Preparation method of nanocellulose with uniform size

Abstract

The invention relates to a preparation method of nanocellulose, which comprises the following steps: grinding and screening microcrystalline cellulose, collecting and screening the microcrystalline cellulose, then adding the microcrystalline cellulose into an acetic acid-sodium acetate buffer solution for sterilization, after sterilization is completed, adding endo-cellulase for enzymolysis, and after enzymolysis is completed, inactivating the endo-cellulase to obtain the nanocellulose. After inactivation, centrifugally washing to obtain enzymolysis cellulose; 2, 2, 6, 6-tetramethylpiperidine-1-oxygen free radical oxidation modification is carried out on the cellulose subjected to enzymolysis, and oxidized cellulose is obtained; carrying out high-speed shearing emulsification on the oxidized cellulose to obtain emulsified cellulose; adding the emulsified cellulose into an aqueous solution of a deep-eutectic solvent DES formed by choline chloride and urea to swell the emulsified cellulose, performing high-pressure homogenization, and performing freeze drying after the high-pressure homogenization is completed to obtain the nano cellulose. According to the method, the size uniformity and dispersity of the nanoscale cellulose can be remarkably improved, and the size of the cellulose can be remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, and specifically relates to a method for preparing nanocellulose with uniform size. Background Technology

[0002] Natural polymer materials, especially cellulose, are ideal alternatives to petroleum-based plastics due to their abundant sources, high renewability, and biodegradability. Nanocellulose, as a novel bio-based nanomaterial, shows great potential in materials science, biomedicine, energy storage, and the food industry due to its high specific surface area, high strength, biodegradability, and functionalizability.

[0003] Existing methods for preparing nanocellulose mainly rely on mechanical and chemical methods. Mechanical methods physically cut cellulose raw materials into nanoscale fibers through high-pressure homogenization, ultrasonic treatment, and ball milling. This method is simple to operate but energy-intensive, and the resulting nanocellulose has a wide particle size distribution. Chemical methods involve hydrolyzing or oxidizing cellulose with acids or alkalis to destroy its crystalline regions and obtain nanocellulose. Common chemical methods include the chloric acid method, the nitric acid method, and the hydrogen peroxide method. Their advantage is that they can obtain relatively uniform particle sizes, but the particle sizes are still relatively large, and they generate large amounts of acidic wastewater, making subsequent treatment complex. Summary of the Invention

[0004] Based on the aforementioned shortcomings and deficiencies in the prior art, one of the objectives of this invention is to at least solve one or more of the aforementioned problems in the prior art. In other words, one of the objectives of this invention is to provide a method for preparing uniformly sized nanocellulose that meets one or more of the aforementioned requirements.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing uniformly sized nanocellulose includes the following steps: (1) Grind the microcrystalline cellulose and then sieve it through a filter screen to collect the sieved microcrystalline cellulose; (2) The sieved microcrystalline cellulose was added to an acetate-sodium acetate buffer solution and then sterilized. After sterilization, an endoglucosidase was added for enzymatic hydrolysis. After enzymatic hydrolysis, the endoglucosidase was inactivated. After inactivation, the cellulose was centrifuged and washed to obtain enzymatically hydrolyzed cellulose. (3) Add enzymatically hydrolyzed cellulose, 2,2,6,6-tetramethylpiperidine-1-oxy free radical and sodium bromide to water, and ultrasonically disperse them under ice bath conditions to obtain a reaction solution. Add sodium hypochlorite aqueous solution to the reaction solution, followed by sodium hydroxide aqueous solution. Stir continuously to carry out the oxidation reaction. After the oxidation reaction is completed, add ethanol to terminate the reaction. Then centrifuge and wash to obtain oxidized cellulose. (4) Add oxidized cellulose to water and disperse it by ultrasonication. After ultrasonic dispersion, perform high-speed shear emulsification to obtain emulsified cellulose. (5) Emulsified cellulose is added to an aqueous solution of DES, a eutectic solvent composed of choline chloride and urea, to swell the emulsified cellulose. After full swelling, it is centrifuged and washed, then added to water for ultrasonic dispersion. After ultrasonic dispersion, it is homogenized under high pressure. After high pressure homogenization, it is freeze-dried to obtain nanocellulose.

[0006] As a preferred embodiment, in step (1), the mesh size of the filter screen is 200 mesh.

[0007] As a preferred embodiment, in step (2), the sieved microcrystalline cellulose is added to an acetate-sodium acetate buffer solution to adjust the pH to 4.8-5.0; The sterilization conditions are: sterilize at 110-130℃ for 10-30 minutes.

[0008] As a preferred embodiment, in step (2), the weight ratio of sieved microcrystalline cellulose to endo-cellulase is 100:(0.5~1.5). The enzymatic hydrolysis temperature is 48–52℃, the time is 2.5–3 hours, and the stirring speed is 100–200 rpm. The inactivation temperature is 90–100℃, and the time is 5–15 min.

[0009] As a preferred embodiment, in step (3), the weight ratio of enzymatically hydrolyzed cellulose, 2,2,6,6-tetramethylpiperidine-1-oxygen radical, sodium bromide, and water is (0.5-1.5):(0.01-0.02):(0.05-0.15):100; The volume ratio of the reaction solution to the 10% sodium hypochlorite aqueous solution is 100:(3-4). The concentration of the sodium hydroxide aqueous solution is 0.1-0.2M. Add the sodium hydroxide aqueous solution dropwise until the pH is adjusted to 10-10.5.

[0010] As a preferred embodiment, in step (3), the oxidation reaction time is 2.5 to 3 hours.

[0011] As a preferred option, in step (3), the ice bath conditions are 2 to 4°C.

[0012] As a preferred embodiment, in step (4), the rotation speed of high-speed shear emulsification is 10000-15000 rpm, the temperature of the cooling circulation system is set to 4-15℃, and the shear emulsification time is 10-15 min; After shearing and emulsification, the mixture was filtered through a 100μm filter to obtain emulsified cellulose.

[0013] As a preferred embodiment, in step (5), the volume concentration of the aqueous solution of the eutectic solvent DES is 20-40%.

[0014] As a preferred option, in step (5), high-pressure homogenization is carried out in a high-pressure homogenizer. The cooling circulation system of the high-pressure homogenizer is set to control the temperature at 4-15°C, the initial pressure is 50-80 MPa, and the homogenization is repeated 1-3 times. Then the pressure is gradually increased by 20-30 MPa each time. After several cycles, samples are taken for testing. The final pressure of pressurization shall not exceed 150 MPa.

[0015] Compared with the prior art, the beneficial effects of this invention are: This invention prepares nanoscale powdered cellulose through enzymatic pretreatment, 2,2,6,6-tetramethylpiperidine-1-oxy radical oxidation modification, ultrasonic dispersion and high-speed shear emulsification, DES aqueous solution lubrication and swelling, high-pressure homogenization, and freeze drying. This method can significantly improve the size uniformity and dispersibility of nanoscale cellulose and reduce its size. At the same time, the enzymatic pretreatment and DES aqueous solution lubrication and swelling can effectively reduce the number of high-pressure homogenization cycles and the homogenization pressure, and support subsequent grafting reactions. It is suitable for drug carriers and fluorescent labeling, and meets the needs of subsequent reprocessing. Attached Figure Description

[0016] Figure 1 This is a Zeta potential diagram of the nanocellulose of Example 1 of the present invention; Figure 2 This is a Zeta potential diagram of the nanocellulose in Example 2 of the present invention; Figure 3 This is a Zeta potential diagram of the nanocellulose of Comparative Example 1 of the present invention; Figure 4 This is a Zeta potential diagram of the nanocellulose of Comparative Example 2 of the present invention; Figure 5 This is a Zeta potential diagram of the nanocellulose of Comparative Example 3 of the present invention; Figure 6 This is a Zeta potential diagram of the nanocellulose of Comparative Example 4 of the present invention; Figure 7 This is a Zeta potential diagram of the nanocellulose of Comparative Example 5 of the present invention; Figure 8 Here is a SEM image of the nanocellulose from Example 1 of this invention; Figure 9 Here is a SEM image of the nanocellulose from Example 2 of this invention; Figure 10 This is a SEM image of the nanocellulose of Comparative Example 1 of the present invention. Figure 11This is a SEM image of the nanocellulose of Comparative Example 2 of the present invention; Figure 12 This is a SEM image of the nanocellulose of Comparative Example 3 of the present invention. Figure 13 This is a SEM image of the nanocellulose of Comparative Example 4 of this invention. Figure 14 This is a SEM image of the nanocellulose of Comparative Example 5 of the present invention. Detailed Implementation

[0017] The preparation method of nanocellulose of the present invention will be described in detail below.

[0018] The method for preparing uniformly sized nanocellulose according to the present invention includes the following steps: (1) Grind the microcrystalline cellulose and then sieve it through a filter screen to collect the sieved microcrystalline cellulose; (2) The sieved microcrystalline cellulose was added to an acetate-sodium acetate buffer solution and then sterilized. After sterilization, an endoglucosidase was added for enzymatic hydrolysis. After enzymatic hydrolysis, the endoglucosidase was inactivated. After inactivation, the cellulose was centrifuged and washed to obtain enzymatically hydrolyzed cellulose. (3) Add enzymatically hydrolyzed cellulose, 2,2,6,6-tetramethylpiperidine-1-oxy free radical and sodium bromide to water, and ultrasonically disperse them under ice bath conditions to obtain a reaction solution. Add sodium hypochlorite aqueous solution to the reaction solution, followed by sodium hydroxide aqueous solution. Stir continuously to carry out the oxidation reaction. After the oxidation reaction is completed, add ethanol to terminate the reaction. Then centrifuge and wash to obtain oxidized cellulose. (4) Add oxidized cellulose to water and disperse it by ultrasonication. After ultrasonic dispersion, perform high-speed shear emulsification to obtain emulsified cellulose. (5) Emulsified cellulose is added to an aqueous solution of DES, a eutectic solvent composed of choline chloride and urea, to swell the emulsified cellulose. After full swelling, it is centrifuged and washed, then added to water for ultrasonic dispersion. After ultrasonic dispersion, it is homogenized under high pressure. After high pressure homogenization, it is freeze-dried to obtain nanocellulose.

[0019] In one or more embodiments, the mesh size of the filter screen in step (1) above is 200 to 300 mesh, which can be determined according to the actual application requirements.

[0020] In one or more embodiments, in step (2) above, sieved microcrystalline cellulose is added to an acetate-sodium acetate buffer solution and the pH is adjusted to 4.8 to 5.0. The specific pH can be determined according to the actual application requirements. The sterilization conditions are: sterilize at 110-130℃ for 10-30 minutes; the specific sterilization temperature and time can be determined according to actual application requirements. In one or more embodiments, in step (2) above, the weight ratio of sieved microcrystalline cellulose to endo-cellulase is 100:(0.5-1.5). The enzymatic hydrolysis temperature is 48–52℃, the time is 2.5–3 hours, and the stirring speed is 100–200 rpm. The inactivation temperature is 90–100℃, and the time is 5–15 min; The above-mentioned weight ratios, enzymatic hydrolysis, and inactivation parameters can be determined according to actual application requirements.

[0021] In one or more embodiments, in step (3) above, the weight ratio of enzymatically hydrolyzed cellulose, 2,2,6,6-tetramethylpiperidine-1-oxy radical, sodium bromide, and water is (0.5-1.5):(0.01-0.02):(0.05-0.15):100; The volume ratio of the reaction solution to the 10% sodium hypochlorite aqueous solution is 100:(3-4). The concentration of the sodium hydroxide aqueous solution is 0.1–0.2 M. Add the sodium hydroxide aqueous solution dropwise until the pH is adjusted to 10–10.5. The above weight ratio, volume ratio, concentration, and pH can be determined according to the actual application requirements.

[0022] In one or more embodiments, the oxidation reaction time in step (3) above is 2.5 to 3 hours, and the specific time can be determined according to the actual application requirements.

[0023] In one or more embodiments, the ice bath conditions in step (3) above are 2 to 4°C, and the specific temperature can be determined according to the actual application requirements.

[0024] In one or more embodiments, the high-speed shear emulsification rotation speed in step (4) above is 10,000 to 15,000 rpm, the cooling circulation system temperature is set to 4 to 15°C, and the shear emulsification time is 10 to 15 minutes; the specific rotation speed, temperature and time can be determined according to the actual application requirements. After shearing and emulsification, the mixture was filtered through a 100μm filter to obtain emulsified cellulose.

[0025] In one or more embodiments, the volume concentration of the aqueous solution of the eutectic solvent DES in step (5) above is 20-40%, and the specific volume concentration can be determined according to the actual application requirements.

[0026] In one or more embodiments, the high-pressure homogenization in step (5) above is carried out in a high-pressure homogenizer. The cooling circulation system of the high-pressure homogenizer is set to control the temperature at 4-15°C, the initial pressure is 50-80 MPa, and the homogenization is repeated 1-3 times. Then the pressure is gradually increased by 20-30 MPa each time. After several cycles, samples are taken for testing. The specific pressure, temperature, pressure increase, and number of homogenization cycles can be determined according to the actual application requirements. The final pressure of pressurization shall not exceed 150 MPa.

[0027] The preparation method of nanocellulose of the present invention will be further explained and illustrated below through specific embodiments.

[0028] Example 1: The preparation method of nanocellulose in this embodiment includes the following steps: (1) Take 2 kg of microcrystalline cellulose and add it to the dry grinding mill. Set the dry grinding particle size to 200 mesh and dry grind for 30 min. After dry grinding, pass it through a 200 mesh filter screen and collect the sieved microcrystalline cellulose. Add the unsieved cellulose back to the dry grinding mill and dry grind it again for 15 min. After dry grinding, pass it through a 200 mesh filter screen and collect the sieved microcrystalline cellulose. (2) Take 1 kg of sieved microcrystalline cellulose and add it to 50 kg of acetate-sodium acetate buffer solution. Adjust the pH to 5.0 and then transfer it to an autoclave. Sterilize at 121℃ for 20 min. After sterilization, allow it to cool to room temperature. Add 10 g of endo-1,4-β-glucanase. Set the stirrer temperature to 52℃ and the speed to 200 rpm. Stir and hydrolyze for 3 h. After hydrolysis, transfer it to a water bath and inactivate the endo-1,4-β-glucanase by water bathing at 95℃ for 10 min. Then centrifuge at 8000 rpm for 10 min. After centrifugation, remove the supernatant and wash the cellulose with deionized water until neutral to obtain hydrolyzed cellulose. (3) Take 800g of enzymatically hydrolyzed cellulose, 12.6g of 2,2,6,6-tetramethylpiperidine-1-oxy free radical, and 80g of sodium bromide and add them to 80kg of deionized water. Then transfer them to an ultrasonic disperser, maintain the temperature at 4℃ in an ice bath, set the ultrasonic frequency to 40kHz, and ultrasonically disperse for 5min. After ultrasonic dispersion, continue to maintain the reaction temperature at 4℃ in an ice bath, turn on the stirring device of the reaction vessel, set the stirring speed to 300rpm, and stir in an ice bath for 15min to obtain the reaction solution. Take 80L of the reaction solution and add 2.976L of 10% sodium hypochlorite aqueous solution dropwise over 5min using an automatic titrator. Then immediately replace the automatic titrator and titrate with 0.1M sodium hydroxide solution to control the pH to 10.5. Continue stirring and oxidize for 3h. After oxidation, add 8L of ethanol to terminate the reaction and continue stirring for 10min. After stirring, transfer the solution to a centrifuge that has been pre-cooled to 4℃ and centrifuge at 8000rpm for 10min. After centrifugation, remove the supernatant and wash with deionized water until neutral to obtain oxidized cellulose. (4) Take 860g of oxidized cellulose and add it to 86kg of deionized water. Transfer it to an ultrasonic disperser and ultrasonically disperse it at 20kHz for 5min. After ultrasonic dispersion, transfer it to a high-speed shear emulsifier. Set the speed of the high-speed shear emulsifier to 10000rpm and the temperature of the cooling circulation system to 15℃. Shear emulsify for 10min to obtain emulsified cellulose. Then filter the emulsified cellulose using a 100μm filter screen. (5) Add the emulsified cellulose obtained by shearing and emulsification and filtration to 4.3 kg of 20% choline chloride: urea DES aqueous solution, stir at 60°C for 30 minutes. The DES aqueous solution gently penetrates into the surface of the amorphous and crystalline regions of cellulose and inserts into the cellulose molecular chains, which acts as a lubricant and swelling agent, making the cellulose swell and loosen the structure and increase the porosity. After stirring, transfer to a centrifuge that has been pre-cooled to 4°C and centrifuge at 8000 rpm for 10 minutes. After centrifugation, remove the supernatant and wash with deionized water until neutral to obtain the swollen emulsified cellulose. (6) Take 850g of swollen emulsified cellulose, add it to 85kg of deionized water, transfer it to an ultrasonic disperser, and ultrasonically disperse it at 20kHz for 5min. After ultrasonic dispersion, transfer the emulsified cellulose to a high-pressure homogenizer, set the pressure of the high-pressure homogenizer to 80MPa, set the temperature of the cooling circulation system to 15℃, and homogenize it under high pressure twice. Then gradually increase the pressure by 30MPa each time. After two cycles, take a sample for testing. Homogenize it for a total of 16 cycles (i.e., homogenize it under 80MPa twice, 110MPa twice, 140MPa twice, and 150MPa ten times). The final pressure is 150MPa. Pause for 10min after every two cycles. After homogenization, immediately transfer it to a 4℃ refrigerator for storage to obtain the cellulose solution. (7) Transfer the cellulose solution to the quick-freezing chamber, set the temperature of the quick-freezing chamber to -80℃, pre-freeze for 30 min, and after the pre-freezing is completed, transfer it to the vacuum drying chamber, verify the integrity of the chamber door seal, ensure the normal operation of the freeze-drying chamber, adjust the vacuum degree to 40 Pa, and maintain the temperature at -80℃ for vacuum freeze-drying for 8 h to obtain powdered nanocellulose.

[0029] Example 2: The preparation method of nanocellulose in this embodiment includes the following steps: (1) Take 1 kg of microcrystalline cellulose and add it to the dry grinding mill. Set the dry grinding particle size to 300 mesh and dry grind for 30 min. After dry grinding, pass it through a 300 mesh filter screen and collect the sieved microcrystalline cellulose. Add the unsieved cellulose back to the dry grinding mill and dry grind it again for 15 min. After dry grinding, pass it through a 300 mesh filter screen and collect the sieved microcrystalline cellulose. (2) Take 500g of sieved microcrystalline cellulose and add it to 25kg of acetate-sodium acetate buffer solution. Adjust the pH to 4.8 and then transfer it to an autoclave. Sterilize at 130℃ for 10min. After sterilization, allow it to cool to room temperature. Add 7.5g of endo-1,4-β-glucanase. Set the stirrer temperature to 52℃ and the speed to 100rpm. Stir and hydrolyze for 2.5h. After hydrolysis, transfer it to a water bath and inactivate the endo-1,4-β-glucanase at 95℃ for 10min. Then centrifuge at 8000rpm for 10min. After centrifugation, remove the supernatant and wash the cellulose with deionized water until neutral to obtain hydrolyzed cellulose. (3) Take 400g of enzymatically hydrolyzed cellulose, 8g of 2,2,6,6-tetramethylpiperidine-1-oxy free radical, and 40g of sodium bromide and add them to 40kg of deionized water. Then transfer them to an ultrasonic disperser, maintain the temperature at 2℃ in an ice bath, set the ultrasonic frequency to 40kHz, and ultrasonically disperse for 5min. After ultrasonic dispersion, continue to maintain the reaction temperature at 2℃ in an ice bath, turn on the stirring device of the reaction vessel, set the stirring speed to 300rpm, and stir in an ice bath for 15min to obtain the reaction solution. Take 40L of the reaction solution and add 1.6L of 10% sodium hypochlorite aqueous solution dropwise over 5min using an automatic titrator. Then immediately replace the automatic titrator and titrate with 0.2M sodium hydroxide solution to control the pH to 10. Continue stirring and oxidize for 2.5h. After oxidation, add 8L of ethanol to terminate the reaction and continue stirring for 10min. After stirring, transfer the solution to a centrifuge that has been pre-cooled to 2℃ and centrifuge at 8000rpm for 10min. After centrifugation, remove the supernatant and wash with deionized water until neutral to obtain oxidized cellulose. (4) Take 440g of oxidized cellulose and add it to 44kg of deionized water. Transfer it to an ultrasonic disperser and ultrasonically disperse it at 20kHz for 5min. After ultrasonic dispersion, transfer it to a high-speed shear emulsifier. Set the speed of the high-speed shear emulsifier to 15000rpm and the temperature of the cooling circulation system to 15℃. Shear emulsify for 10min to obtain emulsified cellulose. Then filter the emulsified cellulose using a 100μm filter screen. (5) Add the emulsified cellulose obtained by shearing and emulsification and filtration to 2.2 kg of 40% choline chloride: urea DES aqueous solution, stir at 60°C for 30 minutes. The DES aqueous solution gently penetrates into the surface of the amorphous and crystalline regions of cellulose, inserts into the cellulose molecular chains, and acts as a lubricant and swelling agent, making the cellulose swell and loosen the structure and increase the porosity. After stirring, transfer to a centrifuge that has been pre-cooled to 2°C, centrifuge at 8000 rpm for 10 minutes, remove the supernatant after centrifugation, and wash with deionized water until neutral to obtain the swollen emulsified cellulose. (6) Take 430g of swollen emulsified cellulose, add it to 43kg of deionized water, transfer it to an ultrasonic disperser, and ultrasonically disperse it at 20kHz for 5min. After ultrasonic dispersion, transfer the emulsified cellulose to a high-pressure homogenizer, set the pressure of the high-pressure homogenizer to 60MPa, set the temperature of the cooling circulation system to 15℃, and homogenize it under high pressure for 3 times. Then gradually increase the pressure by 20MPa each time. After 2 cycles, take a sample for testing. Homogenize it for a total of 16 cycles (i.e., homogenize it under 60MPa for 3 times, under 80MPa for 2 times, under 100MPa for 2 times, under 120MPa for 2 times, under 140MPa for 2 times, and under 150MPa for 5 times). The final pressure is 150MPa. Pause for 10min after every 2 cycles. After homogenization, immediately transfer it to a 4℃ refrigerator for storage to obtain the cellulose solution. (7) Transfer the cellulose solution to the quick-freezing chamber, set the temperature of the quick-freezing chamber to -80℃, pre-freeze for 30 min, and after the pre-freezing is completed, transfer it to the vacuum drying chamber, verify the integrity of the chamber door seal, ensure the normal operation of the freeze-drying chamber, adjust the vacuum degree to 40 Pa, and maintain the temperature at -80℃ for vacuum freeze-drying for 8 h to obtain powdered nanocellulose.

[0030] Comparative Example 1: The preparation method of the nanocellulose in this comparative example differs from that in Example 1 in that: The enzymatic hydrolysis step in step (2) is omitted, that is, the oxidation in step (3) is carried out directly using sieved microcrystalline cellulose; The other steps are the same as in Example 1.

[0031] Comparative Example 2: The preparation method of the nanocellulose in this comparative example differs from that in Example 1 in that: The oxidation step in step (3) is omitted, that is, the enzymatically hydrolyzed cellulose obtained in step (2) is directly used in the shearing emulsification in step (4); The other steps are the same as in Example 1.

[0032] Comparative Example 3: The preparation method of the nanocellulose in this comparative example differs from that in Example 1 in that: The order of steps is reversed between the swelling treatment in step (5) and the shear emulsification in step (4). That is, the oxidized cellulose obtained in step (3) is swollen first, and then shear emulsification is performed. The other steps are the same as in Example 1.

[0033] Comparative Example 4: The preparation method of the nanocellulose in this comparative example differs from that in Example 1 in that: In step (5), the volume concentration of the aqueous solution of choline chloride:urea DES is adjusted to 10%. The other steps are the same as in Example 1.

[0034] Comparative Example 5: The preparation method of the nanocellulose in this comparative example differs from that in Example 1 in that: In step (5), the volume concentration of the aqueous solution of choline chloride:urea DES is adjusted to 60%. The other steps are the same as in Example 1.

[0035] The intermediate and final products in the preparation process of nanocellulose in Examples 1-2 and Comparative Examples 1-5 are tested and analyzed below: I. Cellulose pulp from Examples 1, 2, 1, 2, 3, 4 and 5 before high-pressure homogenization was placed under an atomic force microscope to observe the cellulose diameter distribution, length distribution and fiber state. The results are shown in Table 1.

[0036] Table 1 Results of atomic force microscopy observations

[0037] .

[0038] 2. Nanoscale powdered cellulose prepared in Examples 1, 2, 1, 2, 3, 4 and 5 were placed under an atomic force microscope to observe the diameter distribution, length distribution and morphological characteristics of the cellulose. The results are shown in Table 2.

[0039] Table 2 Results of Atomic Force Microscopy

[0040] .

[0041] Analysis of Tables 1 and 2 shows that, due to the synergistic application of enzymatic hydrolysis and oxidation, combining the high selectivity of biocatalysis and the potent effects of chemical oxidation, enzymatic hydrolysis cleaves long-chain cellulose, exposing more internal hydroxyl groups and providing new reaction sites for oxidation. After shearing and emulsification, a 20% (v / v) concentration of choline chloride:urea DES aqueous solution is used for further lubrication and swelling, making the cellulose structure looser and weakening its internal binding force, thus providing optimal conditions for subsequent high-pressure homogenization. Furthermore, as... Figures 8 to 14 As shown, the nanocellulose prepared in Examples 1 and 2 has a better diameter range, length range, and morphological characteristics than the nanocellulose prepared in Comparative Examples 1, 2, 3, 4, and 5, and its size is more uniform.

[0042] III. Surface chemical property testing; 50 mg of nano-sized powdered cellulose prepared in Examples 1, 2, 1, 2, 3, 4 and 5 were respectively shaken and adsorbed with 0.1 mM methylene blue solution for 30 min. Then, the supernatant was centrifuged at 6000 rpm and the absorbance was measured at 664 nm. At the same time, a standard curve was prepared using standard and a blank control was used with 0.1 mM methylene blue solution. The amount of unadsorbed MB was calculated according to the standard curve to obtain the carboxyl content. Nanoscale powdered cellulose prepared in Examples 1, 2, 1, 2, 3, 4 and 5 was gently mixed with PBS buffer to prepare a 0.01% cellulose solution. The solution was then dialyzed at 4°C for 5 hours to remove free ions. After dialysis, the surface charge was measured using a Malvern Zetasizerdan potentiometer.

[0043] Table 3 Surface chemical property test results

[0044] .

[0045] By analyzing Table 3 and as shown in the figure Figures 1 to 7 As shown, the nano-sized powdered cellulose prepared in Examples 1 and 2 has a good carboxyl content and a moderate zeta potential, making it less prone to agglomeration. In Comparative Example 5, the internal network strength of the fiber weakens due to partial dissolution and gelation, and the structure collapses after drying, resulting in a decrease in negative potential and easy agglomeration.

[0046] IV. Thermal stability test; 10 mg of the nano-sized powdered cellulose prepared in Examples 1, 2, 1, 2, 3, 4 and 5 were taken respectively, spread evenly in a crucible, and the heating rate was set to 10℃ / min. The thermal stability of the nano-sized powdered cellulose was tested by heating, and the results are shown in Table 4.

[0047] Table 4 Thermal stability test results

[0048] .

[0049] Analysis of Table 4 shows that the nano-sized powdered cellulose prepared in Examples 1 and 2 has good crystallinity and thermal stability. Comparative Example 2, because it only involved enzymatic hydrolysis without oxidation, had low crystallinity and a high apparent onset temperature, but poor overall structural strength and was not heat-resistant. The high decomposition temperature was due to the lack of oxidation, resulting in a large number of unstable low-molecular-weight segments, defect sites, and amorphous regions generated by enzymatic hydrolysis. These regions have poor thermal stability, but because they are disordered, they require accumulated heat to trigger a chain decomposition reaction, hence the seemingly higher onset temperature. However, the residual rate shows that Comparative Example 2 ultimately retained more carbon at high temperatures, indicating that after high-temperature "calcination," the defective structures formed more stable carbon. The disordered defective structures generated by enzymatic hydrolysis in Comparative Example 2 are prone to cross-linking and rearrangement at high temperatures, following a deep carbonization path, thus leaving more residual carbon. The sodium carboxylate introduced by the TEMPO oxidation in Comparative Example 1 acts as a catalyst, guiding the decomposition towards a path that preferentially depolymerizes to generate volatile products, thus resulting in the "cleanest" product with the least residual carbon. This invention (enzymatic hydrolysis + oxidation) represents a balance and compromise between two pathways: the defects of enzymatic hydrolysis pre-processing provide conditions for carbonization, but the uniformly distributed sodium carboxylate on the surface simultaneously promotes depolymerization, ultimately resulting in a residual char content between the two. From the perspective of residual rate, through the synergy of enzymatic hydrolysis pre-shearing and oxidative modification, the thermal degradation pathway of nanocellulose is precisely controlled to an ideal balance point, avoiding premature collapse due to excessive catalytic depolymerization and preventing deep carbonization due to structural disorder. This means that the material can form a better interface in composite material processing, while retaining the potential of the carbon skeleton for subsequent functionalization. Therefore, the crystal structure of the above embodiments is more uniform and the decomposition is more thorough. Comparative Example 5, due to the partial dissolution and gelation of DES, is easier to decompose but also easier to form carbon.

[0050] Given that there are numerous embodiments of the present invention, and the raw materials, their dosages, and process parameters involved can all be selected within a limited range according to actual needs, and the experimental data for each embodiment are extensive and numerous, it is not suitable to list and describe them one by one here. However, the content to be verified and the final conclusions obtained in each embodiment are similar. Therefore, the verification content of each embodiment will not be described one by one here.

[0051] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing uniformly sized nanocellulose, characterized in that, Includes the following steps: (1) Grind the microcrystalline cellulose and then sieve it through a filter screen to collect the sieved microcrystalline cellulose; (2) The sieved microcrystalline cellulose was added to an acetate-sodium acetate buffer solution and then sterilized. After sterilization, an endoglucosidase was added for enzymatic hydrolysis. After enzymatic hydrolysis, the endoglucosidase was inactivated. After inactivation, the cellulose was centrifuged and washed to obtain enzymatically hydrolyzed cellulose. (3) Add enzymatically hydrolyzed cellulose, 2,2,6,6-tetramethylpiperidine-1-oxy free radical and sodium bromide to water, and ultrasonically disperse them under ice bath conditions to obtain a reaction solution. Add sodium hypochlorite aqueous solution to the reaction solution, followed by sodium hydroxide aqueous solution. Stir continuously to carry out the oxidation reaction. After the oxidation reaction is completed, add ethanol to terminate the reaction. Then centrifuge and wash to obtain oxidized cellulose. (4) Add oxidized cellulose to water and disperse it by ultrasonication. After ultrasonic dispersion, perform high-speed shear emulsification to obtain emulsified cellulose. (5) Emulsified cellulose is added to an aqueous solution of DES, a eutectic solvent composed of choline chloride and urea, to swell the emulsified cellulose. After full swelling, it is centrifuged and washed, then added to water for ultrasonic dispersion. After ultrasonic dispersion, it is homogenized under high pressure. After high pressure homogenization, it is freeze-dried to obtain nanocellulose.

2. The preparation method according to claim 1, characterized in that, In step (1), the mesh size of the filter sieve is 200 to 300 mesh.

3. The preparation method according to claim 1, characterized in that, In step (2), the sieved microcrystalline cellulose is added to the acetate-sodium acetate buffer solution and the pH is adjusted to 4.8-5.0; The sterilization conditions are: sterilize at 110-130℃ for 10-30 minutes.

4. The preparation method according to claim 1, characterized in that, In step (2), the weight ratio of sieved microcrystalline cellulose to endo-cellulase is 100:(0.5-1.5). The enzymatic hydrolysis temperature is 48–52℃, the time is 2.5–3 hours, and the stirring speed is 100–200 rpm. The inactivation temperature is 90–100℃, and the time is 5–15 min.

5. The preparation method according to claim 1, characterized in that, In step (3), the weight ratio of enzymatically hydrolyzed cellulose, 2,2,6,6-tetramethylpiperidine-1-oxygen radical, sodium bromide, and water is (0.5-1.5):(0.01-0.02):(0.05-0.15):

100. The volume ratio of the reaction solution to the 10% sodium hypochlorite aqueous solution is 100:(3-4). The concentration of the sodium hydroxide aqueous solution is 0.1-0.2M. Add the sodium hydroxide aqueous solution dropwise until the pH is adjusted to 10-10.

5.

6. The preparation method according to claim 1, characterized in that, In step (3), the oxidation reaction takes 2.5 to 3 hours.

7. The preparation method according to claim 1, characterized in that, In step (3), the ice bath conditions are 2-4℃.

8. The preparation method according to claim 1, characterized in that, In step (4), the rotation speed of high-speed shear emulsification is 10,000 to 15,000 rpm, the temperature of the cooling circulation system is set to 4 to 15°C, and the shear emulsification time is 10 to 15 minutes. After shearing and emulsification, the mixture was filtered through a 100μm filter to obtain emulsified cellulose.

9. The preparation method according to claim 1, characterized in that, In step (5), the volume concentration of the aqueous solution of the eutectic solvent DES is 20-40%.

10. The preparation method according to claim 1, characterized in that, In step (5), high-pressure homogenization is carried out in a high-pressure homogenizer. The cooling circulation system of the high-pressure homogenizer is set to control the temperature at 4-15℃, the initial pressure is 50-80MPa, and the homogenization is repeated 1-3 times. Then the pressure is gradually increased by 20-30MPa each time. After several cycles, samples are taken for testing. The final pressure of pressurization shall not exceed 150 MPa.

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