CN113735936A - Method for increasing concentration of silk protein-based aqueous solution and silk protein-based aqueous solution prepared by method - Google Patents
Method for increasing concentration of silk protein-based aqueous solution and silk protein-based aqueous solution prepared by method Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 115
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 114
- 239000007864 aqueous solution Substances 0.000 title claims abstract description 106
- 238000000034 method Methods 0.000 title claims abstract description 50
- 239000000243 solution Substances 0.000 claims abstract description 105
- 238000005119 centrifugation Methods 0.000 claims abstract description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000000502 dialysis Methods 0.000 claims description 38
- 108010022355 Fibroins Proteins 0.000 claims description 27
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims description 24
- 239000002244 precipitate Substances 0.000 claims description 17
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- 238000009835 boiling Methods 0.000 claims description 11
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- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 11
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 11
- 239000000654 additive Substances 0.000 claims description 10
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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Abstract
The invention relates to a method for improving the concentration of a silk protein-based aqueous solution and a silk protein-based aqueous solution prepared by the method. The method comprises the steps of dialyzing and centrifuging the initial silk protein water solution and then performing vacuum centrifugation. The method has the advantages that pure silk protein-based aqueous solution can be provided, and compared with the common preparation method of silk protein-based aqueous solution in the prior art, the method has the advantages of low cost, high efficiency and good controllability.
Description
Technical Field
The invention belongs to the technical field of silk protein-based material treatment, and particularly relates to a method for improving the concentration of a silk protein-based aqueous solution and a silk protein-based aqueous solution prepared by the method.
Background
Protein-based natural high polymer materials (such as collagen, keratin, fibroin and the like) have the characteristics of wide sources, good biocompatibility, biodegradability and the like, are widely concerned in recent years and are applied to the fields of a plurality of biomedicines and bioengineering, such as implant intervention materials, tissue repair, tissue engineering, drug sustained release and the like.
Among many protein-based natural polymer materials, silk protein-based materials have become a hot spot of research in recent years due to their excellent mechanical properties, biocompatibility and biodegradability.
In addition to directly utilizing natural silk fiber, it is one of the main research directions at present to obtain regenerated silk protein by processing natural silk fiber and further to process the regenerated silk protein into various functional materials. In general, the extraction of regenerated silk protein is performed by an aqueous solution method, specifically, natural silk fiber is dissolved in a high-concentration inorganic salt (lithium bromide, sodium thiocyanate, etc.) solution or an alcohol-salt mixed system (calcium chloride-ethanol-water, etc.), and then the solution is dialyzed and purified to obtain a regenerated silk protein aqueous solution. Because the osmotic pressure is very high during dialysis, a large amount of water enters into the dialysis bag, so the concentration of the obtained silk fibroin aqueous solution is usually low (generally about 6 wt.%), and the low-concentration regenerated silk fibroin aqueous solution is greatly limited in material preparation, production and the like. Some other silk protein-based aqueous solutions, such as those of regenerated silk protein derivatives obtained by chemical modification, in addition to the silk protein aqueous solution obtained by the dissolution regeneration method; physically mixing to obtain a regenerated silk protein/additive composite silk protein-based solution; the recombinant silk protein aqueous solution obtained by the gene recombination technique and the like also face similar limitations. How to efficiently and controllably concentrate a silk protein-based aqueous solution with low concentration to obtain a solution with higher concentration becomes a problem to be solved urgently at present.
The prior art methods for concentrating silk protein aqueous solution are mainly divided into two methods, one is dialysis bag osmotic concentration method, which is mainly to put silk protein aqueous solution into a dialysis bag, then dialyze in a concentrated solution of high molecular polymer (such as polyethylene glycol, etc.), during which water in the dialysis bag can seep out to obtain high-concentration silk protein solution (US 8614293B 2). However, this method is time-consuming, requires the use of large amounts of polymer dialysate, and is very costly to concentrate. In addition, some small molecules or impurity ions are left in the polymerization production process of the polyethylene glycol and other polymers used in the dialysis process, and the impurity ions may permeate into the dialysis bag to influence the purity of the prepared silk protein solution. Another method for concentrating silk protein water solution is to put silk protein water solution into dialysis bag, then put into vacuum drying oven and put water absorbent into the drying oven, vacuum drying is carried out at a certain temperature, and silk protein solution (CN 102167724) with different concentration can be obtained by adjusting and controlling the time and vacuum degree of vacuum pumping. The method needs dialysis bag and large amount of water absorbent, and has high concentration cost.
Therefore, a method for preparing a high-concentration silk protein-based aqueous solution with high efficiency, purity, controllability, low cost and industrial prospect is still needed.
Disclosure of Invention
The invention aims to provide a preparation method of a high-concentration silk protein-based aqueous solution, which is efficient, pure, controllable, low in cost and has an industrial prospect.
The invention provides a method for increasing the concentration of a silk protein-based aqueous solution, which comprises the following steps:
(1) putting the initial silk protein aqueous solution into a dialysis bag for dialysis, centrifuging the dialyzed solution at a first centrifugal rotating speed under the environmental pressure, and discarding precipitates to obtain a silk protein-based aqueous solution with the concentration of 4.0-8.0 w/v%, preferably 4.5-6.5 w/v%;
(2) and (2) placing the silk protein-based aqueous solution obtained in the step (1) in a vacuum environment, and performing centrifugal concentration at a second centrifugal rotating speed to obtain the silk protein-based aqueous solution with the concentration of more than 10 w/v%.
In particular embodiments, the starting silk protein aqueous solution in step (1) may be a natural silk protein aqueous solution; an aqueous solution of a regenerated silk protein derivative obtained by chemical modification; a regenerated silk protein/additive (such as medicine, growth factor, antibiotic and the like) composite silk protein base solution obtained by physical mixing; recombinant silk protein aqueous solution, etc.
In a specific embodiment, the starting aqueous native silk protein solution is prepared by: placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 60-100 deg.C for 30-180min to degum Bombyx Bombycis, dissolving dried degummed silk in 9-10M, preferably 9.3M LiBr water solution, and placing in 60-150 deg.C oven for 2-8 hr to completely dissolve.
In a specific embodiment, in step (1), the starting fibroin aqueous solution is dialyzed under the following dialysis conditions: the dialysis time is 2-5 days, the dialysate is deionized water, and the cut-off molecular weight of the dialysis bag is 3500 Dalton (Da).
In a specific embodiment, in step (1), the first centrifugation rotation speed is 5000-.
In a specific embodiment, before step (2), an additive for improving the properties of the silk fibroin-based material product may be added to the silk fibroin-based aqueous solution, the additive being selected from the group consisting of glycerol, inorganic salts, bioactive molecules (e.g., enzymes, antibiotics, drugs, etc.), inorganic materials, organic molecules. The concentration of the additive may be 0.1 to 50 wt%. The properties of the silk fibroin-based material product, such as physicochemical and biological properties, can be improved by adding additives without affecting the concentration effect.
In a specific embodiment, in step (2), the vacuum centrifugation is performed under the following conditions: the second centrifugation rotation speed is 100-.
In a specific embodiment, the concentration of the silk protein based aqueous solution obtained in step (2) is 10-50 w/v%, preferably 25-40 w/v%, and preferably the concentration of the silk protein based aqueous solution obtained in step (2) is 6 times the concentration of the silk protein based aqueous solution obtained in step (1).
In a specific embodiment, the method further comprises: after step (2), a third centrifugation operation at a third centrifugation rotation speed is performed to separate precipitated silk proteins.
In a specific embodiment, the third centrifugation operation is performed under the following conditions: the centrifugation time is 5-30min, the centrifugation temperature is 4-30 ℃, the third centrifugation rotating speed is 5000-.
In another aspect, the present invention provides a silk protein-based aqueous solution prepared by the above method.
In yet another aspect, the present invention provides a silk protein-based material made from the silk protein-based aqueous solution described above.
In a specific embodiment, the silk fibroin-based material can be in the form of any one of a film, a fiber, a sponge, a hydrogel, a powder.
In particular embodiments, the method of preparing the silk fibroin-based material in the form of a film, fiber, sponge, hydrogel, or powder is not limited, and conventional methods in the art for preparing these forms from silk fibroin-based aqueous solutions can be employed.
Advantageous effects
Compared with the method in the prior art, the method for improving the solubility of the silk protein-based aqueous solution has the advantages that: (1) polymers such as polyethylene glycol are not used, so that residual micromolecules or impurity ions in the production process of the polymers are not left in the obtained high-concentration silk protein-based solution, and the solution is purer; (2) and a water absorbent and a dialysis bag are not used in the vacuum concentration process, so that the concentration cost is effectively reduced.
In addition, since the method for increasing the concentration of silk protein-based aqueous solution provided in the present application does not incorporate additional small molecules or foreign ions as impurities, the high-concentration silk protein-based aqueous solution obtained thereby is purer, and thus can be more advantageously applied to silk protein-based materials (e.g., films, fibers, sponges, hydrogels, powders, etc.) in the fields of biomedicine, tissue engineering, textiles, flexible electronics, etc., and thus provides additional advantages over the silk protein silk aqueous solutions of the prior art.
Drawings
FIG. 1: concentration time of silk protein water solution is plotted against concentration.
FIG. 2: the relationship between the shear rate and the shear viscosity of pure silk protein aqueous solutions with different concentrations is shown.
FIG. 3: modulus vs. angular velocity in an aqueous 28 wt.% fibroin solution.
FIG. 4: the initial concentration of silk protein in example 4 is compared with the concentrated solution.
Detailed Description
The process of the present invention is described in detail below by way of specific examples, which are, however, merely representative examples illustrating the process of the present invention and are not intended to limit the scope of the invention.
The terms:
as used herein, the term "highly concentrated silk protein-based aqueous solution" means a silk protein-based aqueous solution having a concentration of greater than 10 w/v%.
In this context, the term "concentration time" refers to the time during which the apparatus is operated when a dilute concentration solution reaches a specified high concentration solution during the centrifugal concentration.
Herein, the term "silk protein" may be understood as "silk fibroin".
The manufacturers of the main experimental reagents and instruments used in the following examples are listed in table 1 below.
TABLE 1 Main test reagents and test instruments
Example 1
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 5.0% (w/v).
(2) And (3) placing the low-concentration silk protein aqueous solution prepared in the above way in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 11 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugation speed is 5000rpm, the centrifugation time is 10min, and the centrifugation temperature is 4 ℃. The finally prepared silk protein aqueous solution has the concentration of 17 wt.% and can be stably stored at low temperature.
Example 2
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 5.4% (w/v).
(2) And (3) placing the low-concentration silk protein aqueous solution prepared in the above way in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 150mL, and the concentration time is 14 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugation speed is 7000rpm, the centrifugation time is 15min, and the centrifugation temperature is 4 ℃. The final silk protein aqueous solution has a concentration of 28 wt.%, and can be stably stored at low temperature.
Example 3
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 6.1% (w/v).
(2) And (3) placing the low-concentration silk protein aqueous solution prepared in the above way in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 13 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugal speed is 9000rpm, the centrifugal time is 15min, and the centrifugal temperature is 4 ℃. The silk protein water solution finally prepared has the concentration of 24 percent and can be stably stored at low temperature.
Example 4
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 6.3% (w/v).
(2) And (3) placing the low-concentration silk protein aqueous solution prepared in the above way in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 45 ℃, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 16 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugal speed is 9000rpm, the centrifugal time is 20min, and the centrifugal temperature is 4 ℃. The concentration of the silk protein aqueous solution finally prepared is 36 wt.%, and the silk protein aqueous solution can be stably stored at low temperature.
A photograph comparing the silk protein based aqueous solution obtained after the above concentration process with the silk protein based aqueous solution before concentration is shown in FIG. 4.
Example 5
(1) Placing cut silkworm cocoonInto 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 5.8% (w/v).
(2) Adding 20 wt% of glycerol into the prepared low-concentration silk protein aqueous solution, and after the two solutions are uniformly mixed, placing the mixture in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 1500rpm, the vacuum degree is 20mbar, the initial solution volume is 150mL, and the concentration time is 17 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation to obtain supernatant so as to remove sediment at the bottom. Wherein the centrifugal speed is 9000rpm, the centrifugal time is 20min, and the centrifugal temperature is 4 ℃. The solid content of the finally prepared composite solution is 31 wt.%, and the composite solution can be stably stored at low temperature.
Example 6
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 5.4% (w/v).
(2) Adding 10 wt% of glycerol into the prepared low-concentration silk protein aqueous solution, and after the two solutions are uniformly mixed, placing the mixture in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 30 ℃, the centrifugal speed is 1500rpm, the vacuum degree is 15mbar, the initial solution volume is 90mL, and the concentration time is 23 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugal speed is 9000rpm, the centrifugal time is 20min, and the centrifugal temperature is 4 ℃. The solid content of the finally prepared composite solution is 37 wt.%, and the composite solution can be stably stored at low temperature.
Example 7
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 6.2% (w/v).
(2) And adding a 5 wt% calcium chloride solution into the prepared low-concentration silk protein aqueous solution, and after the two solutions are uniformly mixed, placing the mixture in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 30 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 10mbar, the initial solution volume is 150mL, and the concentration time is 22 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugal speed is 9000rpm, the centrifugal time is 20min, and the centrifugal temperature is 4 ℃. The solid content of the finally prepared composite solution is 43 wt.%, and the composite solution can be stably stored at low temperature.
Example 8
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. Then removing the dried productDissolving the gel silk in 9.3M LiBr water solution, and putting the solution into a 60 ℃ oven for 4 hours to completely dissolve the degummed silk.
Then the completely dissolved solution was put into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da) in which the dialysate was deionized water and the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure to remove the bottom precipitate to obtain an aqueous fibroin solution with an initial concentration of 4.3% (w/v).
(2) And adding 15 wt% of calcium chloride solution into the prepared low-concentration silk protein aqueous solution, and after the two solutions are uniformly mixed, placing the mixture in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 30 deg.C, the centrifugal rotation speed is 1500rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 25 h.
(3) And (3) putting the concentrated silk protein aqueous solution into a centrifuge for centrifugation, and removing the bottom precipitate for purification for the subsequent processing. Wherein the centrifugal speed is 9000rpm, the centrifugal time is 20min, and the centrifugal temperature is 4 ℃. The solid content of the finally prepared composite solution is 48 wt.%, and the composite solution can be stably stored at low temperature.
Test example 1
To explore the solution concentration as a function of vacuum centrifugation time in vacuum centrifugation, the following test examples were conducted.
(1) Placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 100 deg.C for 30min to degum the Bombyx Bombycis. And then dissolving the dried degummed silk in 9.3M LiBr aqueous solution, and putting the solution into an oven at 60 ℃ for 4 hours to completely dissolve the degummed silk. And then putting the completely dissolved solution into a dialysis bag with the molecular weight cutoff of 3500 daltons (Da), wherein the dialysate is deionized water, the dialysis time is 3 days, and centrifuging the dialyzed solution at the normal pressure of 9000rpm to obtain the silk protein aqueous solution with the initial concentration of 5.0% (w/v).
(2) And placing the prepared low-concentration silk protein aqueous solution in a vacuum environment for centrifugal concentration, wherein the concentration temperature is 45 ℃, the centrifugal rotation speed is 1500rpm, the vacuum degree is 20mbar, and the volume of the initial solution is 120 mL. In the process, a proper amount of solution is taken at certain time intervals, the mass of the solution is weighed as wet weight by using a balance, then the solution is placed in an oven for drying at 60 ℃, and the mass of the sample at the moment is weighed as dry weight by using a balance. The mass fraction of the solution at a certain concentration time is calculated by formula (1). The test results are shown in fig. 1.
Wherein ω is the mass fraction of the solution in wt.%; m is the dry weight of the test sample, M is the wet weight of the test sample, and the units are g.
(3) As can be seen from FIG. 1, the rate of increase in the concentration of the solution gradually increased with the increase in the concentration time. And when the solution concentration reached 31 wt.%, the required concentration time was only 14.5 h. Therefore, compared with other methods in the prior art, the concentration time is greatly shortened, and the concentration efficiency is obviously improved. In addition, the results also show that the silk protein-based aqueous solution with determined concentration can be obtained by adjusting the concentration time by adopting the method disclosed by the invention, so that the method disclosed by the invention has the advantages of good controllability and good repeatability in the concentrated silk protein-based aqueous solution.
Test example 2
The test method comprises the following steps: during the rheological measurements of the solutions, they were measured using an ARES-G2 rotational rheometer (TA-Waters, USA). During testing, a parallel clamping plate with the diameter of 25mm is adopted, the distance between the upper plate clamp and the lower plate clamp is set to be 0.5mm, and the testing temperature is 25 ℃.
In shear rheology experiments, the shear rate was swept from low to high with a shear rate range of 10-2-102s-1. The solutions tested during the experiment were the solutions prepared in the above examples at concentrations of 17 wt.%, 24 wt.%, and 36 wt.%, respectively, and the test results are shown in fig. 2.
In the small-amplitude frequency scanning test process, the strain amplitude in a linear region is adopted to be 1 percent, the frequency scanning is carried out from high to bottom, and the scanning range is 102-10-1rad/s. The mass fraction prepared in the above example was selected to be 28wt% fibroin solution was subjected to a small amplitude frequency sweep test, the test results are shown in fig. 3.
And (4) analyzing results:
as can be seen from FIG. 2, the solution exhibited shear-thinning behavior with increasing shear rate at various concentrations. And the high-concentration solution has higher shear viscosity under the condition of different shear rates compared with the low-concentration solution.
As can be seen from fig. 3, both the storage modulus G' and the loss modulus G ″ increase with increasing frequency. At a frequency of about 3rad/s, the storage modulus G 'and the loss modulus G' intersect. To the left of the intersection (i.e., in the lower frequency range), G '> G' is present; to the right of the intersection point (i.e., in the higher frequency range), G '< G' is shown.
The above results demonstrate that the silk protein solution obtained by the present application has excellent tunable rheological properties relative to conventionally prepared silk protein solutions.
Claims (10)
1. A method of increasing the concentration of a silk fibroin-based aqueous solution, the method comprising the steps of:
(1) putting the initial silk protein aqueous solution into a dialysis bag for dialysis, centrifuging the dialyzed solution at a first centrifugal rotating speed under the environmental pressure, and discarding precipitates to obtain a silk protein-based aqueous solution with the concentration of 4.0-8.0 w/v%, preferably 4.5-6.5 w/v%;
(2) and (2) placing the silk protein-based aqueous solution obtained in the step (1) in a vacuum environment, and performing centrifugal concentration at a second centrifugal rotating speed to obtain the silk protein-based aqueous solution with the concentration of more than 10 w/v%.
2. The method of claim 1, wherein the starting fibroin aqueous solution in step (1) is selected from the following: natural silk protein aqueous solution; an aqueous solution of a regenerated silk protein derivative obtained by chemical modification; physically mixing to obtain a regenerated silk protein/additive composite silk protein-based solution; recombining silk protein aqueous solution.
3. The method of claim 2Wherein, in step (1), the starting natural silk protein aqueous solution is a natural silk protein aqueous solution, and it is prepared by the following method: placing the cut silkworm cocoon in 0.02M Na2CO3Boiling in water solution at 60-100 deg.C for 30-180min to degum Bombyx Bombycis, dissolving dried degummed silk in 9-10M, preferably 9.3M LiBr water solution, and placing in 60-150 deg.C oven for 2-8 hr to completely dissolve.
4. The method according to claim 1, wherein, in step (1), the starting silk protein aqueous solution is dialyzed under the following dialysis conditions: the dialysis time is 2-5 days, the dialysate is deionized water, the cut-off molecular weight of the dialysis bag is 3500Da, and
in step (1), the first centrifugation rotation speed is 5000-11000rpm, preferably 9000 rpm.
5. The method according to claim 1, wherein, prior to step (2), an additive for improving the properties of the silk fibroin-based material product is added to the silk fibroin-based aqueous solution, the additive is selected from the group consisting of glycerol, inorganic salts, bioactive molecules, inorganic materials, organic molecules, and the concentration of the additive is 0.1-50 wt%.
6. The method according to claim 1, wherein, in step (2), the vacuum centrifugation is performed under the following conditions: the second centrifugation rotation speed is 100-.
7. The method according to claim 1, wherein the concentration of the silk protein based aqueous solution obtained in step (2) is 10-50 w/v%, preferably 25-40 w/v%, preferably the concentration of the silk protein based aqueous solution obtained in step (2) is 6 times the concentration of the silk protein based aqueous solution obtained in step (1).
8. The method of claim 1, wherein the method further comprises: after step (2), performing a third centrifugation operation at a third centrifugation rotation speed to separate precipitated silk proteins, preferably the third centrifugation operation is performed under the following conditions: the centrifugation time is 5-30min, the centrifugation temperature is 4-30 ℃, the third centrifugation rotating speed is 5000-.
9. A silk protein based aqueous solution prepared by the method of any one of claims 1-8.
10. A silk fibroin-based material made from the silk fibroin-based aqueous solution of claim 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102167724A (en) * | 2011-01-04 | 2011-08-31 | 复旦大学 | Controllable preparation method for high-concentration regenerated silk protein aqueous solution |
| CN103739691A (en) * | 2013-12-11 | 2014-04-23 | 浙江大学 | Green and environment-friendly method for preparing silk fibroin powder |
| CN103910789A (en) * | 2014-03-27 | 2014-07-09 | 苏州丝美特生物技术有限公司 | Preparation process of high molecular weight silk fibroin freeze-dried powder |
| US20170107264A1 (en) * | 2014-03-27 | 2017-04-20 | Simatech Incorporation | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102167724A (en) * | 2011-01-04 | 2011-08-31 | 复旦大学 | Controllable preparation method for high-concentration regenerated silk protein aqueous solution |
| CN103739691A (en) * | 2013-12-11 | 2014-04-23 | 浙江大学 | Green and environment-friendly method for preparing silk fibroin powder |
| CN103910789A (en) * | 2014-03-27 | 2014-07-09 | 苏州丝美特生物技术有限公司 | Preparation process of high molecular weight silk fibroin freeze-dried powder |
| US20170107264A1 (en) * | 2014-03-27 | 2017-04-20 | Simatech Incorporation | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
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