Nanometer bacterial cellulose wound repair film and preparation method thereof

Abstract

The invention discloses a nano bacterial cellulose wound repair membrane and a preparation method thereof, and relates to the technical field of biomedical materials, the method comprises the following steps: carrying out fermentation culture on acetobacter xylinum to obtain an original bacterial cellulose membrane; immersing into a carboxymethyl chitosan solution for modification treatment, and then cleaning to be neutral; silver nanoparticles are loaded and irradiated by ultraviolet light to be uniformly distributed; and finally, performing vacuum drying and hot-pressing treatment to obtain a finished product. According to the invention, bacterial cellulose and carboxymethyl chitosan are combined and loaded with silver nanoparticles to form a composite material with excellent mechanical properties and a long-acting antibacterial function, and the introduction of carboxymethyl chitosan significantly improves the flexibility and biocompatibility of a bacterial cellulose membrane; meanwhile, the membrane material is endowed with long-acting antibacterial ability through loading of the silver nanoparticles, the mechanical property, flexibility and antibacterial ability of the membrane material can be remarkably improved, and meanwhile, the complexity of the preparation process is optimized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a nano-bacterial cellulose wound repair membrane and a preparation method thereof. BACKGROUND

[0002] With the rapid development of biomedical materials technology, wound repair membranes have gradually become a research hotspot in the medical field due to their excellent performance in promoting tissue regeneration, antibacterial and hemostatic functions. However, the existing wound repair membranes still have some deficiencies in material selection, structure design and preparation process, which affect their clinical application effect and adaptability.

[0003] In the prior art, an artificial tubular biomaterial prepared by combining animal small intestine decellularized matrix membrane with tubular metal mesh can be used as a repair material for patients with damage to tubular organs of the human body. However, in this technical solution, the mechanical strength of the biomaterial mainly depends on the supporting effect of the metal mesh, and the mechanical properties of the decellularized matrix membrane itself are weak, which may not meet the needs of complex wound environments, especially in the application of high tension or dynamic deformation areas. In addition, the presence of the metal mesh may lead to a decrease in the overall flexibility of the material, limiting its applicability in the repair of some flexible tissues.

[0004] In traditional technology, a skin repair biomaterial prepared by decellularizing the tissues of young livestock with mild plant-derived reagents has a high hyaluronic acid content and can promote wound angiogenesis and fibroblast growth. However, in this technical solution, the preparation process of the biomaterial is relatively complex, especially the decellularization step requires high reagent selection and operating conditions, which may lead to an increase in production costs and a decrease in process consistency. In addition, the antibacterial performance of this material depends on the properties of its natural ingredients, and lacks a long-term antibacterial mechanism, which may not be able to continuously exert antibacterial effects in the face of complex infection environments, affecting the stability of wound healing. SUMMARY

[0005] The purpose of the present application is to provide a nano-bacterial cellulose wound repair membrane and a preparation method thereof to solve the problems of the existing wound repair membranes in mechanical properties, antibacterial function and complexity of the preparation process.

[0006] To solve the above technical problems, the present application provides the following technical solutions: A nano-bacterial cellulose wound repair membrane and a preparation method thereof, comprising: Step S10: inoculate the Acetobacter xylinum culture solution into a fermentation medium containing glucose, yeast extract and phosphate buffer, and incubate at 28 to 32℃ for 5 to 7 days to obtain a raw bacterial cellulose film; in the fermentation medium, the mass concentration of glucose is 15 to 25 g / L, the mass concentration of yeast extract is 3 to 6 g / L, and the pH value of the phosphate buffer is 6.8 to 7.2.

[0007] Step S20: immerse the raw bacterial cellulose film into a carboxymethyl chitosan aqueous solution with a mass fraction of 0.5% to 1%, and treat at 40 to 50℃ for 1 to 2 hours, and then wash with deionized water to neutral to obtain a modified bacterial cellulose film; the degree of deacetylation of the carboxymethyl chitosan is 75% to 85%, and the molecular weight is 50,000 to 100,000.

[0008] Step S30: place the modified bacterial cellulose film into a solution containing silver nanoparticles, and irradiate with ultraviolet light for 10 to 20 minutes to uniformly load the silver nanoparticles on the film surface to obtain a silver nanoparticle-loaded bacterial cellulose film; in the solution containing silver nanoparticles, the mass concentration of silver nanoparticles is 0.01 to 0.05 g / L, and the particle size is 10 to 50 nm.

[0009] Step S40: dry the silver nanoparticle-loaded bacterial cellulose film in a vacuum drying oven at 40 to 50℃ for 4 to 6 hours, and then perform hot pressing treatment under nitrogen protection, with a hot pressing temperature of 120 to 140℃, a pressure of 5 to 10 MPa, and a time duration of 2 to 3 minutes to obtain a final nanometer bacterial cellulose wound repair film.

[0010] According to the present application, by combining bacterial cellulose with carboxymethyl chitosan and loading silver nanoparticles, a composite material with excellent mechanical properties and long-acting antibacterial function is formed. The introduction of carboxymethyl chitosan significantly improves the flexibility and biocompatibility of the bacterial cellulose film, and the loading of silver nanoparticles endows the film material with long-acting antibacterial ability. Hot pressing further enhances the structural stability of the film material, making it perform well in high-tension or dynamic deformation areas.

[0011] In the step S10, vitamin C with a mass concentration of 0.1 to 0.5 g / L is further added to the fermentation medium to promote the growth and metabolism of Acetobacter xylinum and increase the yield of bacterial cellulose.

[0012] In the step S20, the carboxymethyl chitosan aqueous solution further contains glycerol with a mass fraction of 0.05% to 0.1% to further improve the flexibility of the film material.

[0013] In the step S30, the wavelength of the ultraviolet light irradiation is 254nm to 365nm, and the power is 10W to 20W, so as to ensure that the silver nanoparticles are uniformly distributed on the film surface.

[0014] In the step S40, before the hot-pressing treatment, the film material is soaked in a polyvinyl alcohol aqueous solution with a mass fraction of 0.1% to 0.5% for 1 to 2 minutes, so as to enhance the interfacial bonding strength of the film material.

[0015] Compared with the prior art, the present application has the beneficial technical effects that: The present application significantly improves the flexibility and biocompatibility of the film material by using carboxymethyl chitosan to modify the original bacterial cellulose film, and the active groups in the carboxymethyl chitosan molecules form stable interaction with the bacterial cellulose fibers, effectively improving the inherent brittleness problem of the cellulose film, so that the finally prepared wound repair film has higher flexibility and better adhesion while maintaining high strength, and can better adapt to the wound contour and provide a more ideal microenvironment for cell growth.

[0016] The present application realizes uniform and firm distribution of silver nanoparticles on the film surface by the technology of ultraviolet light irradiation assisted silver nanoparticle loading, thereby endowing the wound repair film with strong and durable high-efficiency antibacterial performance; the energy of the ultraviolet light promotes the combination of the silver nanoparticles with the modified bacterial cellulose film surface, avoids the agglomeration and shedding of the nanoparticles, so that the film material exhibits high antibacterial rate to common pathogenic bacteria, and by controlling the loading amount and distribution state of the silver nanoparticles, the cytotoxicity possibly caused by silver ions is effectively reduced, and the safety of use is improved.

[0017] The present application significantly enhances the compactness, structural stability and mechanical strength of the film material by using a specific hot-pressing treatment process under nitrogen protection; by hot-treating the bacterial cellulose film loaded with silver nanoparticles under specific temperature, pressure and time parameters, the recombination and crosslinking between the polymer chains are promoted, thereby greatly improving the tensile strength and elongation at break of the film, so that it can withstand various mechanical stresses during application and is not easy to break, while maintaining a suitable porous structure, which is beneficial to the absorption of wound exudate and gas exchange.

[0018] The present application effectively improves the metabolic activity of the Bacillus subtilis and the yield and quality of the bacterial cellulose by optimizing the composition of the fermentation medium and introducing vitamin C as a growth promoter; on the basis of specific proportions of carbon source, nitrogen source and buffer system, the addition of low-concentration vitamin C further stimulates the growth of the bacterial cells, so that the synthesized original bacterial cellulose film has higher purity and more regular three-dimensional nanofiber network structure, laying a solid foundation for the subsequent modification steps, and the specific surface area and pore volume of the final product are significantly improved, which is more beneficial to wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The present application provides a nanometer bacterial cellulose wound repair membrane and a preparation method thereof. DETAILED DESCRIPTION

[0020] The features and exemplary embodiments of various aspects of the present application will be described below in detail, in order to make the purpose, technical solutions and advantages of the present application more clear and apparent, the present application will be further described in detail below in combination with the drawings and specific embodiments. It should be understood that the specific embodiments described herein are only intended to explain the present application, but not to limit the present application. For those skilled in the art, the present application can be implemented without some of these specific details. The following description of the embodiments is only to provide a better understanding of the present application by showing examples of the present application.

[0021] It should be noted that in this paper, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between the entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or equipment. Without more limitations, the elements defined by the statement "include" do not exclude the presence of other identical elements in the process, method, article or equipment including the elements.

[0022] In the embodiments of the present application, the same reference signs represent the same components, and for the sake of brevity, detailed description of the same components is omitted in different embodiments. It should be understood that the thickness, length, width and other dimensions of various components in the embodiments of the present application shown in the drawings, as well as the overall thickness, length, width and other dimensions of the integrated device, are only exemplary and should not constitute any limitation on the present application; "a plurality of" appearing in the present application means two or more (including two).

[0023] A method for preparing a nanometer bacterial cellulose wound repair membrane, comprising the following steps: Step S10: inoculate the Acetobacter xylinum culture solution into a fermentation medium containing glucose, yeast extract and phosphate buffer, and incubate at 28 to 32°C for 5 to 7 days to obtain a raw bacterial cellulose membrane. In the fermentation medium, the mass concentration of glucose is 15 to 25 g / L, the mass concentration of yeast extract is 3 to 6 g / L, and the pH value of the phosphate buffer is 6.8 to 7.2. In some embodiments, vitamin C is also added to the fermentation medium in a mass concentration of 0.1 to 0.5 g / L to promote the growth and metabolism of Acetobacter xylinum and increase the yield of bacterial cellulose.

[0024] Step S20: immerse the raw bacterial cellulose membrane in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.5% to 1% at 40 to 50°C for 1 to 2 hours, and then wash with deionized water until neutral to obtain a modified bacterial cellulose membrane. The degree of deacetylation of the carboxymethyl chitosan is 75% to 85%, and the molecular weight is 50,000 to 100,000. In some embodiments, the carboxymethyl chitosan aqueous solution also contains glycerol in a mass fraction of 0.05% to 0.1% to further improve the flexibility of the membrane material.

[0025] Step S30: place the modified bacterial cellulose membrane in a solution containing silver nanoparticles, and irradiate with ultraviolet light for 10 to 20 minutes to uniformly load silver nanoparticles on the surface of the membrane to obtain a bacterial cellulose membrane loaded with silver nanoparticles. In the solution containing silver nanoparticles, the mass concentration of silver nanoparticles is 0.01 to 0.05 g / L, and the particle size is 10 to 50 nm. In some embodiments, the wavelength of the ultraviolet light is 254 to 365 nm, and the power is 10 to 20 W to ensure uniform distribution of silver nanoparticles on the surface of the membrane.

[0026] Step S40: dry the bacterial cellulose membrane loaded with silver nanoparticles in a vacuum drying oven at 40 to 50°C for 4 to 6 hours, and then perform hot pressing treatment under nitrogen protection, with a hot pressing temperature of 120 to 140°C, a pressure of 5 to 10 MPa, and a time duration of 2 to 3 minutes to obtain a final nanometer bacterial cellulose wound repair membrane. In some embodiments, before hot pressing treatment, the membrane material is soaked in a polyvinyl alcohol aqueous solution with a mass fraction of 0.1% to 0.5% for 1 to 2 minutes to enhance the interfacial bonding strength of the membrane material.

[0027] First, Staphylococcus aureus (ATCC 25923) is used as the main test strain for antibacterial performance evaluation, and Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus epidermidis (ATCC 12228) and Candida albicans (ATCC 10231) are used for broad-spectrum antibacterial performance verification.

[0028] II. Detailed procedure of antibacterial experiment

[0029] Step 1: Strain preparation

[0030] Step 1.1: Strain activation, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus epidermidis (ATCC 12228) and Candida albicans (ATCC 10231) glycerol strains stored at -80℃ were taken out; among them, bacteria were inoculated in nutrient agar slant medium (beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, agar 15 g / L, pH 7.2-7.4), and Candida albicans was inoculated in sabouraud glucose agar slant medium (peptone 10 g / L, glucose 40 g / L, agar 15 g / L, pH 5.6); incubated in a 37℃ constant temperature incubator for 24 hours (Candida albicans was incubated for 48 hours), to obtain activated strains.

[0031] Step 1.2: Preparation of bacterial suspension, fresh culture single colony was picked and inoculated in 10 mL sterile nutrient broth (beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, pH 7.2-7.4) for bacteria, and in sabouraud glucose liquid medium for Candida albicans; incubated in a 37℃, 180 rpm shaking incubator for 16-18 hours (Candida albicans was incubated for 24 hours) to logarithmic growth phase; diluted with sterile normal saline (0.9% NaCl) to a turbidity of 0.5 McFarland standard, which is equivalent to 1.5×10 8 CFU / mL; then diluted with sterile normal saline to a concentration of 1×10 6 CFU / mL to obtain the test bacterial solution.

[0032] Step 2: Determination by inhibition zone method

[0033] Step 2.1: Preparation of plates, nutrient agar medium (beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, agar 15 g / L, pH 7.2-7.4) was autoclaved (121℃, 15 minutes); after cooling to 50-55℃, 20 mL of medium was poured into each sterile culture dish (diameter 90 mm); cooled and solidified in a clean bench to form a flat agar plate. Sabouraud glucose agar medium was used for Candida albicans.

[0034] Step 2.2: Inoculation, 200 μL of test bacterial solution with a concentration of 1×10 6 CFU / mL was uniformly coated on the surface of the entire agar plate with a sterile coating rod; after coating, it was placed in the clean bench for 10 minutes to allow the bacterial solution to be fully absorbed.

[0035] Step 2.3: Membrane sample placement, the prepared nanobacterial cellulose wound repair membrane was cut into a 10 mm diameter circular sample using a sterile punch; each sample was sterilized by soaking in 75% ethanol for 30 seconds, then rinsed with sterile normal saline for 3 times; the membrane sample was gently placed on the center of the agar plate coated with bacterial solution using sterile tweezers, and the membrane sample was gently pressed to ensure full contact with the agar surface without air bubbles; 3 parallel samples were set for each strain, and a blank control group (only coated with bacterial solution without membrane sample) was also set.

[0036] Step 2.4: Cultivation and observation, the inoculated plate was placed in a 37°C constant temperature incubator and cultured for 24 hours; after the incubation was completed, the plate was taken out and the formation of the inhibition zone was observed; the diameter of the inhibition zone was measured from different directions 3 times using a vernier caliper, and the average value was taken (including the diameter of the membrane sample); the area of the inhibition zone was calculated: S inhibition zone = π × (D / 2) 2, where D is the average diameter of the inhibition zone (mm); the antibacterial rate was calculated: antibacterial rate (%) = (inhibition zone area / plate area) × 100%, plate area S plate = π × (90 / 2) 2 = 6361.7 mm 2.

[0037] Step 3: Determination by colony counting method

[0038] Step 3.1: Membrane sample contact with bacterial solution, a 20 mm diameter circular membrane sample (sterilized by 75% ethanol, rinsed with sterile normal saline for 3 times) was placed in a 24-well plate; 1 mL of test bacterial solution with a concentration of 1 × 10 6 CFU / mL was added to the well; incubation was carried out in a 37°C, 100 rpm shaking incubator for a total of 2 hours; a blank control group (without membrane sample, only bacterial solution was added) was also set.

[0039] Step 3.2: Bacterial solution collection and dilution, after the incubation was completed, the bacterial solution in the well was aspirated using a pipette and transferred to a sterile centrifuge tube; 10-fold serial gradient dilution was carried out using sterile normal saline (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ); each dilution was thoroughly mixed using a vortex mixer for 30 seconds.

[0040] Step 3.3: Plate counting, 100 μL of bacterial solution from each of the 10 -3 , 10 -4 , 10 -5 dilutions was spread on the surface of a nutrient agar plate (white Candida albicans used Sabouraud glucose agar plate), and 3 parallel samples were set for each dilution; the control group also underwent serial dilution and plate counting.

[0041] Step 3.4: culture and counting, the plate is inverted in the 37℃ incubator for 24 hours; select the plate with the number of colonies between 30-300 for counting; calculate the number of viable bacteria: CFU / mL = the number of colonies x dilution factor x 10 (conversion coefficient of coating volume); calculate the sterilization rate: sterilization rate (%) = [(control group CFU-experimental group CFU) / control group CFU] x 100%; take the average value of 3 parallel samples in each group ± standard deviation.

[0042] Step 4: detailed mechanism of membrane antibiosis.

[0043] Step 4.1: sustained release mechanism of silver nanoparticles, after the membrane material contacts with the wound or bacterial solution, the water adsorbed on the surface forms a humid microenvironment; silver nanoparticles slowly oxidize under humid conditions, continuously releasing silver ions (Ag + ); the release rate is determined by the particle size of silver nanoparticles (10-50 nm), the loading amount (0.01-0.05 g / L), and the pore structure of the membrane (pore volume 0.90-0.98 cm³ / g); the nanosize effect makes the silver particles have a larger specific surface area, improving the release efficiency of silver ions.

[0044] Step 4.2: multiple antibacterial effects of silver ions, destroy the cell membrane: Ag + binds to the thiol groups (-SH) on the bacterial cell membrane, destroys the integrity of the membrane, and causes the contents of the cell to leak; inhibit DNA replication: Ag + enters the bacterial cell and binds to DNA molecules, preventing DNA unwinding and replication; interfere with the respiratory chain: Ag + binds to the enzyme system in the bacterial respiratory chain, inhibits ATP synthesis, and causes bacterial energy metabolism disorders; produce reactive oxygen: silver nanoparticles catalyze the production of reactive oxygen free radicals (ROS), which oxidize and destroy the structure of bacterial cells.

[0045] Step 4.3: synergistic antibiosis of carboxymethyl chitosan, the positively charged amino groups (-NH 3+ ) on the molecular chain of carboxymethyl chitosan electrostatically adsorb to the negatively charged components such as lipopolysaccharide and phosphomannan on the surface of the bacterial cell membrane; change the permeability and fluidity of the cell membrane, causing the contents of the bacteria to leak; carboxymethyl chitosan can also chelate metal ion cofactors (such as Fe 2+ , Zn 2+ , Mn 2+ ) required for bacterial growth, inhibit bacterial metabolic enzyme activity; carboxymethyl chitosan and silver nanoparticles produce a synergistic effect, improving the overall antibacterial performance.

[0046] Step 4.4: physical barrier effect of nanocellulose structure, the membrane material has a high specific surface area (110-135 m 2 / g) and developed nanoporosity structure (pore volume 0.90-0.98 cm 3 / g); the nanofiber network structure can physically adsorb and capture bacteria, limiting their movement and diffusion on the wound surface; the three-dimensional network structure provides an efficient loading platform for silver nanoparticles, ensuring their uniform distribution on the membrane surface and inside; the hydroxyl groups (-OH) of cellulose form coordinate bonds with silver nanoparticles, enhancing the stability of the loading and achieving the controlled release of silver ions.

[0047] Step 5: Supplementary experiments.

[0048] Step 5.1: Minimum inhibitory concentration (MIC) determination using microbroth dilution method (according to CLSI M07-A10 standard); the membrane material extract was serially diluted by 2-fold (concentration range 0.5-512 μg / mL); 100 μL of the diluted solution and 100 μL of the bacterial solution (final concentration 5×10 5 CFU / mL) were added to a 96-well plate; after incubation at 37°C for 18-24 hours, the turbidity of each well was observed, and the lowest concentration without visible growth was taken as the MIC value; the MIC values of silver nanoparticles for different bacteria were determined to evaluate the differences in antibacterial efficacy.

[0049] Step 5.2: Time-kill curve, the membrane sample (diameter 20 mm) was incubated with the bacterial solution (1×10 6 CFU / mL, 10 mL) at 37°C with shaking; 100 μL was sampled at 0, 1, 2, 4, 6, 8, 12, and 24 hours; after serial dilution, plate counting was performed, and the time-survival curve was plotted; the bactericidal rate at different time points was calculated to evaluate the long-term antibacterial ability and bactericidal kinetic characteristics of the membrane material.

[0050] Step 5.3: Silver ion release kinetics, the membrane sample (1 cm×1 cm) was immersed in 10 mL of phosphate buffer solution (PBS, pH 7.4); incubation was performed in a constant temperature shaking incubator at 37°C; 1 mL was sampled at 1, 2, 4, 8, 12, 24, 48, and 72 hours; the silver ion concentration was determined using inductively coupled plasma mass spectrometry (ICP-MS); the cumulative release curve of silver ions was plotted, and the release kinetics model (zero-order, first-order, or Higuchi diffusion model) was analyzed.

[0051] Step 5.4: Anti-biofilm formation experiment, biofilm culture: 200 μL of bacterial solution (1×10 6CFU / mL) and film sample fragments (5 mm x 5 mm) were incubated at 37°C for 48 hours to form biofilms; a control group (without film samples) was set; crystal violet staining: the culture solution was discarded, and PBS was used to wash 3 times to remove planktonic bacteria; 200 μL of 0.1% crystal violet solution was added for staining for 15 minutes; PBS was used to wash to remove excess dye; 200 μL of 95% ethanol was added to dissolve the crystal violet bound to the biofilm, and the solution was shaken at room temperature for 10 minutes; quantitative detection: the solution was transferred to a new 96-well plate, and the absorbance (OD) at 570 nm was measured by using an enzyme-labeled instrument 570 ); the biofilm inhibition rate was calculated: inhibition rate (%) = [(control group OD 570 - experimental group OD 570 ) / control group OD 570 ] x 100%.

[0052] The technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0053] Example 1: 500 mL of fermentation medium was taken, in which the mass concentration of glucose was 20 g / L, the mass concentration of yeast extract was 5 g / L, and the pH value of phosphate buffer was 7.0; after inoculation of Bacillus acetisaprogen, the medium was incubated at 30°C for 6 days to obtain an original bacterial cellulose film. The film was immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.8%, and treated at 45°C for 1.5 hours, and then washed with deionized water to neutral to obtain a modified bacterial cellulose film. The modified bacterial cellulose film was placed in a solution containing silver nanoparticles with a mass concentration of 0.03 g / L and a particle size of 30 nm, and irradiated with ultraviolet light with a wavelength of 300 nm and a power of 15 W for 15 minutes to uniformly load silver nanoparticles on the surface of the film. Finally, the bacterial cellulose film loaded with silver nanoparticles was dried in a vacuum drying oven at 45°C for 5 hours, and then subjected to hot pressing treatment under nitrogen protection, with a hot pressing temperature of 130°C, a pressure of 8 MPa, and a time duration of 2.5 minutes, to obtain a final nanometer bacterial cellulose wound repair film.

[0054] Example 2: Take 500 mL fermentation medium, wherein the mass concentration of glucose is 25 g / L, the mass concentration of yeast extract is 6 g / L, the pH value of phosphate buffer is 7.2, inoculate Acetobacter xylinum at 32℃ for 7 days to obtain the original bacterial cellulose membrane. The membrane is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 1%, treated at 50℃ for 2 hours, then washed with deionized water to neutral, to obtain the modified bacterial cellulose membrane. The modified bacterial cellulose membrane is placed in a solution containing silver nanoparticles with a mass concentration of 0.05 g / L and a particle size of 50 nm, and is irradiated with ultraviolet light with a wavelength of 365 nm and a power of 20 W for 20 minutes to make the silver nanoparticles uniformly loaded on the surface of the membrane. Finally, the silver nanoparticle-loaded bacterial cellulose membrane is dried in a vacuum drying oven at 50℃ for 6 hours, and then heat-pressed under nitrogen protection, with a heat-pressing temperature of 140℃, a pressure of 10 MPa, and a time duration of 3 minutes, to obtain the final nanometer bacterial cellulose wound repair membrane.

[0055] Example 3: Take 500 mL fermentation medium, wherein the mass concentration of glucose is 15 g / L, the mass concentration of yeast extract is 3 g / L, the pH value of phosphate buffer is 6.8, inoculate Acetobacter xylinum at 28℃ for 5 days to obtain the original bacterial cellulose membrane. The membrane is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.5%, treated at 40℃ for 1 hour, then washed with deionized water to neutral, to obtain the modified bacterial cellulose membrane. The modified bacterial cellulose membrane is placed in a solution containing silver nanoparticles with a mass concentration of 0.01 g / L and a particle size of 10 nm, and is irradiated with ultraviolet light with a wavelength of 254 nm and a power of 10 W for 10 minutes to make the silver nanoparticles uniformly loaded on the surface of the membrane. Finally, the silver nanoparticle-loaded bacterial cellulose membrane is dried in a vacuum drying oven at 40℃ for 4 hours, and then heat-pressed under nitrogen protection, with a heat-pressing temperature of 120℃, a pressure of 5 MPa, and a time duration of 2 minutes, to obtain the final nanometer bacterial cellulose wound repair membrane.

[0056] Example 4: Take 500 mL fermentation medium, the mass concentration of glucose is 22 g / L, the mass concentration of yeast extract is 4 g / L, the pH value of phosphate buffer is 7.1, inoculate Acetobacter xylinum at 31℃ for 6 days to obtain the original bacterial cellulose membrane. The membrane is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.9%, treated at 48℃ for 1.8 hours, then washed with deionized water to neutral, to obtain the modified bacterial cellulose membrane. The modified bacterial cellulose membrane is placed in a solution containing silver nanoparticles with a mass concentration of 0.04 g / L and a particle size of 40 nm, and is irradiated with ultraviolet light with a wavelength of 330 nm and a power of 18 W for 18 minutes to make the silver nanoparticles uniformly loaded on the surface of the membrane. Finally, the silver nanoparticle-loaded bacterial cellulose membrane is dried in a vacuum drying oven at 48℃ for 5.5 hours, and then heat pressed under nitrogen protection, with a heat pressing temperature of 135℃, a pressure of 9 MPa, and a time duration of 2.8 minutes, to obtain the final nanometer bacterial cellulose wound repair membrane.

[0057] Comparative Example 1

[0058] Take 500 mL fermentation medium, the mass concentration of glucose is 22 g / L, the mass concentration of yeast extract is 4 g / L, the pH value of phosphate buffer is 7.1, inoculate Acetobacter xylinum at 31℃ for 6 days to obtain the original bacterial cellulose membrane. The membrane is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.9%, treated at 48℃ for 1.8 hours, then washed with deionized water to neutral, to obtain the modified bacterial cellulose membrane. The modified bacterial cellulose membrane is placed in a solution containing silver nanoparticles with a mass concentration of 0.04 g / L and a particle size of 40 nm, and is irradiated with ultraviolet light with a wavelength of 330 nm and a power of 18 W for 18 minutes to make the silver nanoparticles uniformly loaded on the surface of the membrane. Finally, the silver nanoparticle-loaded bacterial cellulose membrane is dried in a vacuum drying oven at 48℃ for 5.5 hours, and then heat pressed under nitrogen protection, with a heat pressing temperature of 135℃, a pressure of 9 MPa, and a time duration of 2.8 minutes, to obtain the final nanometer bacterial cellulose wound repair membrane.

[0059] Comparative Example 2

[0060] Take 500 mL fermentation medium, the mass concentration of glucose is 20 g / L, the mass concentration of yeast extract is 5 g / L, the pH value of phosphate buffer is 7.0, inoculate Acetobacter xylinum at 30℃ for 6 days to obtain the original bacterial cellulose membrane. The membrane is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.8%, treated at 45℃ for 1.5 hours, then washed with deionized water to neutral, to obtain the modified bacterial cellulose membrane. The modified bacterial cellulose membrane is dried in a vacuum drying oven at 45℃ for 5 hours, then heat pressed under nitrogen protection, the heat pressing temperature is 130℃, the pressure is 8 MPa, and the time lasts for 2.5 minutes, to obtain the final nanometer bacterial cellulose wound repair membrane. The comparative example does not load silver nanoparticles, resulting in a lack of long-acting antibacterial ability of the membrane material.

[0061] Comparative Example 3

[0062] Take 500 mL fermentation medium, the mass concentration of glucose is 20 g / L, the mass concentration of yeast extract is 5 g / L, the pH value of phosphate buffer is 7.0, inoculate Acetobacter xylinum at 30℃ for 6 days to obtain the original bacterial cellulose membrane. The membrane is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.8%, treated at 45℃ for 1.5 hours, then washed with deionized water to neutral, to obtain the modified bacterial cellulose membrane. The modified bacterial cellulose membrane is placed in a solution containing silver nanoparticles with a mass concentration of 0.03 g / L and a particle size of 30 nm, and is irradiated with ultraviolet light with a wavelength of 300 nm and a power of 15 W for 15 minutes to uniformly load silver nanoparticles on the surface of the membrane. Finally, the silver nanoparticle-loaded bacterial cellulose membrane is dried in a vacuum drying oven at 45℃ for 5 hours, but is not heat pressed, resulting in poor structural stability of the membrane material.

[0063] Comparative Example 4

[0064] Take 500 mL of fermentation medium, the mass concentration of glucose is 20 g / L, the mass concentration of yeast extract is 5 g / L, the pH value of phosphate buffer is 7.0, inoculate acetobacter xylinum at 30°C for 6 days, and get the original bacterial cellulose film. The film is immersed in a carboxymethyl chitosan aqueous solution with a mass fraction of 0.8%, treated at 45°C for 1.5 hours, then washed with deionized water to neutral, and the modified bacterial cellulose film is obtained. The modified bacterial cellulose film is placed in a solution containing silver nanoparticles with a mass concentration of 0.03 g / L and a particle size of 30 nm. A wavelength of 300 nm and a power of 15 W are used to irradiate for 15 minutes, so that the silver nanoparticles are uniformly loaded on the surface of the film. Finally, the silver nanoparticle-loaded bacterial cellulose film is dried in air for 5 hours, and then heat pressed under nitrogen protection, with a heat pressing temperature of 130°C, a pressure of 8 MPa, and a time of 2.5 minutes, to obtain the final nanometer bacterial cellulose wound repair film. The comparative example does not use vacuum drying, resulting in a higher water content in the film material, which affects its mechanical properties.

[0065] The nanometer bacterial cellulose wound repair films prepared in Examples 1-4 and Comparative Examples 1-4 were tested for the following properties: Specific surface area and pore volume test: A full-automatic specific surface area and pore size analyzer was used to determine the specific surface area and pore volume of the film material by BET nitrogen adsorption method; after vacuum degassing treatment of the sample at 120°C for 6 hours, nitrogen adsorption-desorption isotherm was determined under the condition of liquid nitrogen at-196°C; the specific surface area was calculated by BET equation, and the pore volume was analyzed by BJH method.

[0066] Antibacterial performance test: The antibacterial performance of the film material on Staphylococcus aureus (ATCC 25923) was determined by inhibition zone method, and the bacterial solution (concentration of 1×10 6 CFU / mL) was uniformly coated on a nutrient agar plate, a circular film sample with a diameter of 10 mm was placed, and the diameter of the inhibition zone was measured after incubation at 37°C for 24 hours, and the antibacterial rate was calculated: antibacterial rate (%)=(inhibition zone area / plate area)×100%.

[0067] Mechanical property test: A universal material testing machine was used to determine the tensile strength and elongation at break of the film material according to GB / T 1040.3-2006 standard, the film sample was cut into dumbbell-shaped samples (gauge length 50 mm, width 10 mm, thickness about 0.1 mm), and the test was carried out at room temperature (25°C) and relative humidity 50% with a tensile speed of 10 mm / min; the tensile strength was the maximum stress when the sample broke, the elongation at break was (elongation at break / original gauge length)×100%, each group of samples was tested in parallel for 5 times, and the average value was taken.

[0068] The following are the comparative experimental data:

[0069] Table 1. Sample performance test results

[0070] According to the above data, the nanometer bacterial cellulose wound repair film prepared in the embodiments of the application has higher specific surface area, pore volume, antibacterial rate, tensile strength and elongation at break compared to the comparative examples, and exhibits more excellent comprehensive performance.

[0071] The above description is merely preferred specific embodiments of the present application, and is not intended to limit the protection scope of the present application, therefore: all equivalent changes made according to the structure, shape, principle of the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing a nano-bacterial cellulose wound repair membrane, characterized in that, Includes the following steps: step S10: Inoculate the Acetobacter xylose culture medium into a fermentation medium containing glucose, yeast extract and phosphate buffer, and incubate statically at 28°C to 32°C for 5 to 7 days to obtain the original bacterial cellulose membrane; the fermentation medium contains glucose at a mass concentration of 15 g / L to 25 g / L, yeast extract at a mass concentration of 3 g / L to 6 g / L, and phosphate buffer at a pH of 6.8 to 7.

2. Step S20: Immerse the original bacterial cellulose membrane in a 0.5% to 1% (w / w) aqueous solution of carboxymethyl chitosan and treat it at 40°C to 50°C for 1 to 2 hours. Then wash it with deionized water until neutral to obtain a modified bacterial cellulose membrane. The degree of deacetylation of the carboxymethyl chitosan is 75% to 85%, and the molecular weight is 50,000 to 100,000. Step S30: The modified bacterial cellulose membrane is placed in a solution containing silver nanoparticles and irradiated with ultraviolet light for 10 to 20 minutes to uniformly load the silver nanoparticles onto the membrane surface, thereby obtaining a bacterial cellulose membrane loaded with silver nanoparticles; the mass concentration of silver nanoparticles in the solution containing silver nanoparticles is 0.01 g / L to 0.05 g / L, and the particle size is 10 nm to 50 nm; Step S40: The bacterial cellulose membrane loaded with silver nanoparticles is dried in a vacuum drying oven at 40°C to 50°C for 4 to 6 hours, and then subjected to hot pressing under nitrogen protection at a temperature of 120°C to 140°C, a pressure of 5MPa to 10MPa, and a duration of 2 to 3 minutes to obtain the final nano-bacterial cellulose wound repair membrane.

2. The method according to claim 1, characterized in that, In step S10, vitamin C with a mass concentration of 0.1 g / L to 0.5 g / L is also added to the fermentation medium.

3. The method according to claim 1, characterized in that, In step S20, the carboxymethyl chitosan aqueous solution also contains 0.05% to 0.1% glycerol by mass.

4. The method according to claim 1, characterized in that, In step S30, the wavelength of the ultraviolet light irradiation is 254nm to 365nm, and the power is 10W to 20W.

5. The method according to claim 1, characterized in that, In step S40, before the hot pressing treatment, the membrane material is immersed in a polyvinyl alcohol aqueous solution with a mass fraction of 0.1% to 0.5% for 1 to 2 minutes.

6. The method according to claim 1, characterized in that, In step S10, the glucose concentration is 20 g / L, the yeast extract concentration is 5 g / L, the phosphate buffer pH is 7.0, the static culture temperature is 30°C, and the culture time is 6 days.

7. The method according to claim 1, characterized in that, In step S20, the mass fraction of the carboxymethyl chitosan aqueous solution is 0.8%, the treatment temperature is 45°C, and the treatment time is 1.5 hours.

8. The method according to claim 1, characterized in that, In step S30, the silver nanoparticles in the solution have a mass concentration of 0.03 g / L and a particle size of 30 nm. The ultraviolet light irradiation has a wavelength of 300 nm, a power of 15 W, and a duration of 15 minutes.

9. The method according to claim 1, characterized in that, In step S40, the vacuum drying temperature is 45°C and the time is 5 hours, and the hot pressing treatment temperature is 130°C, the pressure is 8 MPa, and the time is 2.5 minutes.

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