Method for preparing temperature response type cellulose nanofiber by using endoglucanase

Abstract

The invention discloses a method for preparing temperature response type cellulose nanofibers by using endoglucanase, which comprises the following steps: (1) grafting microcrystalline cellulose with sulfobetaine fragments under an alkaline condition to obtain modified microcrystalline cellulose MCC-S; (2) adding the modified microcrystalline cellulose MCC-S obtained in the step (1) and single endoglucanase into a buffer solution with the pH value of 4-6, and reacting at the temperature of 40-60 DEG C for 3-15 hours to obtain an enzymolysis product; and (3) carrying out ultrasonic treatment on the enzymolysis product obtained in the step (2), carrying out centrifugal separation, and collecting an upper-layer suspension, so as to obtain the cellulose nanofiber CNF-S with the temperature response characteristic. According to the method, the cellulose nanofiber is prepared by hydrolyzing microcrystalline cellulose by utilizing single endoglucanase for the first time, so that the cellulose nanofiber (CNF-S) with temperature-sensitive response and long length-diameter ratio is prepared in a green manner under a mild condition, and UCST response is given to the cellulose nanofiber.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing temperature-responsive cellulose nanofiber by endoglucanase, in particular, a method for preparing cellulose nanofiber with high upper critical solution temperature (UCST) response by endoglucanase catalytic hydrolysis of modified microcrystalline cellulose (MCC-S), belonging to the technical field of new materials. BACKGROUND

[0002] As a kind of surfactant, cellulose nanofiber has many advantages such as high crystallinity, high specific surface area, high Young's modulus and biocompatibility, and is widely used in Pickering emulsion (including temperature-responsive Pickering emulsion). The aspect ratio is the main factor affecting the stability of cellulose nanofiber (CNF) in Pickering emulsion. The three-dimensional network structure formed by particles with large aspect ratio is stable. The CNF used to stabilize the emulsion is generally 5-30 nm wide, and the aspect ratio is greater than 50, which is in the form of filament.

[0003] The common methods for preparing cellulose nanofiber (CNF) include mechanical method, acid hydrolysis method and enzyme hydrolysis method, etc. The cellulose nanofiber is prepared by mechanical method, which is simple to operate and does not need to add additional acid and alkali reagents, but has high energy consumption. The cellulose nanofiber is prepared by acid hydrolysis method, which has relatively low cost and good uniformity of CNF size, but this method needs to add acid and alkali and other chemical reagents, which will cause damage to the equipment and environment. The enzyme hydrolysis method has mild reaction conditions and meets the standard of green preparation.

[0004] The enzyme hydrolysis method for preparing CNF usually uses cellulase, which is a group of enzymes that can degrade cellulose to produce glucose with synergistic effect, and is not a single enzyme. Its multi-component enzyme system mainly includes exoglucanase, endoglucanase and β-glucosidase. Endoglucanase acts on the inside of cellulose chain, randomly cuts glycosidic bond to produce new reducing end and non-reducing end; exoglucanase hydrolyzes from these exposed chain ends (mainly reducing end) to release oligosaccharides such as cellobiose; finally, β-glucosidase hydrolyzes oligosaccharides (mainly cellobiose) to glucose. (See Fungal Cellulases, Chemical Reviews 2015 115 (3), 1308-1448). Therefore, the use of cellulose complex enzyme for hydrolysis to prepare CNF will result in small aspect ratio of CNF, generally less than 50. SUMMARY

[0005] The technical problem solved by the present application is to provide a temperature-responsive cellulose nanofiber preparation method, which uses a single endoglucanase to hydrolyze modified microcrystalline cellulose to prepare cellulose nanofiber with high yield and high aspect ratio, and endows the cellulose nanofiber with temperature-responsive properties.

[0006] To solve the above technical problems, the present application discloses a temperature-responsive cellulose nanofiber preparation method, comprising the following steps:

[0007] (1) Grafting microcrystalline cellulose with sulfobetaine fragments under alkaline conditions to obtain modified microcrystalline cellulose MCC-S;

[0008] (2) Adding the modified microcrystalline cellulose MCC-S obtained in step (1) and a single endoglucanase to a buffer solution with pH=4-6, and stirring at a constant temperature of 40-60℃ for 3-15 hours to obtain an enzymatic product;

[0009] (3) Ultrasonic treatment and centrifugal separation of the enzymatic product obtained in step (2), and collecting the upper suspension to obtain cellulose nanofiber CNF-S with temperature-responsive properties.

[0010] Further, the source of microcrystalline cellulose in step (1) includes commercial microcrystalline cellulose and microcrystalline cellulose extracted from wood, cotton and bamboo; the alkaline conditions are: temperature 40-60℃, pH 11-12, and reaction time 0.5-6 h.

[0011] Further, the mass ratio of sulfobetaine fragments to microcrystalline cellulose in step (1) is 1-5:1.

[0012] Further, the sulfobetaine fragments have the following structure:

[0013] .

[0014] Further, the buffer solution in step (2) is an acetic acid-sodium acetate buffer solution, and the source of the single endoglucanase includes bacteria, fungi and animals.

[0015] Further, the mass ratio of modified microcrystalline cellulose MCC-S to single endoglucanase in step (2) is 2000-10000:1.

[0016] Further, the ultrasonic treatment time in step (3) is 10-50 min.

[0017] Further, the centrifugal conditions in step (3) are: rotation speed 8000-10000 r / min, and single centrifugal time 5-10 min.

[0018] Further, the ultrasonic treatment and centrifugal separation operation of step (3) need to be repeated several times.

[0019] Further, the aspect ratio of the cellulose nanofiber CNF-S obtained in step (3) is greater than or equal to 50, the mass yield is greater than or equal to 40%, and the cellulose nanofiber CNF-S has a UCST temperature response characteristic.

[0020] The beneficial effects of the present application are: the present application first uses endoglucanase to hydrolyze microcrystalline cellulose to prepare cellulose nanofiber, compared with cellulose composite enzyme, the use of single endoglucanase to hydrolyze the amorphous region of microcrystalline cellulose realizes the green preparation of cellulose nanofiber (CNF-S) with temperature-sensitive response and long aspect ratio under mild conditions, the aspect ratio reaches 62.5, the yield is as high as 50.74, and the UCST response is given. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The transmission electron micrograph of the cellulose nanofiber prepared by using endoglucanase;

[0022] Figure 2 The temperature response schematic diagram of 1wt% CNF-S. DETAILED DESCRIPTION

[0023] The present application will be further explained in combination with the following examples. The following examples are only used to illustrate the present application, but not used to limit the scope of the present application.

[0024] The reagents used in the following examples can be purchased from the market. The yield of cellulose nanofiber is determined by measuring the solid content.

[0025] The inventors' previous studies found that grafting a sulfobetaine fragment (SB) to cellulose nanocellulose endows it with temperature-responsive properties (see Synthesis of temperature and pH responsive lignin-grafted sulfobetaine for efficiently recycling cellulase, Bioresource Technology, 2023, 369: 128357, Preparation of temperature-dependent flux controllable cellulose nanofiber-based films and their application in oil-water separation, Carbohydrate Polymers, Volume 357, 2025: 123490). The present application first grafts a temperature-responsive hydrophilic temperature-sensitive fragment (SB) on the surface of microcrystalline cellulose, and then uses a single endoglucanase to hydrolyze the modified microcrystalline cellulose to obtain cellulose nanofibers.

[0026] First, SB can endow cellulose with temperature-responsive properties and improve the hydrophilicity of cellulose, which is a hot research direction for stabilizing cellulose emulsions. Moreover, the UCST response is operable and practical. Therefore, grafting and modifying cellulose nanofibers with SB is expected to endow them with UCST response and hydrophilicity.

[0027] Second, using a single endoglucanase to hydrolyze and prepare cellulose nanofibers can obtain cellulose nanofibers with moderate aspect ratio, which not only have a stable three-dimensional network structure, but also can reduce the size of stable emulsion droplets.

[0028] The present application provides a preparation method of temperature-responsive microcrystalline cellulose, comprising the following steps:

[0029] (1) Grafting sulfobetaine fragments to microcrystalline cellulose under alkaline conditions to obtain modified microcrystalline cellulose MCC-S; the source of the microcrystalline cellulose includes commercial microcrystalline cellulose and microcrystalline cellulose extracted from wood, cotton and bamboo and other plants;

[0030] Specifically: grafting the sulfobetaine fragment (SB) represented by formula (I) on the hydroxyl sites of microcrystalline cellulose under alkaline conditions (adjusting pH=11~12 with 20wt% sodium hydroxide solution) at 60 ℃ for 5h to obtain modified microcrystalline cellulose MCC-S, and after the reaction is completed, wash the fiber with distilled water until the pH value reaches about 7. Among them, the preparation of the acid betaine fragment (SB) of formula (I) (see Synthesis of temperature and pH responsive lignin-grafted sulfobetaine for efficiently recycling cellulase, Bioresource Technology, 2023, 369: 128357).

[0031]

[0032] (I)

[0033] (2) Add the modified microcrystalline cellulose MCC-S obtained in step (1) and a single endoglucanase to a buffer solution with pH=4~6, and stir at a constant temperature of 40~60℃ for 3~15 hours to obtain an enzymatic product; the source of the endoglucanase includes bacteria, fungi and animals; the mass ratio of modified microcrystalline cellulose to endoglucanase is 2000~10000:1;

[0034] Specifically: mix 3g of modified microcrystalline cellulose (MCC-S) obtained in step (1) with 30mL (pH=5) buffer solution, disperse uniformly, and then add endoglucanase, and perform enzymatic hydrolysis in a constant temperature reaction box at 50℃ and a rotation speed of 150r.

[0035] (3) Ultrasonic treatment and centrifugal separation are performed on the enzymatic product obtained in step (2), and the upper suspension is collected to obtain cellulose nanofiber CNF-S with temperature response characteristics.

[0036] Specifically, the enzymatic product obtained in step (2) was placed in an ultrasonic machine for 30 min (power 100 W) to dissolve the biological enzyme. Subsequently, the mixture was placed in a centrifuge (acceleration 10732 g) at 8000 r for 5 min, and the supernatant was poured out. Then, an appropriate amount of distilled water was added and centrifuged to remove enzyme impurities, and this operation was repeated more than 5 times. Then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for 30 min, ultrasonic treated for 30 min and centrifuged at 80000 r for 50 min, repeated several times, the turbid upper layer was collected, and left to stand at room temperature for about two weeks without layering. The suspension contained nanocellulose, the reducing sugar content of the suspension was determined, the remaining fiber was dried and the solid content was measured.

[0037] Comparative Example 1

[0038] The specific experimental steps for preparing cellulose nanofiber are as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution are mixed, and a third generation of cellulase (enzyme protein concentration 2 g / L, pH 4) is added and placed in a constant temperature reaction box at 50 ℃, the rotation speed is set to 150 r, and the enzymolysis is carried out for 9 h.

[0039] Subsequently, the enzymolysis mixture was ultrasonic treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Then, an appropriate amount of distilled water was added and centrifuged to remove enzyme impurities. This was repeated more than 5 times, then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for 30 min, ultrasonic treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, repeated several times, the turbid upper layer was collected, and left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was determined, the remaining fiber was dried and weighed, and the aspect ratio and yield results are shown in the table.

[0040] Comparative Example 2

[0041] The specific experimental steps for preparing cellulose nanofiber are as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution are mixed, and a third generation of cellulase (enzyme protein concentration 2 g / L, pH 5) is added and placed in a constant temperature reaction box at 50 ℃, the rotation speed is set to 150 r, and the enzymolysis is carried out for 9 h.

[0042] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0043] Comparative Example 3

[0044] The specific experimental steps for preparing cellulose nanofibers are as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, and a third generation cellulase (enzyme protein concentration of 2 g / L, pH of 6) was added and placed in a constant temperature reaction box at 50 ℃, with a rotation speed of 150 r for enzymolysis for 9 h.

[0045] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0046] Comparative Example 4

[0047] The specific experimental steps for preparing cellulose nanofibers are as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, and a third generation cellulase (enzyme protein concentration of 2 g / L, pH of 6) was added and placed in a constant temperature reaction box at 50 ℃, with a rotation speed of 150 r for enzymolysis for 9 h.

[0048] Subsequently, the mixture after enzymatic hydrolysis was treated with ultrasound for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, treated with ultrasound for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results were shown in the table.

[0049] Comparative Example 5

[0050] The specific experimental steps for preparing cellulose nanofibers were as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, and a third generation cellulase (enzyme protein concentration was 1 g / L, pH was 5) was added to a constant temperature reaction box at 50 ℃, and the rotation speed was set to 150 r for enzymolysis for 9 h.

[0051] Subsequently, the mixture after enzymatic hydrolysis was treated with ultrasound for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, treated with ultrasound for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results were shown in the table. Comparative Example 6

[0052] The specific experimental steps for preparing cellulose nanofibers were as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, and a third generation cellulase (enzyme protein concentration was 1 g / L, pH was 5) was added to a constant temperature reaction box at 50 ℃, and the rotation speed was set to 150 r for enzymolysis for 9 h.

[0053] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0054] The specific experimental steps for preparing cellulose nanofibers are as follows: 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, and cellulase (enzyme protein concentration of 2 g / L, pH of 5) was added and placed in a constant temperature incubator at 50 ℃, with a rotation speed of 150 r, and enzymolysis was carried out for 12 h.

[0055] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0056] Example 1

[0057] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, and endoglucanase 1 (added amount of 0.1 mg, pH of 5) was added and placed in a constant temperature incubator at 50 ℃, with a rotation speed of 150 r, and enzymolysis was carried out for 9 h.

[0058] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0059] Example 2

[0060] 3g of microcrystalline cellulose (MCC) was mixed with 30 mL of buffer solution, endoglucanase 1 was added (0.2 mg was added, pH 5) and placed in a constant temperature reaction box at 50 °C with a rotation speed of 150 r. The enzymatic hydrolysis was carried out for 9 h.

[0061] Subsequently, the mixture after enzymatic hydrolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then, the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added for centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, repeated several times, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried, and the length-diameter ratio and yield results are shown in the table.

[0062] Example 3

[0063] 3g of microcrystalline cellulose (MCC) was mixed with 30 mL of buffer solution, endoglucanase 1 was added (0.3 mg was added, pH 5) and placed in a constant temperature reaction box at 50 °C with a rotation speed of 150 r. The enzymatic hydrolysis was carried out for 9 h.

[0064] Subsequently, the mixture after enzymatic hydrolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then, the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added for centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, repeated several times, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried, and the length-diameter ratio and yield results are shown in the table.

[0065] Example 4

[0066] 3g of microcrystalline cellulose (MCC) was mixed with 30 mL of buffer solution, endoglucanase 1 was added (0.2 mg was added, pH 4) and placed in a constant temperature reaction box at 50 °C with a rotation speed of 150 r. The enzymatic hydrolysis was carried out for 9 h.

[0067] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0068] Example 5

[0069] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 1 was added (the amount of addition was 0.2 mg, pH was 6), and placed in a constant temperature incubator at 50 ℃, the rotation speed was set to 150 r, and enzymolysis was carried out for 9 h.

[0070] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0071] Example 6

[0072] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 1 was added (the amount of addition was 0.2 mg, pH was 5), and placed in a constant temperature incubator at 50 ℃, the rotation speed was set to 150 r, and enzymolysis was carried out for 6 h.

[0073] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0074] Example 7

[0075] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 1 was added (0.2 mg was added, pH was 5) and placed in a constant temperature reaction box at 50 °C, the rotation speed was set to 150 r, and enzymatic hydrolysis was performed for 12 h.

[0076] Subsequently, the mixture after enzymatic hydrolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then, the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, the turbid upper layer was collected, and was left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0077] Example 8

[0078] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (0.3 mg was added, pH was set to 5) and placed in a constant temperature reaction box at 50 °C, the rotation speed was set to 150 r, and enzymatic hydrolysis was performed for 9 h.

[0079] Subsequently, the mixture after enzymatic hydrolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then, the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, the turbid upper layer was collected, and was left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was determined, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0080] Example 9

[0081] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (0.6 mg was added, pH was set to 5) and placed in a constant temperature reaction box at 50 °C, the rotation speed was set to 150 r, and enzymatic hydrolysis was performed for 9 h.

[0082] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, the turbid upper layer was collected, and was left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results were shown in the table.

[0083] Example 10

[0084] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the amount of addition was 0.9 mg, and the pH was set to 5), and was placed in a constant temperature incubator at 50 ℃ and a rotation speed of 150 r for 9 h.

[0085] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, the turbid upper layer was collected, and was left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results were shown in the table.

[0086] Example 11

[0087] 3 g of microcrystalline cellulose (MCC) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the amount of addition was 0.9 mg, and the pH was set to 5), and was placed in a constant temperature incubator at 50 ℃ and a rotation speed of 150 r for 9 h.

[0088] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, the turbid upper layer was collected, and was left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was determined, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results were shown in the table.

[0089] Example 12

[0090] 3 g of MCC and 30 mL of buffer solution were mixed, endoglucanase 2 (0.6 mg of addition amount, pH set to 5) was added, and placed in a constant temperature incubator at 50 °C, and the rotation speed was set to 150 r for 11 h.

[0091] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then, the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was CNF suspension, the solid content of the suspension was measured, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0092] Example 13

[0093] 3 g of MCC-S and 30 mL of buffer solution were mixed, endoglucanase 2 (0.6 mg of addition amount, pH set to 5) was added, and placed in a constant temperature incubator at 50 °C, and the mass ratio of SB fragment to MCC was set to (1:1), and the rotation speed was set to 150 r for 9 h.

[0094] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then, the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred again at room temperature for 30 min, ultrasonically treated for 30 min. Subsequently, centrifugation was repeated for several times at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was CNF-S suspension, the solid content of the suspension was measured, the remaining fiber was collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0095] Example 14

[0096] 3 g of MCC-S and 30 mL of buffer solution were mixed, endoglucanase 2 (0.6 mg of addition amount, pH set to 5) was added, and placed in a constant temperature incubator at 50 °C, and the mass ratio of SB fragment to MCC was set to (3:1), and the rotation speed was set to 150 r for 9 h.

[0097] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF-S suspension, and the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0098] Example 15

[0099] 3 g of grafted microcrystalline cellulose (MCC-S) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the amount added was 0.6 mg, and the pH was set to 5), and placed in a constant temperature incubator at 50 °C, the mass ratio of SB fragment to MCC was set to (5:1), and the rotation speed was set to 150 r for 9 h.

[0100] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF-S suspension, and the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0101] Example 16

[0102] 3 g of grafted microcrystalline cellulose (MCC-S) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the amount added was 0.6 mg, and the pH was set to 4), and placed in a constant temperature incubator at 50 °C, the mass ratio of SB fragment to MCC was set to (3:1), and the rotation speed was set to 150 r for 9 h.

[0103] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF-S suspension, the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0104] Example 17

[0105] 3 g of grafted microcrystalline cellulose (MCC-S) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the amount added was 0.6 mg, and the pH was set to 6), and placed in a constant temperature incubator at 50 °C, the mass ratio of SB fragment to MCC was set to (3:1), and the rotation speed was set to 150 r for 9 h.

[0106] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was the CNF-S suspension, the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0107] Example 18

[0108] 3 g of grafted microcrystalline cellulose (MCC-S) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the amount added was 0.6 mg, and the pH was set to 6), and placed in a constant temperature incubator at 50 °C, the mass ratio of SB fragment to MCC was set to (3:1), and the rotation speed was set to 150 r for 9 h.

[0109] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was CNF-S suspension, the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0110] Example 19

[0111] 3 g of grafted microcrystalline cellulose (MCCS) and 30 mL of buffer solution were mixed, endoglucanase 2 was added (the addition amount was 1.2 mg, and the pH was set to 5), and the mixture was placed in a 50 ℃ constant temperature incubator, the mass ratio of SB fragment to MCC was set to (3:1), and the rotation speed was set to 150 r. The reaction was carried out for 9 h.

[0112] Subsequently, the mixture after enzymolysis was ultrasonically treated for 30 min to dissolve the cellulase. Then the mixture was centrifuged at 8000 r for 5 min, and the supernatant was poured out. Next, an appropriate amount of distilled water was added to continue centrifugation to remove enzyme impurities. This was repeated more than 5 times, and then the precipitate was collected and diluted with 150 mL of distilled water. The mixture was stirred at room temperature for another 30 min, and ultrasonically treated for 30 min. Subsequently, it was centrifuged at 8000 r for 5 min, and the turbid upper layer was collected and left to stand at room temperature for about two weeks without layering. The suspension was CNF-S suspension, the solid content of the suspension was measured, the remaining fibers were collected and dried to weigh, and the aspect ratio and yield results are shown in the table.

[0113] Table 1 Comparison of aspect ratio and yield of CNF prepared by different enzymes

[0114]

[0115] Table 2 Comparison of aspect ratio and yield of CNF prepared by different pH

[0116]

[0117] Table 3 Comparison of aspect ratio and yield of CNF / CNF-S prepared by different enzyme contents

[0118]

[0119] Table 4 Comparison of aspect ratio and yield of CNF prepared by different enzymolysis times

[0120]

[0121] Table 5. CNF aspect ratio, yield ratio of different grafting degrees

[0122]

[0123] According to Table 1, the CNF prepared by using cellulose complex enzyme has a shorter aspect ratio. After replacing endoglucanase, the aspect ratio is improved.

[0124] According to Table 2, the hydrolysis efficiency of the three enzymes is the highest at pH = 5. According to Table 3, as the enzyme content continuously increases, the yield also continuously increases. However, too high enzyme content leads to intensified hydrolysis, thereby reducing the yield. According to Example 18, when MCC-S is used as the substrate, the yield of cellulose nanofiber reaches a peak value.

[0125] According to the data comparison in Table 4, the optimal enzymolysis time is 9 h. According to Example 14 in Table 5, when the grafting mass ratio is 3:1, the yield reaches the highest.

[0126] In summary, after grafting the temperature-sensitive fragment SB on the surface of microcrystalline cellulose, the CNF is endowed with temperature response performance, as shown in Figure 2 According to Example 18, the cellulose nanofiber prepared by using single endoglucanase hydrolysis has a longer aspect ratio, and the aspect ratio reaches 62.5, as shown in Figure 1 According to Example 18, the cellulose nanofiber prepared by using single endoglucanase hydrolysis has a longer aspect ratio, and the aspect ratio reaches 62.5, as shown in

[0127] The above-mentioned is only a specific embodiment of the present application. The present application is not limited to the above-mentioned embodiment, and can have many variations. All variations directly derived or thought by those skilled in the art from the disclosed content of the present application should be considered as the protection scope of the present application.

Claims

1. A method for preparing a temperature-responsive cellulose nanofiber, characterized by, The method comprises the following steps: (1) grafting microcrystalline cellulose with a sulfobetaine fragment under alkaline conditions to obtain modified microcrystalline cellulose MCC-S; (2) adding the modified microcrystalline cellulose MCC-S obtained in step (1) and a single endoglucanase into a buffer solution with pH=4-6, and stirring at a constant temperature of 40-60℃ for 3-15 hours to obtain an enzymatic product; (3) performing ultrasonic treatment and centrifugal separation on the enzymatic product obtained in step (2), and collecting the upper suspension to obtain cellulose nanofiber CNF-S with temperature response characteristics.

2. The method of claim 1, wherein, The source of the microcrystalline cellulose in step (1) includes commercial microcrystalline cellulose and microcrystalline cellulose extracted from wood, cotton and bamboo; and the alkaline conditions are as follows: temperature 40-60℃, pH 11-12, and reaction time 0.5-6 h.

3. The method according to claim 1 or 2, characterized in that, The mass ratio of the sulfobetaine fragment to the microcrystalline cellulose in step (1) is 1-5:

1.

4. The method of claim 3, wherein, The sulfobetaine fragment has the following structure: 。 5. The method of claim 1, wherein, The buffer solution in step (2) is an acetic acid-sodium acetate buffer solution, and the source of the single endoglucanase includes bacteria, fungi and animals.

6. The method according to claim 1 or 5, characterized in that, The mass ratio of the modified microcrystalline cellulose MCC-S to the single endoglucanase in step (2) is 2000-10000:

1.

7. The method of claim 1, wherein, The ultrasonic treatment time in step (3) is 10-50 min.

8. The method of claim 1, wherein, The centrifugal condition in step (3) is as follows: rotation speed 8000-10000 r / min, and single centrifugal time 5-10 min.

9. The method according to claim 7 or 8, characterized in that, The ultrasonic treatment and centrifugal separation in step (3) need to be repeated for several times.

10. The method of claim 1, wherein, The cellulose nanofiber CNF-S obtained in step (3) has an aspect ratio of ≥50 and UCST temperature response characteristics.

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