A lignin nanoparticle, a preparation method thereof, and an application thereof

Abstract

The present invention discloses a lignin nanoparticle and its preparation method and application, which relate to the technical field of antibacterial materials. The present invention uses only lignin as a raw material, and performs a graded treatment on the lignin using an organic solvent to achieve the distribution adjustment of molecular weight and chemical structure. The graded lignin is then dissolved in a mixed solvent of an organic solvent and water, and added dropwise to distilled water at a uniform speed under stirring conditions, or distilled water is added dropwise to the graded lignin solution at a uniform speed. The lignin nanoparticles are then obtained by rotary evaporation and drying. During the entire process, more active functional groups, phenolic hydroxyl groups and carboxyl groups, are exposed, solving the problems of resource waste and low-value utilization caused by the dispersibility and structural heterogeneity of lignin, as well as the problem that existing lignin antibacterial materials mostly use composite materials, have a complex preparation process, and are costly. The method of the present invention has a simple preparation process and mild conditions. The prepared nanoparticles are safe and non-toxic, have good stability, and have excellent antibacterial properties.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial materials, and in particular to lignin nanoparticles, a preparation method and application thereof. Background Art

[0002] Lignin is a renewable biomass material with a three-dimensional network structure that is widely available and inexpensive. However, due to its structural heterogeneity and dispersibility, it is often treated as waste in the agriculture, forestry, and pulp and paper industries, and its utilization value is low. Considering that lignin is a natural bioactive compound, it is environmentally friendly, biodegradable, and biocompatible, and has broad application prospects in high-value-added fields such as biomedicine. In the development of antibacterial functional materials, lignin itself has been proven to have certain utilization value because it is rich in oxygen-containing functional groups such as phenolic hydroxyl and carboxyl groups. CN 114794149 A discloses a nano-lignin antibacterial material, its preparation method, and application. In the presence of a cosolvent and a metal salt, lignin molecules self-assemble to form lignin nanotubes, while metal ions are reduced by the lignin to form metal nanoparticles, thereby realizing a hybrid nano-antibacterial agent composed of lignin nanotubes loaded with metal nanoparticles, with an antibacterial performance of over 99.9%. However, existing reports on the application of lignin in the development of antibacterial materials mostly use composite materials, with lignin as an auxiliary additive. The main antibacterial effect is mostly exerted by other components such as metal antibacterial materials and polysaccharide antibacterial materials. The material preparation process is complicated and costly, and there is no reasonable guidance on how to exert the antibacterial effect of lignin itself or how to improve its antibacterial properties. Therefore, it is very necessary to provide a low-cost functional material with good antibacterial effect and lignin as the main base material. Summary of the Invention

[0003] In response to the above-mentioned problems existing in the prior art, the first technical problem to be solved by the present invention is to provide a method for preparing lignin nanoparticles; the second technical problem to be solved by the present invention is to provide lignin nanoparticles prepared by this method; and the third technical problem to be solved by the present invention is to provide the application of lignin nanoparticles in antibacterial aspects.

[0004] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0005] A method for preparing lignin nanoparticles comprises the following steps: dissolving graded lignin in an organic solvent or a mixed solvent of an organic solvent and water, adding the solution dropwise at a uniform speed into distilled water under stirring, or adding distilled water dropwise at a uniform speed into the graded lignin solution, and then rotary evaporating and drying to obtain the lignin nanoparticles.

[0006] The graded lignin is prepared by adding washed acid-precipitated lignin into an organic solvent or a mixed solvent of an organic solvent and water, stirring the mixture at room temperature, and then filtering and drying the mixture to obtain the graded lignin.

[0007] Lignin was graded using organic solvents to achieve the distribution adjustment of molecular weight and chemical structure. More active functional groups, phenolic hydroxyl groups and carboxyl groups, were exposed during the self-assembly of lignin molecules to form lignin nanoparticles. The prepared nanoparticles are safe, non-toxic and have good stability.

[0008] The acid-precipitated lignin is prepared by dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH value to 1-2, and obtaining the acid-precipitated lignin; then, the acid-precipitated lignin is washed with distilled water, and the pH value of the washed solution is controlled to be 3-4.

[0009] Furthermore, the concentration of the washed acid-precipitated lignin in the organic solvent or in the mixed solvent of the organic solvent and water is 20 to 70 mg mL -1 .

[0010] Further, the reaction was stirred at room temperature for 8 h.

[0011] Furthermore, the organic solvent used to dissolve the fractionated lignin is consistent with the organic solvent added to the acid-precipitated lignin, and is selected from one of acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol, dichloromethane, tetrahydrofuran, and dioxane; the volume ratio of the organic solvent to water is 1:4 to 4:1.

[0012] Furthermore, the volume ratio of the fractionated lignin solution to distilled water is 1:1 to 1:100.

[0013] Furthermore, under stirring conditions of 400-600 rpm, 2-8 mL·min -1 Add dropwise at a uniform speed.

[0014] Furthermore, the size of the prepared lignin nanoparticles is between 20 and 400 nm.

[0015] The lignin nanoparticles are prepared by the above preparation method.

[0016] The application of the lignin nanoparticles in antibacterial aspects.

[0017] In this application, Escherichia coli and Staphylococcus aureus were selected as model species of Gram-negative bacteria and Gram-positive bacteria, respectively.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention performs graded treatment on lignin and uses nanotechnology to prepare lignin into lignin-based functionalized nanoparticles, which can greatly promote the utilization of lignin and solve the problems of resource waste and low-value utilization caused by its dispersibility and structural heterogeneity.

[0020] (2) The preparation process of the present invention is simple and requires mild conditions. Only lignin is used as a raw material in the preparation process. Lignin is graded using an organic solvent to achieve molecular weight and chemical structure distribution adjustment. During the self-assembly process of lignin molecules to form lignin nanoparticles, more active functional groups, phenolic hydroxyl groups and carboxyl groups, are exposed. The prepared nanoparticles are safe, non-toxic, stable, and have excellent antibacterial properties. This solves the problem that existing lignin antibacterial materials, which are mostly composite materials, have a complex preparation process, and are costly. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Transmission electron microscopy (TEM) morphology of lignin nanoparticles; Figure 1 a, b, c, d, and e are transmission electron microscopy (TEM) images of the lignin nanoparticles prepared in Examples 1, 2, 3, 4, and 5, respectively;

[0022] Figure 2 This is the infrared spectrum of the lignin nanoparticles prepared in Example 1;

[0023] Figure 3 This is the two-dimensional nuclear magnetic resonance image of the lignin nanoparticles prepared in Example 1;

[0024] Figure 4 This is a graph showing the antibacterial effects of the lignin nanoparticles prepared in Example 1 on Escherichia coli and Staphylococcus aureus. In the graph, E. coli represents Escherichia coli, and S. aureus represents Staphylococcus aureus.

[0025] Figure 5 The scanning electron microscope (SEM) morphology of Escherichia coli and Staphylococcus aureus treated with the lignin nanoparticles prepared in Example 1; in the figure, E. coli is Escherichia coli and S. aureus is Staphylococcus aureus;

[0026] Figure 6 Figure 2 is a graph showing the changes in alkaline phosphatase activity, conductivity, and nucleic acid content in the supernatant after co-culture of Escherichia coli and Staphylococcus aureus with lignin nanoparticles over time. Figure a, c, and e are graphs showing the changes in alkaline phosphatase activity, conductivity, and nucleic acid content in the supernatant after co-culture of Escherichia coli with lignin nanoparticles over time. Figure b, d, and f are graphs showing the changes in alkaline phosphatase activity, conductivity, and nucleic acid content in the supernatant after co-culture of Staphylococcus aureus with lignin nanoparticles over time.

[0027] Figure 7 The graph shows the antibacterial effects of the size of lignin nanoparticles on Escherichia coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific examples. These examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or replacements made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. In the following examples, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0029] Example 1

[0030] A method for preparing lignin nanoparticles comprises the following steps:

[0031] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 1, and obtaining acid-precipitated lignin;

[0032] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 3;

[0033] 3) Add the washed acid-precipitated lignin to acetone to a final concentration of 20 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0034] 4) Dissolve the fractionated lignin in acetone to form a fractionated lignin solution, stirring at 400 rpm for 2 mL min -1 Add it dropwise and uniformly into distilled water, with the volume ratio of graded lignin solution to distilled water being 1:10, and then evaporating and drying to obtain lignin nanoparticles. Figure 1 As shown in a, the particles are uniformly dispersed in the range of 20 to 40 nm.

[0035] Taking Example 1 as an example, the structural characteristics of the prepared nanoparticles were tested. Figure 2 As shown in the figure, the infrared spectrum of the prepared lignin nanoparticles retained the basic skeleton of benzene rings compared with the acid-precipitated lignin, proving that the organic solvent fractionation treatment and nanoparticle preparation process will not prematurely destroy the basic structure of lignin. -1 The non-conjugated carbonyl peak is significantly enhanced, 1123 cm -1 The peak intensity at α is weakened, which proves that the number of S-type structural units in the lignin structure is small. Figure 3As shown in the 2D-NMR spectrum of the prepared lignin nanoparticles, compared to acid-precipitated lignin, the polysaccharide component is reduced after organic solvent purification, while the side chain structure remains largely unchanged. The significant change is in the content of the linker. The intensity of the α-CH of the β-O-4 linker decreases after fractionation. As shown in Table 1, the infrared spectrum of the prepared lignin nanoparticles shows a significant decrease in molecular weight and a significant increase in the content of phenolic hydroxyl and carboxyl groups compared to acid-precipitated lignin.

[0036] Table 1

[0037] Mn Mw -OH / % -COOH / % Acid-precipitated lignin 3237 4496 23.46 13.65 Lignin nanoparticles 2496 3338 31.20 24.83

[0038] Example 2

[0039] A method for preparing lignin nanoparticles comprises the following steps:

[0040] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 1, and obtaining acid-precipitated lignin;

[0041] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 3;

[0042] 3) Add the washed acid-precipitated lignin to acetone to a final concentration of 20 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0043] 4) Dissolve the fractionated lignin in acetone to form a fractionated lignin solution, stirring at 400 rpm for 2 mL min -1 The graded lignin solution was added dropwise to the distilled water at a constant speed, with the volume ratio of the graded lignin solution to distilled water being 1:10, and then subjected to rotary evaporation and drying to obtain lignin nanoparticles. Figure 1 As shown in b, the particles are 350 nm and evenly dispersed.

[0044] Example 3

[0045] A method for preparing lignin nanoparticles comprises the following steps:

[0046] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 1, and obtaining acid-precipitated lignin;

[0047] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 3;

[0048] 3) Add the washed acid-precipitated lignin to acetone to a final concentration of 20 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0049] 4) Dissolve the fractionated lignin in acetone to form a fractionated lignin solution, stirring at 400 rpm for 2 mL min -1 Add it dropwise and uniformly into distilled water, with the volume ratio of graded lignin solution to distilled water being 1:1, and then rotary evaporate and dry to obtain lignin nanoparticles. Figure 1 As shown in c, these are semi-hollow particles with a size of 100 to 200 nm and are evenly dispersed.

[0050] Example 4

[0051] A method for preparing lignin nanoparticles comprises the following steps:

[0052] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 1, and obtaining acid-precipitated lignin;

[0053] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 3;

[0054] 3) Add the washed acid-precipitated lignin to acetone to a final concentration of 20 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0055] 4) Dissolve the fractionated lignin in acetone to form a fractionated lignin solution, stirring at 400 rpm for 2 mL min -1 Add it dropwise and uniformly into distilled water, with the volume ratio of graded lignin solution to distilled water being 1:100, and then evaporating and drying to obtain lignin nanoparticles. Figure 1 As shown in d, it is a 60nm uniformly dispersed hollow particle.

[0056] Example 5

[0057] A method for preparing lignin nanoparticles comprises the following steps:

[0058] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 1, and obtaining acid-precipitated lignin;

[0059] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 3;

[0060] 3) Add the washed acid-precipitated lignin to ethanol to a final concentration of 20 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0061] 4) Dissolve the fractionated lignin in ethanol to form a fractionated lignin solution, stirring at 400 rpm for 8 mL min -1Add it dropwise and uniformly into distilled water, with the volume ratio of graded lignin solution to distilled water being 1:100, and then evaporating and drying to obtain lignin nanoparticles. Figure 1 As shown in e, the particles are 60 nm and evenly dispersed.

[0062] Example 6

[0063] A method for preparing lignin nanoparticles comprises the following steps:

[0064] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 2, to obtain acid-precipitated lignin;

[0065] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 4;

[0066] 3) The washed acid-precipitated lignin was added to a mixed solvent of dioxane and water (1:4 v / v) to a final concentration of 70 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0067] 4) Dissolve the fractionated lignin in a mixed solvent of dioxane and water (1:4 v / v) and stir at 400 rpm for 8 mL min -1 The solution was added dropwise and uniformly into distilled water, with the volume ratio of the graded lignin solution to distilled water being 1:10. The solution was then subjected to rotary evaporation and drying to obtain lignin nanoparticles.

[0068] Example 7

[0069] A method for preparing lignin nanoparticles comprises the following steps:

[0070] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 2, to obtain acid-precipitated lignin;

[0071] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 4;

[0072] 3) The washed acid-precipitated lignin was added to a mixed solvent of methanol and water (3:2 v / v) to a final concentration of 70 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0073] 4) Dissolve the fractionated lignin in a mixed solvent of methanol and water (3:2 v / v) and stir at 400 rpm for 4 mL min -1 The solution was added dropwise and uniformly into distilled water, with the volume ratio of the graded lignin solution to distilled water being 1:5. The solution was then subjected to rotary evaporation and drying to obtain lignin nanoparticles.

[0074] Example 8

[0075] A method for preparing lignin nanoparticles comprises the following steps:

[0076] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 2, to obtain acid-precipitated lignin;

[0077] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 4;

[0078] 3) The washed acid-precipitated lignin was added to a mixed solvent of dichloromethane and water (4:1 v / v) to a final concentration of 50 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0079] 4) Dissolve the fractionated lignin in a mixed solvent of dichloromethane and water (4:1 v / v) and stir at 600 rpm for 2 mL min -1 The solution was added dropwise and uniformly into distilled water, with the volume ratio of the graded lignin solution to distilled water being 1:8. The solution was then subjected to rotary evaporation and drying to obtain lignin nanoparticles.

[0080] Example 9

[0081] A method for preparing lignin nanoparticles comprises the following steps:

[0082] 1) dissolving lignin in deionized water, adding hydrochloric acid to adjust the pH to 1, and obtaining acid-precipitated lignin;

[0083] 2) washing the acid-precipitated lignin with distilled water, and controlling the pH value of the solution after washing to be 3;

[0084] 3) Add the washed acid-precipitated lignin to tetrahydrofuran to a final concentration of 20 mg mL -1 The mixture was stirred and reacted at room temperature for 8 h, and then filtered and dried to obtain graded lignin.

[0085] 4) Dissolve the fractionated lignin in tetrahydrofuran and stir at 500 rpm for 4 mL min -1 The solution was added dropwise and uniformly into distilled water, with the volume ratio of the graded lignin solution to distilled water being 1:10. The solution was then subjected to rotary evaporation and drying to obtain lignin nanoparticles.

[0086] Example 10

[0087] Taking the lignin nanoparticle material prepared in Example 1 as an example, the antibacterial properties of the prepared nanoparticles were evaluated.

[0088] Different concentrations of lignin nanoparticles were prepared in physiological saline solution (500 μL). Equal volumes of Escherichia coli and Staphylococcus aureus cultures in their logarithmic growth phase were then added. The mixture was incubated at 37°C for 4 hours with shaking. A 50 μL plate was spread on an LB solid culture plate and incubated in a 37°C incubator for 12 hours. The plates were removed and the colony formation before and after antibacterial treatment was compared. The minimum inhibitory concentration (MIC) was calculated using the plate count method.

[0089] In this example, the colony count before antibacterial treatment refers to the colony count without adding lignin nanoparticles, and the colony count after antibacterial treatment refers to the colony count with adding the lignin nanoparticles prepared in Example 1.

[0090] The results of the antibacterial test on Escherichia coli and Staphylococcus aureus are shown in Figure 4 , at 1 mg·mL -1 When the amount of added was 5 mg·mL, a significant decrease in the number of colonies was observed. -1 The above results show that lignin nanoparticles have a good inhibitory effect on both Escherichia coli and Staphylococcus aureus.

[0091] Example 11

[0092] Taking the lignin nanoparticle material prepared in Example 1 as an example, the antibacterial mechanism of the prepared nanoparticles was analyzed.

[0093] Prepare 10 mg mL with normal saline -1 500uL of lignin nanoparticles was added, and then the same volume of Escherichia coli and Staphylococcus aureus in the logarithmic growth phase was dropped into the culture medium. The culture was incubated at 37°C with shaking for 0h, 4h, and 12h, and samples were taken. After centrifugation at 8000rpm, the supernatant was discarded. The precipitate was washed three times with PBS solution, fixed with glutaraldehyde, and dehydrated with ethanol gradient. The morphology of the bacteria was observed by SEM. The culture was incubated at 37°C with shaking for 0h, 2h, 4h, 6h, 10h, and 12h, and samples were taken. After centrifugation at 8000rpm, the supernatant was taken and tested for alkaline phosphatase activity, conductivity, and nucleic acid content. At the same time, a bacterial solution without lignin nanoparticles was set as a blank control group.

[0094] The morphology of the bacteria photographed by SEM is as follows Figure 5As shown in the figure, at 0 hours, the cells were well-formed, with intact and smooth cell structures. At 4 hours, E. coli cells began to adhere to each other, with some cells deforming, while Staphylococcus aureus cells expanded and developed holes. After 12 hours, a large number of particles appeared on the surface of the E. coli cells, indicating that the cells had ruptured and their contents had spilled out, while the Staphylococcus aureus cells were further damaged and deformed. These results indicate that lignin nanoparticles can adhere to and disrupt the biofilms of E. coli and Staphylococcus aureus, thereby exerting an antibacterial effect.

[0095] The alkaline phosphatase activity, conductivity and nucleic acid content in the supernatant respectively indicate the damage of cell wall, permeability and damage of cell membrane. Figure 6 a in Figure 6 As shown in Figure b, compared with the blank control group, the alkaline phosphatase activity in the supernatant of Escherichia coli co-cultured with lignin nanoparticles increased rapidly within 2 hours and reached a peak value, while the alkaline phosphatase activity in the supernatant of Staphylococcus aureus co-cultured with lignin nanoparticles increased rapidly within 4 hours and reached a peak value. The results show that the destruction of the cell walls of Escherichia coli and Staphylococcus aureus by lignin nanoparticles is concentrated in 2 hours and 4 hours, respectively. The results of conductivity change over time are shown in Figure 3. Figure 6 In the c, Figure 6 As shown in Figure d, compared with the blank control group, the conductivity of the supernatant after co-culture of E. coli and Staphylococcus aureus with lignin nanoparticles increased significantly within 2 to 4 hours, indicating that the permeability of the cell membrane increased significantly during this period. Figure 6 The e in Figure 6 As shown in f. In the initial 2 hours, OD 260 No significant changes were observed. As time went on, the OD values of the control group decreased after 2 h. 260 A significant increase in the level of leukocytes indicates that the cell membrane has been damaged.

[0096] Example 12

[0097] Taking the lignin nanoparticle materials prepared in Examples 1-3 as an example, the antibacterial effects of lignin nanoparticles of different sizes were evaluated.

[0098] Prepare 5 mg mL with normal saline -1500 μL of lignin nanoparticles was added to the sample, followed by equal volumes of Escherichia coli and Staphylococcus aureus cultures in their logarithmic growth phases. The mixture was incubated at 37°C with shaking for 4 hours. 50 μL of the mixture was spread onto LB solid culture plates and incubated in a 37°C incubator for 12 hours. The plates were removed and the colony formation before and after antibacterial treatment was compared. The plate count method was used to calculate the inhibition ratio.

[0099] In this embodiment, the colony count before antibacterial treatment refers to the colony count without adding lignin nanoparticles, and the colony count after antibacterial treatment refers to the colony count with adding the lignin nanoparticles of the present invention.

[0100] The results of the antibacterial test on Escherichia coli and Staphylococcus aureus are shown in Figure 7 , the size of lignin nanoparticles significantly affects their antibacterial properties.

Claims

1. Application of lignin nanoparticles having a size between 20 and 400 nm in the preparation of an antibacterial agent, wherein the preparation method of the lignin nanoparticles comprises the following steps: 1) Dissolve lignin in deionized water and add hydrochloric acid to adjust the pH to 1-2 to obtain acid-precipitated lignin; 2) Wash the acid-precipitated lignin with distilled water, and control the pH value of the solution to be between 3 and 4 after washing; 3) Adding the washed acid-precipitated lignin to an organic solvent or a mixed solution of an organic solvent and water, stirring the reaction at room temperature, and then filtering and drying to obtain graded lignin; the concentration of the washed acid-precipitated lignin in the organic solvent or the mixed solvent of the organic solvent and water is 20-70 mg·mL -1 The reaction was stirred at room temperature for 8 h; the organic solvent was selected from one of acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol, dichloromethane, tetrahydrofuran, and dioxane; the volume ratio of the organic solvent to water was 1:4 to 4:1; 4) dissolving the fractionated lignin in an organic solvent or a mixed solvent of an organic solvent and water to form a fractionated lignin solution, adding the solution dropwise to distilled water at a uniform rate under stirring, or adding distilled water dropwise to the fractionated lignin solution at a uniform rate, and then rotary evaporating and drying to obtain lignin nanoparticles; the organic solvent is selected from one of acetone, methanol, ethanol, isopropanol, n-propanol, n-butanol, dichloromethane, tetrahydrofuran, and dioxane; the volume ratio of the organic solvent to water is 1:4 to 4:1; the organic solvent in step 3) and step 4) is the same; the volume ratio of the fractionated lignin solution to distilled water is 1:1 to 1:100; and the reaction mixture is stirred at 400 to 600 rpm at a rate of 2 to 8 mg·min -1 Add it dropwise into distilled water at a uniform speed, or add distilled water dropwise into the fractionated lignin solution at a uniform speed.

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