CN111875691B - Preparation method of silk fibroin and sericin composite solution - Google Patents
Preparation method of silk fibroin and sericin composite solution Download PDFInfo
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Abstract
The invention provides a preparation method of a silk fibroin and sericin composite solution, which comprises the following steps: A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk; B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution. The silk fibroin and sericin composite solution is prepared by adopting a mild degumming and one-step dissolving method, and the traditional method that silk fibroin and sericin are respectively prepared, respectively dissolved and then mixed for preparing a composite material is broken through; importantly, the molecular weight of silk protein in the composite solution is concentrated above 150 kDa; the molecular weight of the silk fibroin solution or the solution prepared by dissolving and mixing silk fibroin sericin prepared by the reported conventional method is continuously distributed, and the molecular weight is mainly distributed below 85kDa to 15kDa or even smaller.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a preparation method of a silk fibroin and sericin composite solution.
Background
With the rapid development of surgical operations, the dramatic increase and the youth of human diseases and the aging of the world population, the clinical needs and treatments of tissue grafts face more and more pressure. Common tissue repair methods include conventional autografting, allografting and artificial tissue engineering scaffold. Although the autograft repair has the best treatment effect, the autograft repair has serious restrictions on factors such as supply area damage, insufficient supply and complications; allograft faces problems of immunological rejection, disease transmission, etc.; tissue engineering was proposed in the 90 s of the 20 th century, and then research on scaffold materials for tissue engineering was rapidly developed. The synthetic polymer materials in tissue engineering have the advantages of wide sources, controllable physicochemical properties, excellent processing and forming properties and the like, for example, various synthetic polymers such as polyester, polyanhydride, polylactic acid, polyglycolic acid and copolymers thereof lack biological activity, have poor cellular compatibility and do not degrade or degrade products to influence the tissue microenvironment. In situ tissue regeneration is considered to be the most ideal mode for repairing defective or diseased tissues, so natural polymers such as collagen, gelatin, alginate, hyaluronic acid, chitosan and the like are widely favored in the research of tissue engineering scaffolds due to excellent bioactivity and degradability. However, most of the materials have poor mechanical properties, and application research of the materials as tissue (such as bones, teeth and the like) engineering scaffolds with high mechanical property requirements also has huge challenges.
Silk fibroin and sericin have excellent cell compatibility, blood compatibility and tissue compatibility, and can be controllably formed, and have been widely researched in tissue engineering applications such as skin, blood vessels, bones and the like as a tissue engineering scaffold material, but the currently researched regenerated silk (silk fibroin and sericin) protein scaffold material has the same poor mechanical property and is difficult to meet the requirements of hard tissue replacement such as bones, teeth and the like, because the existing degumming method and extraction method have large damage to the macromolecular structures of silk fibroin and sericin, and the polymerization degree (molecular weight) of a macromolecule is an extremely important intrinsic factor for determining the performance of the macromolecule.
Different from other natural polymer materials, especially protein polymer materials, the purity of protein in the silk is very high, the composition is unique, the preparation method can be simple and mild, and small damage to silk macromolecules can be controlled, unlike the complex purification of collagen.
The silk fibroin of the silk inner core accounts for 70-80% of the mass of the silk, macromolecules are connected by 6 heavy chains with 390kDa and 6 light chains with 26kDa in a disulfide bond mode, and then the heavy chains are combined with another glycoprotein/P25 with 25kDa in a hydrophobic effect mode, wherein the heavy chains have a sequence structure basis for forming a high-performance material; sericin outside the silk accounts for 20-30% of the mass of the silk, the molecular weight of sericin is larger, the types of sericin are more than 9, the outer middle layer and the inner middle layer of sericin are indicated from the histological angle by Changlan in relation to Chuanbright, and the small looseness indicates that 4 types of sericin outside and 4 types of sericin inside are provided, namely sericin I, sericin II, sericin III and sericin IV, and the solubility performance from outside to inside is lower and lower, so that the beta conformation is increased.
The regenerated silk fibroin solution is obtained by dissolving strong alkali weak acid salt or enzyme degumming salt, and the methods cause the damage of silk fibroin macromolecules; the regenerated sericin solution is mainly from silkworm cocoons, and the two solutions are generally prepared by independent separation. For example, CN02138129.1 discloses a preparation method of a silk fibroin and sericin composite tissue engineering scaffold, wherein a composite material is prepared by mixing pre-prepared regenerated silk fibroin and regenerated sericin in proportion, a degumming method of silk fibroin adopts a strong base and weak acid salt solvent, and sericin extracts the outer layer of silk sericin coated on cocoon silk. For example, CN201510353363.8 discloses a scaffold material for bone tissue engineering containing silk fibroin and sericin and a preparation method thereof, wherein regenerated silk fibroin and regenerated sericin are obtained respectively step by step and then mixed to prepare the scaffold material, wherein the preparation of silk fibroin does not disclose specific process conditions, and sericin is obtained from silkworm cocoon and treated with organic solvents such as alcohol.
Disclosure of Invention
The invention aims to provide a preparation method of a silk fibroin sericin composite solution, and the silk fibroin sericin composite solution has the characteristics of high viscosity and high molecular weight.
In view of the above, the present application provides a method for preparing a silk fibroin and sericin composite solution, comprising the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;
B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.
Preferably, the temperature of the warm water is 35-60 ℃, and the pretreatment time is 1-10 min.
Preferably, the pretreatment further comprises the following steps: and taking out the pretreated silk, washing in water at 25-30 ℃, and dehydrating.
Preferably, in the step A), the treatment time in the water at 65-95 ℃ is 1-8 h.
Preferably, in the step a), the ratio of the silk to the warm water is 1 g: (40-60) ml, wherein the ratio of the pretreated silk to water is 1 g: (40-60) ml.
Preferably, in step a), before the drying after the treatment, the method further comprises: washing the treated silk in water at 25-30 ℃ and then dehydrating; the drying is carried out by hot air at 40-60 ℃.
Preferably, the neutral salt solution is 8-10M of lithium bromide aqueous solution, and the ratio of the primarily treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.
Preferably, in the step B), the treatment temperature is 50-80 ℃.
Preferably, the purification method specifically comprises:
and (3) filling the obtained silk fibroin sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.
The application provides a preparation method of a silk fibroin and sericin composite solution, which comprises the steps of firstly, placing silk in warm water for pretreatment, and then placing the silk in water for treatment to obtain the primarily treated silk; then treating the silk fibroin/sericin composite solution in a neutral salt solution, and purifying to obtain a silk fibroin/sericin composite solution; the silk fibroin composite solution is prepared by adopting mild degumming and a one-step dissolving method, sericin on the surface of silk is partially or completely removed by pretreatment in warm water and subsequent treatment in water at a specific temperature, silk fibroin macromolecular chains cannot be damaged in the subsequent degumming process of silk fibroin fibers in the whole process of specific warm water treatment, sericin on the outer layer of the silk fibroin fibers is from outside to inside along with a coating layer, the types, the structures and the performances of the sericin are also different, and the silk fibroin composite solution with different compositions and capable of preparing materials with different performances (especially mechanical performances) is obtained by controlling the degumming degree; and ensures high viscosity and molecular weight of the silk fibroin and sericin composite solution.
Detailed Description
For a further understanding of the present invention, reference will now be made to the following preferred embodiments of the invention in conjunction with the examples, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and is not intended to limit the scope of the claims which follow.
Aiming at the problem that silk fibroin macromolecules are seriously damaged or the problem that silk fibroin and sericin composite solutions are respectively prepared in the prior art, the method adopts a deionized water mild degumming and one-step dissolving method to prepare the silk fibroin and sericin composite solution with high macromolecule content and high viscosity, can avoid the damage of the silk fibroin macromolecules, and finally enables the silk fibroin and sericin in the prepared silk fibroin and sericin composite solution to have high viscosity and high molecular weight. Specifically, the embodiment of the invention discloses a preparation method of a silk fibroin and sericin composite solution, which comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk;
B) and (3) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin and sericin composite dissolved solution to obtain the silk fibroin and sericin composite solution.
In the preparation process of the silk fibroin and sericin composite solution, firstly, placing silk in warm water for pretreatment, then placing the silk in water at 65-95 ℃ for treatment, and drying to obtain the primarily treated silk; in the process, sericin of the silk is completely or partially dissolved, the fibroin is still in a fiber state, if undissolved sericin exists, the undissolved sericin is wrapped on the outer surface of the fibroin fiber, and the dissolved sericin exists in water and is in a solution state. The temperature of the warm water is 35-60 ℃, namely the silk can be pretreated in the warm water at 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃, and the ratio of the silk to the warm water is 1 g: (40-60) ml, more specifically, the ratio of the silk to the warm water is 1 g: 42ml, 1 g: 45ml, 1 g: 48ml, 1 g: 52ml, 1 g: 55ml, 1 g: 58 ml. The pretreatment is carried out in the constant-temperature warm water for 1-10 min, and the pretreatment is carried out in stirring all the time so as to ensure that the silk can be preliminarily and completely immersed in the water. And after the pretreatment is finished, taking out the pretreated silk, washing the silk in deionized water at 25-30 ℃ and dehydrating the silk. The dehydrated silk is treated in water at 65-95 deg.C, more specifically, the dehydrated silk is treated in water at 68 deg.C, 70 deg.C, 72 deg.C, 75 deg.C, 78 deg.C, 80 deg.C, 82 deg.C, 84 deg.C, 87 deg.C, 90 deg.C or 93 deg.C. The mode that this application silk was earlier in aquatic preliminary treatment again in aquatic is handled does not relate to any reagent and handles, can dissolve sericin in the while of protection silk fibroin macromolecular structure indestructible. The ratio of the pretreated silk to water is 1 g: (40-60) ml, more specifically, the ratio of the pretreated silk to water is 1 g: 42ml, 1 g: 45ml, 1 g: 48ml, 1 g: 50ml, 1 g: 52ml, 1 g: 55ml, 1 g: 58ml or 1 g: 60 ml. The degumming rate of the silk sericin can be adjusted according to different temperatures and time of water so as to realize the preparation of silk sericin composite solutions with different viscosities and different molecular weights; in the application, the time for treating the silk in the water is preferably 1-8 hours, so that the silk is partially or completely degummed; more specifically, the time for the treatment in water is 1.5h, 2h, 2.5h, 3h, 3.2h, 3.6h, 4.2h, 4.5h, 5.0h, 5.5h, 6.0h, 6.5h, 6.8h, 7.0h or 7.5 h. After treatment, the treated silk is washed in water at 25-30 ℃, dehydrated and dried; the drying is carried out by hot air at 40-60 ℃. The silk is subjected to the treatment to obtain the primary treated silk, the silk still presents a fiber state, the protection of the silk fibroin is realized, and the high molecular weight and the high viscosity of the silk fibroin are favorably ensured.
According to the invention, the silk after primary treatment is treated in a neutral salt solution and purified, and then the silk fibroin sericin composite solution is obtained. In the process, the primary-treated fibrous silk is dissolved to obtain the silk fibroin sericin composite solution, wherein the inner core of the silk is silk fibroin fibers, and the periphery of the silk fibroin fibers is wrapped with sericin with different contents. Neutral salt solution specifically is selected from the lithium bromide aqueous solution in this application, and its concentration is 8 ~ 10M, the silk of preliminary treatment with the proportion of lithium bromide aqueous solution is 1 g: (20-30) ml, more specifically, the ratio of the primary treated silk to the lithium bromide aqueous solution is 1 g: 22ml, 1 g: 24ml, 1 g: 25ml, 1 g: 27ml or 1 g: 29 ml. The treatment temperature is specifically 50-80 ℃, and more specifically, the treatment temperature is specifically 52 ℃, 57 ℃, 62 ℃, 65 ℃, 67 ℃, 68 ℃, 72 ℃, 75 ℃ or 78 ℃.
After the treatment, purifying the obtained silk fibroin sericin dissolving solution to remove lithium bromide and small molecular weight silk fibroin; the purification method specifically comprises the steps of filling the obtained silk fibroin and sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.
The silk fibroin and sericin composite solution is prepared by adopting a mild degumming and one-step dissolving method, and the traditional method that silk fibroin and sericin are respectively prepared, respectively dissolved and then mixed for preparing a composite material is broken through. Importantly, the molecular weight of silk fibroin in the composite solution is concentrated above 150kDa or contains a molecular weight of about 25kDa, and the molecular weight of the silk fibroin solution prepared by the reported conventional method or the solution prepared by dissolving and mixing silk fibroin sericin presents continuous distribution, mainly the molecular weight distribution is below 85kDa to 15kDa or even smaller. In the preparation process of the silk fibroin solution by the mild degumming one-step dissolution method, silk is treated by hot water, so that the macromolecular chains (especially heavy chains) of the silk fibroin cannot be damaged in the degumming process; the sericin of the silk fibroin outer layer is different from the inside and the outside of the coating layer, the type, the structure and the performance of the sericin are different, and silk fibroin composite solutions with different compositions and capable of preparing materials with different performances (especially mechanical properties) are obtained by controlling the degumming degree. The viscosity of the silk protein solution of the one-step dissolution method is far higher than that of the mixed solution reported conventionally. More importantly, the invention provides a novel preparation technology of the high-performance regenerated fibroin material based on the fundamental factor of high molecular weight influencing the mechanical property of the polymer, and solves the technical problem of neck clamping with insufficient mechanical property of the natural biopolymer tissue engineering scaffold capable of inducing the regeneration and repair of hard tissues.
For further understanding of the present invention, the following examples are given to illustrate the preparation method of silk fibroin complex solution provided by the present invention, and the scope of the present invention is not limited by the following examples.
Example 1
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) to preheat to 60 ℃, putting the raw silk into the deionized water, stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk, washing the silk by deionization at the temperature of 25-30 ℃, and dehydrating;
2. placing the dehydrated silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 1 hour at constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. weighing dried silk, dissolving the dried silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a bombyx mori silk fibroin sericin compound solution;
4. filling the silk fibroin sericin composite solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane; and (3) placing the dialysis bag filled with the silk fibroin sericin solution in a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain the purified silkworm fibroin sericin composite aqueous solution.
5. Concentrating silk fibroin by using a rotary evaporator to obtain a composite aqueous solution with the same mass fraction, namely obtaining the high-molecular-weight and high-viscosity silkworm silk fibroin composite aqueous solution. The viscosity value and the extreme value of the viscosity of the silk fibroin sericin composite aqueous solution under the initial shearing action are measured to be 3.5Pa.s and 10Pa.s by using an AR2000 rheometer.
6. Concentrating by using a rotary evaporator or diluting silk fibroin by using deionized water to obtain a composite aqueous solution with a certain mass fraction, and measuring by using a polyacrylamide gel electrophoresis method to obtain the composite aqueous solution, wherein the molecular weight distribution of the composite aqueous solution is mainly more than 150kDa, a small amount of 50-100 kDa and about 25kDa protein chains.
7. And (3) measuring the degumming rate of the silk treated in the steps 1 and 2 by adopting a weighing method, and measuring the degumming rate to be 8%.
Example 2
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) to preheat to 60 ℃, putting the raw silk into the deionized water, stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk, washing the silk by deionization at the temperature of 25-30 ℃, and dehydrating;
2. placing the dehydrated silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 4 hours at a constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. weighing dried silk, dissolving the dried silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a bombyx mori silk fibroin sericin compound solution;
4. and (2) filling the silk fibroin sericin composite solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin sericin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain the purified silkworm fibroin sericin composite aqueous solution.
5. Concentrating the silk fibroin by a rotary evaporator to obtain a composite aqueous solution with the same mass fraction, namely obtaining the high-molecular-weight and high-viscosity silkworm silk fibroin composite aqueous solution. The viscosity value and the extreme value of the viscosity of the silk fibroin sericin composite aqueous solution under the initial shearing action are measured to be 4.5Pa.s and 9Pa.s by using an AR2000 rheometer. Sericin has much higher viscosity (11Pa.s and 40Pa.s) than fibroin, and the presence of sericin increases the viscosity of the solution, so the extreme value of the viscosity of the solution under the shearing action is smaller than that of the solution in the embodiment 1.
6. Concentrating with rotary evaporator or diluting silk fibroin with deionized water to obtain composite water solution with certain mass fraction, and measuring by polyacrylamide gel electrophoresis to obtain composite solution with molecular weight distribution of more than 180kDa and protein chain of about 25 kDa.
7. And (3) measuring the degumming rate of the silk treated in the steps 1 and 2 by adopting a weighing method, and measuring the degumming rate to be 17%.
Example 3
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) to preheat to 60 ℃, putting the raw silk into the deionized water, stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk, washing the silk by deionization at the temperature of 25-30 ℃, and dehydrating;
2. placing the dehydrated silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 4 hours at a constant temperature, taking out the silk containing sericin, fully cleaning the silk with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
3. weighing dried silk, dissolving the dried silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a bombyx mori silk fibroin sericin compound solution;
4. and (2) filling the silk fibroin sericin composite solution into a dialysis bag with the molecular weight cutoff of 50kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin sericin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain the purified silkworm fibroin sericin composite aqueous solution.
5. Concentrating the silk fibroin by a rotary evaporator to obtain a composite aqueous solution with the same mass fraction, namely obtaining the high-molecular-weight and high-viscosity silkworm silk fibroin composite aqueous solution. And measuring the viscosity value and the extreme value of the viscosity of the silk fibroin and sericin composite aqueous solution under the initial shearing action by using an AR2000 rheometer to be 8.0Pa.s and 12 Pa.s. Compared with example 2, the viscosity value of the silk fibroin sericin composite solution is remarkably increased because the solution molecular weight is increased and the small molecular weight is reduced (protein chains of about 25kDa are removed). The presence of sericin increases the viscosity of the solution compared with example 5.
6. The silk fibroin is concentrated by a rotary evaporator or diluted by deionized water to obtain a composite aqueous solution with a certain mass fraction, and the molecular weight distribution of the composite solution is mainly more than 180kDa by adopting a polyacrylamide gel electrophoresis method.
7. And (3) measuring the degumming rate of the silk treated in the steps 1 and 2 by adopting a weighing method, and measuring the degumming rate to be 17%.
Example 4
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) to preheat to 60 ℃, putting the raw silk into the deionized water, stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk, washing the silk by deionization at the temperature of 25-30 ℃, and dehydrating;
2. placing the dehydrated silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 7 hours at a constant temperature, taking out the silk, fully cleaning the silk by using deionized water at 25-30 ℃, dehydrating, and drying by using hot air at 40-60 ℃;
3. weighing dried silk, dissolving the dried silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a bombyx mori silk fibroin sericin compound solution;
4. the silk fibroin sericin compound solution is filled into a dialysis bag with the molecular weight cutoff of 14kDa, and the wall of the dialysis bag is a semipermeable membrane. And (3) placing the dialysis bag filled with the silk fibroin sericin solution in a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain the purified silkworm fibroin sericin composite aqueous solution.
5. Concentrating the silk fibroin by a rotary evaporator to obtain a composite aqueous solution with the same mass fraction, namely obtaining the high-molecular-weight and high-viscosity silkworm silk fibroin composite aqueous solution. And measuring the viscosity value and the extreme value of the viscosity of the silk fibroin and sericin composite aqueous solution under the initial shearing action by using an AR2000 rheometer to be 2.0Pa.s and 5 Pa.s.
6. Concentrating with rotary evaporator or diluting silk fibroin with deionized water to obtain composite water solution with certain mass fraction, and measuring by polyacrylamide gel electrophoresis to obtain composite solution with molecular weight distribution of more than 200kDa and protein chain of about 25 kDa.
7. And (3) determining the degumming rate of the silk treated in the steps 1 and 2 by adopting a weighing method, and determining that the degumming rate is 23%.
Compared with the conventional degumming method of comparative example 1, the degumming rate is the same, namely sericin is completely removed, and the dialysis cut-off molecular weight is the same, but the molecular weight of the silk solution degummed by hot water in the embodiment is not only large in molecular weight and concentrated in distribution, but also small in molecular weight and dispersed in distribution; the viscosity is larger as the molecular weight is larger and the viscosity is larger as the molecular weight polydispersity is smaller, and the viscosity values and viscosity extremes under initial shear measured in this example are 20 times and 12.5 times as large as those of comparative example 1.
Example 5
1. Taking 1 kg of raw silk of silkworms after cocoon cooking and reeling as a raw material, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) to preheat to 60 ℃, putting the raw silk into the deionized water, stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk, washing the silk by deionization at the temperature of 25-30 ℃, and dehydrating;
2. placing the dehydrated silk in deionized water preheated at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 7 hours at a constant temperature, taking out the silk, fully cleaning the silk by using deionized water at 25-30 ℃, dehydrating, and drying by using hot air at 40-60 ℃;
3. weighing dried silk, dissolving the dried silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain a bombyx mori silk fibroin sericin compound solution;
4. and (2) filling the silk fibroin sericin composite solution into a dialysis bag with the molecular weight cutoff of 50kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the silk fibroin sericin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain the purified silkworm fibroin sericin composite aqueous solution.
5. Concentrating the silk fibroin by a rotary evaporator to obtain a composite aqueous solution with the same mass fraction, namely obtaining the high-molecular-weight and high-viscosity silkworm silk fibroin composite aqueous solution. The viscosity value and the extreme value of the viscosity of the silk fibroin and sericin composite aqueous solution under the initial shearing action are measured to be 4.0Pa.s and 8.5Pa.s by using an AR2000 rheometer. Compared with example 4, the viscosity value of the silk fibroin sericin composite solution is remarkably increased because the solution molecular weight is increased and the small molecular weight is reduced (protein chains of about 25kDa are removed).
6. Concentrating with a rotary evaporator or diluting silk fibroin with deionized water to obtain a composite aqueous solution with a certain mass fraction, and measuring by polyacrylamide gel electrophoresis to obtain the composite solution with molecular weight distribution of more than 200 kDa.
7. The degumming rate of the silk treated in the steps 1 and 2 is measured by a weighing method, the degumming rate is 23 percent, and the viscosity value and the extreme value of the viscosity under the initial shearing action measured in the embodiment are 40 times and 21 times of those of the comparative example 1.
Comparative example 1 preparation of conventional pure fibroin solution
1. The method comprises the following steps of taking raw silkworm silks which are reeled by boiling cocoons as raw materials, putting the raw silkworm silks into a sodium carbonate aqueous solution with the concentration of 0.1-0.06% according to the bath ratio of 1:50(g/mL), treating for three times at 98-100 ℃ for 30 minutes each time, taking out, fully cleaning the silks with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
2. weighing the dried silkworm silk, dissolving the dried silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain silkworm fibroin solution;
3. filling the bombyx mori silk fibroin solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified bombyx mori silk fibroin aqueous solution;
4. and then concentrating silk fibroin by adopting a rotary evaporator to obtain an aqueous solution with the same mass fraction, and measuring the viscosity value and the extreme value of the viscosity of the silk fibroin solution under the initial shearing action of the silkworm by using an AR2000 rheometer to be 0.1Pa.s and 0.4 Pa.s.
5. Concentrating with a rotary evaporator or diluting silk fibroin with deionized water to obtain a water solution with a certain mass fraction, and measuring by polyacrylamide gel electrophoresis to obtain a silk fibroin solution with dispersed molecular weight distribution and mainly distributed below 85kDa, wherein although a dialysis bag with a molecular weight cutoff of 14kDa is also used, the silk fibroin content below 15kDa is still large.
6. And (3) determining the degumming rate of the silk treated in the steps 1 and 2 by adopting a weighing method, and determining that the degumming rate is 23%.
Comparative example 2 composite solution prepared by preparing fibroin solution by conventional degumming and mixing with sericin solution
1. The method comprises the following steps of taking raw silkworm silks which are reeled by boiling cocoons as raw materials, putting the raw silkworm silks into a sodium carbonate aqueous solution with the concentration of 0.1-0.06% according to the bath ratio of 1:50(g/mL), treating for three times at 98-100 ℃ for 30 minutes each time, taking out, fully cleaning the silks with deionized water at 25-30 ℃, dehydrating, and drying with hot air at 40-60 ℃;
2. weighing dried silkworm silk, dissolving the dried silkworm silk in 9.3M lithium bromide aqueous solution according to a bath ratio of 1:25(g/mL), and treating at 65 ℃ until the silk is completely dissolved to obtain silkworm fibroin solution;
3. filling the bombyx mori silk fibroin solution into a dialysis bag with the molecular weight cutoff of 14kDa, wherein the wall of the dialysis bag is a semipermeable membrane, placing the dialysis bag filled with the bombyx mori silk fibroin solution into a container filled with deionized water, replacing the water in the container with new deionized water every 2 hours, and continuously dialyzing for 3 days to obtain a purified bombyx mori silk fibroin aqueous solution;
4. taking 1 kg of raw silk of the silkworm reeled by boiling the cocoon, weighing 50L of deionized water according to a bath ratio of 1:50(g/mL) and preheating to 60 ℃, putting the raw silk into the deionized water and stirring at the constant temperature of 60 ℃ for 1-10 minutes, taking out the raw silk and washing and dehydrating the silk by using deionized water at the temperature of 25-30 ℃;
5. placing the dehydrated silk in preheated deionized water at 90 ℃ according to a bath ratio of 1:50(g/mL) for boiling for 7 hours at a constant temperature, removing fibers, collecting a sericin water solution, and freeze-drying to obtain solid sericin;
6. and concentrating the silk fibroin aqueous solution obtained in the step 3 by adopting a rotary evaporator, adding sericin with the content equal to that of the silk sericin in the example 2, dissolving and mixing uniformly, preparing a silk fibroin sericin composite aqueous solution with the mass fraction equal to that of all the examples, and measuring the viscosity value and the extreme value of the viscosity of the solution under the initial shearing action by using an AR2000 rheometer to be 0.45Pa.s and 1.07 Pa.s.
The molecular weight and viscosity of the silk fibroin and sericin composite aqueous solution prepared in the examples 1 to 5 are significantly higher than those of the silk fibroin solution obtained by the conventional degumming method in the comparative examples 1 to 2.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A preparation method of a silk fibroin and sericin composite solution comprises the following steps:
A) placing silk in warm water for pretreatment, then placing the silk in 65-95 ℃ water for treatment, and drying to obtain primary treated silk; the temperature of the warm water is 35-60 ℃;
B) treating the primarily treated silk in a neutral salt solution, and purifying the obtained silk fibroin sericin composite dissolved solution to obtain a silk fibroin sericin composite solution;
the neutral salt solution is a lithium bromide aqueous solution with the concentration of 8-10M, and the ratio of the primarily treated silk to the lithium bromide aqueous solution is 1 g: (20-30) mL.
2. The method according to claim 1, wherein the pretreatment time is 1 to 10 min.
3. The method of claim 1, wherein the pre-treating is followed by: and taking out the pretreated silk, washing in water at 25-30 ℃, and dehydrating.
4. The preparation method according to claim 1, wherein in the step A), the treatment time in the water at 65-95 ℃ is 1-8 h.
5. The method according to claim 1, wherein in step a), the ratio of silk to warm water is 1 g: (40-60) mL, wherein the ratio of the pretreated silk to water is 1 g: (40-60) mL.
6. The method according to claim 1, wherein the post-treatment drying step in step a) further comprises: washing the treated silk in water at 25-30 ℃ and then dehydrating; the drying is carried out by hot air at 40-60 ℃.
7. The method according to claim 1, wherein the temperature of the treatment in step B) is 50 to 80 ℃.
8. The preparation method according to claim 1, wherein the purification method is specifically:
and (3) filling the obtained silk fibroin sericin composite dissolved solution into a dialysis bag with the molecular weight cutoff of 10-50 kDa, and placing the dialysis bag in deionized water for continuous dialysis for 1-3 days.
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| US6175053B1 (en) * | 1997-06-18 | 2001-01-16 | Japan As Represented By Director General Of National Institute Of Sericultural And Entomological Science Ministry Of Agriculture, Forrestry And Fisheries | Wound dressing material containing silk fibroin and sericin as main component and method for preparing same |
| JP2001163899A (en) * | 1999-12-09 | 2001-06-19 | Natl Inst Of Sericultural & Entomological Science | Method for producing functional silk fibroin and its use |
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