CN107022593A - A kind of Colostrinin of Pro-rich polypeptide and preparation method thereof - Google Patents
A kind of Colostrinin of Pro-rich polypeptide and preparation method thereof Download PDFInfo
- Publication number
- CN107022593A CN107022593A CN201710334700.8A CN201710334700A CN107022593A CN 107022593 A CN107022593 A CN 107022593A CN 201710334700 A CN201710334700 A CN 201710334700A CN 107022593 A CN107022593 A CN 107022593A
- Authority
- CN
- China
- Prior art keywords
- pro
- colostrinin
- preparation
- rich polypeptide
- colostrum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Water Supply & Treatment (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a kind of preparation method of the Colostrinin of Pro-rich polypeptide, comprise the following steps：Collect galactopoiesis of the cow of health after childbirth in 72 hours and obtain colostrum；Centrifugal degreasing obtains skimmed milk；By skimmed milk separate after casein it is micro- consider it is degerming or directly by skimmed milk carry out it is micro- consider degerming, plus protease, regulation pH is digested, at 25 DEG C plus ammonium salt precipitation, condensation of pressurizeing obtain condensate product；Condensate product is dissolved in water, concentrate is concentrated by ultrafiltration to obtain, freeze-drying, is produced, the preparation method realizes the Colostrinin industrialized production that Pro-rich polypeptide is isolated and purified from colostrum, the economic benefit of colostrum manufacturer is improved, the offal treatment cost of producer is reduced.The invention also discloses a kind of Colostrinin of Pro-rich polypeptide, purity is high, and the content of proline accounts for major part, and proline content accounts for 27%, and yield is higher.
Description
Technical field
The invention belongs to biological polypeptide extractive technique field, and in particular to a kind of Colostrinin of Pro-rich polypeptide and its
Preparation method.
Background technology
The protein (proline-rich-proteins, PRPs) of Pro-rich represents a class Pro-rich and hydroxyl
The structural proteins of base proline, are widely present in dicotyledon and monocotyledon.Rich proline albuminoid species is various,
The diversity of structure determines the diversity of function, but domestic very few for their research.
1974, scientist in Poland Janusz etc. was when purifying goat's colostrum IgG, it was found that one kind can not be simultaneously by anti-immunity
The material proline polypeptide (PRP) that the antiserum of globulin is precipitated, rich proline polypeptide (PRP) is a kind of immunity substance
Among matter, the polypeptide containing substantial amounts of proline and hydrophobic amino acid, the colostrum for being present in the blood of animal and mammal,
It is water-soluble under the conditions of 4 DEG C, invertibity precipitation occurs at room temperature, with regulation Human immune responses, enhancing skin vascular
Permeability, has prevention and therapeutic action well to Alzheimer disease, while immature thymocyte can be stimulated to turn
The T- cells with functional activity are turned to, can suppress too active or activate sluggish immune system.
Colostrum is one of important sources of rich proline polypeptide, but many colostrum manufacturers are processing filtered now
Cheng Zhong, has filtered rich proline polypeptide, causes rich proline polypeptide loss amount big, not only reduces the economic benefit of manufacturer,
And increase producer's processing cost as discarded object.
In recent years, Great Britain and America country starts gradually heat, but its field is main for the research of just milk-derived Pro-rich polypeptide
For the bioactive functions of some fragments of proline polypeptide (PRP) chymotrypsin hydrolysate, especially anti-oxidant, anti-ageing
Always, effect of anti-nerve retrograde affection, and it is relatively fewer for PRP Purification.
Most of method of currently acquired rich proline polypeptide is obtained by chemical synthesis and genetic engineering, Shao Shuotong
Cross and extract what is obtained from colostrum, specifically, as Chinese patent CN200980128153.1 is disclosed as psychostimulant
Be derived from by the peptide of the rich proline dacryolin (BPLP) of people's alkalescence of Proll gene codes, it is obtained by gene code,
Non- colostrum natural origin；It is derivative as new metal that Chinese patent CN 200580015689.4 discloses BPLP albumen
The peptide of exopeptidase inhibitor, and its coding polynucleotides used, and its antibody diagnostic and therapeutic use.The BPLP
It is also the non-colostrum natural origin by gene code；Chinese patent CN 201310726948.0 discloses a kind of rich dried meat of wheat
Propylhomoserin albuminoid Tazmh122 and its encoding gene and application, wheat richness proline albuminoid are obtained by gene code, non-
Obtained with colostrum natural origin；And commercialized product：Colostrinin、MemoryAid、CogniSure、
Cognate, Cognase, Dyna, it can be seen that, although Pro-rich content of peptides prepared by chemical synthesis and genetic engineering
Height, activity is good, but its cost is high, preparation technology complicated, the cycle is long.Existing report presses two-step method (ethanol extraction method, ammonium sulfate
The precipitation method) Pro-rich polypeptide is prepared from colostrum, its size is the 32 peptides thing mixed above that 0.5-3KDa is constituted, but its
Preparation method is not suitable for industrialized production.
In summary there is provided a kind of suitable for the industrialized colostrum that Pro-rich polypeptide is extracted by raw material of colostrum
Peptide is significant.
The content of the invention
In view of the defect that above-mentioned prior art is present, the purpose of the present invention is to propose to a kind of colostrum of Pro-rich polypeptide
The preparation method of peptide, realizes the Colostrinin industrialized production that Pro-rich polypeptide is isolated and purified from colostrum, improves
The economic benefit of colostrum manufacturer, reduces the offal treatment cost of producer.
Another object of the present invention is to propose a kind of Colostrinin of Pro-rich polypeptide, purity is high, and proline contains
Amount accounts for major part, and proline content accounts for 27%, and yield is higher.
The purpose of the present invention will be achieved by the following technical programs：
A kind of preparation method of the Colostrinin of Pro-rich polypeptide, comprises the following steps：S1. it is on the waiting list colostrum：Collect
Galactopoiesis of the cow of health after childbirth in 72 hours, produces colostrum；S2. by colostrum centrifugal degreasing, skimmed milk is obtained；S3.
By skimmed milk separate after casein it is micro- consider it is degerming or directly by skimmed milk carry out it is micro- consider degerming, adjust pH, plus protease carries out enzyme
Solve enzymolysis product；S4. ammonium salt precipitation, pressurization condensation is added to obtain condensate product at 2-5 DEG C enzymolysis product；S5. condensation is produced
Thing is dissolved in water, concentrate is concentrated by ultrafiltration to obtain；S6. concentrate is freeze-dried, produced.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, centrifugal condition described in step S2
For 40 DEG C~45 DEG C of temperature, rotating speed 5500rpm~6000rpm.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, junket egg is separated described in step S3
White concretely comprises the following steps：By skimmed milk acid for adjusting pH to 4.2-4.6,38-42 DEG C of insulation 25-35min, 2500r/min,
0.4Mpa is centrifuged, and obtains clarified whey.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, it is micro- described in step S3 to consider degerming
From 0.45 μm of the ceramic membrane in aperture；The pH adjusting agent is 4000 mesh calcium hydroxides, and the pH is 8.0~8.2；The enzymolysis
Time is 11-13 hours.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, ammonium salt described in step S4 is selected
40-70% saturated ammonium sulfate salt.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, the condensation time described in step S4
For 7-9 hours.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, condensate product described in step S5
Amount ratio with the water is 1kg: (0.8-1.2) L, and dissolution time is 4~12 hours, and temperature is 12~28 DEG C.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, it is concentrated by ultrafiltration described in step S5
From MWCO pottery films, the aperture of the MWCO potteries film is 10~50KDa, and flow velocity is 3~5m/s, and pressure is 0.3~0.5Mpa.
A kind of preparation method of the Colostrinin of above-mentioned Pro-rich polypeptide, wherein, cryogenic temperature described in step S6
For -50--60 DEG C, freeze-drying time is 4.5-6 hours.
A kind of Colostrinin of the Pro-rich polypeptide prepared according to above-mentioned preparation method.
Compared with prior art, Colostrinin of a kind of Pro-rich polypeptide that the present invention is provided and preparation method thereof, reaches
To have the technical effect that：1. the present invention realizes the industrial metaplasia of Colostrinin that Pro-rich polypeptide is isolated and purified from colostrum
Production；2. the present invention extracts rich proline polypeptide by source of colostrum, the economic benefit of colostrum manufacturer is improved, factory is reduced
The offal treatment cost of family；3. the preparation method operating procedure of the present invention is simple, suitable for industrialized production, and rich extracting
During proline polypeptide, producer can further be reduced cost, increased according to the selective separation casein of respective wish
The revenue source of producer；4. the preparation method defatting step of the present invention is due to contain hormone, antibiosis in the fat in colostrum
Element etc. is unfavorable for the residue of health, the easy corruption of fat and microbial growth, and residual can influence the term of validity of dairy products；5.
The purpose of calcium hydroxide is added in invention not only has regulation pH effects, and reduces enzymolysis time as enzymatic activity agent；6.
The preparation method of invention is progressive, and the science of each parameter selects the colostrum for prepare Pro-rich polypeptide
Peptide, the content of proline accounts for major part, and proline content accounts for 27%, and yield is higher；7. prepared by the present invention is rich in dried meat
The Colostrinin of propylhomoserin polypeptide, which has, strengthens immunocompetent effect, especially with enhancing NK cytoactives effect.
Below just in conjunction with the embodiments and accompanying drawing, the embodiment to the present invention is described in further detail, so that technology
Scheme is more readily understood, grasped.
Brief description of the drawings
Fig. 1 is the SDS-PAGE of the Colostrinin of Pro-rich polypeptide in embodiment 6.
Embodiment
Below by specific embodiment, the present invention will be described, but the invention is not limited in this.In following embodiments
The experimental method, is conventional method unless otherwise specified；The reagent and material, unless otherwise specified, can be from business
Approach is obtained, example below and the scope of the claims for being not used to the limitation present invention, all equivalence enforcements without departing from carried out by the present invention
Or change, it is intended to be limited solely by this patent protection domain.
Embodiment 1
It is on the waiting list colostrum：Galactopoiesis of the cow of health after childbirth in 72 hours is collected, colostrum is produced；
Degreasing：Take the above-mentioned colostrum 100kg being on the waiting list to be placed in continuous disk centrifugal machine to separate, 40 DEG C of temperature, rotating speed
5500rpm, obtains skimmed milk 80kg after centrifugation；
Separate casein：It is 10% vinegar acid for adjusting pH to 4.5,40 DEG C of insulations by the skimmed milk concentration obtained after degreasing
30min, is placed in continuous disc centrifuge and separates, electric current 30A, rotating speed：2500r/min, pressure：0.4Mpa, must clarify breast
Clearly, casein about 8kg is obtained；
It is degerming：By No. 1 ceramic membrane filter system of the above-mentioned clarified whey from the production of TAMI companies of France, ceramic membrane aperture
0.45 μm, progress micro-filtration is degerming, pressure 0.25Mpa, 35 DEG C of temperature, and flow velocity is 4m/s, obtains bacteria-removing liquid about 70kg；
Enzymolysis：Above-mentioned bacteria-removing liquid 70kg is moved into glass heat-insulating buckets of the 100L with screw rod, constant temperature adds 300g to 37 DEG C
4000 mesh calcium hydroxides, adjust pH to 8.0, add chymotrypsin 100g (enzyme activity 1000Iu/mg), and turn on agitator is at a slow speed
Stirring, enzymolysis time is 12 hours, obtains enzymolysis product；
Condensation：Enzymolysis product is cooled to 4 DEG C, 50% saturated ammonium sulfate salt is added after constant temperature, pressure is 0.3Mpa, condensation
Filtered after 8 hours, obtain sediment i.e. condensate product about 24kg；
Quintessence：Gained condensate product 24kg is added into water for injection 24L, and is gently mixed with puddler rush its dissolving, it is molten
The solution time is about 6 hours, 20 DEG C of thermostatic, obtains lysate 48kg；
It is concentrated by ultrafiltration：Aperture is used to be concentrated by ultrafiltration for 30KDa MWCO pottery films above-mentioned lysate 48kg, flow velocity is
3m/s, pressure is 0.3Mpa, and temperature is 20 DEG C, obtains concentrate 40kg；
Low temperature is freezed：Above-mentioned concentrate is used into GLZY vacuum freeze drier temperature：- 55 DEG C, vacuum 110kg conditions
Freeze 5 hours down, produce the Colostrinin 15kg of Pro-rich polypeptide of the present invention, yield is 15%.
Embodiment 2
It is on the waiting list colostrum：Cow galactopoiesis of 68 hours after childbirth of health is collected, colostrum is produced；
Degreasing：Take the above-mentioned colostrum 100kg being on the waiting list to be placed in continuous disk centrifugal machine to separate, temperature 45 C, rotating speed
6000rpm, obtains skimmed milk 81kg after centrifugation；
Separate casein：It is 10% vinegar acid for adjusting pH to 4.2,38 DEG C of insulations by the skimmed milk concentration obtained after degreasing
25min, is placed in continuous disc centrifuge and separates, electric current 30A, rotating speed：2500r/min, pressure：0.4Mpa, must clarify breast
Clearly, casein about 8.2kg is obtained；
It is degerming：By No. 1 ceramic membrane filter system of the above-mentioned clarified whey from the production of TAMI companies of France, ceramic membrane aperture
0.45 μm, progress micro-filtration is degerming, pressure 0.25Mpa, 38 DEG C of temperature, and flow velocity is 5m/s, obtains bacteria-removing liquid about 71kg；
Enzymolysis：Above-mentioned bacteria-removing liquid 71kg is moved into glass heat-insulating buckets of the 100L with screw rod, constant temperature adds 310g to 37 DEG C
4000 mesh calcium hydroxides, adjust pH to 8.2, add chymotrypsin 100g (enzyme activity 1000Iu/mg), and turn on agitator is at a slow speed
Stirring, enzymolysis time is 11 hours, obtains enzymolysis product；
Condensation：Enzymolysis product is cooled to 4 DEG C, 50% saturated ammonium sulfate salt is added after constant temperature, pressure is 0.5Mpa, condensation
Filtered after 8 hours, obtain sediment i.e. condensate product about 25kg；
Quintessence：Gained condensate product 25kg is added into water for injection 25L, and is gently mixed with puddler rush its dissolving, it is molten
The solution time is about 10 hours, 12 DEG C of thermostatic, obtains lysate 49kg；
It is concentrated by ultrafiltration：Aperture is used to be concentrated by ultrafiltration for 30KDa MWCO pottery films above-mentioned lysate 49kg, flow velocity is
5m/s, pressure is 0.5Mpa, and temperature is 20 DEG C, obtains concentrate 41kg；
Low temperature is freezed：Above-mentioned concentrate is used into GLZY vacuum freeze drier temperature：- 55 DEG C, vacuum 110kg conditions
Freeze 5 hours down, produce the Colostrinin 15.8kg of Pro-rich polypeptide of the present invention, yield is 15.9%.
Embodiment 3
It is on the waiting list colostrum：Cow galactopoiesis of 24 hours after childbirth of health is collected, colostrum is produced；
Degreasing：Take the above-mentioned colostrum 100kg being on the waiting list to be placed in continuous disk centrifugal machine to separate, 42 DEG C of temperature, rotating speed
5800rpm, obtains skimmed milk 85kg after centrifugation；
Separate casein：It is 10% vinegar acid for adjusting pH to 4.5,38 DEG C of insulations by the skimmed milk concentration obtained after degreasing
25min, is placed in continuous disc centrifuge and separates, electric current 30A, rotating speed：2500r/min, pressure：0.4Mpa, must clarify breast
Clearly, casein about 8.5kg is obtained；
It is degerming：By No. 1 ceramic membrane filter system of the above-mentioned clarified whey from the production of TAMI companies of France, ceramic membrane aperture
0.45 μm, progress micro-filtration is degerming, pressure 0.25Mpa, 35 DEG C of temperature, and flow velocity is 4m/s, obtains bacteria-removing liquid about 76kg；
Enzymolysis：Above-mentioned bacteria-removing liquid 76kg is moved into glass heat-insulating buckets of the 100L with screw rod, constant temperature is added to 37 DEG C
35094000 mesh calcium hydroxides, adjust pH to 8.0, add chymotrypsin 110g (enzyme activity 1000Iu/mg), and turn on agitator
Mix slowly, enzymolysis time is 12 hours, obtains enzymolysis product；
Condensation：Enzymolysis product is cooled to 4 DEG C, 40% saturated ammonium sulfate salt is added after constant temperature, pressure is 0.4Mpa, condensation
Filtered after 7 hours, obtain sediment i.e. condensate product about 27kg；
Quintessence：Gained condensate product 27kg is added into water for injection 27L, and is gently mixed with puddler rush its dissolving, it is molten
The solution time is about 10 hours, 25 DEG C of thermostatic, obtains lysate 54kg；
It is concentrated by ultrafiltration：Aperture is used to be concentrated by ultrafiltration for 30KDa MWCO pottery films above-mentioned lysate 54kg, flow velocity is
3m/s, pressure is 0.3Mpa, and temperature is 20 DEG C, obtains concentrate 45kg；
Low temperature is freezed：Above-mentioned concentrate is used into GLZY vacuum freeze drier temperature：- 55 DEG C, vacuum 110kg conditions
Freeze 5 hours down, produce the Colostrinin 16.9kg of Pro-rich polypeptide of the present invention, yield is 17%.
Embodiment 4
Casein is not separated in colostrum of the present invention and to obtain Colostrinin industrial making method as follows：
It is on the waiting list colostrum：Galactopoiesis of the cow of health after childbirth in 72 hours is collected, colostrum is produced；
Degreasing：Take the above-mentioned colostrum 100kg being on the waiting list to be placed in continuous disk centrifugal machine to separate, 40 DEG C of temperature, rotating speed
Skimmed milk is obtained after 5500rpm, centrifugation；Obtain 80kg.
It is degerming：By No. 1 ceramic membrane filter system of the above-mentioned skimmed milk from the production of TAMI companies of France, ceramic membrane aperture
0.45 μm, progress micro-filtration is degerming, pressure 0.25Mpa, 35 DEG C of temperature, and flow velocity is 4m/s, obtains bacteria-removing liquid about 80kg；
Enzymolysis：Above-mentioned bacteria-removing liquid is moved into glass heat-insulating buckets of the 100L with screw rod, constant temperature adds 300g4000 to 37 DEG C
Mesh calcium hydroxide, adjusts pH to 8.0, adds chymotrypsin 111g (enzyme activity 1000Iu/mg), and turn on agitator is stirred at a slow speed
Mix, enzymolysis time is 12 hours, obtains enzymolysis product；
Condensation：Enzymolysis product is cooled to 4 DEG C, 50% saturated ammonium sulfate salt is added after constant temperature, pressure is 0.3Mpa, condensation
Filtered after 8 hours, obtain sediment i.e. condensate product about 35kg；
Quintessence：Gained condensate product 35kg is added into water for injection 35L, and is gently mixed with puddler rush its dissolving, it is molten
The solution time is about 6 hours, 20 DEG C of thermostatic, obtains lysate 70kg.
It is concentrated by ultrafiltration：Above-mentioned lysate is concentrated by ultrafiltration using 30KDa MWCO pottery films, flow velocity is 3~5m/s, pressure
Power is 0.3~0.5Mpa, and temperature is 20 DEG C, obtains concentrate 55kg；
Low temperature is freezed：Above-mentioned concentrate is used into GLZY vacuum freeze drier temperature：- 55 DEG C, vacuum 110kg conditions
Freeze 5 hours down, produce the Colostrinin 25kg of Pro-rich polypeptide of the present invention, yield is 25%.
The Colostrinin amino acid composition analysis of embodiment 5
The Colostrinin 2mg for weighing the preparation of embodiment 4 is put into glass tube, adds 2mL, 6mol/L HCl, inflated with nitrogen 5min,
Tube sealing.Glass tube is placed in 80 DEG C of thermostatic drying chambers and hydrolyzes 24h.Hydrolysis is cooled to room temperature after terminating, and is settled to 10mL filterings,
Take 2mL filtrates to put in vacuum desiccator 55 DEG C or so and vacuumize drying, add pure water 2mL dissolvings.0.1mmol/L NaOH are added,
PH value is adjusted to 1.7~2.2,0.22 μm of membrane filtrations, sample bottle is transferred to be measured.Amino is carried out with full-automatic amino-acid analyzer
The analysis of acid composition.
The amino acid composition analysis of the Colostrinin of table 1
| Amino acid | Ratio (%) | Amino acid | Ratio (%) |
| Asp | 4.55 | Ile | 3.05 |
| Thr | 4.4 | Leu | 8.3 |
| Ser | 7.05 | Tyr | 2.05 |
| Glu | 12.15 | Phe | 4.45 |
| Gly | 2.8 | Lys | 4.85 |
| Ala | 3.5 | His | 2.8 |
| Cys | 0.2 | Ary | 1.6 |
| Val | 7.75 | Pro | 26.85 |
| Met | 2.65 |
As shown in Table 1, comprising 17 kinds of amino acid in the Colostrinin that prepared by embodiment 4, wherein, the content of proline is accounted for mainly
Part, proline content is 26.85%.
The Colostrinin molecular weight determination of the Pro-rich polypeptide of embodiment 6
Protein, which determines conventional method, includes SDS-PAGE and gel chromatography technology, solidifying using polyacrylamide
Gel electrophoresis method (SDS-PAGE) checks molecular weight.
Concentrate glue：Voltage is 120V, and electric current is 20mA, and the time is 30min, etc. bromophenol blue pass by concentration glue after, sample quilt
Concentration is into a line；Separation gel：Voltage is 220V, and electric current is 40mA, and the time is 40min.Treat that bromophenol blue is indicated away from plate bottom 3mm
When, stop electrophoresis.Marked on glue, remove concentration glue, film is fallen into dyeing liquor, staining jar is placed on shaking table slowly
Fixed 30min is shaken, dyeing liquor dyeing 1h is changed again, is then taken off using destainer to transparent (decolouring 3 times, the 1st 1h, the 2nd
Secondary 1h, the 3rd 30min), to clearly protein band can be found out.Embodiment 1 and embodiment 4 are detected, such as Fig. 1：Embodiment
The SDS-PAGE of the Colostrinin of the 4 Pro-rich polypeptides prepared；
Learnt through figure, regression equation：Y=10.063x-0.1867；R2=0.9954, with good linear relationship.
Three mass fragments are broadly divided into Colostrinin, first is 5500Da, second is：1500~4000Da, the 3rd is：1000~
1500Da。
The Colostrinin of the Pro-rich polypeptide of embodiment 7 is tested the anxious poison of mouse
Selection health ICR mouse 20, male and female half and half, body weight 18-22g, dose design is 21500mg/kgb.w., is weighed
The Colostrinin 21500mg of Pro-rich polypeptide prepared by embodiment 4, plus distilled water are configured to tested material to 40ml, and mouse is prohibited
After food is stayed overnight, give food within 4 hours after point 2 gavages, each gavage 20ml/kgb.wt, last gavage in 24 hours, freely ingest,
Observation 14 days, the results are shown in Table 2,3.
Influence of the Colostrinin of Pro-rich polypeptide prepared by the embodiment 4 of table 2 to mouse weight
Learnt by table 2, the Colostrinin of Pro-rich polypeptide prepared by embodiment 4 increases without influence on mouse weight.
The Colostrinin of Pro-rich polypeptide prepared by the embodiment 4 of table 3 malicious observation situation anxious to mouse
Learnt by table 3, the Colostrinin of Pro-rich polypeptide prepared by embodiment 4 has no obvious poisoning after mouse stomach
Without animal dead in performance, observation period.
The Colostrinin of the Pro-rich polypeptide of embodiment 8 is to mouse long term toxication
4 week old healthy SD rats are chosen, each 40 of male and female are entered room and adapted to 4, choose body weight and press sex for 70~100
Random packet, every group 10, single cage is raised.Set 0,667,1333 and 2000mg/kg b.w/d. each by 13.3mg/kg b.w.
4 dosage groups, weigh respectively embodiment 4 preparation Pro-rich polypeptide Colostrinin 0,667,1333,2000mg admix feeding
Material is ingested to 100g with daily 100g/kg b.w..Situation of being grown in the mouse test phase is observed, 4 are the results are shown in Table.
Influence of the Colostrinin of Pro-rich polypeptide prepared by the embodiment 4 of table 4 to rat growthing development
As shown in Table 4, each dosage group to rat body weight with compare class value compared with, difference that there are no significant (P ＞ 0.05).
The functional trial of the Colostrinin of the Pro-rich polypeptide of embodiment 9
Health ICR mouse 20 are chosen, male and female half and half, each 40 of male and female are entered room and adapted to 4, and selection body weight is 18~22g
Random packet, every group 10, single cage is raised.Set 0,667,1333 and 2000mg/kg b.w/d. each by 13.3mg/kg b.w.
4 dosage groups, weigh respectively embodiment 4 preparation Pro-rich polypeptide Colostrinin 0,667,1333,2000mg admix feeding
Material to 100g, ingested with daily 100g/kg b.w., after 30 days observe the mouse test phase in mononuclear macrophage function test and
NK cell activity assays, are shown in Table 5, table 6.
5 mouse of table-mononuclear macrophage function test
Each dosage group of Colostrinin of Pro-rich polypeptide prepared by embodiment 4 refers to control group mice phagocytic rate and phagocytosis
Number analyzes to obtain P values ＞ 0.05 through ANONA.
Mouse-NK cytoactive the function tests of table 6
Each dosage group NK cells in mice activity of Colostrinin of Pro-rich polypeptide prepared by embodiment 4 is above control
Group, Analysis of variance and two-by-two comparative statistics handle, in, low dose group it is statistically significant (P ＜ 0.05, P ＜ 0.01).Table
The Colostrinin of Pro-rich polypeptide prepared by bright embodiment 4 has the effect of enhancing NK cytoactives to mouse.
In summary, the experiment through embodiment 6-9 proves, the colostrum peptide molecule of Pro-rich polypeptide prepared by the present invention
Amount is in 1500~4000Da；The Colostrinin of Pro-rich polypeptide prepared by the present invention is learnt by anxious poison and long malicious pharmacological evaluation
Safety, has no toxic side effect；The Colostrinin for proving Pro-rich polypeptide prepared by the present invention by functional trial has to mouse
Play the role of to strengthen NK cell pharmacological activity.
Wherein, the Colostrinin of Pro-rich polypeptide prepared by embodiment 1-3 also passes through colostrum peptide ammino acid group composition
Analysis, protein measure, the anxious poison experiment of mouse, mouse long term toxication and functional trial, its result and embodiment 4
As a result it is similar.
Comparative example 1
It is on the waiting list colostrum：Galactopoiesis of the cow of health after childbirth in 72 hours is collected, colostrum is produced；
Degreasing：Take the above-mentioned colostrum 100kg being on the waiting list to be placed in continuous disk centrifugal machine to separate, 40 DEG C of temperature, rotating speed
Skimmed milk is obtained after 5500rpm, centrifugation；Obtain 80kg.
It is degerming：By No. 1 ceramic membrane filter system of the above-mentioned skimmed milk from the production of TAMI companies of France, ceramic membrane aperture
0.45 μm, progress micro-filtration is degerming, pressure 0.25Mpa, 35 DEG C of temperature, and flow velocity is 4m/s, obtains bacteria-removing liquid about 80kg；
Enzymolysis：Above-mentioned bacteria-removing liquid is moved into glass heat-insulating buckets of the 100L with screw rod, constant temperature adds hydroxide to 37 DEG C
Sodium, adjusts pH to 8.0, adds chymotrypsin 111g (enzyme activity 1000Iu/mg), and turn on agitator is mixed slowly, during enzymolysis
Between be 30 hours, obtain enzymolysis product；
Condensation：Enzymolysis product is cooled to 4 DEG C, 50% saturated ammonium sulfate salt is added after constant temperature, pressure is 0.3Mpa, condensation
Filtered after 8 hours, obtain sediment i.e. condensate product about 35kg；
Quintessence：Gained condensate product 35kg is added into water for injection 35L, and is gently mixed with puddler rush its dissolving, it is molten
The solution time is about 6 hours, 20 DEG C of thermostatic, obtains lysate 70kg.
It is concentrated by ultrafiltration：Above-mentioned lysate is concentrated by ultrafiltration using 30KDa MWCO pottery films, flow velocity is 3~5m/s, pressure
Power is 0.3~0.5Mpa, and temperature is 20 DEG C, obtains concentrate 55kg；
Low temperature is freezed：Above-mentioned concentrate is used into GLZY vacuum freeze drier temperature：- 55 DEG C, vacuum 110kg conditions
Freeze 5 hours down, produce the Colostrinin 25kg of Pro-rich polypeptide of the present invention, yield is 25%.
It is 4000 mesh calcium hydroxides replacing with sodium hydroxide by the difference of comparative example 1 and embodiment 4, its result shows
Enzymolysis time was extended for 30 hours by 12 hours, illustrated that the present invention not only has regulation pH work using the effect of calcium hydroxide
With, and with enzymolysis time is reduced as enzymatic activity agent, improve the effect of enzymolysis efficiency.
Some preferred embodiments of the present invention have shown and described in described above, but as previously described, it should be understood that the present invention
Be not limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and available for various other combinations,
Modification and environment, and above-mentioned teaching or the technology or knowledge of association area can be passed through in invention contemplated scope described herein
It is modified., then all should be in this hair and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of the present invention
In the protection domain of bright appended claims.
Claims (10)
1. a kind of preparation method of the Colostrinin of Pro-rich polypeptide, it is characterised in that comprise the following steps：S1. it is on the waiting list ox
Colostrum：Galactopoiesis of the cow of health after childbirth in 72 hours is collected, colostrum is produced；S2. by colostrum centrifugal degreasing, obtain de-
Fat breast；S3. by skimmed milk separate after casein it is micro- consider it is degerming or directly by skimmed milk carry out it is micro- consider degerming, adjust pH, plus albumen
Enzyme, progress digests to obtain enzymolysis product；S4. ammonium salt precipitation, pressurization condensation is added to obtain condensate product at 2-5 DEG C enzymolysis product；S5.
Condensate product is dissolved in water, concentrate is concentrated by ultrafiltration to obtain；S6. concentrate is freeze-dried, produced.
2. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 1, it is characterised in that step
Centrifugal condition described in S2 is 40 DEG C~45 DEG C of temperature, rotating speed 5500rpm~6000rpm.
3. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 1, it is characterised in that step
Concretely comprising the following steps for casein is separated described in S3：By skimmed milk acid for adjusting pH to 4.2-4.6,38-42 DEG C of insulation 25-
35min, 2500r/min, 0.4Mpa are centrifuged, and obtain clarified whey.
4. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 1, it is characterised in that step
Micro- ceramic membrane for considering degerming 0.45 μm of selection aperture described in S3；The pH adjusting agent is 4000 mesh calcium hydroxides, and the pH is
8.0~8.2；The enzymolysis time is 11-13 hours.
5. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 1, it is characterised in that step
Ammonium salt described in S4 selects 40-70% saturated ammonium sulfate salt.
6. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 5, it is characterised in that step
Condensation time described in S4 is 7-9 hours.
7. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 1, it is characterised in that step
The amount ratio of condensate product described in S5 and the water is 1kg：(0.8-1.2) L, dissolution time is 4~12 hours, and temperature is 12
~28 DEG C.
8. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 7, it is characterised in that step
It is concentrated by ultrafiltration described in S5 from MWCO pottery films, the aperture of the MWCO potteries film is 10~50KDa, and flow velocity is 3~5m/s, pressure
For 0.3~0.5Mpa.
9. a kind of preparation method of the Colostrinin of Pro-rich polypeptide according to claim 1, it is characterised in that step
Cryogenic temperature described in S6 is -50--60 DEG C, and freeze-drying time is 4.5-6 hours.
10. a kind of Colostrinin of Pro-rich polypeptide prepared by preparation method according to claim any one of 1-9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710334700.8A CN107022593B (en) | 2017-05-12 | 2017-05-12 | Colostrum peptide rich in proline polypeptide and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710334700.8A CN107022593B (en) | 2017-05-12 | 2017-05-12 | Colostrum peptide rich in proline polypeptide and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107022593A true CN107022593A (en) | 2017-08-08 |
| CN107022593B CN107022593B (en) | 2021-03-12 |
Family
ID=59529162
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710334700.8A Active CN107022593B (en) | 2017-05-12 | 2017-05-12 | Colostrum peptide rich in proline polypeptide and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107022593B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111868069A (en) * | 2018-02-16 | 2020-10-30 | 国家科学研究中心(Cnrs) | Continuous, solvent-free and non-enzymatic peptide synthesis by reactive extrusion |
| CN113372408A (en) * | 2021-05-21 | 2021-09-10 | 黑龙江康普生物科技有限公司 | Colostrum nano peptide with natural biological activity and preparation method and application thereof |
| PL441959A1 (en) * | 2022-08-05 | 2024-02-12 | Agrapak Spółka Z Ograniczoną Odpowiedzialnością | Method of obtaining bovine colostrum concentrate with increased IgG antibody content |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849903A (en) * | 2006-05-22 | 2006-10-25 | 江南大学 | Method for inductrialized separation of purified lactoferrins from cattle colostrum |
| US20070212367A1 (en) * | 2006-03-13 | 2007-09-13 | Keech Andrew M | Novel immunologically active peptide fragments of a proline-rich polypeptide isolated from colostral mammalian fluids for treatment of viral and non-viral diseases or diseased conditions |
| CN102210374A (en) * | 2010-04-02 | 2011-10-12 | 上海科星生物技术有限公司 | Casein polypeptide of bovine colostrum and extracting method thereof |
| CN104693272B (en) * | 2014-10-16 | 2016-08-24 | 中国农业大学 | Yak milk lactalbumin has the tripeptides of antioxidant activity and application thereof and compositions |
-
2017
- 2017-05-12 CN CN201710334700.8A patent/CN107022593B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070212367A1 (en) * | 2006-03-13 | 2007-09-13 | Keech Andrew M | Novel immunologically active peptide fragments of a proline-rich polypeptide isolated from colostral mammalian fluids for treatment of viral and non-viral diseases or diseased conditions |
| CN1849903A (en) * | 2006-05-22 | 2006-10-25 | 江南大学 | Method for inductrialized separation of purified lactoferrins from cattle colostrum |
| CN102210374A (en) * | 2010-04-02 | 2011-10-12 | 上海科星生物技术有限公司 | Casein polypeptide of bovine colostrum and extracting method thereof |
| CN104693272B (en) * | 2014-10-16 | 2016-08-24 | 中国农业大学 | Yak milk lactalbumin has the tripeptides of antioxidant activity and application thereof and compositions |
Non-Patent Citations (6)
| Title |
|---|
| JANUSZ, M. AND ZABLOCKA, A.: "Colostrinin: a proline-rich polypeptide complex of potential therapeutic interest", 《CELLULAR AND MOLECULAR BIOLOGY》 * |
| 余芳等: "牛初乳中富脯氨酸多肽的纯化与鉴定", 《食品科学》 * |
| 沈芸荪等: "《上海药物实用手册》", 31 July 1992, 文汇出版社 * |
| 牛广财: "《食品科学创新实验技术》", 31 October 2013, 中国质检出版社 * |
| 王立晖: "《生物活性多肽特性与营养学应用研究》", 30 November 2016, 天津大学出版社 * |
| 范青生: "《保健食品配方原理与依据》", 31 January 2007, 中国医药科技出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111868069A (en) * | 2018-02-16 | 2020-10-30 | 国家科学研究中心(Cnrs) | Continuous, solvent-free and non-enzymatic peptide synthesis by reactive extrusion |
| CN113372408A (en) * | 2021-05-21 | 2021-09-10 | 黑龙江康普生物科技有限公司 | Colostrum nano peptide with natural biological activity and preparation method and application thereof |
| PL441959A1 (en) * | 2022-08-05 | 2024-02-12 | Agrapak Spółka Z Ograniczoną Odpowiedzialnością | Method of obtaining bovine colostrum concentrate with increased IgG antibody content |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107022593B (en) | 2021-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8940685B2 (en) | Method for preparing active peptides from corn germ proteins | |
| CN101974589B (en) | Method for preparing immunocompetent soybean peptide by enzymolysis and membrane separation | |
| CN104450839B (en) | The preparation method of the rice bran protein peptide with ACE inhibitory activity | |
| CN109293740A (en) | The ACE in one seed oyster source inhibits and anti-tumor activity peptide | |
| CN107022593A (en) | A kind of Colostrinin of Pro-rich polypeptide and preparation method thereof | |
| CN103290086A (en) | A mung bean protein peptide having ACE inhibitory activity and a preparation method and applications thereof | |
| US12102661B2 (en) | ACE inhibitory peptide composition derived from ginkgo protein and preparation method and application thereof | |
| Mirzaei et al. | Antioxidant, ACE-inhibitory and antimicrobial activities of Kluyveromyces marxianus protein hydrolysates and their peptide fractions | |
| CN103014108A (en) | Preparation method of corn oligopeptide | |
| CN102199647A (en) | Method for separation and extraction of active peptide from domestic fungus by-products | |
| CN105713945A (en) | Preparation method of scallop antioxidant peptide | |
| CN107674905A (en) | Spirulina bioactive peptide, composition and preparation method | |
| CN111961125B (en) | Plukenetia volubilis linneo immune active peptide and preparation method thereof | |
| CN104928339B (en) | A kind of preparation method with the oat protein peptide for inhibiting intestinal inflammatory activity | |
| CN103555800A (en) | Method for preparing immunoactive peptide by taking red pine seed meal as raw material | |
| CN103740797B (en) | Method for preparing high-hydrolysis degree functional oligopeptide by use of high-temperature peanut meal | |
| CN110499351A (en) | A kind of soybean peptide and the preparation method and application thereof with high ACE inhibitory activity | |
| CN116035218A (en) | Pea oligopeptide health care product and preparation method thereof | |
| CN113845565B (en) | Lumbricus bioactive small peptide, and preparation method and application thereof | |
| CN105803024B (en) | A method of ace inhibitory peptide is prepared using coconut cake globulin as raw material | |
| Mohammad et al. | Characteristics of enzymatic hydrolysis of protein from different food sources and potential separation techniques | |
| CN110655553B (en) | ACE inhibitory peptide derived from sesame, preparation method and application thereof in preparation of antihypertensive drugs | |
| CN104928337B (en) | Navodon septentrionalis fish skin zinc chelating peptide | |
| KR20120079977A (en) | Anticancer composition comprising enzymatic hydrolysates of ruditapes philippinarum | |
| CN102277403A (en) | Production technology of yellow wine lees proteins by enzymatic extraction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |