CN112961379B - Nano-cellulose/sodium alginate cryogel and preparation method and application thereof - Google Patents
Nano-cellulose/sodium alginate cryogel and preparation method and application thereof Download PDFInfo
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2301/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives
- C08J2301/02—Cellulose; Modified cellulose
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/04—Alginic acid; Derivatives thereof
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Abstract
The invention discloses a nanocellulose/sodium alginate cryogel and a preparation method and application thereof, which are prepared by utilizing a freezing-induced oxa-Michael reaction, and relates to the technical field of gel preparation. The preparation method comprises the following steps: adding sodium alginate into the nano-cellulose suspension, stirring until the sodium alginate is completely dissolved, adjusting the pH value to 11.0-13.0, adding vinylsulfone, stirring, then placing the mixture at the temperature of-12 to-20 ℃ for continuously reacting for more than 12 hours, and unfreezing and washing the mixture at room temperature after the reaction is finished to obtain the nano-cellulose suspension. The nano-cellulose/sodium alginate cryogel provided by the invention has excellent mechanical properties, does not need to add any catalyst in the preparation process, is simple in preparation method, and provides a new way for designing and developing a chromatographic medium based on natural polysaccharide. Meanwhile, the material is simple in preparation method, has good purification performance on lysozyme, and has wide popularization and application values.
Description
Technical Field
The invention relates to the field of sodium alginate, and in particular relates to a nanocellulose/sodium alginate cryogel and a preparation method and application thereof.
Background
At present, high performance chromatography media that can efficiently separate and adsorb proteins play an important role in the fields of biomedicine and bioseparation. The traditional protein separation and purification method is mainly carried out by a gel bead medium filled chromatographic column. However, the mechanical strength of the gel bead media is weak, which often results in deformation and stacking at high flow rates, which severely hampers the large-scale application of chromatographic techniques. Furthermore, gel bead packed chromatography media cannot handle unclarified viscous feed streams, such as blood, fermentation broths, and egg whites, without high back pressures. Therefore, there is an urgent need for new high performance chromatographic media with high mechanical strength, excellent reusability and macroporous structure to handle viscous feed streams. Monolithic cryogels have excellent mechanical properties due to the presence of interconnected macroporous 3D structures, exhibit great advantages in the separation of cells (mammalian, bacterial and yeast), proteins, viruses and plasmids, and at the same time have high elasticity and compressibility allowing high speed separation at low back pressure. The pores of the frozen gel remain open even at high pressures, allowing the feed stream to flow through without blockage. Currently, various integral cryogels based on natural polysaccharides have attracted extensive attention in academia and industry due to their environmental friendliness, versatility, ready availability, low cost, and the like.
Conventional monolithic polysaccharide-based cryogels can be prepared by a one-time or cyclic freeze-thaw process. Freeze-thaw induced gelation of polysaccharides is primarily due to physical cross-linking of molecular chains, however, physically cross-linked cryogels are unstable and severely limit their use in chromatographic media. However, for chemical cross-linking, the rate of chemical cross-linking plays a crucial role in the manufacture of cryogels. Rapid cross-linking can lead to aggregation and precipitation of the polysaccharide prior to ice crystal formation, thereby forming hydrogels and particles; while slow and insufficient chemical cross-linking will result in poor mechanical properties of the cryogel.
Disclosure of Invention
In view of the above, the present invention provides a controllable chemical crosslinking method for preparing a novel high performance chromatography medium with a macroporous structure, which is excellent in reusability, in order to overcome the problems of the prior art, and particularly provides a preparation method of a nanocellulose/sodium alginate cryogel.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, a method for preparing a nanocellulose/sodium alginate cryogel comprises: adding sodium alginate into the nano-cellulose suspension, stirring until the sodium alginate is completely dissolved, adjusting the pH value to 11.0-13.0, adding vinylsulfone, stirring, then placing the mixture at the temperature of-12 to-20 ℃ for continuously reacting for more than 12 hours, and unfreezing and washing the mixture at room temperature after the reaction is finished to obtain the nano-cellulose suspension.
The invention realizes the chemical crosslinking of the oryzanol, the sodium alginate and the vinyl sulfone crosslinking agent through a freezing induction reaction, namely, the pH of a reaction system is adjusted and is raised through freezing concentration, so that the oxa-Michael reaction between the vinyl sulfone and the nanocellulose and between the vinyl sulfone and the sodium alginate is activated, and the chemical crosslinking of the oryzanol, the sodium alginate and the vinyl sulfone crosslinking agent is realized. During the freezing step, the growing ice crystals force the reactants into the flake layer and chemical cross-linking occurs in the unfrozen/semi-frozen flakes, which is about 5% of the total volume, meaning that the concentration of reactants in the unfrozen/semi-frozen domains is increased more than ten times. In the case of the use of pH modifiers, cryoconcentration leads to an increase in the pH of the reaction zone, which enables the reaction to be activated and thus controlled chemical crosslinking.
The preparation method of the nano-cellulose/sodium alginate cryogel comprises the following steps:
(1) adding sodium alginate into a nano-cellulose suspension with the mass concentration of 0.10-0.75 wt%, and stirring until the sodium alginate is completely dissolved to obtain a mixed solution with the mass concentration of 1.0-2.5 wt%;
(2) adjusting the pH value of the mixed solution obtained in the step (1) to 11.0-13.0, adding vinyl sulfone until the mass concentration of the vinyl sulfone in the mixed solution is 1.00-1.75 wt%, stirring, and then placing the mixed solution at the temperature of-12-20 ℃ for continuous reaction for more than 12 hours;
(3) and (3) after the reaction in the step (2) is finished, unfreezing and washing at room temperature to obtain the nano-cellulose/sodium alginate cryogel.
In a preferred embodiment of the present invention, the nanocellulose is cellulose nanofiber or cellulose nanocrystal.
In a second aspect, a nanocellulose/sodium alginate cryogel wherein the nanocellulose, sodium alginate, respectively, undergo an oxa-Michael reaction with vinylsulphone to form a three-dimensional interconnected porous structure.
In a preferred embodiment of the present invention, the nanocellulose/sodium alginate cryogel is prepared by the above-mentioned preparation method.
In a third aspect, a nanocellulose/sodium alginate cryogel is used for adsorbing and purifying lysozyme.
Compared with the prior art, the invention has the following beneficial effects:
1. the preparation method provided by the invention is simple to operate, does not need any catalyst in the preparation process, is low in raw material price, wide in source, environment-friendly and good in biocompatibility, and meets the requirements of sustainable development.
2. The invention improves the traditional method for preparing the cryogel, and provides the osa-Michael addition reaction triggered by the increase of the pH value between the vinyl sulfone and the hydroxyl groups of the nano-cellulose and the sodium alginate for the first time.
3. The nanocellulose/sodium alginate cryogel prepared by the invention has excellent shape recovery under water and excellent fatigue resistance (almost no plastic deformation after 200 compression cycles under water).
4. The nano-cellulose/sodium alginate cryogel prepared by the invention can be dried under the atmospheric condition after being used for solvent exchange, still can keep the 3D structure thereof, has very low density of only 28kg/m3。
5. The invention also provides the application of the nano-cellulose/sodium alginate cryogel with excellent mechanical properties in the adsorption and purification of lysozyme, and the nano-cellulose/sodium alginate cryogel has good adsorption and purification properties on lysozyme. Has wide popularization and application value in the aspect of biological separation.
Drawings
FIG. 1 is a scanning electron micrograph of a cryogel prepared according to example 1;
FIG. 2 is a chart of the infrared spectrum of the cryogel prepared in example 1;
FIG. 3 is a graph of stress-strain variation versus water circulation 200 times for the cryogel prepared in example 1;
FIG. 4 is a graph showing the relationship between the amount of lysozyme adsorbed on the cryogel prepared in example 1 and the contact time of a 1mg/mL lysozyme solution;
FIG. 5 is a graph showing the adsorption of lysozyme to cryogels prepared at different amounts of Sodium Alginate (SA) based on example 1;
FIG. 6 is a schematic drawing showing the sequential purification of lysozyme from egg white solution using cryogels prepared in example 1.
Detailed Description
In an experiment of the invention, at room temperature, the dispersion liquid of the nano-cellulose, the sodium alginate and the vinyl sulfone with the pH value of 11 does not generate precipitation and gelation, which shows that no oxa-Michael reaction occurs when the pH value is 11; in another experiment of the present invention, aggregation and gelation were observed at room temperature immediately after addition of vinylsulfone to the dispersion by adjusting the dispersion pH to above 13 with NaOH. On the basis of these two experiments, we imagine that the freeze-induced chemical reaction, i.e. the increase in the pH of the system activated by freeze concentration, achieves chemical crosslinking of nanocellulose and sodium alginate with vinyl sulfone crosslinker at pH values higher than 11.
Therefore, the invention provides a preparation method of nano-cellulose/sodium alginate cryogel, which comprises the following steps: adding sodium alginate into the nano-cellulose suspension, stirring until the sodium alginate is completely dissolved, adjusting the pH value to 11.0-13.0, adding vinylsulfone, stirring, then placing the mixture at the temperature of-12 to-20 ℃ for continuously reacting for more than 12 hours, and unfreezing and washing the mixture at room temperature after the reaction is finished to obtain the nano-cellulose suspension.
The preparation method realizes chemical crosslinking of the oryzanol, the sodium alginate and the vinyl sulfone crosslinking agent through a freezing induction reaction, namely, the pH of a reaction system is adjusted and is increased through freezing concentration, so that the oxa-Michael reaction between the vinyl sulfone and the nanocellulose and between the vinyl sulfone and the sodium alginate is activated, and the chemical crosslinking of the oryzanol, the sodium alginate and the vinyl sulfone crosslinking agent is realized. This reaction does not or hardly occur at room temperature, and only when chemical cross-linking occurs at the frozen polysaccharide around the ice crystals, a firm cryogel with interconnected macropores and enhanced mechanical properties is formed. During the freezing step, the growing ice crystals force the reactants into the flake layer and chemical cross-linking occurs in the unfrozen/semi-frozen flakes, which is about 5% of the total volume, meaning that the concentration of reactants in the unfrozen/semi-frozen domains is increased more than ten times. In the case of the use of pH modifiers, cryoconcentration leads to an increase in the pH of the reaction zone, which enables the reaction to be activated and thus controlled chemical crosslinking.
Specifically, the preparation method of the nano-cellulose/sodium alginate cryogel comprises the following steps:
(1) adding sodium alginate into a nano-cellulose suspension with the mass concentration of 0.10-0.75 wt%, and stirring until the sodium alginate is completely dissolved to obtain a mixed solution with the mass concentration of 1.0-2.5 wt%;
(2) adjusting the pH value of the mixed solution obtained in the step (1) to 11.0-13.0, adding vinyl sulfone until the mass concentration of the vinyl sulfone in the mixed solution is 1.00-1.75 wt%, stirring, and then placing the mixed solution at the temperature of-12-20 ℃ for continuous reaction for more than 12 hours;
(3) and (3) after the reaction in the step (2) is finished, unfreezing and washing at room temperature to obtain the nano-cellulose/sodium alginate cryogel.
Preferably, wherein the nanocellulose is cellulose nanofibres or cellulose nanocrystals.
Scanning electron microscopy proves that the nano-cellulose/sodium alginate cryogel prepared by the method has a three-dimensional interconnected porous structure, can be used for treating flowing liquid, and is very suitable for practical application. Therefore, the invention further provides a nano-cellulose/sodium alginate cryogel, wherein the nano-cellulose and the sodium alginate respectively carry out oxa-Michael reaction with vinylsulfone to form a three-dimensional interconnected porous structure. Further experiments prove that the nano-cellulose/sodium alginate cryogel has good regeneration capacity and cycling application stability.
In addition, the invention also provides application of the nano-cellulose/sodium alginate cryogel in adsorption and purification of lysozyme.
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described in detail with reference to the following specific embodiments.
Example 1
(1) Taking 0.5g of sodium alginate in 20mL of 0.75 wt% cellulose nano-fiber (the sodium alginate is 2.5% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to 11.0, adding 0.35mL of vinyl sulfone (the vinyl sulfone is 1.75 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-12 ℃ for reaction for 12 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
FIG. 1 shows the SEM image of the nano-cellulose/sodium alginate cryogel prepared in this example. From the scanning electron micrograph, it can be observed that the interconnected macropores and lamellar walls of several tens of micrometers, in particular, the presence of many irregular micropores in the walls, whose diameter is about 3 to 7 μm, mean that the diffusion of the adsorbate is promoted. The special structure is different from the nano porous structure of the two-dimensional adsorbent hydrogel, the two-dimensional adsorbent cannot treat flowing liquid, and the three-dimensional interconnected porous structure forms a channel allowing the solution to freely flow, so that the method is very suitable for practical application.
FIG. 2 shows an infrared spectrum of the nanocellulose/sodium alginate cryogel produced in this example.
In order to examine the mechanical strength of the nanocellulose/sodium alginate cryogel, the invention performs an underwater cyclic compression experiment: the sample prepared in example 1 was cut into a cylindrical shape (diameter 10mm, height 15mm) and placed in a beaker filled with distilled water and subjected to 200 cycles of compression test in a universal tester at a compression rate of 20 mm/min. Fig. 3 shows a stress-strain variable relationship diagram of 200 times of underwater circulation of the nano-cellulose/sodium alginate cryogel, and it can be seen that the nano-cellulose/sodium alginate cryogel has almost no plastic deformation under 200 times of cyclic compression, and the ultra-strong fatigue resistance of the material is highlighted.
In order to examine the adsorption capacity of the nanocellulose/sodium alginate cryogel, the invention carries out a static lysozyme adsorption experiment: 0.05g of the dried sample of example 1 was taken and placed in 30mL of a lysozyme solution at 1mg/mL and pH 8, and adsorbed on a shaker at a constant temperature at room temperature for 4 hours. FIG. 4 is a graph showing the relationship between the amount of lysozyme adsorbed by the nanocellulose/sodium alginate cryogel and the adsorption time, and it can be seen that the adsorption capacity of the nanocellulose/sodium alginate cryogel to lysozyme increases with the increase of the adsorption time and rapidly reaches an equilibrium within 2 hours.
To examine the effect of the amount of sodium alginate on the adsorption capacity of the finally obtained nanocellulose/sodium alginate cryogel, we varied the amount of Sodium Alginate (SA) on the basis of example 1 to obtain different sodium alginates: (SA)SA) were used, and static lysozyme adsorption experiments were performed using these cryogels, the results of which are shown in fig. 5. As can be seen, the adsorption capacity of the cryogel to the lysozyme is gradually increased along with the increase of the dosage of the Sodium Alginate (SA), and the experimental adsorption capacity can reach 278mg g-1。
To investigate the adsorption stability of nanocellulose/sodium alginate cryogels, we also performed dynamic lysozyme cycling extraction purification experiments:
(1) separating egg white from fresh egg, and adding Na2HPO4Diluting to 50% (v/v) by (20mM, pH 8), and centrifuging at a speed of more than 7000r/min for 10-20 min to obtain an egg white solution.
(2) The cryogel prepared in example 1 was cut into a cylindrical shape having a height of 10mm (diameter of 10mm) and loaded into a plastic syringe, which was connected to a constant flow pump, and an egg white solution was slowly added at a flow rate of 10 mL/h. Subsequently, the frozen gel was washed with Na2HPO4(20mM, pH 8). Thereafter, the adsorbed lysozyme was eluted using a 1.0M NaCl solution, and the eluate was collected.
Then the process is circulated for a total of 6 times. As shown in fig. 6, the adsorption performance of the cryogel remained relatively stable over six cycles, indicating that the cryogel had good reproducibility and cycling application stability.
Example 2
(1) Taking 0.5g of sodium alginate in 20mL of 0.10 wt% cellulose nano-fiber (the sodium alginate accounts for 2.5% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be within the range of 11.0, adding 0.35mL of vinyl sulfone (the vinyl sulfone is 1.75 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-12 ℃ for reaction for 12 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
Example 3
(1) Taking 0.2g of sodium alginate in 20mL of 0.75 wt% cellulose nano-fiber (the sodium alginate is 1.0% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be within the range of 11.0, adding 0.35mL of vinyl sulfone (the vinyl sulfone is 1.75 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-20 ℃ for reaction for 16 h;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
Example 4
(1) Taking 0.5g of sodium alginate in 20mL of 0.75 wt% cellulose nano-fiber (the sodium alginate is 2.5% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be within the range of 11.0, adding 0.2mL of vinyl sulfone (the vinyl sulfone is 1.00 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-20 ℃ for reaction for 24 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
Example 5
(1) Taking 0.2g of sodium alginate in 20mL of 0.75 wt% cellulose nanocrystal (the sodium alginate accounts for 2.0% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be in a range of 13.0, adding 0.3mL of vinyl sulfone (the vinyl sulfone is 1.25 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-16 ℃ for reaction for 12 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
Example 6
(1) Taking 0.3g of sodium alginate in 20mL of 0.75 wt% cellulose nanocrystal (the sodium alginate is 1.5% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be within the range of 12.0, adding 0.35mL of vinyl sulfone (the vinyl sulfone is 1.75 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-12 ℃ for reaction for 30 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
Example 7
(1) Taking 0.1g of sodium alginate in 20mL of 0.10 wt% cellulose nano-fiber (the sodium alginate is 1.0% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be within the range of 11.0, adding 0.20mL of vinyl sulfone (the vinyl sulfone is 1.00 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-12 ℃ for reaction for 12 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
Example 8
(1) Taking 0.5g of sodium alginate in 20mL of 0.75 wt% cellulose nano-fiber (the sodium alginate is 2.5% of the total mass of the solution), and stirring by using a high-speed stirrer until the sodium alginate is completely dissolved;
(2) adjusting the pH value of the obtained mixed solution to be in a range of 13.0, adding 0.35mL of vinyl sulfone (the vinyl sulfone is 1.75 percent of the total mass of the solution), stirring, pouring into a mold, and placing in a low-temperature test box at-20 ℃ for reaction for 12 hours;
(3) and taking out the frozen gel, thawing the frozen gel at room temperature, and washing the frozen gel with a large amount of distilled water to prepare the nano-cellulose/sodium alginate frozen gel.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (6)
1. A preparation method of a nano-cellulose/sodium alginate cryogel is characterized by adding sodium alginate into a nano-cellulose suspension, stirring until the sodium alginate is completely dissolved, adjusting the pH value to be 11.0-13.0, adding vinyl sulfone, stirring, then placing the mixture in a temperature range of-12 to-20 ℃ for continuous reaction for more than 12 hours, and after the reaction is completed, unfreezing and washing at room temperature to obtain the nano-cellulose/sodium alginate cryogel.
2. The process for the preparation of nanocellulose/sodium alginate cryogel according to claim 1, characterized in that it comprises the following steps:
(1) adding sodium alginate into a nano-cellulose suspension with the mass concentration of 0.10-0.75 wt%, and stirring until the sodium alginate is completely dissolved to obtain a mixed solution with the mass concentration of 1.0-2.5 wt%;
(2) adjusting the pH value of the mixed solution obtained in the step (1) to be 11.0-13.0, adding 1.00-1.75 wt% of vinyl sulfone into the mixture, stirring, and then placing the mixed solution at the temperature of-12 to-20 ℃ for continuous reaction for more than 12 hours;
(3) and (3) after the reaction in the step (2) is finished, thawing and washing the mixture at room temperature to prepare the nano-cellulose/sodium alginate cryogel.
3. The process for the preparation of nanocellulose/sodium alginate cryogel according to claim 2, wherein in step (1) said nanocellulose is cellulose nanofibres or cellulose nanocrystals.
4. A nanocellulose/sodium alginate cryogel, wherein nanocellulose, sodium alginate and vinylsulphone undergo an oxa-Michael reaction to form a three-dimensional interconnected porous structure.
5. The nanocellulose/sodium alginate cryogel of claim 4, wherein said nanocellulose/sodium alginate cryogel is prepared by the method of any one of claims 1 to 3.
6. Use of a nanocellulose/sodium alginate cryogel as described in any one of claims 4 to 5 for the adsorption purification of lysozyme.
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