A method for extracting arabinoxylan using corn fiber husk as raw material

Abstract

The present invention discloses a method for extracting arabinoxylan using corn fiber husk as raw material, which belongs to the technical field of polysaccharide preparation, and specifically comprises the following steps: (1) extracting corn fiber husk by alkaline low eutectic solvent, and then precipitating with alcohol to obtain crude polysaccharide; (2) dissolving the crude polysaccharide, destroying the structure by saccharifying enzyme and acidic endoxylanase, and then adding α-D-glucuronidase for modification to obtain arabinoxylan. The method for extracting arabinoxylan of the present invention has a relatively obvious improvement effect on the extraction rate of arabinoxylan, and compared with the use of a single enzyme extraction or low eutectic solvent extraction process, the extraction rate and structural stability of the extraction method of the present invention are also significantly improved; and the extraction equipment and process used by the arabinoxylan extraction method used in the present invention can effectively control the cost of the extraction process, reduce pollution, and improve the efficiency of the extraction work compared with the traditional alkali extraction method.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide preparation, and in particular relates to a method for extracting arabinoxylan by taking corn fiber husk as raw material. Background Art

[0002] Corn is an important food crop and a key raw material for feed, brewing, starch, biopharmaceuticals and other industries. It plays an important role in agricultural production and the national economy. Bran is the main by-product of corn processing. Bran has high nutritional value and is rich in dietary fiber and vitamins. However, only a small part of the bran is used for the development of functional foods. Most of it is used as feed and sold at a relatively low price, wasting precious food resources. With the continuous deepening of research on corn fiber husk, it is gradually found that some active substances in corn fiber husk bran have good medicinal value, such as heteroxylan (about 50%), cellulose (about 20%) and phenolic acid (about 4%), mainly ferulic acid and diferulic acid, among which arabinoxylan (Arabinoxylan, AX) is the main one.

[0003] Arabinoxylan has multiple biological activities such as lowering blood lipids, lowering blood sugar, anti-cancer, anti-oxidation, and improving immunity, and has many advantages, such as low toxicity, wide sources, and simple preparation. Arabinoxylan is mainly composed of two pentoses, arabinose and xylose, and has a complex and diverse structure. At present, there are many technologies for extracting arabinoxylan, among which the common ones are hot water extraction, alkali extraction, enzyme extraction, and microwave, ultrasound and other composite extraction methods; however, different extraction methods have a great influence on its structure, and the solubility, water holding capacity, viscosity, gelation, antioxidant, emulsification, etc. of arabinoxylans with different structures are very different.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide a method for efficiently producing arabinoxylan with a stable structure using corn fiber husk as raw material. Summary of the invention

[0005] In view of this, the present invention provides a method for extracting arabinoxylan using corn fiber husk as raw material, which solves the problems of long extraction time, low extraction yield, unstable structure and the like in the prior art regarding the extraction of arabinoxylan.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for extracting arabinoxylan using corn fiber husk as raw material, specifically comprising the following steps:

[0008] (1) The corn fiber husk is extracted with an alkaline low eutectic solvent and then precipitated with alcohol to obtain crude polysaccharides;

[0009] (2) After the crude polysaccharide is dissolved, the structure is destroyed by saccharifying enzyme and acid endo-xylanase, and then α-D-glucuronidase is added for modification to obtain arabinoxylan.

[0010] In the method of the present invention, firstly, the dissolution of hemicellulose in bran is enhanced by cutting off ester groups, removing carbonyl groups and destroying aromatic ring structures through alkaline low eutectic solvents, which can greatly improve the yield of arabinoxylan. Secondly, multi-enzyme composite modification is adopted, and the difference between starch polysaccharide and arabinoxylan structures is utilized to select hexose in the saccharification enzyme hydrolysis solution to achieve purification. The main chain skeleton of xylan is broken by acidic endoxylanase, and α-D-glucuronidase is coordinated to catalyze the hydrolysis of 4-O-methyl glucuronic acid substituents of the xylan side chain, thereby forming small-molecule arabinoxylan with a higher A / X coefficient. Moreover, after the multi-enzyme composite, the viscosity of the sugar solution can be reduced, and the yield and product purity of arabinoxylan can be improved.

[0011] Preferably, in step (1), the mass volume ratio of the corn fiber husk to the alkaline deep eutectic solvent is 1 g:10-30 ml;

[0012] The extraction conditions are: extraction at 75-90° C. for 2-5 h.

[0013] Preferably, the mass volume ratio of the corn fiber husk to the alkaline deep eutectic solvent is 1 g:20 ml;

[0014] The extraction conditions are: extraction at 80° C. for 4 h.

[0015] Preferably, the corn fiber husk in step (1) has a size of 60-100 mesh.

[0016] Preferably, the corn fiber husk has a size of 60 mesh.

[0017] Preferably, the alkaline deep eutectic solvent in step (1) is an aqueous solution of a hydrogen bond donor and a hydrogen bond acceptor;

[0018] Wherein, the hydrogen bond donor includes any one of butanediol, glycerol and potassium hydroxide;

[0019] The hydrogen bond donor includes choline chloride or sodium hydroxide;

[0020] The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3-7:1;

[0021] The water content of the alkaline deep eutectic solvent is 45-55%.

[0022] Preferably, the alkaline deep eutectic solvent consists of choline chloride and glycerol in a molar ratio of 1:3;

[0023] The preparation method is as follows: after mixing choline chloride, propylene glycol and water in proportion, stirring continuously in a 70° C. water bath until the solution is transparent, an aqueous alkaline ternary low eutectic solvent is obtained.

[0024] The alkaline low eutectic solvent of the present invention is composed of a hydrogen bond acceptor and a hydrogen bond donor, and has properties similar to those of ionic liquids, and is biodegradable, environmentally friendly, low-toxic or non-toxic, thermally stable, easy to use, and recyclable. It has great development prospects in the pretreatment of biomass extraction. At the same time, the low eutectic solvent has a wide polarity range. After arabinoxylan is dissolved in it, the low eutectic solvent will associate with the polysaccharide to form intermolecular hydrogen bonds, fix the polysaccharide inside the solvent, increase the content and stability of arabinoxylan in the solvent, and extract the polysaccharide in the corn husk to the maximum extent.

[0025] Preferably, the specific steps of the alcohol precipitation in step (1) are: concentrating the extracted solution to 1 / 3 of the original volume, slowly adding 3-5 times the volume of anhydrous ethanol, stirring until the flocculation becomes larger, standing at 4°C for 12-15h, separating the polysaccharide and recovering the eutectic solvent.

[0026] Preferably, the mass volume ratio of the crude polysaccharide to water in the dissolution in step (2) is 1 g:10-40 ml, that is, the concentration of the crude polysaccharide solution is 0.1-0.025 g / ml.

[0027] Preferably, the concentration of the crude polysaccharide solution is 0.05 g / ml.

[0028] Preferably, the amount of saccharifying enzyme, α-D-glucuronidase and acid endo-xylanase added in step (2) is: 200-1000 U / ml based on the dissolved solution.

[0029] Preferably, the added amount of the saccharifying enzyme is 200 U / ml, the added amount of the acid endo-xylanase is 800 U / ml, and the added amount of the α-D-glucuronidase is 200 U / ml.

[0030] Preferably, the modification conditions of the saccharifying enzyme and the acidic endoxylanase in step (2) are: enzymolysis at pH 3.5-6.0 and 30-60° C. for 2-5 h;

[0031] The modification conditions of the α-D-glucuronidase are: enzymolysis at pH 5.0-6.5 and 30-60° C. for 2-5 hours.

[0032] Preferably, the modification conditions of the saccharifying enzyme and the acidic endoxylanase are: enzymolysis at pH 4.0 and 45° C. for 4 h;

[0033] The modification conditions of the α-D-glucuronidase are: enzymolysis at 60° C. for 2 h at pH 5.0-6.5.

[0034] The composite enzyme acts on crude polysaccharide, the saccharifying enzyme mainly acts on starch to hydrolyze into glucose, so as to remove starch polysaccharide impurities in the solution for subsequent membrane passing; the acidic endoxylanase can cut the macromolecular xylan skeleton into small molecules, thereby increasing the yield of arabinoxylan, and the alpha-D-glucuronidase is combined to degrade the side chain glucuronic acid and glucose, thereby further controlling the molecular weight distribution of arabinoxylan.

[0035] Preferably, the modified solution in step (2) is sequentially purified, ultrafiltered, concentrated and freeze-dried to obtain purified arabinoxylan.

[0036] Preferably, the purification and impurity removal uses Sevage reagent, and the Sevage reagent is chloroform and n-butanol in a volume ratio of 5:1;

[0037] The ultrafiltration is performed by selecting ultrafiltration membranes with molecular weight cutoffs of 500 Da, 10 kDa and 500 kDa.

[0038] Preferably, the Sevage reagent is used to perform a protein removal operation, and the mixture is filtered cyclically until there is no denatured protein between the organic phase and the inorganic phase in the mixed solution of the Sevage reagent;

[0039] The present invention adopts graded membrane to further separate different impurities, 500kDa can remove macromolecular impurities, 500Da can remove monosaccharides produced when heteropolysaccharides are decomposed, and the target is to collect arabinoxylan with molecular weights of 500-10000Da and 10-500kDa.

[0040] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The method of the invention greatly improves the extraction rate and purity of arabinoxylan, and saves the use cost of reagents and enzyme preparations. For example, the extraction rate of arabinoxylan obtained by the traditional water extraction method is less than 1%; the bran without the enzyme method is directly subjected to alkali extraction, and the extraction rate of the obtained arabinoxylan is 23.07%, and A / X is 0.44; the enzyme method is followed by alkali extraction, and the crude extraction rate of the obtained arabinoxylan is 52.82%, containing 26.96% of arabinoxylan, 26.07% of arabinose and 21.42% of xylose, and the color is darker; and the conventional method needs to use a large amount of alkali solution, which does not meet the national environmental protection requirements, while the total arabinoxylan extraction rate obtained by the method of the invention can reach 38.96% and the purity reaches 98.94%, and the molecular weight of the collected arabinoxylan is concentrated at 9.1kDa and 403kDa, both of which are within the scope of small molecular arabinoxylan; therefore, the extraction rate and purity of arabinoxylan obtained by adopting other different extraction methods are far lower than those of the method of the invention, and the invention has significant progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative work.

[0043] Figure 1 This is a standard curve diagram for calculating the extraction rate of arabinoxylan in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1

[0046] The present invention provides a method for extracting arabinoxylan using corn fiber husk as a raw material, and the specific steps are as follows:

[0047] (1) Preparation of corn fiber husk: Take the corn fiber husk squeezed out of the corn starch processing process, dry it and perform ultrafine grinding to a size of 60 mesh to obtain ground corn fiber husk;

[0048] (2) Alkaline low eutectic solvent extraction: choline chloride and glycerol were prepared in a molar ratio of 1:3, with a water content of 50%; corn fiber husk was mixed with an alkaline low eutectic solvent in a solid-liquid ratio of 1g:20ml, heated at a constant temperature of 85°C, extracted for 4 hours, filtered, and the filtrate was collected. The filtrate was concentrated to 1 / 3 of the original volume by rotary evaporation at 0.1MPa and 60°C, and 4 times the volume of anhydrous ethanol was slowly added. After stirring until the flocculation becomes larger, it was allowed to stand at 4°C for 12 hours to separate the crude polysaccharide and recover the eutectic solvent;

[0049] (3) Enzymatic modification: The crude polysaccharide was redissolved in water at a final concentration of 0.05 g / ml, the pH was adjusted to 4.0, saccharifying enzyme and acid endo-xylanase were added at 200 U / ml, and enzymatic hydrolysis was carried out in a water bath at 45°C for 4 h. The pH was then adjusted to 6.5, α-D-glucuronidase was added at 800 U / ml, and enzymatic hydrolysis was carried out in a water bath at 60°C for 2 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated to terminate the reaction;

[0050] (4) Purification and impurity removal: The crude polysaccharide solution after enzyme inactivation is filtered out with Sevage reagent (chloroform: n-butanol = 5:1 V / V) to remove the protein. The solution is then passed through 500Da, 10kDa, and 500kDa ultrafiltration membranes to separate the low molecular weight polysaccharide solution. The polysaccharide solution is then concentrated and freeze-dried to obtain the finished arabinoxylan product.

[0051] The yield of arabinoxylan was calculated, and the results are shown in Table 1. The calculation was performed according to the following operation:

[0052] Purity of arabinoxylan: Weigh 20 mg of arabinoxylan product and dissolve it in appropriate amount of water, and dilute to 100 mL; then take 2 mL of the solution and add it to a 25 mL stoppered colorimetric tube, add 10 mL of reaction solution, and react in a boiling water bath for 25 min, shake twice during the reaction, and cool in an ice water bath for 5 min after the reaction is completed to terminate the reaction. According to the xylose standard curve (such as Figure 1 ), measure the absorbance of the solution at 552nm and 510nm and calculate the difference, thereby calculating the purity C of the arabinoxylan product AX ;

[0053] Preparation of reaction solution: Mix 110mL of freshly prepared glacial acetic acid, 2mL of concentrated hydrochloric acid, 5mL of 20% phloroglucinol-ethanol solution and 1mL of 1.75% glucose aqueous solution. Prepare as needed and cannot be stored.

[0054] The yield of arabinoxylan was calculated according to the following formula:

[0055] Arabinoxylan yield = C AX ×V×0.88 / M×100%;

[0056] Where:

[0057] C AX -polysaccharide concentration calculated from the xylose standard curve (mg / mL); V-arabinoxylan solution volume; 0.88-ratio coefficient of arabinoxylan to xylose; M-mass of corn bran after pretreatment (g).

[0058] Example 2

[0059] The method is the same as that in Example 1, except that in step (2), the low eutectic solvent is selected to have a ratio of choline chloride to butanediol of 1:4 and a water content of 40%; the corn fiber husk is mixed with the alkaline low eutectic solvent at a solid-liquid ratio of 1g:20ml, and heated at a constant temperature of 75°C for 4h; when separating the polysaccharide, 3 times of anhydrous ethanol is added, and the mixture is filtered after standing for 15h. The yield and other related results are shown in Table 1. The extraction rate of arabinoxylan is low, only 28.00%.

[0060] Example 3

[0061] The method is the same as that in Example 1, except that in step (2), the low eutectic solvent is selected to be choline chloride and potassium hydroxide in a ratio of 1:4, and the water content is 50%; the corn fiber husk and the alkaline low eutectic solvent are mixed at a solid-liquid ratio of 1g:20ml, and heated at a constant temperature of 80°C for 2h. The yield and other related results are shown in Table 1. The total extraction rate is 38.56% and the purity is 98.44%. Compared with Example 1, the extraction rates of different polysaccharide molecular weights are quite different, which affects the production of small molecular weight polysaccharides.

[0062] Example 4

[0063] The method is the same as that in Example 1, except that in step (2), the low eutectic solvent is selected with a ratio of sodium hydroxide to glycerol of 1:3 and a water content of 45%; the corn fiber husk is mixed with the alkaline low eutectic solvent at a solid-liquid ratio of 1g:10ml, and the mixture is heated at a constant temperature of 85°C for 5 hours. The yield and other related results are shown in Table 1. The total extraction rate is 32.71% and the purity is 93.41%. The overall extraction efficiency of the inorganic alkaline low eutectic solvent is lower than that of the choline chloride low eutectic solvent.

[0064] Example 5

[0065] The method is the same as that in Example 1, except that in step (2), the low eutectic solvent is selected to be sodium hydroxide and butanediol in a ratio of 1:4, with a water content of 50%; corn fiber husk is mixed with the alkaline low eutectic solvent at a solid-liquid ratio of 1g:30ml, heated at a constant temperature at 90°C, and extracted for 2h; 5 times of anhydrous ethanol is added when separating polysaccharides, and the mixture is filtered after standing for 12h. The yield and other related results are shown in Table 1. The total extraction rate is 36.00% and the purity is 98.66%. The sodium hydroxide and butanediol low eutectic solvent can effectively extract polysaccharides.

[0066] Example 6

[0067] The method is the same as that in Example 1, except that in step (2), the low eutectic solvent is selected with a ratio of sodium hydroxide to potassium hydroxide of 1:7 and a water content of 50%; corn fiber husk is mixed with an alkaline low eutectic solvent at a solid-liquid ratio of 1g:20ml, heated at a constant temperature of 80°C, and extracted for 2h; 5 times of anhydrous ethanol is added when separating polysaccharides, and filtered after standing for 12h. The yield and other related results are shown in Table 1. The total extraction rate is 25.98% and the purity is 98.11%. Potassium hydroxide is a hydrogen bond donor, and the eutectic reaction with sodium hydroxide will reduce the effective extraction of polysaccharides.

[0068] Comparative Example 1

[0069] The method is the same as that in Example 1, except that in step (2), sodium hydroxide solution is used to extract polysaccharides, and the concentration of the sodium hydroxide solution is 0.5 mol / L; corn fiber husks and sodium hydroxide solution are mixed at a solid-liquid ratio of 1 g:30 ml, and heated at a constant temperature of 85°C for 2 hours. The yield and other related results are shown in Table 1. The total extraction rate of pure alkali solution is 30.64% and the purity is 90.89%. Compared with the above example, it can be shown that the alkaline low eutectic solvent can promote the decomposition of hemicellulose.

[0070] Comparative Example 2

[0071] The method is the same as that in Example 1, except that in step (3), only 200 U / ml of saccharifying enzyme was used, and the enzymatic hydrolysis was carried out at 45°C for 4 h under the condition of pH 4.0. The test results are shown in Table 1. The total extraction rate is 35.06% and the purity is 90.64%. Compared with Example 1, the purity is lower and the molecular weight distribution of arabinoxylan is less stable.

[0072] Comparative Example 3

[0073] The method is the same as that in Example 1, except that in step (3), only 1000 U / ml of α-D-glucuronidase is used, and the enzymatic hydrolysis is carried out at 60°C for 2 h under the condition of pH 5.0. The test results are shown in Table 1. The total polysaccharide extraction rate is 30.77% and the purity is 79.30%. The use of α-D-glucuronidase alone can make the molecular weight distribution of arabinoxylan more uniform, but the overall purity is reduced.

[0074] Comparative Example 4

[0075] The method is the same as that in Example 1, except that in step (3), only 800 U / ml of acidic endoxylanase was used, and the enzymatic hydrolysis was carried out at 45°C for 6 h under the condition of pH 3.5. The test results are shown in Table 1, with a total extraction rate of 36.15% and a purity of 98.30%. When only acidic endoxylanase was used and the reaction time was extended, the yield of polysaccharides with a molecular weight of less than 10 kDa was significantly improved, but the purity decreased, and the reaction hydrolyzed some heteropolysaccharides into small molecular weight polysaccharides.

[0076] Comparative Example 5

[0077] The method is the same as that in Example 1, except that in step (3), 200 U / ml of saccharifying enzyme and 800 U / ml of acidic endo-xylanase are used, and the enzymatic hydrolysis is carried out at 30°C for 4 h at pH 4.0. The test results are shown in Table 1, and the total polysaccharide extraction rate is 35.49% and the purity is 94.87%. The acidic endo-xylanase cuts the polysaccharide, and the molecular weight of the polysaccharide is small, and a higher purity polysaccharide can be obtained.

[0078] Comparative Example 6

[0079] The method is the same as that in Example 1, except that in step (3), 800 U / ml of acidic endo-xylanase is used, and the enzymatic hydrolysis is carried out at 45°C for 4 h under the condition of pH 4.0, and the enzymatic hydrolysis is carried out at 60°C for 2 h under the condition of pH 6.5. The test results are shown in Table 1. The total polysaccharide extraction rate is 36.65% and the purity is 97.62%. When xylanase and α-D-glucuronidase are used in combination, the molecular weight distribution of polysaccharides is relatively uniform, but the purity is reduced.

[0080] Comparative Example 7

[0081] The method is the same as that in Example 1, except that in step (4), the protein removal Sevage reagent is selected to be chloroform: n-butanol = 3:1 (V / V). The test results are shown in Table 1. The total polysaccharide extraction rate is 36.7% and the purity is 96.66%. Compared with Example 1, the polysaccharide purity is reduced.

[0082] Table 1 Analysis results of arabinoxylan composition

[0083]

[0084] By comparing the yields and purity values ​​of the embodiments and comparative examples, it can be seen that the method for extracting arabinoxylan using the present invention has a relatively obvious improvement effect on the extraction rate of arabinoxylan, and compared with the use of a single enzyme extraction or a low eutectic solvent extraction process, the extraction rate and structural stability of the extraction method of the present invention are also significantly improved; on the other hand, the extraction equipment and extraction process used in the arabinoxylan extraction method used in the present invention are compared with the traditional alkali extraction method, which can effectively control the cost of the extraction process and reduce pollution, and the extraction process of the present invention can effectively improve the efficiency of the extraction work.

[0085] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting arabinoxylan using corn fiber husk as raw material, characterized in that: The specific steps include: (1) The corn fiber husk is extracted with an alkaline low eutectic solvent and then precipitated with alcohol to obtain crude polysaccharides; (2) dissolving the crude polysaccharide, destroying the structure by saccharifying enzyme and acid endo-xylanase, and then adding α-D-glucuronidase for modification to obtain arabinoxylan; Wherein, the alkaline deep eutectic solvent in step (1) is an aqueous solution of a hydrogen bond donor and a hydrogen bond acceptor; The hydrogen bond donor includes any one of butanediol and glycerol; The hydrogen bond acceptor includes choline chloride or sodium hydroxide; The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3-7:1; The water content of the alkaline deep eutectic solvent is 45-55%; The amount of saccharifying enzyme, α-D-glucuronidase and acid endo-xylanase added in step (2) is: 200-1000 U / ml based on the dissolved solution; The modification conditions of the saccharifying enzyme and the acidic endoxylanase are: enzymolysis at pH 3.5-6.0 and 30-60° C. for 1-5 hours; The modification conditions of the α-D-glucuronidase are: enzymolysis at pH 5.0-6.5 and 30-60° C. for 1-5 h.

2. The method for extracting arabinoxylan from corn fiber husk as raw material according to claim 1, characterized in that: The mass volume ratio of the corn fiber husk and the alkaline deep eutectic solvent in step (1) is 1 g:10-30 ml; The extraction conditions are: extraction at 80-100° C. for 2-5 hours.

3. The method for extracting arabinoxylan from corn fiber husk as raw material according to claim 1, characterized in that: The size of the corn fiber husk in step (1) is 60-100 mesh.

4. The method for extracting arabinoxylan from corn fiber husk as raw material according to claim 1, characterized in that: The specific steps of the alcohol precipitation in step (1) are: after concentrating the extracted solution, add 3-5 times the volume of anhydrous ethanol, let it stand at 4°C for 12-15 hours and then filter it.

5. The method for extracting arabinoxylan from corn fiber husk as raw material according to claim 1, characterized in that: The mass volume ratio of the crude polysaccharide and water in the dissolution in step (2) is 1 g:10-40 ml.

6. The method for extracting arabinoxylan from corn fiber husk as raw material according to claim 1, characterized in that: The modified solution in step (2) is sequentially purified, ultrafiltered, concentrated and freeze-dried to obtain purified arabinoxylan.

7. The method for extracting arabinoxylan from corn fiber husk as raw material according to claim 6, characterized in that: The purification and impurity removal uses Sevage reagent, and the Sevage reagent is chloroform and n-butanol in a volume ratio of 3-5:1; The ultrafiltration is performed by selecting ultrafiltration membranes with molecular weight cutoffs of 500 Da, 10 kDa and 500 kDa.

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