JP2006325534A - Honeycomb porous body of bacterial cellulose and method for producing the honeycomb porous body - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a functional material usable as a substrate for a cell culture medium or a support for a nanomachine. <P>SOLUTION: A honeycomb porous body of bacterial cellulose is provided. A method for producing the honeycomb porous body is also provided, comprising the step of preparing a solid nutrient medium having an uneven pattern opposite to the surface unevenness of the porous body using itself as a template, the step of inoculating the solid nutrient medium with the bacterial cellulose, and the step of making the microorganisms produce the cellulose under an atmosphere of ≥90% relative humidity and ≥50% carbon dioxide concentration. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a honeycomb porous body made of microbial cellulose produced by microorganisms and a method for producing the same.

  Certain microorganisms are widely known to produce characteristic cellulose called microbial cellulose (BC), which is different from plant-derived cellulose. In particular, it has been reported by Hestrin et al. (Non-patent Document 1) that acetic acid bacteria (Acetobacter bacteria) produce high-quality BC under aerobic culture conditions.

  BC is generally a polysaccharide mainly composed of cellulose having very high crystallinity and uniaxial orientation, and is a polymer having a network structure in which very thin ribbon-like fibers are intertwined in a complicated manner (Non-patent Document 2). ). Examples of applications of this BC to industry include concrete examples such as edible cellulose, acoustic device materials, surgical dressings, and pharmaceutical impregnated pads in various fields such as food, various industrial materials, medical materials, and chemical products. A product has been proposed.

  Further, as one of highly functional polymer materials, a functional polymer material called a honeycomb structure or a honeycomb-like porous body (hereinafter referred to as a honeycomb-like porous body) has attracted attention in recent years. A honeycomb-like porous body is generally a thin film having a (honeycomb-like) structure in which pores or depressions of sub-micron to micron scale size, which are made of a star polymer or a linear polymer, are closely packed. It is understood as a structure.

  As an example of a honeycomb-shaped porous body, a honeycomb-shaped porous material comprising a hydrophilic acrylamide polymer as a main chain skeleton, and an amphiphilic polymer having both a dodecyl group as a hydrophobic side chain and a lactose group or a carboxyl group as a hydrophilic side chain There are a body (Non-patent Document 3), a honeycomb-shaped porous body made of linear polystyrene (Patent Document 1), a honeycomb-shaped porous body made of polyimide (Patent Document 2), and the like.

  As a method for manufacturing a honeycomb-shaped porous body, a special polymer having both a portion having a strong self-aggregating force and a portion exhibiting flexibility is used, and these polymers are dissolved in a hydrophobic organic solvent and cast. A technique (Non-patent Document 4) has been reported. Also, by cooling the polymer solution and condensing vapors in the atmosphere on the solution surface, some of the droplets enter the inside from the surface of the polymer solution, then evaporate the solvent, and then A manufacturing method characterized by removing the condensed droplets has also been reported (Patent Document 1).

A honeycomb-shaped porous body has a shape in which fine pores are regularly arranged. Therefore, it can be used for various applications such as semiconductor low dielectric constant materials, scattering layers for electronic displays, magnetic recording materials, and cell culture substrates. Applications are being studied.
JP-A-8-311231 JP 2003-80538 A Hestrin, S. et al., Nature, 1947, 159, 4028, pp. 64-65 "Advanced Materials Series Microorganisms and Materials", 2001, edited by Japan Society for Materials Science, Tokyo Nishikawa et al., Polymer Journal, 2002, Vol. 34, No. 3, pp. 166-174 Widanski et al., Nature, 1994, 369, 387-389

  If a honeycomb-like porous body, which is expected to have various functions due to structural characteristics, can be manufactured with a promising BC as a polymer material, a honeycomb-like porous body made of BC having the respective advantages (hereinafter referred to as BC) It can open the way for the provision of a new functional material (represented as a honeycomb-like porous body). However, such a BC honeycomb porous body has not yet been manufactured and obtained.

  An object of the present invention is to provide a BC honeycomb porous body and to provide an efficient manufacturing method thereof.

The present inventors prepared a solid nutrient medium having a surface with irregularities opposite to the surface irregularity pattern of the honeycomb-shaped porous body using a honeycomb-shaped porous body prepared by a known production method as a template. The inventors have found that a BC honeycomb-like porous body can be produced by producing BC on a microorganism that produces BC on this solid medium (BC producing microorganism), and the present invention has been completed.
That is, the present invention provides the following inventions 1) to 5).

1) BC honeycomb porous body.

2) Consisting of BC produced by Acetobacter xylinum, Acetobacter pasturinus, Acetobacter aceti, Acetobacter lancensis, Sartina xyloides, Pseudomonas bacteria, Agrobacterium bacteria or Rhizobium bacteria 1) The BC honeycomb-like porous body described.

3) The BC honeycomb porous body according to 1), wherein the pore diameter is 10 nm to 1000 μm and the thickness is 0.1 to 10 μm.

4) A step of preparing a solid nutrient medium having an uneven pattern opposite to the surface unevenness of the porous body using the honeycomb-shaped porous body as a template, a step of inoculating the solid nutrient medium with a microorganism that produces BC, and a relative humidity A method for producing a BC honeycomb porous body, comprising a step of causing the microorganism to produce BC in an atmosphere of 90% or more and a carbon dioxide concentration of 50% or more.

5) The microorganisms that produce BC are Acetobacter xylinum, Acetobacter pasturinus, Acetobacter aceti, Acetobacter lanceens, Sartina xyleudes, Pseudomonas bacteria, Agrobacterium bacteria, or Rhizobium bacteria 4) The manufacturing method as described in.

  The BC honeycomb-shaped porous body of the present invention can be used for various cell culture substrates, breathable wound dressings, nanomachine supports, and the like.

<BC-producing microorganism>
Based on previous studies, various microorganisms such as Acetobacter bacteria, Pseudomonas bacteria, Agrobacterium bacteria, Rhizobium bacteria, etc. have been reported as microorganisms capable of producing BC. In the present invention, any microorganism capable of producing such BC, that is, any BC-producing microorganism should be understood as an available microorganism. Moreover, even if it is a new kind of microorganism that has not been isolated and reported so far, or a known subspecies or mutant of a BC-producing bacterium, any microorganism that can produce BC under the culture conditions in the present invention will also be used. It should be understood that it is available.

  Examples of suitable BC-producing microorganisms for use in the present invention include Acetobactor xylinum, A. pasturianus, A. aceti, A. ransens. ), Sarcinaventriculi, Bacterium xyloides, or their BC-producing subspecies or mutants. In particular, the use of bacteria belonging to the genus Acetobacter, especially A. xylinum, or A. aceti Sp. Xylinum, A. pasturianus, A. ransens is preferable.

<BC>
In general, the chemical structure of BC varies depending on the microorganism that produces it. Cellulose is the main chain, and hexose and pentose such as mannose, fructose, galactose, xylose, arabinose, rhamnose, and uronic acid are used as other constituents. And heteropolysaccharides containing organic acids and the like, and those containing glucans such as β and α. In the present invention, any polysaccharide mainly composed of cellulose produced by microorganisms can be used as the BC constituting the honeycomb porous body of the present invention, regardless of the structural differences as described above. is there.

  BC suitable in the present invention is BC produced by Acetobacter bacteria. This BC has a structure called cellulose Iα structurally, and is advantageous in terms of application to environmentally conscious natural polymer materials because of its biodegradability and strength characteristics. .

<BC honeycomb porous body>
The BC honeycomb-like porous body of the present invention has a (honeycomb-like) structure pattern in which a large number of submicron to micron-scale pores or depressions are closely packed, that is, the pores or depressions are closely packed. It is a thin film structure of BC cellulose having a structure in which fibrous BC is stretched so as to be constructed (FIGS. 1 and 2).

  The diameter of the pore portion of the BC honeycomb porous body of the present invention can generally take a value in the range of 10 nm to 1000 μm, preferably 0.1 μm to 100 μm, more preferably 20 to 30 μm. Moreover, although the height or thickness of the honeycomb-shaped wall portion depends on the amount of BC-producing microorganisms used or the time for producing BC, a value in the range of 0.1 to 10 μm can be taken. These sizes, as will be described later, determine the size of the uneven shape of the surface of the solid nutrient medium used when the BC-producing microorganism produces BC, and the size of the uneven shape of the solid nutrient medium. It can be indirectly controlled by adjusting the unevenness of the honeycomb-like porous body.

  The honeycomb-like porous body can be prepared from an appropriate polymer solution by a known method such as Non-Patent Documents 1 to 4 and Patent Document 1 or 2. For example, by using acrylamide, polystyrene, polycaprolactone, polyimine or a mixture or copolymer thereof and the like, a honeycomb-shaped porous body having a desired pore size and thickness is prepared by following the method disclosed in the above-mentioned literature. can do. Generally, the pore size of the honeycomb-shaped porous body that can be adjusted by a known method is set to 1 μm to 1000 μm. Also, by cooling the polymer solution and condensing vapors in the atmosphere on the solution surface, some of the droplets enter the inside from the surface of the polymer solution, then evaporate the solvent, and then When using a production method (Patent Document 1) characterized by removing condensed droplets, the solvent is evaporated under an air flow having a relative humidity of 30% or more and a flow rate of 10 to 100 L / min. It is also possible to prepare a honeycomb-like porous body having a fine pore size of a submicron level such as 10 nm to 100 nm.

  In the present invention, the honeycomb-like porous body only needs to be usable as a template for preparing a solid nutrient medium described later. That is, any thin film structure having a structure in which vacancies or depressions of sub-micron to micron scale size are closely packed can be used, and the material and preparation method are not limited. Therefore, any honeycomb porous body prepared by improving or modifying a conventionally known method can be used as long as it has a specific structure. Hereinafter, the honeycomb porous body used for the preparation of the solid nutrient medium in the present invention is referred to as a first template.

  The step of preparing a solid nutrient medium having a concavo-convex pattern opposite to the surface concavo-convex of the porous body using the honeycomb-shaped porous body as a template is that the BC-producing microorganism can assimilate the honeycomb-shaped concavo-convex pattern of the first mold Transferred to a solid medium containing an appropriate nutrient source, and has a concave / convex pattern opposite to the concave / convex pattern of the first mold, that is, the hole portion of the first mold is raised, and the concave portion corresponding to the polymer portion of the first mold is formed. This is a step of preparing a solid nutrient medium.

  As a typical example, an agar solution prepared by dissolving a medium developed by Hestrin and Schram (HS medium, Biochem. J., 58, 345 (1954)) is prepared and used as the first template. After pouring from above the concavo-convex surface, or while bringing the concavo-convex surface side of the first mold into contact with the surface of the agar solution, the agar solution is cooled and solidified, and the first mold is peeled off from the solidified agar. Thus, a solid nutrient medium having a desired uneven pattern can be prepared (FIG. 3). Since the size of the unevenness of the solid nutrient medium obtained here is determined by the unevenness of the first mold used, that is, the pore diameter and the hole depth, etc., the hole diameter and the hole depth of the first mold or By adjusting the thickness of the porous body, the size of the unevenness of the solid nutrient medium can be adjusted. In addition, instead of agar used in the above operation, a desired solid nutrient medium can be prepared even if a liquid state and a solid state that can be converted into each other under appropriate conditions, such as alginic acid or gelatin, are used. Is possible.

  When smooth separation of the solid nutrient medium and the first mold is difficult due to problems such as compatibility between the polymer constituting the first mold and the solid nutrient medium, another polymer that does not cause such a problem is used. The uneven pattern of the first template may be transferred to the solid nutrient medium. For example, an appropriate polymer, such as polydimethylsiloxane, is poured into the first mold, solidified, and then peeled off from the first mold, thereby having an uneven pattern opposite to the uneven pattern of the first mold. A hole portion of the first mold rises to prepare a sheet (second mold) having a recess corresponding to the polymer portion of the first mold, and further a polymer, for example, the same polydimethyl as the second mold, is prepared with respect to the second mold. A sheet (third mold) having the same concavo-convex pattern as the first mold is prepared by adding siloxane to solidify and peeling. By using this third template and performing the same operation as the method of transferring the concavo-convex pattern directly from the first template to the solid nutrient medium, the pattern of the concavo-convex pattern of the first template is finally reversed. It is possible to prepare a solid nutrient medium having irregularities, that is, the hole portion of the first mold is raised and has a recess corresponding to the polymer portion of the first mold.

  The step of inoculating a solid nutrient medium with a microorganism that produces microbial cellulose is a step of inoculating a solid nutrient medium with a BC-producing microorganism that has been previously grown in an appropriate medium, for example, an HS liquid medium. Inoculation is preferably performed by adding a cell suspension of a BC-producing microorganism previously grown on a solid nutrient medium.

  The amount of the cell suspension to be added may be such that the unevenness on the surface of the solid nutrient medium is submerged in the added cell suspension. The part may be added to the extent that it is exposed without being immersed in the cell suspension. From the former addition, a BC honeycomb-like porous body in which depressions corresponding to the convex portions are regularly arranged is obtained, and from the latter, one surface having the size of the convex portion immersed in the suspension is obtained. Thus, a BC honeycomb porous body in which holes penetrating through the other surface are regularly arranged can be obtained.

  A cell suspension of a BC-producing microorganism is inoculated with a BC-producing microorganism in a suitable liquid medium, cultured under culture conditions suitable for the microorganism, and an absorbance (OD) at 660 nm of about 0.05 to 0.2. The grown broth may be used as it is, or the cells may be collected by centrifugation or the like and then resuspended in a fresh liquid medium, physiological saline or an appropriate buffer. When cells collected in a fresh liquid medium are resuspended, BC-producing microorganisms may be further grown on the solid nutrient medium after inoculating the cells in the solid nutrient medium.

  The step of causing the microorganism to produce BC in an atmosphere having a relative humidity of 90% or more and a carbon dioxide concentration of 50% or more causes the microorganism inoculated on the solid nutrient medium to produce a large amount of BC, and the BC honeycomb is produced on the solid nutrient medium. This is a step of preparing a porous material. A BC-producing microorganism inoculated on a solid nutrient medium is incubated in an atmosphere having a carbon dioxide concentration of 50% or more, and thus hardly grows itself and produces a larger amount of BC. This phenomenon is not observed under 0-100% oxygen stream or 0-100% nitrogen stream. Many BC-producing microorganisms, especially Acetobacter bacteria, are aerobic microorganisms, and thus BC production has been carried out exclusively in the atmosphere composition. The finding that BC was produced in a larger amount under a high concentration carbon dioxide stream found this time was unexpected. In addition, since the growth of microorganisms is not particularly necessary during production of BC, there is no particular problem even if incubation is performed in an atmosphere in which almost no oxygen is present and the carbon dioxide concentration is substantially 100%. Further, maintaining the relative humidity at 90% or more is a measure for preventing the cell suspension of the BC-producing microorganism inoculated on the solid nutrient medium from drying, and therefore even when the relative humidity is 100%, There is no obstacle to the method.

  When BC is produced by a microorganism in this way, a network-like structure is formed in which very thin ribbon-like fibers are intertwined in a complex manner according to the pattern in which a cell suspension is present on a solid nutrient medium. As a result, a BC honeycomb-like porous structure having a structure in which pores are regularly arranged in a honeycomb shape, that is, a structure in which fibrous BC is extended so that void portions or depressions are regularly (in a honeycomb shape) is formed. A mass is formed.

  The incubation time can be set in the range of 48 to 144 hours. In particular, in the present invention, by adjusting the incubation time, the density of the BC portion of the obtained honeycomb porous body, the film thickness of the porous body, and the like can be adjusted. In general, a denser and thicker honeycomb porous body can be obtained by incubating for a long time. In addition, the incubation temperature of the BC-producing microorganism on the solid nutrient medium may be set in the range of approximately 27 to 29 ° C., depending on the type of BC-producing microorganism to be used.

  The incubation temperature, relative humidity, and carbon dioxide concentration may be adjusted by preparing an appropriate culture device that can arbitrarily adjust each. Moreover, you may use an apparatus as shown in the Example mentioned later.

  The BC honeycomb-like porous body produced on the solid nutrient medium through the above steps can be easily recovered by peeling from the solid nutrient medium or by dissolving the solid nutrient medium again by heating. it can. Depending on the application, the recovered BC honeycomb-like porous body may be used as it is or after it is dried by using a dilute alkali, dilute acid, organic solvent, hot water or the like alone or in combination. .

  In the above production method, nutrient sources that can be included in the solid nutrient medium include nutrient sources or assimilation required by BC-producing microorganisms such as carbon sources, nitrogen sources, inorganic salts, amino acids, vitamins and other organic micronutrients. Any possible nutrient source can be used. In particular, it is convenient to use a preparation medium in which these nutrient sources are combined. Examples of the prepared medium include a normal agar medium and an HS medium. The aforementioned medium can also be used as a medium for growing BC-producing microorganisms in advance for inoculating the solid nutrient medium. A preferred preparation medium in the present invention is an HS medium.

  Moreover, in order to increase the production amount of BC by incubation, it is possible to improve the medium composition by appropriately adding optional components preferable for the growth of the BC-producing microorganism to be used or BC production. For example, inositol, phytic acid, or pyrroloquinoline quinone may be added for the purpose of improving BC productivity. In addition, in order to increase the yield of BC, a small amount of a cellulase preparation can be added while retaining cellulase activity.

  Moreover, what is necessary is just to set the range suitable for every BC production microorganisms to be used for the pH range of a culture medium, but it should just be about pH 4-6, Preferably it is 4.5-5.5. The pH setting can be adjusted by adding a buffer such as citrate to the medium or by adding a sufficient amount of base or acid to the medium to maintain the pH in the desired range.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, it cannot be overemphasized that these description is not for limiting the present invention. Further, “%” in the examples means “wt%” unless otherwise specified.

  5 ml of a chloroform solution in which 10 mg of a mixture of a copolymer of dodecylacrylamide and ω-carboxyhexylacrylamide and polycacrolactone is dissolved in a mixing ratio of 1: 9 is dropped on a glass petri dish, and saturated water vapor is supplied at a flow rate of 2 liters / minute. Were sprayed to prepare a first mold which was a honeycomb-like porous body. From observation with an optical microscope, it was confirmed that a honeycomb film having a structure in which fine pores having a diameter of about 25 μm were closely packed was formed.

  Using a kit made by Toray Dow Corning (Silgard 184) to create a silicone elastomer made of polydimethylsiloxane (PDMS), the first mold and liquid PDMS are brought into contact with each other and maintained at 70 ° C. for 45 minutes to solidify the PDMS. It was. The solidified PDMS was peeled from the first mold to obtain a second mold. The second mold was further brought into contact with liquid PDMS and maintained at 70 ° C. for 45 minutes to solidify the PDMS. The solidified PDMS was peeled from the second mold to obtain a third mold.

  Autoclaving a 1.5% agar solution containing HS medium (0.5% yeast extract, 0.5% peptone, 2.0% glucose, 0.115% citric acid, 0.27% disodium hydrogen phosphate) After sterilization, the sample was transferred to a petri dish, and a third mold was placed thereon to solidify the agar. The third mold was peeled from the agar, and a 1 cm × 1 cm section of the solidified agar was taken out and transferred to another petri dish to obtain the solid nutrient medium of the present invention.

  A 1-liter three-neck separable flask is prepared, a sensor for measuring temperature and humidity is provided at the first port, the second port is connected to a carbon dioxide gas cylinder as a gas inlet, and the third port is used as a gas outlet. used. 500 ml of water was added to the flask to float a piece of wood and placed on a mantle heater.

  Add 30 ml of HS liquid medium to Erlenmeyer flask, inoculate with Acetobacter xylinum (ATCC 53582), perform static culture at 28 ° C for 48 hours under aerobic condition, and culture until reaching 0.094 at 660 nm OD It was. After inoculating 20 μl of this culture broth into a 1 cm × 1 cm solid nutrient medium on a petri dish prepared in advance, the petri dish was placed on a piece of wood in a separable flask and the flask was heated with a mantle heater. The water temperature in the flask was maintained at 28 to 29 ° C., the relative humidity was maintained at 90%, and carbon dioxide was introduced from the bomb into the flask at 0.05 to 0.1 liter / min (carbon dioxide concentration 85%).

  Two days after the start of incubation, white turbidity was observed on the solid nutrient medium. After 6 days of incubation, the solid nutrient medium is recovered, dissolved in boiling water to dissolve the solid nutrient medium, the remaining film is recovered, washed with warm water, and dried to obtain a BC honeycomb porous body. It was.

  When this BC honeycomb-like porous body was observed with an electron microscope, it was confirmed that it had a structure in which pores having a pore diameter of 25 μm were closely packed (FIGS. 1 and 2).

FIG. 1 is an electron micrograph of a honeycomb porous body made of BC of the present invention. FIG. 2 is an electron micrograph (partially enlarged view) of the honeycomb porous body made of BC of the present invention. FIG. 3 is an electron micrograph of the surface of the solid nutrient medium used in the present invention.

Claims (5)

A honeycomb-like porous body made of microbial cellulose. 2. Acetobacter xylinum, Acetobacter pasturinus, Acetobacter aceti, Acetobacter lancensis, Sartina xyleudes, Pseudomonas bacteria, Agrobacterium bacteria or Rhizobium bacteria Honeycomb porous body. The honeycomb-like porous body according to claim 1, wherein the pore diameter is 10 nm to 1000 µm, and the thickness is 0.1 to 10 µm. A step of preparing a solid nutrient medium having a concavo-convex pattern opposite to the surface irregularities of the porous body using the honeycomb porous body as a template, a step of inoculating the solid nutrient medium with microorganisms producing microbial cellulose, and a relative humidity of 90 %, And a method for producing a honeycomb-shaped porous body made of microbial cellulose, comprising a step of causing the microorganism to produce microbial cellulose in an atmosphere having a carbon dioxide concentration of 50% or more. The microorganism that produces microbial cellulose is Acetobacter xylinum, Acetobacter pasturinus, Acetobacter aceti, Acetobacter lanceens, Sartina xyleudes, Pseudomonas bacterium, Agrobacterium bacterium, or Rhizobium bacterium The manufacturing method as described in.