Preparation method of buckwheat shell cellulose nanofiber with controllable size

Abstract

The present application provides a kind of green simple cellulose nanofiber preparation method based on enzyme pretreatment and high pressure homogenization technology, belong to nanometer material preparation technical field, the present application method is prepared from buckwheat shell and other agricultural wastes high quality, average size controllable cellulose nanofiber, enzyme pretreatment reduces the difficulty and energy consumption of subsequent mechanical treatment, while buckwheat shell as low-cost raw material, significantly reduce the production cost;By controlling the number of homogenization cycles, the average size of CNF can be flexibly adjusted to meet the needs of different application fields;Not only solve the deficiency of traditional preparation method, but also promote the resource utilization of agricultural wastes, with significant economic benefits and environmental value.The method has broad prospects in promoting the industrialization application of CNF.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanotechnology and material science, and particularly relates to a preparation method of buckwheat shell cellulose nanofiber with controllable size. BACKGROUND

[0002] Cellulose is one of the natural polymers with the highest content and the widest distribution in nature, accounting for about 50% of natural biological resources, with an annual output of about 1.5 trillion tons. As the main component of plant cell walls, cellulose has the advantages of low cost, renewability, and biodegradability, and is mainly used in the fields of papermaking and textile. Nanocellulose is the smallest physical structural unit of cellulose, which has the "nanoeffect" that ordinary cellulose does not have, such as excellent mechanical properties, low density, high specific surface area, and rich surface functional groups. It is widely used in biomedical, environmental protection, optoelectronics, food industry and other industries.

[0003] At present, the main source of nanocellulose is wood, but the growth cycle of wood is long, and there are other important uses such as building production, so its supply is limited. Agricultural residues, as a potential source of nanocellulose, have the advantages of high yield and strong renewability, but their utilization rate is relatively low. Buckwheat hulls, as waste from buckwheat processing, are currently mainly used as pillow fillers, with a significantly low added value and serious resource waste. However, the lignocellulose content and structure of buckwheat hulls are very similar to those of wood, and the growth cycle is short (2-5 months) and the cellulose yield is high. Therefore, using buckwheat hulls to prepare nanocellulose is an effective supplement to wood production of nanocellulose, and also a high-value utilization of agricultural waste.

[0004] The technology for preparing nanocellulose from agricultural waste mainly focuses on chemical methods (sulfuric acid hydrolysis, 2,2,6,6-tetramethylpiperidine nitrogen oxide-mediated oxidation, etc.), mechanical methods (steam explosion, fine grinding, high-pressure homogenization, ultrasonic crushing, etc.), and biological methods. The chemical method is efficient and mature, but it produces a large amount of acidic wastewater and toxic residues. Although the mechanical method is simple and environmentally friendly, it has limitations such as high energy consumption and uneven particle size distribution of the finished product. Undoubtedly, the biological enzyme hydrolysis method is mild and safe, but it is high in cost, time-consuming, and low in yield. Therefore, combining enzyme hydrolysis with mechanical methods to prepare cellulose nanofiber may benefit from both technologies. Moreover, using a simple, time-saving method to prepare nanocellulose with controllable particle size is a new trend in this field.

[0005] Size control is a key issue in the preparation of nanocellulose. Different application fields have different requirements for the size of nanocellulose, so it is of great significance to develop a method for accurately controlling the size of nanocellulose, which can not only improve the performance and quality of the product, but also expand its application field. SUMMARY

[0006] In view of the current technical situation that the source of nanocellulose is single, the preparation of nanocellulose is high in cost, serious in environmental pollution and uncontrollable in size, the present application aims to provide a green preparation method of buckwheat shell cellulose nanofiber with controllable size.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] The present application provides a preparation method of buckwheat shell cellulose nanofiber with controllable size, comprising:

[0009] S1, preparing buckwheat shell cellulose;

[0010] S2, enzyme pretreatment is carried out on the buckwheat shell cellulose of S1, and after boiling water bath, centrifugal is carried out to discard the supernatant, and then washing is carried out to obtain a suspension;

[0011] S3, after dilution of the buckwheat shell cellulose suspension after enzyme pretreatment of S2, high-pressure homogenization circulation treatment is carried out to prepare buckwheat shell cellulose nanofiber with controllable size.

[0012] In S1, the preparation of buckwheat shell cellulose comprises: after washing and drying of buckwheat shell, crushing, washing and enzyme hydrolysis, enzyme hydrolysis buckwheat is obtained; after soaking in alkali solution, the pH value is adjusted to 3-4 after treatment in NaClO2 solution, and then drying is carried out to obtain buckwheat shell cellulose.

[0013] The enzyme hydrolysis adopts alpha-amylase, and the addition amount is 0.5%-0.7% (mass / volume ratio), and the enzyme hydrolysis is carried out at room temperature for 2-4 hours.

[0014] The enzyme pretreatment adopts cellulose mixed enzyme, the filter paper activity of the cellulose mixed enzyme is 195 FPU / gram, the endoglucanase activity is 2625 U / gram, the beta-glucosidase activity is 7196 U / gram, and the xylan endo-enzyme activity is 2530 U / gram; the cellulose mixed enzyme is added in an amount of 0.4%-0.6% of the substrate mass, and treated at 40-60 DEG C and 250 rpm / min for 4-48 hours.

[0015] Further, the cellulose mixed enzyme is added in an amount of 0.5% of the substrate mass, and treated at 50 DEG C and 250 rpm / min for 24 hours.

[0016] The boiling water bath is treated at 100-120 DEG C for 10-20 minutes.

[0017] The high pressure homogenization is 300-1000 bar, and the treatment is 10-20 times.

[0018] Further, the high pressure homogenization is 300 bar, 2-10 times; 600 bar, 2-10 times; 900 bar, 2-10 times; and 1000 bar, 2-10 times.

[0019] The application provides the size-controllable buckwheat shell cellulose nanofiber obtained by the preparation method of the size-controllable buckwheat shell cellulose nanofiber.

[0020] Further, the diameter of the buckwheat shell cellulose nanofiber can be regulated to be 20-40 nm.

[0021] The application provides application of the size-controllable buckwheat shell cellulose nanofiber in stabilizing emulsion.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] The preparation method provided by the present application realizes efficient conversion from buckwheat shell to cellulose nanofiber by the steps of crushing, alcohol washing, enzymatic hydrolysis, alkali washing, bleaching and high-pressure homogenization, etc. The entire process flow is relatively simple and easy to popularize and apply in industry; the agricultural waste buckwheat shell is converted into high-value nanomaterial, the raw material source is extensive and the price is low, the resource utilization of waste is realized, and the production cost is effectively reduced; through enzymatic pretreatment, cellulose mixed enzymes act specifically on cellulose molecular chains, the crystallinity and polymerization degree of cellulose are reduced, the connection between cellulose molecular chains is partially broken, the fiber structure is exposed and loosened to a certain extent, which is helpful for realizing the refinement and peeling of fibers in the subsequent high-pressure homogenization process, and this step can accelerate the cellulose decomposition process, reduce the time and energy consumption required for subsequent treatment, thereby improving the overall processing efficiency; by adjusting the pressure and cycle number of high-pressure homogenization, the cellulose pretreated by enzymes is further refined, which can make the cellulose nanofiber more uniform and dispersed in the solution, avoiding the aggregation and precipitation of fibers, thereby improving the utilization rate and performance of the fibers; the enzymatic pretreatment combined with high-pressure homogenization treatment plays a synergistic role, effectively exposes the fiber structure, promotes the refinement and peeling of fibers, and can accurately control the diameter of cellulose nanofiber in the range of 5-70nm, and can be more finely controlled to 20-40nm, the size controllability provides greater flexibility for the application of cellulose nanofiber in different fields due to its high specific surface area, good biocompatibility and degradability, etc.; compared with the traditional chemical treatment method, the method provided by the present application is more environmentally friendly and easy to control, and by optimizing the process parameters and improving the processing efficiency, the production cost can be further reduced.

[0024] The present application provides buckwheat shell cellulose nanofibers with controllable size, the diameter of the cellulose nanofibers ranges from 5 to 70 nm, the buckwheat shell cellulose nanofibers have high specific surface area, good biocompatibility and degradability, and can precisely control the stability of food emulsions with different compositions. The buckwheat shell cellulose nanofibers can be used as emulsifiers or stabilizers, and are wrapped on the surface of oil droplets or water droplets in the form of physical adsorption or chemical combination to form a protective layer, thereby preventing the occurrence of phenomena such as separation and flocculation of emulsions; the buckwheat shell cellulose nanofibers can form a uniform dispersion system in food, do not bring obvious particle sensation, and can interact with other ingredients in food, thereby helping to improve the taste and texture of food, and also improving the nutritional value of food and prolonging the shelf life of food; the good biocompatibility makes the buckwheat shell cellulose nanofibers not cause immune response or toxicity reaction in the human body, and can be safely used in food, and the buckwheat shell cellulose nanofibers are an ideal food additive or functional ingredient; the good degradability makes the buckwheat shell cellulose nanofibers gradually decompose in the natural environment, and do not cause environmental pollution; the unique properties of the buckwheat shell cellulose nanofibers can be used to develop new food with special taste, texture and nutritional value; the buckwheat shell cellulose nanofibers with controllable size provided by the present application have significant technical effects in the field of food, can precisely control the stability of food emulsions, prevent the occurrence of phenomena such as separation and flocculation of emulsions, and also improve the taste and texture of food, and improve the nutritional value of food; in addition, the buckwheat shell cellulose nanofibers have the advantages of safety, health, environmental protection and sustainability, and have wide application prospect and great market potential in the food industry. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Scanning electron microscope images of cellulose prepared under different conditions of the present application, wherein A is Example 3, A-1, A-2 and A-3 are different magnifications; B is Example 5, B-1, B-2 and B-3 are different magnifications; C is Example 6, C-1, C-2 and C-3 are different magnifications; D is Example 7, D-1, D-2 and D-3 are different magnifications;

[0026] Figure 2 is an atomic force microscope image and a diameter distribution histogram of nanocellulose fibers prepared under different conditions of the present application, wherein, Figure 2A Example 3, A-1 and A-2 are different magnifications, and A-3 is a particle size distribution; Figure 2B Example 5, B-1 and B-2 are different magnifications, and B-3 is a particle size distribution; Figure 2C Example 6, C-1 and C-2 are different magnifications, and C-3 is a particle size distribution; Figure 2D Example 7, D-1 and D-2 are different magnifications, and D-3 is a particle size distribution; Figure 2EFor Example 8, E-1 and E-2 are different magnifications, and E-3 is the particle size distribution.

[0027] Figure 3 Images of the emulsions of nanocellulose fibers loaded with citral essential oil prepared under different conditions of the present application for different examples stored for 15 days, wherein A is Example 3, A-1 is a CNF addition concentration of 0.15%, A-2 is a CNF addition concentration of 0.35%; B is Example 5, B-1 is a CNF addition concentration of 0.15%, B-2 is a CNF addition concentration of 0.35%; C is Example 6, C-1 is a CNF addition concentration of 0.15%, C-2 is a CNF addition concentration of 0.35%; D is Example 7, D-1 is a CNF addition concentration of 0.15%, D-2 is a CNF addition concentration of 0.35%; E is Example 8, E-1 is a CNF addition concentration of 0.15%, E-2 is a CNF addition concentration of 0.35%. DETAILED DESCRIPTION

[0028] In order to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0029] The present application will be further described in detail as follows:

[0030] Example 1

[0031] 1) Preparation of buckwheat hull cellulose: The buckwheat hull was repeatedly washed with tap water and dried in an oven at 45°C for 24 h. After being ground and passed through a 100-mesh sieve, the buckwheat hull powder was stored in a dry place for later use. The buckwheat hull cellulose (BP) was washed with absolute ethanol for 5 h and repeated twice at room temperature (25°C), and then washed with deionized water and dried. The obtained sample was added to the previously prepared 0.6% (w / v) α-amylase solution for enzymatic hydrolysis for 3 h. After washing and drying, the sample was soaked in a 5% (w / v) NaOH solution, stirred at 70°C for 2 h and repeated twice to remove hemicellulose and other non-cellulose components. Finally, the sample was added to a 1.5% (w / v) NaClO2 solution, and the pH was adjusted to 3-4 using acetic acid, and stirred at 70°C for 2 h, repeated 4 times to remove lignin and other pigments, to obtain a white cellulose sample. The white sample was washed with a large amount of deionized water to neutral, and dried in an oven at 45°C for 24 h to obtain the buckwheat hull cellulose. The solid-liquid ratio of all steps was 1:20 (m / v).

[0032] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH=4.8) with a solid loading of 4% (w / v), and 5 mg protein / g fiber substrate of cellulose mixed enzyme was added; after mixing evenly, the enzyme hydrolysis was carried out at 50°C and 250 rpm / min for 4 h; the mixture was placed in a 100°C boiling water bath for 15 min to inactivate the enzyme; it was centrifuged at 4000 rpm for 15 min to separate the supernatant containing the enzymatic hydrolysate and the solid precipitate, and the supernatant was discarded; the precipitate was washed and centrifuged with deionized water for 4 times to remove the residual enzyme and biomass products.

[0033] Example 2

[0034] 1) Preparation of buckwheat hull cellulose: The buckwheat hull was repeatedly washed with tap water and dried in an oven at 45°C for 24 h, then crushed and sieved through a 100-mesh sieve to obtain buckwheat hull powder, which was stored in a dry place. The buckwheat hull cellulose (BP) was washed with absolute ethanol for 5 h at room temperature (25°C) and repeated twice, then washed clean with deionized water and dried. The obtained sample was added to the previously prepared 0.6% (w / v) a-amylase solution for enzymatic hydrolysis for 3 h; after washing and drying, it was immersed in a 5% (w / v) NaOH solution, stirred at 70°C for 2 h and repeated twice. Finally, the obtained sample was added to a 1.5% (w / v) NaClO2 solution, the pH was adjusted to 3-4 using acetic acid, and it was stirred at 70°C for 2 h; this step was repeated 4 times, and the white sample was washed to neutral with a large amount of deionized water and dried in an oven at 45°C for 24 h to obtain buckwheat hull cellulose; the solid-liquid ratio of all the above steps was 1:20 (m / v).

[0035] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH=4.8) with a solid loading of 4% (w / v), and 5 mg protein / g fiber substrate of cellulose mixed enzyme was added; after mixing evenly, the enzyme hydrolysis was carried out at 50°C and 250 rpm / min for 4 h; the mixture was placed in a 100°C boiling water bath for 15 min to inactivate the enzyme; it was centrifuged at 4000 rpm for 15 min to separate the supernatant containing the enzymatic hydrolysate and the solid precipitate, and the supernatant was discarded; the precipitate was washed and centrifuged with deionized water for 4 times to remove the residual enzyme and biomass products.

[0036] Example 3

[0037] 1) Preparation of buckwheat hull cellulose: Buckwheat hulls were repeatedly washed with tap water and dried in an oven at 45 °C for 24 h. The dried material was ground and sieved through a 100 mesh screen to obtain a fine powder which was stored in a dry place. BP was washed with absolute ethanol for 5 h at room temperature (25 °C) and repeated twice. It was then washed with deionized water and dried. The sample was added to a previously prepared 0.6% (w / v) alpha-amylase solution and incubated for 3 h. After washing and drying, the sample was soaked in a 5% (w / v) NaOH solution and stirred at 70 °C for 2 h and repeated twice. Finally, the sample was added to a 1.5% (w / v) NaClO2 solution and the pH was adjusted to 3-4 using acetic acid and stirred at 70 °C for 2 h. This step was repeated four times. The white sample was washed with a large amount of deionized water until neutral, dried in an oven at 45 °C for 24 h, and buckwheat hull cellulose was obtained. The solid-liquid ratio of all steps was 1:20 (m / v).

[0038] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH 4.8) with a solid loading of 4% (w / v) and 5 mg protein / g of cellulose substrate was added. After mixing, the reaction was carried out at an enzyme hydrolysis temperature of 50 °C and a shaking speed of 250 rpm / min for 24 h. The mixture was placed in a boiling water bath at 100 °C for 15 min to inactivate the enzyme. Then it was centrifuged at 4000 rpm for 15 min and the supernatant was discarded. To remove enzyme impurities and biomass products, the precipitate was washed with deionized water and centrifuged, which was repeated four times.

[0039] Example 4

[0040] 1) Preparation of buckwheat hull cellulose: Buckwheat hulls were repeatedly washed with tap water and dried in an oven at 45 °C for 24 h. The dried material was ground and sieved through a 100 mesh screen to obtain a fine powder which was stored in a dry place. BP was washed with absolute ethanol for 5 h at room temperature (25 °C) and repeated twice. It was then washed with deionized water and dried. The sample was added to a previously prepared 0.6% (w / v) alpha-amylase solution and incubated for 3 h. After washing and drying, the sample was soaked in a 5% (w / v) NaOH solution and stirred at 70 °C for 2 h and repeated twice. Finally, the sample was added to a 1.5% (w / v) NaClO2 solution and the pH was adjusted to 3-4 using acetic acid and stirred at 70 °C for 2 h. This step was repeated four times. The white sample was washed with a large amount of deionized water until neutral, dried in an oven at 45 °C for 24 h, and buckwheat hull cellulose was obtained. The solid-liquid ratio of all steps was 1:20 (m / v).

[0041] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH 4.8) with a solid loading of 4% (w / v), and 5 mg protein / g of fiber substrate of cellulase cocktail was added. After mixing, the reaction was carried out at an enzymatic hydrolysis temperature of 50°C and a shaker speed of 250 rpm / min for 48 h. The mixture was placed in a boiling water bath at 100°C for 15 min to inactivate the enzyme. Then it was centrifuged at 4000 rpm for 15 min and the supernatant was discarded. To remove enzyme impurities and biomass products, the precipitate was washed with deionized water and centrifuged, and this operation was repeated 4 times.

[0042] Example 5

[0043] 1) Preparation of buckwheat hull cellulose: Buckwheat hulls were repeatedly washed with tap water, dried in an oven at 45°C for 24 h, crushed and passed through a 100-mesh sieve to obtain buckwheat hull powder, which was stored in a dry place for use. The BP was washed with absolute ethanol for 5 h at room temperature (25°C) and repeated twice, then washed clean with deionized water and dried. The obtained sample was added to the previously prepared 0.6% (w / v) a-amylase solution for enzymatic hydrolysis for 3 h. After washing and drying, it was soaked in a 5% (w / v) NaOH solution, stirred at 70°C for 2 h and repeated twice. Finally, the obtained sample was added to a 1.5% (w / v) NaClO2 solution, and acetic acid was used to adjust the pH to 3-4, and stirred at 70°C for 2 h. After repeating this step 4 times, the white sample was washed with a large amount of deionized water to neutral, dried in an oven at 45°C for 24 h to obtain buckwheat hull cellulose. The solid-liquid ratio of all steps was 1:20 (m / v).

[0044] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH=4.8) with a solid loading of 4% (w / v), and 5 mg protein / g of fiber substrate of cellulase cocktail was added. After mixing, the reaction was carried out at an enzymatic hydrolysis temperature of 50°C and a shaker speed of 250 rpm / min for 24 h. The mixture was placed in a boiling water bath at 100°C for 15 min to inactivate the enzyme. Then it was centrifuged at 4000 rpm for 15 min and the supernatant was discarded. To remove enzyme impurities and biomass products, the precipitate was washed with deionized water and centrifuged, and this operation was repeated 4 times.

[0045] 3) High-pressure homogenization treatment: The suspension obtained by enzymatic pretreatment was diluted with deionized water to 1% (w / v). Using a high-pressure homogenizer, it was treated once at a pressure of 300 bar to prepare cellulose nanofibers.

[0046] Example 6

[0047] 1) Preparation of buckwheat hull cellulose: Buckwheat hulls were washed repeatedly with tap water and dried in an oven at 45 °C for 24 h. The dried material was ground and sieved through a 100 mesh screen to obtain a fine powder which was stored in a dry place. The BP was washed with absolute ethanol for 5 h at room temperature (25 °C) and repeated twice. It was then washed with deionized water and dried. The sample was added to a pre-prepared solution of 0.6% (w / v) a-amylase and incubated for 3 h. After washing and drying, it was soaked in a 5% (w / v) NaOH solution and stirred at 70 °C for 2 h and repeated twice. Finally, the sample was added to a 1.5% (w / v) NaClO2 solution and the pH was adjusted to 3-4 using acetic acid and stirred at 70 °C for 2 h. This step was repeated four times. The white sample was washed with deionized water until neutral and dried in an oven at 45 °C for 24 h to obtain the buckwheat hull cellulose. The solid-to-liquid ratio for all steps was 1:20 (m / v).

[0048] 2) Enzymatic pretreatment: The buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH 4.8) with a solid loading of 4% (w / v) and 5 mg protein / g of cellulose substrate of cellulase cocktail was added. After mixing well, the reaction was carried out at an enzyme hydrolysis temperature of 50 °C and a shaking speed of 250 rpm / min for 24 h. The mixture was placed in a boiling water bath at 100 °C for 15 min to inactivate the enzymes. It was then centrifuged at 4000 rpm for 15 min and the supernatant was discarded. To remove enzyme impurities and biomass products, the precipitate was washed with deionized water and centrifuged, which was repeated four times.

[0049] 3) High pressure homogenization treatment: The suspension obtained from the enzymatic pretreatment was diluted with deionized water to 1% (w / v). It was treated with a high pressure homogenizer at a pressure of 300 bar for 3 times and 600 bar for 2 times to obtain cellulose nanofibers.

[0050] Example 7

[0051] 1) Preparation of buckwheat hull cellulose: Buckwheat hulls were washed repeatedly with tap water and dried in an oven at 45 °C for 24 h. The dried material was ground and sieved through a 100 mesh sieve to obtain a fine powder which was stored in a dry place. The BP was washed with absolute ethanol for 5 h at room temperature (25 °C) and repeated twice. It was then washed with deionized water and dried. The sample was added to a pre-prepared solution of 0.6% (w / v) a-amylase and incubated for 3 h. After washing and drying, it was soaked in a 5% (w / v) NaOH solution and stirred at 70 °C for 2 h and repeated twice. Finally, the sample was added to a 1.5% (w / v) NaClO2 solution and the pH was adjusted to 3-4 using acetic acid and stirred at 70 °C for 2 h. This step was repeated four times. The white sample was washed with deionized water until neutral and dried in an oven at 45 °C for 24 h to obtain buckwheat hull cellulose. The solid-liquid ratio for all steps was 1:20 (m / v).

[0052] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH 4.8) at a solid loading of 4% (w / v) and 5 mg protein / g of fiber substrate of cellulase cocktail was added. After mixing well, the reaction was carried out at an enzyme hydrolysis temperature of 50 °C and a shaking speed of 250 rpm / min for 24 h. The mixture was placed in a boiling water bath at 100 °C for 15 min to inactivate the enzymes. It was then centrifuged at 4000 rpm for 15 min and the supernatant was discarded. To remove enzyme impurities and biomass products, the precipitate was washed with deionized water and centrifuged, which was repeated four times.

[0053] 3) High pressure homogenization treatment: The suspension obtained from the enzymatic pretreatment was diluted with deionized water to 1% (w / v). It was treated with a high pressure homogenizer at a pressure of 300 bar for 3 times, 600 bar for 3 times, 900 bar for 3 times, and 1000 bar for 1 time to obtain cellulose nanofibers.

[0054] Example 8

[0055] 1) Preparation of buckwheat hull cellulose: Buckwheat hulls were repeatedly washed with tap water and dried in an oven at 45 °C for 24 h. After being ground and sieved through a 100 mesh sieve, the buckwheat hull powder was stored in a dry place for later use. The BP was washed with absolute ethanol for 5 h at room temperature (25 °C) and repeated twice. It was then washed with deionized water and dried. The resulting sample was added to a previously prepared 0.6% (w / v) a-amylase solution and enzymatically hydrolyzed for 3 h. After being washed and dried, it was immersed in a 5% (w / v) NaOH solution, stirred at 70 °C for 2 h and repeated twice. Finally, the resulting sample was added to a 1.5% (w / v) NaClO2 solution and the pH was adjusted to 3-4 using acetic acid. It was stirred at 70 °C for 2 h. This step was repeated four times. The white sample was then washed with a large amount of deionized water until it was neutral, dried in an oven at 45 °C for 24 h, and buckwheat hull cellulose was obtained. The solid-liquid ratio of all steps was 1:20 (m / v).

[0056] 2) Enzymatic pretreatment: Buckwheat hull cellulose was dispersed in 50 mmol / L sodium acetate buffer (pH 4.8) with a solid loading of 4% (w / v), and 5 mg of protein / g of fiber substrate of cellulase cocktail was added. After mixing well, the reaction was carried out at an enzyme hydrolysis temperature of 50 °C and a shaking speed of 250 rpm / min for 24 h. The mixture was placed in a 100 °C boiling water bath for 15 min to inactivate the enzyme. Then it was centrifuged at 4000 rpm for 15 min and the supernatant was discarded. To remove enzyme impurities and biomass products, the precipitate was washed with deionized water and centrifuged, and this operation was repeated four times.

[0057] 3) High-pressure homogenization treatment: The suspension obtained by enzymatic pretreatment was diluted to 1% (w / v) with deionized water. Using a high-pressure homogenizer, it was treated 6 times at 300 bar, 6 times at 600 bar, 6 times at 900 bar, and 2 times at 1000 bar to produce cellulose nanofibers.

[0058] Figure 1 Scanning electron microscope images of microcellulose prepared by pretreatment for different enzymatic hydrolysis times. From left to right: 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h. Figure 1 It can be concluded that for Example 1, when the enzymatic hydrolysis time is 4 h, the fibers are separated layer by layer in a rectangular sheet shape, and the surface appears to have protrusions and wrinkles. This is due to the fact that the amorphous regions of the cellulose chains, which tend to be parallel and randomly distributed, are first attacked by the enzyme. This indicates that in a shorter enzymatic hydrolysis time, the cellulose cocktail first attacks the amorphous regions of the cellulose chains, which are more easily attacked by the enzyme due to the parallel orientation and random distribution of the cellulose chains. However, due to the short time, the enzymatic action has not yet fully penetrated to the deeper layers or crystalline regions of the fiber.

[0059] For Example 2, as the enzymatic time is maintained, the layered structure of the fiber allows the enzyme to access more active sites, thus promoting the progress of the enzymatic reaction. This layer-by-layer unidirectional hydrolysis leads to further thinning of the fiber and the appearance of surface fibrils, indicating an improvement in enzymatic efficiency.

[0060] For Example 3, during the 24 h enzymatic process, the cellulase enzymes work synergistically, not only destroying the amorphous regions of the fiber, but also successfully penetrating the crystalline regions, randomly cutting the β-1,4-glycosidic bonds, leading to fibrillation and breaking of the fiber. This deep enzymatic action causes significant changes in the fiber morphology, forming a network structure of intertwined microfibrils, which is beneficial for subsequent processing and application.

[0061] For Example 4, when the time is extended to 48 h, the fibrillar shape is basically not visible, and the overall shape is granular or agglomerated granular. This is because the hydrolysis time is too long, and the cellulase enzymes excessively cut the cellulose molecules, which are prone to self-assembly into spherical fiber particles of various sizes through interfacial hydrogen bonds. The long enzymatic time leads to excessive cutting of cellulose molecules by cellulase enzymes, damaging the integrity of the fiber. Fiber fragments self-assemble into spherical fiber particles of various sizes through interfacial hydrogen bonds. This structure may not be conducive to certain application requirements, such as the preparation of reinforced materials or the realization of specific biological functions.

[0062] In summary, Example 3 shows the best pretreatment effect when the enzymatic time is 24 h. Under this condition, the cellulase enzymes can effectively penetrate into the fiber, not only destroying the amorphous region but also cutting the β-1,4-glycosidic bonds in the crystalline region, achieving fibrillation and breaking of the fiber, and forming a microfibril network structure that is beneficial for subsequent applications. Therefore, the pretreatment conditions of Example 3 are of great significance for the preparation of microcellulose materials with excellent performance.

[0063] Figure 2 is an atomic force microscope image and diameter distribution histogram of nanocellulose fibers prepared under different embodiment conditions.

[0064] From the data in Figure 2, in Figure 2, A is Example 3, B is Example 5, C is Example 6, D is Example 7, and E is Example 8. Fiber morphology changes: as the number of high-pressure homogenization increases, the fibers gradually appear to be torn and peeled along the longitudinal direction. This is because during high-pressure homogenization, cellulose fibers are subjected to extremely high pressure, and when they pass through the narrow gap, the pressure is released instantaneously, causing strong shear and tensile forces within the fibers, resulting in tearing and peeling of the fibers; this tearing and peeling process actually promotes the nanofibrillation of the fibers, i.e., the fibers are refined into smaller nanoscale units. As the number of homogenization increases, the degree of fiber refinement gradually increases, ultimately resulting in nanocellulose fibers with finer diameters and more uniform distribution.

[0065] Diameter distribution change

[0066] Diameter distribution narrowing and left shift: As can be seen from the diameter distribution histogram, with the increase of the number of high-pressure homogenization, the diameter distribution of cellulose nanofibers gradually narrows and left shifts. This means that the diameter range of the fibers is more concentrated, and the average diameter gradually decreases. This is because the fibers with larger diameters are more easily torn and refined during high-pressure homogenization, thereby reducing the overall average diameter. In Example 7, when the number of homogenization is 10 times, about 48% of the cellulose nanofibers have a diameter of 25-35 nm, and the average diameter is 28.04 ± 0.90 nm, which indicates that after 10 times of homogenization, the fibers have been well refined, but there are still a considerable number of fibers with larger diameters. In Example 8, when the number of homogenization is increased to 20 times, about 40% of the cellulose nanofibers have a diameter of 15-20 nm, and the average diameter is 17.93 ± 0.42 nm, which indicates that after increasing to 20 times of homogenization, the degree of fiber refinement is significantly improved, the proportion of fibers with smaller diameters increases, and the average diameter decreases significantly.

[0067] In summary, the number of high-pressure homogenization has a significant impact on the morphology and diameter distribution of nanocellulose fibers. With the increase of the number of homogenization, the fibers gradually achieve nanofibrillation, the diameter distribution narrows and left shifts, and the average diameter decreases. For application scenarios that require the preparation of nanocellulose fibers with smaller diameters and more uniform distributions (such as high-performance composites, biomedical materials, etc.), increasing the number of high-pressure homogenization is an effective means. In this example, Example 8 shows more excellent nanofibrillation effect when the number of homogenization is 20 times.

[0068] Further statistics of the yield and average diameter of nanocellulose prepared under different example conditions are shown in Table 1.

[0069] Table 1 Yield and average diameter data of nanocellulose fibers prepared under different example conditions

[0070]

[0071] Different superscript letters in the same column indicate significant differences between groups (P < 0.05)

[0072] As shown in Table 1, yield analysis reveals that the yield of CNF decreases with increasing homogenization cycles. From Example 5 to Example 8, the yield gradually decreases from 26.15% to 22.29% as the number of homogenization cycles increases. This is because some cellulose fibers are excessively refined or degraded during multiple homogenization processes, resulting in a reduction in the final collectable CNF mass. Although the yield decreases, even the lowest yield (22.29%) is better than the yield reported by a single method. This indicates that the combined method of enzymatic hydrolysis and high-pressure homogenization has a significant advantage in improving CNF production efficiency. This method may reduce the crystallinity and degree of polymerization of cellulose through enzymatic pretreatment, making the subsequent high-pressure homogenization process more efficient.

[0073] Average diameter analysis: The average diameter of CNF decreased significantly with increasing homogenization cycles. It decreased from 49.98 nm in Example 5 to 17.93 nm in Example 8, indicating that multiple homogenizations can effectively refine cellulose fibers into smaller nanoscale units. The reduction in average diameter not only benefits the application of CNF in composite materials, biomedical materials, and other fields, but also reflects the effectiveness of the enzymatic hydrolysis combined with high-pressure homogenization method in promoting cellulose nanostructuring.

[0074] In summary, enzymatic hydrolysis combined with high-pressure homogenization is an effective method for CNF preparation. This method not only aligns with environmentally friendly principles but also significantly reduces the average diameter of CNFs while maintaining high yields, thus improving production efficiency. Future research should focus on further optimizing process parameters to achieve even higher yields and better CNF quality.

[0075] Figure 3 Images show emulsions of citral essential oil loaded onto nanocellulose fibers prepared under different embodiments of the present invention. To verify the stable emulsifying properties of the nanocellulose prepared under different embodiments of the present invention, the emulsions were prepared using citral as the oil phase (10%, v / v) and CNF suspension as the aqueous phase (90%, v / v). The final CNF concentrations were 0.15% and 0.35% (w / v), respectively.

[0076] From the appendix Figure 3 The data shows that, attached Figure 3In the above, A is Example 3, B is Example 5, C is Example 6, D is Example 7, E is Example 8, -1 is a CNF addition concentration of 0.15%, and -2 is a CNF addition concentration of 0.35%. Since the emulsion is a thermodynamically unstable system, after being stored at room temperature for 15 days, the emulsion shows obvious phase separation. The less water phase layer appearing at the bottom, the more stable the emulsion. It can be observed intuitively that the smaller the diameter of CNF, the better the stability of the emulsion, because smaller CNF particles can more effectively wrap and stabilize oil droplets. At the same time, with the increase of CNF concentration, the effect of stabilizing essential oil is also improved. Under the same CNF diameter, 0.35% CNF concentration can stabilize the emulsion better than 0.15% CNF concentration.

[0077] In this experiment, emulsions of CNF loaded with lemon aldehyde essential oil under different example conditions were prepared, and their stability after being stored at room temperature for 15 days was observed, to verify the stable emulsification performance of CNF. The results show that the diameter and concentration of CNF have a significant effect on the stability of the emulsion. Smaller CNF diameter and higher CNF concentration can more effectively stabilize the essential oil emulsion.

[0078] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. A method for preparing buckwheat shell cellulose nanofiber with controllable size, characterized by, The preparation method comprises the following steps: S1, preparing buckwheat shell cellulose, comprising: washing and drying the buckwheat shell, crushing, washing, adding 0.5% to 0.7% of alpha-amylase by mass volume ratio, and enzymatic hydrolysis at room temperature for 2 to 4 hours to obtain enzymatic hydrolysis buckwheat; soaking in alkali solution, adjusting the pH value to 3 to 4 after treatment in NaClO2 solution, and drying to obtain buckwheat shell cellulose; S2, enzyme pretreatment of buckwheat shell cellulose, after 100 to 120 DEG C boiling water bath for 10 to 20 minutes, centrifugal separation and discarding the supernatant, washing, and obtaining the enzyme pretreated buckwheat shell cellulose suspension; the enzyme pretreatment adopts cellulose mixed enzyme, the filter paper activity of the cellulose mixed enzyme is 195 FPU / g, the endoglucanase activity is 2625 U / g, the beta-glucosidase activity is 7196 U / g, and the xylan endo-enzyme activity is 2530 U / g; the cellulose mixed enzyme is added in an amount of 0.4% to 0.6% of the substrate mass, and treated at 40 to 60 DEG C and 250 rpm / min for 4 to 48 hours; S3, diluting the enzyme pretreated buckwheat shell cellulose suspension, and performing high-pressure homogenization circulation treatment to prepare buckwheat shell cellulose nanofibers with controllable size; the high-pressure homogenization treatment is performed at a pressure of 300 bar for 2 to 10 times, at a pressure of 600 bar for 2 to 10 times, at a pressure of 900 bar for 2 to 10 times, and at a pressure of 1000 bar for 2 to 10 times; the buckwheat shell cellulose nanofibers with controllable size are derived from buckwheat shell cellulose, and the diameter of the buckwheat shell cellulose nanofibers can be controlled in the range of 20 to 40 nm.

2. The method for preparing size-controllable buckwheat hull cellulose nanofibers according to claim 1, characterized in that, The enzyme pretreatment is performed by adding 0.5% of cellulose mixed enzyme by mass of the substrate, and treating at 50 DEG C and 250 rpm / min for 24 hours.

3. The buckwheat shell cellulose nanofibers with controllable size obtained by the preparation method of the buckwheat shell cellulose nanofibers with controllable size according to any one of claims 1 to 2.

4. The use of the size-controllable buckwheat hull cellulose nanofiber according to claim 3 in a stable emulsion, characterized by, The buckwheat shell cellulose nanofibers with an average diameter in the range of 20 to 40 nm are used as stabilizers for stabilizing water-in-oil or oil-in-water emulsions, and when the addition concentration of the buckwheat shell cellulose nanofibers is 0.15% to 0.35%, the stable emulsion can maintain good stability after being stored at room temperature for 15 days.

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