CN113621093A - Method for removing protein in chitosan through electrophoresis - Google Patents
Method for removing protein in chitosan through electrophoresis Download PDFInfo
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- CN113621093A CN113621093A CN202110950967.6A CN202110950967A CN113621093A CN 113621093 A CN113621093 A CN 113621093A CN 202110950967 A CN202110950967 A CN 202110950967A CN 113621093 A CN113621093 A CN 113621093A
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The invention discloses a method for removing protein in chitosan through electrophoresis. Adding chitosan powder into the acidic aqueous solution, and stirring to completely dissolve the chitosan powder to obtain a chitosan acidic aqueous solution; adding alkali into the obtained chitosan acidic aqueous solution, and stirring to separate out chitosan to obtain chitosan alkaline dispersion liquid; adding sodium dodecyl sulfate into the obtained chitosan alkaline dispersion liquid, stirring until the sodium dodecyl sulfate is completely dissolved, and standing for 2 hours; filtering, collecting the filter residue, placing the obtained filter residue into an electrophoresis device, and adding electrophoresis liquid prepared by sodium dodecyl sulfate for electrophoresis; and collecting and filtering the chitosan after electrophoresis, washing with deionized water, and drying to obtain the purified chitosan. The invention removes protein in chitosan by an electrophoresis method, and can ensure that the chitosan reaches the medical standard. Compared with the traditional subtraction method, the method has the advantages of low cost, environmental protection and huge application potential in the medical field.
Description
Technical Field
The invention belongs to the field of medical high polymer materials, relates to a chitosan purification technology, and particularly relates to a method for removing protein in chitosan through electrophoresis.
Background
Chitosan is a natural high molecular alkaline polysaccharide, which not only has good biocompatibility and degradability, but also has the characteristics of bacteriostasis, hemostasis, wound tissue growth promotion and the like, and the excellent properties enable the chitosan to have wide application in the medical field. Because of the immunological rejection of organisms, chitosan used in the medical field needs to be deproteinized, and the residual amount of chitosan protein in the chitosan product of tissue engineering medical instruments (YY/T1699-. The commonly used deproteinization methods mainly include an alkaline method, an enzymatic method and a microbial fermentation method.
The alkali method is the most used method for removing protein from chitosan at present, and usually, high-concentration alkali is used for repeatedly treating chitosan under the condition of high temperature so as to achieve the purpose of removing protein. For example, patent CN110746520A discloses a novel efficient preparation method for chitosan production, which comprises soaking shrimp shell powder in 10 wt% NaOH solution at 40-60 deg.C for 20-24h, and treating twice to remove protein. Although the alkaline method has simple production process and can obtain better deproteinization effect, a large amount of waste water and waste residue generated in the treatment process can cause environmental damage, and the treatment of the waste water and the waste residue can also obviously increase the production cost. In order to solve the problems of the alkaline method, many scholars develop various auxiliary treatment modes, and the auxiliary means such as ultrasound, microwave and the like are utilized to improve the alkaline treatment efficiency and reduce the dosage of alkaline liquor in the deproteinization process. For example, patent CN106995504A discloses a method for extracting chitin from shrimp shells by ultrasonic oscillation and electrical heating, wherein ultrasonic oscillation is used in the deproteinization process, so that the deproteinization rate can be significantly increased. However, the auxiliary means not only increase the treatment cost, but also do not change the essence of the environment pollution caused by the alkaline method.
The enzymatic method is to hydrolyze proteins in chitosan by adding protease, and streptomycete protease, papain, flavourzyme and the like are all studied for removal of proteins. For example, patent CN111138563A discloses a method for preparing chitosan from snow crab shell, which comprises adding flavourzyme into crab shell, reacting at 45-55 deg.C for 2-5 hr, and removing protein from crab shell with flavourzyme. Compared with the alkaline method, the enzymatic method has the advantage of environmental protection, but the method has higher production cost, and about 2g of flavourzyme is consumed for producing 24g of chitosan in the patent.
The biological fermentation method mainly removes protein by fungus or bacteria fermentation, for example, patent CN110004197A discloses a method for preparing chitosan by high-efficiency microbial fermentation, which mixes shrimp shell, potato, glucose and other substances to prepare a potato culture medium, and uses aspergillus oryzae to ferment on the culture medium to remove protein. The biological fermentation method has the advantages of low production cost, environmental protection and the like, but the production time required by the method is long, the fermentation time of aspergillus oryzae in the patent is 3-4 days, and the requirement of industrial production cannot be well met.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for removing protein in chitosan through electrophoresis, which not only solves the problem of high treatment cost, but also solves the problems of environmental pollution and long production period in the treatment process.
The technical scheme provided by the invention is as follows:
a method for removing protein in chitosan by electrophoresis comprises the following steps:
step 4, filtering the mixed solution obtained in the step 3, and collecting filter residues;
and 6, collecting and filtering the chitosan subjected to electrophoresis in the step 5, washing the chitosan with deionized water, and drying to obtain purified chitosan.
Preferably, in step 1, the acidic aqueous solution is hydrochloric acid or acetic acid.
Preferably, in step 2, the base is an alkali metal hydroxide.
Preferably, in the mixed solution in the step 3, the concentration of the sodium dodecyl sulfate is 0.5-2 wt%.
Preferably, in the electrophoretic solution obtained in step 5, the concentration of sodium dodecyl sulfate is 0.05-0.5 wt%.
Preferably, in step 5, the electrophoresis apparatus comprises an electrophoresis tank, a power supply, electrodes and a container with an opening on one side, wherein chitosan is placed in the container, and the opening is sealed by gauze, so that the chitosan is collected after electrophoresis is completed.
Preferably, in the step 5, the distance between the anode material and the anode material is 10-20 cm.
Preferably, in step 5, the electrophoresis uses a constant voltage direct current.
Preferably, in the step 5, the voltage applied between the anode material and the cathode material is 100 to 500 v.
Preferably, in the step 5, the time for electrophoresis is 1-6 h.
The principle of the invention is as follows:
sodium dodecyl sulfate is an anionic surfactant that breaks intramolecular and intermolecular hydrogen bonds and binds to proteins, making the entire protein negatively charged. The negatively charged protein and the uncharged chitosan can be separated under the action of an electric field, so that the aim of removing the protein in the chitosan is fulfilled.
The invention has the following beneficial effects:
the chitosan obtained by the invention completely meets the requirement that the content of chitosan protein is not more than 0.2 wt% in chitosan (YY/T1699-one 2020) of medical instrument products of tissue engineering, can reach the medical level, and can be widely applied in the aspect of medical materials. Meanwhile, a small amount of acid is adopted when the suspension is dissolved, and then the suspension is obtained by adjusting the pH value to be neutral through alkali liquor, so that the acid and alkali used is very small on the whole, and a large amount of environmental pollution and subsequent acid and alkali treatment processes are avoided. The electrophoresis of the invention only needs 1-6 hours, the cycle is short, the process can be started and stopped at any time, and the long preparation cycle of the biological fermentation method is not needed. The electrophoresis is a mature technology in other technical fields, and can be used in the invention after slight modification, so the invention has low production cost and good process stability, and therefore, the invention creates a brand-new process route suitable for production, and the invention can realize large-scale industrial production.
Drawings
FIG. 1 is a schematic view of an electrophoresis apparatus used in the present invention.
FIG. 2 shows the chitosan protein mass fraction after 3h of deproteinization by electrophoresis at different voltages.
1-cathode, 2-anode, 3-container, 4-chitosan, 5-gauze, 6-power supply, 7-electrophoresis solution and 8-electrophoresis tank.
Detailed Description
The invention is further illustrated by the following examples. It should be understood that the examples of the present invention are for illustrative purposes and not intended to limit the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.
As shown in FIG. 1, the electrophoresis apparatus for performing electrophoresis according to the present invention comprises an electrophoresis tank 8, a power source 6, electrodes and a container 3 having a single side opening, the electrode comprises a cathode 1 and an anode 2, the anode and the cathode are respectively connected with the anode and the cathode of a power supply, then the electrophoresis tank is arranged in an electrophoresis tank in parallel, electrophoresis liquid 7 is contained in an electrophoresis tank 8, the container is a container with an opening on one side, can be a horizontal cylindrical shell container or a cuboid container, during electrophoresis, the chitosan 4 is placed in the container 3, then the open end is sealed by a gauze 5 and similar filter screens to prevent the chitosan from diffusing and losing, the container is placed in the electrophoretic fluid and positioned between the cathode and the anode, the open end of the container is aligned with the anode, after the electric field is applied, the charged protein moves to the anode through the gauze under the action of the electric field, and the chitosan is retained in the container due to the blockage of the gauze, so that the removal of the protein on the chitosan is completed.
Example 1
Weighing 2g of chitosan powder in a 250ml conical flask, adding 198ml of deionized water and 2ml of acetic acid, and stirring until the chitosan is completely dissolved; then dropwise adding 1m/L sodium hydroxide solution into the solution while stirring until the pH value of the solution is about 12; adding 2g of sodium dodecyl sulfate into the conical flask, stirring until the sodium dodecyl sulfate is completely dissolved, and standing for 2 hours; filtering the solution in the conical flask, taking filter residues and placing the filter residues in an electrophoresis device, wherein the electrophoresis solution is 0.1 wt% of sodium dodecyl sulfate solution, the distance between a cathode and an anode is set to be 10cm, the voltage between the cathode and the anode is set to be 400v, and electrophoresis is carried out for 3 hours; and (3) washing the electrophoresed chitosan with deionized water, and drying to obtain the product.
Example 2
Weighing 2g of chitosan powder in a 250ml conical flask, adding 198ml of deionized water and 2ml of acetic acid, and stirring until the chitosan is completely dissolved; then dropwise adding 1m/L sodium hydroxide solution into the solution while stirring until the pH value of the solution is about 12; adding 2g of sodium dodecyl sulfate into the conical flask, stirring until the sodium dodecyl sulfate is completely dissolved, and standing for 2 hours; filtering the solution in the conical flask, taking filter residues, placing the filter residues in an electrophoresis device, setting the electrophoresis solution to be 0.1 wt% sodium dodecyl sulfate solution, setting the distance between a cathode and an anode to be 10cm, setting the voltage between the cathode and the anode to be 300v, and carrying out electrophoresis for 3 h; and (3) washing the electrophoresed chitosan with deionized water, and drying to obtain the product.
Example 3
Weighing 2g of chitosan powder in a 250ml conical flask, adding 198ml of deionized water and 2ml of acetic acid, and stirring until the chitosan is completely dissolved; then dropwise adding 1m/L sodium hydroxide solution into the solution while stirring until the pH value of the solution is about 12; adding 2g of sodium dodecyl sulfate into the conical flask, stirring until the sodium dodecyl sulfate is completely dissolved, and standing for 2 hours; filtering the solution in the conical flask, taking filter residues, placing the filter residues in an electrophoresis device, setting the electrophoresis solution to be 0.1 wt% sodium dodecyl sulfate solution, setting the distance between a cathode and an anode to be 10cm, setting the voltage between the cathode and the anode to be 200v, and carrying out electrophoresis for 3 h; and (3) washing the electrophoresed chitosan with deionized water, and drying to obtain the product.
Example 4
Weighing 2g of chitosan powder in a 250ml conical flask, adding 198ml of deionized water and 2ml of acetic acid, and stirring until the chitosan is completely dissolved; then dropwise adding 1m/L sodium hydroxide solution into the solution while stirring until the pH value of the solution is about 12; adding 2g of sodium dodecyl sulfate into the conical flask, stirring until the sodium dodecyl sulfate is completely dissolved, and standing for 2 hours; filtering the solution in the conical flask, taking filter residues, placing the filter residues in an electrophoresis device, setting the electrophoresis solution to be 0.1 wt% sodium dodecyl sulfate solution, setting the distance between a cathode and an anode to be 10cm, setting the voltage between the cathode and the anode to be 200v, and carrying out electrophoresis for 2 h; and (3) washing the electrophoresed chitosan with deionized water, and drying to obtain the product.
The protein content of the chitosan prepared in examples 1-3 was determined by reference to the detection method specified in "Chitosan product of tissue engineering medical devices" (YY/T1699-2020). The experimental results are as follows:
table 1 examples 1-4 protein removal effect table
According to the results of the table, the protein content in the chitosan after removal is lower than 0.2 wt%, and the chitosan reaches the medical level when the protein content of the chitosan is not more than 0.2 wt% in the chitosan product of tissue engineering medical instruments (YY/T1699-. On the whole, the deproteinization method can achieve the effect which is even better than the prior methods such as alkaline method, enzyme method, biological fermentation method and the like, and the method has the advantages of environmental protection, low cost, high efficiency and the like.
The above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is covered by the claims of the present invention.
Claims (9)
1. A method for removing protein in chitosan by electrophoresis is characterized in that: the method comprises the following steps:
step 1, adding chitosan powder into an acidic aqueous solution, and stirring to completely dissolve the chitosan powder to obtain a chitosan acidic aqueous solution;
step 2, adding alkali into the obtained chitosan acidic aqueous solution, and stirring to separate out chitosan to obtain chitosan alkaline dispersion liquid;
step 3, adding sodium dodecyl sulfate into the obtained chitosan alkaline dispersion liquid, stirring until the sodium dodecyl sulfate is completely dissolved, and standing for 1-2.5 hours to obtain a mixed solution;
step 4, filtering the mixed solution obtained in the step 3, and collecting filter residues;
step 5, putting the obtained filter residue into an electrophoresis device, and adding electrophoresis liquid prepared by sodium dodecyl sulfate to carry out electrophoresis for removing protein;
and 6, collecting and filtering the chitosan subjected to electrophoresis in the step 5, washing the chitosan with deionized water, and drying to obtain purified chitosan.
2. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in step 1, the acidic aqueous solution is hydrochloric acid or acetic acid.
3. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in step 2, the alkali is alkali metal hydroxide.
4. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in the mixed solution obtained in the step 3, the concentration of the sodium dodecyl sulfate is 0.5-2 wt%.
5. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in the electrophoresis solution obtained in the step 5, the concentration of the sodium dodecyl sulfate is 0.05-0.5 wt%.
6. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: and 5, in the electrophoresis process, the distance between the anode material and the anode material is 10-20 cm.
7. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in step 5, electrophoresis is performed using constant voltage direct current.
8. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in the electrophoresis process of the step 5, the voltage applied between the anode material and the cathode material is 100-500 v.
9. The method for removing protein from chitosan by electrophoresis as claimed in claim 1, wherein: in the step 5, the time for electrophoresis is 1-6 h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114369273A (en) * | 2022-01-17 | 2022-04-19 | 武汉大学 | Enhanced type electro-deposition chitosan hydrogel and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1608202A (en) * | 2001-12-28 | 2005-04-20 | 独立行政法人科学技术振兴机构 | Method of electrophoresing protein |
| CN111393541A (en) * | 2020-04-13 | 2020-07-10 | 扬州日兴生物科技股份有限公司 | Enzymatic refining process of chitosan |
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- 2021-08-18 CN CN202110950967.6A patent/CN113621093A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1608202A (en) * | 2001-12-28 | 2005-04-20 | 独立行政法人科学技术振兴机构 | Method of electrophoresing protein |
| CN111393541A (en) * | 2020-04-13 | 2020-07-10 | 扬州日兴生物科技股份有限公司 | Enzymatic refining process of chitosan |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114369273A (en) * | 2022-01-17 | 2022-04-19 | 武汉大学 | Enhanced type electro-deposition chitosan hydrogel and preparation method thereof |
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