CN107653291A - The standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system - Google Patents
The standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system Download PDFInfo
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- CN107653291A CN107653291A CN201711126104.7A CN201711126104A CN107653291A CN 107653291 A CN107653291 A CN 107653291A CN 201711126104 A CN201711126104 A CN 201711126104A CN 107653291 A CN107653291 A CN 107653291A
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- 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
Abstract
The invention discloses the standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system, belong to bioengineering field.The present invention is cleaned and lost hair or feathers to yak skin first, ungrease treatment, is then collected by centrifugation in supernatant using the collagen in pepsin extraction yak skin in acid condition and is contained macromolecular collagen, can further be refined by steps such as concentrating and precipitatings；Again through compound protease depth hydrolysis, its enzymolysis liquid is concentrated, dries, and can obtain the Yak-skin Gelatin original polypeptide of small molecule for undegraded complete part.Raw material of the present invention comes from the processing byproduct yak skin of the characteristic resources yak of Tibet region, utilization for Tibetan areas characteristic resources provide new way, the macromolecule collagen and low molecule collagen polypeptide of preparation have important application value in medical and big health field, can realize the efficient of yak skin resource and maximally utilize.
Description
Technical field
The present invention relates to the standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system, belong to biological work
Journey field.
Background technology
Collagen is that mammal in-vivo content is most, is distributed the most wide boiomacromolecule albumen with connective function, glue
Reason has good biocompatibility, biodegradable and bioactivity, therefore in food, medicine, organizational project, change
The fields such as cosmetic are widely applied.Especially field of medicaments, artificial skin, artificial blood vessel, hemostasis are prepared using collagen
Sponge, cardiac valves, diaphragm of burning, operation suture thread, bone material, tissue engineering material, vascular repair material, biological support
Deng.
Small molecule collagen peptide can be directly absorbed by the body without decomposing, therefore collagen existing for small peptide form, can directly be worn
Cross intestinal wall and enter blood circulation, the effect of food is favourable.There is research to think, the collagen polypeptide of small molecule can be directly by intestinal absorption
Participate in the synthesis of human collagen afterwards, prevent calcium loss, and can effective repairing articular cartilage, the profit on repairing articular cartilage surface
It is sliding, reduce friction.In addition, collagen peptide is also used as skin care item composition, can improve relaxation, tightens up skin, reduces wrinkle
Line, calm down microgroove, slowing down skin aging.
Yak is the peculiar ox kind of Qinghai-Tibet Platean, is typical high and cold animal, has cold-resistant, resistance to High aititude, anti anoxia, resists
The advantages that ultraviolet.Research shows that Yak-skin Gelatin is former to have many advantages, such as content is more, and bioactivity is strong, especially wild yak
Ox, its growing environment is pollution-free, is food with grass, prepares Yak-skin Gelatin original by raw material of its skin, its quality is more pure.In recent years
Come, with the strategy implementation of development of the West Regions and a generation all the way, the cultivation of Tibetan area yak and secondary industry development are very fast.Yak skin conduct
The accessory substance of Yak production exploitation, it comprehensively utilizes a major issue as industry development.Traditional is used for skin tanning
The processing of clothes, leather shoes, belongs to devalued processing, and pollutes larger, is not suitable for the environmental protection and development of resources of Tibetan area
It is required that.Therefore, yak skin is comprehensively utilized, develops the collagen of macromolecule collagen and healthy functions with high added value
The products such as peptide, its process can make full use of resource using biotechnology as Main Means, caused of reduced contamination, very
Meet the strategic direction of development of resources.
In current yak collagen extraction technique, include the extraction of conventional kraft collagen, existing more research and report.
Typically, such as " a kind of method that complex enzyme for hydrolyzing prepares cow leather collagen " (application number：201610121685.4), by ox-hide
Soaking and washing, then boiling enzymolysis obtain molecular weight 3050-3300Da collagen, and its process characteristic is handled using boiling,
The complex enzyme for hydrolyzing such as papain, trypsase, Validase TSP Concentrate II are reused, to improve hydrolysis efficiency, reduce
Bitter taste is prepares purpose, but the multiple high temp processing that the invention is related in preparation process is very easy to cause collagen heat inactivation
(such as 90-100 DEG C of activated carbon decolorizing, more than 30min, spray drying inlet temperature are 170 DEG C etc.).In application number
(201610063666.0) in patent, to prepare the yak collagen with skin-care functional as target, water is employed
Putting forward technique makes to extract collagen from yak skin, and compared to conventional acid system, alkaline process, enzyme process, not only time-consuming for single water extraction, consumption
Can be high, or even the raw material corruption apt to deteriorate in preparation process, therefore the fado stays in laboratory research level, and in industry
Middle application is less.In patent application 201610835998.6, a kind of preparation technology for the tire bovine collagen that stops blooding is relate to, it is used
Solvent containing Cyclopropyl Bromide and pyrrolidones, to the ox-hide particulate of the crushing that was freeze-dried (<50um) collagen is carried out to carry
Take.This technique is related to many more manipulations such as solvent pre-treatment, collagen extraction, collagenous degeneration processing, and process is very cumbersome, only used
Solvent processing just needs many more manipulations (be layered and can use after multiple different frequency is ultrasonically treated, heating cools down again), is not suitable for
Quantization processing is carried out to large yak skin resource.In summary, it is overall extraction to be present in existing ox-hide collagen extraction process
Efficiency is low, the collagen of extraction is of poor quality, extraction process complicated difficult to industrialize, the problems such as product is single, in addition, it is existing specially
Property representation almost is not carried out to the collagen for preparing gained in profit.
The present invention is intended to provide it is a kind of can carry out scale processing and extraction collagen quality it is high, resource can be realized
The yak collagen extraction process efficiently utilized, specifically, the extraction of two kinds of yak collagens will be related to, one kind is high
The macromolecular collagen protein of bioactivity, it is characterized in that molecular weight is high, available for biomedical material, such as it is used as artificial organ material
Material or drug delivery materials use, and another is low molecule collagen caused by production process, by compound ferment treatment, form glue
Former protein peptides, the Industry Foundation available for healthy food, skin care item composition, at present existing maturation.
Creative use biology extractive technique of the present invention, it is intended to reduce the pollution of extraction process, improve the bioactivity of product.
Specifically, the removing that keratinase carries out yak hide is employed；The extraction of macromolecular collagen is carried out using pepsin；Adopt
Low molecular collagen peptide preparation is carried out with compound protease.To improve the bioactivity of macromolecular collagen, biology is being carried out
Link is extracted, is prepared using low-temperature treatment, and using freeze-drying.These technologies are applied and its science integrates, and are this items
Purpose core is innovated.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of multi-step enzyme method coordinate system it is standby hide Yak-skin Gelatin original albumen and
The method of collagen polypeptide, this method can obtain medical macromolecular collagen simultaneously and can be prepared into the small molecule glue of nutrient oral liquor
Former peptide, collagen extraction efficiency high, and collagen tertiary structure are stable, can be directly used for biomaterial.
In order to solve the above technical problems, realizing foregoing invention purpose, present invention employs following technical scheme：
(1) enzyme unhairing：Keratinase processing, carries out yak skin depilation；
(2) degreasing：The yak skin that step 1) obtains is passed through into degreaser ungrease treatment；
(3) flaking is freezed：The yak skin that step 2) is obtained digs flakiness or thread；
(4) enzyme process extracts：The yak skin that step 3) place is obtained carries out pepsin enzymolysis, centrifuges supernatant and cortex
Residue；The pepsin in supernatant is inactivated after enzymolysis；
(5) collagen refines：Supernatant obtained by step (4) is further precipitated, dialysed and concentrated, is obtained using freeze-drying method
Must have higher structure and the collagen of bioactivity；
(7) prepared by collagen peptide：Cortex residue obtained by step (4) is further used into compound protease, supernatant is concentrated and dried
Obtain small molecule Yak-skin Gelatin original polypeptide and amino acid.
Concrete technology step is as follows：
(1) enzyme unhairing：Clean up the decontamination of yak skin and repeatedly, shave off subcutaneous fat with knife, be immersed in containing angle
In protein enzyme solution, 1~3h of immersion carries out depilation processing under the conditions of temperature is 40~60 DEG C；
(2) degreasing：Yak skin is subjected to ungrease treatment with the degreaser of 3~5 times of 30~50 DEG C of volumes；
(3) flaking is freezed：12~24h is freezed under the conditions of yak skin after degreasing of losing hair or feathers is placed in into -80 DEG C, by the yak of freezing
Ox-hide digs the thin slice or thread into 0.2~1.0mm；
(4) enzyme process extracts：The yak skin after above-mentioned processing is taken, the 0.05~0.5mol/L acetic acid for adding 3~5 times of volumes is even
5~10min is starched, the pepsin (every 10~25g tissues need 1~2g pepsins) of certain mass is added, in 4 DEG C~40 DEG C
Under conditions of digest 2~5 days, centrifuge filtrate supernatant and undegraded complete cortex and residue；
(5) pH value of supernatant is adjusted with NaOH solution to 9~11,12~24h is stood, inactivates pepsin；
(6) collagen refines：Collagen solution is further precipitated, dialysed and concentrated, freeze-drying method is reused and is had
There are higher structure and the collagen of bioactivity.
(7) prepared by collagen peptide：By undegraded complete cortex, further using pepsin, (every 10~20 grams surplus with residue
Remaining skin slag needs 1~2g pepsins) 6~10h of depth growth under the conditions of 40~60 DEG C, centrifugation, discard residue and collect
Clearly, it is small molecule Yak-skin Gelatin original polypeptide and amino acid after concentrate drying.
The keratinase concentration is 0.1%~0.5% (V/V).
Yak skin in the step (1) derives from the peculiar ox kind yak wildyad in Tibet, is the characteristic yak of Tibetan areas
Ox resource.
Degreaser in the step (2) refers to 8~10% Na2CO3One kind or cooperation in solution, acetone and ether
Use.
The pepsin used in the step (4), the pepsin product of preferential general commercialization, including contain stomach
The compound protease of protease；Its suitable hydrolysis temperature is 4 DEG C~40 DEG C；4 DEG C~10 DEG C of prioritizing selection, can obtain activity compared with
High collagen.
The separation method used in the step (5), using ethanol precipitation, saltout or 40KDa~100KDa milipore filter
Partition method, include the combined process of the above method.
The drying means used in the step (5), using freeze-drying, with ensure the complete structure of macromolecular collagen and
Bioactivity.
The method for concentration used in the step (6), using any one in centrifugal concentrating, ultrafiltration concentration, concentrated by rotary evaporation
Kind.
The drying means used in the step (7), using any one in freeze-drying, spray drying, oven drying
Kind.
Tibet yak skin is successfully prepared into collagen by the present invention due to taking above-mentioned technical proposal by conbined enzymolysis,
It the advantage is that while prepared macromolecular collagen and small molecule collagen peptide, macromolecular collagen can be used as bio-medical material
Material, small molecule collagen peptide can be made into nutrient oral liquor, and the added value for realizing Tibetan areas characteristic resources maximizes exploitation and profit
With.
The present invention has advantages below compared with prior art：
1st, the present invention carries out depilation processing using the keratinase obtained by this laboratory early-stage Study, is pushed away with conventional physics
Physics pushes away a mao method and compared after hair, chemical immersion, and enzymatic isolation method depilation flow is simple, efficiency high, and depilation is more thorough, is lost hair or feathers with chemical method
80% is improved compared to efficiency.
2nd, raw material of the invention comes from characteristic resources-yak of Tibetan areas, by enzymatic isolation method from yak skin substep
Macromolecular collagen and small molecule collagen polypeptide are extracted, wherein macromolecular collagen can be applied to artificial skin, artificial blood vessel, stop
The sea of blood is continuous, cardiac valves, and medical tissue engineering material, small molecule collagen polypeptide and the Freamine Ⅲ such as operation suture thread can be used for
Nutrition oral administration and skin care products are prepared, realizes the value maximization of yak skin resource, the side of providing is develop and useedd for it
Method and approach.
3rd, the present invention using yak leather for collagen tailing as raw material, continue enzymolysis and prepared small molecule collagen polypeptide
And Freamine Ⅲ, enzymolysis liquid are rich in several amino acids, there is higher nutritive value.
4th, products obtained therefrom of the present invention is made using biological method, and technique is simple, and securely and reliably, collagen extraction efficiency is higher than biography
System high temperature enzyme process 20%, collagen quality is high, and tertiary structure is stable, can be used as biomedical material.
5th, the technology such as flaking chopping of the present invention, keratinase depilation, pepsin low temperature hydrolysis, freeze-drying
Method is also substantially distinguished from other collagen preparation technologies.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is that the SEM of macromolecular collagen of the present invention schemes.
Fig. 2 is the infrared analysis figure of macromolecular collagen of the present invention
Fig. 3 is the SDS-PAGE protein electrophoresis figures of macromolecular collagen of the present invention.
The SDS-PAGE protein electrophoresis figures of Fig. 4 small molecule collagen polypeptides of the present invention
Embodiment
The depilation processing of the yak skin of embodiment 1
It is as follows using simultaneously comparative chemistry depilation and the hair removal effect of this patent enzymolysis epilation, method：(1) with reference to patent
Chemical depilation in 201710089525.0, proportionally 100 parts of water add 12 parts of vulcanized sodium and 30 parts of white lime mixed preparings to take off
Hair cream, then by the tiling of yak skin on the ground, surface is downward, and hair side is upward, and the depilatory cream film configured is on hair side, heap
Hair is manually pushed away after putting 5 hours, is rinsed well；(2) keratinase epilation in this patent：By fresh Tibet yak skin decontamination,
Clean up repeatedly, subcutaneous fat is shaved off with knife, be then placed in 0.2% keratin enzyme liquid, 50 DEG C of immersion 1h are lost hair or feathers
Processing；The depilation yak skin obtained after above-mentioned processing is cleaned up repeatedly with clear water, cleans remaining soft flocks, and with 5 times of volumes
40 DEG C of warm water immersion 20min dealkalizes, be repeated 3 times.
The enzyme unhairing that the present invention uses is more thorough, and efficiency improves 80% compared with chemical method loses hair or feathers.
The different epilation comparative results of table 1
The preparation of the yak skin macromolecular collagen of embodiment 2
Collagen extraction method can be summarized as alkaline process, salt method, water extraction, acid system and enzyme process, and wherein alkaline process easily causes peptide bond
Hydrolysis, and if hydrolysis is serious, DL- type amino acid racemic mixtures can be produced, if wherein D- types amino acid is higher than L-type amino
Acid, then it can suppress the absorption of L-type amino acid, and some D- type amino acid are poisonous, and easy teratogenesis is carcinogenic, therefore is applied in extracting
It is less.Salt method technique is not easy stabilization, and obtained collagen crystal is smaller, and accretion rate is fast, thus also not frequently with.Between
More than, we employ water extraction simultaneously, acid carries and invention enzyme process extracts macromolecular collagen protein, and compare production
The difference of thing.
(1) water extraction：With reference to patent 201610063666.0, yak skin is taken, loses hair or feathers after degreasing, cleans up in feed liquid
It is 1 than (g/g):5th, temperature 60 C, extraction time 2 times, water extraction under conditions of 4 hours, obtains extract solution every time, centrifugation, micropore
2 μm of filterings of filter membrane, are freeze-dried after filter vacuum concentration and produce.
(2) sour formulation：Reference literature, yak skin is taken, after degreasing of losing hair or feathers, with 0.05mol/L sodium citrate buffer solutions
(pH3.98) extract, centrifuge, 2 μm of membrane filtrations, be freeze-dried and produce after filter vacuum concentration.
(3) high temperature enzyme formulation：With reference to patent 201710089525.0, the yak skin particle for degreasing of losing hair or feathers is added into 4 times of bodies
2% alkaline matter and 2% enzyme preparation of ponding and skin granular mass, stirred 8 hours with 100 revs/min at 40 DEG C, obtain collagen
Protein liquid, centrifugation, 2 μm of membrane filtrations, it is freeze-dried and produces after filter vacuum concentration
(4) cold-adapted enzyme formulation of the present invention：Yak skin after degreasing is lost hair or feathers freezes 12h under the conditions of being placed in -80 DEG C, will freeze
Hard yak skin uses section power blader into the thin of 0.5mm.The yak skin after above-mentioned processing is taken, adds the 0.5mol/L second of 5 times of volumes
Acid homogenate 5min, the ratio for needing 1g enzymes in every 10g tissues add pepsin, digest 4 days, centrifuge, 2 μ under conditions of 4 DEG C
M membrane filtration separating filtrate supernatants and undegraded complete cortex and residue.It is quiet with the pH value of NaOH solution regulation supernatant to 11
12h is put, inactivates pepsin, is freeze-dried and produces after filter vacuum concentration.
Hydroxyproline is the distinctive amino acid of collagen, and the content of hydroxyproline is directly proportional to collagen content in extract solution, because
This determines collagen yield by calculating the content of hydroxyproline.Calculation formula is as follows：
The total amount of hydroxyproline (%) in the total amount of hydroxyproline/yak skin in recovery rate=extract solution.
The distinct methods of table 2 obtain collagen yield comparative result
The low temperature enzyme process that the present invention uses makes the extraction efficiency of collagen want that educating highest high position enzyme process improves 20%.
The yak macromolecular collagen of embodiment 3 characterizes
Yak skin after degreasing is lost hair or feathers freezes 12h under the conditions of being placed in -80 DEG C, by the yak skin of adfreezing with section power blader into
0.5mm's is thin.The yak skin after above-mentioned processing is taken, the 0.5mol/L acetic acid homogenate 5min of 5 times of volumes is added, is organized by every 10g
Need the ratio of 1g enzymes to add pepsin, digested 4 days under conditions of 4 DEG C, centrifuged, 2 μm of membrane filtration separating filtrate supernatants
With undegraded complete cortex and residue.With the pH value of NaOH solution regulation supernatant to 11,12h is stood, inactivates pepsin,
It is freeze-dried and produces after filter vacuum concentration.The collagen sponge after freeze-drying is taken, is scanned using SEM
Take pictures, observe the filamentary structure and crosslinking situation of collagen.Sem analysis (Fig. 1) is shown, using the collagen of this law acquisition
Aperture is more, preferably remains the original fibrillar meshwork structure of collagen.Collagen be in organism multiple tissues into point it
One, its as a kind of natural polymer biomaterial, have low immunogenicity, excellent biocompatibility, biodegradable,
Many advantages, such as cell growth and anastalsis can be promoted, if the collagen of extraction can retain original fibre structure, can successfully it use
Make guiding material, artificial skin substitute of tissue etc..
The accurate macromolecular collagen that weighs freezes sample 1mg, adds physiological saline 1mL dissolvings, takes out 50 μ L, adds 48 μ L's
2 × SDS buffer solutions and 2 μ L 2 mercapto ethanol are well mixed, and 10min is denatured in 100 DEG C, slightly cold rear brief centrifugation, are taken
It is clear to carry out SDS-PAGE electrophoresis.Electrophoresis result is shown, prepares the macromolecular tropocollagen molecule amount of gained up to 120KDa and the above (figure
2), larger molecular weight further illustrates it and remains the complete triple-helix structure of collagen, structures shape property, therefore these
Peculiar structure remains the good characteristics such as the good biocompatibility of collagen, degradability, low antigenicity, so as to suitable for biology
The exploitation of medical material.
Obtained macromolecular collagen freeze-dried powder is subjected to infrared spectrum analysis.Macromolecular sample has collagen infrared spectrum
Characteristic absorption peak, i.e. acid amides A, B, I, II and III absworption peaks.Acid amides A absworption peak is in 3440-3400cm-1Place, when containing N-H
When the peptide fragment of group participates in hydrogen bond formation, absworption peak can reduce 100cm caused by its stretching vibration-1Left and right, it is seen that macromolecular glue
Raw sample is in 3200cm-1There is absworption peak (Fig. 3) left and right, illustrates that intramolecular has hydrogen bond.Protein molecular methylene group can be sent out
Raw asymmetric stretching vibration so that acid amides B absworption peak is in 3080cm-1Place occurs, it is seen that macromolecular Collagen specimens exist
3000cm-1There is absorption (Fig. 3) at place, illustrates methylene based structures be present, and methylene based structures are the characteristic groups of tertiary structure, explanation
The macromolecular collagen protein obtained by this law preferably maintains the tertiary structure of protein.Amido link I, II, III are reflections
The most important absworption peak of protein peptide chain skeleton structure, wherein acid amides I characteristic absorption peak is in 1700-1600cm-1Place, and absorb
Spike number is bigger, and the degree of order of albumen peptide backbone is higher；Acid amides II absworption peak is usually located at 1600-1500cm-1Between, it is
By alpha-helix, β-spiral, β-corner and absorption band caused by random coil superposition collective effect；Acid amides III absworption peak is usual
Positioned at 1300-1200cm-1Between, acid amides III presence can prove whether the triple-helix structure of collagen keeps complete.From
As a result it is visible, macromolecular collagen is 1650,1550,1250cm-1There is absorption, illustrate acid amides I, II, III key be present, further
Illustrate that macromolecular collagen remains complete triple-helix structure.And exactly this triple-helix structure imparts the good biology of collagen
The good characteristics such as compatibility, degradability, low antigenicity and anthemorrhagic performance, no longer possess these after it loses triple-helix structure
Bioactivity, it cannot also be used as biomedical material.
Illustrate possess distinctive three by the collagen that low temperature enzymatic isolation method obtains with reference to SEM and infrared scan profiling results
Helical structure and original fibre structure, structures shape property, therefore these peculiar structures remain the good biology of collagen
The good characteristics such as compatibility, degradability, low antigenicity, so as to the exploitation suitable for biomedical material.
Collagen polypeptide phenetic analysis in the enzymolysis liquid of embodiment 4
The SDS-PAGE analyses of collagen polypeptide
The accurate collagen polypeptide that weighs freezes sample 1mg, adds physiological saline 1mL dissolvings, takes out 50 μ L, adds the 2 of 48 μ L
× SDS buffer solutions and 2 μ L 2 mercapto ethanol are well mixed, and 10min is denatured in 100 DEG C, slightly cold rear brief centrifugation, take supernatant
Carry out SDS-PAGE electrophoresis.Data shows that collagen polypeptide molecular weight is between 0.5-15KDa, and more than 15KDa macromolecular peptide
Belong to gelatin (molecular weight is from tens of thousands of to hundreds of thousands), it was found from electrophoresis result, peptides are wide in variety in the hydrolyzate of last gained,
It is concentrated mainly on (Fig. 4) between 4-6KDa.
The species and content analysis of Freamine Ⅲ
Enzymolysis liquid is subjected to protein deposition with the TCA of 4 times of volumes, after 12,000rpm centrifuge 5min, takes supernatant through 0.22 μm
Water system membrane filtration, after derivatization carry out gas-chromatography separation and measure.Experimental data proves that Yak skin glue protein contains
Amount is higher than donkey-hide gelatin, and as many as 17 kinds are up to containing amino acid, and the contents of Most amino-acids is higher than donkey-hide gelatin, wherein alanine, tyrosine,
The content of phenylalanine is high more than 4 percentage points, and a variety of nutrition and essential amino acid can be provided for human body, and being compared compared with donkey-hide gelatin has
Higher nutritive value.
The comparison of the extraction of table 1 gained amino acid and donkey-hide gelatin amino acid
Fibrous framework of the collagen as extracellular matrix, it is present in most thin-walled organ interstitial tissues, plays branch
Support and the close phase of formation of armour, the effect of tissue integrity, while collagen and human vas, skin, Google and cartilage
Close, and maintain the important leverage of each organ, tissue elasticity and toughness.Human needs has the main composition amino acid of substantial amounts of collagen
(glycine and proline) can synthesize enough collagen, and (1,000,000 tropocollagen molecules of synthesis need more than 1,000,000,000 glycine
Residue and 6.2 hundred million proline residues), and oral collagen peptide can solve the problems, such as collage synthesis raw material well.In addition big point
Sub- collagen peptide (i.e. gelatin) all has good to hair, nail, skin care and motion orthopaedic disease, degenerative joint disease
Therapeutic effect.
Therefore the extensive gelatin hydrolysate of molecular weight distribution and collagen peptide mixer can obtain by secondary hydrolytic residue,
It can be applied to a variety of scope Orally taken health article exploitations such as beauty, bone and joint diseases.
Claims (11)
1. the standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system, it is characterised in that including following step
Suddenly,
(1) enzyme unhairing：Keratinase processing, carries out yak skin depilation；
(2) degreasing：The yak skin that step 1) obtains is passed through into degreaser ungrease treatment；
(3) flaking is freezed：The yak skin that step (2) is obtained digs flakiness or thread；
(4) enzyme process extracts：The yak skin that step (3) place is obtained carries out protease hydrolyzed, centrifuges supernatant and cortex residue；
The pepsin in supernatant is inactivated after enzymolysis；
(5) collagen refines：Supernatant obtained by step (4) is further precipitated, dialysed and concentrated, freeze-drying, which obtains, has biology living
The macromolecular collagen of property；
(7) prepared by collagen peptide：Cortex residue obtained by step (4) is further used into compound protease, supernatant, which is concentrated and dried, to be obtained
Small molecule Yak-skin Gelatin original polypeptide and amino acid.
2. the method described in claim 1, it is characterised in that comprise the following steps that：
(1) enzyme unhairing：Clean up the decontamination of yak skin and repeatedly, shave off subcutaneous fat with knife, be immersed in containing keratin
In enzyme solutions, 40-60 DEG C of immersion 5-10h carries out depilation processing；
(2) degreasing：Ungrease treatment is carried out by the degreaser of yak cortex 3-5 times of volume of amount, temperature control is at 30-50 DEG C；
(3) flaking is freezed：By -80 DEG C of freezing 12-24h of yak skin after degreasing of losing hair or feathers, the yak skin of freezing is dug into 0.2-
1.0mm thin slice is thread；
(4) enzyme process extracts：The yak skin after above-mentioned processing is taken, adds the 0.05-0.5mol/L acetic acid homogenate 5- of 3-5 times of volume
10min, organize to add the amount of 1-2g pepsins by every 10-25g, add 4 DEG C -40 DEG C of pepsin and digest 2-5 days, centrifugation point
From supernatant and cortex residue；With the pH value of NaOH solution regulation supernatant to 9-11,12-24h is stood, inactivates pepsin；
(5) collagen refines：Step (4) resulting solution is further precipitated, dialysed and concentrated, is had using freeze-drying method
There are higher structure and the collagen of bioactivity；
(6) prepared by collagen peptide：Cortex residue obtained by step (4) is further used into 40-60 DEG C of enzymolysis 6-10h of compound protease,
Centrifugation, discards residue and collects supernatant, and supernatant obtains small molecule Yak-skin Gelatin original polypeptide and amino acid after being concentrated and dried.
3. the method described in claim 1 or 2, it is characterised in that the keratinase concentration is 0.1%-0.5% (V/V).
4. method as claimed in claim 1 or 2, it is characterised in that affiliated yak skin derives from the peculiar ox kind yak in Tibet
Wild yad, it is the characteristic yak resources of Tibetan areas.
5. method as claimed in claim 1 or 2, it is characterized in that, the degreaser in step (2) refers to 8-10% Na2CO3It is molten
One kind in liquid, acetone and ether is used cooperatively.
6. the method as described in claim 1-3 is any, it is characterized in that, the pepsin preferred generic is used in step (4)
Commercialization pepsin product, including the compound protease containing pepsin；Preferably 4 DEG C -10 DEG C of the hydrolysis temperature.
7. method as claimed in claim 2, it is characterized in that, precipitation, dialysis and the method for concentration used in step (5), specifically
Refer to using ethanol precipitation, the milipore filter concentrating and separating method saltoutd with 40KDa-100KDa.
8. method as claimed in claim 1 or 2, it is characterized in that, the drying means used in step (5) is freeze-drying, with
Ensure the complete structure and bioactivity of macromolecular collagen.
9. the method as described in claim 1-3 is any, it is characterized in that, the method for concentration used in step (6) can use centrifugation
Concentration, it is concentrated by ultrafiltration, any one in concentrated by rotary evaporation.
10. the method as described in claim 1, it is characterized in that, the drying means used in step (6) can use freeze-drying,
Spray drying, any one in oven drying.
11. the method as described in claim 1, it is characterized in that, the compound protease used in step (6) includes acidic protein
Enzyme, alkali protease, neutral proteinase, bromelain, papain, trypsase are a kind of or it is combined.
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| CN201711126104.7A CN107653291A (en) | 2017-11-15 | 2017-11-15 | The standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system |
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| CN109295145A (en) * | 2018-10-31 | 2019-02-01 | 西藏央金生态农牧科技有限公司 | A kind of preparation method of Ultra-low molecular weight yak collagen peptide |
| CN109355336A (en) * | 2018-11-09 | 2019-02-19 | 贵州梵净山生态农业股份有限公司 | A kind of extracting method of cow leather collagen |
| CN109371086A (en) * | 2018-11-29 | 2019-02-22 | 沾化恒鑫工业科技有限公司 | A method of bovine collagen albumen is produced using cattle hide gelatin tail road glue |
| CN109406690A (en) * | 2018-11-25 | 2019-03-01 | 山东达因海洋生物制药股份有限公司 | A kind of method in relation to substance in detection chloraldurate |
| CN109730188A (en) * | 2019-01-30 | 2019-05-10 | 西藏央金生态农牧科技有限公司 | Derived from the Antihypertensive Peptides and preparation method thereof of yak skin |
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| CN109295145A (en) * | 2018-10-31 | 2019-02-01 | 西藏央金生态农牧科技有限公司 | A kind of preparation method of Ultra-low molecular weight yak collagen peptide |
| CN109355336B (en) * | 2018-11-09 | 2021-11-16 | 贵州梵净山生态农业股份有限公司 | Method for extracting cow hide collagen |
| CN109355336A (en) * | 2018-11-09 | 2019-02-19 | 贵州梵净山生态农业股份有限公司 | A kind of extracting method of cow leather collagen |
| CN109406690A (en) * | 2018-11-25 | 2019-03-01 | 山东达因海洋生物制药股份有限公司 | A kind of method in relation to substance in detection chloraldurate |
| CN109406690B (en) * | 2018-11-25 | 2022-02-25 | 山东达因海洋生物制药股份有限公司 | Method for detecting related substances in chloral hydrate |
| CN109371086A (en) * | 2018-11-29 | 2019-02-22 | 沾化恒鑫工业科技有限公司 | A method of bovine collagen albumen is produced using cattle hide gelatin tail road glue |
| CN109730188A (en) * | 2019-01-30 | 2019-05-10 | 西藏央金生态农牧科技有限公司 | Derived from the Antihypertensive Peptides and preparation method thereof of yak skin |
| CN109836474A (en) * | 2019-03-19 | 2019-06-04 | 中国科学院西北高原生物研究所 | It is a kind of to have effects that yak skin polypeptide tonified the blood and arrest bleeding and preparation method thereof |
| CN110151664A (en) * | 2019-06-05 | 2019-08-23 | 中国科学院西北高原生物研究所 | A kind of Yak-skin Gelatin skin-protection product and preparation method thereof for dispelling red blood trace on face |
| CN110229863A (en) * | 2019-06-21 | 2019-09-13 | 中国科学院西北高原生物研究所 | A kind of yak glue preparation method enhancing osteoblast ability |
| CN110229864A (en) * | 2019-06-21 | 2019-09-13 | 中国科学院西北高原生物研究所 | A kind of animal glue preparation method promoting platelet activation |
| CN110563834A (en) * | 2019-09-25 | 2019-12-13 | 成都奇璞生物科技有限公司 | Collagen extraction method |
| CN111118093A (en) * | 2020-01-12 | 2020-05-08 | 青海瑞肽生物科技有限公司 | Non-denatured yak skin collagen and preparation method thereof |
| CN111944866A (en) * | 2020-07-31 | 2020-11-17 | 青海瑞肽生物科技有限公司 | Method for preparing yak hide small molecule collagen peptide by continuous rotary evaporation, desolventizing and double enzymolysis |
| CN111944866B (en) * | 2020-07-31 | 2023-06-20 | 青海瑞肽生物科技有限公司 | Method for preparing small molecular collagen peptide of yak leather by continuous rotary steaming desolventizing double enzymolysis |
| CN113912703A (en) * | 2021-03-15 | 2022-01-11 | 陕西医赛尔生物科技有限公司 | Preparation method and application of whitening and spot-lightening active bovine achilles tendon collagen peptide |
| CN113599299A (en) * | 2021-08-25 | 2021-11-05 | 西藏银美科技股份有限公司 | Wrinkle-removing nano cosmetic composite material and preparation thereof |
| CN113599299B (en) * | 2021-08-25 | 2024-02-23 | 西藏银美科技股份有限公司 | Wrinkle-removing nano cosmetic composite material and preparation thereof |
| CN114317661A (en) * | 2022-02-15 | 2022-04-12 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
| CN114317661B (en) * | 2022-02-15 | 2024-04-09 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
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