CN117700513A - Chain extension modification method of sericin, product and application thereof - Google Patents
Chain extension modification method of sericin, product and application thereof Download PDFInfo
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- CN117700513A CN117700513A CN202311723654.2A CN202311723654A CN117700513A CN 117700513 A CN117700513 A CN 117700513A CN 202311723654 A CN202311723654 A CN 202311723654A CN 117700513 A CN117700513 A CN 117700513A
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Abstract
The invention relates to a chain extension modification method of sericin, a product and application thereof, wherein a chain extender is adopted to carry out chain extension reaction on sericin to prepare sericin with increased molecular weight and narrow molecular weight distribution; the chain extender is 2, 2-dimethylolpropionic acid, 2-dimethylolbutyric acid, dimethylol dihydroxyethyl ethylene urea, tris (3-hydroxypropyl) phosphine or tetrakis (hydroxymethyl) phosphonium chloride; the crystallinity of the prepared sericin is 20-50%, and the solubility in water is 0.1-1 g/100g of water; the prepared sericin is used for preparing a functional regenerated sericin material or is used for preparing regenerated protein fibers by copolymerizing with a high molecular monomer. According to the chain extension modification method of the sericin, the average relative molecular mass of the sericin after chain extension is increased and the distribution is more uniform; the product of the invention has low water solubility and high crystallinity, and can be applied to various fields such as drug carriers, tissue engineering clothes, home textiles and the like.
Description
Technical Field
The invention belongs to the technical field of proteins, and relates to a chain extension modification method of sericin, a product and application thereof.
Background
Sericin is one of main components of silk and accounts for about 20% -30% of the total mass of cocoon silk. In the refining of silk fabrics, they are usually ignored and discarded as waste from the textile industry. In recent years, as research is continued, the properties and functions of sericin are receiving more and more attention. Besides good biocompatibility and low immunogenicity which are necessary for biological materials, the modified cellulose has excellent moisture absorption, antibiosis and oxidation resistance, and is widely applied to the fields of foods, cosmetics, biological medicines, textile after-treatment, fiber modified materials and the like. The molecular weight and the distribution of the sericin have great influence on the performance of the sericin protein material, the sericin with large molecular weight and even distribution has good water solubility resistance and mechanical property, and has wide application prospect in the fields of textile fibers, novel composite materials and the like.
Sericin is a globular protein with amphoteric property, and has isoelectric point of about 3.8-4.5. Sericin contains a large amount of polar amino acid, has excellent hydrophilicity, and the secondary structure is mainly random coiled, and then is beta-folded and beta-corner, and contains a small amount of alpha-helix. The degumming method commonly used in industry mainly comprises high-temperature high-pressure water degumming, alkaline solution degumming, acid solution degumming, protease solution degumming and the like; the extraction method mainly comprises chemical coagulation method, organic solvent precipitation method, acid precipitation method, centrifugation method, membrane separation method, ion exchange method, and freezing method. The sericin concentrated solution with higher concentration can be separated and extracted from degumming wastewater, and then sericin powder which is convenient to store or materials used for developing functional products, such as hydrogel, porous bracket, nanometer microsphere, nanometer fiber and the like can be prepared by methods of freezing, drying and the like.
However, in document 1 (sericin is used as a serum substitute or additive in cell culture and cryopreservation and evaluated at [ D ]. University of su.2017.), sericin in silk has at least three layers of layered structure of outside, inside and outside, and there are at least 15 kinds of molecular weights. The molecular weight of sericin is greatly changed from inside to outside, the molecular weight of sericin in the inner layer is large and the distribution is relatively concentrated, and the molecular weight from inside to outside is gradually reduced and the dispersibility is increased. Meanwhile, after the sericin in the nature is separated and purified, degradation of different degrees often occurs, so that the relative molecular mass of the sericin is reduced, the overall distribution is more uneven, and the distribution range is wider. The relative molecular mass of the sericin is 10-400 kDa, and the denaturation or degradation degree of the sericin is greatly different according to different separation and purification methods, and the degumming method with severe conditions can improve the degumming efficiency, but can cause severe degradation of the sericin.
The sericin powder prepared by mass production in the current reeling mill has the relative molecular weight of about 3-30 kDa, lower molecular weight and wider distribution, and is difficult to ensure stable and uniform mechanical properties. The separation and purification efficiency of sericin determines the economic application value of silk industry, and the molecular weight and the distribution of the sericin directly influence the physicochemical property of sericin, so as to determine the mechanical property and the biodegradation rate of the sericin material. The structure and the performance of the sericin material are regulated and controlled by controlling the molecular weight and the distribution of the sericin, so that the sericin material can meet different requirements.
The method of chain-extending modification with dithiothreitol in document 2 (development of waste keratin green modification and regenerated fibers thereof [ D ]. University of east China, 2020.doi: 10.27012/d.cnki.gdhuu.2020.000027) expands disulfide bonds inherent to keratin, enhances flexibility and water stability of regenerated keratin films and prepares regenerated keratin fibers, the chain-extending reaction mechanism of which is a reduction reaction, forms new disulfide bonds with a reducing agent by thiol-disulfide bond exchange, cysteine residues on keratin form one free thiol (-SH) and then the other reducing agent further breaks the new disulfide bonds, releasing the other free thiol. The two ends of Dithiothreitol (DTT) molecule have sulfhydryl groups, which can connect the free sulfhydryl groups on different keratin molecule chains like a bridge, in theory, the reduction reaction can be easily completed only if the reducing agent is excessive, so that the reaction can only improve the average relative molecular mass and can not reduce the distribution width.
Therefore, research on a chain extension modification method of sericin, a product and application thereof realizes the compromise of high molecular weight and narrow molecular weight distribution of sericin, and has very important significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a chain extension modification method of sericin, a product and application thereof.
In order to achieve the above purpose, the invention adopts the following scheme:
a chain extension modification method of sericin adopts a chain extender to carry out chain extension reaction on sericin to prepare sericin with increased molecular weight and narrow molecular weight distribution;
the chain extender is 2, 2-dimethylolpropionic acid, 2-dimethylolbutyric acid, dimethylol dihydroxyethyl ethylene urea, tris (3-hydroxypropyl) phosphine (THPP) or tetrakis (hydroxymethyl) phosphonium chloride (THPC);
the relative molecular mass of the sericin before chain extension is 3-30 kDa, and the polydispersity index (PDI) is 2-8; the relative molecular mass of the sericin after chain extension is 30-80 kDa, and the polydispersity index (PDI) is 0.5-1.8; relative molecular mass test standard reference YY/T1805.2-2021, the relative molecular mass and distribution polydispersity index (PDI) test method is gel chromatography (GPC).
As a preferable technical scheme:
the chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin powder in 0.1-3wt% lithium chloride aqueous solution, regulating pH to 4-11 (chain extension reaction is favorable to be carried out in the pH range), and stirring at constant temperature to obtain sericin solution;
(2) Maintaining the constant temperature condition of the step (1), adding a chain extender into the sericin solution, and reacting for 3-12 hours under the stirring condition;
(3) After the reaction is finished, the pH is regulated to 3.2-5.6 (the pH is close to the isoelectric point of sericin, which is favorable for precipitation and separation of sericin), an ultrafiltration membrane is used for interception, the intercepted solution is further concentrated by a centrifugal method, and then the sericin powder with increased molecular weight and narrow molecular weight distribution is obtained by freeze drying.
The chain extension modification method of sericin comprises the steps of (1) stirring at constant temperature of 30-60 ℃ for 1-3 hours.
According to the chain extension modification method of sericin, the proportion of sericin in the sericin solution in the step (1) is 3-11 wt%.
According to the chain extension modification method of the sericin, the mass ratio of the chain extender to the sericin in the step (2) is 1:1-49.
According to the chain extension modification method of sericin, in the step (3), the pore diameter of the ultrafiltration membrane is 1-100 nm, and the freeze drying time is 48 hours.
The chain extension modification method of sericin comprises the steps of (1) stirring at 60-220 rpm and (2) stirring at 150-300 rpm.
The invention also provides sericin with increased molecular weight and narrow molecular weight distribution, which is prepared by the method, wherein the crystallinity of the sericin is 20-50%, the solubility in water is 0.1-1 g/100g of water, the crystallinity is tested according to YY/T0815-2010, and the solubility in water is tested according to GB/T6370-2012.
The invention also provides application of the sericin with increased molecular weight and narrow molecular weight distribution, which is used for preparing functional regenerated sericin materials with various forms, such as sericin films, hydrogels, porous stents, nano-microspheres, nano-fibers and the like, and is applied to the fields of drug carriers, tissue engineering and the like; or used for preparing regenerated protein fiber by copolymerization with high molecular monomer, and is applied to the fields of clothing, home textile and the like.
The principle of the invention:
sericin contains 18 amino acids, wherein the proportion of polar amino acids is high (> 50%), including serine, aspartic acid, lysine, and the like. These polar amino acids contain a large number of polar groups such as hydroxyl, carboxyl, amino, etc., which determine the biological properties of sericin and also allow it to form blends or copolymers with other polymers by crosslinking, copolymerization or blending, thereby increasing the mechanical resistance of the sericin-based material. However, due to the high temperature, acid and alkali conditions and the like used in the degumming process, the degummed sericin is degraded to different degrees, so that the relative molecular mass of the sericin is reduced, and the molecular weight distribution range is widened.
Sericin of different relative molecular masses has different physicochemical properties, low molecular weight sericin (< 24 kDa) has excellent water solubility, solubility in water several times higher than that of medium molecular weight sericin (24 kDa-98 kDa), high molecular weight sericin (> 98 kDa) has poor water solubility, and crystallinity higher than that of low molecular weight sericin. Sericin with different relative molecular masses has obvious influence on the properties of the material, such as condensed state structure, mechanical property, swelling property, light transmittance and the like. The high molecular weight sericin solution has strong intermolecular force and low motion capacity of chain segment, so that the solution has high viscosity. The higher the molecular weight of sericin, the easier it is to promote the transition of its conformation from a random coil structure to a beta-sheet structure. The molecular chain of the low molecular weight sericin solution is short, the winding among chain segments is not tight, the density of intermolecular hydrogen bonds and hydrophobic interactions is relatively low, the crystallinity is low, the mechanical property of the sericin material is poor, and the water solubility is increased.
Therefore, the invention proposes that the sericin is firstly subjected to chain extension, and the hydroxyl in the adopted chain extender can be subjected to crosslinking reaction with primary amine and secondary amine in the sericin. As shown in FIG. 2, the average relative molecular mass of the small molecular weight sericin can be improved to a large extent by the reaction, so that the average relative molecular mass of the whole sericin is increased; meanwhile, the sericin with large molecular mass has larger steric hindrance and lower movement rate, and is not easy to react with a chain extender, so that the average relative molecular mass of the sericin after chain extension is increased and the distribution is more uniform.
Advantageous effects
(1) According to the chain extension modification method of sericin, the hydroxyl in the adopted chain extender can be subjected to a crosslinking reaction with primary amine and secondary amine in the sericin, and the average relative molecular mass of the sericin after chain extension is increased and the distribution is more uniform;
(2) The sericin with increased molecular weight and narrow molecular weight distribution prepared by the invention has low water solubility and high crystallinity;
(3) The sericin with increased molecular weight and narrow molecular weight distribution can be applied to various fields such as drug carriers, tissue engineering clothes, home textiles and the like.
Drawings
FIG. 1 shows GPC gel permeation chromatograms before and after chain extension of sericin in examples 5 and 6; wherein A-1 is sericin before chain extension of example 5, B-2 is sericin after chain extension of example 5, and B-3 is sericin after chain extension of example 6; the ordinate (AU) represents the percentage of molecular weight corresponding to the abscissa;
FIG. 2 is a schematic diagram of a chain extension mechanism of sericin;
FIG. 3 is a DSC chart of example 5.
Detailed Description
The invention is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
The test criteria/methods involved in the examples are as follows:
crystallinity: test reference standard YY/T0815-2010;
solubility: test reference standard GB/T6370-2012;
relative molecular mass and distribution polydispersity index (PDI): the measurement was performed by a Gel Permeation Chromatography (GPC) method.
Example 1
A chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin with relative molecular mass of 13kDa and polydispersity index of 4 in 0.8wt% lithium chloride aqueous solution, regulating pH to 4, and then stirring at constant temperature of 40 ℃ at a speed of 120rpm for 1.5 hours to obtain sericin solution with sericin accounting for 4wt%;
(2) Maintaining the constant temperature condition of the step (1), adding 2, 2-dimethylolpropionic acid into the sericin solution, and stirring at a speed of 160rpm for reaction for 4 hours; wherein the mass ratio of the 2, 2-dimethylolpropionic acid to sericin is 1:9;
(3) After the reaction, the pH is adjusted to 3.7, the solution is trapped by an ultrafiltration membrane with the pore diameter of 80nm, the trapped solution is further concentrated by a centrifugal method, and then the solution is freeze-dried for 48 hours to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
The final sericin with increased molecular weight and narrow molecular weight distribution has a relative molecular mass of 55kDa and a polydispersity index of 1.3; the crystallinity of sericin with increased molecular weight and narrow molecular weight distribution is 21%, and the solubility in water is 0.7g/100g of water; sericin with increased molecular weight and narrow molecular weight distribution is used for preparing functional regenerated sericin material or is used for preparing regenerated protein fiber by copolymerization with high molecular monomer.
Example 2
A chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin with relative molecular mass of 13kDa and polydispersity index of 4.5 in 1wt% lithium chloride water solution, regulating pH to 5, and then stirring at constant temperature of 120rpm for 1.5 hours at 40 ℃ to obtain sericin solution with sericin accounting for 4wt% of the weight;
(2) Maintaining the constant temperature condition of the step (1), adding 2, 2-dimethylolbutyric acid into the sericin solution, and stirring at a speed of 180rpm for reaction for 4.5 hours; wherein the mass ratio of the 2, 2-dihydroxymethyl butyric acid to the sericin is 1:11;
(3) After the reaction, the pH is adjusted to 3.7, the solution is trapped by an ultrafiltration membrane with the pore diameter of 80nm, the trapped solution is further concentrated by a centrifugal method, and then the solution is freeze-dried for 48 hours to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
The final sericin with increased molecular weight and narrow molecular weight distribution has a relative molecular mass of 60kDa and a polydispersity index of 1.2; the crystallinity of the sericin with increased molecular weight and narrow molecular weight distribution is 23%, and the solubility in water is 0.6g/100g of water; sericin with increased molecular weight and narrow molecular weight distribution is used for preparing functional regenerated sericin material or is used for preparing regenerated protein fiber by copolymerization with high molecular monomer.
Example 3
A chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin with relative molecular mass of 13kDa and polydispersity index of 5 in 1.2wt% lithium chloride water solution, regulating pH to 6, and then stirring at constant temperature of 120rpm for 1.5 hours at 50 ℃ to obtain sericin solution with sericin accounting for 4wt% of the weight;
(2) Maintaining the constant temperature condition of the step (1), adding dimethylol dihydroxyethyl ethylene urea into the sericin solution, and stirring at a speed of 200rpm for reaction for 5 hours; wherein the mass ratio of the dimethylol dihydroxyethyl ethylene urea to the sericin is 1:13;
(3) After the reaction, the pH is adjusted to 3.7, the solution is trapped by an ultrafiltration membrane with the pore diameter of 80nm, the trapped solution is further concentrated by a centrifugal method, and then the solution is freeze-dried for 48 hours to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
The final sericin with increased molecular weight and narrow molecular weight distribution has a relative molecular mass of 65kDa and a polydispersity index of 1.1; the crystallinity of sericin with increased molecular weight and narrow molecular weight distribution is 25%, and the solubility in water is 0.5g/100g of water; sericin with increased molecular weight and narrow molecular weight distribution is used for preparing functional regenerated sericin material or is used for preparing regenerated protein fiber by copolymerization with high molecular monomer.
Example 4
A chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin with relative molecular mass of 13kDa and polydispersity index of 5.5 in 1.4wt% lithium chloride water solution, regulating pH to 7, and stirring at constant temperature of 150rpm for 2 hours at 50 ℃ to obtain sericin solution with sericin proportion of 5wt%;
(2) Maintaining the constant temperature condition of the step (1), adding tris (3-hydroxypropyl) phosphine into the sericin solution, and stirring at a speed of 220rpm for reaction for 5.5 hours; wherein the mass ratio of the tri (3-hydroxypropyl) phosphine to the sericin is 1:15;
(3) After the reaction, the pH is adjusted to 3.8, the solution is trapped by an ultrafiltration membrane with the aperture of 100nm, the trapped solution is further concentrated by a centrifugal method, and then the solution is freeze-dried for 48 hours to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
The final sericin with increased molecular weight and narrow molecular weight distribution has a relative molecular mass of 80kDa and a polydispersity index of 0.8; the crystallinity of sericin with increased molecular weight and narrow molecular weight distribution is 30%, and the solubility in water is 0.2g/100g of water; sericin with increased molecular weight and narrow molecular weight distribution is used for preparing functional regenerated sericin material or is used for preparing regenerated protein fiber by copolymerization with high molecular monomer.
Example 5
A chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin with relative molecular mass of 13kDa and polydispersity index of 6.5 in 1.6wt% lithium chloride aqueous solution, regulating pH to 8, and then stirring at a constant temperature of 60 ℃ at a speed of 150rpm for 2 hours to obtain sericin solution with sericin accounting for 5wt%;
(2) Maintaining the constant temperature condition of the step (1), adding the tetrakis (hydroxymethyl) phosphonium chloride into the sericin solution, and stirring at the speed of 240rpm for reaction for 6 hours; wherein the mass ratio of the tetra-methylol phosphorus chloride to the sericin is 1:17;
(3) After the reaction, the pH is adjusted to 3.8, the solution is trapped by an ultrafiltration membrane with the aperture of 100nm, the trapped solution is further concentrated by a centrifugal method, and then the solution is freeze-dried for 48 hours to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
As shown in FIG. 1, the molecular weight of the sericin after chain extension increases and the distribution becomes narrow. The final sericin with increased molecular weight and narrow molecular weight distribution has a relative molecular mass of 73kDa and a polydispersity index of 0.9; the crystallinity of sericin with increased molecular weight and narrow molecular weight distribution was 29%, and the solubility in water was 0.3g/100g of water; sericin with increased molecular weight and narrow molecular weight distribution is used for preparing functional regenerated sericin material or is used for preparing regenerated protein fiber by copolymerization with high molecular monomer. As shown in FIG. 3, the thermal decomposition temperature of the chain-extended sericin was increased as compared with that of the non-chain-extended sericin, indicating that chemical crosslinking was generated.
Example 6
A chain extension modification method of sericin comprises the following specific steps:
(1) Dissolving sericin with relative molecular mass of 13kDa and polydispersity index of 7 in 1.8wt% lithium chloride water solution, regulating pH to 9, and then stirring at a constant temperature of 60 ℃ at a speed of 150rpm for 2 hours to obtain sericin solution with sericin accounting for 5 wt%;
(2) Maintaining the constant temperature condition of the step (1), adding 2, 2-dimethylolpropionic acid into the sericin solution, and stirring at a speed of 260rpm for reaction for 6.5 hours; wherein the mass ratio of the 2, 2-dimethylolpropionic acid to sericin is 1:19;
(3) After the reaction, the pH is adjusted to 3.8, the solution is trapped by an ultrafiltration membrane with the aperture of 100nm, the trapped solution is further concentrated by a centrifugal method, and then the solution is freeze-dried for 48 hours to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
As shown in FIG. 1, the molecular weight of the sericin after chain extension increases and the distribution becomes narrow. The final sericin with increased molecular weight and narrow molecular weight distribution has a relative molecular mass of 71kDa and a polydispersity index of 1.0; the crystallinity of sericin with increased molecular weight and narrow molecular weight distribution is 28%, and the solubility in water is 0.4g/100g of water; sericin with increased molecular weight and narrow molecular weight distribution is used for preparing functional regenerated sericin material or is used for preparing regenerated protein fiber by copolymerization with high molecular monomer.
Claims (9)
1. A chain extension modification method of sericin is characterized in that: chain extender is adopted to carry out chain extension reaction on sericin to prepare sericin with increased molecular weight and narrow molecular weight distribution;
the chain extender is 2, 2-dimethylolpropionic acid, 2-dimethylolbutyric acid, dimethylol dihydroxyethyl ethylene urea, tris (3-hydroxypropyl) phosphine or tetrakis (hydroxymethyl) phosphonium chloride;
the relative molecular mass of the sericin before chain extension is 3-30 kDa, and the polydispersity index is 2-8; the relative molecular mass of the sericin after chain extension is 30-80 kDa, and the polydispersity index is 0.5-1.8.
2. The method for chain extension modification of sericin according to claim 1, characterized by comprising the following specific steps:
(1) Dissolving sericin in 0.1-3wt% of lithium chloride aqueous solution, regulating pH to 4-11, and stirring at constant temperature to obtain sericin solution;
(2) Maintaining the constant temperature condition of the step (1), adding a chain extender into the sericin solution, and reacting for 3-12 hours under the stirring condition;
(3) After the reaction is finished, regulating the pH to 3.2-5.6, intercepting by using an ultrafiltration membrane, further concentrating the intercepted solution by a centrifugal method, and then freeze-drying to obtain the sericin with increased molecular weight and narrow molecular weight distribution.
3. The method for chain extension modification of sericin according to claim 2, wherein the constant temperature stirring in the step (1) is 30 to 60 ℃ for 1 to 3 hours.
4. The method for chain extension modification of sericin according to claim 2, wherein the sericin in the sericin solution in the step (1) is 3 to 11wt%.
5. The method for modifying the chain extension of sericin according to claim 2, wherein the mass ratio of the chain extender to the sericin in the step (2) is 1:1-49.
6. The method for chain extension modification of sericin according to claim 2, wherein the pore diameter of the ultrafiltration membrane in the step (3) is 1 to 100nm, and the time of freeze-drying is 48 hours.
7. The method for chain extension modification of sericin according to claim 2, wherein the stirring rate in the step (1) is 60 to 220rpm, and the stirring rate in the step (2) is 150 to 300rpm.
8. The sericin having an increased molecular weight and a narrow molecular weight distribution, which is produced by the method according to any one of claims 1 to 7, characterized in that: the crystallinity of sericin is 20% -50%, and the solubility in water is 0.1-1 g/100g of water.
9. Use of sericin of increased molecular weight and narrow molecular weight distribution according to claim 8, characterized in that: is used for preparing a functional regenerated sericin material; or is used for preparing regenerated protein fiber by copolymerization with a high molecular monomer.
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