CN104710526A - Method for separating thrombin inhibition peptide from cow's milk as well as activity determination and mass spectrum determination of thrombin inhibition peptide - Google Patents
Method for separating thrombin inhibition peptide from cow's milk as well as activity determination and mass spectrum determination of thrombin inhibition peptide Download PDFInfo
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Abstract
The invention discloses a method for separating thrombin inhibition peptide from cow's milk as well as activity determination and mass spectrum determination of the thrombin inhibition peptide. The method includes the following steps: after performing enzymolysis and purification to the cow's milk, the obtained protein components are subjected to anticoagulation effect analysis, then the high anticoagulant components are subjected to liquid chromatogram, electrospray mass spectrum determination and bioinformatics analysis, so that thrombin inhibition peptide with the thrombin inhibition ratio reaching to 91.51% in the cow's milk can be effectively separated. The blood coagulation inhibition peptide which is safe and small in toxic and side effects is found out, and lays the foundation for preparation of drugs or health care products for treating and preventing thrombus diseases, guaranteeing the human health and preventing and reducing cardiovascular diseases.
Description
Technical field
The invention belongs to life medical field, be specifically related to be separated in a kind of cow's milk the method for zymoplasm inhibiting peptide and determination of activity thereof and Mass Spectrometric Identification.
Background technology
Along with the development of the change of China's resident living mode, aging population and urbanization, cardiovascular diseases number of the infected continues to increase, and has become the highest disease of China's sickness rate, disability rate and mortality ratio at present.Correspondingly, because of the first place of the total cause of death of urban and rural residents of dead Ye Yizhan China that cardiovascular diseases causes, wherein rural area is 38.7%, and city is 41.1%.Pathologic thrombus wherein has a strong impact on the healthy class cardiovascular disorder of China resident, lung infraction, cerebral thrombosis, retina arteriovenous obstruction, myocardial infarction, four limbs and surrounding blood vessel embolism can be caused, oneself causes a disease through becoming China's height, a high dead class disease, investigation display, just has 1-3 people to suffer from thrombus disease in every 1000 people in recent years.
Research and development anticoagulation medicine, has great importance and clinical value for prevention and minimizing thrombus disease.Although the anticoagulation medicines such as heparin, oral tonka bean camphor, streptokinase and urokinase have applied for some time, these medicines can cause thrombopenia, the side effect such as hemorrhage.In the last few years, the anticoagulant and thrombolytic active substance extracted from leech, tick worm, hook worm, snake venom and bee venom demonstrated good anticoagulant effect, but toxic side effect is large, limited source, expensive.Along with deepening continuously of studying protective foods, functional foodstuff, research and development are safe, effective, side effect anticoagulation product that is little, wide material sources causes increasing concern.
Biologically active peptides is good because of high, the easy absorption of its security, effect, has been widely used in the exploitation of functional product in the last few years, such as anti-oxidation peptide, blood pressure lowering peptide, antifatigue peptide, strengthening immunity peptide and memory peptide.A large amount of biologically active factorss is contained in Ruzhong, as opioid peptides (Opioid peptides), blood pressure lowering peptide (Antihypertensive peptides), antithrombotic peptide (Antithrombolic peptides), immunological enhancement peptide ((Immunositimulating peptodes), the phosphopeptide caseinate (Casein phosphopeptides) etc. of short calcium absorption, these active polypeptide are mainly by organism secretory cell, incretory gland histoorgan, body fluid or kytoplasm produce or obtain, blood coagulation in vital movement or anti-freezing, cytodifferentiation, neurohormone mediator regulates, neoplastic lesion, immunomodulatory, biological clock rhythm, reproduction control etc. are all closely related with active polypeptide.
At present, some peptides have been found to have significant anticoagulant active, can be divided into the large class of non-food source peptide and food source peptide two by its source difference, wherein non-food source anticoagulant peptide has longer research history, and the research of food source anticoagulant peptide just causes in recent years and pays close attention to widely.Coagulation of milk and blood coagulation mechanism have many similarities, cow's milk is except the resist coagulation factor itself existed, some are hidden in the peptide section in protein, discharge through enzymic digestion, also anticoagulating active is shown, these bioactive peptides have good resist coagulation effect equally to blood, thus have antithrombotic physiological function.Cow's milk is as the integral part of food, and the anticoagulant peptide therefrom extracted has the advantage that side effect is little, can directly use.China is livestock industry big country of the world, and within 2012, milk cow amount of livestock on hand has reached 1,493 ten thousand, and cow's milk production reaches 3743.6 ten thousand tons, and become third place in the world and to give milk greatly state, raw material sources are very abundant, have very high researching value.At present, also there is not yet the report of isolation identification both at home and abroad in the research of this one side, urgently further investigate.
Through retrieval, Fu Li etc. adopt trypsinase, papoid, flavor protease, Sumizyme MP, neutral protease and stomach en-to be hydrolyzed to cow's milk protein, and result degree of hydrolysis is between 5.24%-10.58%, and wherein tryptic degree of hydrolysis is the highest; The pH value that the above-mentioned enzyme of this author adopts is respectively 5.88,5.91,6.00,6.32,6.40 and 6.67, obvious slant acidity; This author adopts fresh cow milk to test in addition, and its technical scheme is not easy to technology popularization and Industrialization Progress, also can cause a large amount of Financial cost simultaneously.
Summary of the invention
For above-mentioned present situation, the object of this invention is to provide the method and determination of activity thereof and Mass Spectrometric Identification that are separated zymoplasm inhibiting peptide in a kind of cow's milk.The technical program is by carrying out enzymolysis, purifying to cow's milk, anticoagulant effect analysis is carried out to the protein ingredient obtained, chromatography-electrospray-ionization/mass spectrometry (LC-ESI-MS) qualification and bioinformatic analysis are carried out to highly blood coagulation resistant component, tentatively find out its composition, predict its characteristic and function, for illustrating the anticlotting mechanism of cow's milk further, find safety, the anti-blood coagulation peptide that toxic side effect is little, in order to prepare medicine or the healthcare products for the treatment of and pre-preventing thrombosis, ensure human health, prevention and reduce cardiovascular disorder provide basis.
The present invention is achieved by the following technical solutions:
The invention provides a kind of method being separated zymoplasm inhibiting peptide from cow's milk, described method comprises the steps:
Step one, adopts proteolytic enzyme to carry out enzymolysis to cow's milk, carries out passivation, centrifugal, dialysis, obtain enzymolysis product mixture to enzymolysis product;
Step 2, adopts Sephadex G-50 chromatography column to carry out separation and purification to described enzymolysis product mixture, obtains zymoplasm inhibiting peptide.
Preferably, in step one, described proteolytic enzyme comprises alkali protease2709, flavor protease, Neturase 1.5MG, Alcalase 2.4L, Protex 6L, Protex 7L, neutral protease or trypsinase.
More preferably, described proteolytic enzyme is Alcalase 2.4L.
Preferably, in step one, described cow's milk from whole milk powder, as Shanghai Bright Dairy & Food Co., Ltd. produce whole milk powder (lot number 20140806044132) etc.
Preferably, in step one, described passivation specifically refers to enzymolysis product is placed in 100 DEG C of heating 15min.
Preferably, in step one, the time of described dialysis is 48h.
Preferably, in step one, described enzymolysis product mixture is refrigerated to dry powder, is placed in-20 DEG C of refrigerators for subsequent use.
Preferably, described method also comprise to step 2 gained zymoplasm inhibiting peptide carry out antithrombin activity mensuration, LC-ESI-MS qualification or bioinformatic analysis.
Preferably, described bioinformatic analysis comprises molecular weight, the iso-electric point analysis of zymoplasm inhibiting peptide, Subcellular Localization, signal peptide analysis or functional analysis etc.
Preferably, described Subcellular Localization adopts software to be PSORT II; Signal peptide analysis adopts software to be SignalP 4.1; Functional analysis adopts software to be GO software.
Compared with prior art, the present invention has following beneficial effect:
1, the present invention is on the basis of existing zymolysis technique, is separated, LC-ESI-MS, bioinformatic analysis etc. after purifying to enzymolysis product, be a kind of comprehensively, the zymoplasm inhibiting peptide of system is separated, analytical procedure; Two-dimensional electrophoresis (two-dimensional electrophoresis, 2-DE, also two dimensional electrophoresis is claimed) be the isolation technique used the earliest in existing proteomics research, but it is poor to the albumen sepn effect of alkalescence end, and especially limited to the separating effect of low-abundance protein; And LC-ESI-MS technology is widely used high-throughput, highly sensitive proteomic analytical methods in recent years, more convenient and accurate in the compartment analysis of quantitative analysis and low-abundance protein; The present invention uses the high zymoplasm constituents for suppressing of LC-ESI-MS technology to cow's milk to carry out proteome analysis, result chromatographic fraction liquid chromatography spectrogram shows, albumen sepn is clear, strength of signal is high, albumen sepn is respond well, there is multiple high-abundance proteins, having laid good basis for carrying out ESI mass spectroscopy;
2, selected by the present invention, the pH value of enzyme is respectively 8.0,7.5,8.5,7.0, and environment is gentle;
3, the present invention adopts milk powder to originate as cow's milk, is easier to quantitatively in actually operating, and repeatability is stronger, enhances accuracy of the present invention and science, promotes simultaneously and is beneficial to storage in industrialization, reduce costs in technical solution of the present invention.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the liquid chromatography spectrogram of cow's milk chromatography samples;
Fig. 2 is the Subcellular Localization of cow's milk qualification polypeptide;
Fig. 3 is that the signal peptide of cow's milk qualification polypeptide exists situation;
Fig. 4 is cow's milk chromatographed product qualification protein function distribution plan.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
1 materials and methods
1.1 experiment material
Whole milk powder, Shanghai Bright Dairy & Food Co., Ltd. produces, lot number 20140806044132; Alkali protease2709, Protex 6L, Protex 7L, Neturase 1.5MG, flavor protease are bio tech ltd difficult to understand purchased from Shanghai; Alcalase 2.4L is purchased from letter zymin company of Novi of Denmark; Neutral protease, trypsinase, Tris are purchased from Shanghai Sheng Gong bio-engineering corporation; Zymoplasm (model T4648-1kv, Ox blood plasma), Fibrinogen (model F6755, rat plasma), Sephadex G-50, available from Sigma; Blue dextran Dextran blue 2000 is purchased from Xi Tang bio tech ltd, Shanghai; Other reagent are domestic analytical pure.
The enzymolysis of 1.2 proteolytic enzyme not of the same race to cow's milk compares
Select alkali protease2709, flavor protease, Neturase 1.5MG, Alcalase 2.4L, Protex 6L, Protex 7L, neutral protease and trypsinase totally 8 kinds of proteolytic enzyme carry out enzymolysis respectively and compare.Taking 6.0g whole milk powder is dissolved in 200mL deionized water, magnetic agitation 15min, 90 DEG C of heat pre-treatment 10min.First according to optimum temperuture and the pH value (table 1) of different proteolytic enzyme, add gentle adjusted to ph respectively, then add the protease hydrolyzed 1-2h of quality of milk powder 5%, observe the hydrolysis effect of different proteolytic enzyme.The NaOH adding 0.1mol/L in hydrolytic process is constant to maintain pH value, and every 10min records an alkali consumption.Adopt pH-stat method, by following formulae discovery degree of hydrolysis (degree of hydrolysis, DH): DH%=M/Mh × 100%=[V × C/ (a × m × Mh)] × 100%, wherein V is the volume (mL) consuming alkali; C is paper mill wastewater (mmol/mL); M is protein content (g) in milk powder, and the protein content of cow's milk is 40%; A is amino acid average dissociation degree, 1/a=1.08; Mh is the mmole number of peptide bond in every gram of protein, is 8.3 for powdered milk protein.After reaction terminates, hydrolyzate is heated 15min with passivation proteolytic enzyme in 100 DEG C, after its naturally cooling, with the centrifugal 15min of 4 500r/min, collect supernatant liquor.The milk powder enzymolysis product supernatant 50mL getting degree of hydrolysis the highest adds in dialysis tubing, after the 48h that dialyses, in freeze drier, is refrigerated to dry powder, puts in-20 DEG C of refrigerators for subsequent use in redistilled water.
The separation and purification of 1.3 cow's milk enzymolysis products
Adopt Sephadex G-50 chromatography column separation and purification cow's milk source thrombase inhibiting peptide.Get 2L distilled water, be placed in the ultrasonic 30min of Ultrasonic Cleaners and degas bubble, stand for standby use, the bubble distilled water that all for this reason degass used in subsequent step.Get Sephadex G-50 4.5g, add boiling water bath 1h in distilled water, after swelling equilibrium, naturally cool to room temperature, incline supernatant liquor, after removing broken particle, fill post.Get 0.9 × 50cm chromatography column, cleaning, is vertically placed on iron stand, closes column outlet, adds distilled water, makes bottom be full of liquid, swelling good gel furnishing grout, pours into continuously in post.When the glue bed deposited is to 2-3cm, open the outlet of post, with evenly constant flow velocity encapsulating, until install.Static 10min, opens liquid outlet, gets rid of excessive elutriant, makes, in post, glue face retains 5-6cm eluent.Continue the distilled water continuing an interpolation column volume afterwards, make, in post, glue face retains 5-6cm eluent all the time, with this balance columns layer.Get cryodesiccated purification of samples, be dissolved in respectively in 5mL distilled water and dissolve.Open the outlet of post, in post, eluent stream is to during apart from bed surface 1-2cm, closes outlet.With liquid-transfering gun, 5mL sample is slowly added to post bed surface, open outlet, when sample infiltrates in bed, close outlet close to during the 1cm of bed surface.Add a small amount of distilled water carefully simultaneously, then open the outlet of post, the sample on bed surface is all infiltrated in bed.Add distilled water carefully on the surface of bed, exceed bed surface 5-6cm.Chromatography starts, and constantly adds distilled water, makes, in post, glue face retains 5-6cm eluent, is in charge of collection effluent liquid in the exit of post with centrifuge tube, until sample is all washed down in post, the sample that purifying is good is placed in-20 DEG C of refrigerators for subsequent use.After a sample chromatography is complete, with distilled water cleaning pillars more than two column volumes.
The antithrombin effect measuring of the different purified product of 1.4 cow's milk
Microplate reader method is adopted to measure anticoagulant peptide to the inhibit activities of zymoplasm.The mensuration wavelength of microplate reader is set as 405nm, and fibrinogen solution and thrombin solution are all prepared with distilled water.140 μ L 0.1% (W/V) fibrinogen solutions are added in the aperture of enzyme plate, with the long microplate reader of all-wave (Thermo/MSS BioTek-Epoch) densitometric (optical density, OD) value twice, obtains optical density(OD) difference (ODb); Add 40 μ L sample solutions, reading, then add 10 μ L thrombin solutions (12U/mL) start reaction, reading after 10min, records optical density(OD) difference (ODs).Get 40 μ L distilled water and replace sample solution, other operate same sample hose, record optical density(OD) difference (ODc).
Be calculated as follows sample Trombin inhibiting catalysis fibre proteinogen and form fibrinous ability: Y (inhibiting rate)=[(ODc-ODs)/(ODc-ODb)] × 100%.
The LC-ESI-MS qualification of 1.5 cow's milk highly blood coagulation resistant active ingredients
1.5.1 the FASP enzymolysis of sample
Get cow's milk and be separated the highly blood coagulation resistant enzymic activity sample 50 μ L obtained, add 50 μ L 8M urea, then to add 1 μ L 1MDTT be 10mM to final concentration, 37 DEG C of reductase 12 .5h, are cooled to room temperature.Add 5 μ L 1M IAA solution, lucifuge 30min.Add 200 μ L 25mM ammonium bicarbonate buffers, then add 4 μ g Trypsin, 37 DEG C of enzymolysis spend the night.After enzymolysis sample acidifying, cross the desalination of 3M C18 desalting column, then add 500 μ L 0.1%TFA and cross post 2 times, then add 500 μ L 0.1%TFA, 70% acetonitrile water elution 2 times.By sample freeze-drying, redissolution, for stratographic analysis.
1.5.2 capillary high performance liquid chromatography analysis
0.1% aqueous formic acid (A liquid) balance of chromatographic column 0.15mm × 150mm (RP-C18, Column Technology Inc.) with 95%.Sample is loaded to Zorbax 300SB-C18peptide traps (Agilent Technologies by automatic sampler, Wilmington, DE), be separated through chromatographic column again, related fluid phase gradient is as follows: 0min-50min, and 0.1% formic acid acetonitrile solution (acetonitrile is 84%) (B liquid) linear gradient is from 4% to 50%; 50min-54min, B linear gradient is from 50% to 100%; 54min-60min, B liquid maintains 100%.2.5.3ESI Mass Spectrometric Identification
Enzymolysis product carries out mass spectroscopy through capillary high performance liquid chromatography desalination and after being separated with Q Exactive mass spectrograph (Thermo Fisher).Detection mode is positive ion.The mass-charge ratio of the fragment of polypeptide and polypeptide gathers by the following method: each full scan (full scan) gathers 10 fragment patterns stored (MS afterwards
2scan).
1.6 mass-spectrometric data process and bioinformatic analysis
1.6.1ESI MASS SPECTRAL DATA ANALYSIS
The corresponding database of source document Mascot 2.2 software search, obtains the protein information of qualification.Correlation parameter is as follows: Enzyme=Trypsin; Missed cleavage=2; Fixed modification:Carbamidomethyl (C); Variable modification:Oxidation (M); Search uses Uniprot database, i.e. uniprot_Bos_taurus_31817_20140914.REVERSED.fasta, download date 20140914, sequence 31817.Peptides tolerance:20ppm; MS/MS tolerance:0.1Da; Mascot result filtration parameter is: Mascot score >=20.
1.6.2 the bioinformatic analysis of polypeptide
Protein, iso-electric point are analyzed by proteomics net (http://www.expasy.org/tools) and protein analysis expert software (expert proteins analysis system, ExPASy); Proteins subcellular location is analyzed based on PSORT II software (http://psort.hgc.jp/form2.html); Signal peptide analysis adopts SignalP 4.1 software analysis (http://www.cbs.dtu.dk/services/SignalP/); Functional classification explains software (http://geneontology.org/) analysis based on Gene Ontology (GO).
2 results and analysis
The enzymolysis of 2.1 8 kinds of proteolytic enzyme to cow's milk compares
Adopt 8 kinds of proteolytic ferments, under respective optimum condition, carry out enzymolysis, result to cow's milk protein, the degree of hydrolysis of cow's milk is between 2.95% ~ 14.77%, and that degree of hydrolysis is the highest is Alcalase 2.4L (table 1).
Table 18 kinds of proteolytic ferments are to the enzymolysis result of cow's milk
| Proteolytic ferment | Optimum temperuture (DEG C) | Optimum pH | Alkali consumption (mL) | Degree of hydrolysis (%) |
| Alkali protease2709 | 60 | 8.5 | 23.9 | 11.77 |
| Flavor protease | 52 | 7.5 | 9.6 | 4.73 |
| Neturase 1.5MG | 50 | 7.5 | 10.6 | 5.22 |
| Alcalase 2.4L | 60 | 8.5 | 30 | 14.77 |
| Protex 7L | 50 | 7.5 | 15.5 | 7.63 |
| Protex 6L | 50 | 7.5 | 26 | 12.80 |
| Neutral protease | 50 | 7.0 | 6 | 2.95 |
| Trypsinase | 50 | 8.0 | 7.5 | 3.69 |
2.2 the antithrombin effect measuring of the purifying of cow's milk enzymolysis product and different purified product
Get each 50mL of cow's milk supernatant liquor that Alcalase 2.4L is hydrolyzed, after dialysis, lyophilize, be dissolved in 5mL redistilled water and cross after post, collect different chromatographic solution, measure its Trombin inhibiting active effect, as a result, be numbered 15 cow's milk chromatography samples inhibiting rate the highest, reach 91.51% (table 2).
The inhibiting rate of the different chromatography samples of table 2
Illustrate: "-" represents that this result is suspicious.Note:“-”means these results were doubtful.
The LC-ESI-MS qualification of 2.3 cow's milk highly blood coagulation resistant active ingredients
The cow's milk Alcalase 2.4L enzymolysis purified product being numbered 15# is carried out LC-ESI-MS analysis, and result successfully obtains mass spectrogram (Fig. 1).Cow's milk chromatography samples obtains 147 peptide sequences altogether through ESI qualification, by database search, identifies 70 polypeptide (table 3) altogether.
Table 3 cow's milk chromatography samples LC-ESI-MS qualification result
The molecular weight of 2.4 polypeptide and iso-electric point analysis
Through software analysis, the polypeptide molecular weight identified in milk sample is between 4359.24 ~ 303704.48, and iso-electric point is (table 3) between 4.66 ~ 11.25.
The Subcellular Localization of 2.5 polypeptide
Through PSORT II software analysis, cow's milk qualification polypeptide in be positioned extracellular, cytolemma, tenuigenin, Secretory vesicles polypeptide have 2,7,19,1 respectively, account for 2.86%, 10.00%, 27.14% and 1.43%; The polypeptide be positioned in plastosome, endoplasmic reticulum, cavity, nucleus has 4,8,1 and 23 respectively, accounts for 5.71%, 11.43%, 1.43% and 32.86%; What location was unknown has 5, accounts for 7.14% (Fig. 2).
The signal peptide analysis of 2.6 polypeptide
Through SignalP 4.1 software analysis, in cow's milk qualification polypeptide, what have signal peptide has 29, accounts for 41.43%; No signal peptide have 36, account for 51.43%; Signal peptide the unknown have 5, account for 7.14% (Fig. 3).
The functional classification of 2.7 polypeptide
Through GO software analysis, the function of the polypeptide of cow's milk chromatography samples qualification is broadly divided into 22 classes, comprise and affect albumen (enzyme) active (6), albumen synthesis (4), protein binding (7), extracellular region (12), signal transduction (1), translocator (9), cell adhesion and migration (1), ion metabolism (3), plasmosin (1), apoptosis regulates (2), cytodifferentiation regulates (2), hormonal activity regulates (2), membranin (3), and immune response regulates (1), chemical stimulation detects (1), nucleic acid combines (2), mitochondrial protein and folding (1) thereof, nucleic acid synthesis and metabolism (2), nucleoprotein (1), lipid metabolism (1), structural protein (2) and unknown function albumen (2) (Fig. 4).
The present invention adopts 8 kinds of proteolytic ferments, carries out enzymolysis, found that milk solution, and the degree of hydrolysis of cow's milk is between 2.95% ~ 14.77%, and wherein the degree of hydrolysis of Alcalase 2.4L is the highest, is secondly Sumizyme MP.
The cow's milk supernatant getting Alcalase 2.4L hydrolysis crosses column chromatography, the inhibiting rate of different components to zymoplasm is different, the highest reaches 91.51%, the above results points out this research by the hydrolysis effect of more different proteolytic enzyme, obtain the better component of zymoplasm inhibition, for next step identifies that its protein ingredient, characteristic and function are laid a good foundation.
Bioinformatic analysis is carried out to 70 peptide species of cow's milk ESI Mass Spectrometric Identification, found that, cow's milk qualification polypeptide in be positioned extracellular, cytolemma, tenuigenin, Secretory vesicles polypeptide have 2,7,19,1 respectively, account for 2.86%, 10.00%, 27.14% and 1.43%; Other polypeptide is positioned, in plastosome, endoplasmic reticulum, cavity, nucleus, to be wherein positioned nuclear ratio the highest, to reach 32.86%, also has 5 polypeptide location unknown in addition.Signal peptide analysis shows, and in cow's milk qualification polypeptide, 29 have signal peptide, account for 41.43%; 36 no signal peptides, account for 51.43%; Separately there are 5 the unknowns.GO software analysis shows, the function of cow's milk qualification polypeptide is broadly divided into 22 classes, comprise and affect albumen (enzyme) activity, albumen synthesis, protein binding, extracellular region, signal transduction, translocator, cell adhesion and migration, ion metabolism, plasmosin, apoptosis adjustment, cytodifferentiation adjustment, hormonal activity adjustment, membranin etc., these albumen may participate in the activity of Trombin inhibiting, are worth further investigation further.
Because the zymoplasm inhibiting peptide kind identified at present is still considerably less, especially the zymoplasm inhibiting peptide in cow's milk source is substantially blank, its sequence and constitutional features unclear, this gives and analyses in depth qualification cow's milk source thrombase inhibiting peptide and bring difficulty.Emphasis is positioned in extracellular, cytolemma and tenuigenin to those by next step, there is signal peptide, the polypeptide again with functions such as protein binding, Function protein synthesis and activity is further furtherd investigate, specifically to determine the effect of these polypeptide in cow's milk anticoagulant enzymic activity, for researching and developing safety, effectively, the anticoagulation medicine of little, the wide material sources of side effect or healthcare products lay the foundation.
In sum, the present invention adopts 8 kinds of proteolytic ferments to carry out enzymolysis to cow's milk protein, finds that the degree of hydrolysis of Alcalase 2.4L to cow's milk is the highest, reaches 14.77%.The zymoplasm inhibiting rate of its hydrolysis supernatant liquor Sephadex G-50 column chromatography product can reach 91.51%.The chromatographic fraction the highest to the thrombin-inhibiting activity obtained from cow's milk carries out LC-MSI-MS analysis, identify 70 polypeptide, to the molecular weight of these polypeptide, iso-electric point, Subcellular Localization, signal peptide and possible functional classification have carried out software analysis and prediction, find that they have and affect albumen (enzyme) activity, albumen synthesizes, protein binding, extracellular region, signal transduction, translocator, cell adhesion and migration, ion metabolism, plasmosin, apoptosis regulates, cytodifferentiation regulates, the functions such as hormonal activity adjustment, for identifying definite functions in cow's milk further from now on, the zymoplasm inhibiting peptide of clear in structure, illustrate the anticlotting mechanism of cow's milk, research and development safety, effectively, side effect is little, novel anticoagulation medicine or the healthcare products of wide material sources lay the foundation.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims.
Claims (10)
1. from cow's milk, be separated a method for zymoplasm inhibiting peptide, it is characterized in that, described method comprises the steps:
Step one, adopts proteolytic enzyme to carry out enzymolysis to cow's milk, carries out passivation, centrifugal, dialysis, obtain enzymolysis product mixture to enzymolysis product;
Step 2, adopts Sephadex G-50 chromatography column to carry out separation and purification to described enzymolysis product mixture, obtains zymoplasm inhibiting peptide.
2. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1, it is characterized in that, in step one, described proteolytic enzyme comprises alkali protease2709, flavor protease, Neturase 1.5MG, Alcalase 2.4L, Protex 6L, Protex 7L, neutral protease or trypsinase.
3. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1 and 2, is characterized in that, described proteolytic enzyme is Alcalase 2.4L.
4. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1, is characterized in that, in step one, described cow's milk is selected from whole milk powder.
5. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1, is characterized in that, in step one, described passivation specifically refers to enzymolysis product is placed in 100 DEG C of heating 15min.
6. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1, is characterized in that, in step one, the time of described dialysis is 48h.
7. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1, is characterized in that, in step one, described enzymolysis product mixture is refrigerated to dry powder, is placed in-20 DEG C of refrigerators for subsequent use.
8. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 1, is characterized in that, described method also comprises carries out antithrombin activity mensuration, LC-ESI-MS qualification or bioinformatic analysis to step 2 gained zymoplasm inhibiting peptide.
9. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 8, is characterized in that, described bioinformatic analysis comprises molecular weight, the iso-electric point analysis of zymoplasm inhibiting peptide, Subcellular Localization, signal peptide analysis or functional analysis.
10. the method being separated zymoplasm inhibiting peptide from cow's milk according to claim 9, is characterized in that, described Subcellular Localization adopts software to be PSORT II; Signal peptide analysis adopts software to be SignalP 4.1; Functional analysis adopts software to be GO software.
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2015
- 2015-03-06 CN CN201510100092.5A patent/CN104710526B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| ANNE-MARIE FIAT ET AL.,: "Biologically Active Peptides from Milk Proteins with Emphasis on two examples concerning antithrombotic and immunomodulating activities", 《JOURNAL OF DAIRY SCIENCE》 * |
| B CHABANCE ET AL.,: "Casein peptide release and passage to the blood in humans during digestion of milk or yogurt", 《BIOCHIMIE》 * |
| REBECA ROJAS-RONQUILLO: "Antithrombotic and antihypertensive properties of peptides released from bovine casein by Lactobacillus casei Shirota", 《INTERNATIONAL DAIRY JOURNAL》 * |
| 王子伟等: "菜籽凝血酶抑制肽的酶法制备研究", 《河南工业大学学报（自然科学版）》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106146644A (en) * | 2016-09-07 | 2016-11-23 | 哈尔滨工业大学 | Antithrombotic peptide and directional enzymatic preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN104710526B (en) | 2018-10-02 |
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