Preparation method of biodegradable film based on nanocellulose enhancement

Abstract

The invention discloses a preparation method of a biodegradable film based on nanocellulose enhancement, and relates to the field of films. Hydroxyl groups on the surface of the nano cellulose fiber and Pullulan polysaccharide secreted by aureobasidium pullulans are self-assembled through hydrogen bonds to form a nano network structure; formaldehyde and hydrazide groups of hydrazide hyaluronic acid are subjected to a condensation reaction to generate acylhydrazone bonds, and a highly-crosslinked compact barrier layer is formed on the surface layer of the film; a polyphenol structure of tannic acid and a metal ion Fe < 3 + > fill gaps of nano cellulose fibers through coordination, so that the porosity is remarkably reduced, an oxygen diffusion path is prolonged, and the oxygen permeability is reduced; a hydrogen bond network is formed by the nano cellulose fibers and hydroxyl groups of the Pullulan polysaccharide through surface hydroxyl groups, meanwhile, terminal amino groups of the amino-terminated polycaprolactone and hydroxyl groups of the nano cellulose fibers generate ion-dipole interaction, and a three-dimensional interpenetrating structure is constructed; the tensile strength of the film can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of thin films, and particularly to a preparation method of a biodegradable thin film reinforced by nanocellulose. Background Art

[0002] In recent years, with the increasingly strict environmental protection requirements, the application demand for degradable plastic films in fields such as packaging and agriculture has grown rapidly. In the prior art, a composite film prepared by blending polyolefins (such as LDPE) with degradable materials (such as Pullulan polysaccharide) is one of the common solutions.

[0003] Publication No. CN201410699594.X discloses a preparation method of a degradable plastic composite film with oxygen barrier properties. This preparation method selects the currently mature degradable plastic masterbatch PBM in the market, Pullulan polysaccharide, and film-grade LDPE, and adds antioxidants and ultraviolet absorbers as raw materials. Tannic acid is used to modify Pullulan polysaccharide, and then a three-layer plastic composite film with good oxygen barrier properties, degradability, and mechanical properties is prepared by using a three-layer co-extrusion blown film technology for plastic films. The inner, middle, and outer layers of the composite film of the present invention are LDPE / PBM / Pullulan, PBM / Pullulan, and LDPE / PBM / Pullulan respectively. A degradable plastic composite film with oxygen barrier properties of the present invention will automatically decompose under the action of oxygen-rich and microorganisms, and the discarded film can be completely degraded and digested by microorganisms in nature. The oxygen barrier property can be improved by 70%-80% compared with the LDPE film of the same thickness, and the mechanical properties are basically equivalent to those of the LDPE film of the same thickness. However, there are still the following technical defects: the compatibility between LDPE and polysaccharide is poor, resulting in low tensile strength and toughness of the film, insufficient mechanical properties, and it is difficult to meet the application scenarios with high mechanical property requirements. Relying solely on the improvement effect of the barrier property of Pullulan polysaccharide is limited, and the oxygen transmission rate is still relatively high, making it difficult to meet the high-barrier packaging requirements, such as food preservation. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a biodegradable thin film reinforced by nanocellulose to solve the problems existing in the prior art.

[0005] To solve the above technical problems, the present invention provides the following technical solution: A preparation method of a biodegradable thin film reinforced by nanocellulose, comprising the following preparation steps:

[0006] (1) Inoculate the Aureobasidium pullulans spore suspension into a fermentation medium containing microcrystalline cellulose and cellulase. The volume ratio of the Aureobasidium pullulans spore suspension to the fermentation medium is 1:20 - 50. Ferment at a temperature of 28 - 32 °C and a stirring speed of 180 - 220 rpm for 40 - 50 hours. After centrifugation, take the supernatant for dialysis and freeze-drying to obtain a nanocellulose-Pullulan complex;

[0007] (2) Mix ε-caprolactone and ethylenediamine, add stannous octoate catalyst, and react at 115 - 125 °C for 22 - 26 hours under nitrogen protection. Dissolve the reaction solution in tetrahydrofuran and precipitate it in methanol pre-cooled to -20 °C ± 2 °C. After filtration, dry it under vacuum to obtain amino-terminated polycaprolactone;

[0008] (3) Mix the nanocellulose-Pullulan complex obtained in step (1) with the amino-terminated polycaprolactone obtained in step (2), disperse it in hexafluoroisopropanol, and ultrasonically treat it at a frequency of 38 - 42 kHz for 30 - 35 minutes. Adjust the pH to 5.5 ± 0.5 with an acetic acid-sodium acetate buffer solution with a pH of 5.0–6.0. Then add formaldehyde and hydrazide-modified hyaluronic acid to prepare a spinning solution, stir and react at room temperature for 1–2 hours. Then add the spinning solution into a syringe, and after electrospinning, dry it under vacuum to obtain a fiber membrane;

[0009] (4) Perform ultraviolet irradiation on the fiber membrane with ultraviolet light of 360 - 370 nm; then immerse it in an ethanol / water mixed solvent containing tannic acid and iron citrate for 25 - 35 seconds, ultrasonically treat it, and then hot press it in a flat vulcanizer for 1.5 - 2.5 minutes. After hot pressing, obtain the biodegradable thin film.

[0010] Further, the preparation method of the Aureobasidium pullulans spore suspension in step (1) is: Inoculate Aureobasidium pullulans on a potato dextrose agar medium. The amount of Aureobasidium pullulans inoculated per 1 g of potato dextrose agar medium is 1×10 6 -1×10 7 CFU. Culture at 28 - 32 °C for 5 - 7 days, scrape off the Aureobasidium pullulans spores and dilute them with sterile normal saline to a concentration of 1×10 6 -1×10 8 CFU / mL of Aureobasidium pullulans spore suspension.

[0011] Further, the preparation method of the fermentation medium in step (1) is: Dissolve 8 - 12 g / L of microcrystalline cellulose, 75 - 85 g / L of sucrose, 9 - 11 g / L of yeast extract, 1.8 - 2.2 g / L of potassium dihydrogen phosphate, and 2.8 - 3.2 g / L of disodium hydrogen phosphate in deionized water, sterilize it at 120 - 122 °C under high pressure for 20 minutes, and add 10–15 FPU / g of cellulase under sterile conditions after cooling to room temperature to obtain the fermentation medium.

[0012] Furthermore, in step (2), the mass ratio of ε-caprolactone, ethylenediamine, and stannous octoate catalyst is 100: 0.3–0.5: 0.3–0.7.

[0013] Furthermore, in step (3), the mass ratio of the nanocellulose-Pullulan complex, amino-terminated polycaprolactone, hexafluoroisopropanol, formaldehyde, and hydrazide-modified hyaluronic acid is 10: 3.3–4.3: 55–77: 0.2-0.3: 0.4–0.6.

[0014] Furthermore, the hydrazide-modified hyaluronic acid in step (3) is prepared by the following steps: Dissolve hyaluronic acid with a molecular weight of 1.5×10 5 Da in a phosphate buffer solution with a pH of 7.8-8.2, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, react at room temperature for 30-40 minutes, then add adipic dihydrazide and react for 18-22 hours. Use a dialysis bag with a cut-off molecular weight of 3.5 kDa, dialyze with deionized water, and then freeze-dry to obtain hydrazide-modified hyaluronic acid; wherein, the ratio of hyaluronic acid, phosphate buffer solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and adipic dihydrazide is: 2–4 g: 50–100 mL: 0.96–2.30 g: 0.58–1.73 g: 1.74–3.48 g.

[0015] Furthermore, the parameters of the electrospinning machine in step (3) are: voltage 17-19 kV, receiving distance 14-16 cm, spinning solution flow rate 1.1-1.3 mL / h, needle gauge 21-25G, ambient temperature 25±2 °C, relative humidity 45-55% RH, roller rotation speed 1900-2100 rpm, and spinning time 2.5-3.5 hours.

[0016] Furthermore, in step (4), the irradiation intensity is 48-52 mW / cm 2 and the time is 9-11 minutes.

[0017] Furthermore, in step (4), the frequency of the ultrasonic treatment is 38-42 kHz, the time is 4-6 minutes, the temperature of the flat vulcanizer is 58-62 °C, and the pressure is 4.8-5.2 MPa.

[0018] Furthermore, in step (4), the ratio of tannic acid, ferric citrate, ethanol, and water is: 1.8–2.2 g: 0.4–0.6 g: 650–750 mL: 250–350 mL.

[0019] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0020] 1. In the present invention, during the fermentation stage, microcrystalline cellulose is hydrolyzed by cellulase to obtain nanocellulose fibers. The hydroxyl groups on the surface of the nanocellulose fibers and pullulan polysaccharide secreted by Aureobasidium pullulans self-assemble through hydrogen bonds to form a nano-network structure. Formaldehyde and the hydrazide group of hydrazide-modified hyaluronic acid undergo a condensation reaction to form a hydrazone bond, forming a highly cross-linked dense barrier layer on the surface of the film. The polyphenol structure of tannic acid and metal ion Fe 3 + fill the gaps between nanocellulose fibers through coordination, significantly reducing the porosity, extending the oxygen diffusion path, and effectively reducing the oxygen permeability.

[0021] 2. In the present invention, the nanocellulose fibers form a hydrogen bond network through the hydroxyl groups on their surface and the hydroxyl groups of pullulan polysaccharide. At the same time, the terminal amino group of amino-terminated polycaprolactone and the hydroxyl group of the nanocellulose fibers generate ion-dipole interactions to construct a three-dimensional interpenetrating structure. During the stretching process, the nanocellulose fibers bear the main stress, and the hydrazone bond preferentially breaks and dissipates energy under the action of external force, significantly improving the tensile strength of the film.

[0022] 3. In the present invention, the terminal amino group of amino-terminated polycaprolactone enhances the affinity between the film and soil esterase, accelerating the hydrolysis of ester bonds. The hydrazone bond is protonated and broken in an acidic environment, disintegrating the cross-linked network, thereby increasing the soil degradation rate. Detailed implementation manners

[0023] The following embodiments are provided to better further understand the present invention. They are not limited to the best implementation manners, and do not constitute limitations on the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0024] Aureobasidium pullulans is sourced from Shanghai Xuanya Biotechnology Co., Ltd.; the potato dextrose agar medium is the potato dextrose agar medium in the patent document with the publication number CN112370387B. Cellulase can be Merck Celluclast1.5L.

[0025] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0026] The test methods for the various indicators of the materials obtained in the following examples and comparative examples are as follows: The oxygen transmission rate is measured using a differential pressure gas permeation instrument in accordance with the national standard "GB / T 1038-2000 Test Method for Gas Permeability of Plastic Films and Sheets". The test conditions are a temperature of 23 ± 0.5 °C and a relative humidity of 50 ± 5%. The film sample is fixed on the test chamber, a differential pressure of 0.1 MPa is applied, and the volume of oxygen passing through the film within 24 hours is measured (cm 3 ). The tensile strength is determined in accordance with the national standard "GB / T 1040.3-2006 Determination of Tensile Properties of Plastics". Using a universal material testing machine, the sample is cut into a standard dumbbell shape (width 10 mm, gauge length 50 mm), and tested at a tensile rate of 50 mm / min, and the maximum tensile stress (MPa) is recorded. The soil degradation rate is determined in accordance with the national standard "GB / T 19277.1-2011 Determination of the Ultimate Aerobic Biodegradability of Materials under Controlled Composting Conditions". The sample is buried in a compost environment at 58 ± 2 °C, and the CO2 release amount is measured after 45 days to calculate the degradation rate (%).

[0027] Example 1

[0028] (1) The preparation method of the Aureobasidium pullulans spore suspension is as follows: Aureobasidium pullulans is inoculated on a potato dextrose agar medium, and the amount of Aureobasidium pullulans inoculated per 1 g of potato dextrose agar medium is 1 × 10 6 CFU, cultured at 28 °C for 5 days, the Aureobasidium pullulans spores are scraped and diluted with sterile saline to a concentration of 1 × 10 6 CFU / mL of Aureobasidium pullulans spore suspension.

[0029] Dissolve 8 g / L of microcrystalline cellulose, 75 g / L of sucrose, 9 g / L of yeast extract, 1.8 g / L of potassium dihydrogen phosphate, and 2.8 g / L of disodium hydrogen phosphate in deionized water, autoclave at 120 °C for 20 minutes, and add 10 FPU / g of cellulase under sterile conditions after cooling to room temperature to obtain a fermentation medium.

[0030] The Aureobasidium pullulans spore suspension is inoculated into the fermentation medium containing microcrystalline cellulose and cellulase. The volume ratio of the Aureobasidium pullulans spore suspension to the fermentation medium is 1:20, fermented at a temperature of 28 °C and a stirring speed of 180 rpm for 40 hours, and the supernatant is taken after centrifugation, dialyzed, and freeze-dried to obtain a nanocellulose-Pullulan composite.

[0031] (2) Mix ε-caprolactone and ethylenediamine, add stannous octoate catalyst, react at 115 °C for 22 hours under nitrogen protection, dissolve the reaction solution in tetrahydrofuran, precipitate in methanol pre-cooled to -20 °C, filter and dry under vacuum to obtain amino-terminated polycaprolactone; the mass ratio of ε-caprolactone, ethylenediamine, and stannous octoate catalyst is 100:0.3:0.3.

[0032] (3) Dissolve hyaluronic acid with a molecular weight of 1.5×10 5 Da in phosphate buffer at pH 7.8, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, react at room temperature for 30 minutes, then add adipic dihydrazide and react for 18 hours. Use a dialysis bag with a molecular weight cut-off of 3.5 kDa, dialyze with deionized water and then freeze-dry to obtain hydrazide-modified hyaluronic acid; wherein, the ratio of hyaluronic acid, phosphate buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and adipic dihydrazide is: 2 g: 50 mL: 0.96 g: 0.58 g: 1.74 g.

[0033] Mix the nanocellulose-Pullulan complex obtained in step (1) with the amino-terminated polycaprolactone obtained in step (2), disperse in hexafluoroisopropanol, sonicate at a frequency of 38 kHz for 30 minutes, adjust the pH to 5.0 with acetic acid-sodium acetate buffer at pH 5.0, then add formaldehyde and hydrazide-modified hyaluronic acid to prepare a spinning solution, stir and react at room temperature for 1 hour, then add the spinning solution into a syringe, perform electrospinning and then vacuum dry to obtain a fibrous membrane; the mass ratio of nanocellulose-Pullulan complex, amino-terminated polycaprolactone, hexafluoroisopropanol, formaldehyde, and hydrazide-modified hyaluronic acid is 10: 3.3: 55: 0.2: 0.4. The parameters of the electrospinning machine are voltage 17 kV, receiving distance 14 cm, spinning solution flow rate 1.1 mL / h, needle gauge 21G, environmental temperature 23 °C, relative humidity 45% RH, roller rotation speed 1900 rpm, and spinning time 2.5 hours.

[0034] (4) Perform ultraviolet irradiation on the fibrous membrane with 360 nm ultraviolet light; then immerse it in an ethanol / water mixed solvent containing tannic acid and ferric citrate for 25 seconds, sonicate, and then hot press in a flat vulcanizer for 1.5 minutes to obtain the biodegradable film after hot pressing. The irradiation intensity is 48 mW / cm 2 , the time is 9 minutes. The frequency of sonication is 38 kHz, the time is 4 minutes, the temperature of the flat vulcanizer is 58 °C, and the pressure is 4.8 MPa. The ratio of tannic acid, ferric citrate, ethanol, and water is: 1.8 g: 0.4 g: 650 mL: 250 mL.

[0035] Example 2

[0036] (1) The preparation method of Aureobasidium pullulans spore suspension is: inoculate Aureobasidium pullulans on potato dextrose agar medium, the inoculation amount of Aureobasidium pullulans per 1 g of potato dextrose agar medium is 5×10 6 CFU, culture at 30 °C for 6 days, scrape the Aureobasidium pullulans spores and dilute them with sterile normal saline to a concentration of 5×10 7Auspicullaria pullulans spore suspension of CFU / mL.

[0037] Dissolve 10 g / L of microcrystalline cellulose, 80 g / L of sucrose, 10 g / L of yeast extract, 2.0 g / L of potassium dihydrogen phosphate, and 3.0 g / L of disodium hydrogen phosphate in deionized water, sterilize at 121 °C under high pressure for 20 minutes, and add 12.5 FPU / g of cellulase under sterile conditions after cooling to room temperature to obtain a fermentation medium.

[0038] Inoculate the Auspicullaria pullulans spore suspension into the fermentation medium containing microcrystalline cellulose and cellulase. The volume ratio of the Auspicullaria pullulans spore suspension to the fermentation medium is 1:35, ferment at 30 °C and a stirring speed of 200 rpm for 45 hours, take the supernatant after centrifugation, dialyze and freeze-dry to obtain a nanocellulose-Pullulan complex.

[0039] (2) Mix ε-caprolactone and ethylenediamine, add stannous octoate catalyst, and react at 120 °C for 24 hours under nitrogen protection. Dissolve the reaction solution in tetrahydrofuran, precipitate it in methanol pre-cooled to -20 °C, filter and dry under vacuum to obtain amino-terminated polycaprolactone; the mass ratio of ε-caprolactone, ethylenediamine, and stannous octoate catalyst is 100:0.4:0.5.

[0040] (3) Dissolve hyaluronic acid with a molecular weight of 1.5×10 5 Da in phosphate buffer solution with pH 8.0, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, react at room temperature for 35 minutes, then add adipic dihydrazide and react for 20 hours. Use a dialysis bag with a molecular weight cut-off of 3.5 kDa, dialyze with deionized water and then freeze-dry to obtain hydrazide-modified hyaluronic acid; among them, the ratio of hyaluronic acid, phosphate buffer solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and adipic dihydrazide is: 3 g: 75 mL: 1.63 g: 1.15 g: 2.61 g.

[0041] Mix the nanocellulose-Pullulan composite obtained in step (1) with the amino-terminated polycaprolactone obtained in step (2), disperse it in hexafluoroisopropanol, sonicate it at a frequency of 40 kHz for 32 minutes, adjust the pH to 5.5 with acetic acid-sodium acetate buffer solution at pH 5.5, then add formaldehyde and hydrazide-modified hyaluronic acid to prepare a spinning solution, stir and react at room temperature for 1.5 hours, then add the spinning solution into a syringe, and after electrospinning, dry it in vacuum to obtain a fiber membrane; the mass ratio of nanocellulose-Pullulan composite, amino-terminated polycaprolactone, hexafluoroisopropanol, formaldehyde and hydrazide-modified hyaluronic acid is 10:3.8:66:0.25:0.5. The parameters of the electrospinning machine are voltage 18 kV, receiving distance 15 cm, spinning solution flow rate 1.2 mL / h, needle gauge 23G, ambient temperature 25 °C, relative humidity 50% RH, roller speed 2000 rpm, and spinning time 3 hours.

[0042] (4) Perform ultraviolet irradiation on the fiber membrane with 365 nm ultraviolet light; then immerse it in an ethanol / water mixed solvent containing tannic acid and ferric citrate for 30 seconds, sonicate it, and then hot press it in a flat vulcanizer for 2 minutes to obtain the biodegradable film after hot pressing. The irradiation intensity is 50 mW / cm 2 , and the time is 10 minutes. The frequency of sonication is 40 kHz and the time is 5 minutes. The temperature of the flat vulcanizer is 60 °C and the pressure is 5.0 MPa. The ratio of tannic acid, ferric citrate, ethanol and water is: 2.0 g: 0.5 g: 700 mL: 300 mL.

[0043] Example 3

[0044] (1) The preparation method of the Aureobasidium pullulans spore suspension is as follows: Inoculate Aureobasidium pullulans on potato dextrose agar medium, and the amount of Aureobasidium pullulans inoculated per 1 g of potato dextrose agar medium is 1×10 7 CFU, culture at 32 °C for 7 days, scrape the Aureobasidium pullulans spores and dilute them with sterile normal saline to a concentration of 1×10 8 CFU / mL of Aureobasidium pullulans spore suspension.

[0045] Dissolve microcrystalline cellulose 12 g / L, sucrose 85 g / L, yeast extract 11 g / L, potassium dihydrogen phosphate 2.2 g / L, and disodium hydrogen phosphate 3.2 g / L in deionized water, autoclave at 122 °C for 20 minutes, and add 15 FPU / g cellulase under sterile conditions after cooling to room temperature to obtain a fermentation medium.

[0046] Inoculate Aureobasidium pullulans spore suspension into a fermentation medium containing microcrystalline cellulose and cellulase. The volume ratio of the Aureobasidium pullulans spore suspension to the fermentation medium is 1:50. Ferment at 32°C and a stirring speed of 220 rpm for 50 hours. After centrifugation, take the supernatant for dialysis and freeze-drying to obtain a nanocellulose-Pullulan complex.

[0047] (2) Mix ε-caprolactone and ethylenediamine, add stannous octoate catalyst, and react at 125°C for 26 hours under nitrogen protection. Dissolve the reaction solution in tetrahydrofuran and precipitate it in methanol pre-cooled to -22°C. Filter and dry in vacuo to obtain amino-terminated polycaprolactone; the mass ratio of ε-caprolactone, ethylenediamine, and stannous octoate catalyst is 100:0.5:0.7.

[0048] (3) Dissolve hyaluronic acid with a molecular weight of 1.5×10 5 Da in phosphate buffer solution with pH 8.2. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and react at room temperature for 40 minutes. Then add adipic dihydrazide and react for 22 hours. Use a dialysis bag with a molecular weight cut-off of 3.5 kDa, dialyze with deionized water and then freeze-dry to obtain hydrazide-modified hyaluronic acid; among them, the ratio of hyaluronic acid, phosphate buffer solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and adipic dihydrazide is: 4 g:100 mL:2.30 g:1.73 g:3.48 g.

[0049] Mix the nanocellulose-Pullulan complex obtained in step (1) with the amino-terminated polycaprolactone obtained in step (2), disperse it in hexafluoroisopropanol, and ultrasonically treat it at a frequency of 42 kHz for 35 minutes. Adjust the pH to 6.0 with acetic acid-sodium acetate buffer solution with pH 6.0. Then add formaldehyde and hydrazide-modified hyaluronic acid to prepare a spinning solution, stir and react at room temperature for 2 hours. Then add the spinning solution into a syringe, and after electrospinning, dry in vacuo to obtain a fiber membrane; the mass ratio of the nanocellulose-Pullulan complex, amino-terminated polycaprolactone, hexafluoroisopropanol, formaldehyde, and hydrazide-modified hyaluronic acid is 10:4.3:77:0.3:0.6. The parameters of the electrospinning machine are voltage 19 kV, receiving distance 16 cm, spinning solution flow rate 1.3 mL / h, needle gauge 25G, environmental temperature 27°C, relative humidity 55%RH, roller speed 2100 rpm, and spinning time 3.5 hours.

[0050] (4) Perform ultraviolet irradiation on the fiber membrane with 370 nm ultraviolet light; then immerse it in an ethanol / water mixed solvent containing tannic acid and iron citrate for 35 seconds, ultrasonically treat it, and then hot-press it in a flat vulcanizing machine for 2.5 minutes. After hot-pressing, obtain the biodegradable thin film. The irradiation intensity is 52 mW / cm 2, The time is 11 minutes. The frequency of ultrasonic treatment is 42 kHz and the time is 6 minutes. The temperature of the flat vulcanizer is 62 °C and the pressure is 5.2 MPa. The ratio of tannic acid, iron citrate, ethanol and water is: 2.2 g: 0.6 g: 750 mL: 350 mL.

[0051] Comparative Example 1

[0052] The difference between Comparative Example 1 and Example 1 is only that a nanocellulose-Pullulan complex is used, while conventional nanocellulose (nanocellulose with a diameter of 2 - 20 nm and a length of 0.1 - 100 μm prepared by mechanical grinding of cotton fibers in this comparative example) is used.

[0053] Comparative Example 2

[0054] The difference between Comparative Example 2 and Example 1 is only that formaldehyde is not added.

[0055] Comparative Example 3

[0056] The difference between Comparative Example 3 and Example 1 is only that hydrazide-modified hyaluronic acid is not added.

[0057] Amino-terminated polycaprolactone

[0058] Comparative Example 4

[0059] The difference between Comparative Example 4 and Example 1 is only that amino-terminated polycaprolactone is not added.

[0060] The performance analysis results of the samples prepared in the examples and comparative examples of the present invention are given in Table 1 below.

[0061] Table 1

[0062] Group <![CDATA[Oxygen transmission rate (cm 3 )]]> Tensile strength (MPa) Soil degradation rate (%) Example 1 120 35 85 Example 2 95 42 78 Example 3 80 50 70 Comparative example 1 200 20 60 Comparative example 2 150 25 72 Comparative example 3 180 18 65 Comparative example 4 250 15 50

[0063] From the experimental data of the examples and comparative examples, it can be found that in the present invention, the hydroxyl groups on the surface of nanocellulose fibers self-assemble with Pullulan polysaccharide secreted by Aureobasidium pullulans through hydrogen bonds to form a nano-network structure; formaldehyde undergoes a condensation reaction with the hydrazide groups of hydrazide-modified hyaluronic acid to form a hydrazone bond, forming a highly cross-linked dense barrier layer on the surface layer of the film; the polyphenol structure of tannic acid and metal ion Fe 3+Filling the gaps between nanocellulose fibers through coordination significantly reduces the porosity, extends the oxygen diffusion path, and decreases the oxygen permeability; the nanocellulose fibers form a hydrogen bond network with the hydroxyl groups of Pullulan polysaccharide through the surface hydroxyl groups, and at the same time, the terminal amino groups of the terminal amino polycaprolactone generate ion-dipole interactions with the hydroxyl groups of the nanocellulose fibers to construct a three-dimensional interpenetrating structure, improving the tensile strength of the film. Moreover, the terminal amino groups of the terminal amino polycaprolactone enhance the affinity between the film and soil esterase, accelerating the hydrolysis of ester bonds. The acylhydrazone bond is protonated and broken in an acidic environment, disrupting the cross-linked network, thereby increasing the soil degradation rate of the film.

[0064] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be construed as limiting the claims involved.

Claims

1. A preparation method of a nanocellulose-reinforced biodegradable film, characterized in that, It includes the following preparation steps: (1) Inoculate the Aureobasidium pullulans spore suspension into the fermentation medium containing microcrystalline cellulose and cellulase. The volume ratio of the Aureobasidium pullulans spore suspension to the fermentation medium is 1:20 - 50. Ferment at a temperature of 28 - 32°C and a stirring speed of 180 - 220 rpm for 40 - 50 hours. After centrifugation, take the supernatant for dialysis and freeze-drying to obtain the nanocellulose-Pullulan composite; (2) Mix ε-caprolactone and ethylenediamine, add stannous octoate catalyst, and react at 115 - 125°C for 22 - 26 hours under nitrogen protection. Dissolve the reaction solution in tetrahydrofuran and precipitate it in methanol pre-cooled to -20°C ± 2°C. After filtration, dry it under vacuum to obtain amino-terminated polycaprolactone; (3) Mix the nanocellulose-Pullulan composite obtained in step (1) with the amino-terminated polycaprolactone obtained in step (2), disperse it in hexafluoroisopropanol, and ultrasonically treat it at a frequency of 38 - 42 kHz for 30 - 35 minutes. Adjust the pH to 5.5 ± 0.5 through an acetic acid-sodium acetate buffer solution with a pH of 5.0–6.

0. Then add formaldehyde and hydrazide-modified hyaluronic acid to prepare a spinning solution, stir and react at room temperature for 1–2 hours. Then add the spinning solution into a syringe, and after electrospinning, dry it under vacuum to obtain a fiber membrane; (4) Perform ultraviolet irradiation on the fiber membrane with ultraviolet light of 360 - 370 nm; then immerse it in an ethanol / water mixed solvent containing tannic acid and ferric citrate for 25 - 35 seconds, perform ultrasonic treatment, and then hot-press it in a flat vulcanizer for 1.5 - 2.5 minutes. After hot-pressing, obtain the biodegradable film.

2. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: The preparation method of Aureobasidium pullulans spore suspension in step (1) is as follows: inoculate Aureobasidium pullulans on potato dextrose agar medium, and the inoculation amount of Aureobasidium pullulans per 1 g of potato dextrose agar medium is 1×10 6 -1×10 7 CFU, culture at 28 - 32 °C for 5 - 7 days, scrape the Aureobasidium pullulans spores and dilute them with sterile normal saline to a concentration of 1×10 6 -1×10 8 CFU / mL of Aureobasidium pullulans spore suspension.

3. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: The preparation method of the fermentation medium in step (1) is: Dissolve 8 - 12 g / L of microcrystalline cellulose, 75 - 85 g / L of sucrose, 9 - 11 g / L of yeast extract, 1.8 - 2.2 g / L of potassium dihydrogen phosphate, and 2.8 - 3.2 g / L of disodium hydrogen phosphate in deionized water, sterilize it at 120 - 122°C under high pressure for 20 minutes, and add 10–15 FPU / g of cellulase under sterile conditions after cooling to room temperature to obtain the fermentation medium.

4. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: In step (2), the mass ratio of ε-caprolactone, ethylenediamine, and stannous octoate catalyst is 100:0.3–0.5:0.3–0.

7.

5. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: In step (3), the mass ratio of the nanocellulose-Pullulan composite, amino-terminated polycaprolactone, hexafluoroisopropanol, formaldehyde, and hydrazide-modified hyaluronic acid is 10:3.3–4.3:55–77:0.2 - 0.3:0.4–0.

6.

6. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, wherein: The hydrazide-modified hyaluronic acid described in step (3) is prepared by the following steps: Dissolve hyaluronic acid with a molecular weight of 1.5×10 5 Da in a phosphate buffer solution with a pH of 7.8 - 8.2, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, react at room temperature for 30 - 40 minutes, then add adipic dihydrazide and react for 18 - 22 hours. Use a dialysis bag with a molecular weight cut-off of 3.5 kDa, dialyze with deionized water and then freeze-dry to obtain the hydrazide-modified hyaluronic acid; wherein, the ratio of hyaluronic acid, phosphate buffer solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and adipic dihydrazide is: 2–4 g: 50–100 mL: 0.96–2.30 g: 0.58–1.73 g: 1.74–3.48 g.

7. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, wherein: The parameters of the electrospinning machine in step (3) are: voltage 17 - 19 kV, receiving distance 14 - 16 cm, spinning solution flow rate 1.1 - 1.3 mL / h, needle gauge 21 - 25G, environmental temperature 25 ± 2°C, relative humidity 45 - 55%RH, roller rotation speed 1900 - 2100 rpm, and spinning time 2.5 - 3.5 hours.

8. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: In the step (4), the irradiation intensity is 48 - 52 mW / cm 2 , and the time is 9 - 11 minutes.

9. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: In step (4), the frequency of the ultrasonic treatment is 38 - 42 kHz, the time is 4 - 6 minutes, the temperature of the flat vulcanizer is 58 - 62°C, and the pressure is 4.8 - 5.2 MPa.

10. The preparation method of a nanocellulose-reinforced biodegradable film according to claim 1, characterized in that: In step (4), the ratio of tannic acid, ferric citrate, ethanol and water is: 1.8–2.2 g : 0.4–0.6 g : 650–750 mL : 250–350 mL.