Preparation method and application of modified bacterial cellulose medical dressing membrane material

Abstract

The invention provides a preparation method and application of a modified bacterial cellulose medical dressing membrane material, and relates to the technical field of medical membrane material preparation. According to the medical dressing membrane material, rosa roxburghii tratt is subjected to wall breaking, juicing and multiple freeze thawing to serve as a fermentation substrate, glucose and yeast extract powder serve as a culture medium in an auxiliary mode, acetobacter xylinum, lactic acid bacteria and saccharomycetes are cultured to produce high-quality and high-yield BC, and meanwhile, chitosan quaternary ammonium salt is adopted to be compounded with a BC membrane subsequently to improve the antibacterial property of the material. The defects in the prior art are overcome, the roxburgh rose juice is adopted as a culture medium raw material, the glucose and the yeast extract powder are assisted for culturing bacteria, the yield of bacterial cellulose is effectively increased, the basic performance of the bacterial cellulose is guaranteed, and the effect of the bacterial cellulose serving as a medical dressing is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical film materials, and particularly relates to a preparation method and application of a modified bacterial cellulose medical dressing film material. Background Art

[0002] Bacterial cellulose (BC) is a natural polymer material secreted and synthesized by bacteria on the surface of a static liquid culture medium. Bacterial cellulose (BC) itself has good mechanical properties, a 3D nano-network structure, high water retention capacity, and biocompatibility, showing great potential in the field of wound dressing substrates. However, BC itself does not have antibacterial properties, and endowing BC with antibacterial functions through physical or chemical modification is particularly important for its application in the field of medical dressings.

[0003] Currently, the production of bacterial cellulose mainly involves liquid cultivation of corresponding strains. The culture media selected are mostly HS medium, modified HS medium, mannitol medium, sucrose medium, etc. Different culture media are often selected for different strains and different situations. However, at present, the yield of bacterial cellulose cultivated with these conventional culture media is low, and the overall production cost is high for actual production and use. Therefore, how to optimize the corresponding culture conditions to improve the yield of bacterial cellulose and the effect of bacterial cellulose as a medical dressing in the end is an important research direction at present. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a preparation method and application of a modified bacterial cellulose medical dressing film material. Using prickly pear juice as a culture medium raw material, and assisting with glucose and yeast extract powder to cultivate bacteria can effectively improve the yield of bacterial cellulose, ensure the basic properties of bacterial cellulose, and improve the effect as a medical dressing.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0006] A preparation method of a modified bacterial cellulose medical dressing film material, the preparation method comprising the following steps:

[0007] S1. After breaking the wall of prickly pear mixed with deionized water, centrifuge to collect the supernatant, and freeze-thaw it 3 - 5 times and then filter it to obtain a fermentation substrate;

[0008] S2. Add glucose with a w / v of 2% - 8% and yeast extract powder with a w / v of 0.75% - 1.2% to the fermentation substrate, mix evenly and sterilize to obtain a pear juice culture medium for standby;

[0009] S3. Compound acetic acid bacteria, lactic acid bacteria, and yeast into a compound bacterial liquid for standby;

[0010] S4. Inoculate the complex bacterial liquid into the above-mentioned pear juice medium in an amount of 10% (v / v) for static fermentation. After obtaining the BC membrane, terminate the culture, take out the BC membrane and freeze-dry it to obtain the freeze-dried BC membrane.

[0011] S5. Add chitosan quaternary ammonium salt to the glacial acetic acid solution and stir to dissolve it. Control the concentration of chitosan quaternary ammonium salt to be 0.2% - 2.5% to obtain a chitosan quaternary ammonium salt solution for standby.

[0012] S6. Place the freeze-dried BC membrane in the chitosan quaternary ammonium salt solution and let it stand at room temperature. Then take out the BC membrane to remove the residue of chitosan quaternary ammonium salt, and then freeze-dry it to obtain the medical dressing membrane material.

[0013] Preferably, the specific preparation method of the fermentation substrate is as follows: First, remove the cores of Rosa roxburghii fruits, wash them, add deionized water with a mass ratio of 10 - 12 times, carry out cell wall breaking and juicing, then filter under pressure to remove the suspended fruit residues. The filtrate is centrifuged at 6000 - 8000 r / min for 10 - 15 min, and then the supernatant is collected and frozen at -30°C to -40°C for 8 - 10 h and then melted in a water bath at 25 - 35°C. The freeze-thaw treatment is repeated 3 - 5 times and then filtered. The filtrate is used as the fermentation substrate.

[0014] Preferably, the sterilization method in step S2 is the moist heat sterilization method at 115°C for 15 - 25 min, and then it is naturally cooled to 25 ± 1°C for standby.

[0015] Preferably, the content of Acetobacter xylinum in the complex bacterial liquid in step S3 is 1.35×10 8 CFU / mL, the content of lactic acid bacteria is 1.8×10 5 CFU / mL, and the content of yeast is 2.8×10 5 CFU / mL.

[0016] Preferably, the fermentation method in step S4 is to culture in the dark at a temperature of 27 - 35°C; and the culture is terminated when the thickness of the BC membrane reaches 2 - 8 mm.

[0017] Preferably, the mass concentration of glacial acetic acid in the glacial acetic acid solution in step S5 is 0.3% - 0.8%.

[0018] Preferably, the chitosan quaternary ammonium salt in step S5 is hydroxypropyltrimethylammonium chloride chitosan.

[0019] Preferably, the standing time at room temperature in step S6 is 18 - 36 h.

[0020] And the above-prepared membrane material can be prepared into a medical antibacterial dressing.

[0021] The present invention provides a preparation method and application of a modified bacterial cellulose medical dressing membrane material, and the advantages compared with the prior art are as follows:

[0022] The present invention uses the filtered and centrifuged product of broken-wall juicing of Rosa roxburghii tratt followed by repeated freeze-thawing and filtration as the basal medium for bacteria to produce bacterial cellulose. Then, a certain amount of glucose and yeast extract powder are added to effectively improve the yield of bacterial cellulose, further enhance the quality of bacterial cellulose, and increase the water retention rate of bacterial cellulose, facilitating the subsequent processing of bacterial cellulose into medical dressing film materials. Description of the Drawings

[0023] Figure 1 It is a line graph showing the effect of different glucose concentrations on the yield of bacterial cellulose in Example 1 of the present invention;

[0024] Figure 2 It is a macroscopic schematic diagram of different membranes in Example 3 of the present invention, where (a) is the BC membrane, (b) is the HBC-1 membrane, (c) is the HBC-2 membrane, and (d) is the HBC-3 membrane;

[0025] Figure 3 It is a microscopic schematic diagram of different membranes in Example 3 of the present invention, where (a) is the BC membrane, (b) is the HBC-1 membrane, (c) is the HBC-2 membrane, and (d) is the HBC-3 membrane; (e) is the cross-section of the BC membrane; (f) is the cross-section of the HBC-3 membrane;

[0026] Figure 4 It is the FT-IR spectra of different membranes and HACC in Example 3 of the present invention;

[0027] Figure 5 In (a), it is the XPS full spectrum of BC and HBC-3 in Example 3 of the present invention; (b) is the peak-fitting spectrum of Cl 2p of HBC-3 in Example 3 of the present invention;

[0028] Figure 6 It is a microscopic schematic diagram of the BC-1 membrane prepared using soybean milk medium in the examples of the present invention;

[0029] Figure 7 It is the stress-strain curve graph of different membranes in Example 3 of the present invention;

[0030] Figure 8 It is a schematic diagram of the antibacterial effects of the control group, BC, and HBC-3 in Example 3 of the present invention, where the bacteria in (a) are Escherichia coli; the bacteria in (b) are Staphylococcus aureus; the bacteria in (c) are Pseudomonas aeruginosa; the bacteria in (d) are Streptococcus mutans. Detailed Embodiments

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following describes the technical solutions in the embodiments of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0032] The mixed bacterial solution used in the following examples is composed of Acetobacter xylinum (ATCC23767), Lactobacillus (CGMCC No. 15703), and Saccharomyces cerevisiae (CICC 1270). Among them, the content of Acetobacter xylinum is 1.35×10 8 cells / mL, the content of Lactobacillus is 1.8×10 5 cells / mL, and the content of Saccharomyces cerevisiae is 2.8×10 5 cells / mL; and Acetobacter xylinum, Lactobacillus, and Saccharomyces cerevisiae are all purchased from Shandong Weibao Bacteria Biotechnology Co., Ltd.

[0033] The formula of the HS medium (pH = 6.0) used below is: glucose: 20 g / L, peptone: 5 g / L, yeast extract: 5 g / L, sodium dihydrogen phosphate: 2.7 g / L, citric acid: 1.15 g / L.

[0034] Example 1:

[0035] Effect of different glucose contents in the medium on BC membrane production:

[0036] (1) Select 10 kg of fresh pear fruits. After removing the cores and washing, put them into a wall-breaking juicer with 11 times the amount (w / w) of deionized water to obtain the initial pear juice extract.

[0037] Separate with a plate and frame filter press. After removing the suspended fruit residues, place the filtrate in a high-speed centrifuge and centrifuge at 8000 r / min for 10 min. Collect the supernatant, freeze it at -35 °C for 9 h, then thaw it in a water bath at 30 °C, and perform the freeze-thaw treatment 4 times. Then filter to obtain the filtrate as the fermentation substrate.

[0038] Add 0%-10% (w / v) glucose and 4% (w / v) yeast extract solution to each liter of the filtrate, adjust and mix well, and then use the moist heat sterilization method at 115 °C for 20 min to sterilize the medium. After the sterilization procedure ends, let it cool naturally to 25 ± 1 °C to obtain the Rosa roxburghii juice medium 1 for standby.

[0039] (2) Transfer the composite bacterial solution aseptically to the Rosa roxburghii juice medium 1 at an inoculation amount of 10% (v / v) to ensure full contact between the bacterial strains and the medium. Seal it with micro-oxygen and keep it at a constant temperature of 30 °C, and perform static fermentation for 7 days under light-shielded conditions.

[0040] The addition amounts of glucose were set to 0%, 2%, 4%, 6%, 8%, and 10% respectively, and the BC yields after 7 days of cultivation in each medium were detected. The specific results are as Figure 1 shown. Among them, when the addition amount of glucose was 6%, the BC yield reached the highest at 0.88 g / 100 mL; and when the content was 2 - 8%, good BC yields were obtained.

[0041] Example 2:

[0042] Effect of different culture media on BC membrane production:

[0043] 1. Preparation of culture media:

[0044] (1) Rosa roxburghii juice medium 1: As shown in Example 1 above, control the glucose content to be 6%, and prepare Rosa roxburghii juice medium 1;

[0045] (2) Rosa roxburghii juice medium 2: Select 10 kg of fresh pear fruits, after removing the cores and washing, put them into a wall-breaking juicer with 11 times the amount (w / w) of deionized water to obtain the initial pear juice extract;

[0046] Separate with a plate and frame filter press, remove the suspended fruit residues, and then place the filtrate in a high-speed centrifuge and centrifuge at 8000 r / min for 10 min. Collect the supernatant as the fermentation substrate;

[0047] Add 6% (w / v) glucose and 4% (w / v) yeast extract solution to each liter of supernatant, adjust and mix well, and then use the moist heat sterilization method at 115 °C for 20 min for the aseptic treatment of the medium. After the sterilization program ends, naturally cool to 25 ± 1 °C to obtain Rosa roxburghii juice medium 2;

[0048] (3) Soybean milk medium: Select soybeans without diseases and pests, wash them clean, and put them into a wall-breaking juicer with 11 times the amount (w / w) of deionized water to obtain the initial soybean extract;

[0049] Separate with a plate and frame filter press, remove the suspended soybean dregs, and then place the filtrate in a high-speed centrifuge and centrifuge at 8000 r / min for 10 min. Collect the supernatant as the fermentation substrate;

[0050] Add 6% (w / v) glucose and 4% (w / v) yeast extract solution to each liter of filtrate, adjust and mix well, and then use the moist heat sterilization method at 115 °C for 20 min for the aseptic treatment of the medium. After the sterilization program ends, naturally cool to 25 ± 1 °C to obtain the soybean milk medium for standby;

[0051] (4) HS medium.

[0052] 2. Cultivation:

[0053] Transfer the compound bacterial liquid aseptically to each of the above media at an inoculation amount of 10% (v / v) to ensure sufficient contact between the bacterial strain and the medium. Seal it under microaerobic conditions and incubate at a constant temperature of 30 °C for 7 days of static fermentation under light-shielded conditions.

[0054] Detect the yield of BC in each medium after 7 days of cultivation. The specific results are shown in Table 1 below:

[0055] Table 1

[0056]

[0057] 3. Select the BC membranes produced by each group of the above media for the following detections:

[0058] (1) Use an American INSTRON 5982 universal material testing machine to measure the mechanical properties of the material. Preparation of test samples: Cut wet film strips of samples with a specification of 10 mm × 50 mm, measure their thickness with an electronic vernier caliper, and the test tensile speed is 5 mm·min -1 , and test with 3 parallel samples in each group;

[0059] The tensile strength of the specimen is expressed as σ (N / mm 2 ), and the calculation formula is as follows:

[0060]

[0061] Among them, P—the maximum load, N; b—the width of the specimen, mm; d—the thickness of the specimen, mm; the elongation at break of the specimen is expressed as ε (%), and the calculation formula is as follows:

[0062]

[0063] In the formula: G0—the original marked distance of the specimen, mm; G—the distance between the marks at the break of the specimen, mm.

[0064] The specific detection results are shown in Table 2 below:

[0065] Table 2

[0066]

[0067]

[0068] (2) Detect the water holding rate, rehydration rate and moisture permeability of the BC membranes obtained from each medium:

[0069] Water holding rate test: After all samples absorb the surface moisture with filter paper, weigh them and record as M1. Then put the samples into a vacuum freeze dryer for drying, and then take them out and weigh them, and record as M2. The moisture content (W 持 ) of the wet bacterial cellulose membrane is calculated according to the following formula:

[0070]

[0071] Rehydration rate test: The sample was placed in a beaker filled with distilled water, taken out after soaking for 24 h, weighed after absorbing the surface moisture with filter paper, and recorded as M3. The rehydration rate (W 复 ) of the bacterial cellulose dry film was calculated according to the following formula:

[0072]

[0073] Moisture permeability test: Pure water was poured into a 50 mL experimental beaker, and the water level was controlled to be about 10 mm from the bottom of the sample. The material to be tested completely covered the opening of the beaker and was tightened with an elastic fixing band. The blank control group was sealed with a waterproof plastic film. The experimental parameters were set as: constant temperature 38 °C, constant humidity 50%, continuous action for 24 hours, and effective action area 0.001963 m 2 . The moisture permeability (WVT) was calculated according to the following formula:

[0074]

[0075] In the formula: WVT is the moisture permeation amount, g / (m 2 ·24 h); Δm is the difference between two weighings of the same test sample combination, g; Δm′ is the difference between two weighings of the blank sample combination, g; A is the effective test area, m 2 ; T is the test time, h.

[0076] The specific results are shown in Table 3 below:

[0077] Table 3

[0078] Culture medium Water holding rate (%) Rehydration rate (%) <![CDATA[Water vapor permeability (g / m 2 *24h)]]> Roselle juice medium 1 98.02±0.10 87.35±0.58 5335.84±2.39 Roselle juice medium 2 97.49±0.21 85.32±0.49 5273.69±2.01 Soybean milk medium 95.36±0.19 82.71±0.62 5215.64±3.05 HS medium 96.72±0.12 83.54±0.49 5303.41±2.10

[0079] In summary, the BC film obtained by culturing with the Rosa roxburghii Tratt juice medium 1 has a high yield and high quality.

[0080] Example 3:

[0081] Preparation of hydroxypropyltrimethylammonium chloride chitosan (HACC)-bacterial cellulose:

[0082] An aqueous solution of glacial acetic acid and deionized water was prepared at a volume ratio of 0.5%. After ultrasonic treatment for 8 min to uniformly disperse the solution, HACC was added to the system respectively to prepare HACC solutions with three concentrations of 0.2%, 1.2%, and 2.5%. The mixed solution was placed in a constant temperature magnetic stirrer at 30 °C and continuously stirred for 5 h to ensure that HACC was fully dissolved to form a clear and stable HACC solution.

[0083] Select the BC membranes cultured with the above-mentioned roselle juice medium 1. After freeze-drying, immerse three BC membranes into 50 mL of three HACC solutions with different concentrations respectively, and let them stand at room temperature for 30 h to allow the quaternary ammonium salt molecules to penetrate into the internal pores of the BC membranes through diffusion. After the loading is completed, take out the membrane pieces and rinse them three times with sterile deionized water to remove the unbound HACC residues on the surface. Then transfer them to a freeze dryer and dry them at -50 °C for 72 h to achieve the stable immobilization of HACC by intermolecular hydrogen bonding and electrostatic adsorption. Finally, three HBC composite antibacterial dressings with different HACC concentrations are obtained, labeled as HBC-1 (concentration: 0.2%), HBC-2 (concentration: 1.2%), and HBC-3 (concentration: 2.5%) respectively.

[0084] (1) Observe the macroscopic appearance ( Figure 2 ) and microscopic appearance ( Figure 3 ) and spectrograms ( Figures 4 - 5 ) of the BC membrane (not immersed in HACC solution) cultured with roselle juice medium 1, the HBC-1 membrane, the HBC-2 membrane, and the HBC-3 membrane, as well as the microscopic images ( Figure 6 ) of the BC-1 membrane cultured with soymilk medium;

[0085] It can be seen from Figure 3 and Figure 7 that the BC membrane cultured with roselle juice medium 1 and the HBC-1 membrane, the HBC-2 membrane, and the HBC-3 membrane all have good 3D network structures, while the nano-appearance morphology of the BC-1 membrane cultured with soymilk medium is slightly worse than that of the BC membrane.

[0086] (2) Mechanical property testing:

[0087] Conduct tensile strength, specimen elongation at break, and Young's modulus tests on the BC membrane and the HBC-1 membrane, the HBC-2 membrane, and the HBC-3 membrane, and plot the stress-strain curves ( Figure 7 ), and the specific mechanical properties are shown in Table 4 below:

[0088] Table 4

[0089] Performance BC HBC - 1 HBC - 2 HBC - 3 Tensile strength (MPa) 29.43±0.71 22.68±0.88 18.97±0.47 17.44±0.77 Elongation at break (%) 22.76±1.40 16.37±0.72 18.16±1.03 21.15±0.43 Young's modulus (MPa) 18.59±0.82 16.58±1.36 20.92±1.93 10.27±1.06

[0090] (3) Water-holding property testing:

[0091] Conduct water-holding rate, rehydration rate, and moisture permeability tests on the BC membrane and the HBC-1 membrane, the HBC-2 membrane, and the HBC-3 membrane, and the basic results are shown in Table 5 below:

[0092] Table 5

[0093] Group Dry weight (g) Water holding rate (%) Rehydration rate (%) <![CDATA[Water vapor permeability (g / m 2 *24h)]]> BC 0.88±0.21 98.02±0.10 87.35±0.58 5335.84±2.39 HBC - 1 0.84±0.30 97.82±0.03 84.58±0.63 6106.76±2.88 HBC - 2 0.85±0.43 96.22±0.02 82.16±0.46 6428.36±4.12 HBC - 3 0.89±0.33 94.22±0.04 78.65±0.57 6849.27±3.42

[0094] (4) Antibacterial property testing:

[0095] The bacterial suspension concentrations of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were serially diluted to 10 6 CFU / mL using sterile PBS buffer. 100 μL of each diluted solution was taken for inoculation on LB agar plates, and then the sterilized BC membrane and HBC-3 membrane test samples were symmetrically placed on the surface of the culture medium. After the petri dishes were incubated at a constant temperature of 37°C for 24 h, an electronic vernier caliper was used to measure the diameter of the inhibition zone. Three replicate experiments were set for each group and the mean value was calculated. Streptococcus mutans was treated in the same way and cultured at 37°C in an incubator for 48 h. The presence or absence of an inhibition zone was observed. If an inhibition zone appeared, it indicated that the material had antibacterial effects.

[0096] The specific results are as Figure 8 shown. An obvious inhibition zone appeared in the HBC-3 membrane group, that is, the HBC-3 membrane had antibacterial effects.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a modified bacterial cellulose medical dressing film material, characterized in that, The preparation method includes the following steps: S1. After mixing Rosa roxburghii tratt with deionized water for cell wall breaking, centrifuge to collect the supernatant, subject it to repeated freeze-thaw cycles 3 - 5 times, and filter it to obtain the fermentation substrate. S2. Add glucose with a w / v of 2% - 8% and yeast extract powder with a w / v of 0.75% - 1.2% to the fermentation substrate, mix evenly, and sterilize to obtain the pear juice medium for standby. S3. Compound Acetobacter xylinum, lactic acid bacteria, and yeast into a compound bacterial solution for standby. S4. Inoculate the compound bacterial solution into the above-mentioned pear juice medium at a volume ratio (v / v) of 10% for static fermentation. After obtaining the BC membrane, terminate the culture. Take out the BC membrane and freeze-dry it to obtain the freeze-dried BC membrane. S5. Add quaternary ammonium salt chitosan to an acetic acid solution and stir to dissolve it, controlling the concentration of quaternary ammonium salt chitosan to be 0.2% - 2.5% to obtain the quaternary ammonium salt chitosan solution for standby. S6. Place the freeze-dried BC membrane in the quaternary ammonium salt chitosan solution, let it stand at room temperature, take out the BC membrane, remove the residue of quaternary ammonium salt chitosan, and then freeze-dry it to obtain the medical dressing membrane material.

2. The preparation method according to claim 1, wherein: The specific preparation method of the fermentation substrate is as follows: First, remove the cores of Rosa roxburghii tratt, wash them, add deionized water with a mass ratio of 10 - 12 times, perform cell wall breaking and juice extraction, then filter under pressure to remove suspended fruit residues. The filtrate is centrifuged at 6000 - 8000 r / min for 10 - 15 min, and then the supernatant is collected and frozen at -30°C to -40°C for 8 - 10 h, and then melted in a water bath at 25 - 35°C. Repeat the freeze-thaw treatment 3 - 5 times and then filter. The filtrate is used as the fermentation substrate.

3. The preparation method according to claim 1, characterized in that: The sterilization method in step S2 is moist heat sterilization at 115°C for 15 - 25 min, and then naturally cool to 25 ± 1°C for standby.

4. The preparation method according to claim 1, characterized in that: In the step S3, the content of Gluconacetobacter xylinus in the compound bacterial liquid is 1.35×10 8 cells / mL, the content of lactic acid bacteria is 1.8×10 5 cells / mL, and the content of yeast is 2.8×10 5 cells / mL.

5. The preparation method according to claim 1, characterized in that: The fermentation method in step S4 is to culture in the dark at a temperature of 27 - 35°C; and terminate the culture when the thickness of the BC membrane reaches 2 - 8 mm.

6. The preparation method according to claim 1, characterized in that: The mass concentration of acetic acid in the acetic acid solution in step S5 is 0.3% - 0.8%.

7. The preparation method according to claim 1, characterized in that: The quaternary ammonium salt chitosan in step S5 is hydroxypropyl trimethyl ammonium chloride chitosan.

8. The preparation method according to claim 1, wherein: The standing time at room temperature in step S6 is 18 - 36 h.

9. Application of the modified bacterial cellulose medical dressing membrane material as described in any one of claims 1 - 8 in the preparation of a medical antibacterial dressing.

Images