CN1821267A - Method for refining sik fibroin - Google Patents
Method for refining sik fibroin Download PDFInfo
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- CN1821267A CN1821267A CN 200610049999 CN200610049999A CN1821267A CN 1821267 A CN1821267 A CN 1821267A CN 200610049999 CN200610049999 CN 200610049999 CN 200610049999 A CN200610049999 A CN 200610049999A CN 1821267 A CN1821267 A CN 1821267A
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- fibroin
- refining
- sik
- described step
- fimbrin
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Abstract
The present invention discloses the preparation process of refined fimbrin. The technological scheme includes the following steps: mixing the material and 0.2-6 mol/L sulfuric acid solution in the weight ratio of 0.01-0.1 and hydrolysis in 60-80deg.c for 2-48 hr; neutralizing the hydrlysate with alkali solution to pH 6.8-7.2, filtering the supernatant, concentrating the filtrate, drying and grinding to obtain the refined fimbrin powder. The refined fimbrin powder is used as the precursor for deep processing in regenerated fiber, food, cosmetics, biomedicine material and other fields. The process of the present invention obtains refined fimbrin powder product of molecular weight smaller than 4 KD, and adopts non-volatile sulfuric acid resulting in stable product quality.
Description
Technical field
The present invention relates to the reprocessing field of silky fibre, particularly a kind of method that fibrous fibroin or rough silk fibroin is become silk fibroin with the physico-chemical process processing and refining.
Background technology
Silk fibroin is a kind of natural protein that accounts for fibroin weight 70～80%, is the main body of silk, becomes silk through filature, after knitting processing such as silk fabric.The husks that produces in throwing and silk weaving production process, waste cocoon, soup cocoon, pelettes and waste silk etc., processing can obtain fibrous silk fibroin through coming unstuck.Fibrous fibroin by the neutral salt of high density (as CaCl
2, LiBr etc.) become the higher silk fibroin protein solution of purity after the dissolving, desalination.Thereby the recycling of the useless silk of realization etc.Silk fibroin can partly be degraded, and also can be degraded to amino acid or water-soluble oligopeptide.One of gordian technique that fibroin utilizes again is how to obtain satisfactory silk fibroin solution.And the size of molecular weight has determined the purposes of fibroin.Molecular weight can be used as the additive of makeup at thousands of fibroins; And when the fibroin hydrolysis becomes hundreds of oligopeptide of molecular weight or amino acid, just be suitable for foodstuffs industry.
Neutral salt is (as CaCl
2) preparation and obtain molecular weight at tens thousand of silk fibroins, still belong to high molecular weight protein; Basic hydrolysis causes racemization; A little less than the enzymic hydrolysis effect, need to adopt graded hydrolysis or with other method (as neutral salt etc.) combination, could obtain ideal effect; The hydrochloric acid hydrolysis main drawback is contaminate environment, etching apparatus, and the desalination difficulty; And phosphoric acid hydrolysis, though gentle, produce also relatively safety, the difficult control of quality.
Summary of the invention
At the deficiencies in the prior art part, the invention provides a kind of fibrous fibroin or promptly make the polymer silk fibroin becomes silk fibroin with the physico-chemical process processing and refining method.
The present invention includes following steps:
(1) be that 0.2～6mol/L sulphuric acid soln mixed by weight 1: 100～10: 100 with raw material and concentration, hydrolysis is 2～48 hours in 60～80 ℃ of water-baths;
(2) being neutralized to pH with alkaline solution after the hydrolysis is 6.8～7.2, gets supernatant liquid filtering, then filtrate is concentrated, dry, grind refined fimbrin powder.
Raw material in the described step (1) is that fibrous fibroin or other are promptly made the polymer silk fibroin.
Sulphuric acid soln concentration in the described step (1) is 2～6mol/L.
In the described step (2) alkaline solution be Ba (OH)
2Or Ca (OH)
2Alkaline solution.
As a kind of improvement of the present invention, used alkaline solution is Ba (OH)
2Alkaline solution.
Supernatant is filtered with the middling speed qualitative filter paper in the described step (2).
Concentrated in the described step (2) is in 40 ℃ of water-baths.
Drying in the described step (2) is under-56 ℃ of temperature.
Filtrate volume ml after concentrated finger concentrates in the described step (2) is 4: 1 with the ratio of raw material weight g.
The present invention also provides the application as the deep processing precursor in fields such as regenerated fibre, food, makeup, biomedical materials of this refining sik fibroin.
Advantage of the present invention: the product of gained of the present invention all be molecular weight less than the 4kD refining sik fibroin, used sulfuric acid does not have the volatility of hydrochloric acid, and constant product quality.
Description of drawings
Fig. 1 is in the embodiment of the invention under the different condition, H
2SO
4The SDS-PAGE figure of dissolving preparation silk fibroin;
Among Fig. 1: 1-6mol/LH
2SO
4, 60 ℃, 4h; 2-4mol/LH
2SO
4, 60 ℃, 6h; 3-2mol/L H
2SO
4, 70 ℃, 24h; 4-1mol/L H
2SO
4, 70 ℃, 24h; 5-0.6mol/L H
2SO
4, 70 ℃, 24h; 6-0.2mol/L H
2SO
4, 70 ℃, 24h;
Fig. 2 is the 2mol/L H of the embodiment of the invention
2SO
4The SDS-PAGE figure of dissolving preparation silk fibroin in the different time;
Among Fig. 2: 1-2mol/L H
2SO
4, 70 ℃, 4h; 2-2mol/L H
2SO
4, 70 ℃, 12h;
Fig. 3 obtains the SDS-PAGE figure of silk fibroin for the neutral salt preparation of the embodiment of the invention;
Among Fig. 3: M-protein standard molecular weight; 1-50%CaCl
22H
2The silk fibroin of O preparation; The silk fibroin of 2-10mol/L LiBr preparation.
Embodiment
Below the present invention is further described by example: percentage concentration is weight percent concentration in following examples.
Embodiment 1:
(1) with the husks 165g that produces in the fiber tow production process, joining 8250ml concentration is 0.5%Na
2CO
3In the solution;
(2) boil and come unstuck 30 minutes, use 60 ℃ of hot water injections then;
(3) repeating step (2) once obtains the fibrous fibroin of 100g.
Embodiment 2:
(1) with the leftover bits and pieces cocoon layer 140g that produces in the fiber tow production process, joining 7000ml concentration is 0.5%Na
2CO
3In the solution;
(2) boil and come unstuck 30 minutes, then with washing in 70 ℃ of hot water;
(3) repeating step (2) once obtains the fibrous fibroin of 100g.
Embodiment 3:
(1) with the soup cocoon, pelettes, the waste silk that produce in the fiber tow production process, or the waste silk 125g that produces in the silk weaving production process, joining 6250ml concentration is 0.5%Na
2CO
3In the solution;
(2) boil and come unstuck 30 minutes, then with washing in 80 ℃ of hot water;
(3) repeating step (2) once obtains the fibrous fibroin of 100g.
Embodiment 4:
Fibrous fibroin 100g is joined in the sulphuric acid soln that 1000mL concentration is 4mol/L, and hydrolysis obtained hydrolyzed solution in 4 hours in 70 ℃ water-bath.Hydrolyzed solution Ba (OH)
28H
2It is 6.8 that O is neutralized to the pH value, gets supernatant liquor then and filters with the middling speed qualitative filter paper, and filtrate is used rotatory evaporator, is concentrated into 400ml in 40 ℃ of water-baths, is placed in the refrigerator freezing more than 4 hours in-20 ℃ with concentrating good solution.Take out and put into freeze drier in-56 ℃ of dryings.Grind to form refined fimbrin powder with shredder again.Obtain refined fimbrin powder 88.5g.The SDS-PAGE detected result: the refined fimbrin powder molecular weight of acquisition is less than 4kD.
The yield of present embodiment refined fimbrin powder is 88.5%.
Embodiment 5:
To promptly make polymer silk fibroin powder 100g, joining 1000ml concentration is in the 2mol/L sulphuric acid soln, and hydrolysis is 24 hours in 60 ℃ water-bath; Then hydrolyzed solution Ba (OH)
28H
2It is 7.2 that O is neutralized to the pH value, gets supernatant liquor then and filters with the middling speed qualitative filter paper, and filtrate is used rotatory evaporator, is concentrated into 400ml in 40 ℃ of water-baths, and it is freezing more than 4 hours in-20 ℃ that concentrated good solution is placed into refrigerator.Take out and put into freeze drier in-56 ℃ of dryings.Grind to form refined fimbrin powder with shredder again.Obtain refined fimbrin powder 94g.The SDS-PAGE detected result: the refined fimbrin powder molecular weight of acquisition is less than 4kD.
The yield of present embodiment refined fimbrin powder is 94%.
Embodiment 6:
Fibrous fibroin 100g is joined in the sulphuric acid soln that 1000mL concentration is 6mol/L, and hydrolysis obtained hydrolyzed solution in 2 hours in 80 ℃ water-bath.Hydrolyzed solution Ba (OH)
28H
2It is 7 that O is neutralized to the pH value, gets supernatant liquor then and filters with the middling speed qualitative filter paper, and filtrate is used rotatory evaporator, is concentrated into 400ml in 40 ℃ of water-baths, is placed in the refrigerator freezing more than 4 hours in-20 ℃ with concentrating good solution.Take out and put into freeze drier in-56 ℃ of dryings, grind to form refined fimbrin powder with shredder again.Obtain refined fimbrin powder 58.5g.The SDS-PAGE detected result: the refined fimbrin powder molecular weight of acquisition is less than 4kD.
The yield of present embodiment refined fimbrin powder is 58.5%.
Embodiment 7:
Fibrous fibroin 10g is joined in the sulphuric acid soln that 1000mL concentration is 0.2mol/L, and hydrolysis obtained hydrolyzed solution in 48 hours in 70 ℃ water-bath.Hydrolyzed solution Ba (OH)
28H
2It is 7 that O is neutralized to the pH value, gets supernatant liquor then and filters with the middling speed qualitative filter paper, and filtrate is used rotatory evaporator, is concentrated into 40ml in 40 ℃ of water-baths, is placed in the refrigerator freezing more than 4 hours in-20 ℃ with concentrating good solution.Take out and put into freeze drier in-56 ℃ of dryings, grind to form refined fimbrin powder with shredder again.Obtain refined fimbrin powder 7.38g.The SDS-PAGE detected result: the refined fimbrin powder molecular weight of acquisition is less than 4kD.
The yield of present embodiment refined fimbrin powder is 73.8%.
Embodiment 8:
With fibrous fibroin 100g, putting into 1000ml concentration is 50%CaCl
22H
2In the O solution, boil dissolving 5 minutes.With the lysate specification of packing into is in the dialysis tubing of 8-12ku, and dialysis obtained neutral salt throwing fibroin solution after 3 days in distilled water, was concentrated into 400ml, and joining 600ml concentration is in the 2mol/L sulphuric acid soln, and hydrolysis is 24 hours in 60 ℃ water-bath; Then hydrolyzed solution Ba (OH)
28H
2It is 7 that O is neutralized to the pH value, gets supernatant liquor then and filters with the middling speed qualitative filter paper, and filtrate is used rotatory evaporator, is concentrated into 400ml in 40 ℃ of water-baths, and it is freezing more than 4 hours in-20 ℃ that concentrated good solution is placed into refrigerator.Take out and put into freeze drier in-56 ℃ of dryings.Grind to form refined fimbrin powder with shredder again.Obtain silk fibroin powder 70.8g.The SDS-PAGE detected result: the silk fibroin powder molecular weight of acquisition is greater than 20kD.
The yield of present embodiment macromolecule silk fibroin powder is 70.8%.
In the foregoing description 4～8, the drying of silk fibroin protein solution has adopted freeze-drying method.If when the silk fibroin protein solution amount of preparation is big, can adopt spray drying process.Specifically be with the silk fibroin protein solution after concentrating, be placed into and carry out drying in the spray-drier.Inlet temperature is controlled at 80～85 ℃, and temperature out is controlled at 170～185 ℃.
With reference to Fig. 1: fibrous fibroin is at 6mol/L H
2SO
4In in 60 ℃ of hydrolysis 4 hours; At 4mol/LH
2SO
4In in 60 ℃ of hydrolysis 6 hours; At 2mol/L H
2SO
4In in 70 ℃ of hydrolysis 24 hours; At 1mol/LH
2SO
4In in 70 ℃ of hydrolysis 24 hours; At 0.6mol/L H
2SO
4In in 70 ℃ of hydrolysis 24 hours; At 0.2mol/L H
2SO
4In in 70 ℃ of hydrolysis 24 hours; The SDS-PAGE figure of dissolving preparation silk fibroin.
With reference to Fig. 2: fibrous fibroin is at 2mol/L H
2SO
4In in 70 ℃ of hydrolysis 4 hours; At 2mol/LH
2SO
4In in 70 ℃ of hydrolysis 12 hours; The SDS-PAGE figure of dissolving preparation silk fibroin.
By Fig. 1 and Fig. 2 as can be known, H
2SO
4The silk fibroin solution that hydrolysis prepares is used different concns H with the molecular weight that SDS-PAGE detects silk fibroin
2SO
4The hydrolysis fibroin, under different temperature, the time that hydrolysis is different, or the H of same concentration
2SO
4, under the same temperature, the hydrolysis different time, the maximum molecular weight of silk fibroin all is 4kD in the resulting silk fibroin solution, having obtained molecular weight is the following refined fimbrin powder of 4kD.
With reference to Fig. 3: fibrous fibroin is 50%CaCl in concentration
22H
2Among the O, be among the 10mol/L LiBr in concentration, the SDS-PAGE figure of dissolving preparation silk fibroin.
As shown in Figure 3, with the silk fibroin SDS-PAGE detection molecules amount that different neutral salt prepares, the result shows that the molecular weight of the silk fibroin that obtains is more than 20kD.
The average digestibility of silk fibroin protein solution is 47.4%, and the average digestibility of silk fibroin powder is 27.6%.Illustrate that silk fibroin can be absorbed by human consumption.Silk fibroin has the ethanol concn that reduces in the blood, the effect that reduces cholesterol in the blood, can promote the effect such as synthetic of insulin secretion and Dopamine HCL, so, can be used as the raw material of health functional food.As according to a certain percentage with refined fimbrin powder, mix with auxiliary materials such as dextrin, make health functional food.And for example silk fibroin has nutrition, preserves moisture and function such as oxidation-resistance, can be used as the raw material of high-grade skin protection cosmetics, makes protective skin cream, Cosmetic pack, shampoo and body wash etc.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (10)
1, a kind of method of refining sik fibroin, its feature may further comprise the steps successively:
(1) be that 0.2～6mol/L sulphuric acid soln mixed by weight 1: 100～10: 100 with raw material and concentration, hydrolysis is 2～48 hours in 60～80 ℃ of water-baths;
(2) being neutralized to pH with alkaline solution after the hydrolysis is 6.8～7.2, gets supernatant liquid filtering, then filtrate is concentrated, dry, grind refined fimbrin powder.
2, the method for refining sik fibroin according to claim 1 is characterized in that: the raw material in the described step (1) is that fibrous fibroin or other are promptly made the polymer silk fibroin.
3, the method for refining sik fibroin according to claim 1 is characterized in that: the sulphuric acid soln concentration in the described step (1) is 2～6mol/L.
4, the method for refining sik fibroin according to claim 1 is characterized in that: in the described step (2) alkaline solution be Ba (OH)
2Or Ca (OH)
2Alkaline solution.
5, according to the method for claim 1 or 4 described refining sik fibroins, it is characterized in that: used alkaline solution is Ba (OH)
2Alkaline solution.
6, the method for refining sik fibroin according to claim 1 is characterized in that: supernatant is filtered with the middling speed qualitative filter paper in the described step (2).
7, the method for refining sik fibroin according to claim 1 is characterized in that: concentrated in the described step (2) is in 40 ℃ of water-baths.
8, the method for refining sik fibroin according to claim 1 is characterized in that: the drying in the described step (2) is under-56 ℃ of temperature.
9, the method for refining sik fibroin according to claim 1 is characterized in that: the filtrate volume ml after concentrated finger concentrates in the described step (2) is 4: 1 with the ratio of raw material weight g.
10, the refining sik fibroin of the described method gained of claim 1, it is characterized in that: this refining sik fibroin is the application as the deep processing precursor in fields such as regenerated fibre, food, makeup, biomedical materials.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610049999 CN1821267A (en) | 2006-03-24 | 2006-03-24 | Method for refining sik fibroin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610049999 CN1821267A (en) | 2006-03-24 | 2006-03-24 | Method for refining sik fibroin |
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|---|---|
| CN1821267A true CN1821267A (en) | 2006-08-23 |
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ID=36922856
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1985791B (en) * | 2006-12-27 | 2010-05-19 | 浙江大学 | Preparing process of skin care silk protein gel cream |
| CN102219844A (en) * | 2011-04-02 | 2011-10-19 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN103816105A (en) * | 2014-03-12 | 2014-05-28 | 西南大学 | Complete multifunctional hand cream and preparation method thereof |
| CN103897021A (en) * | 2013-02-04 | 2014-07-02 | 中国科学院上海有机化学研究所 | Silk peptide as well as preparation method and application thereof |
| CN108244657A (en) * | 2018-01-12 | 2018-07-06 | 海南蛛王药业有限公司 | A kind of method for hydrolysis of spider silk |
-
2006
- 2006-03-24 CN CN 200610049999 patent/CN1821267A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1985791B (en) * | 2006-12-27 | 2010-05-19 | 浙江大学 | Preparing process of skin care silk protein gel cream |
| CN102219844A (en) * | 2011-04-02 | 2011-10-19 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN102219844B (en) * | 2011-04-02 | 2013-04-17 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN103897021A (en) * | 2013-02-04 | 2014-07-02 | 中国科学院上海有机化学研究所 | Silk peptide as well as preparation method and application thereof |
| CN103897021B (en) * | 2013-02-04 | 2016-06-29 | 中国科学院上海有机化学研究所 | Silk peptide, preparation method and application |
| CN103816105A (en) * | 2014-03-12 | 2014-05-28 | 西南大学 | Complete multifunctional hand cream and preparation method thereof |
| CN103816105B (en) * | 2014-03-12 | 2015-12-02 | 西南大学 | A kind of full effect Multifunctional handguard frost and preparation method thereof |
| CN108244657A (en) * | 2018-01-12 | 2018-07-06 | 海南蛛王药业有限公司 | A kind of method for hydrolysis of spider silk |
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Open date: 20060823 |