CN108192941A - A kind of method of quality control of biologically active collagen - Google Patents
A kind of method of quality control of biologically active collagen Download PDFInfo
- Publication number
- CN108192941A CN108192941A CN201810189372.1A CN201810189372A CN108192941A CN 108192941 A CN108192941 A CN 108192941A CN 201810189372 A CN201810189372 A CN 201810189372A CN 108192941 A CN108192941 A CN 108192941A
- Authority
- CN
- China
- Prior art keywords
- collagen
- quality control
- tendon
- acetic acid
- molecular weight
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- 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
Abstract
The invention discloses a kind of method of quality control of biologically active collagen.The present invention realizes the high efficiency extraction to Cowhells tendon type i collagen using the condition of acid enzymolysis, optimizes the production technology of collagen, by changing enzyme reaction time, dialysis time and bag filter molecular cut off size, controls the molecular weight and molecular weight distribution of collagen；By the way that end peptide is gone to control the immunogenicity of collagen；The bioactivity of collagen is determined by the triple-helix structure of transmission electron microscope characterization collagen, prepared active type I collagen has reached international standards, and the further application for collagen provides the foundation.
Description
Technical field
The present invention relates to collagen extraction techniques fields, and in particular to a kind of quality control side of biologically active collagen
Method.
Background technology
Collagen be animal in-vivo content at most, the most wide protein of distribution, be widely present in connective tissue, skin,
The positions such as bone, internal organ and tendon, ligament, sclera are the main constituents of extracellular matrix, and wherein Type I collagen is using most
It is extensive.Collagen has low immunogenicity, controlled degradation, catabolite nontoxic, includes and promotes cell adherence and life
Long RGD (is made of) structural domain arginine, glycine and aspartic acid, can induce cell migration, stimulate cellular proliferation, be compared with
One of early biomaterial ratified by FDA and SFDA.Active collagen can be with fibrin, glycosaminoglycan, chitosan, alginic acid
Salt etc. is built into compound rest or growth factor-loaded such as bFGF, BMP or loaded gene, stem cell etc., for promoting bone, flesh
The reparation of the tissues such as meat, artificial blood vessel, nerve, cornea has non-in fields such as wound dressing, artificial skin and drug releases
Often wide application prospect.
The extracting method of collagen mainly includes acid extraction method, alkaline process, salt method and enzyme process, and collagenous biological activity and its molecule
It measures quality size and triple helix structure is closely related, how to keep the bioactivity of collagen is the key that extraction process, therefore,
It is necessary to the extraction process to collagen to optimize, and improves collagenous biological activity.
Invention content
It is an object of the invention to provide a kind of biologically active collagen in place of overcome the deficiencies in the prior art
Method of quality control optimizes collagen extraction technological parameter, improves the bioactivity of collagen.
To achieve the above object, the technical solution that the present invention takes is as follows：
A kind of method of quality control of biologically active collagen, includes the following steps：
(1) tendon pre-processes：Degreasing is carried out to tendon, is sliced and disinfects；
(2) extraction of collagen：It will be crushed through step (1) pretreated tendon, 0.5 is added in the tendon of crushing
The acetic acid of~1.0mol/L and the pepsin of 1000~3000U/L are digested, and remove end peptide, and hydrolysis temperature is 10 DEG C~30
DEG C, the enzyme digestion reaction time is 48~144h, and supernatant is taken after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, will precipitation again with 0.1~
The acetic acid of 0.5mol/L or the hydrochloric acidolysis of 0.05~0.1mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen
Solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 50~100kDa, with purifying
The acetate buffer of water or pH 4.5~5.0 dialyse 2~5 days as extracellular fluid dialysis, obtain the biologically active glue
Stoste.
Above-mentioned technical proposal realizes the high efficiency extraction to tendon type i collagen using the condition of acid enzymolysis, passes through change
The enzyme reaction time, dialysis time and bag filter molecular cut off size, optimized production process, control collagen molecular weight and
Molecular weight distribution；By the end peptide structure of the effect excision collagen of pepsin under acid condition, so as to control the immune of collagen
Originality.
The preferred embodiment of method of quality control as collagen of the present invention, further including to described there is biology to live
Property collagen carry out molecular weight determination, end peptide removal detection and triple-helix structure identify the step of.
Research shows that albumen of the collagen molecules amount obtained according to the technical program in more than 90kDa is no less than
80%, collagen has higher bioactivity.
When collagen is applied to titanium alloy surface coating, collagenic coating is in the homogeneity of titanium alloy surface and tropocollagen molecule amount point
Cloth is related, and the distribution of tropocollagen molecule amount is narrower, and collagenic coating is coated with more uniform in titanium alloy surface.Therefore the molecular weight of collagen is controlled
It is of great significance to the homogeneity and bioactivity of collagenic coating.
The main immunogenic site of collagen is the non-helical amino acid region at collagen both ends, is called end peptide.For will be low
The immunogenicity of collagen selectively cuts off end peptide when extracting collagen using pepsin.Therefore, the end peptide for detecting collagen is gone
It is extremely important to its safety except situation.
Complete triple helix structure has decisive influence to the bioactivity of collagen, possesses complete triple helix structure
Tropocollagen molecule has the site that cell can combine on its strand, and cell is according to the space at tropocollagen molecule binding site
Structure and functional group identify.So verification collagen is most important with triple-helix structure.
The preferred embodiment of method of quality control as collagen of the present invention, in the step (2), enzyme digestion reaction
Time is 72~120h.
The preferred embodiment of method of quality control as collagen of the present invention, in the step (2), hydrolysis temperature
It is 20 DEG C~25 DEG C.
The preferred embodiment of method of quality control as collagen of the present invention, in the step (2), acetic acid it is dense
It spends for 0.8~1.0mol/L.
The preferred embodiment of method of quality control as collagen of the present invention, in the step (2), tendon：Second
Acid：The mass ratio of pepsin is 1：1.3~2.8：0.006~0.022.
The preferred embodiment of method of quality control as collagen of the present invention, in the step (3), acetic acid it is dense
It spends for 0.1~0.25mol/L, a concentration of 0.06~0.08mol/L of hydrochloric acid.
The preferred embodiment of method of quality control as collagen of the present invention, in the step (4), bag filter
Molecular cut off is 90~100kDa, and dialysis time is 3 days.
The preferred embodiment of method of quality control as collagen of the present invention, the tendon are Cowhells tendon.
Compared with prior art, advantage of the invention is that：
The present invention realizes the high efficiency extraction to Cowhells tendon type i collagen using the condition of acid enzymolysis, anti-by changing enzyme
Between seasonable, the time of dialysis and bag filter molecular cut off size, control the molecular weight and molecular weight distribution of collagen；By going
Hold the immunogenicity of peptide control collagen；The bioactivity of collagen, institute are determined by the triple-helix structure of transmission electron microscope characterization collagen
The active type I collagen of preparation has reached international standards, and the application for collagen provides the foundation.
Description of the drawings
The SDS-PAGE collection of illustrative plates of collagen that Fig. 1 is embodiment 1, prepared by embodiment 2 and embodiment 5, (1.Takara eggs
White molecular weight marker (height)；2. 20150501B batches；3. 20150502B batches；4. 20150503B batches；5.sigma reference substances
CAS 9007-34-5)；
Fig. 2 is 20150501B batches of tropocollagen molecule amount distribution maps；
Fig. 3 is 20150502B batches of tropocollagen molecule amount distribution maps；
Fig. 4 is 20150503B batches of tropocollagen molecule amount distribution maps；
Fig. 5 is collagenous fibres transmission electron microscope picture.
Specific embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with the drawings and specific embodiments pair
The present invention further illustrates.It will be appreciated by those skilled in the art that specific embodiment described herein is only explaining this hair
It is bright, it is not intended to limit the present invention.
Test method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.
Embodiment 1
A kind of embodiment of the preparation method of method of quality control as collagen of the present invention, described in the present embodiment
The preparation method of method of quality control of collagen include the following steps：
(1) tendon pre-processes：Degreasing is carried out to tendon, is sliced and disinfects；
(2) extraction of collagen：Freezing smashing will be carried out through the pretreated tendon of step (1), added in the tendon of crushing
Enter acetic acid and 3000U/L pepsins are digested, a concentration of 0.75mol/L of acetic acid, tendon：Acetic acid：The matter of pepsin
Amount is than being 1：1.3：0.006, end peptide is removed, hydrolysis temperature is 30 DEG C, and enzymolysis time 48h takes supernatant after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, precipitation is used again
The acetic acid acidolysis of 0.2mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 50kDa, by the use of purified water as
Extracellular fluid dialysis dialyses 5 days, obtains the biologically active collagen stoste；
(5) molecular weight determination, end peptide removal detection and three spiral knots are carried out to the biologically active collagen stoste
Structure is identified.
Embodiment 2
A kind of embodiment of the preparation method of method of quality control as collagen of the present invention, described in the present embodiment
The preparation method of method of quality control of collagen include the following steps：
(1) tendon pre-processes：Degreasing is carried out to tendon, is sliced and disinfects；
(2) extraction of collagen：Freezing smashing will be carried out through the pretreated tendon of step (1), added in the tendon of crushing
The pepsin for entering acetic acid and 2500U/L is digested, a concentration of 1.0mol/L of acetic acid, tendon：Acetic acid：The matter of pepsin
Amount is than being 1：2.5：0.02, end peptide is removed, hydrolysis temperature is 20 DEG C, and enzymolysis time 120h takes supernatant after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, precipitation is used again
The hydrochloric acidolysis of 0.06mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 100kDa, with pH's 5.0
Acetate buffer dialyses 3 days as extracellular fluid dialysis, obtains the biologically active collagen stoste；
(5) molecular weight determination, end peptide removal detection and three spiral knots are carried out to the biologically active collagen stoste
Structure is identified.
Embodiment 3
A kind of embodiment of the preparation method of method of quality control as collagen of the present invention, described in the present embodiment
The preparation method of method of quality control of collagen include the following steps：
(1) tendon pre-processes：Degreasing is carried out to tendon, is sliced and disinfects；
(2) extraction of collagen：Freezing smashing will be carried out through the pretreated tendon of step (1), added in the tendon of crushing
Enter acetic acid and 3000U/L pepsins are digested, a concentration of 0.5mol/L of acetic acid, tendon：Acetic acid：The quality of pepsin
Than being 1：1.6：0.015, end peptide is removed, hydrolysis temperature is 30 DEG C, and enzymolysis time 72h takes supernatant after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, precipitation is used again
The hydrochloric acidolysis of 0.06mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 90kDa, by the use of purified water as
Extracellular fluid dialysis dialyses 2 days, obtains the biologically active collagen stoste；
(5) molecular weight determination, end peptide removal detection and three spiral knots are carried out to the biologically active collagen stoste
Structure is identified.
Embodiment 4
A kind of embodiment of the preparation method of method of quality control as collagen of the present invention, described in the present embodiment
The preparation method of method of quality control of collagen include the following steps：
(1) tendon pre-processes：Degreasing is carried out to tendon, is sliced and disinfects；
(2) extraction of collagen：Freezing smashing will be carried out through the pretreated tendon of step (1), added in the tendon of crushing
The pepsin for entering acetic acid and 1000U/L is digested, a concentration of 0.8mol/L of acetic acid, tendon：Acetic acid：The matter of pepsin
Amount is than being 1：1.5：0.01, end peptide is removed, hydrolysis temperature is 25 DEG C, and enzymolysis time 84h takes supernatant after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, precipitation is used again
The acetic acid acidolysis of 0.5mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 80kDa, with the vinegar of pH 4.5
Phthalate buffer dialyses 4 days as extracellular fluid dialysis, obtains the biologically active collagen stoste；
(5) molecular weight determination, end peptide removal detection and three spiral knots are carried out to the biologically active collagen stoste
Structure is identified.
Embodiment 5
A kind of embodiment of the preparation method of method of quality control as collagen of the present invention, described in the present embodiment
The preparation method of method of quality control of collagen include the following steps：
(1) tendon pre-processes：Degreasing is carried out to tendon, is sliced and disinfects；
(2) extraction of collagen：Freezing smashing will be carried out through the pretreated tendon of step (1), added in the tendon of crushing
Enter acetic acid and 2000U/L pepsins are digested, a concentration of 0.9mol/L of acetic acid, tendon：Acetic acid：The quality of pepsin
Than being 1：2.8：0.022, end peptide is removed, hydrolysis temperature is 10 DEG C, and enzymolysis time 144h takes supernatant after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, precipitation is used again
The acetic acid acidolysis of 0.4mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 90kDa, with the vinegar of pH 5.0
Phthalate buffer dialyses 5 days as extracellular fluid dialysis, obtains the biologically active collagen stoste；
(5) molecular weight determination, end peptide removal detection and three spiral knots are carried out to the biologically active collagen stoste
Structure is identified.
Collagen performance measurement
1st, molecular weight determination
Embodiment 1 (lot number be 20150501B batch) is measured by SDS-PAGE methods, (lot number is embodiment 2
20150502B crowd) and the collagen that is prepared of embodiment 5 (lot number is that 20150503B is criticized).In polyacrylamide gel system
In, a certain amount of lauryl sodium sulfate (SDS) is added in, SDS is a kind of anionic detergent, in 100 DEG C of heating, in β-sulfydryl
In the presence of ethyl alcohol, the quaternary structure of most of polymer proteins can be made to disintegrate, and be securely joined with each subunit, make each Asia
Negative electrical charge in base band, and quantity is considerably beyond the original quantity of electric charge of protein molecule, so as to mask variety classes protein
Between original charge difference.At this point, the electrophoretic mobility of protein molecule depends primarily on its molecular size range, in certain model
In enclosing, the logarithm of molecular weight and relative mobility proportion relation.
LgM=a-bde/do(1)
deFor sample migration distance, doFor frontal migration distance (dyestuff), de/doAs relative mobility
Make standard curve using the reference substance of known molecular amount or the constant of standard curve is determined with calculating method, you can be obtained
The molecular weight of unknown sample.
Agents useful for same is shown in Table 1 with instrument.
1 molecule measuring test instrument of table and reagent
(1) preparation of reagents
1) collagenase digesting liquid：With 20mM NaH2PO4(pH 7.4 includes 0.1mM CaCl to solution2) dissolving clostridiopetidase A, match
Into liquid containing collagenase digesting.
2) acrylamide gel liquid storage：29g acrylamides, 1g methylene diacrylamides add water to be settled to 100mL, filtering,
It is protected from light in brown bottle, 4 DEG C of preservations.
3) separation gel buffer solution (1.5M Tris-HCl)：18.15g Tris with 1mol/L HCl tune pH to 8.8, add water
It is settled to 100mL.
4) concentration glue buffer solution (0.5M Tris-HCl)：6g Tris with 1mol/L HCl tune pH to 6.8, add water constant volume
To 100mL.
5) 10%SDS：10g SDS are settled to 100mL, room temperature preservation.
6) 10% ammonium persulfate：0.1g ammonium persulfates are settled to 1mL, now with the current.
7) sample-loading buffer：0.5M Tris-HCl 2.5mL, glycerine 2.0mL, beta -mercaptoethanol 2.0mL, bromine are measured respectively
Phenol indigo plant 0.1mg, ultra-pure water are settled to 10mL.
8) electrophoretic buffer：Tris 15.1g are weighed respectively, and glycine 72.0g, SDS 5.0g is dissolved in ultra-pure water, fixed
Hold to 1000mL, use preceding 5 times of dilution.
9) coomassie brilliant blue staining liquid：Coomassie brilliant blue R250 1g is weighed, adds in methanol 200mL, glacial acetic acid 50mL, water
250mL mixings.
10) destainer：Glacial acetic acid 100mL is measured respectively, and methanol 150mL is dissolved to 1000mL with pure water.
(2) experimental method
1) separation gel (7%) and concentration glue (4%) are made by table 2, it is to be solidified.
2 separation gel of table and concentration glue formula
2) sample presses 1 with sample-loading buffer：Point sample after 3~5min, 20 μ L (molecular weight marks of per pass are heated after 1 mixing for 100 DEG C
Quasi- product require loading according to specification).
3) constant pressure 110V or constant current 20mA/ glue (reference) are adjusted, electrophoresis is carried out, stops when bromophenol blue is close to offset plate bottom
Only electrophoresis.
4) it dyes, decoloration.
5) each band migration rate is measured.
6) it is recorded with image analysis system and analyzes band gray scale.
(3) interpretation of result
The collagen (lot number is respectively 20150501B batches, 20150502B batches and 20150503B batches) of three batches is carried out
SDS-PAGE electrophoresis, the results are shown in Figure 1.
As shown in Fig. 2, calculate the percentage that 20150501B batches of molecular weight account for total protein abundance in the albumen of more than 90kDa：
The molecular weight of Takara molecular weight of albumen marker (height) each band and mobility are substituted into formula (1), calculated
Constant a=2.36, b=2.33.According to the band abundance of Software Integration, the protein band (1 that molecular weight is less than 90kDa is calculated
~6 protein peaks) abundance and total protein abundance ratio (%), result 82.63%.I.e. molecular weight is accounted in the albumen of more than 90kD
The 82.63% of total protein.
Such as Fig. 3, calculate 20150502B batches of molecular weight account for the 93.94% of total protein in the albumen of more than 90kDa.
Such as Fig. 4, calculate 20150503B batches of molecular weight account for the 93.21% of total protein in the albumen of more than 90kDa.
Comprehensive analysis result above, albumen of the 3 batch tropocollagen molecule amounts in more than 90kDa are no less than 80%, and between batch
Stablize, significant change does not occur between characteristic bands and molecular weight batch.The result shows that during by the reaction that changes pepsin
Between, dialysis time and bag filter molecular cut off size, to control the molecular weight of collagen and molecular weight distribution, using the present invention
Method of quality control obtain collagen have higher bioactivity.
2nd, peptide removal in end measures
End peptide is carried out to the collagen that embodiment 3 is prepared to measure.Using the structure of the various amino acid compositions of sample it is different,
Acid-base property, polarity and molecular size are different, detach them on cation exchange column, are carried out using ninhydrin post-column derivation method
Chromogenic reaction analyzes the constituent content of protein hydrolyzate and various free amino acids.
This experiment agents useful for same is shown in Table 3 with instrument.
3 end peptide content test agent of table and instrument
(1) preparation of reagents
Sodium citrate buffer solution (pH2.2)：Trisodium citrate 19.6g is weighed, with adding in organic pure hydrochloric acid after water dissolution
16.5mL adds water to be settled to 1000mL, shakes up.
Alkaline hydrolysis agent-lithium hydroxide solution, c (LiOH)=4mol/L：A hydronium(ion) lithia 167.8g is weighed, uses water dissolution
And it is diluted to 1000mL.
Hydrochloric acid solution, c (HCl)=6mol/L：Pure hydrochloric acid is mixed in equal volume with water.
Test specimen
((molecular cut off is reaction solution for 20150707B, non-refined solution (20150706B), collagen stoste
The collagen purification liquid of 140000Da) and bovine serum albumin(BSA) (BSA) control.
(2) experimental method
Sample 1.5mL is taken in hydrolyzing in pipe, precise weighing.Addition 4mol/L LiOH solution (consider solubility, it can volume
Outer addition solid) 1.5mL.Nitrogen is passed through not less than 2min, tube sealing.Hydrolysis pipe is placed in 110 DEG C of constant temperature hydrolysis 20h.Take out water
Solution pipe is cooled to room temperature, and is transferred in 25mL volumetric flasks with the sodium citrate buffer solution of pH 2.2, adds in 2mL 6mol/L HCl
PH is adjusted to neutrality.With the buffer solution constant volume of pH 2.2.0.22 μm of membrane filtration takes clear liquid as alkaline hydrolyzate, upper amino acid point
Analyzer is analyzed.
(6) experimental result and analysis
Tryptophan is the amino acids characteristic of collagen albumen end peptide structure, can pass through the measure control of tryptophane
The removing of end peptide processed.By the meso sample to reaction process and purification process, the hydrolysis release glue under the action of 110 DEG C, alkali
Tryptophan in former albumen, by amino-acid analyzer (Hitachi's L-8800 types), using tryptophan standards product as control (middle inspection institute,
140686, CAS：73-22-3), quantitative analysis is carried out, determines content of the tryptophan in reaction solution and refined solution.
Test specimen is as shown in Table 4 below：
4 end peptide content testing experiment sample of table
Note：Calculation formula X (mg/L)=C × 1/20 × F × V/V'
Tryptophane measurement result is as shown in table 5 below in each sample：
The tryptophane quantitative analysis results of 5 each sample of table
The above results find that bovine serum albumin (BSA) is positive control, and it is 0.66% to detect its tryptophane, reaction
It can detect the tryptophan of lower content (＜ 0.1%) in liquid and non-refined solution, and the tryptophan in collagen stoste
It does not detect, this illustrates that collagen extraction technique can remove collagen end peptide completely, ensures collagen finished product low immunogenicity.
3rd, collagen structure is verified
The triple-helix structure of collagen prepared by embodiment 4 is characterized by transmission electron microscope (TEM) analysis.This experiment is adopted
Sample is collagen stoste (20150315B).
Reagent selected by this experiment is shown in Table 6 with instrument.
6 transmission electron microscope observing reagent of table and instrument
(1) preparation of reagents
1) mother liquor
0.2M Na2HPO4：Weigh 71.6g Na2HPO4·12H2O, is dissolved in 800mL distilled water, and heating stirring to dissolving is treated
After cooling, volumetric flask constant volume is transferred to 1L, room temperature saves backup.
0.2M NaH2PO4：Weigh 31.2g NaH2PO4·2H2O, is dissolved in 800mL distilled water, and heating stirring is cold to dissolving
But after, volumetric flask constant volume is transferred to 1L, room temperature saves backup.
2) PBS is prepared
The PBS of 0.01M, pH 7.0：Take 0.2M Na2HPO4Mother liquor 31mL and 0.2M NaH2PO4Mother liquor 19mL, is transferred to
In volumetric flask, it is settled to 1L.
The PBS of 0.2M, pH 7.2：Take 0.2M Na2HPO4Mother liquor 72mL and 0.2M NaH2PO4Mother liquor 28mL, after mixing often
Temperature saves backup.
(2) experimental method
Sample is opened, takes 10mL collagen stostes, dialyses in 0.01M, the PBS solution of pH 7.0 to neutrality, takes bag filter
Middle collagen solution presses 1:2 ratios are mixed with 0.2M pH value 7.2PBS and are shaken up, and are placed in constant temperature waters in 30 DEG C of water-bath and for 24 hours, are carried out
Self assembly, the collagen solution after self assembly are stored to 4 DEG C.
To tile for the copper mesh of TEM sample preparation to filter paper, be added dropwise on copper mesh using liquid-transfering gun 5 μ L from group
Collagen solution is filled, after 30s, moisture on copper mesh is blotted from copper mesh edge using filter paper, stands 10min, 5 μ L are added dropwise on copper mesh
2% tungstophosphoric acid is blotted moisture from copper mesh edge using filter paper after 1min, 5 μ L distilled water is added dropwise, after 10s, filter paper is in copper
Network edge suck dry moisture, be stored at room temperature it is dry after, be placed in drying box, wait to be tested, use Flied emission transmission electron microscope (JEM-
2100F) it is observed.
Collagen stoste is placed in PBS and carries out self assembly, forms collagenous fibres.Collagenous fibres are added dropwise on copper mesh, are used
Tungstophosphoric acid (2%) dyes, and is observed using Flied emission transmission electron microscope (JEM-2100F).
Complete triple helix structure has decisive influence to the bioactivity of collagen, possesses complete triple helix structure
Tropocollagen molecule has the site that cell can combine on its strand, and cell is according to the space at tropocollagen molecule binding site
Therefore structure and functional group identify, verification collagen has the triple-helix structure most important.
Tropocollagen molecule with triple helix structure is under certain temperature, ion concentration and pH value condition, it may occur that poly-
Collect phenomenon, tropocollagen molecule is not only axially extending, but also laterally assembles, and is self-assembly of collagenous fibres.The parallel laterally assembled
The tropocollagen molecule of row is not flush neat tail, but the mutually front and rear a certain distance that is staggered, and forms special light and dark band,
This special band is the strong evidence that tropocollagen molecule has complete triple helix structure.
Collagenous fibres transmission electron microscope picture is shown in Fig. 5.Black arrow meaning is collagenous fibres in Fig. 5, and collagenous fibres length can
More than 1 μm, width is between 100~300nm, and collagenous fibres have light and dark band, and band is with triple helix knot
The tropocollagen molecule of structure laterally assembles generation, and transmission electron microscope results illustrate that the collagen prepared has triple helix structure.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (9)
1. a kind of method of quality control of biologically active collagen, which is characterized in that include the following steps：
(1) tendon pre-processes：Degreasing is carried out to animal tendon, is sliced and disinfects；
(2) extraction of collagen：To be crushed through step (1) pretreated tendon, in the tendon of crushing add in 0.5~
The acetic acid of 1.0mol/L and the pepsin of 1000~3000U/L are digested, and remove end peptide, and hydrolysis temperature is 10 DEG C~30
DEG C, the enzyme digestion reaction time is 48~144h, and supernatant is taken after centrifugation；
(3) purifying of collagen：Supernatant in step (2) is saltoutd and precipitation process, will precipitation again with 0.1~
The acetic acid of 0.5mol/L or the hydrochloric acidolysis of 0.05~0.1mol/L, centrifuging and taking supernatant, and desalting processing is carried out, obtain collagen
Solution；
(4) it dialyses：Collagen solution is fitted into bag filter, the molecular cut off of bag filter is 50~100kDa, with purified water or
The acetate buffer of pH 4.5~5.0 dialyses 2~5 days as extracellular fluid dialysis, it is former to obtain the biologically active collagen
Liquid.
2. the method for quality control of collagen according to claim 1, which is characterized in that further include has bioactivity to described
Collagen stoste carry out molecular weight determination, end peptide removal detection and triple-helix structure identify the step of.
3. the method for quality control of collagen according to claim 1, which is characterized in that in the step (2), during enzyme digestion reaction
Between be 72~120h.
4. the method for quality control of collagen according to claim 1, which is characterized in that in the step (2), hydrolysis temperature is
20 DEG C~25 DEG C.
5. the method for quality control of collagen according to claim 1, which is characterized in that in the step (2), the concentration of acetic acid
For 0.8~1.0mol/L.
6. the method for quality control of collagen according to claim 1, which is characterized in that in the step (2), tendon：Acetic acid：
The mass ratio of pepsin is 1：1.3~2.8：0.006~0.022.
7. the method for quality control of collagen according to claim 1, which is characterized in that in the step (3), the concentration of acetic acid
For 0.1~0.25mol/L, a concentration of 0.06~0.08mol/L of hydrochloric acid.
8. the method for quality control of collagen according to claim 1, which is characterized in that in the step (4), bag filter is cut
Molecular weight is stayed as 90~100kDa, dialysis time is 3 days.
9. the method for quality control of collagen according to claim 1, which is characterized in that the tendon is Cowhells tendon.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810189372.1A CN108192941A (en) | 2018-03-07 | 2018-03-07 | A kind of method of quality control of biologically active collagen |
| PCT/CN2018/078680 WO2019169646A1 (en) | 2018-03-07 | 2018-03-12 | Quality control method for biologically active collagen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810189372.1A CN108192941A (en) | 2018-03-07 | 2018-03-07 | A kind of method of quality control of biologically active collagen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108192941A true CN108192941A (en) | 2018-06-22 |
Family
ID=62595293
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810189372.1A Pending CN108192941A (en) | 2018-03-07 | 2018-03-07 | A kind of method of quality control of biologically active collagen |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108192941A (en) |
| WO (1) | WO2019169646A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876190A (en) * | 2019-04-11 | 2019-06-14 | 北京大学口腔医学院 | The preparation method and applications of three dimensional biological marking ink |
| CN110638684A (en) * | 2019-09-02 | 2020-01-03 | 深圳兰度生物材料有限公司 | Injectable collagen composition and preparation method thereof |
| CN114058664A (en) * | 2021-11-16 | 2022-02-18 | 武汉轻工大学 | N-terminal precise grafting modification method of natural collagen |
| CN117205306A (en) * | 2023-07-31 | 2023-12-12 | 成都维德医疗器械有限责任公司 | Collagen extract, composition, preparation method and application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980121B (en) * | 2021-10-22 | 2023-05-23 | 四川大学 | Method for regulating and controlling molecular weight distribution of reconstructed collagen |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544097A (en) * | 2003-11-27 | 2004-11-10 | 四川大学 | Biologic material for medical use and its preparing process and usage |
| CN101126104A (en) * | 2007-05-22 | 2008-02-20 | 上海市食品研究所 | Method for preparing natural active collagen by using acid-enzyme composite |
| CN101570772A (en) * | 2008-05-04 | 2009-11-04 | 中国肉类食品综合研究中心 | Method for preparing natural ossein |
| CN102391374A (en) * | 2011-11-15 | 2012-03-28 | 无锡贝迪生物工程有限公司 | Preparation method of active collagen with triple-helix structure |
| CN103260628A (en) * | 2010-10-07 | 2013-08-21 | 成功大学 | Use of hyaluronan for promoting angiogenesis |
| CN104307050A (en) * | 2014-11-10 | 2015-01-28 | 江苏德威兰医疗器械有限公司 | Artificial skin and preparation method thereof |
| CN104721877A (en) * | 2015-03-18 | 2015-06-24 | 广州创尔生物技术股份有限公司 | Sterile collagen dressing and preparation method thereof |
| CN106701879A (en) * | 2017-03-29 | 2017-05-24 | 武汉医佳宝生物材料有限公司 | Method for extracting type I collagen |
| CN107684532A (en) * | 2017-09-28 | 2018-02-13 | 武汉医佳宝生物材料有限公司 | A kind of active collagen facial mask and preparation method |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363040A (en) * | 2008-09-19 | 2009-02-11 | 无锡贝迪生物工程有限公司 | Method for preparing collagen protein |
| US7964704B2 (en) * | 2009-07-27 | 2011-06-21 | Life Fusion, Llc | Preparation of high purity collagen |
| US10119131B2 (en) * | 2011-03-16 | 2018-11-06 | Biospecifics Technologies Corp. | Compositions and methods for producing clostridial collagenases |
| CN103571901A (en) * | 2012-08-06 | 2014-02-12 | 江苏奥普莱医疗用品有限公司 | Preparation method of macromolecular active collagen |
| CN102836462A (en) * | 2012-09-20 | 2012-12-26 | 绍兴好士德医用品有限公司 | Preparation method for collagen tissue engineering skin |
| CN104073536A (en) * | 2013-03-25 | 2014-10-01 | 林琳 | Multi-means cooperated extraction method for macro-molecular active collagen |
| CN104017073A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Method for preparing collagen |
| CN105985429A (en) * | 2015-02-09 | 2016-10-05 | 广州创尔生物技术股份有限公司 | Aseptic collagen liquid with biological activity and preparation method thereof |
| CN105983094A (en) * | 2015-02-09 | 2016-10-05 | 广州创尔生物技术股份有限公司 | Preparation method of sterile collagen liquid having bio-activity and preparation method of sterile collagen dressing having bio-activity |
| CN107354192A (en) * | 2017-08-18 | 2017-11-17 | 广东医科大学 | A kind of method for purifying NTx albumen |
-
2018
- 2018-03-07 CN CN201810189372.1A patent/CN108192941A/en active Pending
- 2018-03-12 WO PCT/CN2018/078680 patent/WO2019169646A1/en active Application Filing
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544097A (en) * | 2003-11-27 | 2004-11-10 | 四川大学 | Biologic material for medical use and its preparing process and usage |
| CN101126104A (en) * | 2007-05-22 | 2008-02-20 | 上海市食品研究所 | Method for preparing natural active collagen by using acid-enzyme composite |
| CN101570772A (en) * | 2008-05-04 | 2009-11-04 | 中国肉类食品综合研究中心 | Method for preparing natural ossein |
| CN103260628A (en) * | 2010-10-07 | 2013-08-21 | 成功大学 | Use of hyaluronan for promoting angiogenesis |
| CN102391374A (en) * | 2011-11-15 | 2012-03-28 | 无锡贝迪生物工程有限公司 | Preparation method of active collagen with triple-helix structure |
| CN104307050A (en) * | 2014-11-10 | 2015-01-28 | 江苏德威兰医疗器械有限公司 | Artificial skin and preparation method thereof |
| CN104721877A (en) * | 2015-03-18 | 2015-06-24 | 广州创尔生物技术股份有限公司 | Sterile collagen dressing and preparation method thereof |
| CN106701879A (en) * | 2017-03-29 | 2017-05-24 | 武汉医佳宝生物材料有限公司 | Method for extracting type I collagen |
| CN107684532A (en) * | 2017-09-28 | 2018-02-13 | 武汉医佳宝生物材料有限公司 | A kind of active collagen facial mask and preparation method |
Non-Patent Citations (4)
| Title |
|---|
| DEFU LI ET AL.: ""Ultrasonic irradiation in the enzymatic extraction of collagen"", 《ULTRASONICS SONOCHEMISTRY》 * |
| 于爱鸣: "《生物化学》", 31 July 2011, 上海科学技术出版社 * |
| 左伋主编: "《医学分子细胞生物学》", 30 September 2005, 上海：复旦大学出版社 * |
| 王晓波: "《药物运释系统》", 31 August 2007, 中国医药科技出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876190A (en) * | 2019-04-11 | 2019-06-14 | 北京大学口腔医学院 | The preparation method and applications of three dimensional biological marking ink |
| CN110638684A (en) * | 2019-09-02 | 2020-01-03 | 深圳兰度生物材料有限公司 | Injectable collagen composition and preparation method thereof |
| CN114058664A (en) * | 2021-11-16 | 2022-02-18 | 武汉轻工大学 | N-terminal precise grafting modification method of natural collagen |
| CN114058664B (en) * | 2021-11-16 | 2023-05-26 | 武汉轻工大学 | N-terminal precise grafting modification method of natural collagen |
| CN117205306A (en) * | 2023-07-31 | 2023-12-12 | 成都维德医疗器械有限责任公司 | Collagen extract, composition, preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019169646A1 (en) | 2019-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108192941A (en) | A kind of method of quality control of biologically active collagen | |
| US8512756B2 (en) | Collagen preparation and method of isolation | |
| EP2644692B1 (en) | High-strength collagen fiber membrane and method for producing same | |
| US20060210601A1 (en) | Stretchable collagen material and manufacturing method and use thereof | |
| EP2231205B1 (en) | Colloidal collagen burn wound dressing produced from jellyfish | |
| Liu et al. | Alginate/Gelatin‐Based Hydrogel with Soy Protein/Peptide Powder for 3D Printing Tissue‐Engineering Scaffolds to Promote Angiogenesis | |
| CN103732614A (en) | Hyaluronic acid-binding synthetic peptidoglycans, preparation, and methods of use | |
| CN108578771B (en) | Preparation method and products thereof with the FGF1 sericin gel for promoting cell-proliferation activity | |
| Chen et al. | Highly active porous scaffolds of collagen and hyaluronic acid prepared by suppression of polyion complex formation | |
| US8877500B2 (en) | Thermally induced gelation of collagen hydrogel and method of thermally inducing gelling a collagen hydrogel | |
| CN103599567A (en) | Temperature-sensitive composite as well as preparation method and application thereof | |
| CN106032546A (en) | Method for extracting medical material-based collagen from fish scales | |
| CN109627326B (en) | Extraction method of non-denatured collagen and identification method of collagen | |
| Seyer et al. | The identification of two types of collagen in the articular cartilage of postnatal chickens | |
| CN1235947C (en) | Collagen fibre extracting method and manufacture of tissue filling material therefrom | |
| Wong et al. | Pre-crosslinked polymeric collagen in 3-D models of mechanically stiff tissues: Blended collagen polymer hydrogels for rapid layer fabrication | |
| JP2014100110A (en) | High-melting point gelatin composition, production method thereof and use thereof | |
| CN111632026A (en) | Self-assembled short peptide hydrogel and application thereof | |
| US8389010B2 (en) | Stretchable collagen material and manufacturing method and use thereof | |
| Jiang et al. | Determination of the bands of four common animal collagens by SDS-PAGE electrophoresis and the comparative study of their protein functional regions | |
| CN114106148B (en) | Medical-grade non-denatured collagen with fish skin source and preparation method thereof | |
| CN114099530B (en) | New application of 2-amino-2 '-fluoro-2' -deoxyadenosine | |
| CN112840013A (en) | Cell culture substrate containing collagen derived from caocho fish scale | |
| CN109265535A (en) | The preparation method of collagen | |
| Yao | Research on extraction and properties of antler plate collagen protein |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180622 |