CN106701879A - Method for extracting type I collagen - Google Patents
Method for extracting type I collagen Download PDFInfo
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- CN106701879A CN106701879A CN201710196069.XA CN201710196069A CN106701879A CN 106701879 A CN106701879 A CN 106701879A CN 201710196069 A CN201710196069 A CN 201710196069A CN 106701879 A CN106701879 A CN 106701879A
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- 238000000034 method Methods 0.000 title claims abstract description 47
- 102000012422 Collagen Type I Human genes 0.000 title claims abstract 5
- 108010022452 Collagen Type I Proteins 0.000 title claims abstract 5
- 102000008186 Collagen Human genes 0.000 claims abstract description 56
- 108010035532 Collagen Proteins 0.000 claims abstract description 56
- 229920001436 collagen Polymers 0.000 claims abstract description 56
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000000502 dialysis Methods 0.000 claims abstract description 17
- 238000000605 extraction Methods 0.000 claims abstract description 17
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 238000005238 degreasing Methods 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 229940088598 enzyme Drugs 0.000 claims abstract description 7
- 239000000047 product Substances 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 69
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 50
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 27
- 239000012498 ultrapure water Substances 0.000 claims description 27
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 230000029087 digestion Effects 0.000 claims description 14
- 239000012535 impurity Substances 0.000 claims description 13
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 13
- 210000001519 tissue Anatomy 0.000 claims description 13
- 102000057297 Pepsin A Human genes 0.000 claims description 11
- 108090000284 Pepsin A Proteins 0.000 claims description 11
- 229940111202 pepsin Drugs 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 10
- 239000004744 fabric Substances 0.000 claims description 10
- 235000013372 meat Nutrition 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 229910001220 stainless steel Inorganic materials 0.000 claims description 10
- 239000010935 stainless steel Substances 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 238000013019 agitation Methods 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
- 238000001556 precipitation Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 229910000831 Steel Inorganic materials 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 239000012670 alkaline solution Substances 0.000 claims description 5
- 210000003722 extracellular fluid Anatomy 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 239000010959 steel Substances 0.000 claims description 5
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 108090000270 Ficain Proteins 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 2
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 claims description 2
- 235000019836 ficin Nutrition 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 238000000859 sublimation Methods 0.000 claims description 2
- 230000008022 sublimation Effects 0.000 claims description 2
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims 3
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 1
- 239000003513 alkali Substances 0.000 claims 1
- 238000004140 cleaning Methods 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 210000002435 tendon Anatomy 0.000 abstract description 5
- 210000002919 epithelial cell Anatomy 0.000 abstract description 3
- 206010020718 hyperplasia Diseases 0.000 abstract description 3
- 241000283690 Bos taurus Species 0.000 abstract 2
- 102000029816 Collagenase Human genes 0.000 abstract 1
- 108060005980 Collagenase Proteins 0.000 abstract 1
- 229960002424 collagenase Drugs 0.000 abstract 1
- 230000000249 desinfective effect Effects 0.000 abstract 1
- 230000006862 enzymatic digestion Effects 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 238000005185 salting out Methods 0.000 abstract 1
- 239000011550 stock solution Substances 0.000 abstract 1
- 238000009461 vacuum packaging Methods 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 230000003946 protein process Effects 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000012620 biological material Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 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 description 1
- 241000124008 Mammalia Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000515 collagen sponge Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
Classifications
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- 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]
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- 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)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (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)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to a method for extracting type I collagen. The method comprises the following steps: cleaning bovine tendon, refrigerating and slicing; disinfecting and degreasing the sliced bovine tendon with 75-percent alcohol and normal hexane; adding a proper amount of active enzyme into homogenate for performing enzymatic digestion after removing impure proteins and smashing the homogenate; performing salting-out and dialysis treatment on supernatant; performing freeze drying and vacuum packaging on a collagen stock solution obtained after the dialysis to obtain a type I collagen product. The method is simple in preparation process, can be used for realizing large-scale production, and is an economical and effective extraction method. The type I collagen extracted by the method has superior biocompatibility and bioactivity, can be applied to biomedicine, and has an important application value in the field of makeups; meanwhile, epithelial cells can be activated, the hyperplasia of the epithelial cells and the generation of collagenase are facilitated, and the skin becomes tight and elastic.
Description
Technical field
The present invention relates to biomaterial for medical purpose field, specially a kind of method for extracting I-type collagen.
Background technology
Collagen is mammal in-vivo content most rich in protein, accounts for the 25% ~ 30% of vivo protein total amount.Glue
Former albumen is the composition of connective tissue generally existing, is organ and the essential Tectonic Framework of tissue, it has now been found that 27
Different types of collagen more than kind.I-type collagen is widely present in the tissue such as bone, cornea, skin, tendon of animal
In, molecular weight about 300KD, its molecular length is about 300nm, diameter about 1.5nm, in bar-shaped.I-type collagen in microstructure
It is made up of three peptide chains, including two α (I) chains and α (II) chain.Because I-type collagen has degradability, biofacies
Capacitive and many excellent biological characteristicses such as hemostatic, thus by as natural biologic material be widely used in it is biomedical and
Organizational engineering field.
The I-type collagen sold in the market is mainly prepared from from Animal Skin and with being extracted in tendinous tissue, due to
The I-type collagen that existing extracting method is prepared is influenceed by process conditions, and extraction efficiency is relatively low, and the collagen for extracting
The foreign protein contained in albumen is more, has a strong impact on the quality of collagen, and in fields such as biomedical and cosmetic industries
Extensive use I-type collagen demand it is growing day by day, therefore, therefore design a kind of I-type collagen extracting method with
It is aobvious in the application in fields such as biomedical and cosmetic industries to meet I-type collagen to improve the extraction efficiency of collagen
Obtain very necessary.
The present invention is organized as raw material with animal heel string, and a kind of I-type collagen is prepared using acidicenzym law technology.The party
Method can not only improve the extraction efficiency of I-type collagen, and the foreign protein content of the I-type collagen extracted is relatively low.This
Inventing the I-type collagen extracted has excellent biocompatibility and bioactivity, can adapt to the application of biomedicine, together
When can activated epithelial, promote epithelial hyperplasia and clostridiopetidase A generation, make skin-tightening elastic, in cosmetic field
With important application value.
The content of the invention
The invention aims to provide it is a kind of extract I-type collagen sponge method, the preparation process is simple,
The extraction efficiency of I-type collagen can be improved, and the foreign protein content of the I-type collagen extracted is relatively low.
The purpose of the present invention can be achieved through the following technical solutions.
The present invention is organized as raw material with animal heel string, and a kind of I-type collagen is prepared using acidicenzym law technology, and it is carried
Taking process includes following seven steps：（1）Freezing microtome section after ox heel string is cleaned；（2）Configuration solution is de- by heel string section sterilization
Fat；（3）Heel string section is washed to neutrality after soaking in sodium carbonate solution sloughs foreign protein；（4）Heel string is cut using refiner
Piece is carried out will be broken；（5）To pepsin is added in homogenate, digestion is stirred at 15 ~ 25 DEG C；（6）Supernatant is carried out into salt
Analysis is processed, then will carry out dialysis treatment after precipitation dissolving；（7）Collagen stoste after dialysis is carried out into freeze-drying and obtains final product I type glue
Former albumen, Vacuum Package after taking-up.
The concrete technical scheme of implementation is as follows.
Step one：Cleaning
Fresh ox heel string is cleaned up with ultra-pure water, manadesma, broken Gu, Of-thin meat and fat is rejected using knife blade, -20
Ox heel string is refrigerated to hardening in DEG C refrigerator, ox heel string is cut into the heel string piece of 1mm thickness after taking-up along tissue fibers direction.
Step 2：Sterilization degreasing process
Weigh 100g heel string pieces and be placed in sterilization degreasant solution and soak, mechanical agitation filters off solution using stainless steel cloth, takes out
Cleaned several times with ultra-pure water afterwards.
Step 3：Removing impurities protein Process
Heel string piece after degreasing of sterilizing is soaked with alkaline solution carries out removing impurities albumen, mechanical agitation, and 6 ~ 8h of interval is changed once
Alkaline solution, solution is filtered off using stainless steel cloth, and it is several times neutral to heel string piece surface to be cleaned with ultra-pure water after taking-up（pH
Be worth is 7）.
Step 4：Crushing process
Heel string piece after removing impurities albumen is put into meat grinder and is rubbed, and by rubbing after the acidity that 1000mL is placed in tendinous tissue
In solution, it is homogenized using refiner is crushed several times, 5min is homogenized every time.
Step 5：Enzymolysis, digestion technique
Homogenate is placed in the rustless steel container of Large Copacity, is continued to addition 1000mL acid solutions in homogenate, then to it
Middle to add appropriate organized enzyme, stirring digestion is centrifuged 15 ~ 30min.Take supernatant standby.
Step 6：Saltout dialysis technique
Supernatant is neutralized to solution in neutrality using sodium hydroxide solution, is added sodium chloride to be stirred and is saltoutd, centrifugation 15 ~
30min, precipitation is I-type collagen first product, is precipitated several times with ultrapure water, drains.By being precipitated and dissolved in for draining
Dialysed in dialyzate.
Step 7：Drying process
Collagen stoste after dialysis is dried treatment at -50 DEG C, Vacuum Package after taking-up.
The solution and ultrapure coolant-temperature gage that a kind of all steps of method of described extraction I-type collagen are used are 15
~25℃。
Sterilization degreasant solution in a kind of method and step two of described extraction I-type collagen for 75% alcohol and just oneself
The mixed solution of alkane, 75% ethanol is 1 with the volume ratio of n-hexane:1~5:1, preferably 75% ethanol：N-hexane=3:1；Sterilization degreasing
Time is 6 ~ 24h, and the preferably time is 12h.
The alkaline solution of the removing impurities albumen in a kind of method and step three of described extraction I-type collagen is to select hydroxide
The solution that any one is prepared in sodium, oxidized potassium, sodium carbonate, potassium carbonate and sodium acid carbonate, preferably sodium carbonate；Sodium carbonate liquor
Concentration be 0.01 ~ 1.0mol/L, preferred concentration is 0.05mol/L.
Acid solution in a kind of method and step four of described extraction I-type collagen is that acetic acid solution and citric acid are molten
Any one in liquid or two kinds, preferably acetic acid solution；The concentration of solution is 0.01% ~ 2.0%（Mass fraction）, preferred concentration is
0.5%（Mass fraction）.
Organized enzyme in a kind of method and step five of described extraction I-type collagen is pepsin, trypsase and
In ficin any one or two kinds and more than, preferred pepsin；The concentration of organized enzyme is 20 ~ 1000mg/L, excellent
It is 100mg/L to select concentration；12 ~ 72h of digestion time, preferably digestion time are 24h.
Sodium chloride concentration in a kind of method and step six of described extraction I-type collagen is 1 ~ 5mol/L, preferably dense
Spend 2 mol/L；The time is saltoutd for 1 ~ 24h, the time of preferably saltouing is for 12h.
Dialyzate in a kind of method and step six of described extraction I-type collagen is acetic acid solution, and concentration is
0.01mol/L；Extracellular fluid dialysis are the acetic acid solution or ultra-pure water of 0.001 mol/L, preferably ultra-pure water.
Drying mode in a kind of method and step seven of described extraction I-type collagen is that radiant drying or freezing are dry
It is dry, preferably freeze drying；Freeze-drying temperature is -50 DEG C；Sublimation drying is 24 ~ 72h, and the preferably time is 48h.
The advantage that the present invention is provided is as follows：（1）A kind of method of extraction I-type collagen that the present invention is provided improves I
The extraction efficiency of collagen type, and in collagen foreign protein content it is less.（2）The I-type collagen that the present invention is extracted
With good biocompatibility and bioactivity, additionally it is possible to promote the generation of the hyperplasia and clostridiopetidase A of epithelial cell, satisfaction is faced
The demand of bed biomedical engineering field and cosmetic industry.（3）Preparation process is simple of the invention, achievable extensive life
Produce, be a kind of cost-effective extracting method.
Specific embodiment
With reference to embodiment, the invention will be further described.
Case study on implementation 1
（1）Cleaning：Fresh ox heel string is cleaned up with ultra-pure water, manadesma, broken Gu, Of-thin meat are rejected using knife blade
And fat, ox heel string is refrigerated to hardening in -20 DEG C of refrigerators, ox heel string is cut into 1mm along tissue fibers direction after taking-up
The heel string piece of thickness.
（2）Sterilization degreasing process：100g heel strings piece is weighed in 75% ethanol：N-hexane=3:Soaked in 1 mixed solution
12h, 15 ~ 25 DEG C of solution temperature, mechanical agitation filters off solution using stainless steel cloth, is cleaned with ultra-pure water 5 times after taking-up.
（3）Removing impurities protein Process：With 0.05mol/L soaking in sodium carbonate solution 24h, 15 ~ 25 DEG C of solution temperature, machinery is stirred
Mix, interval 8h changes a sodium carbonate liquor, and solution is filtered off using stainless steel cloth, clean with ultra-pure water after taking-up for several times extremely with
Tendon piece surface is neutrality（PH value is 7）.
（4）Crushing process：Heel string piece after removing impurities albumen is put into meat grinder and is rubbed, by rubbing after put with tendinous tissue
In the 0.5% of 1000mL（Mass fraction）Acetic acid solution, 15 ~ 25 DEG C of solution temperature is homogenized broken 5 times to it, often using refiner
Secondary homogenate 5min.
（5）Enzymolysis, digestion technique：In rustless steel container by homogenate as Large Copacity, continue to be added in homogenate
1000mL 0.5%（Mass fraction）Acetic acid solution, then be added thereto to pepsin, pepsin concn is 100mg/L,
Digestion 24h is stirred at 15 ~ 25 DEG C, 15 ~ 30min is centrifuged.Take supernatant standby.
（6）Saltout dialysis technique：Supernatant is neutralized to solution in neutrality using 1mol/L sodium hydroxide solutions, is added
Sodium chloride to sodium chloride concentration is 2mol/L, stirs the 12h that saltouts, and 15 ~ 30min is centrifuged, and precipitation is I-type collagen first product,
Precipitated 3 times with ultrapure water, it is drained and standby.It is precipitated and dissolved in what is drained in 0.01mol/L acetic acid solutions, is made with ultra-pure water
For extracellular fluid dialysis are dialysed, 15 ~ 25 DEG C of dialysate temperature.
（7）Drying process：Collagen stoste after dialysis is placed in freeze drier the freeze-drying 48h at -50 DEG C, is taken
Go out rear Vacuum Package.
The I-type collagen extracted to case study on implementation 1 carries out mechanics yield detection, after testing, I types glue in embodiment 1
The extraction efficiency of former albumen reaches 82.6%.
Case study on implementation 2
（1）Cleaning：Fresh ox heel string is cleaned up with ultra-pure water, manadesma, broken Gu, Of-thin meat are rejected using knife blade
And fat, ox heel string is refrigerated to hardening in -20 DEG C of refrigerators, ox heel string is cut into 1mm along tissue fibers direction after taking-up
The heel string piece of thickness.
（2）Sterilization degreasing process：100g heel strings piece is weighed in 75% ethanol：N-hexane=3:Soaked in 1 mixed solution
12h, 15 ~ 25 DEG C of solution temperature, mechanical agitation filters off solution using stainless steel cloth, is cleaned with ultra-pure water 5 times after taking-up.
（3）Removing impurities protein Process：With 0.5mol/L soaking in sodium carbonate solution 24h, 15 ~ 25 DEG C of solution temperature, mechanical agitation,
Interval 8h changes a sodium carbonate liquor, and solution is filtered off using stainless steel cloth, is cleaned for several times to heel string with ultra-pure water after taking-up
Piece surface is neutrality（PH value is 7）.
（4）Crushing process：Heel string piece after removing impurities albumen is put into meat grinder and is rubbed, by rubbing after put with tendinous tissue
In the 0.5% of 1000mL（Mass fraction）Acetic acid solution, 15 ~ 25 DEG C of solution temperature is homogenized broken 5 times to it, often using refiner
Secondary homogenate 5min.
（5）Enzymolysis, digestion technique：In rustless steel container by homogenate as Large Copacity, continue to be added in homogenate
1000mL 0.5%（Mass fraction）Acetic acid solution, then be added thereto to pepsin, pepsin concn is 100mg/L,
Digestion 24h is stirred at 15 ~ 25 DEG C, 15 ~ 30min is centrifuged.Take supernatant standby.
（6）Saltout dialysis technique：Supernatant is neutralized to solution in neutrality using 1mol/L sodium hydroxide solutions, is added
Sodium chloride to sodium chloride concentration is 2mol/L, stirs the 12h that saltouts, and 15 ~ 30min is centrifuged, and precipitation is I-type collagen first product,
Precipitated 3 times with ultrapure water, it is drained and standby.It is precipitated and dissolved in what is drained in 0.01mol/L acetic acid solutions, is made with ultra-pure water
For extracellular fluid dialysis are dialysed, 15 ~ 25 DEG C of dialysate temperature.
（7）Drying process：Collagen stoste after dialysis is placed in freeze drier the freeze-drying 48h at -50 DEG C, is taken
Go out rear Vacuum Package.
Case study on implementation 3
（1）Cleaning：Fresh ox heel string is cleaned up with ultra-pure water, manadesma, broken Gu, Of-thin meat are rejected using knife blade
And fat, ox heel string is refrigerated to hardening in -20 DEG C of refrigerators, ox heel string is cut into 1mm along tissue fibers direction after taking-up
The heel string piece of thickness.
（2）Sterilization degreasing process：100g heel strings piece is weighed in 75% ethanol：N-hexane=3:Soaked in 1 mixed solution
12h, 15 ~ 25 DEG C of solution temperature, mechanical agitation filters off solution using stainless steel cloth, is cleaned with ultra-pure water 5 times after taking-up.
（3）Removing impurities protein Process：With 0.05mol/L soaking in sodium carbonate solution 24h, 15 ~ 25 DEG C of solution temperature, machinery is stirred
Mix, interval 8h changes a sodium carbonate liquor, and solution is filtered off using stainless steel cloth, clean with ultra-pure water after taking-up for several times extremely with
Tendon piece surface is neutrality（PH value is 7）.
（4）Crushing process：Heel string piece after removing impurities albumen is put into meat grinder and is rubbed, by rubbing after put with tendinous tissue
In the 0.5% of 1000mL（Mass fraction）Acetic acid solution, 15 ~ 25 DEG C of solution temperature is homogenized broken 5 times to it, often using refiner
Secondary homogenate 5min.
（5）Enzymolysis, digestion technique：In rustless steel container by homogenate as Large Copacity, continue to be added in homogenate
1000mL 0.5%（Mass fraction）Acetic acid solution, then be added thereto to pepsin, pepsin concn is 200mg/L,
Digestion 24h is stirred at 15 ~ 25 DEG C, 15 ~ 30min is centrifuged.Take supernatant standby.
（6）Saltout dialysis technique：Supernatant is neutralized to solution in neutrality using 1mol/L sodium hydroxide solutions, is added
Sodium chloride to sodium chloride concentration is 2mol/L, stirs the 12h that saltouts, and 15 ~ 30min is centrifuged, and precipitation is I-type collagen first product,
Precipitated 3 times with ultrapure water, it is drained and standby.It is precipitated and dissolved in what is drained in 0.01mol/L acetic acid solutions, is made with ultra-pure water
For extracellular fluid dialysis are dialysed, 15 ~ 25 DEG C of dialysate temperature.
（7）Drying process：Collagen stoste after dialysis is placed in freeze drier the freeze-drying 48h at -50 DEG C, is taken
Go out rear Vacuum Package.
Above content is to combine specific embodiment detailed description made for the present invention, it is impossible to assert that the present invention is specific
Implementation is only limitted to described above.Under without departing substantially from design of the invention, those skilled in the art according to the present invention make it is each
Plant modification and replace, should all be considered as protection scope of the present invention.
Claims (9)
1. it is a kind of extract I-type collagen method, it is characterised in that preparation method include following five steps：
Step one：Fresh ox heel string is cleaned up with ultra-pure water, manadesma, broken Gu, Of-thin meat and fat are rejected using knife blade
Fat, hardening is refrigerated in -20 DEG C of refrigerators by ox heel string, and ox heel string is cut into 1mm thickness along tissue fibers direction after taking-up
Heel string piece；
Step 2：Weigh appropriate heel string piece and be placed in sterilization degreasant solution and soak, mechanical agitation is filtered off molten using stainless steel cloth
Liquid, is cleaned several times after taking-up with ultra-pure water；
Step 3：Heel string piece after step 2 is processed is soaked with alkaline solution and carries out removing impurities albumen, and mechanical agitation is spaced 6 ~ 8h
Primary alkali solution is changed, solution is filtered off using stainless steel cloth, cleaned several times to heel string piece surface with ultra-pure water after taking-up
It is neutrality（PH value is 7）；
Step 4：Heel string piece after removing impurities albumen in step 3 is put into meat grinder and is rubbed, and by rubbing after with tendinous tissue
It is placed in the acid solution of 1000mL, it is homogenized using refiner is crushed several times, 5min is homogenized every time；
Step 5：Homogenate in step 4 is placed in the rustless steel container of Large Copacity, continues to be added in homogenate
1000mL acid solutions, then appropriate organized enzyme is added thereto to, stirring digestion is centrifuged 15 ~ 30min, takes supernatant standby；
Step 6：The supernatant in step 5 is neutralized to solution in neutrality using sodium hydroxide solution, adds sodium chloride to carry out
Stirring is saltoutd, and 15 ~ 30min is centrifuged, and precipitation is I-type collagen first product, is precipitated several times with ultrapure water, drains, will
What is drained is precipitated and dissolved in dialyzate, is being dialysed；
Step 7：Collagen stoste after being dialysed in step 6 is dried treatment, Vacuum Package after taking-up.
2. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that described type i collagen egg
Solution that all steps are used is extracted in vain and ultrapure coolant-temperature gage is 15 ~ 25 DEG C.
3. according to a kind of method of the extraction I-type collagen described in claim 1, it is characterised in that disappear in described step two
Malicious degreasant solution is the mixed solution of 75% alcohol and n-hexane, and 75% ethanol is 1 with the volume ratio of n-hexane:1~5:1, preferably
75% ethanol：N-hexane=3:1；Sterilization degreasing time is 6 ~ 24h, and the preferably time is 12h；.
4. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that in described step three
The alkaline solution of removing impurities albumen is that any one is prepared in selecting NaOH, potassium hydroxide, sodium carbonate, potassium carbonate and sodium acid carbonate
Solution, preferred sodium carbonate；The concentration of sodium carbonate liquor is 0.01 ~ 1.0mol/L, and preferred concentration is 0.05mol/L.
5. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that in described step four
Acid solution is any one in acetic acid solution and citric acid solution or two kinds, preferably acetic acid solution；The concentration of solution is
0.01%~2.0%（Mass fraction）, preferred concentration is 0.5%（Mass fraction）.
6. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that in described step five
Organized enzyme be in pepsin, trypsase and ficin any one or two kinds and more than, preferred pepsin；It is living
Property enzyme concentration be 20 ~ 1000mg/L, preferred concentration is 100mg/L；12 ~ 72h of digestion time, preferably digestion time are 24h.
7. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that in described step six
Sodium chloride concentration is 1 ~ 5mol/L, the mol/L of preferred concentration 2；The time is saltoutd for 1 ~ 24h, the time of preferably saltouing is for 12h.
8. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that in described step six
Dialyzate is acetic acid solution, and concentration is 0.01mol/L；Extracellular fluid dialysis are the acetic acid solution or ultra-pure water of 0.001 mol/L, preferably
Ultra-pure water.
9. it is according to claim 1 it is a kind of extract I-type collagen method, it is characterised in that in described step seven
Drying means be radiant drying or freeze-drying, preferably freeze drying；Freeze-drying temperature is -50 DEG C；Sublimation drying
It is 24 ~ 72h, the preferably time is 48h.
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