CN106946987B - High-concentration acid-soluble collagen solution and preparation method of water-soluble collagen solution - Google Patents
High-concentration acid-soluble collagen solution and preparation method of water-soluble collagen solution Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 146
- 108010035532 Collagen Proteins 0.000 title claims abstract description 146
- 229920001436 collagen Polymers 0.000 title claims abstract description 146
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000000515 collagen sponge Substances 0.000 claims abstract description 37
- 238000000502 dialysis Methods 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 28
- 239000000243 solution Substances 0.000 claims description 154
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 84
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 50
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 32
- 239000011259 mixed solution Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000008367 deionised water Substances 0.000 claims description 18
- 229910021641 deionized water Inorganic materials 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- 239000004202 carbamide Substances 0.000 claims description 17
- 238000011049 filling Methods 0.000 claims description 15
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- 239000000725 suspension Substances 0.000 claims description 8
- 230000010355 oscillation Effects 0.000 claims description 7
- 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
- 238000004108 freeze drying Methods 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
- 238000005185 salting out Methods 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 239000000084 colloidal system Substances 0.000 claims description 4
- 239000000126 substance Substances 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 14
- 229920000642 polymer Polymers 0.000 abstract description 4
- 238000010008 shearing Methods 0.000 abstract description 3
- 238000001704 evaporation Methods 0.000 description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 17
- 150000002148 esters Chemical class 0.000 description 11
- 239000002253 acid Substances 0.000 description 8
- 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
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108010020346 Polyglutamic Acid Proteins 0.000 description 4
- 229920002643 polyglutamic acid Polymers 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
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- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
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- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 238000009777 vacuum freeze-drying Methods 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 125000003277 amino group Chemical group 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
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The invention relates to a preparation method of a high-concentration acid-soluble and water-soluble collagen solution, which comprises the following steps: shearing collagen sponge into thin strips → dissolving in solution containing hydrogen bond disruptor to reach required concentration → dialyzing to replace the hydrogen bond disruptor → obtaining high concentration collagen solution. The technical method dissolves the collagen sponge in the solvent containing the hydrogen bond disruptor, and the hydrogen bond disruptor can disrupt the hydrogen bonds formed among the collagen, so that the collagen in the solution is prevented from forming collagen polymers, the viscosity of the collagen solution is reduced, the solubility of the collagen in the solution is improved, the concentration of the collagen in the solution is increased to more than 30g/L, and the solvent containing the hydrogen bond disruptor is replaced by dialysis, so that the high-concentration uniform collagen solution is obtained. The proposal can prepare high-concentration acid-soluble collagen solution and water-soluble collagen solution with uniform concentration in a short time and in a large scale by using the collagen sponge.
Description
Technical Field
The invention relates to the field of preparation of collagen solutions, in particular to a high-concentration acid-soluble collagen solution and a preparation method of the water-soluble collagen solution.
Background
Collagen is the major structural protein of extracellular matrix, the most abundant protein in the organism, and it is widely present in the human and animal bodies. As a natural polymer material with a wide variety of sources, collagen has many excellent properties, such as high affinity with cells, hemagglutination with platelets, promotion of wound healing and creeping coverage of epidermal cells, biodegradability, and the like, and thus is widely used in the field of medicine and health. Collagen is usually extracted from animal skin in the form of a solution, and then lyophilized to obtain a refined collagen sponge, which is soluble in an acidic solution and is called acid-soluble collagen. Chinese patent application "a water-soluble undenatured collagen and its preparation method" (201610348502.2). According to the method, acid-soluble collagen is modified by polycarboxylic acid-NHS ester, the NHS ester group reacts with collagen amino groups, so that the collagen and the polycarboxylic acid are connected, and a large number of carboxyl groups on the polycarboxylic acid can reduce the isoelectric point of the collagen and enhance the solubility of the collagen under a neutral condition. After modification, the collagen was freeze-dried to obtain a water-soluble collagen sponge. The collagen sponge can be dissolved in solvent again, and can be used in the form of solution, or the collagen solution can be further prepared into collagen fiber, collagen gel, etc.
In many applications, it is desirable to have a relatively high concentration of collagen solution to facilitate processing into various desired product forms. However, due to the nature of collagen molecules, intermolecular hydrogen bonds are easily formed in the solution, and further aggregates are formed, so that the collagen solution becomes thick, and the concentration of the collagen solution is difficult to further increase. Generally, the collagen solution is very viscous at a concentration of about 20g/L, and the collagen solution is difficult to continue to dissolve after the concentration of 20g/L is reached, so that the concentration of the collagen solution is greatly limited. The prior literature reports that the concentration of collagen solution is thickened by a concentration method, namely a method of evaporating the solvent naturally or evaporating the solvent under negative pressure is adopted, so that the concentration of collagen can be effectively improved, and the requirement of high-concentration (more than 30g/L) collagen solution is met. However, the method of natural evaporation concentration has a long cycle time, which usually takes several weeks to reach the target concentration, and the throughput of this method is too small to realize mass production. The solvent evaporation under negative pressure can slightly shorten the preparation period, but the preparation period is still too long. And the evaporation concentration method causes uneven concentration of the inner layer and the outer layer of the collagen solution, and even causes the surface layer collagen to be dried and formed into a film under extreme conditions.
Disclosure of Invention
For this reason, the inventors have provided a solution that can efficiently prepare a high-concentration collagen solution having a uniform concentration by breaking hydrogen bonds of collagen molecules. To achieve the above object, the inventors provide a method for preparing a high-concentration acid-soluble collagen solution, comprising the steps of:
a1: cutting acid-soluble collagen sponge into thin strips to prepare first sponge thin strips;
a2: adding acetic acid-urea solution or acetic acid-thiourea solution into the first sponge thin strip, and stirring until the first sponge thin strip is completely dissolved to prepare a first mixed solution, wherein the concentration of the first sponge thin strip is 40-80 g/L;
a3: and filling the first mixed solution into a first dialysis bag, and dialyzing the first dialysis bag in an acetic acid solution for 24-72 hours to prepare a high-concentration acid-soluble collagen solution.
The acid-soluble collagen sponge is dissolved in an acid solvent containing urea or thiourea, and the urea and the thiourea are hydrogen bond damaging agents, so that hydrogen bonds formed among collagen can be damaged, the collagen in the solution is prevented from forming collagen polymers, the viscosity of the collagen solution is reduced, the solubility of the collagen in the solution is improved, the concentration of the collagen in the solution is increased, and the urea or the thiourea is replaced through dialysis, so that the high-concentration uniform acid-soluble collagen solution is obtained.
Further, in the step a1, the acid-soluble collagen sponge is prepared by the following method:
b1: cutting animal skin into pieces, and mincing to obtain a first colloid;
b2: adding pepsin with the mass percentage of 3% and an acetic acid solution with the concentration of 0.5mol/L into the first colloid substance to prepare a first suspension; placing the first suspension in a constant-temperature shaking table at 4 ℃ for oscillation, wherein the rotating speed of the shaking table is 180r/min, and the oscillation time is 72 hours to prepare a second mixed solution;
b3: centrifuging the second mixed solution to obtain a supernatant, salting out the supernatant to prepare a first crude collagen suspension, centrifuging the first crude collagen suspension, and removing the supernatant to prepare first crude collagen;
b4: adding the first crude collagen into 0.1mol/L acetic acid solution, and stirring until the first crude collagen is completely dissolved to prepare a third mixed solution;
b5: filling the third mixed solution into a second dialysis bag, and putting the second dialysis bag into deionized water for dialysis for 72 hours to prepare a fourth mixed solution;
b6: and (4) freeze-drying the fourth mixed solution for 72 hours to obtain the acid-soluble collagen sponge.
Further, in the step A2, the acetic acid-urea solution and the acetic acid-thiourea solution have the acetic acid molar concentration of 0.01-0.5mol/L and the urea and thiourea concentration of 5-30 g/L.
Further, in the step A2, the temperature is controlled to be 4-10 ℃, and the dissolving time is controlled to be 30-180 minutes.
Further, in the step A3, the concentration of the acetic acid solution is 0.01-0.1mol/L, and the solution is changed for 1 time every 12 hours.
In addition, the inventor also provides a preparation method of the high-concentration water-soluble collagen solution, which comprises the following steps:
c1: cutting the water-soluble collagen sponge into strips, and preparing second sponge strips;
c2: adding the second sponge thin strip into a urea solution or a thiourea solution, and stirring until the second sponge thin strip is completely dissolved to prepare a fifth mixed solution, wherein the concentration of the second sponge thin strip is 40-80 g/L;
c3: and filling the fifth mixed solution into a third dialysis bag, and dialyzing the third dialysis bag in deionized water for 24-72 hours to prepare a high-concentration water-soluble collagen solution.
The water-soluble collagen sponge is dissolved in a urea or thiourea solvent, and the urea and the thiourea are hydrogen bond damaging agents, so that hydrogen bonds formed among the collagen can be damaged, the collagen in the solution is prevented from forming a collagen polymer, the viscosity of the collagen solution is reduced, the solubility of the collagen in the solution is improved, the concentration of the collagen in the solution is increased, and the urea or the thiourea is replaced through dialysis, so that the high-concentration uniform water-soluble collagen solution is obtained.
Further, the concentration of the urea solution and the thiourea solution in the step C2 is 5-30 g/L.
Further, in the step C2, the temperature is controlled to be 4-10 ℃, and the dissolving time is controlled to be 60-180 minutes.
Further, in the step C3, deionized water was changed 1 time every 12 hours.
Different from the prior art, above-mentioned technical scheme dissolves the collagen sponge in the solvent that contains the hydrogen bond breaker, because the hydrogen bond breaker can destroy the hydrogen bond that forms between the collagen, avoids the collagen in the solution to form the collagen polymer to reduce the viscosity of collagen solution, improve the solubility of collagen in the solution, make the concentration of collagen in the solution increase to more than 30g/L, the solvent that contains the hydrogen bond breaker is replaced out through the dialysis again, thereby obtain the even unanimous collagen solution of high concentration. According to the scheme, the collagen sponge can be used for preparing high-concentration acid-soluble collagen solution and water-soluble collagen solution with uniform concentration in a large scale in a short time, and the conditions that the high-concentration acid-soluble collagen solution and water-soluble collagen solution in the current industry are small in preparation yield, long in preparation period and difficult to obtain uniform high-concentration collagen solution are changed.
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the technical means in detail, the following detailed description is given with reference to specific embodiments.
The preparation method of the high-concentration acid-soluble collagen solution and the water-soluble collagen solution has the technical route that: shearing collagen sponge into thin strips → dissolving in solution containing hydrogen bond disruptor to reach required concentration → dialyzing to replace the hydrogen bond disruptor → obtaining high concentration collagen solution.
The collagen described in this embodiment is natural collagen that has not been subjected to degradation treatment.
Example 1:
(1) cutting cowhide into small pieces, and mincing in meat mincer. Putting minced cowhide with wet weight of 10g into a conical flask, adding pepsin of 0.3g and acetic acid of 500mL with concentration of 0.5mol/L, then putting the conical flask into a constant temperature shaking table with temperature of 4 ℃ to extract collagen by oscillation, setting the rotation speed of the shaking table at 180r/min, and working for 72 hours. Centrifuging the solution after shaking extraction, taking supernatant, adding sodium chloride to the concentration of 0.7mol/L, salting out, centrifuging again, removing the supernatant, dissolving the obtained crude collagen in 0.1mol/L acetic acid for dissolving, filling the dissolved collagen solution into a dialysis bag, dialyzing with deionized water for 72 hours, and finally freeze-drying in vacuum for 72 hours to obtain the acid-soluble collagen sponge.
(2) Taking 0.01mol of acetic acid and 30g of urea, preparing 1L of acetic acid-urea solution (wherein the molar concentration of the acetic acid is 0.01mol/L, and the concentration of the urea is 30g/L), shearing 80g of acid-soluble collagen sponge into strips, gradually adding the strips into the solvent, and stirring for 120 minutes at 4 ℃ until the sponge is completely dissolved. Filling the dissolved mixed solution into a dialysis bag, dialyzing in dialysate (0.01mol/L acetic acid solution) for 72 hours, changing the solution every 12 hours for 1 time, and obtaining the high-concentration transparent acid-soluble collagen solution after dialysis.
(3) After the transparent acid-soluble collagen solution was diluted 10-fold with 0.01mol/L acetic acid solution, the concentration of the diluted collagen solution was 7.82g/L by measurement at 540nm by the Biurets method, and the concentration of the acid collagen solution obtained in example 1 was 78.2 g/L.
Example 1 the time required for preparing an acidic collagen solution having a concentration of 78.2g/L using an acid-soluble collagen sponge was 74 hours; the time required by the traditional natural evaporation method is 27-29 days, and the time required by the negative pressure evaporation method is 16-17 days.
Example 2
(1) Cutting pigskin into small pieces, and mincing in a meat mincer. Putting minced pork skin with wet weight of 10g into a 1L conical flask, adding 0.3g of pepsin and 500mL of acetic acid with concentration of 0.5mol/L, and then putting the conical flask into a constant-temperature shaking table with 4 ℃ for oscillation at the working speed of 180r/min for 72 hours. Centrifuging the oscillated solution, taking supernatant, adding sodium chloride to the concentration of 0.7mol/L, salting out, centrifuging again, removing the supernatant, dissolving the obtained crude collagen in 0.1mol/L acetic acid, filling the dissolved collagen solution into a dialysis bag, dialyzing for 72 hours against deionized water, and finally carrying out vacuum freeze drying for 72 hours to obtain the acid-soluble collagen sponge.
(2) Taking 0.05mol of acetic acid and 5g of thiourea, preparing 1L of acetic acid-thiourea solution (the molar concentration of the acetic acid is 0.5mol/L, the concentration of the thiourea is 5g/L), cutting 60g of acid-soluble collagen sponge into thin strips, gradually adding the solution into the solvent, and stirring the solution at 7 ℃ for 60 minutes until the sponge is completely dissolved. And filling the mixed solution into a dialysis bag, dialyzing in dialysate (0.1mol/L acetic acid solution) for 24 hours, changing the dialysate 1 time every 12 hours, and obtaining the high-concentration transparent acid-soluble collagen solution after dialysis.
(3) The transparent acid-soluble collagen solution was diluted 10-fold with 0.1mol/L acetic acid solution and then measured at 540nm by the Biurets method, and it was found that the concentration of the diluted collagen solution was 5.90g/L and the concentration of the acid-soluble collagen solution obtained in example 2 was 59.0 g/L.
Example 2 the time required for preparing an acidic collagen solution with a concentration of 59.0g/L using an acid soluble collagen sponge was 25 hours; the time required by the natural evaporation method is 20-21 days, and the time required by the negative pressure evaporation method is 12-13 days.
Example 3
(1) Cutting fish skin into small pieces, and mincing in a meat mincer. Putting minced fish skin with wet weight of 10g into a conical flask, adding 0.3g of pepsin and 500mL of acetic acid with concentration of 0.5mol/L, then putting the conical flask into a constant-temperature shaking table with 4 ℃ for oscillation, wherein the working speed is 180r/min, and the working time is 72 hours. Centrifuging the oscillated solution, taking supernatant, adding sodium chloride to the concentration of 0.7mol/L, salting out, centrifuging again, removing the supernatant, dissolving the obtained crude collagen in 0.1mol/L acetic acid, filling the dissolved collagen solution into a dialysis bag, dialyzing for 72 hours against deionized water, and finally carrying out vacuum freeze drying for 72 hours to obtain the acid-soluble collagen sponge.
(2) Taking 0.25mol of acetic acid and 17.5g of urea, preparing 1L of acetic acid-urea solution (the molar concentration of the acetic acid is 0.25mol/L, the concentration of the urea is 17.5g/L), cutting 40g of acid-soluble collagen sponge into strips, gradually adding the strips into the solvent, and stirring for 180 minutes at 10 ℃ until the sponge is completely dissolved. And (3) filling the mixed solution into a dialysis bag, dialyzing in dialysate (0.05mol/L acetic acid solution) for 48 hours, changing the dialysate every 12 hours for 1 time, and obtaining the high-concentration transparent acid-soluble collagen solution after dialysis.
(3) After the transparent acid-soluble collagen solution was diluted 10-fold with 0.05mol/L acetic acid solution and examined at 540nm by the Biurets method, the concentration of the diluted collagen solution was found to be 3.92g/L, and the concentration of the acid-soluble collagen solution obtained in example 3 was found to be 39.2 g/L.
Example 3 the time required for preparing an acidic collagen solution with a concentration of 39.2g/L using an acid soluble collagen sponge was 51 hours, the time required for the conventional natural evaporation method was 15-16 days, and the time required for the negative pressure evaporation method was 8-9 days.
Example 4
(1) Adding 500mg of polyglutamic acid into 10mL of dimethyl sulfoxide solution, and stirring until the polyglutamic acid is completely dissolved; adding 100mg of succinimide, stirring, adding 33mg of carbodiimide, reacting for 9 hours at normal temperature, and adding absolute ethyl alcohol to separate out a product; washing with anhydrous ethanol for 2 times, washing with n-hexane for 2 times, and vacuum drying to obtain polyglutamic acid-NHS ester.
(2) Dissolving natural collagen in hydrochloric acid solution (pH 3.0) at a concentration of 10 mg/ml; dissolving polyglutamic acid-NHS ester in DMSO at a concentration of 50 mg/mL; adding 1200 mu L (1/5) of polyglutamic acid-NHS ester solution into 40ml of collagen solution, stirring, and reacting for 12 hours at normal temperature; dialyzing with deionized water for 1 day, and freeze drying to obtain water soluble collagen sponge.
(3) 1L of urea solution with the concentration of 5g/L is prepared, 40g of collagen sponge is cut into thin strips, the strips are gradually added into the solvent, and the mixture is stirred for 120 minutes at the temperature of 7 ℃ until the sponge is completely dissolved. And (3) filling the mixed solution into a dialysis bag, dialyzing in deionized water for 24 hours, changing the solution for 1 time every 12 hours, and obtaining the transparent water-soluble collagen solution with high concentration after dialysis.
(4) After diluting the transparent water-soluble collagen solution 10 times with deionized water, the concentration of the diluted collagen solution was 3.95g/L and the concentration of the water-soluble collagen solution obtained in example 4 was 39.5g/L, as measured by the Biurets method at 540 nm.
Example 4 the time required for preparing an aqueous collagen solution having a concentration of 39.5g/L using a water-soluble collagen sponge was 26 hours, whereas the time required for the conventional natural evaporation method was 16 to 17 days and the time required for the negative pressure evaporation method was 9 to 10 days.
Example 5
(1) 1000mg of polymalic acid was added to 20mL of DMSO solution, and stirred until the polymalic acid was completely dissolved. Adding 50mg of NHS, stirring, adding 50mg of EDC, reacting for 18h at normal temperature, and adding absolute ethyl alcohol to precipitate a product. Washing with anhydrous ethanol for 2 times, washing with n-hexane for 1 time, and vacuum drying to obtain polymalic acid-NHS ester.
(2) Dissolving natural collagen in hydrochloric acid solution (pH 2.5) to obtain collagen solution with concentration of 5 mg/ml; dissolving polymalic acid-NHS ester in DMSO at a concentration of 100 mg/mL; adding 500 μ L (1/2) of polymalic acid-NHS ester solution into 20ml of collagen solution, stirring, and reacting at normal temperature for 18 h; dialyzing with deionized water for 2d, and freeze-drying to obtain water-soluble collagen sponge.
(3) Preparing 1L of 30g/L thiourea solution, cutting 60g of water-soluble collagen sponge into strips, gradually adding into the solvent, and stirring at 4 ℃ for 60 minutes until the sponge is completely dissolved. And (3) filling the mixed solution into a dialysis bag, dialyzing in deionized water for 48 hours, changing the solution for 1 time every 12 hours, and obtaining the transparent water-soluble collagen solution with high concentration after dialysis.
(4) After diluting the transparent water-soluble collagen solution 10 times with deionized water, concentration detection was performed at 540nm by the Biurets method, and it was found that the concentration of the diluted collagen solution was 5.93g/L and the concentration of the water-soluble collagen solution obtained in example 5 was 59.3 g/L.
Example 5 the time required for preparing 59.3g/L of an aqueous collagen solution using a water-soluble collagen sponge was 49 hours, whereas the time required for the conventional natural evaporation method was 21 to 22 days and the time required for the negative pressure evaporation method was 14 to 15 days.
Example 6
(1) Adding 2000mg of polyglutamic acid into 50mL of DMSO solution, and stirring until the polyglutamic acid is completely dissolved; adding 1000mg of NHS, stirring, adding 800mg of EDC, reacting for 15h at normal temperature, and adding acetone to separate out a product; washing with anhydrous ethanol for 3 times, washing with n-hexane for 2 times, and vacuum drying to obtain polyglutamic acid-NHS ester. The degree of esterification of the polyglutamic acid-NHS ester was determined to be 15.3%.
(2) Dissolving natural collagen in hydrochloric acid solution (pH 4.5) at a concentration of 1 mg/ml; dissolving polyglutamic acid-NHS ester in DMSO at a concentration of 10 mg/mL; 30ml of collagen solution was added to 150. mu.L (1/20) of polyglutamic acid-NHS ester solution, and the mixture was stirred and reacted at room temperature for 24 hours. Dialyzing with deionized water for 3 days, and freeze-drying to obtain water-soluble collagen sponge.
(3) Preparing 1L of 17.5g/L thiourea solution, cutting 80g of collagen sponge into strips, gradually adding the strips into the solution, and stirring for 180 minutes at 10 ℃ until the sponge is completely dissolved. And (3) filling the mixed solution into a dialysis bag, dialyzing in deionized water for 72 hours, changing the solution for 1 time every 12 hours, and obtaining the transparent water-soluble collagen solution with high concentration after dialysis.
(4) The transparent water-soluble collagen solution was diluted 10-fold with deionized water and then examined at 540nm by the Biurets method, whereby the concentration of the diluted collagen solution was 7.97g/L and the concentration of the water-soluble collagen solution obtained in example 6 was 79.7 g/L.
Example 6 the time required for 79.7g/L aqueous collagen solution prepared using water-soluble collagen sponge was 75 hours, whereas the time required for the conventional natural evaporation method was 28-30 days and the time required for the negative pressure evaporation method was 18-19 days.
By combining the above examples, the concentration of the acid-soluble collagen solution prepared by the invention is 39.2-78.2g/L, and the concentration of the water-soluble collagen solution prepared by the invention is 39.5-79.7 g/L; the concentration of the collagen solution exceeds 39g/L, so that the requirement of high-concentration collagen solution (the concentration of the collagen solution exceeds 30g/L) is met, and the requirements of various processed products can be met. The preparation time of the high-concentration collagen solution is 75 hours at most, which is far shorter than that of the traditional natural evaporation method and negative pressure evaporation method with the same concentration. Meanwhile, due to the principle of the evaporation method, a few times of low-concentration collagen solution needs to be prepared for evaporation when high-concentration collagen solution is produced, so that the production batch using the preparation method of the invention is several times of that of the evaporation method under the same production conditions.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all the modifications of the equivalent structure or equivalent flow path using the present specification, or the direct or indirect application to other related fields are included in the scope of the present invention.
Claims (9)
1. A preparation method of a high-concentration acid-soluble collagen solution is characterized by comprising the following steps:
a1: cutting acid-soluble collagen sponge into thin strips to prepare first sponge thin strips;
a2: adding acetic acid-urea solution or acetic acid-thiourea solution into the first sponge thin strip, and stirring until the first sponge thin strip is completely dissolved to prepare a first mixed solution, wherein the concentration of the first sponge thin strip is 40-80 g/L;
a3: and filling the first mixed solution into a first dialysis bag, and dialyzing the first dialysis bag in an acetic acid solution for 24-72 hours to prepare a high-concentration acid-soluble collagen solution.
2. The method according to claim 1, wherein in the step A1, the acid-soluble collagen sponge is prepared by the following steps:
b1: cutting animal skin into pieces, and mincing to obtain a first colloid;
b2: adding pepsin with the mass percentage of 3% and an acetic acid solution with the concentration of 0.5mol/L into the first colloid substance to prepare a first suspension; placing the first suspension in a constant-temperature shaking table at 4 ℃ for oscillation, wherein the rotating speed of the shaking table is 180r/min, and the oscillation time is 72 hours to prepare a second mixed solution;
b3: centrifuging the second mixed solution to obtain a supernatant, salting out the supernatant to prepare a first crude collagen suspension, centrifuging the first crude collagen suspension, and removing the supernatant to prepare first crude collagen;
b4: adding the first crude collagen into 0.1mol/L acetic acid solution, and stirring until the first crude collagen is completely dissolved to prepare a third mixed solution;
b5: filling the third mixed solution into a second dialysis bag, and putting the second dialysis bag into deionized water for dialysis for 72 hours to prepare a fourth mixed solution;
b6: and (4) freeze-drying the fourth mixed solution for 72 hours to obtain the acid-soluble collagen sponge.
3. The method of claim 1, wherein: in the step A2, the acetic acid-urea solution and the acetic acid-thiourea solution have the acetic acid molar concentration of 0.01-0.5mol/L and the urea and thiourea concentration of 5-30 g/L.
4. The method of claim 1, wherein: in the step A2, the temperature is controlled to be 4-10 ℃, and the dissolving time is controlled to be 30-180 minutes.
5. The method of claim 1, wherein: in the step A3, the concentration of the acetic acid solution is 0.01-0.1mol/L, and the solution is changed for 1 time every 12 hours.
6. A preparation method of a high-concentration water-soluble collagen solution is characterized by comprising the following steps:
c1: cutting the water-soluble collagen sponge into strips, and preparing second sponge strips;
c2: adding the second sponge thin strip into a urea solution or a thiourea solution, and stirring until the second sponge thin strip is completely dissolved to prepare a fifth mixed solution, wherein the concentration of the second sponge thin strip is 40-80 g/L;
c3: and filling the fifth mixed solution into a third dialysis bag, and dialyzing the third dialysis bag in deionized water for 24-72 hours to prepare a high-concentration water-soluble collagen solution.
7. The method of claim 6, wherein: the concentration of the urea solution and the thiourea solution in the step C2 is 5-30 g/L.
8. The method of claim 6, wherein: in the step C2, the temperature is controlled to be 4-10 ℃, and the dissolving time is controlled to be 60-180 minutes.
9. The method of claim 6, wherein: in the step C3, deionized water is changed every 12 hours for 1 time.
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