CN104072809A - Preparation method of graphene oxide/bacterial cellulose antibacterial compound material - Google Patents
Preparation method of graphene oxide/bacterial cellulose antibacterial compound material Download PDFInfo
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Abstract
The invention discloses a preparation method of a graphene oxide/bacterial cellulose antibacterial compound material. The preparation method comprises the following steps: (1) inoculating activated bacterial cellulose into a seed medium to culture, then, inoculating the activated bacterial cellulose into a fermentation medium according to an inoculum size of 5-10%, and after fully and uniformly mixing, putting in a constant-temperature culture box at 30+/-2 DEG C, standing and fermenting for 1-2 weeks to obtain a bacterial cellulose wet film; (2) after alkali-washing the bacterial cellulose wet film, pulping to a homogeneous phase suspension liquid by virtue of a mechanical method; (3) mixing a graphene oxide solution with the bacterial cellulose homogeneous phase suspension liquid, and carrying out ultrasonic dispersion to prepare a homogeneous phase mixed liquid; (4) decompressing and filtering the homogeneous phase mixed liquid, and freezing and drying to separate the graphene oxide/bacterial cellulose antibacterial compound material. The preparation method of the graphene oxide/bacterial cellulose antibacterial compound material disclosed by the invention is simple, few in step and easy to operate, and the prepared graphene oxide/bacterial cellulose antibacterial compound material has excellent antibacterial performance and can be widely applied to the fields such as medical dressing and artificial skins.
Description
Technical field
The present invention relates to technical field of composite materials, particularly a kind of preparation method of graphene oxide/bacteria cellulose antimicrobial composite material.
Background technology
Graphene by carbon atom with sp
2the monoatomic layer that hydridization connects forms, and is the thinnest two-dimensional material of finding at present, and this special structure makes it contain many peculiar physicochemical property, as high specific surface area and good heat conductivility, mechanical property and electronics transmission capacity.Graphene oxide (Graphene Oxide, GO) be a kind of derivative of Graphene, to be peeled off and the single or multiple lift graphite oxide that forms by graphite oxide, there is typical accurate two-dimensional space structure, on its lamella, contain a lot of oxy radicals, there is wetting ability and mechanical property that higher specific surface energy is good, in water and most of polar organic solvent, there is good dispersion stabilization.The polar functional group on graphene oxide surface easily and some polarity organic molecules and strong interaction or the chemical bond of polymer formation.The preparation method of graphene oxide has a lot, and at present three kinds of conventional basic skills are Brodie method, Staudenmaier method and Hummers method, and additive method be all on these three kinds of method bases, carry out improved.
Bacteria cellulose (Bacterial cellulose, BC) is the extracellular fiber element being produced by certain microorganism (being mainly bacterium), and within 1866, British scientist Brown has reported acetobacter xylinum energy synthetic cellulose first.The Pseudomonas of fermented-producing bacteria cellulose mainly contains, acetic acid Pseudomonas, aerobacter, Agrobacterium, rhizobium, Sarcina, Alcaligenes, pseudomonas, Zoogloea and achromobacter, acetobacter xylinum (Acetobacter xylinum) is that produce at present bacteria cellulose main used bacterium.The primitive fiber diameter of bacteria cellulose only has 40-60nm, is the naturally occurring meticulous nano material of occurring in nature, has unique character: (1) has high chemical purity and high-crystallinity, there is no the association products such as xylogen, pectin and hemicellulose; (2) have very strong water-holding power, the bacteria cellulose water-holding power of undried reaches more than 1000%, and after lyophilize, water-holding power still reaches 600%; (3) there is higher biocompatibility and biodegradability, in physical environment, under the conditions such as acidity, microorganism and cellulase catalysis, can finally be degraded into the small-molecule substances such as monose; (4) Fibre diameter is between 0.01-0.1 μ m, and more than several times to ten that Young's modulus is general vegetable fibre times, and tensile strength is high; (5) there is Modulatory character during bacteria cellulose biosynthesizing, by regulating culture condition, can obtain the discrepant bacteria cellulose of chemical property.At present, bacteria cellulose has a wide range of applications at aspects such as food, acoustical vibration film, papermaking, medical materials.
Although bacteria cellulose has the performance of a lot of excellences, himself does not have germ resistance, has affected its application and development in medical dressing field.Because graphene oxide has superior germ resistance, and cytotoxicity is little, can produce in batches, with low cost, and this novel carbon nanomaterial is expected to be widely used at environment and clinical field.Therefore, the graphene oxide with germ resistance is added the matrix material of preparing in bacteria cellulose will be expected to obtain novel antibacterial medical dressing.
Summary of the invention
Goal of the invention: directed toward bacteria Mierocrystalline cellulose does not have the problem of germ resistance, the preparation method who the object of this invention is to provide a kind of graphene oxide/bacteria cellulose antimicrobial composite material, bacteria cellulose and graphene oxide are carried out compound, raw material obtains 100% utilization, the matrix material of preparation had both kept the character of bacteria cellulose uniqueness, had again germ resistance.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is as follows:
A preparation method for graphene oxide/bacteria cellulose composite material, comprises following operation steps:
(1) bacteria cellulose after activation being produced to bacterium access seed culture medium cultivates, according in the inoculum size access fermention medium of 5-10%, after fully mixing, be placed in 30 ± 2 ℃ of constant incubators again, in standing for fermentation 1-2 week, obtain bacterial cellulose wet-coating;
(2) will after bacterial cellulose wet-coating alkali cleaning, by mechanical means, pull an oar into homogeneous suspension liquid;
(3) graphene oxide solution is mixed with bacteria cellulose homogeneous suspension liquid, after ultrasonic dispersion, make homogeneous phase mixed solution;
(4) by homogeneous phase mixed solution filtration under diminished pressure, after lyophilize, isolate graphene oxide/bacteria cellulose composite material.
Described bacterial classification is the acetobacter xylinum in acetic acid Pseudomonas.
Described bacteria cellulose homogeneous suspension liquid concentration is 50-100g/L.
The quality volume fraction of described graphene oxide solution is 0.5-2g/L.
When preparing homogeneous phase mixed solution, the volume ratio of bacteria cellulose homogeneous suspension liquid and graphene oxide solution is 200～50:1.
The filtration of homogeneous phase mixed solution is to the filtration under diminished pressure under-0.1MPa at-0.08MPa.
Described preparation method will obtain graphene oxide/bacteria cellulose composite material.
Beneficial effect: compared with prior art, the preparation method of graphene oxide/bacteria cellulose antimicrobial composite material of the present invention is simple, and step is few, easy to operate, does not include malicious chemical reagent and high temperature, process is simple, green, easy to operate, can suitability for industrialized production.Bacteria cellulose and graphene oxide are carried out compound, raw material obtains 100% utilization, and resulting graphene oxide/bacteria cellulose composite material had both kept the character of bacteria cellulose uniqueness, had again superior germ resistance, can be widely used in the fields such as medical dressing, artificial skin.
Accompanying drawing explanation
Fig. 1 is that graphene oxide/bacteria cellulose composite material amplifies the SEM photo figure of 15000 times.Wherein, figure a is the prepared graphene oxide/bacteria cellulose composite material of embodiment 1, figure b is the prepared graphene oxide/bacteria cellulose composite material of embodiment 2, figure c is the prepared graphene oxide/bacteria cellulose composite material of embodiment 3, and figure d is the prepared graphene oxide/bacteria cellulose composite material of embodiment 4;
Fig. 2 is the SEM figure of the prepared graphene oxide/bacteria cellulose composite material of embodiment 2.Wherein, figure a is for amplifying the SEM photo of 60000 times, and figure b is the SEM photo in graphene oxide/bacteria cellulose composite material transverse section;
Fig. 3 is the XRD figure of bacteria cellulose and graphene oxide/bacteria cellulose composite material.In figure, a is the XRD figure of pure bacteria cellulose, b is the XRD figure of embodiment 1 graphene oxide/bacteria cellulose composite material, c is the XRD figure of embodiment 2 graphene oxides/bacteria cellulose composite material, d is the XRD figure of embodiment 3 graphene oxides/bacteria cellulose composite material, and e is the XRD figure of embodiment 4 graphene oxides/bacteria cellulose composite material;
Fig. 4 is the uv-visible absorption spectroscopy figure of graphene oxide/bacteria cellulose composite material.In figure, the uv-visible absorption spectroscopy figure that a is pure bacteria cellulose, b is the uv-visible absorption spectroscopy figure of embodiment 1 graphene oxide/bacteria cellulose composite material;
Fig. 5 is the antibiotic rate result figure of graphene oxide/bacteria cellulose composite material to streptococcus aureus;
Fig. 6 is that graphene oxide/bacteria cellulose composite material is to colibacillary antibiotic rate result figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but the present invention is not subject to the restriction of these examples of implementation.
Embodiment 1
Yeast extract (5g/L), glucose (5g/L), potassium primary phosphate (1g/L), bitter salt (15g/L) and peptone (5g/L) are mixed with respectively to the seed culture medium of 100mL and the fermention medium of 200mL, need not regulate pH value.Seed culture medium and fermention medium are put into Autoclave, and temperature is that 121 ℃, pressure are 0.1MPa, and sterilizing 20 minutes adds ethanol (20mL/L) after nutrient solution is cooling.Acetobacter xylinum after activation is seeded in seed culture medium, in the shaking table of 30 ℃ and 160rpm/min, cultivates 24h.The inoculum size of (volume ratio, within the scope of 5-10% all can) is inoculated in fermention medium fully concussion by seed culture fluid it is mixed according to 6%, and then standing cultivations 1-2 is all at 30 ± 2 ℃, obtains oyster white bacteria cellulose film.With the residue on deionized water rinsing bacteria cellulose surface, be immersed in the NaOH solution of 0.1M, 80 ℃ of heated and stirred 1h, then use vinegar acid for adjusting pH to 7.0, finally use deionized water rinsing bacteria cellulose film.5g bacterial cellulose wet-coating is put into deionized water, by mechanical means, pull an oar into 50mL homogeneous suspension liquid, obtaining bacteria cellulose homogeneous suspension liquid concentration is 100g/L, mix with the volume ratio of 200:1 with graphene oxide solution (2g/L), supersound process 10min after stirring (power is 300W), obtains a homogeneous mixture.By carry out-0.1MPa of mixture negative pressure filtration, last-80 ℃ of vacuum lyophilizations, obtain graphene oxide/bacteria cellulose composite material.
Embodiment 2
Method is with embodiment 1, and difference is, bacteria cellulose homogeneous suspension liquid is with graphene oxide solution by volume for 100:1 mixes, and after stirring, supersound process becomes uniform mixture.By carry out-0.1MPa of mixture negative pressure filtration, last-80 ℃ of vacuum lyophilizations, obtain graphene oxide/bacteria cellulose composite material.
Embodiment 3
Method is with embodiment 1, and difference is only, bacteria cellulose homogeneous suspension liquid is with graphene oxide solution by volume for 200:3 mixes, and after stirring, supersound process becomes uniform mixture.By carry out-0.1MPa of mixture negative pressure filtration, last-80 ℃ of vacuum lyophilizations, obtain graphene oxide/bacteria cellulose composite material.
Embodiment 4
Method is with embodiment 1, and difference is only, bacteria cellulose homogeneous suspension liquid is with graphene oxide solution by volume for 50:1 mixes, and after stirring, supersound process becomes uniform mixture.By carry out-0.1MPa of mixture negative pressure filtration, last-80 ℃ of vacuum lyophilizations, obtain graphene oxide/bacteria cellulose composite material.
Embodiment 5
Method is with embodiment 1, and difference is only, bacteria cellulose homogeneous suspension liquid concentration is 80g/L, and the quality volume fraction of graphene oxide solution is 1g/L.Bacteria cellulose homogeneous suspension liquid is with graphene oxide solution by volume for 100:1 mixes, and after stirring, supersound process becomes uniform mixture.By carry out-0.08MPa of mixture negative pressure filtration, last-80 ℃ of vacuum lyophilizations, obtain graphene oxide/bacteria cellulose composite material.
Embodiment 6
Method is with embodiment 1, and difference is, bacteria cellulose homogeneous suspension liquid concentration is 50g/L, and the quality volume fraction of graphene oxide solution is 0.5g/L.Bacteria cellulose homogeneous suspension liquid is with graphene oxide solution by volume for 200:3 mixes, and after stirring, supersound process becomes uniform mixture.By carry out-0.08MPa of mixture negative pressure filtration, last-80 ℃ of vacuum lyophilizations, obtain graphene oxide/bacteria cellulose composite material.
Performance test:
(1) scanning electronic microscope (SEM) test
Graphene oxide/bacteria cellulose composite material amplifies the SEM photo of 15000 times, as shown in Figure 1.Figure a is the graphene oxide/bacteria cellulose composite material of embodiment 1 preparation, figure b is the graphene oxide/bacteria cellulose composite material of embodiment 2 preparations, figure c is the graphene oxide/bacteria cellulose composite material of embodiment 3 preparations, and figure d is the graphene oxide/bacteria cellulose composite material of embodiment 4 preparations.As shown in Figure 2, figure a is that the graphene oxide/bacteria cellulose composite material of embodiment 2 preparations amplifies the SEM photo of 60000 times, and figure b is the SEM photo in graphene oxide/bacteria cellulose composite material transverse section of embodiment 2 preparations.From Fig. 1 and Fig. 2, can observe graphene oxide and be evenly distributed in the surperficial and inner of bacteria cellulose.
(2) X-ray diffraction (XRD) test
The XRD figure of bacteria cellulose and graphene oxide/bacteria cellulose composite material, as shown in Figure 3.In figure, a is the XRD figure of pure bacteria cellulose, b is the XRD figure of the graphene oxide/bacteria cellulose composite material of embodiment 1 preparation, c is the XRD figure of the graphene oxide/bacteria cellulose composite material of embodiment 2 preparations, d is the XRD figure of the graphene oxide/bacteria cellulose composite material of embodiment 3 preparations, and e is the XRD figure of the graphene oxide/bacteria cellulose composite material of embodiment 4 preparations.From figure, a can find out that the characteristic diffraction peak of pure bacteria cellulose appears at 2 θ=14.46 °, 16.64 ° and 22.66 °, they belong to respectively (
), (110) and (200) crystal face; For graphene oxide/bacteria cellulose composite material, can observe equally three diffraction peaks that belong to bacteria cellulose; And there is the characteristic diffraction peak of graphene oxide in the position in 2 θ=9.64 °.
(3) uv-visible absorption spectroscopy test
The uv-visible absorption spectroscopy figure of bacteria cellulose and graphene oxide/bacteria cellulose composite material, as shown in Figure 4.In figure, a is the uv-visible absorption spectroscopy figure of the graphene oxide/bacteria cellulose of embodiment 1 preparation, there is no obvious absorption peak; B is the uv-visible absorption spectroscopy figure of the graphene oxide/bacteria cellulose of embodiment 2 preparations, and c is the uv-visible absorption spectroscopy figure of the graphene oxide/bacteria cellulose of embodiment 3 preparations.There is the maximum absorption band of graphene oxide in matrix material as we can see from the figure when wavelength is 240nm.
(4) antibacterial experiment:
Experiment adopts liquid culture system, with pure bacteria cellulose film, opposes in the same old way, and the bacteria colony count that is adsorbed onto test sample adopts colony counting method to characterize.
Concrete steps are:
A. bacterium solution preparation
The solid medium of cultivating staphylococcus glucose coccus (Staphylococcus aureus) and intestinal bacteria (Escherichia coli) employing is trypticase soy agar substratum Tryptose Soya Agar (TSA), substratum consists of: pancreatic digest of casein 15.0g/L, soyflour papain digestion thing 5.0g/L, sodium-chlor 5.0g/L, agar 15.0g/L, pH value 7.2,121 ℃ of sterilizings of high pressure steam, after 20 minutes, are down flat plate.Staphylococcus glucose coccus and the intestinal bacteria bacterial classification of with disinfection inoculation ring, getting freezing preservation are inoculated into respectively on TSA culture plate, be placed in 37 ℃ of constant temperature culture 24h of incubator, with disinfection inoculation ring picking colony, add respectively in the aseptic Erlenmeyer flask of the pancreas peptone soybean broth substratum (TSB) that fills 100mL sterilizing, cultivate 4-6h, adopt turbidimetry measurement to make its bacterial concentration 1 * 10
7cFU/mL left and right.
B. adsorption experiment
By cultured Bacteria liquid centrifugal 5min under-4 ℃ and 5000rpm/min respectively, remove supernatant liquor, with phosphoric acid buffer (PBS), the thalline of precipitation is disperseed respectively again, obtaining bacterial concentration is 1 * 10
7two kinds of different bacterium liquid of CFU/mL, and add respectively in the glass jar of sterilizing.Sample to be tested (1cm * 1cm) is put into the glass jar that contains bacterium liquid, glass jar is placed in respectively in the shaking table of 37 ℃ and 20rpm/min and cultivates 1h.
C. sterilizing rate calculates
Take out sample, put into the ultrasonic 10min of 10mL sterilized water, the bacterium that is adsorbed onto sample is shaken in deionized water.Adopt stepwise dilution to be coated with dull and stereotyped method, respectively the bacterium of test specimens product absorption is counted, sterilizing rate is calculated by following formula: R (%)=(N
contrast-N
sample)/N
sample* 100%, wherein, N
contrastand N
samplebe respectively the colony number of the matrix material absorption of pure bacteria cellulose film and interpolation graphene oxide.
The antibiotic rate result of graphene oxide/bacteria cellulose composite material to streptococcus aureus, as shown in Figure 5.Graphene oxide/bacteria cellulose composite material has the effect of very good anti-Staphylococcus aureus, and when graphene oxide content is 2g/L, matrix material can reach 65% to the antibiotic rate of streptococcus aureus.
Graphene oxide/bacteria cellulose composite material is to colibacillary antibiotic rate result, as shown in Figure 6.Graphene oxide/bacteria cellulose composite material and pure bacteria cellulose are compared and are had very superior anticolibacillary effect, and when graphene oxide content is 2g/L, matrix material can reach 97% to colibacillary antibiotic rate.
Claims (7)
1. a preparation method for graphene oxide/bacteria cellulose antimicrobial composite material, is characterized in that, comprises following operation steps:
(1) bacteria cellulose after activation being produced to bacterium access seed culture medium cultivates, according in the inoculum size access fermention medium of 5-10%, after fully mixing, be placed in 30 ± 2 ℃ of constant incubators again, in standing for fermentation 1-2 week, obtain bacterial cellulose wet-coating;
(2) will after bacterial cellulose wet-coating alkali cleaning, by mechanical means, pull an oar into homogeneous suspension liquid;
(3) graphene oxide solution is mixed with bacteria cellulose homogeneous suspension liquid, after ultrasonic dispersion, make homogeneous phase mixed solution;
(4) by homogeneous phase mixed solution filtration under diminished pressure, after lyophilize, isolate graphene oxide/bacteria cellulose composite material.
2. the preparation method of graphene oxide/bacteria cellulose composite material according to claim 1, is characterized in that: described bacterial classification is the acetobacter xylinum in acetic acid Pseudomonas.
3. the preparation method of graphene oxide/bacteria cellulose composite material according to claim 1, is characterized in that: described bacteria cellulose homogeneous suspension liquid concentration is 50-100g/L.
4. the preparation method of graphene oxide/bacteria cellulose composite material according to claim 1, is characterized in that: the quality volume fraction of described graphene oxide solution is 0.5-2g/L.
5. the preparation method of graphene oxide/bacteria cellulose composite material according to claim 1, is characterized in that: when preparing homogeneous phase mixed solution, the volume ratio of bacteria cellulose homogeneous suspension liquid and graphene oxide solution is 200 ~ 50:1.
6. the preparation method of graphene oxide/bacteria cellulose composite material according to claim 1, is characterized in that: the filtration of homogeneous phase mixed solution is to the filtration under diminished pressure under-0.1MPa at-0.08 MPa.
7. graphene oxide/the bacteria cellulose composite material of the prepared acquisition of preparation method of the graphene oxide/bacteria cellulose composite material described in any one in claim 1-6.
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