CN107354192A - A kind of method for purifying NTx albumen - Google Patents
A kind of method for purifying NTx albumen Download PDFInfo
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- CN107354192A CN107354192A CN201710712842.3A CN201710712842A CN107354192A CN 107354192 A CN107354192 A CN 107354192A CN 201710712842 A CN201710712842 A CN 201710712842A CN 107354192 A CN107354192 A CN 107354192A
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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
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- 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]
Abstract
The present invention relates to the technical field of purification of collagen, and in particular to a kind of method for purifying NTx albumen.The method of the purifying NTx albumen, including：Step 1, NTx albumen is extracted using suitable method；Step 2, obtain NTx albumen weak solution；Step 3, obtain NTx albumen after purification.The method of the purifying NTx albumen compared with prior art, can substantially shorten the purification time of NTx protein liquid, reach preferable purification effect.The present invention, which is purified by ultrafiltration technology Purified collagen, only needs 12h ~ 24h cans to reach required purity and concentration, and uses the dialysis purifying of prior art then to need the time of 1 week or so to can be only achieved identical purity and concentration.It is provided by the invention it is a kind of purify NTx albumen method, have method it is simple, easy to operate, production cost is low, and can be applied to industrialization large-scale application the characteristics of.
Description
Technical field
The present invention relates to the technical field of purification of collagen, and in particular to a kind of method for purifying NTx albumen.
Background technology
I-type collagen low immunogenicity, can promote cellular activities and growth, rush because having good biocompatibility
Enter the advantages such as coagulating platelets and biodegradable.NTx albumen is in operation suture thread, stanch fibre, artificial skin, artificial
Blood vessel and artificial bone etc. are clinically widely applied.Made of I-type collagen operation suture thread it is existing with
The similar high intensity of natural silk, there is absorbability again, not easy damaged body tissue in operating process, have to the surface of a wound glutinous well
Attached property.Powdery, flat-shaped and spongiform styptic is made in I-type collagen, only needs the compressing of short period just to can reach satisfaction
Haemostatic effect.Simultaneously as containing substantial amounts of hydroxyl in collagen molecules, there is fairly good moisture-keeping function.Type i collagen egg
White pure solution can resist a variety of radiation well, keep the integrality of cuticula moisture and fibre structure, improve Skin Cell life
Dis environment and the metabolism for promoting skin histology, the effect of to reach skin care, anti-aging.Thus I-type collagen
Using also quite varied in terms of medical and beauty treatment with health care.
NTx albumen is in triple helix structure, is made up of three α peptide chains, molecular weight is about 300kDa.Type i collagen egg
Contain 2 α in vain1（α3）Peptide chain and 1 α2Peptide chain, it is to have two bands of a spectrum, a bands of a spectrum at 100kDa in gel electrophoresis figure middle-molecular-weihydroxyethyl
It is due to two α1（α3）Peptide chain overlaps resulting, the α of another bands of a spectrum expression wall scroll2Peptide chain.It is in molecular weight in collection of illustrative plates
Also there are a bands of a spectrum at 200kDa, corresponding to the β chains in collagen structure, β chains are the dimers of α peptide chains.
I-type collagen prepares operation suture thread, stanch fibre, styptic sponge, dressing, artificial skin and artificial blood vessel etc.
During biomaterial for medical purpose, it is necessary to reach high-purity requirement.The common isolation and purification method of collagen includes salting out method, dialysis
Method, centrifugal process, electrophoresis and chromatography.But these purification process have the shortcomings that certain, as dialysis purifying needs 7 days or so
Time, time-consuming, easy cooperating microorganisms, is unfavorable for industrialized production.The purification process of I-type collagen then has no too many
Reported in literature.Due to traditional collagen purification technique, generally time-consuming longer, operating process is cumbersome, increases commodity production
Cost, therefore, it is necessary to seek new purification technique to purify I-type collagen.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of method for purifying NTx albumen, this is pure
Changing the method for NTx albumen has the advantages of time-consuming short, operating process is simple low with production cost.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of method for purifying NTx albumen is provided, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with certain density aqueous slkali, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；
Step 3: NTx albumen weak solution made from step 2 under pressure, is purified by milipore filter,
Purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type after purification between 4 ~ 7
Collagen.
In above-mentioned technical proposal, in the step 1, the animal tissue rich in I-type collagen from ox, pig,
One kind or any two or more composition in sheep, chicken, fish, donkey, deer, jellyfish or sea cucumber, the animal tissue is skin, flesh
One kind or any two or more composition in tendon, bone, cartilage or basilar memebrane.
In above-mentioned technical proposal, in the step 1, the protease is clostridiopetidase A, pepsin, trypsase, pawpaw
One kind or any two or more composition in protease, bromelain, chymotrypsin, bovine trypsin.
In above-mentioned technical proposal, in the step 1, the acid is organic acid, inorganic acid or buffers one kind of centering or appoint
Anticipate two or more compositions.
In above-mentioned technical proposal, the organic acid is acetic acid, formic acid, malic acid or citric acid；The inorganic acid be hydrochloric acid,
Sulfuric acid or phosphoric acid；The buffering to for Acetic acid-sodium acetate buffering to, citric acid-sodium citrate buffering to, phosphate-buffered pair or
Trishydroxymethylaminomethane-hydrochloride buffer pair.
In above-mentioned technical proposal, in the step 1, the cryogenic conditions are 5 DEG C ~ 10 DEG C.
In above-mentioned technical proposal, in the step 2, the aqueous slkali is sodium hydroxide solution, potassium hydroxide solution, carbon
Acid sodium solution or sodium bicarbonate solution, the concentration of the aqueous slkali is 5%-40%.
In above-mentioned technical proposal, in the step 2, the concentration of the NTx albumen weak solution for 0.01mg/ml ~
3mg/ml。
In above-mentioned technical proposal, in the step 3, the size of the pressure is 0.05Mpa ~ 1Mpa.
In above-mentioned technical proposal, in the step 3, the molecular weight that dams of the milipore filter is 100000 dalton, described
Milipore filter is hollow-fibre membrane or tubular ultra-filtration membrane, and the milipore filter is inner pressed milipore filter or external pressure type ultra-filtration membrane.
Compared with prior art, beneficial effect is the present invention：
（1）A kind of method for purifying NTx albumen provided by the invention, compared with prior art, can substantially shorten NTx
The purification time of protein liquid, reach preferable purification effect.The present invention, which is purified by ultrafiltration technology Purified collagen, only to be needed
12h ~ 24h cans reach required purity and concentration, and use prior art dialysis purifying then need 1 week or so when
Between can be only achieved identical purity and concentration.
（2）A kind of method for purifying NTx albumen provided by the invention, it is simple with method, it is easy to operate, it is produced into
This is low, and the characteristics of industrialization large-scale application can be applied to.
Brief description of the drawings
Fig. 1 is a kind of milipore filter purification schematic diagram of the method for purifying NTx albumen of the present invention.
Fig. 2 is the NTx Protein S DS-PAEG gel electrophoresis figures of the present invention.Wherein, 1 is Marker, and 2 be NTx
Albumen.
Embodiment
In order that technical problem solved by the invention, technical scheme and beneficial effect are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only explaining
The present invention, it is not intended to limit the present invention.
Embodiment 1.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen derives from ox, pig, sheep, chicken, fish, donkey, deer, jellyfish or sea cucumber
In one kind or any two or more composition, the animal tissue be one in skin, tendon, bone, cartilage or basilar memebrane
Kind or any two or more composition；In the present embodiment, protease is clostridiopetidase A, pepsin, trypsase, Papain
One kind or any two or more composition in enzyme, bromelain, chymotrypsin, bovine trypsin；The present embodiment
In, acid is organic acid, inorganic acid or buffers one kind of centering or any two or more composition；Wherein, organic acid be acetic acid,
Formic acid, malic acid or citric acid；Inorganic acid is hydrochloric acid, sulfuric acid or phosphoric acid；Buffering to for Acetic acid-sodium acetate buffering to, citric acid-
Buffered sodium citrate is to, phosphate-buffered pair or trishydroxymethylaminomethane-hydrochloride buffer pair；In the present embodiment, cryogenic conditions
For 5 DEG C ~ 10 DEG C；
Step 2: the aqueous slkali that the supernatant that step 1 obtains is 5%-40% with concentration is adjusted to neutrality, filter, insoluble matter is
NTx albumen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen
There is Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali
For sodium hydroxide solution, potassium hydroxide solution, sodium carbonate liquor or sodium bicarbonate solution；In the present embodiment, I obtained type glue
The concentration of former albumen weak solution is 0.01mg/ml ~ 3mg/ml；
Step 3: NTx albumen weak solution made from step 2 is passed through into milipore filter（As shown in Figure 1）, 0.05Mpa ~
Under 1Mpa pressure, purified, purified water is continuously added in purge process, until collagen solution pH value after purification is 4 ~ 7
Between, that is, obtain NTx albumen after purification.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, is surpassed
Filter membrane is hollow-fibre membrane or tubular ultra-filtration membrane, and milipore filter is inner pressed milipore filter or external pressure type ultra-filtration membrane.
Embodiment 2.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen derives from ox, and the animal tissue is skin；The present embodiment
In, protease is clostridiopetidase A；In the present embodiment, acid is organic acid；Wherein, organic acid is acetic acid；In the present embodiment, cryogenic conditions
For 8 DEG C；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with the aqueous slkali that concentration is 20%, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali is
Sodium hydroxide solution；In the present embodiment, the concentration of obtained NTx albumen weak solution is 2mg/ml；
Step 3: NTx albumen weak solution made from step 2 under 0.5Mpa pressure, is carried out pure by milipore filter
Change, purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type after purification between 5
Collagen.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, and milipore filter is hollow-fibre membrane, ultrafiltration
Film is inner pressed milipore filter.
A kind of method of purifying NTx albumen of the present embodiment, ultrafiltration purification technology is applied to NTx protein liquid
Purifying, hence it is evident that shorten NTx protein liquid purification time, reach preferable purification effect.The present invention combines centrifugation, filtering
NTx albumen is purified with ultrafiltration purification technology, is advantageous to prepare the I-type collagen of high-purity.I-type collagen
It is widely used in terms of medical and beauty treatment with health care, the pure solution of collagen can resist a variety of radiation well, keep cuticlar water
Point and fibre structure integrality, improve Skin Cell living environment and promote skin histology metabolism, to reach moist
The effect of skin, anti-aging.Injectivity collagen, hemostasis collagen sponge made of I-type collagen, and orthopedic surgery
Collagen charges etc. many important effects are all played in medical cosmetology field.
Embodiment 3.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen derives from pig, and the animal tissue is tendon；The present embodiment
In, protease is pepsin；In the present embodiment, acid is inorganic acid；Wherein, inorganic acid is hydrochloric acid；In the present embodiment, low temperature bar
Part is 5 DEG C；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with the aqueous slkali that concentration is 5%, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali is
Potassium hydroxide solution；In the present embodiment, the concentration of obtained NTx albumen weak solution is 0.01mg/ml；
Step 3: NTx albumen weak solution made from step 2 under 0.05Mpa pressure, is carried out pure by milipore filter
Change, purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type after purification between 4
Collagen.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, and milipore filter is tubular ultra-filtration membrane, ultrafiltration
Film is external pressure type ultra-filtration membrane.
A kind of method of purifying NTx albumen of the present embodiment, ultrafiltration purification technology is applied to NTx protein liquid
Purifying, hence it is evident that shorten NTx protein liquid purification time, reach preferable purification effect.The present invention combines centrifugation, filtering
NTx albumen is purified with ultrafiltration purification technology, is advantageous to prepare the I-type collagen of high-purity.I-type collagen
It is widely used in terms of medical and beauty treatment with health care, the pure solution of collagen can resist a variety of radiation well, keep cuticlar water
Point and fibre structure integrality, improve Skin Cell living environment and promote skin histology metabolism, to reach moist
The effect of skin, anti-aging.Injectivity collagen, hemostasis collagen sponge made of I-type collagen, and orthopedic surgery
Collagen charges etc. many important effects are all played in medical cosmetology field.
Embodiment 4.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen derives from the composition of sheep, chicken and fish, and the animal tissue is
The composition of bone and cartilage；In the present embodiment, protease is trypsase；In the present embodiment, acid is buffering pair；Wherein, buffer
To for Acetic acid-sodium acetate buffering pair；In the present embodiment, cryogenic conditions are 10 DEG C；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with the aqueous slkali that concentration is 40%, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali is
Sodium carbonate liquor；In the present embodiment, the concentration of obtained NTx albumen weak solution is 3mg/ml；
Step 3: NTx albumen weak solution made from step 2 under 1Mpa pressure, is purified by milipore filter,
Purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type glue after purification between 7
Former albumen.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, and milipore filter is tubular ultra-filtration membrane, milipore filter
For external pressure type ultra-filtration membrane.
A kind of method of purifying NTx albumen of the present embodiment, ultrafiltration purification technology is applied to NTx protein liquid
Purifying, hence it is evident that shorten NTx protein liquid purification time, reach preferable purification effect.The present invention combines centrifugation, filtering
NTx albumen is purified with ultrafiltration purification technology, is advantageous to prepare the I-type collagen of high-purity.I-type collagen
It is widely used in terms of medical and beauty treatment with health care, the pure solution of collagen can resist a variety of radiation well, keep cuticlar water
Point and fibre structure integrality, improve Skin Cell living environment and promote skin histology metabolism, to reach moist
The effect of skin, anti-aging.Injectivity collagen, hemostasis collagen sponge made of I-type collagen, and orthopedic surgery
Collagen charges etc. many important effects are all played in medical cosmetology field.
Embodiment 5.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen derives from jellyfish, and the animal tissue is cartilage；The present embodiment
In, protease is the composition of bromelain, chymotrypsin and bovine trypsin；In the present embodiment, acid is organic acid
With the composition of inorganic acid；Wherein, organic acid is formic acid；Inorganic acid is sulfuric acid；In the present embodiment, cryogenic conditions are 9 DEG C；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with the aqueous slkali that concentration is 10%, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali is
Sodium bicarbonate solution；In the present embodiment, the concentration of obtained NTx albumen weak solution is 1mg/ml；
Step 3: NTx albumen weak solution made from step 2 under 0.01Mpa pressure, is carried out pure by milipore filter
Change, purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type after purification between 6
Collagen.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, and milipore filter is tubular ultra-filtration membrane, ultrafiltration
Film is external pressure type ultra-filtration membrane.
A kind of method of purifying NTx albumen of the present embodiment, ultrafiltration purification technology is applied to NTx protein liquid
Purifying, hence it is evident that shorten NTx protein liquid purification time, reach preferable purification effect.The present invention combines centrifugation, filtering
NTx albumen is purified with ultrafiltration purification technology, is advantageous to prepare the I-type collagen of high-purity.I-type collagen
It is widely used in terms of medical and beauty treatment with health care, the pure solution of collagen can resist a variety of radiation well, keep cuticlar water
Point and fibre structure integrality, improve Skin Cell living environment and promote skin histology metabolism, to reach moist
The effect of skin, anti-aging.Injectivity collagen, hemostasis collagen sponge made of I-type collagen, and orthopedic surgery
Collagen charges etc. many important effects are all played in medical cosmetology field.
Embodiment 6.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen is from the composition of ox, pig, sheep and chicken, the animal groups
It is woven to counterdie；In the present embodiment, protease is bromelain；In the present embodiment, acid is organic acid；Wherein, organic acid is lemon
Lemon acid；In the present embodiment, cryogenic conditions are 6 DEG C；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with the aqueous slkali that concentration is 30%, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali is
Sodium bicarbonate solution；In the present embodiment, the concentration of obtained NTx albumen weak solution is 0.05mg/ml；
Step 3: NTx albumen weak solution made from step 2 under 0.8Mpa pressure, is carried out pure by milipore filter
Change, purified water is continuously added in purge process, until collagen solution pH value after purification obtains I after purification between 5.5
Collagen type.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, and milipore filter is hollow-fibre membrane, is surpassed
Filter membrane is inner pressed milipore filter.
A kind of method of purifying NTx albumen of the present embodiment, ultrafiltration purification technology is applied to NTx protein liquid
Purifying, hence it is evident that shorten NTx protein liquid purification time, reach preferable purification effect.The present invention combines centrifugation, filtering
NTx albumen is purified with ultrafiltration purification technology, is advantageous to prepare the I-type collagen of high-purity.I-type collagen
It is widely used in terms of medical and beauty treatment with health care, the pure solution of collagen can resist a variety of radiation well, keep cuticlar water
Point and fibre structure integrality, improve Skin Cell living environment and promote skin histology metabolism, to reach moist
The effect of skin, anti-aging.Injectivity collagen, hemostasis collagen sponge made of I-type collagen, and orthopedic surgery
Collagen charges etc. many important effects are all played in medical cosmetology field.
Embodiment 7.
A kind of method for purifying NTx albumen, it comprises the following steps：
Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin
Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant
Liquid；In the present embodiment, the animal tissue rich in I-type collagen derives from donkey, and the animal tissue is the group of skin and tendon
Compound；In the present embodiment, protease be chymotrypsin,；In the present embodiment, acid is buffering pair；Wherein, buffer to for phosphoric acid
Salt buffer pair；In the present embodiment, cryogenic conditions are 7 DEG C；
Step 2: the supernatant that step 1 obtains is adjusted to neutrality with the aqueous slkali that concentration is 8%, filtering, insoluble matter is I type
Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out
Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；In the present embodiment, aqueous slkali is
Sodium hydroxide solution；In the present embodiment, the concentration of obtained NTx albumen weak solution is 1.5mg/ml；
Step 3: NTx albumen weak solution made from step 2 under 0.08Mpa pressure, is carried out pure by milipore filter
Change, purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type after purification between 5
Collagen.In the present embodiment, the molecular weight that dams of milipore filter is 100000 dalton, and milipore filter is hollow-fibre membrane, ultrafiltration
Film is inner pressed milipore filter.
A kind of method of purifying NTx albumen of the present embodiment, ultrafiltration purification technology is applied to NTx protein liquid
Purifying, hence it is evident that shorten NTx protein liquid purification time, reach preferable purification effect.The present invention combines centrifugation, filtering
NTx albumen is purified with ultrafiltration purification technology, is advantageous to prepare the I-type collagen of high-purity.I-type collagen
It is widely used in terms of medical and beauty treatment with health care, the pure solution of collagen can resist a variety of radiation well, keep cuticlar water
Point and fibre structure integrality, improve Skin Cell living environment and promote skin histology metabolism, to reach moist
The effect of skin, anti-aging.Injectivity collagen, hemostasis collagen sponge made of I-type collagen, and orthopedic surgery
Collagen charges etc. many important effects are all played in medical cosmetology field.
Purity detecting
The collagen purity of SDS-PAEG gel electrophoresis detection after purification：
It is 1mg/m L to prepare NTx protein liquid concentration, and electrophoresis uses the mass fraction to be for 5% concentration glue and mass fraction
10% separation gel.Constant pressure electrophoresis, voltage are 80 V, treat that bromophenol blue is run to glue bottom and stop electrophoresis.Separation gel is put into coomassie
30min is dyed in light blue R-250 solution, uses acetic acid-ethanol decolorization liquid to be decolourized to separation gel background colour to be transparent（Fig. 2
It is shown）.
The structure of NTx albumen is in triple helix structure, is made up of three α peptide chains, molecular weight is about 300kDa.It is driven
The I-type collagen extracted in thing tendon contains 2 α1（α3）Peptide chain and 1 α2Peptide chain, it is in gel electrophoresis figure middle-molecular-weihydroxyethyl
There are two bands of a spectrum at 100kDa, a bands of a spectrum are due to two α1（α3）Peptide chain overlaps resulting, another bands of a spectrum expression wall scroll
α2Peptide chain.In collection of illustrative plates molecular weight be 200kDa at also have a bands of a spectrum, corresponding to the β chains in collagen structure, β chains are α peptides
The dimer of chain.When showing three bands of a spectrum in gel electrophoresis figure, represent that NTx albumen preserves complete triple helix knot
Structure.Remaining miscellaneous band is had no in Fig. 2, illustrates that collagen impurity content after purification is few, purity is high, and Chun Du≤96%.
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 scope is protected, although being explained with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent substitution, without departing from the reality of technical solution of the present invention
Matter and scope.
Claims (10)
- A kind of 1. method for purifying NTx albumen, it is characterised in that：It comprises the following steps：Step 1: the animal tissue that will be enriched in NTx albumen carries out degreasing, removes the pretreatment of manadesma and fat, Ran Houjin Row crushes, and is then extracted under cryogenic using acid and protease associated methods, after high speed centrifugation, collects supernatant Liquid；Step 2: the supernatant that step 1 obtains is adjusted to neutrality with certain density aqueous slkali, filtering, insoluble matter is I type Collagen, then with substantial amounts of pure water rinsing insoluble matter, refilter, and repeat the above steps repeatedly, until collagen goes out Existing Swelling, after then adding appropriate diluted acid dissolving, obtain NTx albumen weak solution；Step 3: NTx albumen weak solution made from step 2 under pressure, is purified by milipore filter, Purified water is continuously added in purge process, until collagen solution pH value after purification obtains I type after purification between 4 ~ 7 Collagen.
- A kind of 2. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 1, institute The animal tissue rich in I-type collagen is stated to derive from one kind in ox, pig, sheep, chicken, fish, donkey, deer, jellyfish or sea cucumber or appoint Anticipate two or more compositions, the animal tissue is one kind or any two kinds in skin, tendon, bone, cartilage or basilar memebrane Composition above.
- A kind of 3. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 1, institute Protease is stated as clostridiopetidase A, pepsin, trypsase, papain, bromelain, chymotrypsin, ox pancreas egg One kind or any two or more composition in white enzyme.
- A kind of 4. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 1, institute Acid is stated as organic acid, inorganic acid or buffers one kind of centering or any two or more composition.
- A kind of 5. method for purifying NTx albumen according to claim 4, it is characterised in that：The organic acid is second Acid, formic acid, malic acid or citric acid；The inorganic acid is hydrochloric acid, sulfuric acid or phosphoric acid；The buffering for Acetic acid-sodium acetate to delaying Punching is to, citric acid-sodium citrate buffering to, phosphate-buffered pair or trishydroxymethylaminomethane-hydrochloride buffer pair.
- A kind of 6. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 1, institute Cryogenic conditions are stated as 5 DEG C ~ 10 DEG C.
- A kind of 7. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 2, institute It is sodium hydroxide solution, potassium hydroxide solution, sodium carbonate liquor or sodium bicarbonate solution to state aqueous slkali, the concentration of the aqueous slkali For 5%-40%.
- A kind of 8. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 2, institute The concentration for stating NTx albumen weak solution is 0.01mg/ml ~ 3mg/ml.
- A kind of 9. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 3, institute The size for stating pressure is 0.05Mpa ~ 1Mpa.
- A kind of 10. method for purifying NTx albumen according to claim 1, it is characterised in that：In the step 3, The molecular weight that dams of the milipore filter is 100000 dalton, and the milipore filter is hollow-fibre membrane or tubular ultra-filtration membrane, described Milipore filter is inner pressed milipore filter or external pressure type ultra-filtration membrane.
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| WO2019169646A1 (en) * | 2018-03-07 | 2019-09-12 | 广州创尔生物技术股份有限公司 | Quality control method for biologically active collagen |
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| CN112608968A (en) * | 2021-01-14 | 2021-04-06 | 山东恒鑫生物科技有限公司 | Method for producing fish collagen peptide by using tilapia mossambica scale as raw material |
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| CN113230892A (en) * | 2021-05-27 | 2021-08-10 | 广州创尔生物技术股份有限公司 | Ultrafiltration membrane for purifying collagen by ultrafiltration and method for purifying collagen by ultrafiltration |
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