
Compared with prior art, the directional enzymatic preparation method of antithrombotic peptide of the present invention comprises following beneficial effect:
1, with casein for development of raw materials anticoagulant peptide, at present and have no Patents document；Through identifying, according to this
Antithrombotic acitivity peptide prepared by bright described enzymolysis preparation is different from the like product of report at present.Pressing down of thrombin peptide for inhibiting
Rate processed is higher, and the thrombin suppression ratio of this 2mg antithrombotic peptide dry powder is 60%～95%.
2, using Lac Bovis seu Bubali or casein is development of raw materials biologically active peptide, and raw material is the abundantest, cheap, can meet
Large-scale industrial production.For Lac Bovis seu Bubali product diversification, casein biologically active peptide functional food, health food or
Exploitation in medicine have great importance.
Detailed description of the invention
Detailed description of the invention one: comprise following multiple polypeptides in the antithrombotic peptide described in present embodiment, every peptide species
Aminoacid sequence be respectively His-Gln-Gly-Leu-Pro-Gln-Glu-Val-Leu-Asn-Glu-Asn-Leu-Leu-Arg,
Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys、Glu-Asp-Val-Pro-Ser-Glu-Arg、
Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg、Asn-Met-Ala-Ile-Asn-Pro-Ser-Lys、Asn-
Ala-Val-Pro-Ile-Thr-Pro-Thr-Leu-Asn-Arg、Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys、Val-
Leu-Pro-Val-Pro-Gln-Lys、Ala-Val-Pro-Tyr-Pro-Gln-Arg、Gly-Pro-Phe-Pro-Ile-Ile-
Val。
Detailed description of the invention two: the directional enzymatic preparation method of present embodiment antithrombotic peptide follows these steps to implement:
One, casein is dissolved in the aqueous solution that pH is 7.0～8.0, is heated to 50～90 DEG C and processes 5～10min, cooling
Casein solution is obtained to room temperature；
Two, the pH to 7.0～8.5 of alkaline conditioner regulation casein solution system is used, then at 30～45 DEG C of constant temperature
Under water bath condition, addition E/S 2.0%～5.0% trypsin is hydrolyzed and reacts 0.5～4h, and enzyme denaturing obtains enzymolysis after processing
Solution；
Three, it is centrifuged enzymolysis solution processing, collects supernatant, then use the film of molecular cut off 1K～10KDa
Carry out ultra-filtration filters, the enzymolysis solution after being filtered；
Four, the enzymolysis solution after filtering carries out homogenizing process under conditions of 50～75 DEG C, 15～30MPa, obtains all
Enzymolysis solution after matter；
Five, use the mode of reverse osmosis to carry out desalting processing the enzymolysis solution after homogenizing, through sterilization, divided after drying
The son amount peptide masses percentage composition less than 5000 accounts for the antithrombotic peptide of 40%～80%.
Present embodiment in advance can be based on ncbi database, BIOPEP data base, Peptide Cutter database
One or several combinations in different kind organism data base, it is also possible to be based on according to having having of definite research conclusion anti-at present
The comparison result of the bio-active peptide sequence of thrombosis activity；Use analysis of biological information technology, determine respectively in casein molecule
The amino acid sites of antithrombotic acitivity peptide and sequence, and carry out Virtual water solution, determine the product composition of different enzyme hydrolysis and live
Property, lay the foundation for enzyme hydrolysis process and parameter determination.
Casein in present embodiment step one can be being that milk obtains cheese egg for raw material by the way of acid adjustment precipitates
In vain, it is also possible to casein or the sodium caseinate product of commercialization.In step one, pH is the aqueous solution of 7.0～8.0, refers to
The aqueous solution of sodium hydroxide.E/S described in step 2 refers to the ratio of enzyme and casein usage amount.
Present embodiment makes the antithrombotic peptide product middle-molecular-weihydroxyethyl polypeptide less than 5000Da contain by hydrolysis and ultra-filtration filters
Measure the most:
1, by the directional enzymatic method of controlling, make full use of trypsin specific amino acids hydrolytic sites, use and close
Suitable enzyme addition (2%～5%), controlled enzymatic hydrolysis time (0.5～4h), control effective system temperature (30～45 DEG C), body
It is the state modulator enzymolysis process such as pH (7.0～8.5)；
2, by centrifuging and taking supernatant after hydrolyzed solution enzyme denaturing, carry out hyperfiltration treatment, by control ultrafilter membrane pore diameter range (1k～
10KDa) etc. method realizes the effective molecular weight of product and retains.
Present embodiment finds that the relatively low polypeptide of molecular weight has more preferable biologic activity.For antithrombotic acitivity
Speech, the polypeptide of molecular weight preferably can combine with the active site of thrombin, and the peptide fragment that molecular weight is bigger is sterically hindered
Relatively conference affects the combination of itself and thrombin.Therefore, the molecular weight content of peptides less than 5KDa is the highest, the biology of product
Activity is the best.
Detailed description of the invention three: caseic described in present embodiment step one unlike detailed description of the invention two
Preparation process is as follows: processed through centrifugal degreasing by milk, prepares skimmed milk, and regulation skimmed milk pH is 4.5～4.8, quiet
Put centrifugal collecting precipitation, obtain casein.Other step and parameter are identical with detailed description of the invention two.
Detailed description of the invention four: the cheese egg described in present embodiment step one unlike detailed description of the invention two or three
The concentration of white solution is 1wt%～10wt%.Other step and parameter are identical with detailed description of the invention two or three.
Detailed description of the invention five: described in present embodiment step 2 unlike one of detailed description of the invention two to four
Alkaline conditioner is NaOH solution, KOH solution, sodium bicarbonate solution or potassium bicarbonate solution.Other step and parameter are with concrete
One of embodiment two to four is identical.
Detailed description of the invention six: described in present embodiment step 5 unlike one of detailed description of the invention two to five
Sterilization processing is to carry out ultra high temperature short time sterilization 2～8s with the temperature of 130～142 DEG C in tubing heat exchanger.Other step
And parameter is identical with one of detailed description of the invention two to five.
Detailed description of the invention seven: described in present embodiment step 5 unlike one of detailed description of the invention two to five
Sterilization processing, by the way of membrane filtration degerming combination pasteurize, the most first uses the inorganic ceramic film in 0.2～1.2 μm apertures to enter
Row filtration sterilization, then pasteurize at a temperature of 62～65 DEG C, the process time is 20～30min.Other step and parameter
Identical with one of detailed description of the invention two to five.
Detailed description of the invention eight: described in present embodiment step 5 unlike one of detailed description of the invention two to seven
Dried is to be initially charged the β-ring-type of the maltodextrin of the enzymolysis solution weight after accounting for sterilization 1%～3% and 0.5%～2%
Dextrin, then it is dried process by spray drying process, inlet temperature during spray drying is 145～200 DEG C, temperature of outgoing air
It it is 70～95 DEG C.Other step and parameter are identical with one of detailed description of the invention two to seven.
Detailed description of the invention nine: described in present embodiment step 5 unlike one of detailed description of the invention two to eight
Dried is carried out by 120～160 DEG C of (low temperature) spray drying methods.Other step and parameter and detailed description of the invention two
Identical to one of eight.
Detailed description of the invention ten: present embodiment is the egg of antithrombotic peptide unlike one of detailed description of the invention two to nine
White matter content (purity) is more than 95%.Other step and parameter are identical with one of detailed description of the invention two to nine.
Detailed description of the invention 11: described in present embodiment step 5 unlike one of detailed description of the invention two to ten
Antithrombotic peptide middle-molecular-weihydroxyethyl be 2000～5000 peptide masses percentage composition account for 50%～80%.Other step and parameter and tool
One of body embodiment two to ten is identical.
Embodiment one: the directional enzymatic preparation method of the present embodiment antithrombotic peptide follows these steps to implement:
One, casein is dissolved in the aqueous solution that pH is 7.0, is heated to 50 DEG C and processes 20min, obtain after being cooled to room temperature
Casein solution；
Two, the pH to 7.8 of alkaline conditioner regulation casein solution system is used, then under the conditions of 40 DEG C of waters bath with thermostatic control
Addition E/S 3% trypsin is hydrolyzed and reacts 3h, and enzyme denaturing obtains enzymolysis solution after processing；
Three, it is centrifuged enzymolysis solution processing, collects supernatant, then use the film of molecular cut off 5KDa to surpass
Filter filter, the enzymolysis solution after being filtered；
Four, the enzymolysis solution after filtering 60 DEG C, carry out homogenizing process under conditions of 25MPa, obtain the enzyme after homogenizing
Solve solution；
Five, the mode of reverse osmosis is used to carry out desalting processing the enzymolysis solution after homogenizing, after sterilization, dried
To the molecular weight antithrombotic peptide that peptide masses content is 65% less than 5000.
The present embodiment uses Q-TOF mass-spectrometric technique to identify ten kinds of peptides that content is bigger, antithrombotic peptide in polypeptide products
Including the aminoacid sequence of polypeptide be HQGLPQEVLNENLLR, FFVAPFPEVFGK, EDVPSER, YLGYLEQLLR come from α-
S1-casein (Alpha-S1-casein)；Or NMAINPSK, NAVPITPTLNR, FALPQYLK come from α-S2-casein
(Alpha-S2-casein)；Or VLPVPQK, AVPYPQR, GPFPIIV come from beta-casein (Beta-casein).
The present embodiment dependence test is carried out in the following manner:
1, polypeptide (protein) assay:
Use polypeptide (protein) content in Kjeldahl nitrogen determination sample.
2, degree of hydrolysis assay method
Use pH-stat method to measure caseic degree of hydrolysis, start timing after adding protease, add in hydrolytic process
The NaOH of 0.5mol/L maintains pH constant, and every 20min records an alkali consumption, until hydrolysis terminates.Hydrolysis is calculated by following formula
Degree.
DH=(CV/ α Mh) × 100%
V: alkali consumption, ml.
C:NaOH molar concentration.
M: protein quality.
The average degree of dissociation of α: Argine Monohydrochloride, α=10pH-pK/(l+10pH-pK)。
The peptide bond mM number that h: protein has, for casein separation albumen, h=7.75.
3, thrombin suppression ratio measures
Use microplate reader method, the temperature of microplate reader is set as 37 DEG C, measure wavelength and be set as 405nm.Fibrinogen
Solution and thrombin solution are all prepared with Tris-HCl buffer (0.05mol/L, pH=7.2).Add in the aperture of ELISA Plate
Enter 140 μ L 2mg/ml (W/V) fibrinogen solutions and 40 μ L sample solution, reading (A after mixingSB).Add 10 μ L blood coagulations
Enzymatic solution (12U/mL) starts reaction, reading (A after 10minS).Take 40 μ L Tris-HCl buffer and replace sample solution, other
Operate same sample cell, record light absorption value (ACBAnd AC)。
Suppression ratio=[(AC-ACB)-(AS-ASB)]/(AC-ACB) × 100%
4, Q-TOF carries out polypeptide mass spectrum Sequence Identification
It is to derive according to the fragment ion in its mass spectrum by the sequence of mass spectrometric determination polypeptide.Mass spectrum occurs
Sequence information fragment is mainly formed by peptide bond fission.Cataclasis during polypeptide collision induced dissociation preferentially occurs at acyl
On amine key, generate single-minded sequence ions.Based on this principle, the aminoacid sequence of the bioactive peptide of product of the present invention uses high score
Distinguish that mass spectrometry method carries out sequence analysis qualification.First with the product after mass spectral analysis enzyme action, then Swiss-is carried out by mass spectrometric data
The database searchs such as Prot, and then obtain bioactive peptide amino acid sequence information.
5, the distribution tests of polypeptide molecular weight
The molecular weight of the casein anticoagulant peptide that the present embodiment obtains first uses SDS-PAGE electrophoresis and double-deck Tricine-
SDS-PAGE identifies, determines peptide fragment molecular weight in gained hydrolyzed solution by standard protein Marker；Wherein, SDS-PAGE
Separation gel and concentration glue are respectively 12% and 5%, and the separation gel of Tricine-SDS-PAGE and concentration glue are respectively 16% He
4%.Further it is determined by MALDI-TOF.The protein content of the casein anticoagulant peptide that the present embodiment obtains is (pure
Degree) more than 95%, degree of hydrolysis is 12.5%, and dried product is casein anticoagulant peptide product, and the present invention obtains after testing
The thrombin suppression ratio of 2mg dry powder (being dissolved in 1 milliliter of water) be 82%.
The present invention is that casein the biologically active peptide especially deep development of antithrombotic acitivity peptide, casein active peptide are in merit
Certain basis has been established in the application etc. of energy property field of food.
Claims (10)
1. antithrombotic peptide, it is characterised in that comprising following polypeptide in this antithrombotic peptide, the aminoacid sequence of polypeptide is respectively His-
Gln-Gly-Leu-Pro-Gln-Glu-Val-Leu-Asn-Glu-Asn-Leu-Leu-Arg、Phe-Phe-Val-Ala-Pro-
Phe-Pro-Glu-Val-Phe-Gly-Lys、Glu-Asp-Val-Pro-Ser-Glu-Arg、Tyr-Leu-Gly-Tyr-Leu-
Glu-Gln-Leu-Leu-Arg、Asn-Met-Ala-Ile-Asn-Pro-Ser-Lys、Asn-Ala-Val-Pro-Ile-Thr-
Pro-Thr-Leu-Asn-Arg、Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys、Val-Leu-Pro-Val-Pro-Gln-
Lys、Ala-Val-Pro-Tyr-Pro-Gln-Arg、Gly-Pro-Phe-Pro-Ile-Ile-Val。
2. the directional enzymatic preparation method of an antithrombotic peptide, it is characterised in that be to follow these steps to realize:
One, casein is dissolved in the aqueous solution that pH is 7.0～8.0, is heated to 50～90 DEG C and processes 5～10min, be cooled to room
Casein solution is obtained after temperature；
Two, the pH to 7.0～8.5 of alkaline conditioner regulation casein solution system is used, then 30～45 DEG C of waters bath with thermostatic control
Under the conditions of add E/S 2.0%～5.0% trypsin be hydrolyzed reaction 0.5～4h, enzyme denaturing process after obtain enzymolysis solution；
Three, it is centrifuged enzymolysis solution processing, collects supernatant, then use the film of molecular cut off 1K～10KDa to carry out
Ultra-filtration filters, the enzymolysis solution after being filtered；
Four, the enzymolysis solution after filtering carries out homogenizing process, after obtaining homogenizing under conditions of 50～75 DEG C, 15～30MPa
Enzymolysis solution；
Five, the mode of reverse osmosis is used to carry out desalting processing the enzymolysis solution after homogenizing, through sterilizing, obtaining molecular weight after drying
Peptide masses percentage composition less than 5000 accounts for the antithrombotic peptide of 40%～80%.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that described in step one
Caseic preparation process as follows: being processed through centrifugal degreasing by milk, prepare skimmed milk, regulation skimmed milk pH is
4.5～4.8, stand centrifugal collecting precipitation, obtain casein.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that described in step one
The concentration of casein solution be 1wt%～10wt%.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that described in step 2
Alkaline conditioner be NaOH solution, KOH solution, sodium bicarbonate solution or potassium bicarbonate solution.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that described in step 5
Sterilization processing be to carry out ultra high temperature short time sterilization 2～8s with the temperature of 130～142 DEG C in tubing heat exchanger.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that described in step 5
Dried be the β-ring of the maltodextrin being initially charged the enzymolysis solution weight after accounting for sterilization 1%～3% and 0.5%～2%
Shape dextrin, then it is dried process by spray drying process, inlet temperature during spray drying is 145～200 DEG C, air draft temperature
Degree is 70～95 DEG C.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that described in step 5
Dried carried out by 120～160 DEG C of spray drying methods.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that antithrombotic peptide
Protein content is more than 95%.
The directional enzymatic preparation method of a kind of antithrombotic peptide the most according to claim 2, it is characterised in that in antithrombotic peptide
Molecular weight be 2000～5000 peptide masses percentage composition account for 50%～80%.
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Cited By (4)
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| CN107163133A (en) * | 2017-06-26 | 2017-09-15 | 大连工业大学 | A kind of biologically active peptide and preparation method thereof |
| CN107880105A (en) * | 2017-12-11 | 2018-04-06 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide VPITPTLNR and its preparation method and application |
| CN110669125A (en) * | 2019-10-31 | 2020-01-10 | 大连工业大学 | Polypeptide with antithrombotic activity and application thereof |
| CN114235932A (en) * | 2021-12-20 | 2022-03-25 | 长春中医药大学 | Method for extracting and researching wild goose blood polypeptide by enzymolysis method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107163133A (en) * | 2017-06-26 | 2017-09-15 | 大连工业大学 | A kind of biologically active peptide and preparation method thereof |
| CN107880105A (en) * | 2017-12-11 | 2018-04-06 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide VPITPTLNR and its preparation method and application |
| CN107880105B (en) * | 2017-12-11 | 2020-06-16 | 浙江辉肽生命健康科技有限公司 | Bioactive polypeptide VPITPTLNR, and preparation method and application thereof |
| CN110669125A (en) * | 2019-10-31 | 2020-01-10 | 大连工业大学 | Polypeptide with antithrombotic activity and application thereof |
| CN114235932A (en) * | 2021-12-20 | 2022-03-25 | 长春中医药大学 | Method for extracting and researching wild goose blood polypeptide by enzymolysis method |
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