CN102634068A - Method and device for preparing functional nanoparticle/bacterial cellulose composite membranes - Google Patents
Method and device for preparing functional nanoparticle/bacterial cellulose composite membranes Download PDFInfo
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Abstract
The invention relates to a method and device for preparing functional nanoparticle/bacterial cellulose composite membranes by an inverted culturing method. The method comprises the steps of preparing a culture medium, carrying out activation and accessing on strains, and carrying out culturing and post-processing on bacterial cellulose membranes, wherein in the step of preparing the culture medium, functional nanoparticles are added. Inverted culturing refers to inversely place a culturing vessel on an oxygen chamber, the culturing vessel and the oxygen chamber are separated by using an oxygen permeating membrane, and a seam between the culturing vessel and the oxygen chamber is sealed. The device disclosed by the invention comprises a base and a culturing vessel, wherein the base is an open vessel with a step at the upper part thereof, the culturing vessel is inversely placed on the step of the vessel, the base and the culturing vessel are separated by using an oxygen permeating membrane, the oxygen permeating membrane and the lower part of the base form an oxygen chamber, and the lower part of the base is provided with an air outlet hole and an air inlet hole. The method and device disclosed by the invention integrate the culturing and functionalization of bacterial cellulose membranes, and are simple in operation, short in cycle and low in cost; and according to the invention, on the premise of not damaging a three-dimensional network structure in a bacterial cellulose membrane, a functionalized bacterial cellulose composite membrane which is good in functional-nanoparticle adsorption property and excellent in performance is prepared.
Description
Technical field
The present invention relates to a kind of method and device thereof for preparing function nano particle/bacteria cellulose composite membrane, particularly relate to a kind of method and device thereof that culture method prepares function nano particle/bacteria cellulose composite membrane of being inverted.
Background technology
As a kind of novel biomaterial; Bacteria cellulose has the character of many uniquenesses;, high Young's modulus strong like superfine reticular fiber structure, the high chemical purity of high-crystallinity, retentiveness ventilation property, excellent biological compatibility and biodegradability etc., these performances make bacteria cellulose all be widely used in every field.Through with bacteria cellulose and the compound multifunction that can realize bacteria cellulose of various functionalized particles.
The method of bacteria cellulose functionalization mainly contains following several kinds at present: in-situ compositing, physisorphtion, physical blending method.In-situ compositing be exactly through chemical reaction on the hydroxyl group sites of bacteria cellulose film directly the complex functionality particle from realizing the functionalization of bacteria cellulose; Physisorphtion is immersed in bacteria cellulose film in the functional particles solution exactly, utilizes the hydroxyl hydrogen bond to the absorption of functional particles and realize the functionalization of bacteria cellulose film; The physical blending method is earlier bacteria cellulose film to be pulled an oar, and then with bacteria cellulose slurries and functional particles blend, realizes the functionalization of bacteria cellulose film at last again through the appropriate means film forming.More than three kinds of methods significant disadvantage is all arranged; Only be distributed in the surface of bacteria cellulose film by its functional particles of bacteria cellulose composite membrane of in-situ compositing and physisorphtion preparation; Have only the functional particles of very small amount can get into the inside of bacteria cellulose film, so the utilization ratio of bacteria cellulose film is low.Bacteria cellulose composite membrane by the physisorphtion preparation is also insecure for the absorption of functional particles, is difficult to make the functional permanent maintenance that obtains.To therefore bacteria cellulose film making beating earlier have been destroyed the tridimensional network of bacteria cellulose film when physisorphtion prepares composite package, can't obtain the mould material of excellent mechanical, thereby limit the application of bacteria cellulose composite membrane.
Summary of the invention
In order to overcome the deficiency of above several method; The invention provides a kind of method and device thereof for preparing bacteria cellulose composite membrane; Be a kind of method and device thereof for preparing function nano particle/bacteria cellulose composite membrane; Particularly a kind ofly be inverted method and the device thereof that culture method prepares function nano particle/bacteria cellulose composite membrane, the cultivation that is about to bacteria cellulose film combines with the functionalization that realizes bacteria cellulose film, and a step accomplishes.This method is in the insufficient while that has overcome traditional method, and is simple to operate in addition, the cycle short, low cost and other advantages.
Specific practice is that the function nano particle is added in the bacteria cellulose fermention medium; Method through stirring and interpolation tensio-active agent obtains stable function nano particle suspension liquid; In such fermention medium, cultivating the bacteria cellulose that obtains can coat the function nano particle fully; And through suitable dispersion means; Can make function nano evengranular be distributed in bacteria cellulose film the surface and inner; Thereby under the prerequisite of not destroying the inner three-dimensional structure of bacteria cellulose film, obtain the functionalization bacteria cellulose composite membrane of excellent property; Solved the common problem for preparing the functionalization bacteria cellulose film through ordinary method, that is, functional particles can only be in the bacteria cellulose film surface recombination and inside and the functional particles and the bacteria cellulose film that are difficult for getting into film to the not strong problem of function nano particle adsorptive power.Because general nano particle proportion is greater than the proportion of substratum, therefore use a kind of special method to come the culturing bacterium Mierocrystalline cellulose---be inverted culture method.
A kind of method for preparing function nano particle/bacteria cellulose composite membrane of the present invention; Comprise preparation seed culture medium, preparation fermention medium, actication of culture, bacterial classification access, cultivation and post-processing step, add the function nano particle in the described preparation fermention medium step; The biological culture process is according to the biological culture process of bacteria cellulose film; The cultivation of bacteria cellulose film is with reference to the biological culture of the bacteria cellulose film in Ph D dissertation " preparation of bacteria cellulose/carbon nano tube compound material and structure properties research " and the Master's thesis " preparation and the sign of novel medical biotechnology material---bacteria cellulose "; Perhaps articles of reference " Synthesis of cellulose by acetobacter xylinum ", the biological culture of the bacteria cellulose film among " In situ modification of bacterial cellulose network structure by adding interfering substances during fermentation " and " the Nano-biomaterials application:In situ modification of bacterial cellulose structure by adding HPMC during fermentation ".
Described cultivation is to cultivate after culture vessel is inverted, and culture vessel is inverted on the oxygen room, separate with the oxygen flow material membrane between culture vessel and the oxygen room, and sealed vessel and oxygen room's seam crossing.Because function nano particulate proportion is bigger than fermention medium, the function nano particle can form function nano particles dispersed layer at fermention medium and silica gel intersection after leaving standstill.Under 30 ℃, bacteria cellulose was carried out static cultivation 7～14 days.Because the bacterial classification that adopts is an aerobic bacteria, so bacteria cellulose film must be to generate at the interface that culture medium solution contacts with oxygen.At liquid-gas interface, the bacteria cellulose filament is constantly secreted in the secretion terminal of bacterium, and filament is assembled tow, and tow is gathered into some cotton-shaped tow group.The stack of rolling into a ball along with tow finally forms bacteria cellulose film at gas-liquid surface.Tow group generation, gathering, synergetic simultaneously can the adsorption function nano particle, make the function nano uniform particles be distributed in the bacteria cellulose film.Utilize the function nano particle/bacteria cellulose composite membrane of present method preparation can make the function nano particle mainly be distributed in the inside of bacteria cellulose film; Therefore the function nano particle is made the time spent difficult drop-off receiving external force, thereby makes that the function of function nano particle/bacteria cellulose composite membrane is more lasting.
The step of actication of culture is that transfering loop is burnt heat on spirit lamp, repeatedly several times, then transfering loop is stretched into inclined-plane in vitro, hooks up to insert in the Erlenmeyer flask that seed culture medium is housed after bacterial classification 1 encircles to carry out actication of culture.
Bacterial classification is meant acetobacter xylinum, produces acetobacter, acetify bacillus, Acetobacter pasteurianus, glucose bacillus, Agrobacterium, root nodule bacterium, sarcina, pseudomonas cepacia or Pseudomonas cocovenenans.
The step that bacterial classification inserts inserts in the fermention medium for preparing for from seed culture medium, take out bacterial classification with transfer pipet, must be near spirit lamp in the bacterial classification migration process.
Fermention medium also need add the function nano particle except basal component.
As optimized technical scheme:
Aforesaid a kind of method for preparing function nano particle/bacteria cellulose composite membrane; Described solid function nano particle is meant the nano-particle material with various appointed functions, is that wall material and cholesteryl liquid crystal are that the temperature of core causes reversible color change microcapsule or has the nano-Ag particles of anti-microbial property for ferroferric oxide nano granules with superparamagnetism, with gum arabic and gelatin.
Aforesaid a kind of method for preparing function nano particle/bacteria cellulose composite membrane, described function nano particle are that grain diameter is the solid function nano particle of 1～1000nm; Described function nano particulate quality accounts for 1.0～6.0wt% of fermention medium total mass.
Aforesaid a kind of method for preparing function nano particle/bacteria cellulose composite membrane; Also can add corresponding dispersion agent in the described fermention medium according to the difference of the function nano grain type that is added; When the function nano particle is nonpolar nano particle, use ionic dispersant, when the function nano particle is charged nano particle, use non-ionic dispersing agent; The quality of dispersion agent is 1.5～2.0: 1 with the function nano particulate mass ratio that is added;
Described function nano particle is that charged nano particle is a ferroferric oxide nano granules, and non-ionic dispersing agent adopts polyoxyethylene glycol.Polyoxyethylene glycol is the non-ionic type superpolymer that contains the ether chain; Its group neutral; Polyoxyethylene glycol can adsorb with generations interactions such as the weak electrostatic attraction of ferroferric oxide nano granules through hydrogen bond, dipole, Van der Waals forces when in fermention medium, adding polyoxyethylene glycol; Form integument on ferroferric oxide nano granules surface, make to form space steric effect between ferroferric oxide nano granules and mutually exclusive, reach ferroferric oxide nano granules is carried out the dispersive purpose.
Described function nano particle be charged nano particle be that wall material and cholesteryl liquid crystal are that the temperature of core causes reversible color change microcapsule with gum arabic and gelatin, the mass ratio of gum arabic and gelatin is 1: 1, the mass ratio of wall material and core is 2: 1.Non-ionic dispersing agent adopts tween 80.Tween 80 is the polyethenoxy sorbitan oleic acid ester; Can form hydrogen bond between the hydroxyl of ester bond and microcapsule wall material, thereby make tween 80 be adsorbed on the microcapsule wall material surface, because the tween 80 molecular volume is very big; Therefore its space steric effect is obvious, makes the microcapsule stable dispersion.
Described function nano particle is nonpolar nano granule silver particle, and ionic dispersant adopts X 2073.The nano-Ag particles surface is electronegative, and after anionic surfactant sodium dodecylbenzene sulfonate absorption, the nano-Ag particles negative charge increases, and repulsion increases each other, from but nano-Ag particles be easy to disperse.
The method for preparing function nano particle/bacteria cellulose composite membrane as stated, described cultivation is cultivated for being inverted, and promptly is to cultivate after culture vessel is inverted.Culture vessel is inverted on the airtight oxygen room, separates with the oxygen flow pellosil between oxygen room and the culture vessel, and with paraffin sealed vessel and airtight oxygen room seam crossing.Begin aerating oxygen in oxygen room after leaving standstill 1h, carry out 7～14 days the cultivation of leaving standstill.
Aforesaid a kind of method for preparing function nano particle/bacteria cellulose composite membrane, controlled initiative oxygen supply mode is adopted in described cultivation, and described controlled initiative oxygen supply is meant the oxygen supply in oxygen room through the external impetus system.
Aforesaid a kind of method for preparing function nano particle/bacteria cellulose composite membrane, the material of described sealing culture vessel and oxygen room's seam is paraffin or silicone oil.
Aforesaid a kind of method for preparing function nano particle/bacteria cellulose composite membrane, described oxygen flow material membrane is a pellosil.
The method for preparing function nano particle/bacteria cellulose composite membrane as stated, described aftertreatment are meant through after 7～14 days the static cultivation, and the function nano particulate bacteria cellulose film that contains that generates is taken out from substratum; It is immersed in the NaOH solution of 4.0～5.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At air drying or dry in vacuum freeze drier, can obtain function nano particle/bacteria cellulose composite membrane at last.
The present invention also provides a kind of device for preparing function nano particle/bacteria cellulose composite membrane; Described device mainly is made up of base and culture vessel; Described base is the open container that a step is arranged at top; Described culture vessel is upside down on the step of said base, separates with the oxygen flow material membrane between described base and the described culture vessel, and airtight oxygen room is formed at described oxygen flow material membrane and described base bottom; Described base bottom has production well and air inlet port.
Aforesaid device, described base and described culture vessel seam crossing seal with sealing material.Sealing material is chosen as paraffin or silicone oil usually.
Aforesaid device, described air inlet port connects inlet pipe, and said inlet pipe is connected the oxygen supply pipe of external impetus system.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the present invention has realized the cultivation of bacteria cellulose film is combined with the functionalization that realizes bacteria cellulose film through adopting the inversion best cultivation; Make bacteria cellulose film accomplish from cultivating functionalization one step, simple to operate, the cycle short, low cost and other advantages.
(2) the present invention has overcome the shortcoming that adopts traditional method to prepare the function bacteria cellulose composite membrane, can under the prerequisite of not destroying bacteria cellulose film interior three-dimensional structure, prepare the functionalization bacteria cellulose composite membrane fabulous to the absorption of function nano particle, that performance is splendid through the method for preparing function nano particle/bacteria cellulose composite membrane of the present invention.
(3) the present invention has adopted a kind of special device to control the growth of bacteria cellulose film.Adopt controlled initiative oxygen supply mode through of the control of control oxygen flow, can prepare the bacteria cellulose film of different densities, different aperture size to control the oxygen concentration in the airtight oxygen room and then to realize bacteria cellulose film is grown.
Description of drawings
Accompanying drawing is to be inverted the culture apparatus synoptic diagram
Wherein 1 is culture vessel, the 2nd, and function nano particle, the 3rd, oxygen flow material membrane, the 4th, base, the 5th, production well, the 6th, sealing material, the 7th, oxygen room, the 8th, air inlet port.
Embodiment
Below in conjunction with embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Shown in accompanying drawing; A kind of device for preparing function nano particle/bacteria cellulose composite membrane of the present invention; Described device mainly is made up of base 4 and culture vessel 1, and described base 4 is open containers that a step is arranged at top, and described culture vessel 1 is upside down on the step of said base; Separate with oxygen flow material membrane 3 between described base 4 and the described culture vessel 1, described oxygen flow material membrane 3 forms airtight oxygen room 7 with described base 4 bottoms; Described base 4 bottoms have production well 5 and air inlet port 8.
Described base and described culture vessel seam crossing are with sealing material 6 sealings.Sealing material 6 is chosen as paraffin or silicone oil usually.
Described air inlet port 8 connects inlet pipe, and said inlet pipe is connected the oxygen supply pipe of external impetus system.
1. the preparation seed culture medium is used for the activation of product acetobacter.The composition of seed culture medium is: glucose 7.0w/v%, peptone 0.6w/v%, monohydrate potassium 0.3w/v%, potassium primary phosphate 0.2w/v%, disodium hydrogen phosphate dodecahydrate 0.3w/v%, yeast extract 0.5w/v%.The pH value of control seed culture medium is 7.0, and at 121 ℃ of following high-temperature sterilization 30min.
2. take out and be stored in the product acetobacter in the refrigeration chamber.Transfering loop is burnt heat on spirit lamp, transfering loop is stretched into inclined-plane in vitro, hook up bacterial classification one ring back access and be equipped with in the Erlenmeyer flask of the seed culture medium described in step 1.The Erlenmeyer flask that inserts bacterial classification is sealed with gauze, put into shaking table and under 30 ℃, carry out activation 24h, shaking speed 160rpm.
3. in culture vessel, prepare the fermention medium that contains ferroferric oxide nano granules, be used to cultivate function nano particle/bacteria cellulose composite membrane.The composition of fermention medium is: glucose 7.0w/v%; Peptone 0.6w/v%, monohydrate potassium 0.3w/v%, potassium primary phosphate 0.3w/v%; Disodium hydrogen phosphate dodecahydrate 0.4w/v%; Yeast extract 0.6w/v%, ferroferric oxide nano granules 1.0wt%, the particle diameter of ferriferrous oxide nano-particle are 1～20nm.Mechanical stirring 300r/min stirs 1h.The pH value of control fermention medium is 7.0, and at 121 ℃ of following high-temperature sterilization 30min.
4. be inverted on the airtight oxygen room after culture vessel being sealed with the oxygen flow pellosil,, leave standstill 1h with the joint seal of paraffin with culture vessel and oxygen room.Aerating oxygen in oxygen room then carries out 14 days static cultivation under 30 ℃.
5. through after the cultivation like step 4, will contain function nano particulate bacteria cellulose composite membrane and from culture vessel, take out, it will be immersed in the NaOH solution of 5.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At last at air drying or dry in vacuum freeze drier, the Z 250/bacteria cellulose composite membrane that can obtain having superparamagnetism.
6. resulting Z 250/bacteria cellulose composite membrane has good superparamagnetism, and remanent magnetism and coercive force all level off to zero, and maximum saturation susceptibility reaches 10.63emu/g; Mechanical property is excellent, and ultimate tensile strength reaches 20.6MPa.
1. the preparation seed culture medium is used for the activation of acetify bacillus.The composition of seed culture medium is: glucose 5.0w/v%, peptone 0.4w/v%, monohydrate potassium 0.1w/v%, potassium primary phosphate 0.1w/v%, disodium hydrogen phosphate dodecahydrate 0.1w/v%, yeast extract 0.4w/v%.The pH value of control seed culture medium is 6.0, and at 121 ℃ of following high-temperature sterilization 30min.
2. take out and be stored in the acetify bacillus in the refrigeration chamber.Transfering loop is burnt heat on spirit lamp, transfering loop is stretched into inclined-plane in vitro, hook up bacterial classification one ring back access and be equipped with in the Erlenmeyer flask of the seed culture medium described in step 1.The Erlenmeyer flask that inserts bacterial classification is sealed with gauze, put into shaking table and under 30 ℃, carry out activation 24h, shaking speed 160rpm.
In culture vessel preparation to contain with gum arabic and gelatin be that wall material and cholesteryl liquid crystal are the fermention medium that the temperature of core causes reversible color change microcapsule, be used to cultivate function nano particle/bacteria cellulose composite membrane.The composition of fermention medium is: glucose 5.0w/v%, peptone 0.5w/v%, monohydrate potassium 0.1w/v%; Potassium primary phosphate 0.1w/v%; Disodium hydrogen phosphate dodecahydrate 0.2w/v%, yeast extract 0.5w/v% is that wall material and cholesteryl liquid crystal are that the temperature of core causes reversible color change microcapsule 6.0wt% with gum arabic and gelatin; Its particle diameter is 700～1000nm, adds non-ionic dispersing agent tween 80 9.0wt%.Magnetic force is mixed 400r/min and is stirred 1h.The pH value of control fermention medium is 7.0, and at 121 ℃ of following high-temperature sterilization 30min.
4. be inverted on the airtight oxygen room after culture vessel being sealed with the oxygen flow pellosil,, leave standstill 1h with the joint seal of silicone oil with culture vessel and airtight oxygen room.Aerating oxygen in airtight oxygen room then carries out 14 days static cultivation under 30 ℃.
5. through after the cultivation like step 4, will contain function nano particulate bacteria cellulose composite membrane and from culture vessel, take out, it will be immersed in the NaOH solution of 4.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At air drying or dry in vacuum freeze drier, can obtain having microcapsule/bacteria cellulose composite membrane that temperature causes the reversible color performance at last.
6. resulting have microcapsule/bacteria cellulose composite membrane that temperature causes the reversible color performance and can be implemented in 35～42 ℃ of reversible colors, and variable color sensitivity is 0.2 ℃, and can reuse, and discoloration is lasting.
1. according to the described culturing process of Ph D dissertation " preparation of bacteria cellulose/carbon nano tube compound material and structure properties research ", preparation seed culture medium and fermention medium are respectively applied for the activation of acetobacter xylinum and the cultivation of acetobacter xylinum bacteria cellulose.
2. in preparation fermention medium step, in fermention medium, add nano-Ag particles, its particle diameter is 60～120nm, adds the 5.0wt% that quality accounts for the fermention medium total mass.
3. when cultivating culture vessel sealed with the oxygen flow pellosil and be inverted on the airtight oxygen room afterwards, with the joint seal of paraffin with culture vessel and airtight oxygen room.Leave standstill behind the 1h aerating oxygen in oxygen room.Under 30 ℃, carry out 14 days static cultivation.
4. through after the cultivation like step 3, the bacteria cellulose composite membrane that will contain nano-Ag particles takes out from culture vessel, and it is immersed in the NaOH solution of 4.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At air drying or dry in vacuum freeze drier, can obtain having the nanometer silver/bacteria cellulose composite membrane of good anti-microbial property at last.
5. resulting nanometer silver/bacteria cellulose composite membrane has good antimicrobial property; To four kinds of common bacteriums: intestinal bacteria, streptococcus aureus; Candida albicans and Pseudomonas aeruginosa all have excellent anti-microbial property; 24h discharges the silver ions amount at 5～300mg/L, and antibiotic rate is all greater than 99.98%.
Embodiment 4
1. the preparation seed culture medium is used for the Acetobacter pasteurianus activation.The composition of seed culture medium is: glucose 7.0w/v%, peptone 0.5w/v%, monohydrate potassium 0.2w/v%, potassium primary phosphate 0.1w/v%, disodium hydrogen phosphate dodecahydrate 0.2w/v%, yeast extract 0.5w/v%.The pH value of control seed culture medium is 7.0, and at 121 ℃ of following high-temperature sterilization 30min.
2. take out and be stored in the Acetobacter pasteurianus in the refrigeration chamber.Transfering loop is burnt heat on spirit lamp, transfering loop is stretched into inclined-plane in vitro, hook up bacterial classification one ring back access and be equipped with in the Erlenmeyer flask of the seed culture medium described in step 1.The Erlenmeyer flask that inserts bacterial classification is sealed with gauze, put into shaking table and under 30 ℃, carry out activation 24h, shaking speed 160rpm.
3. in culture vessel, prepare the fermention medium that contains ferroferric oxide nano granules, be used to cultivate Z 250/bacteria cellulose composite membrane.The composition of fermention medium is: glucose 7.0w/v%, peptone 0.5w/v%, monohydrate potassium 0.2w/v%; Potassium primary phosphate 0.2w/v%; Disodium hydrogen phosphate dodecahydrate 0.2w/v%, yeast extract 0.5w/v%, ferroferric oxide nano granules 5.0wt%; The particle diameter of ferriferrous oxide nano-particle is 1～20nm, adds ionic dispersant polyoxyethylene glycol 7.5wt%.Mechanical stirring 300r/min stirs 1h.The pH value of control fermention medium is 6.0, and at 121 ℃ of following high-temperature sterilization 30min.
4. be inverted on the airtight oxygen room after culture vessel being sealed with the oxygen flow pellosil,, leave standstill 1h with the joint seal of silicone oil with culture vessel and oxygen room.Aerating oxygen in oxygen room then carries out 14 days static cultivation under 30 ℃.
5. through after the cultivation like step 4, will contain function nano particulate bacteria cellulose composite membrane and from culture vessel, take out, it will be immersed in the NaOH solution of 4.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At last at air drying or dry in vacuum freeze drier, the Z 250/bacteria cellulose composite membrane that can obtain having superparamagnetism.
6. resulting Z 250/bacteria cellulose composite membrane has good superparamagnetism, and remanent magnetism and coercive force all level off to zero, and maximum saturation susceptibility reaches 38.51emu/g; Mechanical property is excellent, and ultimate tensile strength reaches 15.6MPa.
1. the preparation seed culture medium is used for the activation of glucose bacillus.The composition of seed culture medium is: glucose 6.0w/v%, peptone 0.5w/v%, monohydrate potassium 0.3w/v%, potassium primary phosphate 0.2w/v%, disodium hydrogen phosphate dodecahydrate 0.2w/v%, yeast extract 0.5w/v%.The pH value of control seed culture medium is 7.0, and at 121 ℃ of following high-temperature sterilization 30min.
2. take out and be stored in the glucose bacillus in the refrigeration chamber.Transfering loop is burnt heat on spirit lamp, transfering loop is stretched into inclined-plane in vitro, hook up bacterial classification one ring back access and be equipped with in the Erlenmeyer flask of the seed culture medium described in step 1.The Erlenmeyer flask that inserts bacterial classification is sealed with gauze, put into shaking table and under 30 ℃, carry out activation 24h, shaking speed 160rpm.
3. in culture vessel, prepare the fermention medium that contains nano-Ag particles, be used to cultivate nanometer silver/bacteria cellulose composite membrane.The composition of fermention medium is: glucose 7.0w/v%, peptone 0.5w/v%, monohydrate potassium 0.2w/v%; Potassium primary phosphate 0.2w/v%; Disodium hydrogen phosphate dodecahydrate 0.2w/v%, yeast extract 0.5w/v%, nano-Ag particles 3.0wt%; The particle diameter of nano-Ag particles is 60～120nm, adds ionic dispersant X 2073 6.0wt%.Magnetic agitation 300r/min stirs 1h.The pH value of control fermention medium is 7.0, and at 121 ℃ of following high-temperature sterilization 30min.
4. be inverted on the airtight oxygen room after culture vessel being sealed with the oxygen flow pellosil,, leave standstill 1h with the joint seal of silicone oil with culture vessel and oxygen room.Aerating oxygen in oxygen room then carries out 14 days static cultivation under 30 ℃.
5. through after the cultivation like step 4, will contain function nano particulate bacteria cellulose composite membrane and from culture vessel, take out, it will be immersed in the NaOH solution of 4.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At air drying or dry in vacuum freeze drier, can obtain having the nanometer silver/bacteria cellulose composite membrane of good anti-microbial property at last.
6. resulting nanometer silver/bacteria cellulose composite membrane has good antimicrobial property; To four kinds of common bacteriums: intestinal bacteria, streptococcus aureus; Candida albicans and Pseudomonas aeruginosa all have excellent anti-microbial property; 24h discharges the silver ions amount at 6～250mg/L, and antibiotic rate is all greater than 99.98%.
1. according to the described culturing process of Master's thesis " preparation and the sign of novel medical biotechnology material---bacteria cellulose ", preparation seed culture medium and fermention medium are respectively applied for the activation of Agrobacterium and the cultivation of Agrobacterium bacteria cellulose.
2. in preparation fermention medium step, in fermention medium, add nano-Ag particles, its particle diameter is 60～120nm, adds the 4.0wt% that quality accounts for the fermention medium total mass.Add the ionic dispersant X 2073, add the 8.0wt% that quality accounts for the fermention medium total mass.
3. when cultivating culture vessel sealed with the oxygen flow pellosil and be inverted on the airtight oxygen room afterwards, with the joint seal of silicone oil with culture vessel and airtight oxygen room.Leave standstill behind the 1h aerating oxygen in oxygen room.Under 30 ℃, carry out 7 days static cultivation.
4. through after the cultivation like step 3, the bacteria cellulose composite membrane that will contain nano-Ag particles takes out from culture vessel, and it is immersed in the NaOH solution of 4.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At air drying or dry in vacuum freeze drier, can obtain having the nanometer silver/bacteria cellulose composite membrane of good anti-microbial property at last.
5. resulting nanometer silver/bacteria cellulose composite membrane has good antimicrobial property; To four kinds of common bacteriums: intestinal bacteria, streptococcus aureus; Candida albicans and Pseudomonas aeruginosa all have excellent anti-microbial property; 24h discharges the silver ions amount at 3～280mg/L, and antibiotic rate is all greater than 99.98%.
1. according to the described culturing process of paper " Synthesis ofcelluloseby acetobacterxylinum ", preparation seed culture medium and fermention medium are respectively applied for the activation of root nodule bacterium and the cultivation of root nodule bacterium bacteria cellulose.
2. in preparation fermention medium step; In fermention medium, adding with gum arabic and gelatin is that wall material and cholesteryl liquid crystal are that the temperature of core causes reversible color change microcapsule; Its particle diameter is 400～700nm, adds the 5.0wt% that quality accounts for the fermention medium total mass.Add the non-ionic dispersing agent tween 80, add the 8.0wt% that quality accounts for the fermention medium total mass.
3. when cultivating culture vessel sealed with the oxygen flow pellosil and be inverted on the airtight oxygen room afterwards, with the joint seal of silicone oil with culture vessel and airtight oxygen room.Leave standstill behind the 1h aerating oxygen in oxygen room.Under 30 ℃, carry out 10 days static cultivation.
4. through after the cultivation like step 3; To contain with gum arabic and gelatin is that wall material and cholesteryl liquid crystal are that the bacteria cellulose composite membrane that the temperature of core causes reversible color change microcapsule takes out from culture vessel; It is immersed in the NaOH solution of 5.0wt%, and in 100 ℃ water-bath, heats 1h, wash with Hydrogen chloride then; Extremely neutral with deionized water rinsing then; Remove residual substratum on the composite package, at air drying or dry in vacuum freeze drier, can obtain having microcapsule/bacteria cellulose composite membrane that temperature causes the reversible color performance at last.
5. resulting have microcapsule/bacteria cellulose composite membrane that temperature causes the reversible color performance and can be implemented in 35～42 ℃ of reversible colors, and variable color sensitivity is 0.2 ℃, and can reuse, and discoloration is lasting.
Embodiment 8
1. according to the described culturing process of paper " In situ modification of bacterial cellulose network structure by adding interfering substances during fermentation ", preparation seed culture medium and fermention medium are respectively applied for the activation of sarcina and the cultivation of sarcina bacteria cellulose.
2. in preparation fermention medium step; In fermention medium, adding with gum arabic and gelatin is that wall material and cholesteryl liquid crystal are that the temperature of core causes reversible color change microcapsule; Its particle diameter is 400～700nm, adds the 6.0wt% that quality accounts for the fermention medium total mass.
3. when cultivating culture vessel sealed with the oxygen flow pellosil and be inverted on the airtight oxygen room afterwards, with the joint seal of silicone oil with culture vessel and airtight oxygen room.Leave standstill behind the 1h aerating oxygen in oxygen room.Under 30 ℃, carry out 12 days static cultivation.
4. through after the cultivation like step 3; To contain with gum arabic and gelatin is that wall material and cholesteryl liquid crystal are that the bacteria cellulose composite membrane that the temperature of core causes reversible color change microcapsule takes out from culture vessel; It is immersed in the NaOH solution of 4.0wt%, and in 100 ℃ water-bath, heats 1h, wash with Hydrogen chloride then; Extremely neutral with deionized water rinsing then; Remove residual substratum on the composite package, at air drying or dry in vacuum freeze drier, can obtain having microcapsule/bacteria cellulose composite membrane that temperature causes the reversible color performance at last.
5. resulting have microcapsule/bacteria cellulose composite membrane that temperature causes the reversible color performance and can be implemented in 35～42 ℃ of reversible colors, and variable color sensitivity is 0.2 ℃, and can reuse, and discoloration is lasting.
Embodiment 9
1. according to the described culturing process of paper " Nano-biomaterials application:In situ modification of bacterial cellulose structure by adding HPMC during fermentation ", preparation seed culture medium and fermention medium are respectively applied for the activation of pseudomonas cepacia and the cultivation of pseudomonas cepacia bacteria cellulose.
2. in preparation fermention medium step, in fermention medium, add ferroferric oxide nano granules, its particle diameter is 100～300nm, adds the 3.0wt% that quality accounts for the fermention medium total mass.Add the ionic dispersant polyoxyethylene glycol, add the 6.0wt% that quality accounts for the fermention medium total mass
3. when cultivating culture vessel sealed with the oxygen flow pellosil and be inverted on the airtight oxygen room afterwards, with the joint seal of paraffin with culture vessel and airtight oxygen room.Leave standstill behind the 1h aerating oxygen in oxygen room.Under 30 ℃, carry out 14 days static cultivation.
4. through after the cultivation like step 3, the bacteria cellulose composite membrane that will contain ferroferric oxide nano granules takes out from culture vessel, and it is immersed in the NaOH solution of 5.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At last at air drying or dry in vacuum freeze drier, the Z 250/bacteria cellulose composite membrane that can obtain having superparamagnetism.
5. resulting Z 250/bacteria cellulose composite membrane has good superparamagnetism, and remanent magnetism and coercive force all level off to zero, and maximum saturation susceptibility reaches 40.73emu/g; Mechanical property is excellent, and ultimate tensile strength reaches 13.3MPa.
Embodiment 10
1. the preparation seed culture medium is used for the Pseudomonas cocovenenans activation.The composition of seed culture medium is: glucose 5.0w/v%, peptone 0.4w/v%, monohydrate potassium 0.1w/v%, potassium primary phosphate 0.1w/v%, disodium hydrogen phosphate dodecahydrate 0.1w/v%, yeast extract 0.4w/v%.The pH value of control seed culture medium is 6.0, and at 121 ℃ of following high-temperature sterilization 30min.
2. take out and be stored in the Pseudomonas cocovenenans in the refrigeration chamber.Transfering loop is burnt heat on spirit lamp, transfering loop is stretched into inclined-plane in vitro, hook up bacterial classification one ring back access and be equipped with in the Erlenmeyer flask of the seed culture medium described in step 1.The Erlenmeyer flask that inserts bacterial classification is sealed with gauze, put into shaking table and under 30 ℃, carry out activation 24h, shaking speed 160rpm.
3. in culture vessel, prepare the fermention medium that contains nano-Ag particles, be used to cultivate function nano particle/bacteria cellulose composite membrane.The composition of fermention medium is: glucose 5.0w/v%, peptone 0.5w/v%, monohydrate potassium 0.1w/v%; Potassium primary phosphate 0.1w/v%; Disodium hydrogen phosphate dodecahydrate 0.2w/v%, yeast extract 0.5w/v%, nano-Ag particles 5.0wt%; Its particle diameter is 60～120nm, adds ionic dispersant X 2073 8.0wt%.Magnetic force is mixed 400r/min and is stirred 1h.The pH value of control fermention medium is 6.0, and at 121 ℃ of following high-temperature sterilization 30min.
4. be inverted on the airtight oxygen room after culture vessel being sealed with the oxygen flow pellosil,, leave standstill 1h with the joint seal of silicone oil with culture vessel and airtight oxygen room.Aerating oxygen in airtight oxygen room then carries out 7 days static cultivation under 30 ℃.
5. through after the cultivation like step 4, will contain function nano particulate bacteria cellulose composite membrane and from culture vessel, take out, it will be immersed in the NaOH solution of 5.0wt%; And in 100 ℃ water-bath, heat 1h; With the Hydrogen chloride flushing, extremely neutral with deionized water rinsing then then, remove residual substratum on the composite package; At air drying or dry in vacuum freeze drier, can obtain having the nanometer silver/bacteria cellulose composite membrane of good anti-microbial property at last.
6. resulting nanometer silver/bacteria cellulose composite membrane has good antimicrobial property; To four kinds of common bacteriums: intestinal bacteria, streptococcus aureus; Candida albicans and Pseudomonas aeruginosa all have excellent anti-microbial property; 24h discharges the silver ions amount at 5～350mg/L, and antibiotic rate is all greater than 99.98%.
Claims (10)
1. a method for preparing function nano particle/bacteria cellulose composite membrane comprises preparation seed culture medium, preparation fermention medium, actication of culture, bacterial classification access, cultivation and post-processing step, it is characterized in that:
Add the function nano particle in the described preparation fermention medium step;
Described cultivation is to cultivate after culture vessel is inverted, and culture vessel is inverted on the airtight oxygen room, separate with the oxygen flow material membrane between culture vessel and the oxygen room, and sealed vessel and oxygen room's seam crossing.
2. a kind of method for preparing function nano particle/bacteria cellulose composite membrane according to claim 1 is characterized in that, described function nano particle is that grain diameter is the solid function nano particle of 1～1000nm; Described function nano particulate quality accounts for 1.0～6.0wt% of fermention medium total mass.
3. a kind of method for preparing function nano particle/bacteria cellulose composite membrane according to claim 2; It is characterized in that; Described solid function nano particle is meant the nano particle with various appointed functions, is that wall material and cholesteryl liquid crystal are that the temperature of core causes reversible color change microcapsule or has the nano-Ag particles of anti-microbial property for ferroferric oxide nano granules with superparamagnetism, with gum arabic and gelatin.
4. a kind of method for preparing function nano particle/bacteria cellulose composite membrane according to claim 3; It is characterized in that; Also add corresponding dispersion agent in the described fermention medium according to the difference of the function nano grain type that is added; When the function nano particle is nonpolar nano particle, use ionic dispersant, when the function nano particle is charged nano particle, use non-ionic dispersing agent; The quality of dispersion agent is 1.5～2.0: 1 with the function nano particulate mass ratio that is added;
Described function nano particle is the charged nano particle of Z 250, and non-ionic dispersing agent adopts polyoxyethylene glycol;
Described function nano particle is for being that wall material and cholesteryl liquid crystal are that the temperature of core causes the charged nano particle of reversible color change microcapsule with gum arabic and gelatin, non-ionic dispersing agent employing tween 80;
Described function nano particle is the nonpolar nano particle of nanometer silver, and ionic dispersant adopts X 2073.
5. a kind of method for preparing function nano particle/bacteria cellulose composite membrane according to claim 1; It is characterized in that; Controlled initiative oxygen supply mode is adopted in described cultivation, and described controlled initiative oxygen supply is meant the oxygen supply in oxygen room through the external impetus system.
6. a kind of method for preparing function nano particle/bacteria cellulose composite membrane according to claim 1 is characterized in that the material of described sealing culture vessel and oxygen room's seam is paraffin or silicone oil.
7. a kind of method for preparing function nano particle/bacteria cellulose composite membrane according to claim 1 is characterized in that described oxygen flow material membrane is a pellosil.
8. device that method adopted for preparing function nano particle/bacteria cellulose composite membrane as claimed in claim 1; It is characterized in that: described device mainly is made up of base and culture vessel; Described base is the open container that a step is arranged at top; Described culture vessel is upside down on the step of said base, separates with the oxygen flow material membrane between described base and the described culture vessel, and airtight oxygen room is formed at described oxygen flow material membrane and described base bottom; Described base bottom has production well and air inlet port.
9. device according to claim 8 is characterized in that, described base and described culture vessel seam crossing seal with sealing material.
10. device according to claim 8 is characterized in that, described air inlet port connects inlet pipe, and said inlet pipe is connected the oxygen supply pipe of external impetus system.
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