JP2011056456A - Method for producing bio-nanofiber - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient production method of a nanofiber that refines cellulose and chitin chitosan uniformly when biomass resources are utilized effectively, and to provide a method for efficiently decomposing and saccharifying a nanofiber using a hydrolase. <P>SOLUTION: The method for producing the bio-nanofiber aims at enhancing the shearing effect at the time of jetting the biomass (cellulose and chitin chitosan) to continuously produce the high crystalline bio-nanofiber that has been refined with a uniform width based on the water jet technology by combining the single jetting chamber of which the cavitation effect limiting the Reynolds number and the collision effect to a hard object are optimized with a refining apparatus equipped with a circuit corresponding to the high concentration and high-viscosity treatment. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing bionanofiber and its use as a composite material, pharmaceutical, food cosmetic or enzyme substrate.

  Cellulose accounts for the majority of biomass on the earth, and chitin is the next most abundant biomass after cellulose, and its effective use is expected. However, since these biopolymers form a strong crystal structure, they are difficult to be decomposed and miniaturized, and their use is stagnant. If glucose can be obtained by decomposing cellulose, it can be used as a raw material in a biorefinery for producing alcohols such as ethanol, propanol and butanol and organic acids such as succinic acid in combination with a fermentation method. In addition, N-acetylglucosamine obtained by hydrolyzing chitin is not only a health food additive but also a raw material for hyaluronic acid which has recently become a hot topic as a moisturizing ingredient, and thus the cosmetics industry is also drawing attention. N-acetylglucosamine is also expected as a therapeutic agent for dermatitis, osteoarthritis and the like, and its use is expected in a wide range of industrial fields. In addition, research is underway to obtain cellulose, chitin, and chitosan that are uniformly refined (nanofiberized) at the nano level for the purpose of developing composite materials derived from biomass (green composites) and biocompatible materials in the medical field. Has been done.

  Attempts have been made to obtain nanofibrous cellulose using a water jet (Patent Documents 1 and 2), but these methods do not sufficiently refine the cellulose.

JP-A-2005-270891 JP 2007-185117 A

  Biomass, especially cellulose and chitin, has been lagging in effective use due to its strong crystal structure. For effective utilization of these, it is essential to develop a highly efficient and continuous refinement method for crystalline biopolymers (cellulose and chitin).

  An object of the present invention is to efficiently convert biomass such as cellulose, chitin, and chitosan into nanofibers and effectively use the biomass.

  The present inventors have succeeded in developing a new miniaturization technology specialized in biomass based on the water jet technology. By using this technology, nanofibers of cellulose, chitin and chitosan can be made. Cellulose made into nanofibers can be used to develop green composites as reinforcing agents for various resins that utilize properties such as high strength and low thermal expansion. In addition, chitin and chitosan made into nanofibers can develop biocompatible materials in the medical field using their antibacterial properties, wound healing properties, and biodegradability.

The present invention provides the following method for producing bio-nanofibers.
Item 1. A method for producing a bio-nanofiber, characterized in that a biomass dispersion fluid is injected at a high pressure of 100 to 245 MPa so as to collide with a hard body for collision.
Item 2. Item 2. The method for producing bio-nanofibers according to Item 1, wherein a single injection chamber in which the number of lay nozzles is limited to 2266 to 3885 and the impact effect on a hard body is optimized is used.
Item 3. Combining the single injection chamber with a miniaturization device equipped with a high-concentration (10 to 20% by weight) biomass dispersion fluid increases the shear effect during injection and reduces the nanofiber fiber width (minor axis). A method for producing a bio-nanofiber, characterized in that it is refined to 10 to 100 nm, preferably 10 to 40 nm.
Item 4. The bio-nanofiber obtained by the manufacturing method according to Items 1 to 3, having a crystallinity of 40% or more.
Item 5. Item 5. A film comprising a composite material or bionanofiber comprising the bionanofiber according to item 4.
Item 6. Item 5. A bionanofiber saccharification method, wherein the bionanofiber according to item 4 is efficiently hydrolyzed using an enzyme.

  The cellulose or chitin / chitosan nanofiber according to the present invention can be used effectively as a green composite or a biocompatible material. Furthermore, an effective biomass decomposition method using a combination of this technology and an enzyme is provided. By utilizing the method according to the present invention, it can be expected that the decomposition of biomass and the research and development of new functional composite materials will gain momentum.

Dispersed state of chitin treated in a single injection chamber (after 15,000 rpm centrifugation) Dispersion state of cellulose nanofibers by transmission electron microscope Solution dispersion state of chitin nanofibers by transmission electron microscope Field emission scanning electron micrograph of cellulose nanofibers Field emission scanning electron micrograph of chitin nanofibers Powder X-ray diffraction pattern of cellulose nanofibers treated in a single injection chamber X-ray powder diffraction pattern of chitin nanofibers processed in single injection chamber Comparison of chitin degradation rate after 24 hours reaction with yatalase Comparison of chitin degradation rate after 24 hours reaction with thermostable chitinase enzyme Comparison of glucose degradation rates of Trichoderma novozyme cellulase enzyme-treated crystalline cellulose Comparison of glucose degradation rate of thermostable cellulase enzyme-treated crystalline cellulose Chitin film Schematic diagram of the device of the present invention Schematic diagram of the device of the present invention The improvement of the apparatus of this invention is shown. The improvement of the apparatus of this invention is shown.

  The average diameter (short axis) of the nanofiber produced by the method of the present invention is about 10 to 100 nm, preferably about 10 to 50 nm, more preferably about 10 to 40 nm, still more preferably about 15 to 25 nm, most preferably. It is about 20 nm.

  In the present invention, “biomass” to be subjected to high-pressure injection treatment means a biological polymer, particularly a polymer that is hardly soluble in water, and specifically includes cellulose, chitin, chitosan and the like. Cellulose, chitin, and chitosan are materials that are difficult to be enzymatically degraded and difficult to process. According to the present invention, a crystalline or sparingly water-soluble natural polymer such as cellulose, chitin, or chitosan can be converted into a nanofiber by high-pressure jet treatment using a water-dispersed fluid, and the obtained nanofiber is converted into glucose by enzymatic degradation. , N-acetylglucosamine, monomers such as glucosamine, or oligomers such as dimers and trimers thereof. Here, the “dispersion fluid” is a dispersion of biomass in water. When the concentration is low, it becomes a fluid dispersion, but the viscosity increases as the biomass becomes finer. When it becomes high, it becomes a property close to paste. The concentration of the dispersion fluid of biomass nanofibers is preferable because the treatment efficiency increases as the concentration increases, but in particular, in the case of fine fibers at the nano level, the viscosity becomes too high and high-pressure injection becomes difficult when the paste is formed. In the present invention, an apparatus capable of high-pressure injection even when the biomass fiber (preferably biomass nanofiber) is high in concentration has been developed, so the concentration of biomass in the dispersion fluid is, for example, about 1 to 20% by weight, preferably 5 It may be about -20% by weight, more preferably about 10-20% by weight, and still more preferably about 11-20% by weight.

  The biomass raw material used in the present invention may be in any form such as fibrous or granular. Regarding the decomposition (saccharification) of biomass up to the constituent unit sugar according to the present invention, in the case of cellulose, in addition to a wide range of plant raw materials (rice straw, rice husk, wheat straw, corn cob, etc., wood, forest land, lumber mill residue, construction In the case of chitin, biomass such as shells of crustaceans such as shrimp and crab is preferably used directly as a raw material. On the other hand, in the case of nanofibers, in the case of cellulose, crystalline cellulose from which lignin and hemicellulose have been removed is used. In the case of chitin and chitosan, purified chitin and chitosan that have been deproteinized and decalcified by a generally known method are used as raw materials. It is preferable to use as. In the case of nanofiber formation, commercially available raw materials may be used for both cellulose and chitin / chitosan. When biomass is subjected to high-pressure injection processing with the apparatus according to the present invention, cellulose and chitin / chitosan are thinned by untangling of fibers while maintaining the length of the fibers, but the processing conditions such as the injection pressure and the number of processing are changed. It is also possible to cut the fiber or reduce the molecular weight. In the present specification, the “nanofiber” means a fiber having a nano width. For example, when the fibers of the cellulose are unwound and become one minimum unit fiber by carrying out the method of the present invention, the diameter is about 10 to 50 nm. The diameter (width) of the biomass material or nanofiber can be measured by an electron micrograph. Such a fiber is not nano-sized in length, but has a diameter (width) of nano-size, and is therefore referred to as nanofiber in this specification.

  The average diameter of the nanofibers obtained by treatment by the method of the present invention is about 10 to 100 nm, preferably about 10 to 40 nm, and most preferably about 15 to 25 nm. Since the nanofiber of the present invention has a large fiber length / fiber width (aspect ratio), it can be easily formed into a film or sheet in which nanofibers are entangled like a nonwoven fabric while maintaining strength, and various materials. Can be suitably used. The nonwoven fabric obtained by forming the nanofiber of the present invention into a film / sheet is hereinafter referred to as “nanofiber film”. The nanofiber film of the present invention is characterized by a very narrow fiber width and high transparency.

  The dispersed fluid of biomass can be jetted from a nozzle at a high pressure using an ultrafine device using a water jet developed by Sugino Machine Co., Ltd. The pressure of the high pressure injection is about 100 to 245 MPa. The injection speed is about 440 to 700 m / s.

  The dispersed fluid of biomass that has been injected with high pressure and collided with the collision hard body is collected and again injected with high pressure from the nozzle toward the collision hard body, and this operation is performed a required number of times, for example, about 1 to 50 times, preferably Repeat about 1 to 40 times, more preferably about 1 to 30 times, still more preferably about 1 to 20 times, and particularly preferably about 1 to 10 times. When the biomass collides with the collision hard body, the fibers are untangled, the fiber diameter is reduced, and the nano size is reduced. In addition, as a hard body for collision, shapes, such as ball shape and flat plate shape, are mentioned. Examples of the diameter of the nozzle that jets the dispersion fluid at a high pressure include about 0.1 to 0.8 mm.

  Increasing the specific surface area through the conversion of biomass into nanofibers can increase the accessibility of enzymes that hydrolyze them. It became clear that cellulose and chitin refined by this technology are also suitable as enzyme substrates. That is, glucose and N-acetylglucosamine, which are solubilized sugars, can be extracted from cellulose and chitin with high efficiency. In particular, the process of extracting glucose from cellulose has become a bottleneck in biorefinery, and therefore effective use of this apparatus is expected. Similarly, in the case of chitin / chitosan, it is possible to extract N-acetylglucosamine and glucosamine, which are solubilized sugars, with high efficiency. In the case of chitosan, chitosan of about 6 sugars is excellent in biological activity such as antibacterial properties, and the value as a functional material can be increased by increasing the proportion of chitosan having such a length. N-acetylglucosamine is a component of hyaluronic acid and has a characteristic that it is easy to ingest because it has sweet taste. Furthermore, in the case of cellulose, by reducing the fiber diameter (fiber width), it is possible to expect an effect that the fibers are entangled with each other at high density and the strength is increased. Moreover, the function as a heat insulating material or a filtering agent can be improved by increasing the space | gap between fibers. When bio-nanofiber is used as a cosmetic material, it has a very smooth feel and can be expected to have a moisturizing effect, a skin care effect, an antibacterial effect, and a metabolism promoting effect.

In order to use biomass such as cellulose, chitin, and chitosan as industrial resources, pretreatment using strong acid, supercritical method, mechanochemical method, ionic liquid, etc. has been studied. Practical use has been delayed due to problems such as high cost and production efficiency.
In addition, a mortar (disc mill) or an electrospinning method is used to convert biomass into nanofibers, but production efficiency is low, and there is a problem in uniformity of refinement.
The present invention provides a low-environmental low-load biomass decomposition and nanofiber technology using only water without using any catalyst or harmful chemicals.

  The nanofibers obtained by the present invention have a fiber diameter of 100 nm or less, more preferably 80 nm or less, still more preferably 60 nm or less, particularly 40 nm or less. Since the nanofiber of the present invention has a very thin fiber diameter, it has an appearance close to a transparent solution when dispersed in water, and it is visually recognized that the nanofiber is dispersed in water. Alternatively, a transparent dispersion liquid on the right side of FIG. 1 can be obtained. FIG. 1 is a photograph of the state of each solution of chitin after centrifugation (15,000 rpm). Crystalline chitin that has not been subjected to water jet treatment precipitates without the need for centrifugation (Fig. 1 left), whereas crystal chitin that has been treated with water jet is uniformly dispersed after centrifugation (15,000 rpm). Formed) (center of FIG. 1). A transparent gel state can also be obtained by changing the number of water jet treatments (right in FIG. 1). When the same treatment is performed with a water jet, a similar paste (gel form) can be obtained with cellulose and chitosan. When the thus obtained bionanofiber gel is cast in a petri dish and dried, a translucent or transparent film is obtained. For example, in the case of chitin nanofibers, a transparent film can be obtained by casting the transparent gel on the right side of FIG. Cellulose and chitosan nanofibers can produce a translucent or transparent film in the same manner as chitin nanofibers. The nanofiber film thus obtained can be used as a transparent substrate, filter material, packaging material, etc. for displays such as organic EL and liquid crystal.

The bionanofiber of the present invention is useful as a UV care cosmetic because it has the property of passing visible light but cutting ultraviolet rays. Since bionanofiber also has a moisturizing action, it can be blended into cosmetics and applied to the skin as a film or sheet. In particular, chitin / chitosan is known to give moisture and elasticity to damaged hair and skin, and the chitin / chitosan nanofiber of the present invention is suitable as a cosmetic.
Chitosan, which is a cationic polymer, is considered to exhibit antibacterial activity by attracting positively charged amino groups to negative charges on the bacterial cell wall and inhibiting the freedom of the bacteria. Thus, by incorporating the chitosan nanofiber of the present invention into fibers, antibacterial activity can be imparted to clothing, daily necessities, interiors, and the like.

It is known that the elastic modulus and strength of cellulose nanofibers composed of extended chain crystals reach 140 Gpa and 3 Gpa, respectively, which are equivalent to typical high-strength fibers and aramid fibers, and have higher elasticity than glass fibers. Moreover, the coefficient of linear thermal expansion is 1.0 × 10 ⁻⁷ / ° C., which is as low as quartz glass. Cellulose nanofibers are used as reinforcing fibers for composites because of these properties. Since the cellulose nanofabric of the present invention can maintain high crystallinity, it can achieve high functionality (improvement in heat resistance and strength characteristics) by compounding with rubber or resin. In visible light, an object having a size of 1/10 or less of the wavelength of 400 to 800 nm does not scatter even when mixed with a material. Therefore, by uniformly dispersing the cellulose nanofibers of the present invention having excellent heat resistance and strength characteristics due to high crystal in the transparent resin, the heat resistance and strength characteristics can be improved while maintaining the transparency of the resin. Is possible. For example, by mixing the cellulose nanofibers of the present invention with rubber, plastic, transparent resin, etc., the effects of both reinforcement and weight reduction of automobile tires, bumpers, windshields and the like can be obtained.

The transparent film made using the cellulose nanofiber of the present invention has the same meaning as transparent paper. The principle of paper transparency is to reduce the frequency of light refraction (scattering) in the paper layer (between fibers) and reduce opacity in principle. While the refractive index of cellulose constituting the paper fiber is 1.49, the refractive index of air is significantly different from 1.00. General high quality paper contains about 50% of air in its volume, and there are many fine voids between the fibers. For this reason, the light is complicatedly refracted at the interface between the countless number of the two, and the paper looks white and opaque. Since the wavelength of visible light is 400 to 800 nm, an object sufficiently smaller than that (1/10 or less) can be regarded as causing no light scattering. Therefore, in order to make the paper transparent, not only the fibers are made thinner than the visible light wavelength (1/10 or less), but also the gaps between the fibers must be made sufficiently thinner than the visible light wavelength. Methods for making existing paper transparent are roughly classified into the following three types.
a. Beating method A method of reducing the voids in the paper by increasing the binding area between fibers by highly beating cellulose. Glassine paper (Oji Paper product name: Graphan)
b. Sulfuric acid method A method of gelling cellulose by chemically modifying it with sulfuric acid. Parchment paper.
c. Composite A method that fills voids between fibers with a material having a refractive index equal to that of cellulose. Impregnated paper with resin and oil.
Thus, a great deal of labor is required to make paper transparent, but the cellulose nanofibers of the present invention have a large aspect ratio and can increase the entanglement between fibers, making it easy to make transparent paper. it can. Conventionally, transparent resin films have been used for envelope window materials, but since resin films are a contraindication and prevent recycling, they have recently become recyclable paper materials such as glassine paper and coated paper impregnated with aqueous wax. Transitioning. Such a movement is similar not only in the envelope window material but also in various packaging material fields. In order to replace the conventional transparent film with a recyclable paper material, attention is focused on the transparent paper. Since the cellulose nanofiber film of the present invention is derived from 100% biomass, it can be used as a recyclable envelope window material, coated paper, high-quality paper, and various packaging materials.

  Chitin / chitosan nanofibers can also be cast to create a sheet. By applying this, it is possible to create an artificial skin and an anti-organ adhesion sheet at the time of surgery. The chitin contact lens is obtained by dissolving a chitin in an amide solvent to obtain a transparent and viscous dope, and then removing the solvent with a special coagulating liquid and drying to obtain a transparent lens. When nanofibers are used, a special solvent is not required, and a lens-like transparent molded body can be produced only by drying.

  In the dispersion fluid for high-pressure injection in the present invention, biomass may be dispersed only in water, but a small amount of acid such as phosphoric acid, citric acid, acetic acid, malic acid may be added, sodium hydroxide, potassium hydroxide, etc. A small amount of alkali such as alkali carbonate, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate may be added. In particular, regarding the decomposition of biomass, the efficiency can be increased by adding a small amount of acid / alkali.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, it cannot be overemphasized that this invention is not limited to these Examples.

Example 1
FIG. 13 shows an example of a high-pressure injection device used in the present invention.
(1) A slurry-like fluid in which biomass (cellulose, chitin / chitosan) is mixed with a liquid is pressurized to an ultra-high pressure of 100 to 245 MPa, and sprayed at a high pressure from a fine orifice nozzle (φ0.1 to 0.8 mm). Get nanofibers.
(2) The discharge flow from the orifice nozzle is a high-speed jet of 440 to 700 m / s, but a high shearing force is generated in the orifice accelerated to that speed. Since the thickness of the orifice nozzle used here is extremely thin at 0.4 mm, almost 100% of the pressure energy can be converted into the velocity energy of injection. That is, inside the orifice, a narrow gap of 0.1 to 0.8 mm and an ultrahigh speed of 440 to 700 m / s are satisfied, and the components for obtaining a high shearing force are satisfied.
[Shearing force] = [Slurry viscosity] × [Speed] / [Gap]
As for the viscosity of the slurry, by improving each part of this processing circuit, it becomes possible to process a slurry with a higher concentration, that is, a higher viscosity, which also increases the shearing force (shear stress) of the slurry itself. It was.
(3) Further, cavitation bubbles are generated in a high-speed jet flow (high pressure injection state) of 440 to 700 m / s, and a strong impact force is generated by the disappearance of the bubbles. By providing the “impact enhancement region” after the orifice nozzle, cavitation can be efficiently generated.
(4) The Reynolds number of the processing conditions of the present embodiment takes a range of 2266 ≦ Re ≦ 3885 immediately after the orifice nozzle injection.
Re = [orifice nozzle diameter] × [injection speed] × [raw material density] / [raw material viscosity]
The conditions for determining Re are
Orifice nozzle diameter: 0.5 mm
Injection speed: 440 to 700 m / s (injection pressure 100 to 245 MPa)
Raw material density: 1030-1110 kg / m ³ (concentration 10-20 wt%)
Raw material viscosity: 1000-10000 mPas (concentration 10-20 wt%)

(5) A ball-shaped or flat ceramic hard body is provided as a structural jet receiver. In order to further reduce the crystallinity, a region where the jet pressure is increased and the jet velocity is high is used, and the impact force against the hard body is also used for pulverization.

Example 2
Development of high-concentration (high-viscosity) compatible circuit Conventionally, the concentration of 2 wt% has been the limit in the conversion of cellulose into nanofibers. Nanofiberization of high concentration (high viscosity) biomass up to -20 wt% was made possible.
(1) The feed pipe for supplying biomass to the pressure intensifier (high pressure pump) has an inner diameter of 40 to 50 mm, and the raw material tank and the feed pump are integrated to generate the raw material suction. Pressure loss due to piping could be eliminated, and high concentration (high viscosity) biomass could be supplied to the pressure booster. There is the following relationship between pressure loss, pipe inner diameter, pipe length, and raw material viscosity.
[Pressure loss] ∝ [Pipe length] x [Raw material viscosity] / ([Pipe inner diameter] to the fourth power)
Normally, an air-type diaphragm pump capable of a deadline operation is used as a liquid supply pump for a pressure booster type high-pressure pump of 100 MPa or more. However, since the air pressure uses general factory air 0.5 MPa, the pushing force is It is limited to 0.5 MPa. When the deadline operation can be performed and high concentration (high viscosity) biomass is supplied to the intensifier at an indentation pressure of 0.5 MPa, the above pressure loss reducing method is effective.
(2) The check valve (check valve) existing in the middle of the supply to the pressure booster also had a large pressure loss structure in order to obtain a high-pressure seal, but the pressure loss could be reduced by simplifying the internal structure. . In general check valves, the ball-shaped valve of the sealing mechanism is pressed with a spring in order to emphasize sealing performance. For this reason, a high-concentration raw material and a fibrous material become resistance when passing through the passage in the spring, and the raw material cannot be supplied. By the way, when the raw material becomes highly viscous, it is rather difficult for leakage to occur and the sealing property tends to be improved. The relationship between leakage and viscosity is as follows.

Therefore, the spring for pressing the ball valve was eliminated, and the pressing force was determined by the pressure of the fluid itself.
In addition, in order to improve the sealing performance only with the pressure of the fluid itself, the intermediate strength between the resin and rubber, which gives more flexibility to the resin sheet used in the slurry (particle-mixed) seal with more emphasis on sealing performance. The material was changed. The sealing property improves with flexibility. Conventional resin sheets with hardness are used for highly abrasive slurries, and the material used this time is biomass, which is not highly abrasive, so even soft materials have sufficient wear resistance. It was.
(3) The high viscosity slurry of 10-20 wt% causes crosslinking in the raw material tank and forms a rat hole at the suction port under the tank. In that case, since air mixes into the pressure booster, a pressure increase failure is caused in the high-pressure pump. With propeller sales and impact knockers on the tank wall, it is impossible to flatten high-concentration (high-viscosity) biomass below the tank and feed it into the suction port. Therefore, the tank is sealed with a lid, the lid is sealed so as not to leak to the outside, and the raw material is fed into the tank while keeping the state filled with biomass. At the tank outlet, biomass is sucked by the feed pump, so that a constant amount is always maintained in the tank. By maintaining this state, high concentration (high viscosity) biomass can be processed multiple times. Inside the raw material tank, the agitator is rotated for mixing.

Example 3
Observation of bionanofiber by electron microscope
Freeze-etch replica method electron microscope observation Freeze-etch replicas were prepared by Heuser, J. et al. (Heuser, J. 1981). First, a 2% (w / v) sample dispersion (chitin dispersion or cellulose dispersion) was rapidly frozen by a metal pressure bonding method using liquid helium. Next, the frozen sample is fixed to a sample stage in a vacuum chamber of a freeze fracture apparatus (model BAF 400D, Balzers Union, Balzers, Liechtenstein), and the sample is placed at a portion immediately below the rapid freezing surface under the condition of minus 90 ° C. Then, it was cut with a knife. Furthermore, the fracture | rupture cross-section etching process was performed by leaving still at minus 90 degreeC for 10 minutes. The mixture of platinum and carbon was deposited with a film thickness of about 6 nanometers by an electron beam gun placed at an angle of about 25 ° while rotating the sample in a horizontal plane (about 50 rpm) (rotary shadow method) ). Subsequently, in order to reinforce the replica film, carbon was vapor-deposited with a film thickness of about 25 nanometers from an electron beam gun placed above (angle of about 90 °). The sample after vapor deposition was taken out from the freeze fracture apparatus, and the sample organic matter was removed by washing. When the sample was chitin, chitin was removed from the replica membrane with dimethylacetamide containing 8% (w / v) lithium chloride, and when the sample was cellulose, cellulose was removed from the replica membrane with phosphoric acid. These replica films were further washed with water and then placed on a sample grid for electron microscope (Veco, copper, 150 mesh) coated with a collodion film. For electron microscope observation, an image obtained by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM method) was used with a Tecnai G2 F20 manufactured by FEI (Hillsboro, Oregon) under an acceleration voltage of 200 kV. By applying the quick freezing replica method, the shape of bionanofiber dispersed in water can be clearly confirmed with a transmission electron microscope. Thereby, the fiber width of each nanofiber dispersed in water can be accurately known. Regarding the fiber width (minor axis) of nanofibers, the film thickness of about 20 to 40 nm randomly extracted from the photographed photo was measured in consideration of the deposited film thickness (6 nm). It turned out that it was made into nanofiber. HAADF-STEM photographs of cellulose and chitin nanofiberized by water jet treatment are shown in FIGS.
Heuser, J.M. 1981. Preparing biosamples for stereo-microscopy by the quick-freeze, deep-etch, rotary-replication technique. Methods Cell Biol. 22: 97-122.
Field emission scanning electron microscope (FE-SEM) observation Water-jet treated cellulose and chitin dispersions were replaced with t-butanol and lyophilized. A mixture of platinum and palladium was deposited on the dried sample with a film thickness of about 3 nanometers. The electron microscope was observed under the condition of an acceleration voltage of 2.0 kV using JSM-6700 of JEOL. FE-SEM photographs of cellulose and chitin nanofiberized by water jet treatment are shown in FIGS.

Example 4
Crystallinity of bionanofiber The crystallinity of bionanofiber was determined by powder X-ray diffraction. Prior to the X-ray diffraction experiment, a dry powder sample was obtained by freeze-drying the sample subjected to the water jet treatment. The X-ray diffraction experiment was performed using CuKα rays (A = 1.542) that passed through a Ni filter at an acceleration voltage of 40 kV and an acceleration current of 150 mA using a rotary anti-cathode X-ray generator Rotorflex RU-200B (Rigaku) manufactured by Rigaku Corporation. And measured with a horizontal goniometer for powder X-ray diffraction. The diffraction intensity was measured in the range of the diffraction angle 2θ from 5 ° to 35 °. The degree of crystallinity was determined by removing the background scattering from each scattering pattern, obtaining the integrated intensity of the peak derived from the crystal plane, and taking the intensity ratio relative to the untreated water jet treatment. In the case of cellulose, the crystallinity at each number of collisions with respect to the untreated was 40 to 83%, and that of chitin was 48 to 73%. Other physical pulverization methods such as ball mills and disk mills are characterized by a decrease in crystallinity as processing continues, whereas water jets increase again after a decrease in crystallinity. . That is, the degree of crystallinity can be adjusted by the number of treatments, and the bio-nanofiber of the present invention is suitable as an enzyme substrate that requires low crystallinity or a composite material that requires high crystallinity. 6 and 7 show powder X-ray diffraction patterns of cellulose and chitin nanofiberized by water jet treatment.

Example 5
Enzymatic degradation of chitin nanofiber <br/> Experimental method
-Preparation method of chitinase 1) Yatalase (manufactured by Takara Co., Ltd., Corynebacterium sp.)
The powder sample was dissolved in distilled water, put into a dialysis tube (fraction size MW3500), and dialyzed with distilled water at 4 ° C. for 24 hours. After dialysis, lyophilization was carried out to prepare a final concentration of 48 mg / ml with distilled water.

    2) Preparation of a thermostable chitinase enzyme (derived from Pyrococcus furiosus) was published in the literature (Bioscience, Biotechnology, and Biochemistry, Vol. 70, No. 7 pp. 1696-1701 (2006), Acta Crystal 8). 791-3 (2006)). The protein concentration was prepared using 20 mM Tris buffer so as to be 150 μM.

-Enzyme reaction experiment method After preparing chitin nanofibers to a substrate concentration of 0.25% and an acetate buffer solution of 20 mM (pH 5.5), it was reacted with yatalase or thermostable chitinase, and after 24 hours, Somody-Nelson The amount of reducing sugar in solubilized sugar was quantified by the method to determine the chitin degradation rate.
3 ml of the prepared 0.25% chitin was put into a test tube, stirred with a stirrer in a constant temperature bath maintained at 37 ° C., 25 μl of yatalase (4.8 mg / ml) was added, and the reaction was started (using thermostable chitinase) In this case, the reaction temperature was measured by adding 20 μl of thermostable enzyme to 3 ml of 0.25% chitin at 85 ° C.).
1, 3, 4, 5, 24 hours after putting the enzyme, 200 μl of the reaction solution was taken out into a 1.5 ml tube, centrifuged at 15,000 rpm for 3 minutes, and the supernatant was collected.
Free degradation products were quantified using the Sommoji-Nelson method. The collected reaction solution supernatant was added to a test tube containing 400 μl of Sommoji copper solution, mixed well, and boiled at 100 ° C. for 20 minutes. After cooling in ice for 5 minutes, 400 μl of Nelson's solution was added and stirred well. After standing for 15 minutes, the absorbance at 500 nm was measured.
Separately, an N-acetylglucosamine standard solution was similarly operated to prepare a calibration curve between the N-acetylglucosamine concentration and the absorbance. Using this calibration curve, the amount of reducing sugar in the sample was determined.

・ Results and discussion <br/> Effects of enzymes on chitin nanofibers Refined treatment and enzymatic reaction of untreated chitin were performed, and the solubilized sugar released by the enzyme reaction was quantified by the Sommoji-Nelson method, and the degradation rate of chitin Asked. (It is known by HPLC quantification that 100% of the product is N-acetylglucosamine when chitin is decomposed with yatalase, and 90% is decomposed to dimer in the case of thermostable enzyme.)
As shown in FIG. 8, the degradation rate of chitin nanofibers by yatalase was compared, and the effect of chitinase derived from room temperature was examined.
As a result, the chitin nanofibers were improved in decomposition rate by about 3 times compared to the untreated, and the effect of the nanofibers appeared. The reaction efficiency of the enzyme increased as the number of passes increased.

As shown in FIG. 9, the degradation rate of chitin nanofibers when treated with a thermostable enzyme was compared, and the effect of chitinase derived from thermostable was investigated.
As a result, the chitin nanofibers improved the decomposition rate by a factor of 5 compared to the untreated, and the reaction efficiency of the enzyme increased as the number of passes increased.

Example 6
Enzymatic degradation of cellulose nanofibers
Enzyme preparation method 1) Trichoderma cellulase enzyme (manufactured by Sigma, Cellulase mixture; derived from Trichoderma sp.)
Novozyme cellulase enzyme (manufactured by Novozyme, BGL; derived from Aspergillus sp)
2) Preparation of thermostable cellulase enzyme (AIST, EG; derived from Pyrococcus horikoshii) (AIST, BGL; derived from Pyrococcus furiosus) was in accordance with the literature (9: 37-43 Extrememoles (2005)). The protein concentration was adjusted with ultrapure water so that EG was 20 μM and BGl was 10 μM.

Enzyme reaction experiment method After preparing crystalline cellulose to a substrate concentration of 1% and acetate buffer 20 mM (pH 5.5), it is reacted with Trichoderma novozyme cellulase enzyme or thermostable cellulase enzyme, 1, 2, 3, 4, After 24 hours, the solubilized glucose was quantified and the decomposition rate of crystalline cellulose was determined.

  For the measurement, untreated crystalline cellulose and cellulose nanofiber (number of collisions 10 times) were used. 3 ml of the prepared 1% crystalline cellulose was put into a test tube, stirred with a stirrer in a thermostat kept at 50 ° C., and 7.5 μl of Trichoderma novozyme cellulase enzyme (1: 1) was added to start the reaction (heat resistance When the enzyme was used, the reaction temperature was 85 ° C., measured by adding 45 μl of thermostable enzyme (EG: BGL = 20 μM: 10 μM = 2: 1) to 3 ml of 1% crystalline cellulose). 1, 2, 3, 4, 24 hours after putting the enzyme, 200 μl of the reaction solution was taken out into a 1.5 ml tube, centrifuged at 15,000 rpm for 3 minutes, and the supernatant was collected.

Glucose was measured based on the CII-Test Wako manual.
8 μl of the collected supernatant and 1.2 ml of glucose coloring solution were placed in a 1.5 ml tube and mixed well. Next, it heated with the block heater for 5 minutes at 37 degreeC. Absorbance at 505 nm was measured. Separately, a glucose standard solution was similarly operated to prepare a calibration curve between the glucose concentration and the absorbance. Using this calibration curve, the glucose concentration in the sample was determined.

·Results and discussion
Enzyme effect on cellulose nanofibers Enzymatic reaction of untreated, refined, and phosphoric acid-treated crystalline cellulose is performed, and the solubilized sugar released by enzymatic hydrolysis reaction is quantified with glucose CII-Test Wako to decompose crystalline cellulose. The rate was determined.

  As shown in FIG. 10, the degradation rates by Trichoderma novozyme cellulase enzyme were compared. As a result, it is understood that the cellulose nanofibers have a degradation rate of about twice as much as that of untreated during the reaction for 24 hours, and there is an enzyme degradation effect by the nanofiber formation.

  As shown in FIG. 11, when treated with a thermostable enzyme, a degradation rate of about 5 times that of untreated was obtained.

  As a result, it was found that a sufficient effect of decomposing crystalline cellulose was obtained with 10 collisions using a single injection chamber, and the effect of nanofiber formation could be confirmed at room temperature and high temperature. It can be concluded that there are effects of enzymatic degradation by nanofibrosis after 10 collisions.

  Although nanofibrous chitin and cellulose retain a crystallinity of 40% or more with respect to untreated fibers, the increase in specific surface area due to the nanofibrosis increases the accessibility of the enzyme that hydrolyzes them and It is thought that the decomposition reaction is promoted.

Example 7
Film formation of bionanofibers refined using a single injection chamber A bionanofiber film can be obtained by pouring (drying) the bionanofibers refined using a single injection chamber into a petri dish and drying. There is no restriction | limiting in particular in the method of film-forming, A film can be produced by making it dry after operations, such as a film applicator generally used for film production, and suction filtration. Drying can be performed by heating or room temperature drying. The transparency of the film can be adjusted according to the degree of micronization (nanofiber) of the biomass by the single injection chamber. According to the physical principle that an object having a size of 1/10 or less of visible light wavelength (400 to 800 nm) does not cause light scattering, a bio-nanofiber film miniaturized with a uniform width of 20 nm is very highly transparent. Showing gender. FIG. 12 shows a chitin film produced by the above method. Similar films can be produced for cellulose and chitosan refined using a single injection chamber.

  According to the present invention, cellulose and chitin / chitosan can be made into nanofibers by a single injection chamber for miniaturization using a water jet as a core technology, so that these biomass can be effectively used widely. This device does not use any harmful chemicals such as acids and alkalis, and is harmonized with the environment using only water.

  Examples of the synthetic polymer resin when the cellulose nanofiber of the present invention is used as a filler include vinyl resins such as polyolefin, polycondensation resins such as polyamide, and the like. A new functional transparent film / resin can be synthesized by compositing with a transparent substrate (resin) such as epoxy having a refractive index equal to that of cellulose. In particular, the cellulose nanofibers of the present invention can change strength or surface characteristics by being combined with a polymer such as a phenol resin capable of forming a hydrogen bond, polyethylene glycol, polyethylene terephthalate, or polyvinyl alcohol.

  Furthermore, the cellulose nanofibers of the present invention can be improved in properties such as strength and heat resistance of these resins by being combined with biodegradable resins such as polylactic acid, polybutylene succinate and polycaprolactone.

  By surface modification (chemical modification) of cellulose nanofibers, resins with properties other than those listed above, for example cellulose nanofibers, are acetylated to give hydrophobic properties, so that they can be combined with hydrophobic resins such as acrylic resins. Is also possible.

A cosmetic (sunscreen agent) and a liquid crystal substrate that make use of optical properties obtained by making cellulose nanofibers, and a filter that makes use of nano-order voids formed between cellulose fibers can be produced. In particular, by substituting the hydroxyl group of cellulose with various functional groups, it is possible to produce separation / filter materials having different characteristics and functions. Moreover, the size of the gap can be changed by controlling the diameter of the nanofiber.
The compounding with the various resins described above can be applied not only to cellulose nanofibers but also to chitin / chitosan nanofibers.
Chitosan is also a natural polymer material that can be variously modified because it has highly reactive amino groups and hydroxyl groups in the molecule. Chitosan is known to adsorb many substances such as proteins and metals. The chitosan nanofiber of the present invention is suitable as an adsorbent because of its increased specific surface area, and the film formed by drying the chitosan nanofiber is a porous film having voids controlled at the nano level, such as enzymes. It can be used as a carrier for immobilizing a biocatalyst, a carrier for chromatography such as separation and purification, or a cell culture substrate.
Chitosan is known to produce excellent acoustic properties when embedded in woody materials or paper as powder particles or mixed in adhesives. When the chitosan nanofiber of the present invention is applied to the surface of an acoustic diaphragm such as a speaker, a uniform and hard film is formed, and the same effect can be obtained. As a feature related to the vibration characteristics of the acoustic plate, in order to obtain excellent acoustic characteristics, a rigid, high-strength material having a high strength and a high elastic modulus that vibrates as a whole is required. Since the cellulose nanofiber of the present invention can be miniaturized while maintaining high crystallinity, it can be used as a material for an acoustic diaphragm capable of exhibiting a high elastic modulus in the same manner as chitosan nanofiber.
Since chitosan exhibits antibacterial and deodorant properties, the antibacterial and deodorant effects can be imparted to these products by blending the chitosan nanofibers of the present invention into fibers such as clothing, daily necessities, and interiors.

  In addition, by combining the nanofibers of chitosan, which is a cationic polymer, and cellulose, which is an anionic polymer, it is expected to reinforce electrostatic attraction by the charge of anions and cations, making it possible to create stronger nanofibers. is there. The most important factor involved in the strength of paper is the bond strength between fibers, which is determined by hydrogen bonding between the hydroxyl groups of cellulose. In order to increase the strength of the paper, it is only necessary to increase the hydrogen bonds. In addition to the above properties, chitosan has a chemical structure similar to cellulose and is linear, so it has a high affinity with cellulose and is used as a paper strength enhancer and an ink-jet ink receptor. -It must be solubilized by chemical modification such as hydroxylation. Since the chitosan nanofiber of the present invention is already uniformly dispersed in water, it does not need to be chemically modified, and an ink jet printing paper or the like can be enhanced by simply applying or blending.

  Furthermore, since chitosan is a cationic polymer, it has the power to retain pharmaceuticals and is known to exhibit sustained release as a pharmaceutical carrier. When the medicine taken is absorbed into the body, it is sent to the liver to be detoxified, and the rest goes into the blood, travels around the body, reaches the affected area, and becomes effective. Therefore, the medicine to be taken is administered in anticipation of the amount detoxified in the liver. When the chitosan nanofiber of the present invention is used as a pharmaceutical carrier, sustained drug efficacy can be increased by gradually releasing the drug, and side effects can be reduced by showing effects in a small amount.

  Contact lenses using chitin as a raw material have actually been commercialized. In this case, as with the manufacture of sutures, the gelled chitin dissolved in an amide solvent is removed with a special coagulation solution and dried. And getting a transparent lens. Chitin nanofiberized with a water jet can be easily formed into a lens shape simply by giving it a thickness and drying, so it can be used not only as a contact lens but also as an optical / transparent material derived from biomass.

  The cellulose and chitin / chitosan nanofiber of the present invention can be carbonized by high-temperature treatment in the absence of oxygen (in an inert gas atmosphere such as nitrogen or argon). That is, a biomass-derived nanocarbon fiber can be produced. Porous carbon containing a large amount of small pores at the nano level is used as a deodorizing agent, decoloring agent, or filter for water purification, and the bio-nano carbon fiber produced by carbonizing the bio-nanofiber of the present invention is also such a field. Can be used. Porous carbon not only captures odors and dirt molecules in its small pores, but it can also capture ions (charged atoms and molecules) with the help of electricity. Since the captured charge can be taken out, a large-capacity capacitor (capacitor) has been developed using this. Since such a capacitor can be used as an auxiliary power source for a fuel cell vehicle and a storage for storing surplus power at night, it has attracted much attention in recent years. Since the capacitance of the electric double layer capacitor is determined by the amount of charge stored in the electric double layer, the larger the surface area of the electrode, the larger the capacitance can be obtained, so activated carbon having high conductivity and specific surface area. Is used as an electrode material. The bio-nanocarbon fiber produced by carbonizing the bio-nanofiber of the present invention is a mesoporous activated carbon having a high specific surface area and controlled pores at the nano level. It can be used as an electrode material to enhance.

Claims (6)

A method for producing a bio-nanofiber, characterized in that a biomass dispersion fluid is injected at a high pressure of 100 to 245 MPa so as to collide with a hard body for collision. 2. The method for producing bio-nanofiber according to claim 1, wherein a single injection chamber in which the number of lay nozzles is limited to 2266 to 385 and the impact effect on a hard body is optimized is used. Combining the single injection chamber with a miniaturization device equipped with a high-concentration (10 to 20% by weight) biomass dispersion fluid increases the shear effect during injection and reduces the nanofiber fiber width (minor axis). A method for producing a bio-nanofiber, characterized in that it is refined to 10 to 100 nm, preferably 10 to 40 nm. A bionanofiber having a crystallinity of 40% or more obtained by the production method according to claim 1. The film which consists of a composite material or bio-nanofiber containing the bio-nanofiber of Claim 4. The bio-nanofiber saccharification method of efficiently hydrolyzing the bio-nanofiber according to claim 4 using an enzyme.

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