WO2021138837A1 - 动态发酵制备核壳结构细菌纤维素复合材料的装置及方法 - Google Patents

动态发酵制备核壳结构细菌纤维素复合材料的装置及方法 Download PDF

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WO2021138837A1
WO2021138837A1 PCT/CN2020/070900 CN2020070900W WO2021138837A1 WO 2021138837 A1 WO2021138837 A1 WO 2021138837A1 CN 2020070900 W CN2020070900 W CN 2020070900W WO 2021138837 A1 WO2021138837 A1 WO 2021138837A1
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fermentation
heating
bacterial cellulose
core
core material
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PCT/CN2020/070900
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English (en)
French (fr)
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钟宇光
钟春燕
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钟春燕
钟宇光
海南椰国食品有限公司
海南光宇生物科技有限公司
南京椰国食品有限公司
保定光宇水果加工食品有限公司
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Priority to PCT/CN2020/070900 priority Critical patent/WO2021138837A1/zh
Priority to JP2022542054A priority patent/JP7383160B2/ja
Publication of WO2021138837A1 publication Critical patent/WO2021138837A1/zh
Priority to US17/858,403 priority patent/US20220348975A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/02—Cellulose; Modified cellulose
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D101/00—Coating compositions based on cellulose, modified cellulose, or cellulose derivatives
    • C09D101/02—Cellulose; Modified cellulose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/14—Rotation or movement of the cells support, e.g. rotated hollow fibers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
    • C12M41/24—Heat exchange systems, e.g. heat jackets or outer envelopes inside the vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2207/00—Properties characterising the ingredient of the composition
    • C08L2207/53—Core-shell polymer

Definitions

  • the invention belongs to the technical field of dynamic fermentation, and relates to a device and method for preparing a core-shell structure bacterial cellulose composite material by dynamic fermentation.
  • Bacterial cellulose is a natural cellulose obtained by microbial fermentation. It is a polymer compound formed by connecting glucose with ⁇ -1,4-glycoside chains. Bacterial cellulose and natural cellulose produced by plants or seaweeds have the same molecular structural units, and they have many unique properties: 1 High crystallinity and degree of polymerization.
  • Bacterial cellulose fibers are composed of microfibers with a diameter of 3 to 4 nanometers combined into fiber bundles with a thickness of 40 to 60 nanometers, which are interwoven to form a developed ultra-fine nano network structure; 3High elastic modulus and tensile strength. The elastic modulus of bacterial cellulose is several to ten times that of general plant fibers, and its tensile strength is high; 4 strong water retention values (WRV).
  • WRV water retention values
  • the WRV value of undried bacterial cellulose is as high as 1000%, and the water holding capacity after freeze-drying still exceeds 600%.
  • the swelling capacity of the bacterial cellulose dried at 100°C in water is equivalent to that of cotton linters; 5Excellent biocompatibility, adaptability and biodegradability; 6Synthesis controllability. It is precisely because of these excellent properties of bacterial cellulose that it is widely used in food, biomedicine, medical equipment, tissue engineering materials and many other fields.
  • bacterial cellulose can be used as a food forming agent, thickener, dispersant, anti-solubilizing agent, improving taste, and used as the skeleton of casings and certain foods. It has become a kind of New important food base material and dietary fiber. For example, it has been widely used in jelly, milk tea, jam, dessert and other foods, and it is currently one of the most popular raw materials in commercial food. Due to the good biocompatibility of bacterial cellulose, high mechanical strength in wet state, and good liquid and gas permeability, bacterial cellulose can be used as an excellent biological material in various fields of biomedicine.
  • Biofill and Gengiflex are two typical bacterial cellulose products, which have been widely used as surgical and dental materials. For secondary and tertiary burns, ulcers, etc., Biofill has been successfully used as a temporary substitute for artificial skin. Gengiflex has been used for the repair of root membrane tissue. A new biomaterial BASYC designed based on the in-situ plasticity of bacterial cellulose is expected to be used as an artificial blood vessel in microsurgery. At the same time, the reported applications also include the repair of bone, cornea, cartilage, tendon and other tissues.
  • the fermentation preparation technology of bacterial cellulose is mainly divided into static fermentation and dynamic fermentation, and the preparation of bacterial cellulose composite material is mainly based on static fermentation.
  • various water-soluble polymers such as carboxymethyl cellulose, hemicellulose, chitosan, and gelatin are added to the culture broth to obtain different types of composite materials.
  • breathable material as the mold, bacterial cellulose materials of different shapes can be obtained during the static fermentation process.
  • the patent UK Patent 12,169,543 uses an oxygen-permeable hand mold to prepare glove-shaped artificial skin; the patent EP Patent 0,396,344; the patent JP Patent 3,272,772, which uses an oxygen-permeable cavity tube to inject a culture solution containing bacteria in Artificial blood vessels are prepared under static fermentation conditions.
  • conventional paddle agitation can only produce granular bacterial cellulose.
  • the Rotating Disc Fermentor designed by Krystynowicz A et al. solves the problem that bacterial cellulose is difficult to form a film during dynamic culture, and obtains a disc-shaped bacterial cellulose membrane (Journal of Industrial Microbiology & Biotechnology 2002(29): 189-195 ).
  • Chinese patent CN2937138Y discloses a rotating disk type fermentation reaction device, but this type of device can only obtain a disc-shaped bacterial cellulose membrane.
  • Chinese Patent CN101914434A designs a dynamically prepared special-shaped cavity bacterial cellulose material, which can obtain a cavity bacterial cellulose material with a certain cross-sectional shape.
  • the mold must pass through the rotating shaft, and a completely sealed bacterial cellulose coating material cannot be obtained.
  • the bacterial cellulose fermentation and culture device with reasonable design, simple and easy design can make the method for preparing the composite material with a certain morphology and completely covering the required composite material has great practical significance and commercial prospects.
  • the present invention provides a device for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, the device comprising:
  • Both ends of the rotating shafts of the two drums are respectively movably connected to the inner wall of the fermentation culture container, the two drums are arranged in parallel in the horizontal direction, a gap is provided between the drums, and the distance between the rotating shafts of the drums is adjustable;
  • Two heating deflectors are parallel to the rotating shafts of the two drums.
  • One end of one heating deflector is movably connected to the fermentation culture vessel (preferably connected by fasteners), and the other end extends diagonally downward to the two drums.
  • the fermentation culture vessel preferably connected by fasteners
  • one end of the other heating deflector is movably connected with the fermentation culture container, and the other end extends diagonally downward to above the gap between the two rollers and abuts against the other roller.
  • the device provided by the present invention can realize dynamic fermentation and coating, and can be coated to obtain a bacterial cellulose composite material with controllable shape and size.
  • the device is simple, convenient to operate, and has high yield, and is suitable for industrial production; the obtained coated bacteria Cellulose composite materials can be widely used in the fields of biomedicine and medical devices such as the coating of bacterial cellulose on the surface of implant medical devices, the coating of sustained-release drugs or micro-devices, and can also be used as the coating of food or food materials.
  • the device further includes a driving component that drives the two rollers to rotate; more preferably, the driving component is used to drive the two rollers to rotate in the same direction (which can be clockwise in the same direction or counterclockwise in the same direction). Turn).
  • the device further includes a heating component for heating the two heating baffles.
  • the heating component may be an external heating device that causes the heating deflector to generate heat or a heating component that is built in the heating deflector itself.
  • the two rollers are cylindrical rollers with the same shape and size.
  • the angle between the two heating baffles and the inner side wall of the fermentation culture container is 15-60 degrees.
  • the two heating deflectors are movable and detachable movable connection fittings. Fasteners can be used to fix them on the top of the fermentation culture vessel, and the inclination angle of the two heating deflectors can be adjusted by the fasteners. Adjust the flow rate of the heating liquid of the thermoplastic polymer.
  • the present invention also provides a method for preparing a core-shell structure bacterial cellulose composite material by dynamic fermentation, which adopts the above-mentioned device for dynamic fermentation, and includes the following steps:
  • thermoplastic polymer is evenly coated on the core material-bacterial cellulose The surface of the composite, thereby preparing a bacterial cellulose composite with a core-shell structure; or,
  • thermoplastic polymer heating liquid down the heating baffles at both ends respectively.
  • the thermoplastic polymer is evenly coated on the surface of the core material to obtain the core material-thermoplastic polymer composite;
  • the bacterial cellulose is prepared to obtain a bacterial cellulose composite material with a core-shell structure.
  • the coating amount of the bacterial cellulose and the coating amount of the thermoplastic polymer material are set reasonably according to actual needs.
  • the obtained core-shell bacterial cellulose composite material can have a structure of "core material-bacterial cellulose-thermoplastic polymer” from the inside to the outside, or “core material-thermoplastic polymer-bacteria”. Cellulose” structure and so on.
  • the method further comprises repeating the step of dynamic fermentation coating bacterial cellulose and/or the step of coating thermoplastic polymer to obtain more hierarchical core-shell structure bacterial cellulose composite material. Taking such repeated steps can obtain bacterial cellulose composites with different hierarchical structures; for example, from the inside to the outside, it can be "core material-bacterial cellulose-thermoplastic polymer-bacterial cellulose-thermoplastic polymer" structure, "core material” -Thermoplastic polymer-bacterial cellulose-thermoplastic polymer-bacterial cellulose” structure and so on.
  • the core material includes one or a combination of inorganic materials, organic polymer materials, and metal materials.
  • the shape of the core material is spherical, quasi-spherical, cylindrical, rod-shaped or any irregular body.
  • the thermoplastic polymer material includes one or a combination of one or more of polyethylene, polypropylene, polystyrene, polymethyl methacrylate, nylon, polyurethane, polyester, and polylactic acid .
  • the heating temperature of the heating deflector is 50-300°C.
  • the strains include Acetobacter xylinum, Rhizobium, Sarcina, Pseudomonas, Achromobacter, Alcaligenes, Aerobacter, and Azotobacter. One or more combinations.
  • the added amount of the strain seed mash is 1 to 5% by weight of the fermentation broth.
  • the time for dynamic fermentation is 3-30 days; the fermentation temperature is 20-30°C.
  • the rotation speed of the two drums is the same, both being 0.1-60 rpm; preferably 4-20 rpm.
  • the flow rate of the culture solution of the bacterial cellulose must be controlled within a certain range during the dynamic culture process. If the flow rate is too fast, the strains are easy to mutate, and it is difficult for the bacterial cellulose to adhere to the core material, and only granular bacterial cellulose can be obtained in the culture solution. . When the flow rate is slow, bacterial cellulose will be produced in large quantities on the gas-liquid interface of the culture container, which affects the coating of the core material by the bacterial cellulose.
  • the flow rate of the culture fluid will also affect the production of bacterial cellulose, the flatness of the surface of the core material, and the three-dimensional network structure of nanofibers that constitute the bacterial cellulose; the better condition is to control the rotation rate of the drum at 4-20 rpm range.
  • the dynamic fermentation process further includes adding 0.1 to 5 wt% of soluble additives to the fermentation mixture;
  • the soluble additives include gelatin, sodium hyaluronate, starch, pectin, chitosan A combination of one or more of sugar, sodium alginate and soluble cellulose derivatives.
  • the inventors discovered through research that adding soluble polymers in situ during the bacterial cellulose cultivation process can change the physical and chemical properties of the bacterial cellulose itself, and obtain composite materials that meet various applications.
  • the dynamic fermentation method adopted by the present invention can compound with the soluble polymer in the fermentation and culture process while the bacterial cellulose covers the core material, thereby expanding the application range of the bacterial cellulose composite material.
  • the step of purifying the bacterial cellulose-coated compound is further included, specifically: at a temperature of 70-100°C, the content of the compound is 4%-8% by mass. Wash in% NaOH aqueous solution for 4-6 hours, and then repeatedly rinse with distilled water until it is neutral. Through purification, the bacterial protein in the fermentation product and the residual culture medium adhered to the bacterial cellulose membrane are removed, so that the coated bacterial cellulose composite material can reach medical or edible conditions.
  • the present invention also provides the core-shell bacterial cellulose composite material prepared by the above method, the core layer of which is a core material, and the core material is coated with a single layer or multiple layers of bacterial cellulose and/or thermoplastic polymers material.
  • the device provided by the present invention can realize dynamic fermentation and coating, and can be coated to obtain a bacterial cellulose composite material with controllable shape and size.
  • the device is simple, convenient to operate, and has a high yield, and is suitable for industrial production;
  • the coated bacterial cellulose composite material can be widely used in the fields of biomedicine and medical equipment such as the coating of bacterial cellulose on the surface of implant medical equipment, the coating of sustained-release drugs or micro-devices, and it can also be used as the coating of food or food materials. Wait.
  • the dynamic fermentation method of the present invention does not need to use any toxic solvents, will not cause problems such as environmental pollution and ecological hazards, and meets the requirements of medical or edible use; various water-soluble polymers can be added during the fermentation process to obtain various A variety of bacterial cellulose composite materials with different properties, and have excellent biocompatibility and biosafety.
  • the dynamic fermentation method of the present invention can quickly obtain bacterial cellulose composite materials with different levels of core-shell structure, which can be widely used in many fields such as biomedicine, medical equipment, and food.
  • FIG. 1 is a schematic diagram of the structure of an apparatus for preparing a core-shell bacterial cellulose composite material by dynamic fermentation in an embodiment of the present invention.
  • Fermentation culture vessel 2. Roller, 3. Fermentation culture liquid, 4. Core material to be coated, 5. Heating baffle, 6. Fasteners.
  • This embodiment provides a device for preparing a core-shell bacterial cellulose composite material by dynamic fermentation. As shown in FIG. 1, the device includes:
  • the deflector 5 is parallel to the rotating shafts of the two drums 2, and one end of the heating deflector 5 is movably connected to the fermentation culture vessel 1 through the fastener 6, and the other end extends diagonally downward to above the gap between the two drums.
  • one end of the other heating deflector is movably connected with the fermentation culture container through fasteners 6, and the other end extends diagonally downward to above the gap between the two rollers and abuts against the other roller 2 .
  • the two rollers are preferably cylindrical rollers with the same shape and size.
  • the core material 4 to be coated is located above the gap between the two drums 2 and abuts against the drums; the fermentation culture liquid 3 is loaded in the fermentation culture container 1.
  • the device also includes a driving component for driving the two rollers to rotate, for driving the two rollers to rotate in the same direction.
  • the device also includes a heating component for heating the two heating baffles.
  • this embodiment also provides a method for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, which adopts the above-mentioned device of this embodiment to perform dynamic fermentation, and includes the following steps:
  • Two cylindrical glass drums with the same shape and size, which can rotate clockwise in the same direction, are arranged in parallel in the fermentation culture container with the opening facing upwards.
  • the spherical polyurethane can realize vibration without horizontal displacement with the rotation of the drum.
  • Acetobacter xylinum which can secrete bacterial cellulose, is activated to prepare a seed mash, and then the seed mash with a strain concentration of 1 wt% and a fermentation medium are mixed to obtain a fermentation mixture; wherein the fermentation medium is high-temperature sterilization
  • the components of the culture medium are commonly used in the fermentation of bacterial cellulose in the field.
  • thermoplastic polymer polyethylene is evenly coated on the core material-
  • the surface of the bacterial cellulose composite is prepared to obtain a core-shell structure composite material with a polyethylene/bacterial cellulose uniformly coated on the surface of a spherical polyurethane material.
  • This embodiment provides a method for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, which adopts the device of the above-mentioned embodiment 1 to perform dynamic fermentation, and includes the following steps:
  • Two cylindrical glass drums with the same shape and size, which can rotate clockwise in the same direction, are arranged in parallel in the fermentation culture container with the opening facing upwards.
  • a bioceramic (material core) with a diameter of 50mm and a length of 70mm is placed between the two rollers, as shown in Figure 1, the distance between the two rollers is adjusted to 40mm and the rotation rate is 8rpm.
  • the bioceramics can realize vibration without horizontal displacement with the rotation of the drum.
  • thermoplastic polymer polypropylene is evenly coated on the bioceramics- The surface of the bacterial cellulose composite, thereby preparing a core-shell structure composite material with the surface of the bioceramic uniformly coated with polypropylene/bacterial cellulose.
  • This embodiment provides a method for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, which adopts the device of the above-mentioned embodiment 1 to perform dynamic fermentation, and includes the following steps:
  • Two cylindrical stainless steel drums with the same shape and size, which can rotate clockwise in the same direction, are arranged in parallel in the fermentation culture container with the opening facing upward. Place a 6.5 mm titanium alloy orthopedic implant screw (core material) between the two rollers, as shown in Figure 1, adjust the distance between the two rollers to 5 mm and the rotation rate to 12 rpm. The screw can realize vibration without horizontal displacement with the rotation of the drum.
  • the Sarcina species that can secrete bacterial cellulose to activate and prepare seed mash, and then mix the seed mash with a strain concentration of 3 wt% and the fermentation medium to obtain a fermentation mixture; wherein the fermentation medium is a high-temperature mash Bacterized culture medium.
  • the components of the culture medium are commonly used in the field of bacterial cellulose fermentation.
  • the fermentation mixture also includes 1wt% sodium hyaluronate and sodium alginate. The mass ratio of sodium hyaluronate and sodium alginate is 1:1.
  • thermoplastic polymer polypropylene ethylene is evenly coated on the screw-bacteria The surface of the cellulose composite, thereby preparing a core-shell structure composite material with the surface of the screw uniformly coated with polypropylene/bacterial cellulose.
  • This embodiment provides a method for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, which adopts the device of the above-mentioned embodiment 1 to perform dynamic fermentation, and includes the following steps:
  • the rod-shaped material (core material) of nano-hydroxyapatite filled with polymethyl methacrylate with a diameter of 2 mm is placed between the two rollers, as shown in Figure 1, the distance between the two rollers is adjusted to 1.5 mm and the rotation rate is 2 rpm.
  • the rod can be rotated with the drum to achieve vibration without horizontal displacement.
  • thermoplastic polymer nylon As the drum rotates, the thermoplastic polymer is evenly coated on the rod-bacteria
  • the surface of the cellulose composite is prepared to obtain a core-shell structure composite material with a nylon/bacterial cellulose uniformly coated on the surface of the rod.
  • This embodiment provides a method for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, which adopts the device of the above-mentioned embodiment 1 to perform dynamic fermentation, and includes the following steps:
  • Two cylindrical plastic drums with the same shape and size, which can rotate clockwise in the same direction, are arranged in parallel in the fermentation culture container with the opening facing upward. Place the silicone rubber (core material) for filling irregular breasts between the two rollers, as shown in Figure 1, adjust the distance between the two rollers to 70 mm and the rotation rate to 30 rpm.
  • the silicone rubber can realize vibration without horizontal displacement with the rotation of the drum.
  • the fermentation medium is a high-temperature sterilized medium, and the components of the medium are commonly used components for the fermentation of bacterial cellulose in the field.
  • the fermentation mixture also includes 3wt% pectin.
  • thermoplastic polymer polyethylene is evenly coated on the silicone rubber- The surface of the bacterial cellulose composite, thereby preparing a core-shell structure composite material with the surface of the silicone rubber uniformly coated with polyethylene/bacterial cellulose.
  • This embodiment provides a method for preparing a core-shell bacterial cellulose composite material by dynamic fermentation, which adopts the device of the above-mentioned embodiment 1 to perform dynamic fermentation, and includes the following steps:
  • Two cylindrical plastic drums with the same shape and size, which can rotate clockwise in the same direction, are arranged in parallel in the fermentation culture container with the opening facing upward. Place the irregularly shaped cobalt-chromium-molybdenum alloy artificial hip joint head (core material) between the two rollers, as shown in Figure 1, adjust the distance between the two rollers to 50mm and the rotation rate to 15rpm.
  • the alloy can realize vibration without horizontal displacement with the rotation of the drum.
  • the base is a high-temperature sterilized medium.
  • the components of the medium are commonly used in the field of bacterial cellulose fermentation.
  • the fermentation mixture also includes 5wt% of chitosan and sodium carboxymethyl cellulose, chitosan and carboxymethyl cellulose.
  • the mass ratio of sodium methylcellulose is 1:3.
  • Biocompatibility test referring to the biological evaluation of GB/T 16886 medical devices, the composite material (Example 1) and the polyurethane material (the core material of Example 1) were respectively subjected to cytotoxicity, delayed contact sensitization, and guinea pig sensitization. Evaluation of skin irritation.
  • the cytotoxicity of the polyurethane material was level 2, with skin sensitization; the composite material (Example 1) had cytotoxicity less than level 2, without skin sensitization, and no intradermal irritation. , Has good biological safety. It shows that the use of this patent improves the biocompatibility of the material.

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Abstract

一种动态发酵制备核壳结构细菌纤维素复合材料的装置及方法。该装置包括发酵培养容器（1）和设置在发酵培养容器中两个滚筒（2）和两块加热导流板（5）；两个滚筒的转轴两端分别与发酵培养容器的内壁活动连接，在水平方向上平行排列，滚筒之间设有间隙，且滚筒的转轴之间距离可调；两块加热导流板（5）与两个滚筒（2）转轴平行，一加热导流板（5）的一端与发酵培养容器（1）活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与其中一滚筒（2）抵接；另一加热导流板一端与发酵培养容器活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与另一滚筒抵接。该装置能够实现动态发酵和包覆，获得形状尺寸可控、生物相容性和安全性优良的细菌纤维素复合材料。

Description

动态发酵制备核壳结构细菌纤维素复合材料的装置及方法 技术领域
本发明属于动态发酵技术领域，涉及一种动态发酵制备核壳结构细菌纤维素复合材料的装置及方法。
背景技术
纤维素是地球上最丰富、发展潜力巨大的生物聚合物，它不仅是纺织工业和造纸工业的传统原料，还可以用来制造高分子复合材料和高性能材料，在许多高新科技领域发挥着重要作用。细菌纤维素是经微生物发酵得到的天然纤维素，由葡萄糖以β-1,4-糖苷链连接而成的高分子化合物。细菌纤维素和植物或海藻产生的天然纤维素具有相同的分子结构单元，且其具有许多独特的性质：①高结晶度和聚合度。细菌纤维素与植物纤维素相比无木质素、果胶和半纤维素等伴生产物，具有高结晶度(可达95％，植物纤维素的为65％)和高的聚合度(DP值2000～8000)；②超精细纳米网状结构。细菌纤维素纤维是由直径3～4纳米的微纤组合成40～60纳米粗的纤维束，并相互交织形成发达的超精细纳米网络结构；③高弹性模量和抗张强度。细菌纤维素的弹性模量为一般植物纤维的数倍至十倍以上，并且抗张强度高；④强持水能力(water retention values，WRV)。未经干燥的细菌纤维素的WRV值高达1000％以上，冷冻干燥后的持水能力仍超过600％。经100℃干燥后的细菌纤维素在水中的再溶胀能力与棉短绒相当；⑤优良的生物相容性、适应性和生物可降解性；⑥合成可控性。正是由于细菌纤维素这些优良性能，使其广泛应用于食品、生物医药、医疗器械、组织工程材料等诸多领域。
由于细菌纤维素具有很强的亲水性、黏稠性和稳定性，可作为食品成型剂、增稠剂、分散剂、抗溶化剂、改善口感作为肠衣和某些食品的骨架，已成为一种新型重要的食品基料和膳食纤维。如在果冻、奶茶、果酱、甜点等食品中已经得到广泛应用，是目前商业食品中颇受欢迎的原材料之一。由于细菌纤维素良好的生物相容性、湿态时高的机械强度、良好的液体和气体透过性，细菌纤维素可作为一种优良的生物材料应用于生物医用各领域。如Biofill和Gengiflex就是两个典型的细菌纤维素产品，已广泛用作外科和齿科材料。对于二级和三级烧伤、溃疡等，Biofill已被成功地用作人造皮肤的临时替代品。Gengiflex已用于齿根膜组织的修复。基于细菌纤维素的原位可塑性设计出的一种新型生物材料BASYC可望在显微外科中用作人造血管。同时，已有报道的应用还包括骨、 角膜、软骨、肌腱等组织的修复。
目前细菌纤维素的发酵制备技术主要分为静态发酵和动态发酵两类，其中细菌纤维素复合材料的制备主要以静态发酵为主。静态发酵过程是在培养液中添加羧甲基纤维素、半纤维素、壳聚糖、明胶等各种水溶性高分子获得不同类型的复合材料。同时通过采用透气材料作为模具，在静态发酵过程中可以获得不同形状的细菌纤维素材料。如专利UK Patent 12，169，543，采用透氧的手型模具制备手套形状的人工皮肤；专利EP Patent0,396,344；专利JP Patent 3,272,772，利用透氧空腔圆管通过注入含细菌的培养液在静态发酵条件下制备人工血管。在动态发酵方面，常规的桨叶式搅拌仅能制备颗粒状细菌纤维素。Krystynowicz A等人设计的转盘发酵装置(Rotating disc fermentor)解决了细菌纤维素在动态培养时难以成膜的问题，得到了圆片状细菌纤维素膜(Journal ofIndustrial Microbiology&Biotechnology 2002(29):189-195)。在此基础上中国专利CN2937138Y公开了一种转盘式发酵反应装置，但这一类型的装置只能得到圆片状细菌纤维素膜。为了进一步得到具有形状可控的细菌纤维素材料，中国专利CN101914434A设计了一种动态制备异型空腔细菌纤维素材料，可以得到具有一定横截面形状的空腔细菌纤维素材料。但这种方法模具必须穿过转轴，不能得到完全密闭的细菌纤维素包覆材料。
综上所述，设计合理、简单、易行的细菌纤维素发酵培养装置，能够使其制备具有一定形貌并且完全包覆所需复合材料的方法具有巨大的现实意义和商业化前景。
发明内容
本发明的一个目的在于提供一种动态发酵制备核壳结构细菌纤维素复合材料的装置；本发明的另一目的在于提供动态发酵制备核壳结构细菌纤维素复合材料的方法。本发明的再一目的在于提供该方法制备获得的核壳结构细菌纤维素复合材料。
本发明的目的通过以下技术方案得以实现：
一方面，本发明提供一种动态发酵制备核壳结构细菌纤维素复合材料的装置，该装置包括：
发酵培养容器和设置在发酵培养容器中的两个滚筒和两块加热导流板；
两个滚筒的转轴两端分别与所述发酵培养容器的内壁活动连接，两个滚筒在水平方向上平行排列，滚筒之间设有间隙，且滚筒的转轴之间距离可调；
两块加热导流板与两个滚筒的转轴平行，其中一个加热导流板的一端与所述发酵培养容器活动连接(优选通过紧固件连接)，另一端向斜下方延伸至两个滚筒的间隙上方 并与其中一个滚筒抵接；另一块加热导流板一端与所述发酵培养容器活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与另一个滚筒抵接。
本发明提供的装置能够实现动态发酵和包覆，能够包覆获得形状尺寸可控的细菌纤维素复合材料，该装置简单，操作方便，产率高，适用于工业化生产；得到的包覆型细菌纤维素复合材料可以广泛应用于植入物医疗器械的表面细菌纤维素包覆、缓释药物或微型器械的包覆等生物医药、医疗器械领域，也可以作为食品或食材的包覆等。
上述的装置中，优选地，所述装置还包括驱动两个滚筒转动的驱动部件；更加优选地，所述驱动部件用于驱动两个滚筒同向转动(可以同向顺时针或同向逆时针转动)。
上述的装置中，优选地，所述装置还包括对两块加热导流板进行加热的加热部件。加热部件可以是使加热导流板生热的外界加热装置或者是加热导流板本身自带的加热部件。
上述的装置中，优选地，所述两个滚筒为形状大小相同的圆柱状滚筒。
上述的装置中，优选地，两块加热导流板分别与所述发酵培养容器内侧壁的夹角为15～60度。所述的两块加热导流板是可移动可拆除的活动连接配件，可使用紧固件将其固定于发酵培养容器的顶部，并通过紧固件调节两块加热导流板的倾斜角度以调整热塑性高分子的加热液流速。
另一方面，本发明还提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其是采用上述的装置进行动态发酵，包括以下步骤：
调整滚筒间距使其间隙最小宽度小于芯材的直径或尺寸(该尺寸满足芯材能够夹持于两个滚动的上方)，将待包覆的芯材灭菌后置于两个滚筒的间隙上方，保证芯材能够在间隙上方分别与两个滚筒相抵接；设置滚筒的转动速度和转动方向，使芯材随滚筒的转动能够实现无水平位移的震动(包括转动)；
设置加热板的长度和调节加热板的角度，保证两个加热板的一端位于芯材与滚筒抵接的缝隙处(这样既可以保证滚筒带着芯材转动，又可以使热塑性高分子沿加热导流板流下，通过滚筒转动包覆于芯材表面)；
配制细菌纤维素发酵培养液并高压灭菌，然后与菌株种子醪液混合获得发酵混合液；
将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，发酵结束后，排出发酵液，此时芯材外层包覆有菌株发酵获得的细菌纤维素，形成芯材-细菌纤维素复合物；启动加热导流板，将热塑性高分子的加热液分别沿两端的加热导 流板流下，伴随滚筒转动，热塑高分子均匀涂覆在芯材-细菌纤维素复合物的表面，从而制备获得核壳结构细菌纤维素复合材料；或者，
启动加热导流板，将热塑性高分子的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在芯材的表面，获得芯材-热塑性高分子复合物；将发酵混合液倒入发酵培养容器中，浸没滚筒，启动滚筒同向转动进行动态发酵，发酵结束后，排出发酵液，此时芯材-热塑性高分子复合物的外层包覆有菌株发酵获得的细菌纤维素，从而制备获得核壳结构细菌纤维素复合材料。
本发明中，细菌纤维素的包覆量、热塑性高分子材料的包覆量根据实际所需进行合理设置。
本发明的动态发酵方法中，获得的核壳结构细菌纤维素复合材料由内到外可以为：“芯材-细菌纤维素-热塑性高分子”结构，或者为“芯材-热塑性高分子-细菌纤维素”结构等。
上述的方法中，优选地，该方法还包括重复动态发酵包覆细菌纤维素的步骤和/或包覆热塑性高分子的步骤，获得更多层次核壳结构细菌纤维素复合材料。采取此种重复步骤可以获得不同层次结构的细菌纤维素复合材料；例如，从内到外可以为“芯材-细菌纤维素-热塑性高分子-细菌纤维素-热塑性高分子”结构、“芯材-热塑性高分子-细菌纤维素-热塑性高分子-细菌纤维素”结构等等。
上述的方法中，优选地，所述芯材包括无机材料、有机高分子材料和金属材料中的一种或多种的组合。
上述的方法中，优选地，所述芯材的形状为球型、类球形、圆柱形、棒状体或任意不规则体。
上述的方法中，优选地，所述热塑性高分子材料包括聚乙烯、聚丙烯、聚苯乙烯、聚甲基丙烯酸甲酯、尼龙、聚氨酯、聚酯和聚乳酸中的一种或多种的组合。
上述的方法中，优选地，所述加热导流板的加热温度为50～300℃。
上述的方法中，优选地，所述菌株包括木醋杆菌、根瘤菌属、八叠球菌属、假单胞菌属、无色杆菌属、产碱菌属、气杆菌属和固氮菌属中的一种或多种的组合。
上述的方法中，优选地，菌株种子醪液的添加量为发酵培养液的1～5wt％。
上述的方法中，优选地，进行动态发酵的时间为3～30天；发酵温度为20～30℃。
上述的方法中，优选地，进行动态发酵时，两个滚筒的转速相同，均为0.1～60rpm；优选为4～20rpm。
细菌纤维素在动态培养过程中培养液流速必须控制在一定范围内，流速过快菌种容易变异，并且细菌纤维素很难在芯材上附着，仅能在培养液中得到颗粒状细菌纤维素。流速较慢时，细菌纤维素会在培养容器的气液界面上大量生成，影响细菌纤维素对芯材的包覆。另外，培养液流速也会影响细菌纤维素的产量、包覆在芯材表面的平整度，以及构成细菌纤维素的纳米纤维三维网络结构；较佳的条件为控制滚筒的旋转速率在4～20rpm范围。
上述的方法中，优选地，在进行动态发酵过程中，还包括向发酵混合液中添加0.1～5wt％的可溶性添加剂；所述可溶性添加剂包括明胶、透明质酸钠、淀粉、果胶、壳聚糖、海藻酸钠和可溶性纤维素衍生物中的一种或多种的组合。
发明人研究发现，在细菌纤维素培养过程中原位添加可溶性高分子能够改变细菌纤维素本身的物理化学性能，得到满足各种应用的复合材料。本发明所采用的一种动态发酵方法能够在细菌纤维素包覆芯材的同时，与可溶性高分子在发酵培养过程中复合，扩大了细菌纤维素复合材料的应用范围。
上述的方法中，优选地，动态发酵结束后还包括对包裹有细菌纤维素的复合物进行纯化的步骤，具体为：在70～100℃的温度下，在质量百分含量为4％～8％的NaOH水溶液中洗涤4～6h，再用蒸馏水反复冲洗至中性。通过纯化除去了发酵产物中菌体蛋白和粘附在细菌纤维素膜上的残余培养基，使得包覆型细菌纤维素复合材料达到医用或食用条件。
再一方面，本发明还提供上述方法制备获得的核壳结构细菌纤维素复合材料，其内核层为芯材，芯材外包覆有单层或多层的细菌纤维素和/或热塑性高分子材料。
本发明的有益效果：
(1)本发明提供的装置能够实现动态发酵和包覆，能够包覆获得形状尺寸可控的细菌纤维素复合材料，该装置简单，操作方便，产率高，适用于工业化生产；得到的包覆型细菌纤维素复合材料可以广泛应用于植入物医疗器械的表面细菌纤维素包覆、缓释药物或微型器械的包覆等生物医药、医疗器械领域，也可以作为食品或食材的包覆等。
(2)本发明的动态发酵方法无需使用任何有毒溶剂，不会带来环境污染以及生态危害等问题，符合医用或者食用的使用要求；在发酵过程中添加各种水溶性高分子，能够获得各种不同性能的细菌纤维素复合材料，且具备优良的生物相容性和生物安全性。
(3)采用本发明的动态发酵方法能够快速获得不同层次的核壳结构的细菌纤维素复合材料，可广泛满足生物医药、医疗器械、食品等诸多领域。
附图说明：
图1为本发明实施例中动态发酵制备核壳结构细菌纤维素复合材料的装置结构示意图。
附图符号说明：
1、发酵培养容器，2、滚筒，3、发酵培养液，4、待包覆芯材，5、加热导流板，6、紧固件。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施提供一种动态发酵制备核壳结构细菌纤维素复合材料的装置，如图1所示，该装置包括：
发酵培养容器1和设置在发酵培养容器中两个滚筒2和两块加热导流板5。两个滚筒2的转轴两端分别与所述发酵培养容器的内壁活动连接，两个滚筒在水平方向上平行排列，滚筒之间设有间隙，且滚筒的转轴之间距离可调；两块加热导流板5与两个滚筒2的转轴平行，其中一个加热导流板5的一端与发酵培养容器1通过紧固件6实现活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与其中一个滚筒抵接；另一块加热导流板一端与所述发酵培养容器通过紧固件6实现活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与另一个滚筒2抵接。两个滚筒优选为形状大小相同的圆柱状滚筒。
在进行动态发酵时，待包覆芯材4位于两个滚筒2间隙的上方并与滚筒相抵接；发酵培养液3装载于发酵培养容器1中。所述装置还包括驱动两个滚筒转动的驱动部件，用于驱动两个滚筒同向转动。所述装置还包括对两块加热导流板进行加热的加热部件。
另一方面，本实施例还提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其采用本实施上述的装置进行动态发酵，包括以下步骤：
(1)开口朝上的发酵培养容器中平行排列两个形状大小相同，可同向顺时针匀速旋转的圆柱形玻璃滚筒。将直径为30mm的球形聚氨酯材料(芯材)放置在两个滚筒间，如图1所示，调节两滚筒间距为26mm、旋转速率为4rpm。使球形聚氨酯可以随滚筒旋 转而实现无水平位移的震动。
(2)在发酵培养容器顶部固定两块加热导流板，调节两块加热导流板与发酵培养容器侧面的角度均为30度，保证两个加热板的一端位于球形聚氨酯与滚筒抵接的缝隙处。
(3)选取能分泌细菌纤维素的木醋杆菌活化制备成种子醪液，然后将菌株浓度为1wt％的种子醪液和发酵培养基混合得到发酵混合液；其中，发酵培养基为高温灭菌过的培养基，培养基的成分为本领域细菌纤维素发酵的常用成分。
(4)将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，于35℃下发酵3天，发酵结束后，排出发酵液，此时球形聚氨酯外层包覆有菌株发酵获得的细菌纤维素，将产物浸泡在质量百分含量为4％的NaOH水溶液中，在100℃的温度下加热6h，再用蒸馏水反复冲洗至中性，形成球形聚氨酯-细菌纤维素复合物。
(5)启动加热导流板，调整加热温度为190℃，将热塑性高分子聚乙烯的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在芯材-细菌纤维素复合物的表面，从而制备获得球形聚氨酯材料表面均匀包覆有聚乙烯/细菌纤维素的核壳结构复合材料。
实施例2
本实施例提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其采用上述实施例1的装置进行动态发酵，包括以下步骤：
(1)开口朝上的发酵培养容器中平行排列两个形状大小相同，可同向顺时针匀速旋转的圆柱形玻璃滚筒。将直径为50mm，长为70mm的生物陶瓷(料芯)放置在两个滚筒间，如图1所示，调节两滚筒间距为40mm、旋转速率为8rpm。使生物陶瓷可以随滚筒旋转而实现无水平位移的震动。
(2)在发酵培养容器顶部固定两块加热导流板，调节两块加热导流板与发酵培养容器侧面的角度均为45度，保证两个加热板的一端位于生物陶瓷与滚筒抵接的缝隙处。
(3)选取能分泌细菌纤维素的根瘤菌属活化制备成种子醪液，然后将菌株浓度为2wt％的种子醪液和发酵培养基混合得到发酵混合液；其中，发酵培养基为高温灭菌过的培养基，培养基的成分为本领域细菌纤维素发酵的常用成分，发酵混合液中还包括0.1wt％的明胶。
(4)将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，于20℃下发酵30天，发酵结束后，排出发酵液，此时生物陶瓷外层包覆有菌株 发酵获得的细菌纤维素，将产物浸泡在质量百分含量为5％的NaOH水溶液中，在90℃的温度下加热5h，再用蒸馏水反复冲洗至中性，形成生物陶瓷-细菌纤维素复合物。
(5)启动加热导流板，调整加热温度为230℃，将热塑性高分子聚丙烯的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在生物陶瓷-细菌纤维素复合物的表面，从而制备获得生物陶瓷表面均匀包覆有聚丙烯/细菌纤维素的核壳结构复合材料。
实施例3
本实施例提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其采用上述实施例1的装置进行动态发酵，包括以下步骤：
(1)开口朝上的发酵培养容器中平行排列两个形状大小相同，可同向顺时针匀速旋转的圆柱形不锈钢滚筒。将6.5mm的钛合金骨科植入螺钉(芯材)放置在两个滚筒间，如图1所示，调节两滚筒间距为5mm、旋转速率为12rpm。使螺钉可以随滚筒旋转而实现无水平位移的震动。
(2)在发酵培养容器顶部固定两块加热导流板，调节两块加热导流板与发酵培养容器侧面的角度均为45度，保证两个加热板的一端位于螺钉与滚筒抵接的缝隙处。
(3)选取能分泌细菌纤维素的八叠球菌属活化制备成种子醪液，然后将菌株浓度为3wt％的种子醪液和发酵培养基混合得到发酵混合液；其中，发酵培养基为高温灭菌过的培养基，培养基的成分为本领域细菌纤维素发酵的常用成分，发酵混合液中还包括1wt％的透明质酸钠和海藻酸钠，透明质酸钠和海藻酸钠质量比为1：1。
(4)将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，于25℃下发酵5天，发酵结束后，排出发酵液，此时螺钉外层包覆有菌株发酵获得的细菌纤维素，将产物浸泡在质量百分含量为6％的NaOH水溶液中，在80℃的温度下加热4h，再用蒸馏水反复冲洗至中性，形成螺钉-细菌纤维素复合物。
(5)启动加热导流板，调整加热温度为200℃，将热塑性高分子聚丙乙烯的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在螺钉-细菌纤维素复合物的表面，从而制备获得螺钉表面均匀包覆有聚丙乙烯/细菌纤维素的核壳结构复合材料。
实施例4
本实施例提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其采用上述 实施例1的装置进行动态发酵，包括以下步骤：
(1)开口朝上的发酵培养容器中平行排列两个形状大小相同，可同向顺时针匀速旋转的圆柱形聚四氟乙烯滚筒。将直径为2mm的纳米羟基磷灰石填充聚甲基丙烯酸甲酯的棒状材料(芯材)放置在两个滚筒间，如图1所示，调节两滚筒间距为1.5mm、旋转速率为2rpm。使棒材可以随滚筒旋转而实现无水平位移的震动。
(2)在发酵培养容器顶部固定两块加热导流板，调节两块加热导流板与发酵培养容器侧面的角度均为60度，保证两个加热板的一端位于棒材与滚筒抵接的缝隙处。
(3)选取能分泌细菌纤维素的假单胞菌属活化制备成种子醪液，然后将菌株浓度为4wt％的种子醪液和发酵培养基混合得到发酵混合液；其中，发酵培养基为高温灭菌过的培养基，培养基的成分为本领域细菌纤维素发酵的常用成分，发酵混合液中还包括2wt％的可溶性淀粉。
(4)将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，于30℃下发酵7天，发酵结束后，排出发酵液，此时棒材外层包覆有菌株发酵获得的细菌纤维素，将产物浸泡在质量百分含量为7％的NaOH水溶液中，在70℃的温度下加热6h，再用蒸馏水反复冲洗至中性，形成棒材-细菌纤维素复合物。
(5)启动加热导流板，调整加热温度为270℃，将热塑性高分子尼龙的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在棒材-细菌纤维素复合物的表面，从而制备获得棒材表面均匀包覆有尼龙/细菌纤维素的核壳结构复合材料。
实施例5
本实施例提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其采用上述实施例1的装置进行动态发酵，包括以下步骤：
(1)开口朝上的发酵培养容器中平行排列两个形状大小相同，可同向顺时针匀速旋转的圆柱形塑料滚筒。将不规则体乳房填充用硅橡胶(芯材)放置在两个滚筒间，如图1所示，调节两滚筒间距为70mm、旋转速率为30rpm。使硅橡胶可以随滚筒旋转而实现无水平位移的震动。
(2)在发酵培养容器顶部固定两块加热导流板，调节两块加热导流板与发酵培养容器侧面的角度均为45度，保证两个加热板的一端位于硅橡胶与滚筒抵接的缝隙处。
(3)选取能分泌细菌纤维素的无色杆菌属和产碱菌属混合活化制备成种子醪液，然后将菌株浓度为5wt％的种子醪液和发酵培养基混合得到发酵混合液；其中，发酵培养基为高温灭菌过的培养基，培养基的成分为本领域细菌纤维素发酵的常用成分，发酵混 合液中还包括3wt％的果胶。
(4)将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，于31℃下发酵15天，发酵结束后，排出发酵液，此时硅橡胶外层包覆有菌株发酵获得的细菌纤维素，将产物浸泡在质量百分含量为8％的NaOH水溶液中，在70℃的温度下加热5h，再用蒸馏水反复冲洗至中性，形成硅橡胶-细菌纤维素复合物。
(5)启动加热导流板，调整加热温度为190℃，将热塑性高分子聚乙烯的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在硅橡胶-细菌纤维素复合物的表面，从而制备获得硅橡胶表面均匀包覆有聚乙烯/细菌纤维素的核壳结构复合材料。
实施例6
本实施例提供一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其采用上述实施例1的装置进行动态发酵，包括以下步骤：
(1)开口朝上的发酵培养容器中平行排列两个形状大小相同，可同向顺时针匀速旋转的圆柱形塑料滚筒。将不规则形状的钴铬钼合金人工髋关节头(芯材)放置在两个滚筒间，如图1所示，调节两滚筒间距为50mm、旋转速率为15rpm。使合金可以随滚筒旋转而实现无水平位移的震动。
(2)在发酵培养容器顶部固定两块加热导流板，调节两块加热导流板与发酵培养容器侧面的角度均为60度，保证两个加热板的一端位于合金与滚筒抵接的缝隙处。
(3)选取能分泌细菌纤维素的气杆菌属和固氮菌属混合活化制备成种子醪液，然后将菌株浓度为3wt％的种子醪液和发酵培养基混合得到发酵混合液；其中，发酵培养基为高温灭菌过的培养基，培养基的成分为本领域细菌纤维素发酵的常用成分，发酵混合液中还包括5wt％的壳聚糖和羧甲基纤维素钠，壳聚糖和羧甲基纤维素钠质量比为1：3。
(4)将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，于32℃下发酵10天，发酵结束后，排出发酵液，此时合金外层包覆有菌株发酵获得的细菌纤维素，将产物浸泡在质量百分含量为6％的NaOH水溶液中，在90℃的温度下加热4h，再用蒸馏水反复冲洗至中性，形成合金-细菌纤维素复合物。
(5)启动加热导流板，调整加热温度为190℃，将热塑性高分子聚乙烯的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在合金-细菌纤维素复合物的表面，从而制备获得合金表面均匀包覆有聚乙烯/细菌纤维素的核壳结构复合材料。
性能测试实验：
对实施例1制备的球形聚氨酯材料表面均匀包覆有聚乙烯/细菌纤维素的核壳结构复合材料进行了如下性能测试：
生物相容性实验：参照GB/T 16886医疗器械的生物学评价，分别对复合材料(实施例1)和聚氨酯材料(实施例1的芯材)进行细胞毒性、豚鼠迟发接触性致敏、皮肤刺激等评价。
结果表明：聚氨酯材料(实施例1的芯材)，细胞毒性为2级，有皮肤致敏反应；复合材料(实施例1)细胞毒性小于2级，无皮肤致敏反应，无皮内刺激反应，具有良好的生物安全性。说明本专利的使用提高了材料的生物相容性能。

Claims (13)

  1. 一种动态发酵制备核壳结构细菌纤维素复合材料的装置，该装置包括：
    发酵培养容器和设置在发酵培养容器中的两个滚筒和两块加热导流板；
    两个滚筒的转轴两端分别与所述发酵培养容器的内壁活动连接，两个滚筒在水平方向上平行排列，滚筒之间设有间隙，且滚筒的转轴之间距离可调；
    两块加热导流板与两个滚筒的转轴平行，其中一个加热导流板的一端与所述发酵培养容器活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与其中一个滚筒抵接；另一块加热导流板一端与所述发酵培养容器活动连接，另一端向斜下方延伸至两个滚筒的间隙上方并与另一个滚筒抵接。
  2. 根据权利要求1所述的装置，其中，所述装置还包括驱动两个滚筒转动的驱动部件，优选地，所述驱动部件用于驱动两个滚筒同向转动。
  3. 根据权利要求1所述的装置，其中，所述装置还包括对两块加热导流板进行加热的加热部件。
  4. 根据权利要求1所述的装置，其中，所述两个滚筒为形状大小相同的圆柱状滚筒。
  5. 根据权利要求1所述的装置，其中，两块加热导流板分别与所述发酵培养容器内侧壁的夹角为15～60度。
  6. 一种动态发酵制备核壳结构细菌纤维素复合材料的方法，其是采用权利要求1～5任一项所述的装置进行动态发酵，包括以下步骤：
    调整滚筒间距使其间隙最小宽度小于芯材的直径或长度，将待包覆的芯材灭菌后置于两个滚筒的间隙上方，保证芯材能够在间隙上方分别与两个滚筒相抵接；设置滚筒的转动速度和转动方向，使芯材随滚筒的转动能够实现无水平位移的震动；
    设置加热板的长度和调节加热板的角度，保证两个加热板的一端位于芯材与滚筒抵接的缝隙处；
    配制细菌纤维素发酵培养液并高压灭菌，然后与菌株种子醪液混合获得发酵混合液；
    将发酵混合液倒入发酵培养容器中，浸没芯材，启动滚筒同向转动进行动态发酵，发酵结束后，排出发酵液，此时芯材外层包覆有菌株发酵获得的细菌纤维素，形成芯材-细菌纤维素复合物；启动加热导流板，将热塑性高分子的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在芯材-细菌纤维素复合物的表面，从而制备获得核壳结构细菌纤维素复合材料；或者，
    启动加热导流板，将热塑性高分子的加热液分别沿两端的加热导流板流下，伴随滚筒转动，热塑高分子均匀涂覆在芯材的表面，获得芯材-热塑性高分子复合物；将发酵混合液倒入发酵培养容器中，浸没滚筒，启动滚筒同向转动进行动态发酵，发酵结束后，排出发酵液，此时芯材-热塑性高分子复合物的外层包覆有菌株发酵获得的细菌纤维素，从而制备获得核壳结构细菌纤维素复合材料。
  7. 根据权利要求6所述的方法，其中，该方法还包括重复动态发酵包覆细菌纤维素的步骤和/或包覆热塑性高分子的步骤，获得更多层次核壳结构细菌纤维素复合材料。
  8. 根据权利要求6所述的方法，其中，所述芯材包括无机材料、有机高分子材料和金属材料中的一种或多种的组合；
    优选地，所述芯材的形状为球型、类球形、圆柱形、棒状体或任意不规则体。
  9. 根据权利要求6所述的方法，其中，所述热塑性高分子材料包括聚乙烯、聚丙烯、聚苯乙烯、聚甲基丙烯酸甲酯、尼龙、聚氨酯、聚酯和聚乳酸中的一种或多种的组合；
    优选地，所述加热导流板的加热温度为50～300℃。
  10. 根据权利要求6所述的方法，其中，所述菌株包括木醋杆菌、根瘤菌属、八叠球菌属、假单胞菌属、无色杆菌属、产碱菌属、气杆菌属和固氮菌属中的一种或多种的组合；
    优选地，菌株种子醪液的添加量为发酵培养液的1～5wt％；
    优选地，进行动态发酵的时间为3～30天；发酵温度为20～30℃；
    优选地，进行动态发酵时，两个滚筒的转速相同，均为0.1～60rpm；优选为4～20rpm。
  11. 根据权利要求6所述的方法，其中，在进行动态发酵过程中，还包括向发酵混合液中添加0.1～5wt％的可溶性添加剂；所述可溶性添加剂包括明胶、透明质酸钠、淀粉、果胶、壳聚糖、海藻酸钠和可溶性纤维素衍生物中的一种或多种的组合。
  12. 根据权利要求6所述的方法，其中，动态发酵结束后还包括对包裹有细菌纤维素的复合物进行纯化的步骤，具体为：在70～100℃的温度下，在质量百分含量为4％～8％的NaOH水溶液中洗涤4～6h，再用蒸馏水反复冲洗至中性。
  13. 权利要求6～12任一项所述方法制备获得的核壳结构细菌纤维素复合材料，其内核层为芯材，芯材外包覆有单层或多层的细菌纤维素和/或热塑性高分子材料。
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