CN111549087A - Preparation method and application of high molecular weight keratin - Google Patents
Preparation method and application of high molecular weight keratin Download PDFInfo
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Abstract
The invention discloses a preparation method and application of high molecular weight keratin. The method can extract and crosslink the wool keratin from the waste wool, and the molecular weight of the wool keratin can reach 120 kDa. The preparation method of the high molecular weight keratin has the advantages of simple process and easily controlled conditions, the raw material is waste wool, the waste is recycled, no chemical reagent is introduced, the method is green and environment-friendly, and the energy is saved; the invention also provides application of the high molecular weight keratin to a biological material, and the nano material pad has the effects of biocompatibility and wound healing promotion.
Description
Technical Field
The invention relates to a preparation method and application of high molecular weight keratin, and belongs to the technical field of biology.
Background
China is a big country for producing and using wool, a large amount of non-spinnable short fibers and thick fibers exist every year, and damaged wool in the pretreatment process is discarded, so that resources are wasted, and the environmental pressure is increased. How to reuse the waste wool is always a hot point of research.
The processing and hydrolyzing process of the wool keratin comprises a chemical treatment method, a physical processing method and a biological enzyme hydrolysis method. The chemical treatment method adopts strong acid and strong base for matching treatment, the nutrition value of the hydrolysis product is damaged more, and the environmental pollution is serious and the application is minimum. The physical processing method, i.e. the high-temperature high-pressure hydrolysis method, has high requirements on equipment and energy consumption, so the production cost is high. Chemical treatment and physical processing can only obtain polypeptide with very low molecular weight; the biological enzyme hydrolysis method is to degrade wool into soluble wool by adopting high-activity proteolytic enzyme or microorganism under mild and proper conditions, the method has the advantages of less pollution, low energy consumption and mild reaction conditions, but the problems of difficult reaction control, poor enzyme action specificity and the like exist in the use process of the keratinase, and the prepared keratin has lower molecular weight (<60kDa) and poor mechanical performance.
Disclosure of Invention
In order to solve the technical problems, the invention uses the combination of keratinase and glutamine transaminase, hydrolyzes wool by the keratinase to obtain soluble keratin hydrolysate, adds Tgase into supernatant fluid by centrifugation to crosslink keratin, and obtains macromolecular keratin powder by dialysis and freeze drying. The method provided by the invention is simple to operate, and the molecular weight of the obtained wool keratin is 120 kDa.
The first object of the present invention is to provide a process for the preparation of high molecular weight keratin, comprising the steps of:
(1) cleaning wool fibers, degreasing for 12-24 hours by using a degreasing solvent, cleaning to remove the degreasing solvent remained on the surface, drying, and shearing to obtain degreased wool fibers;
(2) adding the degreased wool fibers obtained in the step (1) into water, adjusting the pH value to 9-10, adding keratinase, hydrolyzing at 45-55 ℃ for 12-24 h, and centrifuging to obtain keratin hydrolysate;
(3) adjusting the pH value of the keratin hydrolysate obtained in the step (2) to 6-8, adding glutamine transaminase, reacting at 45-55 ℃ for 24-48 h, and inactivating enzyme to obtain a keratin solution;
(4) and (4) dialyzing, freezing and drying the keratin solution obtained in the step (3) to obtain the high molecular weight keratin.
Furthermore, the addition amount of the keratinase is 15,000-20,000U/g wool.
Further, the amount of the transglutaminase added is 15-20U/g keratin.
Further, in the step (2), the bath ratio of the degreased wool fibers to the water is 4-6: 100.
Further, in the step (1), the degreasing solvent is one or more of ether, benzene, carbon disulfide, acetone and chloroform.
Further, in the step (1), the degreasing is carried out for 12-24 hours at 65-75 ℃.
Further, in the step (4), the dialysis is carried out for 2-4 days by adopting a dialysis bag with the molecular interception amount of 8,000-14,000, and water is changed for 4-6 h in the dialysis process.
Further, in the step (2), the centrifugation is carried out at 6000-10000 rpm for 5-10 min.
The second purpose of the invention is to provide the keratin prepared by the method.
The third purpose of the invention is to provide the application of the keratin in the biological nano-material pad.
Further, the application specifically includes: weighing wool keratin powder, dissolving the wool keratin powder in hexafluoroisopropanol under a room temperature condition, stirring the solution under a sealed condition until the wool keratin powder is uniformly dissolved, weighing poly (beta-hydroxybutyrate valerate) and adding the poly (beta-hydroxybutyrate valerate) into the solution to prepare macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution, sealing the solution for 3 hours, and stirring the solution for later use, wherein the mass fraction of the macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution is 4-6 wt%, and the mass ratio of the macromolecular keratin to the poly (beta-hydroxybutyrate valerate) is 3:7-5: 5; extracting the dissolved macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution by using an injector, and fixing the macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution on a constant flow pump, wherein the flow rate of the constant flow pump is 0.5-1.0 mL/h; then connecting the positive electrode of the high-voltage electrostatic generator to the needle head, connecting the negative electrode of the high-voltage electrostatic generator to a receiving plate, paving a layer of aluminum foil paper on the receiving plate for receiving the electrostatic spinning fiber, starting electrostatic spinning, and performing under the condition of room temperature, wherein the humidity is lower than 75%, the distance is controlled to be 15-25cm, and the voltage is controlled to be 10-20 kV; spinning for 4 hours each time, taking down the aluminum foil paper after the spinning is finished, and storing under a dry condition.
The invention has the beneficial effects that:
(1) the keratin prepared by the method has a molecular weight of up to 120kDa, belongs to soluble protein, and has improved heat stability.
(2) The invention utilizes keratinase and TGase when preparing the keratin with large molecular weight, has mild reaction condition and small pollution to the environment.
(3) The high molecular weight keratin prepared by the invention improves the survival rate of cells and promotes cell migration.
(4) The high molecular weight keratin prepared by the method can improve the mechanical property of the keratin nano fiber, wherein the Young modulus is improved by 41 percent.
Drawings
FIG. 1 is an SDS-PAGE pattern of wool keratin before and after crosslinking
FIG. 2 is an XRD diffractogram of wool and wool keratin before and after crosslinking
FIG. 3 is an infrared spectrum of wool and wool keratin before and after crosslinking
FIG. 4 is a circular dichroism chart of wool keratin before and after crosslinking
FIG. 5 shows the distribution of the structural content of wool keratin before and after crosslinking
FIG. 6 shows the results of the thermodynamic analysis of wool and wool keratin before and after crosslinking
FIG. 7 shows the effect of crosslinked keratin on cell viability
FIG. 8 is a graph showing the effect of cross-linking keratin on cell migration
FIG. 9 is SEM image and diameter distribution of nanofibers prepared by composite electrospinning of keratin and poly (beta-hydroxybutyrate valerate) (PHBV) before and after crosslinking, AB, CD and EF are PHBV, PHBV/30% hydrolyzed keratin and PHBV/30% crosslinked keratin respectively
FIG. 10 is an infrared spectrum of nanofibers obtained by electrospinning
FIG. 11 is an XRD diffractogram of electrospun nanofibers
FIG. 12 is a stress-strain curve of nanofibers prepared by composite electrospinning of keratin and poly (β -hydroxybutyrate valerate) before and after crosslinking
Fig. 13 shows the effect of electrospun nanofibers on wound healing in mice.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1:
the preparation method of high molecular weight wool keratin described in this embodiment includes the following steps:
(1) degreasing the waste wool by using acetone at the temperature of 72 ℃ for 15h, washing the degreased wool for 3 times by using a deionized beam, and drying the degreased wool in an oven at the temperature of 60 ℃;
(2) adding 5g of cut wool into water, adjusting the pH value of the solution to 9, adding 20,000U/g of wool keratinase, hydrolyzing at 50 ℃ for 12 h;
(3) and (3) carrying out centrifugal separation on the solution treated in the step (2) to obtain wool keratin hydrolysate. The speed of centrifugation was 8,000rpm, and the time of centrifugation was 10 min.
(4) Adding TGase into the supernatant of the hydrolyzed keratin solution obtained in the step (3) to crosslink keratin, adjusting the pH to 8.0, adding the TGase in an amount of 15U/g keratin, reacting at 45 ℃ for 24 hours;
(5) and (3) putting the supernatant obtained in the step (4) into a dialysis bag with the molecular cut-off of 8,000-14,000, changing water every 4 hours in the dialysis process, and dialyzing for 2 days.
(6) Freezing the macromolecular keratin solution obtained in the step (5) in a refrigerator at the temperature of-80 ℃ for 12 hours, and then drying the macromolecular keratin solution on a freeze dryer for 48 hours to obtain keratin solid; the dried sample was ground to obtain wool keratin powder.
The hydrolysis rate of wool is 63.1% by adopting the scheme, and the molecular weight distribution of keratin prepared by the double-enzyme method is 120.1 kDa. The infrared spectrum shows that no new chemical bond is generated in the keratin before and after crosslinking. XRD, circular dichroism results show that the alpha-helix proportion of the crosslinked keratin is reduced, and the beta-sheet, turn angle and irregular coil proportion are increased. Thermodynamic experiments show that the maximum thermal decomposition temperature of the crosslinked keratin is slightly higher than that of the hydrolyzed keratin, which indicates that the thermal stability of the keratin is improved by crosslinking. In addition, cross-linking keratin can increase cell survival and promote cell migration.
Example 2:
the preparation method of high molecular weight wool keratin described in this embodiment includes the following steps:
(1) degreasing the waste wool by using acetone at the temperature of 72 ℃ for 15h, washing the degreased wool for 3 times by using a deionized beam, and drying the degreased wool in an oven at the temperature of 60 ℃;
(2) adding 5.5g of cut wool into water, adjusting the pH value of the solution to 9.3, adding 18,000U/wool keratinase, hydrolyzing at 50 ℃ for 15 h;
(3) and (3) carrying out centrifugal separation on the solution treated in the step (2) to obtain wool keratin hydrolysate. The speed of centrifugation was 8,000rpm, and the time of centrifugation was 10 min.
(4) Adding TGase into the supernatant of the hydrolyzed keratin solution obtained in the step (3) to perform crosslinking on keratin, adjusting the pH of the hydrolysate to 7.0, adding the TGase in an amount of 20U/g keratin, reacting at 49 ℃ for 32 hours;
(5) and (3) putting the supernatant obtained in the step (4) into a dialysis bag with the molecular cut-off of 8,000-14,000, changing water every 5 hours in the dialysis process, and dialyzing for 3 days.
(6) Freezing the high molecular weight keratin solution obtained in the step (5) in a refrigerator at the temperature of-80 ℃ for 24 hours, and then drying the solution on a freeze dryer for 60 hours to obtain keratin solid; the dried sample was ground to obtain wool keratin powder.
The wool hydrolysis rate is 69.2% by adopting the scheme, and the keratin molecular weight distribution prepared by the double-enzyme method is 121.5 kDa. The infrared spectrum shows that no new chemical bond is generated in the keratin before and after crosslinking. XRD, circular dichroism results show that the alpha-helix proportion of the crosslinked keratin is reduced, and the beta-sheet, turn angle and irregular coil proportion are increased. Thermodynamic experiments show that the maximum thermal decomposition temperature of the crosslinked keratin is slightly higher than that of the hydrolyzed keratin, which indicates that the thermal stability of the keratin is improved by crosslinking. In addition, cross-linking keratin can increase cell survival and promote cell migration.
Example 3:
the preparation method of high molecular weight wool keratin described in this embodiment includes the following steps:
(1) degreasing the waste wool by using acetone at the temperature of 72 ℃ for 24 hours, washing the degreased wool for 3 times by using a deionized beam, and drying the degreased wool in an oven at the temperature of 60 ℃;
(2) adding 5.8g of cut wool into water, adjusting the pH value of the solution to 10, adding 25,000U/wool keratinase, hydrolyzing at 50 ℃ for 24 hours;
(3) and (3) carrying out centrifugal separation on the solution treated in the step (2) to obtain wool keratin hydrolysate. The speed of centrifugation was 8,000rpm, and the time of centrifugation was 10 min.
(4) And (3) adding TGase into the supernatant of the hydrolyzed keratin solution obtained in the step (3) to crosslink keratin, and adjusting the pH value of the hydrolysate to 6.0. The addition amount of TGase is 25U/g keratin, the reaction temperature is 52 ℃, and the hydrolysis time is 48 h;
(5) and (3) putting the supernatant obtained in the step (4) into a dialysis bag with the molecular cut-off of 8,000-14,000, changing water every 6 hours in the dialysis process, and dialyzing for 4 days.
(6) Freezing the high molecular weight keratin solution obtained in the step (5) in a refrigerator at the temperature of-80 ℃ for 24 hours, and then drying the solution for 72 hours on a freeze dryer to obtain a keratin solid; the dried sample was ground to obtain wool keratin powder.
The wool hydrolysis rate is 72.6% by adopting the scheme, and the keratin molecular weight distribution prepared by the double-enzyme method is 107.5 kDa. The infrared spectrum shows that no new chemical bond is generated in the keratin before and after crosslinking. XRD, circular dichroism results show that the alpha-helix proportion of the crosslinked keratin is reduced, and the beta-sheet, turn angle and irregular coil proportion are increased. Thermodynamic experiments show that the maximum thermal decomposition temperature of the crosslinked keratin is slightly higher than that of the hydrolyzed keratin, which indicates that the thermal stability of the keratin is improved by crosslinking. In addition, cross-linking keratin can increase cell survival and promote cell migration.
Example 4:
the application method of the high molecular weight keratin comprises the following steps:
(1) weighing the high molecular weight keratin powder prepared in the example 1 and dissolving the powder in hexafluoroisopropanol at room temperature, stirring the mixture under a sealed condition until the powder is uniformly dissolved, weighing poly (beta-hydroxybutyrate valerate) and adding the poly (beta-hydroxybutyrate valerate) into the solution to prepare a high molecular weight keratin/poly (beta-hydroxybutyrate valerate) spinning solution, sealing the solution for 3 hours and stirring the solution for later use; wherein the mass fraction of the high molecular weight keratin/poly (beta-hydroxybutyrate valerate) spinning solution is 4-6 wt%, and the mass ratio of the high molecular weight keratin to the poly (beta-hydroxybutyrate valerate) is 3: 7.
(2) Extracting the dissolved high molecular weight keratin/poly (beta-hydroxybutyrate valerate) spinning solution by using an injector, and fixing the spinning solution on a constant flow pump, wherein the flow rate of the constant flow pump is 1.0 mL/h;
(3) connecting the positive pole of a high-voltage electrostatic generator to a needle head, connecting the negative pole of the high-voltage electrostatic generator to a receiving plate, paving a layer of aluminum foil paper on the receiving plate for receiving electrostatic spinning fibers, starting electrostatic spinning, and performing under the condition of room temperature, wherein the humidity is lower than 75%, the distance is controlled at 15cm, and the voltage is controlled at 20 kV.
(4) And 4h of spinning for each time, taking down the aluminum-foil paper after the spinning is finished, and storing under a dry condition.
SEM results show that the fiber membranes of all structures present three-dimensional structures, and the surfaces of the nanofibers are smooth and have no beading. The average fiber diameter of the PHBV nano-fiber template is about 512 +/-210 nm. After the addition of keratin, the fiber diameter was slightly reduced, with mean diameters of 355. + -.118 nm (PHBV/30% hydrolysed keratin) and 353. + -.111 nm (PHBV/30% cross-linked keratin), respectively. The infrared spectrum and the X-ray diffraction result show that the peak shape and the position of the characteristic peak of the PHBV/30% crosslinked keratin nanofiber membrane are not obviously changed, which indicates that the two are mixed in a physical form and do not have chemical change. And (3) performance test results: the young's modulus of the hydrolyzed keratin/poly (beta-hydroxybutyrate valerate) nanofiber mat was 30.78 MPa; the young's modulus of the high molecular weight keratin/poly (beta-hydroxybutyrate valerate) nanofiber mat was 43.98 MPa. Mouse wound healing experiments prove that the PHBV/crosslinked keratin nanofiber membrane has a promoting effect on mouse wound healing.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A method for producing high molecular weight keratin, comprising the steps of:
(1) cleaning wool fibers, degreasing for 12-24 hours by using a degreasing solvent, cleaning to remove the degreasing solvent remained on the surface, drying, and shearing to obtain degreased wool fibers;
(2) adding the degreased wool fibers obtained in the step (1) into water, adjusting the pH value to 9-10, adding keratinase, hydrolyzing at 45-55 ℃ for 12-24 h, and centrifuging to obtain keratin hydrolysate;
(3) adjusting the pH value of the keratin hydrolysate obtained in the step (2) to 6-8, adding glutamine transaminase, reacting at 45-55 ℃ for 24-48 h, and inactivating enzyme to obtain a keratin solution;
(4) and (4) dialyzing, freezing and drying the keratin solution obtained in the step (3) to obtain the high molecular weight keratin.
2. The method according to claim 1, wherein the amount of keratinase added is 15,000 to 20,000U/g wool.
3. The method according to claim 1, wherein said transglutaminase is added in an amount of 15-20U/g keratin.
4. The method according to claim 1, wherein in step (2), the bath ratio of the degreased wool fibers to the water is 4-6: 100.
5. The method according to claim 1, wherein in step (1), the degreasing solvent is one or more of ether, benzene, carbon disulfide, acetone, and chloroform.
6. The method according to claim 1, wherein in the step (1), the degreasing is performed at 65-75 ℃ for 12-24 h.
7. The method of claim 1, wherein in the step (4), the dialysis is performed for 2-4 days by using a dialysis bag with a molecular cut-off of 8,000-14,000, and the water is changed for 4-6 h during the dialysis.
8. The method according to claim 1, wherein in the step (2), the centrifugation is carried out at 6000 to 10000rpm for 5 to 10 min.
9. A keratin produced by the method of any one of claims 1 to 8.
10. Use of the keratin of claim 9 in a bio-nanomaterial pad.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114351285A (en) * | 2022-03-09 | 2022-04-15 | 宜宾惠美纤维新材料股份有限公司 | Cheese protein fiber and preparation method thereof |
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| CN114351285A (en) * | 2022-03-09 | 2022-04-15 | 宜宾惠美纤维新材料股份有限公司 | Cheese protein fiber and preparation method thereof |
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