CN107163133A - A kind of biologically active peptide and preparation method thereof - Google Patents
A kind of biologically active peptide and preparation method thereof Download PDFInfo
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- CN107163133A CN107163133A CN201710493904.6A CN201710493904A CN107163133A CN 107163133 A CN107163133 A CN 107163133A CN 201710493904 A CN201710493904 A CN 201710493904A CN 107163133 A CN107163133 A CN 107163133A
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- 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
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- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a kind of biologically active peptide and preparation method thereof, using cow's milk or casein as raw material, choose suitable protease, digest under certain condition, then concentrated using membrane technology, desalination is carried out by the way of counter-infiltration, and the polypeptide powder prepared rich in biologically active peptide is dried using Vacuum Freezing ＆ Drying Technology or low temperature spray drying technology.Also it can be fished again by molecule and polypeptide of the screening with anticoagulant active and be sequenced and obtain amino acid sequence, and synthesize by way of synthesis in solid state the polypeptide products of final high-purity.By identification, the inhibiting rate of peptide angiotonin conversion enzyme inhibition rate and blood coagulation enzyme inhibition peptide is higher, and the blood coagulation enzyme inhibition rate of the 1mg antithrombotic peptide dry powder is respectively 30~80% and 70~98%.Cow's milk or casein are used for development of raw materials biologically active peptide, raw material enriches very much, cheap, can meet large-scale industrial production.
Description
Technical field
The invention belongs to food-borne biologically active peptide development technique field, and in particular to using cow's milk or casein as raw material
Prepare the preparation method with hypotensive and antithrombotic acitivity polypeptide.
Background technology
Because modern people's operating pressure is big, rhythm of life it is fast, and sleep insufficiency, excessive diet, thermophilic cigarette and excessive drinking etc.
Thrombotic diseases in the influence of bad habits and customs, cardiovascular and cerebrovascular disease turn into the number one killer of the mankind, at present, the heart
Vascular diseases cause millions of people's premature deaths every year in Europe, and in China, angiocardiopathy fashion trend is notable all the more.According to tune
Look into, China angiocardiopathy illness rate male is 1.78%, and women 1.10%, and illness rate is raised and increased with the age, China is every
Year has nearly 3,500,000 people to die from angiocardiopathy, accounts for more than the 41% of total death toll.
Current thrombotic diseases belong to one of cardiovascular and cerebrovascular disease of main harm China resident living health, and hypertension
It is a main inducing of angiocardiopathy.Mainly there are heparin, warfarin etc. for the medicine for the treatment of thrombus, though effect is preferably
It is all the presence of certain side effect, such as decrease of platelet and causes bleeding.Equally, the medicine for treating hypertension is mainly
ACE inhibitor（ ACEI）Class medicine, such as enalapril, captopril, lisinopril, though it can effectively suppress ACE
Activity, reduce blood pressure, but have obvious side effect, long-term taking can cause decompression excessively, urinary system disease, cough,
The side effect such as sense of taste distortion and angioneurotic edema.The shortcoming of these medicines is recognized, people, which begin one's study, develops safer
The hypotensive of health, antithrombotic reagent.Under this background, biologically active peptide is due to good, the safe characteristic of its absorbability
Cause the concern of numerous researchers.At present, studies have reported that some peptides can significantly inhibit vasotonia
Plain converting Enzyme or thrombin activity, with significant hypotensive and antithrombotic acitivity.Can be by it by the source difference of active peptide
It is divided into non-food-borne peptide and the major class of food-borne peptide two, wherein the longer research history of non-food-borne anticoagulant peptide, and food-borne
The research of anticoagulant peptide just causes extensive concern in recent years.
At present, for the angle of prevention, the activity for suppressing relevant physiological system core enzyme or key factor is exploitation
The main point of penetration of hypotensive and antithrombotic product, is generally acknowledged ideal selection.For hypotensive and antithrombotic acitivity
The most critical action target spot molecule generally acknowledged for product is Angiotensin-Converting and fibrin ferment respectively.
Angiotensin-Converting is containing Zn2+Metallopeptidase, Zn2+Binding site is that Angiotensin-Converting is urged
Change position where the active group of reaction.Angiotensin converting enzyme-inhibiting peptide (blood pressure lowering peptide) energy and Angiotensin-Converting
The Zn of active site2+With reference to being allowed to inactivate, so that line artery Converting Enzyme catalyzing hydrolysis angiotensinⅠ turns into blood vessel
Angiotensin Ⅱ, and catalyzing hydrolysis bradykinin turn into two kinds of biochemical reaction processes of inactive fragments, play the work of hypotensive
With.From initial Ferreira in 1965 etc. from America thatch head pallas pit viper snake venom in find first blood pressure lowering peptide to the later stage with
Milk protein, wheat plantule protein, PINPROL, Fish protein, rice residue protein, sesame protein etc. are that raw material uses vitro enzyme
The modes such as solution, biological enzymolysis, internal digestion obtain more than thousand kinds blood pressure lowering peptides.
Hirudin be it is relatively early studied can suppress the polypeptide of thrombin activity, hirudin with prothrombin molecule surface
The fibrinogen recognition site of basic amino acid composition is combined, make fibrin ferment configuration occur it is slight change, and then hirudin with
The enzyme active center of fibrin ferment is combined, so as to suppress the catalytic activity of fibrin ferment, prevents fibrin ferment 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 its serpin identified with oneself and other natural anticoagulants without homology.Tick anticoagulant peptide TAP
The single-stranded acidity peptide being made up of 60 amino acid, can be combined, molecular structure and hirudin class with the catalytic site of fibrin ferment
Seemingly, it with Xa factor by being combined, so that the formation of thromboplastin compound is prevented, to suppress the activation of factor.Nematode
Anticoagulant peptide is a series of micromolecule polypeptides, by being combined with coagulation factor activity site, is closed to suppress blood coagulation protoenzyme or fibrin ferment
Into.Bivalirudin is a kind of thrombin inhibitor of synthesis, belongs to the micromolecule polypeptide that hirudin is derived.Bivalirudin is with solidifying
Hemase combines to form 1:1 compound, directly suppresses thrombin activity, so that anticoagulation is played, but it suppresses blood coagulation enzyme activity
The duration of property is shorter.Melagatran is a kind of fibrin dipeptide analog, and its anticoagulant mechanism is and thrombin activity position
Point is combined, and competitive can directly suppress fibrin ferment.All kinds of active peptides with anticoagulant functions reported at present, some are originally
Body is the active ingredient of animal saliva or venom, although anticoagulant effect is good, may also have strong side effect simultaneously, deposit
In potential safety hazard；Some anticoagulant peptides are few in natural biological in-vivo content, and separation and purifying cost are high, so most of polypeptides
It is difficult to large-scale production；Anticoagulant peptide activity cycle also is short, can not orally waiting of having.These drawbacks promote researchers pole
Power develops the anticoagulant peptide of food-borne, and the exploitation of these anticoagulative substances is carried out from the angle of clinical medicine.It is right
In the early prevention of thrombus, it is ideal means to start with from the angle of functional food component.
All there is bio-active peptide sequence in most food proteins, can be by food proteins sequence by enzymolysis
Peptide is discharged, and obtains a variety of active peptides with biological function or physiological effect.Food-borne anticoagulant active peptide refers to day
Right animal or plant albumen is precursor substance, directly extracts or carry out the polypeptide with relevant biological activity of reprocessing acquisition.
Related application basic research and product development highlight significance.
In recent years, from different food proteins find with hypotensive, anticoagulant functions protolysate research gradually
Attract attention.Rafik etc. uses multiple protein enzyme hydrolysis using cuttlefish muscle as raw material, utilizes Gel-filtration and anti-phase
Isolated 3 kinds of high performance liquid chromatography has the polypeptides of hypotensive activity, identified, and ACE inhibitory activity is most strong to be
Met-Ala-Trp, IC50For 16.32 μm of ol/L.Tsai etc., with composite protease hydrolysis, is had using Clam Meat as raw material
The dipeptides of ACE inhibitory activity, amino acid sequence is Tyr-Asn, its IC50For 51 μm of ol/L.Shimizu etc. utilizes pawpaw egg
White enzyme (Papain) hydrolysis pork, isolated peptide composition shows preferable antithrombotic acitivity after rat is administered.Yang Wangen
Albumen is hydrolyzed Deng using L the and Protease N of Alcalase 2.4, the hydrolysate with anticoagulant active has been obtained.
Rajapakse etc. obtains with anticoagulation and suppressed the single-stranded monomeric protein of platelet aggregation from fish protein zymolyte.
Nie Yilei etc. uses trypsin hydrolysis gelatin, it is found that the anticoagulant effect of gelatin hydrolysied matter is notable.Also studies have found that to be derived from
The antithrombotic acitivity peptide of k-casein can reduce platelet aggregation caused by fibrin ferment, and in significant dose-effect relationship.But this
The control of a little research hydrolytic processes has certain blindness, and mostly rests on laboratory stage, and this is that such product is opened
Technical bottleneck in hair.
At present adopt obtain in various manners have hypotensive concurrently and the polypeptide of antithrombotic acitivity is rarely reported, only 2012
Rojas-Ronquillo etc. are identified by the bovine casein that ferments from beta-casein has hypotensive and antithrombotic acitivity concurrently
17 p277 QEPVLGPVRGPFPIIV（aa192-209）.It is to utilize the specific of proteolytic enzyme protolysate that enzymatic isolation method, which prepares active peptides,
Amino acid sites, so as to obtain the polypeptide with bioactivity.This method operation is relatively easy, working condition is gentle, product peace
Quan Xinggao, is the common method for obtaining food-grade organism active peptide.China's cow's milk aboundresources, casein is multiple biological activities
The good source of peptide, therefore be that development of raw materials identifies that having hypotensive and antithrombotic acitivity peptide concurrently has positive important by casein
Meaning.The attention that blood coagulation enzyme inhibition peptide is just being increasingly subject to domestic and foreign scholars is prepared using enzymatic hydrolysis, it is further developed and made
It is used to prevent cardiovascular and cerebrovascular disease for functional food ingredient, with wide market prospects.
The content of the invention
Present invention aim to address technical problem, the present invention present in a variety of functional activity peptides exploitation of casein
Prepared there is provided a kind of while having the preparation method of hypotensive and antithrombotic acitivity peptide.The present invention is for cow's milk multi-function health-care
The exploitation and utilization of food and medicine are significant.
The technical proposal of the invention is realized in this way：Preparation method step is as follows：
First, casein is dissolved in the aqueous solution that pH is 7.0~8.0, is heated to 50~90 DEG C of 5~10min of processing, is cooled to room
Casein solution is obtained after temperature；
The 2nd, step one sample is cooled to the pH of regulation system after room temperature, protease is then added and reaction is hydrolyzed, at the enzyme that goes out
Obtain digesting solution after reason；
3rd, centrifugal treating is carried out to enzymolysis solution, collects supernatant, then carried out using molecular cut off 1K~10KDa film
Ultra-filtration filters, the enzymolysis solution after being filtered；
4th, the enzymolysis solution after filtering is subjected to homogenization under conditions of 50~75 DEG C, 15~30MPa, obtained after homogeneous
Enzymolysis solution；
5th, the enzymolysis solution after homogeneous is subjected to desalting processing by the way of counter-infiltration, molecular weight is obtained after sterilizing, drying
Peptide masses percentage composition less than 5000 accounts for 40%~80% hypotensive and antithrombotic acitivity polypeptide.
6th, the mode such as fished using molecule, obtain the polypeptide of target sequence；
1st, it is which peptide carries out interaction point with fibrin ferment actually using being screened by the way of molecule is fished in enzymolysis mixture
Analysis.Using biomembrane interference technique（BLI）Analyzed.The principle of this method is that a branch of visible ray passes through optical fiber, in sensor
Two interfaces of the optical film of end can form two beam reflectance spectrums, and superposition forms a branch of interference spectrum, and molecule, which is combined, to be caused
Thicknesses of layers changes, and is embodied by the shift value of interference spectrum.
2nd, fibrin ferment is fixed to " universal sensor " such as Streptavidin (SA), Super Streptavidin
(SSA)、High Precision Streptavidin (SAX)、Amine Reactive 2nd Generation (AR2G)、
Aminopropylsilane(APS)；Or " trap-type " sensor such as Anti-Penta-HIS (HIS), Ni-NTA (HIS),
On the sensors such as Anti-GST.
3rd, sensor is eluted using adsorption-buffering solution, by under uncombined or combination unstable fibrin ferment elution
Come.
4th, obtained mixtures of polypeptides concentration control will be digested in 1~1000ug/mL, and with a suitable flow velocity stream
Cross sensor surface.
5th, sensor is eluted using cushioning liquid, will be not associated with or be eluted with reference to unstable polypeptide.
6th, the polypeptide combined in sensor is eluted using desorption cushioning liquid, collects each component and carry out UPLC-Q-
TOF-MS detection, it is determined that the amino acid sequence Asn-Met-Ala-Ile- of the multiple active peptides powerful with fibrin ferment affinity
Asn-Pro-Ser-Lys-Glu-Asn-Leu-Cys-Ser-Thr-Phe-Cys-Lys。
7th, the above-mentioned polypeptide filtered out is further according to the active size of molecular docking, and synthetic determination is finally filtered out and conformed to
The polypeptide asked.
7th, polypeptide products are obtained by the way of synthesis in solid state.
Further, when step 2）When described protease is trypsase, casein is adjusted using alkaline conditioner molten
The pH of liquid system to 7.0~8.5, then adds 500~10000u/g trypsase under the conditions of 45~65 DEG C of waters bath with thermostatic control and enters
5~180min of row hydrolysis.
Further, when step 2）When described protease is bromelain protease enzyme, hydrolysis reaction is to use alkali
Property conditioning agent regulation system pH to 6.0~8.0, under the conditions of 50~60 DEG C of waters bath with thermostatic control add 500~10000u/g pineapples
5~180min is hydrolyzed in protease.
Further, when step 2）When described protease is papain, hydrolysis reaction is with alkalescence regulation
The pH of agent regulation system to 5~7, adds the progress of 500~10000u/g papains under the conditions of 50~65 DEG C of waters bath with thermostatic control
5~240min of hydrolysis.
The preparation method of biologically active peptide of the present invention with hypotensive and anticoagulant active, with cow's milk or junket
Albumen is raw material, chooses suitable protease, enzymolysis prepares casein hypotensive under certain condition and anticoagulant active peptide is thick
Product, is then concentrated using membrane technology, carries out desalination by the way of counter-infiltration, and use Vacuum Freezing ＆ Drying Technology or
Person's low temperature spray drying technology, finally prepares hypotensive and antithrombotic peptide.
Beneficial effects of the present invention are：1st, there is hypotensive and anticoagulant active peptide fragment, mesh by development of raw materials of casein
It is preceding and have no Patents documents；By identification, the tool hypotensive prepared according to enzymolysis preparation of the present invention and anti-blood
Thrombus activity peptide is different from the like product reported at present.The peptide angiotonin changes the suppression of enzyme inhibition rate and blood coagulation enzyme inhibition peptide
Rate processed is higher, and the blood coagulation enzyme inhibition rate of the 1mg antithrombotic peptide dry powder is respectively 30~80% and 70~98%.2nd, using cow's milk or
Casein is development of raw materials biologically active peptide, and raw material enriches very much, cheap, can meet large-scale industrial production.For
Cow's milk product diversification, exploitation of the casein biologically active peptide in functional food, health food or medicine have
Important meaning.
Embodiment
Explanation is further explained to the present invention with reference to specific embodiment.
Embodiment one
The amino acid sequence with hypotensive and antithrombotic acitivity polypeptide described in present embodiment is Asn-Met-Ala-Ile-
Asn-Pro-Ser-Lys-Glu-Asn-Leu-Cys-Ser-Thr-Phe-Cys-Lys。
The active peptide enzymolysis preparation of hypotensive of the present invention and antithrombotic follows these steps to implement：
First, casein is dissolved in the aqueous solution that pH is 7.0~8.0, is heated to 50~90 DEG C of 5~10min of processing, is cooled to room
Casein solution is obtained after temperature；
The 2nd, step one sample is cooled to the pH of regulation system after room temperature, protease is then added and reaction is hydrolyzed, at the enzyme that goes out
Obtain digesting solution after reason；
3rd, centrifugal treating is carried out to enzymolysis solution, collects supernatant, then carried out using molecular cut off 1K~10KDa film
Ultra-filtration filters, the enzymolysis solution after being filtered；
4th, the enzymolysis solution after filtering is subjected to homogenization under conditions of 50~75 DEG C, 15~30MPa, obtained after homogeneous
Enzymolysis solution；
5th, the enzymolysis solution after homogeneous is subjected to desalting processing by the way of counter-infiltration, molecular weight is obtained after sterilizing, drying
Peptide masses percentage composition less than 5000 accounts for 40%~80% hypotensive and antithrombotic acitivity polypeptide.
6th, the synthesis in solid state of peptide：
1 synthesis order：From sequence C end to N-terminal, step is as follows：A. n equivalent resins are weighed and are put into reactor, DCM (dichloros are added
Methane) be swelled half an hour, then take out DCM, add first amino acid 2n equivalent in sequence, plus 2n equivalents DIEA, in right amount
DMF, DCM, DIEA（Diisopropylethylamine）、DMF（Dimethylformamide）, DCM, nitrogen bubble reaction 60min.Then add
About 5n equivalents of methanol, reacts half an hour, takes out reaction solution, is cleaned with DMF, MEOH；B. toward in addition sequence in reactor second
Individual amino acid（Also it is 2n equivalents）, 2n equivalents HBTU（1- hydroxyls, benzo, three chlorazol tetramethyl hexafluorophosphates）And DIEA, N2
Blistering reaction half an hour, wash liquid off, then ninhydrin detection is blocked with pyridine and acetic anhydride.Finally clean, add in right amount
Liquid of raising one's hat removes Fmoc（9-fluorenylmethyloxycarbonyl）Protection group, is cleaned, ninhydrin detection；C. sequence is sequentially added according to step b mode
Different amino acid and various modifications are carried out in row；D. removed after resin is dried up with nitrogen from reaction column, pour into flask
In, then toward in flask plus a certain amount of（Cutting liquid and resin are about with 10ml/ grams of ratio）Cutting liquid（Composition is 95%
TFA, 2% dithioglycol, 2% tri isopropyl silane, 1% water）, shake, filter resin；E. filtrate is obtained, is then added into filtrate
Enter a large amount of ether, separate out crude product, be then centrifuged for, cleaning can obtain the crude product of sequence；2 peptide purifications：Develop new technology
Crude product is purified to high performance liquid chromatography and requires purity.3 polypeptides are freezed：Purified liquid be put into freeze dryer carry out it is dense
Contracting, is lyophilized into white powder.
Casein in the step of the present embodiment one can be that milk obtains junket egg for raw material by way of acid adjustment is precipitated
In vain, the casein or casein sodium product of commercialization can also be used.PH is 7.0~8.0 aqueous solution in step one, is referred to
The aqueous solution of sodium hydroxide.
When the protease described in step 2 is trypsase, the pH of casein solution system is adjusted using alkaline conditioner
To 7.0~8.5,500~10000u/g trypsase is then added under the conditions of 45~65 DEG C of waters bath with thermostatic control reaction 5 is hydrolyzed
~180min；
When the protease described in step 2 is bromelain protease enzyme, hydrolysis reaction is to adjust body with alkaline conditioner
The pH of system to 6.0~8.0, addition 500~10000u/g bromelains are hydrolyzed 5 under the conditions of 50~60 DEG C of waters bath with thermostatic control
~180min；
When the protease described in step 2 is papain, hydrolysis reaction is the pH with alkaline conditioner regulation system
To 5~7, under the conditions of 50~65 DEG C of waters bath with thermostatic control add 500~10000u/g papains be hydrolyzed reaction 5~
240min；
Present embodiment makes the content of peptides that antithrombotic peptide product middle-molecular-weihydroxyethyl is less than 5000Da by enzyme hydrolysis and ultra-filtration filters
It is as many as possible：
1st, by using suitable enzyme addition（500~10000u/g）, the controlled enzymatic hydrolysis time（5~240min）, control it is effective
System temperature（40~65 DEG C）, system pH（6.0~9）Deng state modulator enzymolysis process；
2nd, hydrolyzate is gone out centrifuging and taking supernatant after enzyme, hyperfiltration treatment is carried out, by the pore diameter range for controlling milipore filter（1k~
10KDa）Etc. the effective molecular weight retention that method realizes product.
Present embodiment finds that the relatively low polypeptide of molecular weight has more preferable biological activity.For antithrombotic acitivity
Speech, the less polypeptide of molecular weight can preferably be combined with the active site of fibrin ferment, the larger peptide fragment steric hindrance of molecular weight
Compared with the combination that conference influences itself and Angiotensin-Converting and fibrin ferment.Therefore, polypeptide of the molecular weight less than 5KDa contains
Amount is higher, and the biological activity of product is better.
Embodiment two：The concentration of present embodiment and the casein solution described in step one unlike embodiment one
For 4wt%~12wt%.Other steps and parameter are identical with embodiment one.
Embodiment three：Present embodiment is from the alkaline conditioner unlike embodiment one or two described in step 2
NaOH solution, KOH solution, sodium bicarbonate solution or potassium bicarbonate solution.Other steps and parameter are identical with embodiment one or two.
Example IV：The preparation process of present embodiment and the casein described in step one unlike embodiment two is such as
Under：Milk is handled by centrifugal degreasing, skimmed milk is prepared, regulation skimmed milk pH is 4.5~4.8, and standing is collected by centrifugation
Precipitation, obtains casein.Other steps and parameter are identical with embodiment two.
Embodiment five：Present embodiment and the protease unlike embodiment one described in step 2 be pepsin,
Two kinds and multiple protein enzyme complex enzyme hydrolysis between trypsase, papain, bromelain and pancreatin.Other steps and ginseng
Number is identical with embodiment one.
Embodiment six：Present embodiment is from working as protease pepsin, pancreas described in step 2 unlike embodiment one
When protease, papain, the compound protease of bromelain and pancreatin, destroy the enzyme treatment is the enzyme 5 that gone out at 80~100 DEG C
~20min.Other steps and parameter are identical with embodiment one.
Embodiment seven：When the protease described in step 2 is stomach unlike one of present embodiment and embodiment one to six
Protease, trypsase, papain, bromelain and pancreatin, destroy the enzyme treatment are that acid regulator or alkalescence tune is added dropwise
Save pH to 4.0~5.0 or 10-12 that agent adjusts enzymolysis liquid.Other steps and parameter are identical with one of embodiment one to six.
Embodiment eight：Sterilization processing unlike one of present embodiment and embodiment one to seven described in step 5 be
2~8s of ultra high temperature short time sterilization is carried out with 130~142 DEG C of temperature in tubing heat exchanger.Other steps and parameter and implementation
One of example one to seven is identical.
Present embodiment step 5 can also be by way of the degerming combination pasteurize of membrane filtration.Using 0.2~1.2 μm
The inorganic ceramic membrane in aperture carries out filtration sterilization, then carries out 62~65 DEG C of pasteurize, and processing time is 20~30min.
Embodiment nine：Drying process unlike one of present embodiment and embodiment one to eight described in step 5 is logical
Crossing vacuum freeze-drying method, control condenser temperature -73~-40 DEG C, vacuum is 0.01~0.5mbar, drying time is 3~
24h.Other steps and parameter are identical with one of embodiment one to eight.
Embodiment ten：Drying process unlike one of present embodiment and embodiment one to nine described in step 5 is first
The maltodextrin and 0.5%~2% cycloheptaamylose of the enzymolysis solution weight 2%~3% accounted for after sterilization are added, then it is dry by spraying
Processing is dried in drying method, and EAT during spray drying is 145~200 DEG C, and temperature of outgoing air is 70~95 DEG C.Other steps
Rapid and parameter is identical with one of embodiment one to nine.
Embodiment 11：Drying process unlike one of present embodiment and embodiment one to ten described in step 6 is
Pass through 120~160 DEG C（Low temperature）What low temperature spray drying method was carried out.One of other steps and parameter and embodiment one to ten phase
Together.
The present embodiment is identified with hypotensive and antithrombotic acitivity polypeptide in polypeptide products using Q-TOF mass-spectrometric techniques
NMAINPSKENLCSTFCK comes from α-S2- caseins（Alpha-S2-casein）.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
SEQUENCE LISTING
<110>Dalian Polytechnic University
<120>A kind of biologically active peptide and preparation method thereof
<130> 2017.6.12
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> PRT
<213>It is artificial synthesized
<400> 1
Asn Met Ala Ile Asn Pro Ser Lys Glu Asn Leu Cys Ser Thr Phe Cys
1 5 10 15
Lys
Claims (9)
1. a kind of biological polypeptide, it is characterised in that the amino acid sequence of the polypeptide is：Shown in SEQ ID No.1.
2. application of the biological polypeptide of amino acid sequence as claimed in claim 1 in terms of hypotensive and antithrombotic reagent.
3. a kind of preparation method of biological polypeptide as claimed in claim 1, it is characterised in that step is as follows：With cow's milk or
Casein is raw material, chooses suitable protease, digests under certain condition, is then concentrated using membrane technology, using anti-
The mode of infiltration carries out desalination, then using Vacuum Freezing ＆ Drying Technology or low temperature spray drying technology, obtains polypeptide mixing
Thing.
4. the preparation method of a kind of biological polypeptide according to claim 3, it is characterised in that obtained mixtures of polypeptides is again
By the way of molecule is fished, the multiple active peptides powerful with fibrin ferment affinity are screened, amino acid sequence is then carried out
The determination of row, target polypeptides are synthesized by obtained amino acid sequence by way of synthesis in solid state.
5. the preparation method of a kind of biological polypeptide according to claim 3, it is characterised in that step is as follows：
1）Casein is dissolved in the aqueous solution that pH is 7.0~8.0, is heated to 50~90 DEG C of 5~10min of processing, is cooled to room
Casein solution is obtained after temperature；
2）Step one sample is cooled to the pH of regulation system after room temperature, protease is then added and reaction is hydrolyzed, at the enzyme that goes out
Obtain digesting solution after reason；
3）Centrifugal treating is carried out to enzymolysis solution, supernatant is collected, is then surpassed using molecular cut off 1K~10KDa film
Filtration filter, the enzymolysis solution after being filtered；
4）Enzymolysis solution after filtering is subjected to homogenization under conditions of 50~75 DEG C, 15~30MPa, obtained after homogeneous
Digest solution；
5）Enzymolysis solution after homogeneous is subjected to desalting processing by the way of counter-infiltration, molecular weight is obtained after sterilizing, drying
Peptide masses percentage composition less than 5000 accounts for 40%~80% hypotensive and antithrombotic acitivity polypeptide.
6）The mode such as fished using molecule, obtain the polypeptide of target sequence.
6. a kind of preparation method of biological polypeptide according to claim 5, it is characterised in that the protease described in step 2
During for trypsase, the pH to 7.0~8.5 of casein solution system is adjusted using alkaline conditioner, then in 45~65 DEG C of perseverances
500~10000u/g trypsase is added under the conditions of tepidarium 5~180min of reaction is hydrolyzed.
7. the preparation method of a kind of biological polypeptide according to claim 5, it is characterised in that when the albumen described in step 2
When enzyme is bromelain protease enzyme, hydrolysis reaction is the pH to 6.0~8.0 with alkaline conditioner regulation system, 50
500~10000u/g bromelains are added under the conditions of~60 DEG C of waters bath with thermostatic control 5~180min is hydrolyzed.
8. the preparation method of a kind of biological polypeptide according to claim 5, it is characterised in that when the albumen described in step 2
When enzyme is papain, hydrolysis reaction is the pH to 5~7 with alkaline conditioner regulation system, in 50~65 DEG C of constant temperature
500~10000u/g papains are added under water bath condition 5~240min of reaction is hydrolyzed.
9. the preparation method of biological polypeptide according to claim 4, it is characterised in that the solid phase synthesis process is：Close
Into order：From sequence C end to N-terminal, step is as follows：
A. n equivalent resins are weighed and are put into reactor, DCM is added and is swelled half an hour, then take out DCM, add in sequence first
Amino acid 2n equivalents, plus 2n equivalents DIEA, appropriate DMF, DCM, DIEA, DMF, DCM, nitrogen bubble reaction 60min, then
About 5n equivalents of methanol is added, half an hour is reacted, takes out reaction solution, is cleaned with DMF, MEOH；B. added toward in reactor in sequence
Second amino acid, 2n equivalents HBTU and DIEA, N2 blistering reaction half an hour, wash liquid off, then ninhydrin detection uses pyridine
With acetic anhydride end-blocking, finally clean, add appropriate liquid of raising one's hat and remove Fmoc protection groups, clean, ninhydrin detection；C. according to step
Rapid b mode sequentially adds amino acid different in sequence and carries out various modifications；D. from reaction after resin is dried up with nitrogen
Remove, poured into flask in post, then toward in flask plus a certain amount of cutting liquid, concussion, filter resin；E. filtrate is obtained, so
A large amount of ether are added in backward filtrate, crude product is separated out, is then centrifuged for, cleaning can obtain the crude product of sequence.
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| CN108004287A (en) * | 2017-11-13 | 2018-05-08 | 江苏大学 | A kind of preparation method of the macromolecular polypeptides based on gastro-intestinal digestion |
| CN108003231A (en) * | 2017-11-13 | 2018-05-08 | 江苏大学 | A kind of method of free aminoacid content in reduction small-molecular peptides |
| CN108586604A (en) * | 2018-06-19 | 2018-09-28 | 大连工业大学 | Facilitate bone bioactivity peptide and its screening technique |
| CN110669125A (en) * | 2019-10-31 | 2020-01-10 | 大连工业大学 | Polypeptide with antithrombotic activity and application thereof |
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Cited By (4)
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| CN108004287A (en) * | 2017-11-13 | 2018-05-08 | 江苏大学 | A kind of preparation method of the macromolecular polypeptides based on gastro-intestinal digestion |
| CN108003231A (en) * | 2017-11-13 | 2018-05-08 | 江苏大学 | A kind of method of free aminoacid content in reduction small-molecular peptides |
| CN108586604A (en) * | 2018-06-19 | 2018-09-28 | 大连工业大学 | Facilitate bone bioactivity peptide and its screening technique |
| CN110669125A (en) * | 2019-10-31 | 2020-01-10 | 大连工业大学 | Polypeptide with antithrombotic activity and application thereof |
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