CN102532253A - Method for extracting silk protein - Google Patents
Method for extracting silk protein Download PDFInfo
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- CN102532253A CN102532253A CN2012100381752A CN201210038175A CN102532253A CN 102532253 A CN102532253 A CN 102532253A CN 2012100381752 A CN2012100381752 A CN 2012100381752A CN 201210038175 A CN201210038175 A CN 201210038175A CN 102532253 A CN102532253 A CN 102532253A
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Abstract
The invention relates to a method for extracting a silk protein. At present, a method for extracting the silk protein, in which the extraction condition is mild and which facilitates reducing the influence of the extraction condition on the structure and performance of the silk protein, is not provided. The method comprises the following steps of: placing the raw materials for preparing the silk protein in a CaCl2 aqueous solution at the temperature of between 95 and 100 DEG C, and dissolving until an undissolved part is jelly to obtain a dissolution treatment solution; filtering the dissolution treatment solution by using filter paper to obtain a silk protein/CaCl2 mixed aqueous solution and the undissolved jelly; washing the undissolved jelly to obtain silk protein colloid; and/or placing the silk protein/CaCl2 mixed aqueous solution in a dialysis bag and dialyzing and purifying to obtain a silk protein solution; and processing the silk protein colloid and/or the silk protein solution to obtain a finished product silk protein. According to the method, the extraction condition is mild, the extraction efficiency of the silk protein is improved conveniently, the waste of the silk protein resources is reduced, and the influence of the extraction condition on the structure and performance on the silk protein is reduced.
Description
Technical field
The present invention relates to a kind of process for extracting of fibroin, belong to a kind of complete processing of fibroin.
Background technology
Silk mainly is made up of silk gum and two kinds of protein of fibroin, is generically and collectively referred to as fibroin.Silk gum is enclosed in the fibroin fiber top layer, plays adhesive action.Silk gum and fibroin have various structure and performance: silk gum is globular preteins, and secondary structure is main with random coil, and poor stability dissolves in the solution such as hot water, acid, alkali, proteolytic enzyme and lower concentration yellow soda ash; Fibroin is a fibrous protein, contains a large amount of crystalline texture, and stable performance dissolves in high calcium chloride concentration (CaCl
2), calcium chloride ethanol water (CaCl
2/ CH
3CH
2OH/H
2O) in mixed solution and the lithiumbromide solution such as (LiBr).Therefore; Can be according to solvability difference and the practical application request of fibroin in different solutions; Select suitable lysate to extract processing; Obtain various fibroins, be used for different shape functional materialss such as processing and preparing makeup and foodstuff additive, biological tissue's repair materials, high-absorbent material, pharmaceutical carrier and cell culture medium with different structure and performance.
At present, traditional fibroin extracts thinking and is: 1. cocoon layer or silk are handled and purifying with silk gum lysate (like boiling water, HTHP, acid, alkali, liquid of protease etc.), obtained part or most of sericin; 2. earlier with cocoon layer or silk with the silk gum lysate (like 0.5% w/w Na
2CO
3Solution etc.) processing of coming unstuck fully, fibroin fiber; Then, with this fibroin fiber with the fibroin lysate (like 50% w/w CaCl
2Solution etc.) processing, dialysis purifying obtain silk fibroin.
For example open day is on 03 11st, 2009, and publication number is in the Chinese patent of CN101381758, discloses a kind of method by silk extraction silk peptide, and this method comprises following process: silk is added Na
2CO
3After the refining, leach silk and again it is added CaCl in the solution
2Dissolve in the solution, the molten silk liquid that obtains after the dissolving carries out enzymatic hydrolysis reaction with proteolytic enzyme, hydrolyzed solution after ultrafiltration and nanofiltration separation, the spray-dried silk peptide powder that obtains of the feed liquid that obtains.And for example open day is on 02 16th, 2011; Publication number is in the Chinese patent of CN101974596A; A kind of process for extracting of silk peptide is disclosed; It is main raw material that this method adopts useless silk, through removal of impurities, water distribution and interpolation S-WAT intensification boiling, filtration, protease hydrolyzed, the enzyme that goes out, decolouring, membrane sepn, the filtrating that obtains is promptly obtained silk peptide after drying.These traditional fibroin process for extracting exist that fibroin is prone to degrade (the particularly extraction of sericin), leaching process complicated (obtaining silk fibroin need proceed in two phases), wasting of resources shortcomings such as (the come unglued liquid degraded severity in the fibroin leaching process, more difficult purifying and utilizations).
Also do not have at present a kind of technology is simple, extraction conditions is gentle, help improving fibroin extraction efficiency, reduce the fibroin wasting of resources, alleviate the process for extracting of extraction conditions the fibroin of fibroin structure and performance impact.
Summary of the invention
The objective of the invention is to overcome the above-mentioned deficiency that exists in the prior art, and provide that a kind of technology is simple, extraction conditions is gentle, help improving fibroin extraction efficiency, reduce the fibroin wasting of resources, alleviate the process for extracting of extraction conditions the fibroin of fibroin structure and performance impact.
The present invention addresses the above problem the technical scheme that is adopted: the characteristics of the process for extracting of this fibroin are: this process for extracting in turn includes the following steps:
(1), is cut into fragment or the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material with the cocoon layer of silk cocoon stripping lamellar or with the cocoon layer of silk cocoon;
(2), it is 95-100 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid;
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly;
(4), with dissolving jelly water cleaning many times in the step (3), removing adherent calcium chloride molecule, thereby make the fibroin colloid; And/or with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution;
(5), the fibroin colloid that makes in the step (4) and/or fibroin solution are processed and made the finished product fibroin.The present invention adopts and directly places calcium chloride water to extract processing the fibroin raw material; Can obtain two kinds of fibroins simultaneously with different structure and performance; Promptly can obtain fibroin colloid and fibroin solution simultaneously; Fibroin colloidal composition is the sericin colloid among the present invention, and the staple of fibroin solution is a silk fibroin.The situation that fibroin is prone to degraded can not take place in fibroin of the present invention in leaching process; Leaching process is simple; Only need to obtain the fibroin solution that silk fibroin is the master, improved the fibroin utilization ratio of raw materials greatly, make fibroin be convenient to purifying and utilization once going on foot.
As preferably, in the step according to the invention (5), the fibroin colloid that makes in the step (4) and/or fibroin solution are processed and made Powdered, membranaceous, porous support shape or fibrous finished product fibroin.Make that thus the applicable surface of the finished product fibroin that the present invention is prepared from is wider.
As preferably, the silk in the step according to the invention (1) comprises cocoon layer silk, husks and pelettes.
As preferably, the mass concentration of calcium chloride is 50% (w/w) in the step according to the invention (2), and bath raio is 1:25.Make the present invention better thus to the solute effect of fibroin raw material.
As preferably, in the step according to the invention (4), it is three times with the number of times that does not dissolve the cleaning of jelly water in the step (3).Thereby make that preparation fibroin colloidal technology is simpler, the fibroin colloidal performance that is prepared from is more reliable.
As preferably, in the step according to the invention (4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 3 days.It is simpler to make that thus the present invention prepares the technology of fibroin solution, and the performance of the fibroin solution that is prepared from is more reliable.
As preferably, in the step according to the invention (3), the dissolution process liquid that step (2) makes is used filter paper filtering, with the less water flushing, filtering separation obtains fibroin/CaCl then
2Mixed aqueous solution and molten jelly.Make fibroin/CaCl among the present invention thus
2Mixed aqueous solution can separate with not molten jelly more fully.
The present invention compared with prior art, have the following advantages and effect: extraction process is simple, extraction conditions is gentle, helps improving the extraction efficiency of fibroin, reduces the waste of fibroin resource, alleviates extraction conditions to fibroin structure and Effect on Performance.The present invention extracts the finished product fibroin that forms and can be used for being processed into variform functional materialss such as solution, powder, support, film, gel and regenerated fibre, and applicable surface is very wide, and new thinking and method can be provided for the processing and utilization of fibroin.
The present invention adopts solution such as the fibroin raw material directly being placed calcium chloride to extract processing; Can obtain two kinds of fibroins simultaneously with different structure and performance; Promptly can obtain fibroin colloid and fibroin solution simultaneously, fibroin colloidal composition is the sericin colloid among the present invention, and the staple of fibroin solution is a silk fibroin; Make that whole extraction process is simple, extraction efficiency is high.
Description of drawings
Fig. 1 is the SDS-PAGE synoptic diagram of finished product fibroin in the embodiment of the invention 2.
Embodiment
Below in conjunction with accompanying drawing and through embodiment the present invention is done further detailed description, following examples are to explanation of the present invention and the present invention is not limited to following examples.
Embodiment 1.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 0.5g silk cocoon, the cocoon layer of silk cocoon is shelled to become to approach cocoon layer and make the fibroin raw material.The present invention can be cut into fragment or the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material with the cocoon layer of silk cocoon stripping lamellar or with the cocoon layer of silk cocoon, and wherein, used silk comprises cocoon layer silk, husks and pelettes.
(2), it is that 98 ℃ mass concentration is the CaCl of 50% (w/w) that the fibroin raw material that step (1) is made places temperature
2Dissolve CaCl in the aqueous solution
2The aqueous solution keeps slight boiling condition, and bath raio is 1:25, carries out about dissolution process 5min, does not dissolve part until fibroin and is jelly, thereby make dissolution process liquid.It is 95-100 ℃ CaCl that the present invention can place the fibroin raw material that step (1) makes temperature
2Dissolving is several minutes in the aqueous solution, normally dissolves 0.5-5 minute, does not dissolve part until the fibroin raw material and is jelly, thereby make dissolution process liquid.The mass concentration of the calcium chloride that the present invention is used is generally 35%-60% (w/w), is preferably 45%-55% (w/w), and the best is 50% (w/w); Bath raio is generally 1: (20-30), be preferably 1: (22-28), the best is 1:25.Temperature is 95-100 ℃ CaCl among the present invention
2The aqueous solution is in slight boiling condition.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and uses a small amount of pure water rinsing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with dissolving jelly water cleaning many times in the step (3), removing adherent calcium chloride molecule, thereby make the fibroin colloid, fibroin colloidal composition is the sericin colloid in the present embodiment.Water of the present invention clean do not dissolve jelly number of times usually at 2-6 time, preferred three times.The present invention also can be with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution, and the time of dialysis is generally 2-6 days, is preferably 3 days.The staple of fibroin solution is a silk fibroin among the present invention.
(5), with the processing of the fibroin colloid that makes in the step (4) and make the finished product fibroin, that this fibroin colloid can be processed into is Powdered, membranaceous, porous support shape or fibrous finished product fibroin.
Embodiment 2.
Referring to Fig. 1, the process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 0.5g silk cocoon, with the cocoon layer stripping lamellar of this silk cocoon and make the fibroin raw material.
(2), it is 96 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, the mass concentration of calcium chloride is 50% (w/w), and bath raio is 1:25, about dissolution process 5min, does not dissolve part until the fibroin raw material and is jelly, thereby make dissolution process liquid.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and uses a small amount of pure water rinsing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis purifying is 3 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.Fibroin colloidal composition is the sericin colloid in the present embodiment.
(5), the fibroin solution that makes in the step (4) is processed and made the finished product fibroin, that the fibroin solution among the present invention can be processed into is Powdered, membranaceous, porous support shape or fibrous finished product fibroin.The finished product fibroin content that present embodiment is prepared from has the polypeptide of higher molecular weight (greater than 120KD) in a large number, and major ingredient is a silk fibroin, and accompanying drawing 1 is the SDS-PAGE synoptic diagram of the finished product fibroin that is prepared from of present embodiment.
Embodiment 3.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 1g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 100 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, about dissolving 5min, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 50% (w/w) in the present embodiment, and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and uses a small amount of pure water rinsing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with dissolving jelly water cleaning many times in the step (3), removing adherent calcium chloride molecule, thereby make the fibroin colloid.Fibroin colloidal composition is the sericin colloid in the present embodiment.
(5), the fibroin colloid that makes in the step (4) is tiled in the plastic culture dish; Place seasoning under the room temperature; Obtain the finished product fibroin of fibroin film, thereby the fibroin colloid processing that makes in the step (4) is made the finished product fibroin.
Embodiment 4.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 1g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 97 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, about dissolving 5min, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 50% (w/w) in the present embodiment, and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and uses a small amount of pure water rinsing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 3 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.The staple of fibroin solution is a silk fibroin in the present embodiment.
(5), the fibroin solution that makes in the step (4) is poured in the plastic culture dish; Place seasoning under the room temperature; Obtain the finished product fibroin of fibroin film, the fibroin solution that makes in the step (4) is processed and made the purpose of finished product fibroin thereby reach.
Embodiment 5.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 5g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 98 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, about dissolving 5min, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 50% (w/w) in the present embodiment, and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and uses a small amount of pure water rinsing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), take off and dissolve jelly on the filter paper; Use the pure water cleaning many times, be about to the not molten jelly water cleaning many times in the step (3), to remove adherent calcium chloride molecule; Thereby make the fibroin colloid, this fibroin colloidal composition is the sericin colloid.
(5), the fibroin colloid that makes in the step (4) is tiled in the plastic culture dish; Place under-20 ℃ the condition and be refrigerated to complete glaciation; It is dry to put into freeze drier then, thereby obtains the finished product fibroin of fibroin powder, powder is weighed again.Calculate the yield of fibroin powder according to following formula: yield (%)=protein powder weight/cocoon layer weight * 100%, the yield of fibroin powder is 25.45 ± 3.7% in the present embodiment.
Embodiment 6.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 5g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 98 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, about dissolving 5min, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 50% (w/w) in the present embodiment, and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and uses a small amount of pure water rinsing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), take out filtrating, place dialysis tubing, with pure water dialysis purifying 3 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution gets fibroin solution, is about to the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying and makes fibroin solution, and the staple of this fibroin solution is a silk fibroin.
(5), the fibroin solution that makes in the step (4) is poured in the plastic culture dish; Then plastic culture dish is placed under-20 ℃ the condition and be refrigerated to complete glaciation; Again this plastic culture dish is put into freeze drier and carry out drying; Thereby obtain the finished product fibroin of fibroin powder, again the fibroin powder is weighed.Calculate the yield of fibroin powder according to following formula: yield (%)=protein powder weight/cocoon shell weight * 100%.The yield of fibroin powder is 66.32 ± 6.4% in the present embodiment.
Embodiment 7.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 10g silk cocoon, the cocoon layer of silk cocoon is cut into fragment and makes the fibroin raw material.
(2), it is 99 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after several minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 35% (w/w), and bath raio is 1:20.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.When the present invention uses filter paper filtering at the dissolution process liquid that step (2) is made, also can wash with less water, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), the jelly water that dissolves in the step (3) is cleaned 4 times, removing adherent calcium chloride molecule, thereby make the fibroin colloid, this fibroin colloidal composition is the sericin colloid; And with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 3 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution, and the staple of this fibroin solution is a silk fibroin.
(5), the fibroin colloid that makes in the step (4) and fibroin solution are processed and made the finished product fibroin, this finished product fibroin can be Powdered, membranaceous, porous support shape or fibrous.
Embodiment 8.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 20g silk cocoon, the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material, this silk comprises cocoon layer silk, husks and pelettes.
(2), it is 98 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after several minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 40% (w/w), and bath raio is 1:22.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be five times, removing adherent calcium chloride molecule, thereby make the fibroin colloid, this fibroin colloidal composition is the sericin colloid.
(5), the fibroin colloid that makes in the step (4) is processed and made the finished product fibroin, the finished product fibroin can be Powdered, membranaceous, porous support shape or fibrous.
Embodiment 9.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 8g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 95 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 4 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 55% (w/w), and bath raio is 1:26.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and with the less water flushing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be six times, removing adherent calcium chloride molecule, thereby make the fibroin colloid, this fibroin colloidal composition is the sericin colloid; With the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 5 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution, and the staple of this fibroin solution is a silk fibroin.
(5), the fibroin colloid that makes in the step (4) and fibroin solution are processed and made the finished product fibroin.
Embodiment 10.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 15g silk cocoon, the cocoon layer of silk cocoon is cut into fragment and makes the fibroin raw material.
(2), it is 99 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 3 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 45% (w/w), and bath raio is 1:28.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 2 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution, and the staple of this fibroin solution is a silk fibroin.
(5), the fibroin solution that makes in the step (4) processed and made the finished product fibroin, this finished product fibroin is the porous support shape.
Embodiment 11.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 4g silk cocoon, the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material.
(2), it is 96 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 4 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 52% (w/w), and bath raio is 1:24.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and with the less water flushing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be three times, removing adherent calcium chloride molecule, thereby make the fibroin colloid, this fibroin colloidal composition is the sericin colloid.
(5), the fibroin colloid that makes in the step (4) is processed and made fibrous finished product fibroin.
Embodiment 12.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 3g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 97 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 2 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 49% (w/w), and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 4 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.
(5), the fibroin solution that makes in the step (4) is processed and made fibrous finished product fibroin.
Embodiment 13.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 12g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 99 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 30 seconds, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 53% (w/w), and bath raio is 1:22.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be three times, removing adherent calcium chloride molecule, thereby make the fibroin colloid.
(5), the fibroin colloid that makes in the step (4) is made pulverous finished product fibroin.
Embodiment 14.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 18g silk cocoon, the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material, this silk comprises cocoon layer silk, husks and pelettes.
(2), it is 98 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 4 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 35% (w/w), and bath raio is 1:20.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and with the less water flushing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 6 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.
(5), the fibroin solution that makes in the step (4) is processed and made fibrous finished product fibroin.
Embodiment 15.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 25g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 98 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 1 minute, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 50% (w/w), and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be four times, removing adherent calcium chloride molecule, thereby make the fibroin colloid; And with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 4 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.
(5), the fibroin colloid that makes in the step (4) processed and made the finished product fibroin of porous support shape, the fibroin solution that makes in the step (4) processed and made the finished product fibroin of porous support shape.
Embodiment 16.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 6g silk cocoon, the cocoon layer of silk cocoon is cut into fragment and makes the fibroin raw material.
(2), it is 97 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after several minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 48% (w/w), and bath raio is 1:26.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and with the less water flushing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 5 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.
(5), the fibroin solution that makes in the step (4) is processed and made the finished product fibroin of porous support shape.
Embodiment 17.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 30g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 95 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after several minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 53% (w/w), and bath raio is 1:26.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be three times, removing adherent calcium chloride molecule, thereby make the fibroin colloid.
(5), the fibroin colloid that makes in the step (4) is processed and made fibrous finished product fibroin.
Embodiment 18.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), takes by weighing the cocoon layer of 7g silk cocoon, with the cocoon layer stripping lamellar of silk cocoon and make the fibroin raw material.
(2), it is 98 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 3 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 38% (w/w), and bath raio is 1:29.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be three times, removing adherent calcium chloride molecule, thereby make the fibroin colloid; And with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 5 days, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution.
(5), the fibroin colloid that makes in the step (4) processed and made membranaceous finished product fibroin, the fibroin solution that makes in the step (4) processed and made membranaceous finished product fibroin.
Embodiment 19.
The process for extracting of fibroin in turn includes the following steps in the present embodiment.
(1), take by weighing the cocoon layer of 100g silk cocoon, the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material, this silk comprises cocoon layer silk, husks and pelettes.
(2), it is 99 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, dissolve after 4 minutes, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid.The mass concentration of used calcium chloride is 50% (w/w), and bath raio is 1:25.
(3), dissolution process liquid that step (2) is made uses filter paper filtering, and with the less water flushing, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly.
(4), with in the step (3) to dissolve the number of times that the jelly water cleans be three times, removing adherent calcium chloride molecule, thereby make the fibroin colloid.
(5), the fibroin colloid that makes in the step (4) processed and made the finished product fibroin, this finished product fibroin is the porous support shape.
Though the present invention with embodiment openly as above; But it is not in order to limit protection scope of the present invention; Any technician who is familiar with this technology, change and the retouching in not breaking away from design of the present invention and scope, done all should belong to protection scope of the present invention.
Claims (7)
1. the process for extracting of a fibroin, it is characterized in that: this process for extracting in turn includes the following steps:
(1), is cut into fragment or the silk of silk cocoon is cut into staple fibre and makes the fibroin raw material with the cocoon layer of silk cocoon stripping lamellar or with the cocoon layer of silk cocoon;
(2), it is 95-100 ℃ CaCl that the fibroin raw material that step (1) is made places temperature
2Dissolve in the aqueous solution, do not dissolve part until the fibroin raw material and be jelly, thereby make dissolution process liquid;
(3), dissolution process liquid that step (2) is made uses filter paper filtering, filtering separation obtains fibroin/CaCl
2Mixed aqueous solution and molten jelly;
(4), with dissolving jelly water cleaning many times in the step (3), removing adherent calcium chloride molecule, thereby make the fibroin colloid; And/or with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, to remove fibroin/CaCl
2Calcium chloride molecule in the mixed aqueous solution, thus make fibroin solution;
(5), the fibroin colloid that makes in the step (4) and/or fibroin solution are processed and made the finished product fibroin.
2. the process for extracting of fibroin according to claim 1; It is characterized in that: in the said step (5), the fibroin colloid that makes in the step (4) and/or fibroin solution are processed and made Powdered, membranaceous, porous support shape or fibrous finished product fibroin.
3. the process for extracting of fibroin according to claim 1 and 2, it is characterized in that: the silk in the said step (1) comprises cocoon layer silk, husks and pelettes.
4. the process for extracting of fibroin according to claim 1 and 2 is characterized in that: the mass concentration of calcium chloride is 50% (w/w) in the said step (2), and bath raio is 1:25.
5. the process for extracting of fibroin according to claim 1 and 2 is characterized in that: in the said step (4), be three times with the number of times that does not dissolve the cleaning of jelly water in the step (3).
6. the process for extracting of fibroin according to claim 1 and 2 is characterized in that: in the said step (4), with the fibroin/CaCl in the step (3)
2Mixed aqueous solution places dialysis tubing water dialysis purifying, and the time of dialysis is 3 days.
7. the process for extracting of fibroin according to claim 1 and 2 is characterized in that: in the said step (3), the dissolution process liquid that step (2) makes is used filter paper filtering, with the less water flushing, filtering separation obtains fibroin/CaCl then
2Mixed aqueous solution and molten jelly.
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| CN102731796A (en) * | 2012-07-06 | 2012-10-17 | 广西工学院 | Preparation method of protein-based surfactant fibroin acid calcium |
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| CN104592376A (en) * | 2014-12-29 | 2015-05-06 | 无锡蚕魅科技有限公司 | Extraction method of fibroin |
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| CN105017382A (en) * | 2015-08-14 | 2015-11-04 | 海安县南莫制丝有限公司 | Method for preparing soluble silk protein solution through waste silk |
| CN106596820A (en) * | 2016-12-12 | 2017-04-26 | 浙江理工大学 | Method for analyzing influence of carbon-nanotube-reinforced silk on fibroin amino acid sequences based on proteomics |
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| CN102731796A (en) * | 2012-07-06 | 2012-10-17 | 广西工学院 | Preparation method of protein-based surfactant fibroin acid calcium |
| CN104447973A (en) * | 2014-11-24 | 2015-03-25 | 浙江大学 | Method for extracting silk gland silk protein |
| CN104447973B (en) * | 2014-11-24 | 2017-06-30 | 浙江大学 | A kind of extracting method of sericterium fibroin |
| CN104592376B (en) * | 2014-12-29 | 2018-02-27 | 无锡蚕魅科技有限公司 | A kind of extracting method of fibroin |
| CN104592376A (en) * | 2014-12-29 | 2015-05-06 | 无锡蚕魅科技有限公司 | Extraction method of fibroin |
| CN104761616A (en) * | 2015-04-15 | 2015-07-08 | 海安县南莫制丝有限公司 | Method for preparing soluble silk protein liquid employing waste silk |
| CN105017382A (en) * | 2015-08-14 | 2015-11-04 | 海安县南莫制丝有限公司 | Method for preparing soluble silk protein solution through waste silk |
| CN106596820A (en) * | 2016-12-12 | 2017-04-26 | 浙江理工大学 | Method for analyzing influence of carbon-nanotube-reinforced silk on fibroin amino acid sequences based on proteomics |
| CN106860189A (en) * | 2017-03-23 | 2017-06-20 | 佛山圣婕妮生物科技有限公司 | A kind of zero-emission Hydrolyze method extracts the method for making protein surface activating agent |
| CN107982079A (en) * | 2017-12-01 | 2018-05-04 | 黄彬彬 | A kind of haze amino acid is without silicone oil shampoo and preparation method thereof |
| CN108498861A (en) * | 2018-06-28 | 2018-09-07 | 河南省肿瘤医院 | A kind of preparation method of hydroxyapatite-fibroin-titanium dioxide biomaterial |
| CN114504546A (en) * | 2020-11-16 | 2022-05-17 | 西南大学 | Preparation method of sericin nanogel |
| CN114504546B (en) * | 2020-11-16 | 2024-02-02 | 西南大学 | Preparation method of sericin nanogel |
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