Method for extracting enteromorpha polysaccharide by using ultrasonic-assisted deep-eutectic solvent

Abstract

The invention discloses a method for extracting enteromorpha polysaccharide by using an ultrasonic-assisted deep-eutectic solvent, which comprises the following steps: rinsing silt on the surface of fresh enteromorpha with clear water, drying, freeze-drying, crushing, filling enteromorpha powder into a sealed bag, and refrigerating in a refrigerator for later use; the preparation method comprises the following steps: by taking 1, 2-propylene glycol as a hydrogen bond donor and choline chloride as a hydrogen bond receptor, adding deionized water to prepare a deep-eutectic solvent; the method comprises the following steps: weighing enteromorpha powder, mixing with a deep eutectic solvent, carrying out water bath and ultrasonic-assisted extraction, and centrifuging to obtain an enteromorpha polysaccharide leaching solution; and adding absolute ethyl alcohol into the enteromorpha polysaccharide extract, uniformly mixing, standing at 4 DEG C for 12 hours, centrifuging, taking precipitate, redissolving, carrying out rotary evaporation on the precipitate, deproteinizing by adopting a Sevage method, dialyzing, decoloring, taking supernate, and freeze-drying, so as to obtain the enteromorpha polysaccharide. The method disclosed by the invention has the advantages of high efficiency, short time consumption, high yield, simplicity in operation and the like, is mild in extraction condition, safe, green and environment-friendly, and lays a foundation for extraction of enteromorpha polysaccharide in food industry.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for extracting enteromorpha polysaccharide by utilizing ultrasound-assisted low eutectic solvent. Background Art

[0002] Polysaccharides are a class of high-molecular carbohydrates composed of sugar chains bound by glycosidic bonds. They are one of the three major biological molecules that maintain the activities of living organisms. Their complex molecular structure gives them a variety of biological activities. Polysaccharides play a significant role in regulating immune function, have a certain effect on lowering blood sugar, and show strong antioxidant activity. In addition, polysaccharides also have anti-depressant, anti-tumor and anti-radiation effects. Based on their rich biological activities, polysaccharides have broad application prospects in the fields of food and medicine. Not only as edible materials, they can also be used in food preservation, medical care and other fields, making positive contributions to improving human health and quality of life.

[0003] Enteromorpha prolifera is a widely distributed large green algae, belonging to the Chlorophyta, Chlorophyceae, Ulvales, Ulvaceae, and finally to the genus Enteromorpha. It grows in a wide range, especially in coastal areas of my country, where traces of Enteromorpha can often be seen. Studies have found that Enteromorpha, as a potential natural resource, contains rich nutrients, including polysaccharides, proteins, minerals and vitamins, among which the most striking is its high content of crude polysaccharides, which accounts for 35% to 50% of the dry weight. Therefore, Enteromorpha polysaccharides are considered to be a natural antioxidant with important development and utilization potential. Research on Enteromorpha polysaccharides will not only help develop new natural antioxidants, but also help promote the sustainable use of marine resources and make positive contributions to human health and environmental protection.

[0004] Deep eutectic solvent is a multi-component mixture composed of hydrogen bond acceptors and hydrogen bond donors. Its remarkable feature is that the freezing point is much lower than the melting point of the pure components. This feature gives it unique advantages in many fields. DES can be divided into two categories: binary DES and ternary DES. Binary DES is composed of two substances, while ternary DES contains three substances. Common hydrogen bond acceptors include quaternary ammonium salts, quaternary phosphonium salts, etc., while hydrogen bond donors include glycerol, propylene glycol, etc. Deep eutectic solvents have many unique properties, such as good solubility, low volatility, high thermal stability, biodegradability, etc.

[0005] Hot-dip extraction is the most common polysaccharide extraction method, which is simple to operate and mild in reaction, but the yield is relatively low. It also consumes a lot of cost and time, and cannot meet the needs of the rapidly developing food industry. Driven by the concept of "circular economy", green solvents are gradually considered to be a more efficient and environmentally friendly extraction method. As an environmentally friendly and efficient green solvent, low eutectic solvent is an alternative to traditional organic solvents. At present, there is no report on the use of low eutectic solvents to extract Enteromorpha polysaccharides from Enteromorpha. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for extracting Enteromorpha polysaccharides using ultrasound-assisted low eutectic solvents. The extraction method has the advantages of high efficiency, short time consumption, high yield, and simple operation. Not only are the extraction conditions mild and safe, but the use of toxic solvents in the extraction and separation process is avoided, which greatly reduces environmental pollution and is green and environmentally friendly. The extraction method lays a foundation for the extraction of Enteromorpha polysaccharides in the food industry.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: a method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent, comprising the following steps:

[0008] (1) taking fresh Enteromorpha, first rinsing the mud and sand on the surface of Enteromorpha with clean water, drying in an oven, freeze-drying, and crushing to obtain Enteromorpha powder, and then putting the obtained Enteromorpha powder into a sealed bag and putting it in a refrigerator for later use;

[0009] (2) using 1,2-propylene glycol as a hydrogen bond donor, using choline chloride as a hydrogen bond acceptor, and adding deionized water to prepare a deep eutectic solvent;

[0010] (3) Weighing an appropriate amount of Enteromorpha powder and mixing it with the prepared low eutectic solvent to obtain a mixture, then subjecting the mixture to water bath and ultrasound-assisted extraction, cooling the filtrate obtained by extraction to room temperature, and centrifuging the filtrate to obtain an Enteromorpha polysaccharide extract;

[0011] (4) adding anhydrous ethanol to the Enteromorpha polysaccharide extract, mixing evenly, standing at 4° C. for 12 h, centrifuging, re-dissolving the precipitate, and performing rotary evaporation to obtain Enteromorpha crude polysaccharide;

[0012] (5) The crude Enteromorpha polysaccharide was deproteinized by the Sevage method, dialyzed and decolorized, and the supernatant was freeze-dried to obtain Enteromorpha polysaccharide.

[0013] Increasing the molar ratio of the low eutectic solvent can enhance the hydrogen bonding between components, but an excessive amount will enhance the interaction between components, resulting in an increase in the viscosity of the low eutectic solvent, which will affect the yield of polysaccharides; most low eutectic solvents form a hydrogen bond network, and the increased viscosity hinders the diffusion movement of molecules. Adding a certain amount of water can effectively reduce the viscosity, thereby increasing the yield of polysaccharides. Therefore, as a preference, in step (2), the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2, and the volume water content of the prepared low eutectic solvent is 20-60%.

[0014] The low eutectic solvent is a viscous liquid with relatively poor fluidity. As the solid-liquid ratio gradually decreases, the contact area between the Enteromorpha powder and the low eutectic solvent increases, promoting the combination of the Enteromorpha powder and the low eutectic solvent, which helps to increase the yield of polysaccharides. When the amount of the low eutectic solvent is too much, the extract has been fully combined with the Enteromorpha powder, the extraction amount reaches a saturated state, and other impurities are dissolved, resulting in the inhibition of polysaccharide dissolution, which in turn leads to a decrease in the yield of Enteromorpha polysaccharides. Therefore, as a preferred method, in step (3), the solid-liquid ratio of the Enteromorpha powder and the low eutectic solvent is 1g:20~60mL.

[0015] Preferably, in step (3), the temperature of the water bath is 60-100° C., and the power of the ultrasound is 450 W. If the temperature of the water bath is too high, the dissolved substances will increase, the viscosity will increase, the dissolution of the polysaccharide will be inhibited, and the yield of Enteromorpha polysaccharide will be low. Therefore, the temperature of the water bath is controlled at 60-100° C. in the present invention. When the temperature of the water bath is 91° C., the yield of Enteromorpha polysaccharide is the highest.

[0016] Preferably, in step (4), the Enteromorpha polysaccharide extract and anhydrous ethanol are evenly mixed in a volume ratio of 1:4 to ensure the extraction effect and obtain a polysaccharide sample with higher purity.

[0017] Preferably, in step (5), the Sevage reagent used for deproteinizing Enteromorpha crude polysaccharide by the Sevage method is prepared by mixing n-butanol and chloroform in a volume ratio of 1:4, and the specific process of step (5) is: after mixing the Enteromorpha crude polysaccharide with the Sevage reagent, fully oscillate for 15 minutes and then centrifuge, aspirate the upper layer solution after centrifugation, repeatedly use the Sevage method to deproteinize Enteromorpha crude polysaccharide until no protein is produced, take the final upper layer solution for dialysis and decolorization, and then freeze-dry to obtain powdered Enteromorpha polysaccharide. The above-mentioned deproteinization method has mild conditions and will not cause the denaturation of polysaccharides.

[0018] Preferably, in step (5), the dialysis conditions are: dialysis bag pore size 3500Da, time 3 days. This dialysis operation is simple and convenient, can remove impurities and improve the purity of polysaccharides.

[0019] Preferably, in step (5), the decolorization is performed by using macroporous resin AB-8 to adsorb the pigment. This decolorization operation has a high decolorization rate and polysaccharide retention rate.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1) The present invention proposes for the first time a method for extracting Enteromorpha polysaccharides using an ultrasound-assisted low eutectic solvent. The green and environmentally friendly low eutectic solvent is used to separate Enteromorpha polysaccharides through ultrasound-assisted extraction. The extraction method has the advantages of high efficiency, short time consumption, high yield, simple operation, etc., not only mild and safe extraction conditions, but also avoids the use of toxic solvents in the extraction and separation process, greatly reducing environmental pollution, and being green and environmentally friendly. The extraction method lays a foundation for the extraction of Enteromorpha polysaccharides in the food industry;

[0022] 2) The method of the present invention uses a low eutectic solvent as an extraction solvent, and extracts polysaccharides with the low eutectic solvent through ultrasound. During the ultrasound process, the violent vibration of the surface of the Enteromorpha powder helps to mix the Enteromorpha powder and the low eutectic solvent, making it dissolve better. Ultrasound can also destroy the cell walls of the Enteromorpha cells, accelerating the release of polysaccharides in the cells in water, thereby increasing the yield of Enteromorpha polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results are obtained by scanning the protein-free Enteromorpha polysaccharide in Example 1 at 200-600 nm using an ultraviolet spectrophotometer;

[0024] Figure 2 is a comparison diagram of Enteromorpha polysaccharide before and after decolorization in Example 1;

[0025] Figure 3 The single factor results (volume water content of the deep eutectic solvent) of the yield of Enteromorpha polysaccharide obtained in Examples 1 to 5;

[0026] Figure 4 The single factor results (solid-liquid ratio of Enteromorpha powder to deep eutectic solvent) of the Enteromorpha polysaccharide yields obtained in Examples 1 and 6 to 9 are as follows;

[0027] Figure 5 The single factor results (extraction temperature) of the yield of Enteromorpha polysaccharide obtained in Examples 1 and 10 to 13;

[0028] Figure 6 The response surface 3D diagram results of the yield determination of Enteromorpha polysaccharide obtained in Examples 1 to 5 (volume water content of the deep eutectic solvent);

[0029] Figure 7 The response surface contour map results of the Enteromorpha polysaccharide yield determination obtained in Examples 1 to 5 (volume water content of the deep eutectic solvent);

[0030] Figure 8 The response surface 3D diagram results of the Enteromorpha polysaccharide yield determination obtained in Examples 1 and 6 to 9 (the solid-liquid ratio of Enteromorpha powder to the deep eutectic solvent);

[0031] Fig. 9 The response surface contour map results of the Enteromorpha polysaccharide yield determination obtained in Examples 1 and 6 to 9 (the solid-liquid ratio of Enteromorpha powder to the deep eutectic solvent);

[0032] Fig.10 The response surface 3D diagram results (extraction temperature) of the Enteromorpha polysaccharide yield obtained in Examples 1 and 10 to 13;

[0033] Fig.11 The response surface contour map results (extraction temperature) of the Enteromorpha polysaccharide yield obtained in Examples 1 and 10 to 13. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to the accompanying drawings.

[0035] Embodiment 1:

[0036] (1) taking fresh Enteromorpha, first rinsing the mud and sand on the surface of Enteromorpha with clean water, drying in an oven, freeze-drying, and crushing to obtain Enteromorpha powder, and then putting the obtained Enteromorpha powder into a sealed bag and putting it in a refrigerator for later use;

[0037] (2) using 1,2-propylene glycol as a hydrogen bond donor and choline chloride as a hydrogen bond acceptor, and adding deionized water to prepare a low eutectic solvent. The specific preparation method of the low eutectic solvent is as follows: mixing the hydrogen bond acceptor and the hydrogen bond donor in a molar ratio of 1:2 and then mixing with deionized water, stirring in a water bath at 80° C. until clear and transparent, to prepare a low eutectic solvent with a volume water content of 20%;

[0038] (3) Weighing 1 g of Enteromorpha powder and mixing it with 30 mL of a low eutectic solvent to obtain a mixture, and then subjecting the mixture to a 60° C. water bath (i.e., extraction temperature) and 450 W ultrasonic-assisted extraction for 1 h. After the filtrate obtained by extraction was cooled to room temperature, the filtrate was centrifuged at 7000 r / min for 15 min to obtain an Enteromorpha polysaccharide extract;

[0039] (4) adding 4 times the volume of anhydrous ethanol to the Enteromorpha polysaccharide extract, mixing evenly, standing at 4° C. for 12 h, centrifuging, re-dissolving the precipitate, and performing rotary evaporation to obtain Enteromorpha crude polysaccharide;

[0040] (5) The Sevage method was used to remove protein from the crude polysaccharide of Enteromorpha. The Sevage reagent used was prepared by mixing n-butanol and chloroform in a volume ratio of 1:4. After mixing the crude polysaccharide of Enteromorpha with the Sevage reagent, the mixture was fully shaken for 15 minutes and then centrifuged. After centrifugation, the upper layer solution was aspirated and the Sevage method was repeatedly used to remove protein from the crude polysaccharide of Enteromorpha until no protein was produced. The final upper layer solution was dialyzed and decolorized. The dialysis conditions were: the pore size of the dialysis bag was 3500Da, the time was 3 days, and the macroporous resin AB-8 was used for decolorization to adsorb the pigment. After decolorization, the mixture was freeze-dried to obtain a powdered Enteromorpha polysaccharide, which was the purified Enteromorpha polysaccharide.

[0041] The calculation formula of the yield of Enteromorpha polysaccharide is:

[0042] Wherein, E is the yield of Enteromorpha polysaccharide (%); M1 is the mass of freeze-dried Enteromorpha polysaccharide (g); and M2 is the mass of Enteromorpha powder (g).

[0043] The total sugar content of Enteromorpha polysaccharide obtained in Example 1 was determined by phenol-sulfuric acid method, and the determination process was as follows:

[0044] Preparation of standard curve: Accurately weigh 0.1g of anhydrous glucose powder and dilute it to a 100mL volumetric flask to obtain a 0.1mg / mL glucose standard solution. Accurately pipette 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of the prepared glucose standard solution into test tubes, respectively, add distilled water to 2mL and shake well to prepare 5 glucose reference solutions of different concentrations. Pipette 1mL of each of the 5 reference solutions, add 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid prepared on the same day, shake well immediately, let stand for 30min to fully react and cool to room temperature. Distilled water is used as a blank control, and the absorbance value (A) of the crude polysaccharide is measured at 490nm. The standard curve is drawn with glucose concentration (C) as the horizontal axis and absorbance value (A) as the vertical axis. Glucose standard curve A=13C+0.021, R 2 =0.9991, where A is the absorbance value, C is the glucose concentration (mg / mL), R 2 is the correlation coefficient.

[0045] Determination of total sugar content of Enteromorpha polysaccharide: Take 1 mL of Enteromorpha polysaccharide extract and add 1 mL of distilled water, then add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid in sequence, shake well, let stand for 30 minutes to fully react, cool to room temperature, measure the absorbance value A at 490 nm, and calculate the polysaccharide content in the Enteromorpha polysaccharide extract according to the standard curve.

[0046] The total sugar content of Enteromorpha polysaccharide was calculated according to the following formula:

[0047]

[0048] Wherein, C is the concentration of Enteromorpha polysaccharide extract (mg / mL), V is the volume of Enteromorpha polysaccharide extract (mL), D is the dilution multiple of Enteromorpha polysaccharide extract, and M is the mass of Enteromorpha powder (g).

[0049] The protein content of Enteromorpha polysaccharide obtained in Example 1 was determined by Coomassie Brilliant Blue method, and the determination process was as follows:

[0050] Preparation of standard curve: Prepare 0.04, 0.08, 0.12, 0.16, 0.2 mg / mL bovine serum albumin (BSA) respectively, add appropriate amount of Coomassie Brilliant Blue G250 staining solution, react for 3-5 min, measure the absorbance at A595 nm, draw the standard curve, and the regression equation is: y = 4.1875x + 0.8576 (R 2 =0.9992).

[0051] Determination of protein content of Enteromorpha polysaccharide: Take 1 mL of Enteromorpha polysaccharide extract, add 1 mL of the above-mentioned Coomassie Brilliant Blue G250 staining solution, mix well, measure the absorbance at 595 nm with a UV-visible spectrophotometer, and calculate the protein content according to the above regression equation.

[0052] The measurement results of Enteromorpha polysaccharide obtained in Example 1 are shown in Table 1.

[0053] Table 1

[0054]

[0055] Example 2: The extraction method is the same as that of Example 1 except that the volumetric water content of the deep eutectic solvent used is 30%.

[0056] Example 3: The extraction method is the same as that of Example 1 except that the volumetric water content of the deep eutectic solvent used is 40%.

[0057] Example 4: The extraction method is the same as that of Example 1 except that the volumetric water content of the deep eutectic solvent used is 50%.

[0058] Example 5: The extraction method is the same as that of Example 1 except that the volumetric water content of the deep eutectic solvent used is 60%.

[0059] Example 6: The extraction method is the same as that of Example 1 except that the solid-liquid ratio of Enteromorpha powder to the low eutectic solvent is 1 g:20 mL.

[0060] Example 7: The extraction method is the same as that of Example 1 except that the ratio of Enteromorpha powder to low eutectic solvent is 1 g:40 mL.

[0061] Example 8: The extraction method is the same as that of Example 1 except that the ratio of Enteromorpha powder to low eutectic solvent is 1 g:50 mL.

[0062] Example 9: The extraction method is the same as that of Example 1 except that the ratio of Enteromorpha powder to low eutectic solvent is 1 g:60 mL.

[0063] Example 10: Except that the extraction temperature used is 70°C, the rest of the extraction method is the same as that of Example 1.

[0064] Example 11: Except that the extraction temperature used is 80°C, the rest of the extraction method is the same as that of Example 1.

[0065] Example 12: Except that the extraction temperature used is 90°C, the rest of the extraction method is the same as that of Example 1.

[0066] Example 13: Except that the extraction temperature used is 100°C, the rest of the extraction method is the same as that of Example 1.

[0067] The results of scanning the protein-free Enteromorpha polysaccharide in Example 1 at 200-600 nm using an ultraviolet spectrophotometer are shown in FIG. Figure 1 ; The comparison diagram of Enteromorpha polysaccharide before and after decolorization in Example 1 is shown in Figure 2 , before bleaching Figure 2 Middle A, after decolorization Figure 2 Middle Figure B.

[0068] Figure 3 The single factor results (volume water content of the deep eutectic solvent) of the yield of Enteromorpha polysaccharide obtained in Examples 1 to 5; Figure 4 The single factor results (solid-liquid ratio of Enteromorpha powder to deep eutectic solvent) of the Enteromorpha polysaccharide yields obtained in Examples 1 and 6 to 9 are as follows; Figure 5 The single factor results (extraction temperature) of the yield of Enteromorpha polysaccharide obtained in Examples 1 and 10 to 13; Figure 6 The response surface 3D diagram results of the yield determination of Enteromorpha polysaccharide obtained in Examples 1 to 5 (volume water content of the deep eutectic solvent); Figure 7 The response surface contour map results of the Enteromorpha polysaccharide yield determination obtained in Examples 1 to 5 (volume water content of the deep eutectic solvent); Figure 8 The response surface 3D diagram results of the Enteromorpha polysaccharide yield determination obtained in Examples 1 and 6 to 9 (the solid-liquid ratio of Enteromorpha powder to the deep eutectic solvent); Fig. 9 The response surface contour map results of the Enteromorpha polysaccharide yield determination obtained in Examples 1 and 6 to 9 (the solid-liquid ratio of Enteromorpha powder to the deep eutectic solvent); Fig.10The response surface 3D diagram results (extraction temperature) of the Enteromorpha polysaccharide yield obtained in Examples 1 and 10 to 13; Fig.11 The response surface contour map results (extraction temperature) of the Enteromorpha polysaccharide yield obtained in Examples 1 and 10 to 13.

Claims

1. A method for extracting Enteromorpha polysaccharides using ultrasound-assisted deep eutectic solvents, It is characterized in that The following steps are involved: (1) taking fresh Enteromorpha, first rinsing the mud and sand on the surface of Enteromorpha with clean water, drying in an oven, freeze-drying, and crushing to obtain Enteromorpha powder, and then putting the obtained Enteromorpha powder into a sealed bag and putting it in a refrigerator for later use; (2) using 1,2-propylene glycol as a hydrogen bond donor, using choline chloride as a hydrogen bond acceptor, and adding deionized water to prepare a deep eutectic solvent; (3) Weighing an appropriate amount of Enteromorpha powder and mixing it with the prepared low eutectic solvent to obtain a mixture, then subjecting the mixture to water bath and ultrasound-assisted extraction, cooling the filtrate obtained by extraction to room temperature, and centrifuging the filtrate to obtain an Enteromorpha polysaccharide extract; (4) adding anhydrous ethanol to the Enteromorpha polysaccharide extract, mixing evenly, standing at 4° C. for 12 h, centrifuging, re-dissolving the precipitate, and performing rotary evaporation to obtain Enteromorpha crude polysaccharide; (5) The crude Enteromorpha polysaccharide was deproteinized by the Sevage method, dialyzed and decolorized, and the supernatant was freeze-dried to obtain Enteromorpha polysaccharide.

2. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (2), the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2, and the volume water content of the prepared low eutectic solvent is 20-60%.

3. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (3), the material-liquid ratio of the Enteromorpha powder and the low eutectic solvent is 1 g: 20-60 mL.

4. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (3), the temperature of the water bath is 60-100° C., and the power of the ultrasound is 450W.

5. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (4), the Enteromorpha polysaccharide extract and anhydrous ethanol are evenly mixed in a volume ratio of 1:

4.

6. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (5), the Sevage reagent used for deproteinizing the Enteromorpha crude polysaccharide by the Sevage method is prepared by mixing n-butanol and chloroform in a volume ratio of 1:

4. The specific process of step (5) is as follows: after mixing the Enteromorpha crude polysaccharide with the Sevage reagent, fully oscillate for 15 minutes and then centrifuge, aspirate the upper solution after centrifugation, repeatedly use the Sevage method to deproteinize the Enteromorpha crude polysaccharide until no protein is produced, take the final upper solution for dialysis and decolorization, and then freeze-dry to obtain powdered Enteromorpha polysaccharide.

7. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (5), the dialysis conditions are: dialysis bag pore size 3500Da, time 3 days.

8. The method for extracting Enteromorpha polysaccharide using ultrasound-assisted deep eutectic solvent according to claim 1, It is characterized in that In step (5), the macroporous resin AB-8 is used for decolorization to adsorb the pigment.

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