CN107217085A - A kind of method that neutral proteinase extracts collagen - Google Patents
A kind of method that neutral proteinase extracts collagen Download PDFInfo
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- CN107217085A CN107217085A CN201710656008.7A CN201710656008A CN107217085A CN 107217085 A CN107217085 A CN 107217085A CN 201710656008 A CN201710656008 A CN 201710656008A CN 107217085 A CN107217085 A CN 107217085A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 39
- 108010035532 Collagen Proteins 0.000 title claims abstract description 39
- 229920001436 collagen Polymers 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 22
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 title claims abstract description 14
- 239000000284 extract Substances 0.000 title claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 238000004140 cleaning Methods 0.000 claims abstract description 25
- 239000002253 acid Substances 0.000 claims abstract description 21
- 150000002500 ions Chemical class 0.000 claims abstract description 13
- 238000009938 salting Methods 0.000 claims abstract description 7
- 108090000145 Bacillolysin Proteins 0.000 claims abstract description 3
- 102000035092 Neutral proteases Human genes 0.000 claims abstract description 3
- 108091005507 Neutral proteases Proteins 0.000 claims abstract description 3
- 238000007598 dipping method Methods 0.000 claims abstract description 3
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 3
- 238000000502 dialysis Methods 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 150000003839 salts Chemical class 0.000 claims description 23
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 102000005927 Cysteine Proteases Human genes 0.000 claims description 12
- 108010005843 Cysteine Proteases Proteins 0.000 claims description 12
- 229960002152 chlorhexidine acetate Drugs 0.000 claims description 12
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- MCSINKKTEDDPNK-UHFFFAOYSA-N propyl propionate Chemical compound CCCOC(=O)CC MCSINKKTEDDPNK-UHFFFAOYSA-N 0.000 claims description 12
- 239000000376 reactant Substances 0.000 claims description 11
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- WBZKQQHYRPRKNJ-UHFFFAOYSA-L disulfite Chemical compound [O-]S(=O)S([O-])(=O)=O WBZKQQHYRPRKNJ-UHFFFAOYSA-L 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 239000000460 chlorine Substances 0.000 claims 1
- 229910052801 chlorine Inorganic materials 0.000 claims 1
- 238000002386 leaching Methods 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 235000021419 vinegar Nutrition 0.000 claims 1
- 239000000052 vinegar Substances 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 abstract description 11
- 239000012535 impurity Substances 0.000 abstract 1
- 238000005406 washing Methods 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 18
- 238000002791 soaking Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 12
- 239000000835 fiber Substances 0.000 description 11
- 239000008213 purified water Substances 0.000 description 11
- 241000283725 Bos Species 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 210000002435 tendon Anatomy 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000002734 Collagen Type VI Human genes 0.000 description 1
- 108010043741 Collagen Type VI Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 239000000515 collagen sponge Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
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
- 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 or cold insoluble globulin [CIG]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method that neutral proteinase extracts collagen.This method comprises the following steps, and first ox heel string is cut into slices；The cleaning and dipping that will cut into slices again soaks 0.5 ~ 24h in 0.1% 40% salting liquid.Then it is immersed in neutral protease enzymolysis liquid, neutral proteinase：Phosphate buffer is 1:5~1:20000（W/V）, PH is between 2 ~ 8.1 ~ 72h is reacted under 4 ~ 50 DEG C of environment；Centrifugation, takes purpose collagen, is that 0.01 ~ 5.0mol/L aqueous slkalis adjust PH to 7 ~ 12 with molar concentration, 2 ~ 48h is kept at 2 ~ 8 DEG C, finally pH value is adjusted to 4 ~ 7 for 0.01 ~ 5.0mol/L acid solution with molar concentration, collects product, dialysis, cleaning remove excess ions and impurity, obtain collagen.The method that collagen is extracted from ox heel string using the neutral proteinase of the invention provided maintains the triple-helix structure of collagen, can prepare a variety of collagen solutions for meeting use requirement, security is good, simple to operate without high-end devices.
Description
Technical field
The present invention relates to biology medical material technical field, more particularly to a kind of neutral proteinase extracts glue from ox heel string
Former method.
Background technology
Collagen types are more, and common type is I type, II type, III type, V type and Ⅺ type, and collagen is because with good biology
Compatibility, biodegradable and bioactivity, and it is widely used in food, health products, biomaterial for medical purpose, tissue work
The fields such as journey, cosmetics.In medical domain, collagen is due to high with low immunogenicity, the re-forming property of fiber, mechanical performance
And the advantages of biodegradability, medical collagen sponge, film biological sticking patch, artificial skin etc. has successively been made.I in collagen
Collagen Type VI is prevalent in organism, is accounted for the 90% of organism collagen total amount, is accounted for the 20% of total protein quality, is divided extensively
Cloth rises in the tissue such as tendon, ligament, cornea, bone, skin to the mechanical strength and biological function for maintaining these connective tissues
Key effect.
On space structure, collagen shows special triple-helix structure, and three separate peptide chains rely on glycine
Between the hydrogen bond that is formed maintain the structure that triple helix is mutually wound.This special triple helix structure of collagen ensure that its
Mechanical strength.Collagenous fibres are the grown forms that collagen exercises physiological action, and collagenous fibres are woven into rich in machine in vivo
The network structure of tool intensity and elasticity turns into the most basic constituent of connective tissue.
Current collagen extraction method is mainly Mechanical Crushing raw material to be extracted, and protease digests under acid environment and obtains purpose
Collagen, such method is broken into single-stranded in most three spirals of preparation process, and restriction enzyme site easily occurs in enzymolysis process
Non-selectivity is so as to uncontrolled phenomenon.Therefore one kind is needed to have in the case where not destroying the intrinsic triple-helix structure of collagen
Effect removes the preparation extracting method of the terminal peptide of the non-helical structure of collagenous fibres.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of method that neutral proteinase extracts collagen, it ensure that
Terminal peptide is removed in the case of whole triple helix structure, and the collagen prepared by the method can prepare a variety of use requirements
Collagen solution.Technical scheme is as follows：
The invention provides a kind of method that neutral proteinase extracts collagen, comprise the following steps,
First ox heel string is cut into slices；
The cleaning and dipping that will cut into slices again soaks 0.5 ~ 24h in 0.1% ~ 40% salting liquid.Drain excessive moisture.
It is immersed in neutral protease enzymolysis liquid, enzymolysis liquid is neutral proteinase：Phosphate buffer presses 1:5~1:20000
（W/V）It is formulated, enzymolysis liquid PH is between 2 ~ 8.1 ~ 72h is reacted under 4 ~ 50 DEG C of environment；
By the reactant centrifugation after enzymolysis, supernatant is taken, is that 0.01 ~ 5.0mol/L aqueous slkalis adjust PH to 7 ~ 12 with molar concentration,
2 ~ 48h is kept under 2 ~ 8 DEG C of environment, finally pH value is adjusted to 4 ~ 7 for 0.01 ~ 5.0mol/L acid solution with molar concentration, receives
Collect product, the acid ion of clean water soaking and washing to salt meets standard requirement, and dewater treatment obtains collagen.
In the preferred embodiment of the present invention, the neutral proteinase is cysteine proteinase, cysteine
The mass fraction of protease is 0.05% ~ 0.2%.
In the preferred embodiment of the present invention, the pH value of the phosphate buffer solution is 2 ~ 8, and phosphate radical rubs
Your concentration is 0.001 ~ 0.5mol/L.
In the preferred embodiment of the present invention, the gross mass of the salting liquid, the mass fraction of the salt is
0.1%~40%。
In a kind of more preferably embodiment of the present invention, the salt in the salting liquid is selected from sodium chloride, sodium sulphate, chlorination
At least one of calcium, potassium chloride.
In a kind of more preferably embodiment of the present invention, the solute molality of the aqueous slkali is 0.01 ~ 5.0mol/
L。
In a kind of more preferably embodiment of the present invention, the solute in the aqueous slkali is sodium hydroxide, sodium acid carbonate,
One or more in sodium citrate.
In a kind of more preferably embodiment of the present invention, the solute molality of the acid solution is 0.01 ~ 5.0mol/
L。
In a kind of more preferably embodiment of the present invention, the solute in the acid solution is hydrochloric acid, sulfuric acid, glacial acetic acid,
One or more in citric acid.
In the preferred embodiment of the present invention, to ox heel string carry out collagen extraction before, first to the ox with
Tendon is pre-processed；The pretreatment is cut into slices, then be with mass fraction afterwards to be rinsed repeatedly to ox heel string with water
0.05% ~ 20% chlorhexidine acetate solution soaks 0.5 ~ 24h, then is rinsed repeatedly with water.
In the preferred embodiment of the present invention, the section is freezing microtome section.
Brief description of the drawings
Fig. 1 is extracted the infared spectrum of collagen by the present embodiment 1.
Embodiment
It should be noted that water used in the present invention is the purified water or water for injection used in pharmaceuticals industry, meet
Standards of pharmacopoeia.
Reagent and instrument
Reagent is commercially available.
Embodiment 1
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 30min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string after cleaning is cut into slices, and is immersed in 0.9% sodium chloride
In solution, 2h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:150 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 4.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 72h is reacted under 4 DEG C of environment.
It is that 2.0mol/L sodium hydroxide solutions adjust PH to 10 ± 0.2 with molar concentration, 10h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 5 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 2
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 16min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string section after cleaning, the sodium sulphate for being immersed in 15% is molten
In liquid, 4h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:100 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 4.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 1h is reacted under 37 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 5000rp/min, time 15min will be digested.Take supernatant.
It is that 1.0mol/L sodium hydroxide solutions adjust PH to 9 ± 0.2 with molar concentration, 24h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 6 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 3
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh and 2h is soaked in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05%, purified water soaking and washing 5 times,
Cleaning is all exposed to fiber, drains excessive moisture standby.Ox heel string after cleaning is cut into slices, and is immersed in 15% metabisulfite solution
In, soak 4h.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:200 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.002mol/L
In salt buffer solution, phosphate buffer solution PH is 3.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 0.5h is reacted under 40 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 4000rp/min, time 30min will be digested.Take supernatant.
It is that 0.5mol/L sodium carbonate liquors adjust PH to 8 ± 0.2 with molar concentration, 24h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 4 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 4
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 30min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string after cleaning is cut into slices, and is immersed in 0.7% sodium chloride
In solution, 2h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:150 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 2.5.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 48h is reacted under 4 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 6000rp/min, time 15min will be digested.Take supernatant.
It is that 0.5mol/L sodium hydroxide solutions adjust PH to 8 ± 0.2 with molar concentration, 2h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 6 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 5
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 30min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string after cleaning is cut into slices, and is immersed in 0.7% potassium chloride
In solution, 2h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:150 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 6.0.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 24h is reacted under 25 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 8000rp/min, time 15min will be digested.Precipitation is taken, salting liquid disperses.
It is that 0.5mol/L sodium hydroxide solutions adjust PH to 10 ± 0.2 with molar concentration, 2h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 5 ± 0.2 for 0. 01mol/L hydrochloric acid solution with molar concentration, collects product, clean water
Soaking and washing to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 6
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 30min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string after cleaning is cut into slices, and is immersed in 0.7% sodium chloride
In solution, 2h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:150 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 5.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 72h is reacted under 30 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 6000rp/min, time 15min will be digested.Take supernatant.
It is that 0.5mol/L sodium hydroxide solutions adjust PH to 8 ± 0.2 with molar concentration, 2h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 6 ± 0.2 for 0. 1mol/L sulfuric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 7
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh and 6h is soaked in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05%, purified water soaking and washing 5 times,
Cleaning is all exposed to fiber, drains excessive moisture standby.Ox heel string section after cleaning, the sodium chloride for being immersed in 0.85% is molten
In liquid, 15h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:160 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 6.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 72h is reacted under 4 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 6000rp/min, time 15min will be digested.Take supernatant.
It is that 0.5mol/L sodium bicarbonate solutions adjust PH to 10 ± 0.2 with molar concentration, 12h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 4.5 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, collects product, clean water
Soaking and washing to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 8
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 30min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string after cleaning is cut into slices, and is immersed in 0.9% calcium chloride
In solution, 2h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:150 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.02mol/L
In salt buffer solution, phosphate buffer solution PH is 2.5.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 48h is reacted under 4 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 6000rp/min, time 15min will be digested.Take supernatant.
It is that 0.5mol/L sodium hydroxide solutions adjust PH to 9 ± 0.2 with molar concentration, 12h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 7 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 9
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.05% and soak 30min, purified water soaking and washing 5
It is secondary, clean all exposed to fiber, drain excessive moisture standby.Ox heel string section after cleaning, the sodium chloride for being immersed in 20% is molten
In liquid, 2h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:50 weigh cysteine proteinase, are dissolved into the phosphate that molar concentration is 0.02mol/L
In cushioning liquid, phosphate buffer solution PH is 3.5.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 48h is reacted under 15 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 6000rp/min, time 15min will be digested.Take supernatant.
It is that 0.05mol/L sodium citrate solutions adjust PH to 8 ± 0.2 with molar concentration, 24h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 5 ± 0.2 for 0. 01mol/L sulfuric acid solution with molar concentration, collects product, clean water
Soaking and washing to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
Embodiment 10
Fresh ox is chosen with tendinous tissue 0.5kg, water is cleaned to being visible by naked eyes foreign matter；It is non-that manadesma, fat etc. are rejected with scissors
Ox heel string fibr tissue, after washing, -20 DEG C of freeze overnights；Microtome, slice thickness about 1mm is standby；
Weigh and 5h is soaked in the section of 100g oxen heel string, the chlorhexidine acetate solution for being immersed in 0.1%, purified water soaking and washing 5 times,
Cleaning is all exposed to fiber, drains excessive moisture standby.Ox heel string section after cleaning, the sodium chloride for being immersed in 0.85% is molten
In liquid, 5h is soaked.Drain excessive moisture.
Enzymolysis liquid is prepared, by 1:200 weigh cysteine proteinase, are dissolved into the phosphoric acid that molar concentration is 0.05mol/L
In salt buffer solution, phosphate buffer solution PH is 2.5.
The ox that soaks will be invaded to pour into enzymolysis liquid with tendinous fibres, dispersed with stirring, 48h is reacted under 4 DEG C of environment.
The reactant centrifugation after terminating, rotating speed 6000rp/min, time 15min will be digested.Take supernatant.
It is that 2mol/L sodium hydroxide solutions adjust PH to 8 ± 0.2 with molar concentration, 2h is kept under 2 ~ 8 DEG C of environment.
PH value finally is adjusted to 6 ± 0.2 for 0. 1mol/L hydrochloric acid solution with molar concentration, is collected product, is cleaned water logging
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
It is described above to be for only for ease of it will be understood by those skilled in the art that technical scheme, not to limit
The present invention.Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in this
Within the protection domain of invention.
Claims (10)
1. a kind of method that neutral proteinase extracts collagen, it is characterised in that comprise the following steps,
First ox heel string is cut into slices；The cleaning and dipping that will cut into slices again is soaked 0.5 ~ 24h, drained in 0.1% ~ 40% salting liquid
Excessive moisture；It is immersed in neutral protease enzymolysis liquid, enzymolysis liquid is neutral proteinase：Phosphate buffer presses 1:5~1:
20000（W/V）It is formulated, enzymolysis liquid PH is between 2 ~ 8；1 ~ 72h is reacted under 4 ~ 50 DEG C of environment；By the reactant after enzymolysis from
The heart, takes purpose collagen, with molar concentration be 0.01 ~ 5.0mol/L aqueous slkalis adjust PH to 7 ~ 12, under 2 ~ 8 DEG C of environment keep 2 ~
48h, finally adjusts pH value to 4 ~ 7 with molar concentration for 0.01 ~ 5.0mol/L acid solution, collects product, clean water dialysis, leaching
Bubble cleaning to the acid ion of salt meets standard requirement, and dewater treatment obtains collagen.
2. according to the method described in claim 1, it is characterised in that the neutral proteinase is the one of cysteine proteinase
Kind.
3. according to the method described in claim 1, it is characterised in that the mass fraction of the neutral proteinase is 0.05% ~
0.2%。
4. according to the method described in claim 1, it is characterised in that the pH value of the phosphate buffer solution is 2 ~ 8, phosphate radical
Molar concentration be 0.001 ~ 0.2mol/L.
5. according to the method described in claim 1, it is characterised in that the salting liquid is sodium chloride solution, metabisulfite solution, chlorine
Change calcium solution, the one or more of Klorvess Liquid.
6. according to the method described in claim 1, it is characterised in that the solute molality based on the aqueous slkali is 0.01 ~
5.0mol/L。
7. method according to claim 6, it is characterised in that the solute in the aqueous slkali is sodium hydroxide, bicarbonate
One or more in sodium, sodium citrate.
8. according to the method described in claim 1, it is characterised in that the solute molality based on the acid solution is 0.01 ~
5.0mol/L。
9. method according to claim 8, it is characterised in that the solute in the acid solution is hydrochloric acid, sulfuric acid, ice vinegar
One or more in acid, citric acid.
10. according to the method described in claim 1, it is characterised in that before collagen extraction is carried out to ox heel string, first to described
Ox heel string is pre-processed；The pretreatment is cut into slices, then be with mass fraction afterwards to be rinsed repeatedly to ox heel string with water
0.05% ~ 20% chlorhexidine acetate solution soaks 0.5 ~ 24h, then is rinsed repeatedly with water.
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| CN107929795A (en) * | 2017-11-16 | 2018-04-20 | 北京华信佳音医疗科技发展有限责任公司 | A kind of preparation and its application of antibacterial anti hemorrhagic material |
| CN110563834A (en) * | 2019-09-25 | 2019-12-13 | 成都奇璞生物科技有限公司 | Collagen extraction method |
| CN112625122A (en) * | 2020-12-30 | 2021-04-09 | 福建纽伯尔生物科技有限公司 | Collagen fiber extraction method |
| CN114032623A (en) * | 2022-01-10 | 2022-02-11 | 天新福（北京）医疗器材股份有限公司 | Preparation process of high-yield collagen fibers |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107929795A (en) * | 2017-11-16 | 2018-04-20 | 北京华信佳音医疗科技发展有限责任公司 | A kind of preparation and its application of antibacterial anti hemorrhagic material |
| CN110563834A (en) * | 2019-09-25 | 2019-12-13 | 成都奇璞生物科技有限公司 | Collagen extraction method |
| CN112625122A (en) * | 2020-12-30 | 2021-04-09 | 福建纽伯尔生物科技有限公司 | Collagen fiber extraction method |
| CN114032623A (en) * | 2022-01-10 | 2022-02-11 | 天新福（北京）医疗器材股份有限公司 | Preparation process of high-yield collagen fibers |
| CN114032623B (en) * | 2022-01-10 | 2022-03-29 | 天新福（北京）医疗器材股份有限公司 | Preparation process of high-yield collagen sponge |
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Application publication date: 20170929 |