CN106946987A - The preparation method of high concentrated acid dissolubility collagen solution and water-soluble glue original solution - Google Patents
The preparation method of high concentrated acid dissolubility collagen solution and water-soluble glue original solution Download PDFInfo
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 126
- 102000008186 Collagen Human genes 0.000 title claims abstract description 126
- 229920001436 collagen Polymers 0.000 title claims abstract description 126
- 239000003292 glue Substances 0.000 title claims abstract description 28
- 239000002253 acid Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000000515 collagen sponge Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000000502 dialysis Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 120
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 45
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 45
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 24
- 239000011259 mixed solution Substances 0.000 claims description 24
- 239000004202 carbamide Substances 0.000 claims description 20
- 239000008367 deionised water Substances 0.000 claims description 18
- 229910021641 deionized water Inorganic materials 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 12
- 241001421714 Olynthus Species 0.000 claims description 11
- 239000000725 suspension Substances 0.000 claims description 8
- WFAJBYGUONQMCI-UHFFFAOYSA-N acetic acid urea Chemical compound NC(=O)N.NC(=O)N.NC(=O)N.NC(=O)N.NC(=O)N.NC(=O)N.C(C)(=O)O WFAJBYGUONQMCI-UHFFFAOYSA-N 0.000 claims description 6
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- ACOWUXLCNNHFCQ-UHFFFAOYSA-N [amino(sulfanyl)methylidene]azanium;acetate Chemical compound CC(O)=O.NC(N)=S ACOWUXLCNNHFCQ-UHFFFAOYSA-N 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 239000000084 colloidal system Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 238000000859 sublimation Methods 0.000 claims description 2
- 230000008022 sublimation Effects 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 abstract description 18
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 18
- 239000002904 solvent Substances 0.000 abstract description 15
- 239000003795 chemical substances by application Substances 0.000 abstract description 12
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 16
- 238000001704 evaporation Methods 0.000 description 15
- 230000008020 evaporation Effects 0.000 description 14
- 150000002148 esters Chemical class 0.000 description 10
- 239000010813 municipal solid waste Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108010020346 Polyglutamic Acid Proteins 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 229920002643 polyglutamic acid Polymers 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108010077465 Tropocollagen Proteins 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000512 collagen gel Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000003067 hemagglutinative effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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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 or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- General Health & Medical Sciences (AREA)
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Abstract
The present invention relates to a kind of high concentrated acid dissolubility and water-soluble glue original solution preparation method, comprise the following steps：Collagen sponge is shredded to slice → dissolved in the solution of hydrogen bonds disrupting agent, reaches that required concentration → dialysis displaces hydrogen bond disrupting agent → obtains highly concentrated collagen solution.Above-mentioned technical method is dissolved collagen sponge in the solvent of hydrogen bonds disrupting agent, because hydrogen bond disrupting agent can destroy the hydrogen bond formed between collagen, avoid the collagen formation collagen condensate in solution, so as to reduce the viscosity of collagen solution, improve the solubility of collagen in the solution, so that the concentration of collagen in the solution increases to more than 30g/L, then displaces the solvent of hydrogen bonds disrupting agent by dialysis, so as to obtain the collagen solution of the uniformity of high concentration.The program can be prepared on a large scale the high concentrated acid dissolubility collagen solution and water-soluble glue original solution of even concentration with collagen sponge in the short time.
Description
Technical field
The present invention relates to the preparation field of collagen solution, more particularly to high concentrated acid dissolubility collagen solution and water-soluble collagen
The preparation method of solution.
Background technology
Collagen is the major structural protein of extracellular matrix, is biological in-vivo content highest protein, it is widely present
In in human body and animal body.As a kind of natural macromolecular material of wide material sources, collagen has many excellent performances, such as with
The high-affinity of cell, produces hemagglutinative function with blood platelet, can promote the covering of creeping of wound convergence and epidermal cell, can be biological
Degraded etc., thus it is widely used in pharmaceutical sanitary field.Collagen is typically to extract to obtain from Animal Skin with the form of solution,
Carry out obtaining refined collagen sponge after frozen dried again, this collagen can dissolve in an acidic solution, referred to as acid -soluble collagen.China
Patent application " a kind of water-soluble undenatured collagen and preparation method thereof " (201610348502.2).The method utilizes polycarboxylic acids-NHS
Ester is modified to acid-soluble collagen, and NHS ester groups react with collagenous amino, collagen is connected with polycarboxylic acids, on polycarboxylic acids
A large amount of carboxyls of institute's band can reduce collagen isoelectric point, the solubility property of enhancing collagen in neutral conditions.After modification, by collagen
Freeze-drying, obtains water-soluble glue olynthus.Collagen sponge can be redissolved in solvent, be applied with the form of solution,
Also collagen solution can be further made to the product form such as collagenous fibres, collagen gel to be applied.
In many application scenarios, it is desirable to which collagen solution has higher concentration, in favor of being processed into various required productions
Product form.However, due to the characteristic of tropocollagen molecule, it is easily formed intermolecular hydrogen bonding in the solution, and then is further formed poly-
Collective, so that collagen solution retrogradation, the concentration of collagen solution is difficult to further raising.Generally collagen solution is about
It is very sticky during 20g/L concentration, up to 20g/L concentration after add collagen be difficult to continue dissolve, make its concentration by
Greatly limitation.Existing literature report is thickened by the method concentrated to collagen solution, i.e., using natural evaporation solvent or
The method of evaporation solvent under negative pressure, can effectively improve the concentration of collagen, reach wanting for high concentration (being more than 30g/L) collagen solution
Ask.But the method cycle of natural evaporation concentration is oversize, it usually needs several weeks can be only achieved aimed concn, and the place of this method
Reason amount is too small, it is difficult to realize a large amount of prepare.Evaporation solvent can slightly shorten manufacturing cycle under negative pressure, but manufacturing cycle still mistake
It is long.And evaporation concentration method causes to even result in top layer collagen drying under the ectonexine density unevenness one of collagen solution, extreme case
Film forming.
The content of the invention
Therefore, inventor providing the hydrogen bond by destroying tropocollagen molecule, the high concentration of even concentration can be efficiently carried out
Scheme prepared by collagen solution.To achieve the above object, a kind of high concentrated acid dissolubility collagen solution preparation side is inventor provided
Method, comprises the following steps：
A1：Acid-soluble collagen sponge is shredded to fine strip shape, the first sponge slice is made；
A2：First sponge slice is added into acetic acid-urea liquid or acetic acid-thiourea solution, stirring to the first sponge slice
It is completely dissolved, the first mixed solution is made, wherein the concentration of the first sponge slice is 40-80g/L；
A3：First mixed solution is loaded into the first bag filter, first bag filter is placed in acetum and dialysed
24-72 hours, high concentrated acid dissolubility collagen solution is made.
Acid-soluble collagen sponge is dissolved in the acid flux material containing urea or thiocarbamide, because urea and thiocarbamide are hydrogen
Key disrupting agent, can destroy the hydrogen bond formed between collagen, it is to avoid the collagen formation collagen condensate in solution, so as to reduce collagen
The viscosity of solution, improves the solubility of collagen in the solution so that the concentration increase of collagen in the solution, then is replaced by dialysing
Go out urea or thiocarbamide, so as to obtain the acid-soluble collagen solution of the uniformity of high concentration.
Further, in the A1 steps, acid-soluble being prepared by the following method for collagen sponge and obtain：
B1：It will be rubbed after Animal Skin stripping and slicing, the first colloid be made；
B2：The acetic acid that the pepsin and concentration that first colloid is added into mass percent 3% are 0.5mol/L
Solution, is made the first suspension；First suspension is placed in 4 DEG C of constant-temperature tables and vibrated, shaking speed is 180r/min,
Duration of oscillation is 72 hours, and the second mixed solution is made；
B3：Second mixed solution is subjected to centrifuging and taking supernatant, the supernatant is saltoutd, and is made first rough
Collagen suspension, the first rough collagen suspension is centrifuged, and abandons supernatant, the first rough collagen is made；
B4：Described first rough collagen is added into 0.1mol/L acetums, stirs to being completely dissolved, the 3rd mixing is made
Solution；
B5：3rd mixed solution is loaded into the second bag filter, second bag filter is placed in deionized water and carried out
Dialysis, dialysis time is 72 hours, and the 4th mixed solution is made；
B6：4th mixed solution is freeze-dried, sublimation drying is 72 hours, and acid-soluble collagen is made
Sponge.
Further, acetic acid-urea liquid and acetic acid-thiourea solution described in the A2 steps, acetic acid molar concentration is
The concentration of 0.01-0.5mol/L, urea and thiocarbamide is 5-30g/L.
Further, in the A2 steps, temperature control is at 4-10 DEG C, and dissolution time was controlled at 30-180 minutes.
Further, in the A3 steps, the concentration of acetum is 0.01-0.1mol/L, and acetum is small every 12
When change liquid 1 time.
In addition, inventor additionally provides a kind of high-concentration water-soluble collagen solution preparation method, comprise the following steps：
C1：Water-soluble glue olynthus is shredded to fine strip shape, the second sponge slice is made；
C2：Second sponge slice is added into urea liquid or thiourea solution, stirring to the second sponge slice is completely dissolved, and is made
Into the 5th mixed solution, wherein the concentration of the second sponge slice is 40-80g/L；
C3：5th mixed solution is loaded into the 3rd bag filter, the 3rd bag filter is placed in deionized water and dialysed
24-72 hours, high-concentration water-soluble collagen solution is made.
Water-soluble glue olynthus is dissolved in urea or thiocarbamide solvent, because urea and thiocarbamide are hydrogen bond disrupting agent,
The hydrogen bond formed between collagen can be destroyed, it is to avoid the collagen formation collagen condensate in solution, so as to reduce the viscous of collagen solution
Degree, improves collagen solubility in the solution so that the concentration increase of collagen in the solution, then by dialysis displace urea or
Thiocarbamide, so as to obtain the water-soluble glue original solution of the uniformity of high concentration.
Further, the concentration of urea liquid and thiourea solution described in the C2 steps, urea and thiocarbamide is 5-30g/
L。
Further, in the C2 steps, temperature control is at 4-10 DEG C, and dissolution time was controlled at 60-180 minutes.
Further, in the C3 steps, deionized water changed liquid every 12 hours 1 time.
Prior art is different from, above-mentioned technical proposal is dissolved collagen sponge in the solvent of hydrogen bonds disrupting agent,
Because hydrogen bond disrupting agent can destroy the hydrogen bond formed between collagen, it is to avoid the collagen formation collagen condensate in solution, so as to drop
The viscosity of low collagen solution, improves collagen solubility in the solution so that the concentration of collagen in the solution increase to 30g/L with
On, then the solvent of hydrogen bonds disrupting agent displaced by dialysis, so as to obtain the collagen solution of high concentration uniformity.The program
The high concentrated acid dissolubility collagen solution and water-soluble collagen of even concentration can be prepared on a large scale within a short period of time with collagen sponge
Solution, changes the acid-soluble collagen solution of high concentration and water-soluble glue original solution in current industry and prepares that yield is smaller, prepare
Excessive cycle, and it is difficult to the situation of the highly concentrated collagen solution of acquisition uniformity.
Embodiment
To describe the technology contents of technical scheme in detail, feature, the objects and the effects being constructed, below in conjunction with specific reality
Example is applied to be explained in detail.
The preparation method technology path of high concentrated acid dissolubility collagen solution and water-soluble glue original solution of the present invention is：Collagen sea
Silk floss is shredded to slice → dissolved in the solution of hydrogen bonds disrupting agent, reaches that required concentration → dialysis displaces hydrogen bond destruction
Agent → obtain highly concentrated collagen solution.
Collagen described in present embodiment is the natural collagen without degradation treatment.
Embodiment 1：
(1) ox-hide is cut into small pieces, is put into meat grinder and rubs.Weight in wet base 10g rubbing ox-hide is put into conical flask, plus
The pepsin and 500mL concentration for entering 0.3g are 0.5mol/L acetic acid, and then conical flask is put into 4 DEG C of constant-temperature tables and vibrated
Collagen is extracted, shaking speed is set to 180r/min, 72 hours working times.Solution after mechanical shaking extraction takes supernatant after centrifugation
Liquid, adds after sodium chloride is saltoutd to 0.7mol/L concentration and centrifuges again, abandon supernatant, the rough collagen of gained is dissolved in
Dissolved in 0.1mol/L acetic acid, the collagen solution dissolved be fitted into bag filter, dialysed 72 hours with deionized water,
Acid-soluble collagen sponge is made in 72 hours in last vacuum freeze drying.
(2) take acetic acid 0.01mol and urea 30g, prepare 1L acetic acid-urea liquid (wherein acetic acid molar concentration
0.01mol/L, urea concentration is 30g/L), the acid-soluble collagen sponges of 80g are shredded to fine strip shape, are gradually added into the solvent,
Stir 120 minutes, be completely dissolved to sponge at 4 DEG C.Mixed solution after dissolving is loaded into bag filter, in dialyzate
Dialysis 72 hours, change liquid 1 time in during which every 12 hours, the saturating of high concentration are produced after the completion of dialysis in (0.01mol/L acetums)
The bright acid-soluble collagen solution of shape.
(3) the acid-soluble collagen solution of transparence is diluted after 10 times with 0.01mol/L acetums, existed with Biurets methods
540nm carries out Concentration Testing, and it is 7.82g/L, the gained acid collagen solution concentration of embodiment 1 to measure collagen solution concentration after dilution
For 78.2g/L.
Embodiment 1 is 74 small the time required to the acid collagen solution that concentration is 78.2g/L is prepared with acid-soluble collagen sponge
When；And the time required to traditional natural evaporation it is 27-29 days, it is 16-17 days the time required to Municipal Solid Waste Landfillies.
Embodiment 2
(1) pigskin is cut into small pieces, is put into meat grinder and rubs.Weight in wet base 10g rubbing pigskin is put into 1L conical flask
In, the pepsin and 500mL concentration for adding 0.3g are 0.5mol/L acetic acid, and conical flask then is put into 4 DEG C of constant-temperature tables
Middle vibration, working speed is 180r/min, and the working time is 72 hours.Solution after vibration takes supernatant after centrifugation, adds
Sodium chloride is centrifuged again after being saltoutd to 0.7mol/L concentration, abandons supernatant, and the rough collagen of gained is dissolved in 0.1mol/L's
In acetic acid, the collagen solution dissolved is fitted into bag filter deionized water is dialysed 72 hours, last vacuum freeze drying 72
Hour, acid-soluble collagen sponge is made.
(2) acetic acid 0.05mol and thiocarbamide 5g are taken, 1L acetic acid-thiourea solution (acetic acid molar concentration 0.5mol/L, sulphur is prepared
Urea concentration is 5g/L), the acid-soluble collagen sponges of 60g are shredded to fine strip shape, are gradually added into the solvent, in 7 DEG C of agitating solutions
60 minutes, it is completely dissolved to sponge.Mixed solution is loaded into bag filter, the dialysis 24 in dialyzate (0.1mol/L acetums)
Hour, change liquid 1 time within during which every 12 hours, the acid-soluble collagen solution of transparence of high concentration is produced after the completion of dialysis.
(3) the acid-soluble collagen solution of transparence is diluted after 10 times with 0.1mol/L acetums, existed with Biurets methods
540nm is detected that it is 5.90g/L to measure collagen solution concentration after dilution, and the acid-soluble collagen solution concentration of the gained of embodiment 2 is
59.0g/L。
Embodiment 2 is 25 the time required to the acid collagen solution that concentration is 59.0g/L is prepared with acid-soluble collagen sponge
Hour；And the time required to natural evaporation method it is 20-21 days, it is 12-13 days the time required to Municipal Solid Waste Landfillies.
Embodiment 3
(1) fish-skin is cut into small pieces, is put into meat grinder and rubs.Weight in wet base 10g rubbing fish-skin is put into conical flask, plus
The pepsin and 500mL concentration for entering 0.3g are 0.5mol/L acetic acid, and then conical flask is put into 4 DEG C of constant-temperature tables and shaken
Swing, working speed is 180r/min, 72 hours working times.Solution after vibration takes supernatant after centrifugation, adds sodium chloride
Centrifuged again after being saltoutd to 0.7mol/L concentration, abandon supernatant, the rough collagen of gained is dissolved in 0.1mol/L acetic acid,
The collagen solution dissolved is fitted into bag filter deionized water is dialysed 72 hours, last vacuum freeze drying 72 hours, system
Obtain acid-soluble collagen sponge.
(2) acetic acid 0.25mol and urea 17.5g are taken, 1L acetic acid-urea liquid (acetic acid molar concentration 0.25mol/ is prepared
L, urea concentration 17.5g/L), 40g acid-soluble collagen sponge is shredded to fine strip shape, is gradually added into the solvent and is stirred at 10 DEG C
Mix 180 minutes, be completely dissolved to sponge.Mixed solution is loaded into bag filter, in dialyzate (0.05mol/L acetums) thoroughly
Analysis 48 hours, changes liquid 1 time in during which every 12 hours, the acid-soluble collagen solution of transparence of high concentration is produced after the completion of dialysis.
(3) the acid-soluble collagen solution of transparence is diluted after 10 times with 0.05mol/L acetums, existed with Biurets methods
540nm is detected that it is 3.92g/L to measure collagen solution concentration after dilution, and the acid-soluble collagen solution concentration of the gained of embodiment 3 is
39.2g/L。
It is small for 51 that embodiment 3 prepares the required time of concentration 39.2g/L acid collagen solution with acid-soluble collagen sponge
When, traditional natural evaporation required time is 15-16 days, and Municipal Solid Waste Landfillies required time is 8-9 days.
Embodiment 4
(1) 500mg polyglutamic acid is added in 10mL dimethyl sulphoxide solution, stirring, until polyglutamic acid is complete
Dissolving；100mg succimide is added, stirring adds and 9h is reacted under 33mg carbodiimides, normal temperature, adds anhydrous
Ethanol separates out product；With absolute ethanol washing 2 times, after n-hexane is washed 2 times, vacuum drying obtains polyglutamic acid-NHS esters.
(2) natural collagen is dissolved in hydrochloric acid solution (pH 3.0), concentration is 10mg/ml；Polyglutamic acid-NHS esters are molten
Solution is in DMSO, and concentration is 50mg/mL；Collagen solution 40ml is taken, the μ L (1/5) of polyglutamic acid-NHS ester solutions 1200 is added, stirs
Mix, reacted 12 hours under normal temperature；Dialysed 1 day with deionized water, water-soluble glue olynthus is obtained after freeze-drying.
(3) compound concentration is 5g/L urea liquid 1L, and 40g collagen sponge is shredded to fine strip shape, this is gradually added into
In solvent, in temperature, 7 DEG C are stirred 120 minutes, are completely dissolved to sponge.Mixed solution is loaded into bag filter, in deionized water
Dialysis 24 hours, changes liquid 1 time in during which every 12 hours, the transparence water-soluble glue original solution of high concentration is produced after the completion of dialysis.
(4) transparence water-soluble glue original solution is diluted after 10 times with deionized water, carried out with Biurets methods in 540nm
Detection, it is 3.95g/L to measure collagen solution concentration after dilution, and the gained water-soluble glue original solution concentration of embodiment 4 is 39.5g/L.
It is small for 26 that embodiment 4 prepares the required time of concentration 39.5g/L aqueous collagen solution with water-soluble glue olynthus
When, and the time required to traditional natural evaporation it is 16-17 days, it is 9-10 days the time required to Municipal Solid Waste Landfillies.
Embodiment 5
(1) 1000mg polymalic acid is added in 20mL DMSO solution, stirring, until polymalic acid is completely dissolved.
50mg NHS is added, stirring adds and 18h is reacted under 50mg EDC, normal temperature, and adding absolute ethyl alcohol separates out product.With nothing
Water-ethanol is washed 2 times, after n-hexane is washed 1 time, and vacuum drying obtains polymalic acid-NHS esters.
(2) natural collagen is dissolved in hydrochloric acid solution (pH 2.5), obtains the collagen solution that concentration is 5mg/ml；By poly- apple
Tartaric acid-NHS esters are dissolved in DMSO, and concentration is 100mg/mL；Collagen solution 20ml is taken, polymalic acid-NHS ester solutions are added
18h is reacted under 500 μ L (1/2), stirring, normal temperature；With deionized water dialysis 2d, water-soluble glue olynthus is obtained after freeze-drying.
(3) 30g/L thiourea solution 1L is prepared, 60g water-soluble glue olynthus is shredded to fine strip shape, this is gradually added into
In solvent, stir 60 minutes, be completely dissolved to sponge at 4 DEG C.Mixed solution is loaded into bag filter, 48 are dialysed in deionized water
Hour, change liquid 1 time within during which every 12 hours, the transparence water-soluble glue original solution of high concentration is produced after the completion of dialysis.
(4) transparence water-soluble glue original solution is diluted after 10 times with deionized water, carried out with Biurets methods in 540nm
Concentration Testing, it is 5.93g/L to measure collagen solution concentration after dilution, and the gained water-soluble glue original solution concentration of embodiment 5 is
59.3g/L。
The required time that embodiment 5 prepares 59.3g/L aqueous collagen solution with water-soluble glue olynthus is 49 hours, and
It it is 14-15 days the time required to being 21-22 days, Municipal Solid Waste Landfillies the time required to traditional natural evaporation.
Embodiment 6
(1) 2000mg polyglutamic acid is added in 50mL DMSO solution, stirring, until polyglutamic acid is completely dissolved；
1000mg NHS is added, stirring adds and 15h is reacted under 800mg EDC, normal temperature, and adding acetone separates out product；With anhydrous
Ethanol is washed 3 times, after n-hexane is washed 2 times, and vacuum drying obtains polyglutamic acid-NHS esters.After measured, polyglutamic acid-NHS esters
Esterification degree is 15.3%.
(2) natural collagen is dissolved in hydrochloric acid solution (pH 4.5), concentration is 1mg/ml；Polyglutamic acid-NHS esters dissolve
In DMSO, concentration is 10mg/mL；Collagen solution 30ml is taken, the μ L (1/20) of polyglutamic acid-NHS ester solutions 150 is added, stirs,
Reacted 24 hours under normal temperature.Dialysed 3 days with deionized water, water-soluble glue olynthus is obtained after freeze-drying.
(3) 17.5g/L thiourea solution 1L is prepared, 80g collagen sponge is shredded to fine strip shape, the solvent is gradually added into
In, stir 180 minutes, be completely dissolved to sponge at 10 DEG C.Mixed solution is loaded into bag filter, dialysis 72 is small in deionized water
When, change liquid 1 time within during which every 12 hours, the transparence water-soluble glue original solution of high concentration is produced after the completion of dialysis.
(4) transparence water-soluble glue original solution is diluted after 10 times with deionized water, carried out with Biurets methods in 540nm
Detection, it is 7.97g/L to measure collagen solution concentration after dilution, and the gained water-soluble glue original solution concentration of embodiment 6 is 79.7g/L.
The required time for the aqueous collagen solutions of 79.7g/L that embodiment 6 is prepared with water-soluble glue olynthus is 75 hours, and
It it is 18-19 days the time required to being 28-30 days, Municipal Solid Waste Landfillies the time required to traditional natural evaporation.
In summary embodiment, the concentration of the acid-soluble collagen solution prepared by the present invention is 39.2-78.2g/L, this hair
The concentration of bright prepared water-soluble glue original solution is 39.5-79.7g/L；The concentration of collagen solution reaches height more than 39g/L
The requirement of concentration collagen solution (concentration of collagen solution is more than 30g/L), the need for various converted products can be met.It is of the invention high
Concentration collagen solution preparation time is up to 75 hours, far below the preparation of same concentration traditional natural evaporation and Municipal Solid Waste Landfillies
Time.Simultaneously as the principle of evaporation, to produce high concentration collagen solution and need to configure the low concentration collagen solutions of several times of amounts and
Row evaporation, therefore under identical working condition, evaporation is several times as much as using the production batch of preparation method of the present invention.
It should be noted that herein, such as first and second or the like relational terms are used merely to a reality
Body or operation make a distinction with another entity or operation, and not necessarily require or imply these entities or deposited between operating
In any this actual relation or order.Moreover, term " comprising ", "comprising" or its any other variant are intended to
Nonexcludability is included, so that process, method, article or terminal device including a series of key elements not only include those
Key element, but also other key elements including being not expressly set out, or also include being this process, method, article or end
The intrinsic key element of end equipment.In the absence of more restrictions, limited by sentence " including ... " or " including ... "
Key element, it is not excluded that also there is other key element in the process including the key element, method, article or terminal device.This
Outside, herein, " being more than ", " being less than ", " exceeding " etc. are interpreted as not including this number；" more than ", " following ", " within " etc. understand
It is to include this number.
Although the various embodiments described above are described, those skilled in the art once know basic wound
The property made concept, then can make other change and modification to these embodiments, so embodiments of the invention are the foregoing is only,
Not thereby the scope of patent protection of the present invention, equivalent structure or equivalent process that every utilization description of the invention is made are limited
Conversion, or is directly or indirectly used in other related technical fields, be similarly included in the present invention scope of patent protection it
It is interior.
Claims (9)
1. a kind of high concentrated acid dissolubility collagen solution preparation method, it is characterised in that comprise the following steps：
A1：Acid-soluble collagen sponge is shredded to fine strip shape, the first sponge slice is made；
A2：First sponge slice is added into acetic acid-urea liquid or acetic acid-thiourea solution, stirred complete to the first sponge slice
Dissolving, is made the first mixed solution, wherein the concentration of the first sponge slice is 40-80g/L；
A3：First mixed solution is loaded into the first bag filter, first bag filter is placed in acetum the 24-72 that dialyses
Hour, high concentrated acid dissolubility collagen solution is made.
2. preparation method according to claim 1, it is characterised in that in the A1 steps, the acid-soluble collagen sponge
Be prepared by the following method and obtain：
B1：It will be rubbed after Animal Skin stripping and slicing, the first colloid be made；
B2：The acetum that the pepsin and concentration that first colloid is added into mass percent 3% are 0.5mol/L,
The first suspension is made；First suspension is placed in 4 DEG C of constant-temperature tables and vibrated, shaking speed is 180r/min, vibration
Time is 72 hours, and the second mixed solution is made；
B3：Second mixed solution is subjected to centrifuging and taking supernatant, the supernatant is saltoutd, and the first rough collagen is made
Suspension, the first rough collagen suspension is centrifuged, and abandons supernatant, the first rough collagen is made；
B4：Described first rough collagen is added into 0.1mol/L acetums, stirred to being completely dissolved, the 3rd mixing is made molten
Liquid；
B5：3rd mixed solution is loaded into the second bag filter, second bag filter, which is placed in deionized water, is dialysed,
Dialysis time is 72 hours, and the 4th mixed solution is made；
B6：4th mixed solution is freeze-dried, sublimation drying is 72 hours, acid-soluble collagen sea is made
It is continuous.
3. preparation method according to claim 1, it is characterised in that：Acetic acid-urea liquid described in the A2 steps and
Acetic acid-thiourea solution, acetic acid molar concentration is 0.01-0.5mol/L, and the concentration of urea and thiocarbamide is 5-30g/L.
4. preparation method according to claim 1, it is characterised in that：In the A2 steps, temperature control is molten at 4-10 DEG C
Time control is solved at 30-180 minutes.
5. preparation method according to claim 1, it is characterised in that：In the A3 steps, the concentration of acetum is
0.01-0.1mol/L, acetum changed liquid every 12 hours 1 time.
6. a kind of high-concentration water-soluble collagen solution preparation method, it is characterised in that comprise the following steps：
C1：Water-soluble glue olynthus is shredded to fine strip shape, the second sponge slice is made；
C2：Second sponge slice is added into urea liquid or thiourea solution, stirring is completely dissolved to the second sponge slice, be made the
Five mixed solutions, wherein the concentration of the second sponge slice is 40-80g/L；
C3：5th mixed solution is loaded into the 3rd bag filter, the 3rd bag filter is placed in deionized water the 24-72 that dialyses
Hour, high-concentration water-soluble collagen solution is made.
7. preparation method according to claim 6, it is characterised in that：Urea liquid and thiocarbamide are molten described in the C2 steps
The concentration of liquid, urea and thiocarbamide is 5-30g/L.
8. preparation method according to claim 6, it is characterised in that：In the C2 steps, temperature control is molten at 4-10 DEG C
Time control is solved at 60-180 minutes.
9. preparation method according to claim 6, it is characterised in that：In the C3 steps, deionized water was every 12 hours
Change liquid 1 time.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109337383A (en) * | 2018-09-30 | 2019-02-15 | 福建工程学院 | A kind of collagen-based selfreparing hydrogel and preparation method thereof |
| CN113583109A (en) * | 2021-08-03 | 2021-11-02 | 美尔健（深圳）生物科技有限公司 | Jellyfish active protein and preparation method and application thereof |
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| EP0628573A1 (en) * | 1993-05-24 | 1994-12-14 | Kao Corporation | Process for producing solubilized protein |
| DE102006026591B4 (en) * | 2006-05-31 | 2008-09-04 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Method for isolating collagen from collagen-containing tissue |
| CN104672316B (en) * | 2015-02-11 | 2018-06-26 | 苏州丝美特生物技术有限公司 | A kind of silk fibroin protein solution prepares and identification method |
| CN104017073A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Method for preparing collagen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109337383A (en) * | 2018-09-30 | 2019-02-15 | 福建工程学院 | A kind of collagen-based selfreparing hydrogel and preparation method thereof |
| CN109337383B (en) * | 2018-09-30 | 2021-05-25 | 福建工程学院 | Collagen-based self-repairing hydrogel and preparation method thereof |
| CN113583109A (en) * | 2021-08-03 | 2021-11-02 | 美尔健（深圳）生物科技有限公司 | Jellyfish active protein and preparation method and application thereof |
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