Network structure and method for producing the same, and β-1,3 glucan beads and method for producing the same

Abstract

Provided are: a novel network structure body formed from sub-micron fibrils containing β-1,3-glucan; and a method for producing same. Provided are: β-1,3-glucan beads; and a structure thereof.　This network structure body includes a network structure in which fibrils, which contain β-1,3-glucan and have an average fiber diameter of more than 100 nm but less than 1000 nm, are connected to each other and extend in a linear manner to demarcate a network. The fibrils preferably extend continuously connected in a linear or curved manner to demarcate a network.

Description

[Technical Field]

[0001] The present invention relates to a network structure and a method for producing the same, as well as β-1,3 glucan beads and a method for producing the same. [Background technology]

[0002] In recent years, fibers made from β-1,3-glucans, such as paramylon, have been attracting attention as a naturally occurring fiber with a low environmental impact. Paramylon is a storage polysaccharide produced by Euglena. It is composed of approximately 2,000 glucose molecules linked by β-1,3 bonds, and exists within Euglena cells as particles several micrometers in diameter. Patent Document 1 proposes fibrous paramylon obtained by fiberizing paramylon granules using shear force. Non-Patent Document 1 describes a technique (dilution method) in which β-1,3-glucan is converted into a single-stranded state (random coil state) in an aqueous sodium hydroxide solution, and then the concentration of sodium hydroxide is reduced with water to establish a more stable triple helix structure, resulting in nanofibers.

[0003] [Patent Document 1] International Publication No. 2018 / 116936

[0004] [Non-Patent Document 1] Carbohydrate Polymers, 2013, Volume 93, Pages 499-505 Summary of the Invention

[0005] The fibrous paramylon in Patent Document 1 is said to be in a state where multiple fibrous materials are entangled and gathered together. It is also said to have a mesh-like structure where multiple fibrous materials are aggregated into a network. The nanofibers in Non-Patent Document 1 are aggregates based on the self-organizing ability inherent to paramylon. Random coil-shaped paramylon dispersed in a solution gathers together to form nanofibers, but the fiber diameter is limited to a few tens of nanometers, making it difficult to form submicron fibrils with a fiber diameter exceeding 100 nm. Furthermore, no branched structure is observed in each nanofiber, and each nanofiber does not share part of its structure with other nanofibers, so no mesh structure is formed.

[0006] An object of the present invention is to provide a novel network structure formed of submicron fibrils containing β-1,3 glucan and a method for producing the same. Another object of the present invention is to provide β-1,3 glucan beads and a method for producing the same.

[0007] The present invention has the following aspects. [1] A network structure comprising a network structure in which fibrils containing β-1,3-glucan and having an average fiber diameter of more than 100 nm and less than 1000 nm extend linearly while connecting with each other to define the network. [2] The network structure according to [1], wherein the fibrils are connected in a continuous manner and extend in straight or curved lines to define the mesh. [3] The network structure according to [1] or [2], wherein the fibrils extend linearly while branching to define the network. [4] The network structure according to [3], wherein the fibrils are separated from each other between the branches. [5] The network structure according to any one of [1] to [4], wherein the network structure is formed by connecting two or more of the networks. [6] The network structure according to any one of [1] to [5], wherein the network structure is formed by three or more of the networks being in contact with each other. [7] A network structure according to any one of [1] to [6], wherein the number of ends of the fibrils in the network structure is 10 or less in one field of view observed at 2000x magnification using a scanning electron microscope. "8] A method for producing a network structure according to any one of [1] to [7], A manufacturing method comprising: adding dropwise a β-1,3-glucan solution containing raw material β-1,3-glucan and a good solvent to a poor solvent to obtain β-1,3-glucan beads. [9] The manufacturing method described in [8], further comprising defibrating the β-1,3-glucan beads.

[10] The production method according to [8] or [9], wherein the good solvent comprises one or more solvents selected from aprotic solvents and ionic liquids.

[11] The method according to any one of [8] to

[10] , wherein the antisolvent comprises an alcohol.

[12] A manufacturing method according to any one of [8] to

[11] , wherein the defibration treatment includes shearing in a poor solvent.

[13] β-1,3-glucan beads comprising a mesh structure in which fibrils containing β-1,3-glucan and having an average fiber diameter of more than 100 nm and less than 1000 nm are connected to each other and extend linearly to define the mesh.

[14] A method for producing β-1,3-glucan beads, comprising dropping a β-1,3-glucan solution containing raw material β-1,3-glucan and a good solvent into a poor solvent.

[0008] According to the present invention, a novel network structure formed of submicron fibrils containing β-1,3 glucan and a method for producing the same can be provided. According to the present invention, β-1,3 glucan beads and a method for producing the same can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] This is a schematic diagram showing how a network structure of submicron fibrils is formed starting from paramylon particles. [Figure 2]Photographs showing an example of β-1,3-glucan beads: (a) is a photograph taken with a home digital camera, and (b) is a scanning electron microscope photograph at 50x magnification. [Figure 3] Scanning electron micrographs of the cross section of β-1,3-glucan beads: (a) 50x magnification, (b) 2000x magnification, (c) 3000x magnification, (d) 5000x magnification, and (e) 10000x magnification. [Figure 4] 4 is a scanning electron microscope photograph at 2000 magnification of a cross section of a region of β-1,3-glucan beads located closer to the center than the region in FIG. 3. [Figure 5] These are scanning electron micrographs of an example of a network structure obtained after defibrating β-1,3-glucan beads: (a) 100x magnification, (b) 1000x magnification, and (c) 2000x magnification. [Figure 6] 5 shows 500x scanning electron microscope photographs of the network structure obtained in Example 1. (a) is a photograph of the network structure obtained by immersing paramylon beads in ethanol for 1 hour and then defibrating them for 20 minutes, and (b) is a photograph of the network structure obtained by immersing paramylon beads in ethanol for 3 hours and then defibrating them for 10 minutes. [Figure 7] 1 is a scanning electron microscope photograph of the network structure obtained in Example 2 at 500 magnifications. [Figure 8] 1 is a scanning electron microscope photograph of the network structure obtained in Example 3 at 500 magnifications. [Figure 9] 1 is a scanning electron microscope photograph of the network structure obtained in Example 4 at 500 magnifications. [Figure 10] 1 is a scanning electron microscope photograph of the sample obtained in Comparative Example 1 at 2000 magnifications. [Figure 11] 1 is a scanning electron microscope photograph of the sample obtained in Comparative Example 2 at 500x magnification. [Figure 12] 1 is a scanning electron microscope photograph of the sample obtained in Comparative Example 3 at 500x magnification. [Figure 13]1 is a graph showing the frequency dependence of the storage modulus and loss modulus of a polylactic acid-paramylon submicron fibril network composite. DETAILED DESCRIPTION OF THE INVENTION

[0010] An embodiment of the present invention will be described in detail below. The present invention is not limited to the following embodiment, and can be implemented by making appropriate modifications within the scope that does not impair the effects of the present invention. When a specific description given for one embodiment also applies to other embodiments, that description may be omitted in the other embodiments.

[0011] [Net structure] The network structure according to this embodiment includes a mesh structure (hereinafter also referred to as a "network") in which fibrils containing β-1,3-glucan and having an average fiber diameter of more than 100 nm and less than 1000 nm (herein also referred to as "submicron fibrils" or simply "fibrils") are connected to each other and extend linearly to define the mesh. No network structure with such characteristics has been known to date. The term "network structure" refers to an article having, at least on the surface and / or part of the interior thereof, a mesh structure that can be observed with a scanning electron microscope at a magnification of at least 100 to 10,000 times. A "mesh structure" is made up of two or more meshes. The "mesh" is composed of fibrils and pores surrounded by the fibrils when observed under a scanning electron microscope at a magnification of at least 100 to 10,000 times. In a scanning electron microscope photograph, the fibrils are observed as linear regions, and the pores are observed as dark shadows. The shape of the pores in the mesh is not limited and may include a circular shape, an elongated circular shape, a cocoon shape, a polygonal shape with rounded corners, and the like. The phrase "extending linearly while connected" means that the fibrils share parts of each other and extend linearly. When fibrils are merely physically connected by gathering or entangling, the fibrils are not connected to each other. By "compartmentalized" we mean that the fibrils separate regions: adjacent networks share, at least in part, one or more fibrils.

[0012] The network structure in which fibrils are connected to each other and extend linearly to define the mesh is a strong network that is resistant to collapse even when dispersed in water or a resin, and can therefore be a network structure with excellent strength. Furthermore, since the fibrils have already formed a network, they do not further aggregate and are less likely to aggregate when dispersed in water or a resin. As a result, a network structure with excellent dispersibility in water or a resin can be obtained.

[0013] In contrast, paramylon particles are known to be composed of independent fibrous structures with a fiber diameter of approximately 4 nm arranged in parallel (e.g., Amer. J. Bot., 74(6), 877 (1987)), and the fibrous structures do not form a dense network within the paramylon particles. Therefore, fibrous paramylon produced by the conventional top-down method, in which the fibrous structures are simply physically entangled, is constantly changing when dispersed in an aqueous solution, for example, and has low mechanical stability against external forces.

[0014] (Fibril) The fibrils contain β-1,3-glucan and have an average fiber diameter of more than 100 nm and less than 1000 nm. Fibrils with an average fiber diameter of more than 100 nm and less than 1000 nm are also called submicron fibrils. The fibrils are preferably made by fibrous β-1,3-glucan.

[0015] β-1,3-glucan refers to a polysaccharide having a structure in which glucose units are linked by β-1,3 bonds. That is, β-1,3-glucan has a structure in which the 1-position of one glucose unit and the 3-position of another glucose unit form a β-1,3-glucoside bond. The term "β-1,3-glucan" includes β-1,3-glucan and its derivatives. In one embodiment, the derivative may be one in which the hydrogen atoms of one or more hydroxyl groups of β-1,3-glucan are substituted with other groups. β-1,3-glucan is mainly produced by algae, fungi, etc.

[0016] In one embodiment, the β-1,3-glucan has the following formula (1): It is preferred that the structure be as shown in TIFF0007789401000001.tif26170.

[0017] In chemical formula (1), n ​​represents an integer of 60 to 3,000. n is preferably 500 to 2,800, more preferably 700 to 2,500, even more preferably 800 to 2,200, and particularly preferably 1,000 to 2,000. Paramylon synthesized and accumulated by Euglena is typically a β-1,3-glucan in which 1,500 to 2,000 glucose molecules are linked by β-1,3 bonds. In one embodiment, the β-1,3-glucan preferably contains paramylon. In one embodiment, the β-1,3-glucan is paramylon. In one embodiment, the β-1,3-glucan may contain paramylon and a β-1,3-glucan other than paramylon.

[0018] The average fiber diameter of the fibrils is more than 100 nm and less than 1000 nm, preferably 200 to 990 nm, more preferably 300 to 950 nm, even more preferably 400 to 950 nm, still more preferably 430 to 900 nm, and particularly preferably 430 to 700 nm. The average fiber diameter is determined by measuring the fiber width at three points for any 20 fibrils in a 2000x field of view using a scanning electron microscope, and averaging the results. In this specification, a fibril extending between one branch and another branch in a network structure is counted as one fibril.

[0019] Nanofibers produced by a conventional bottom-up method such as that described in Non-Patent Document 1 are formed by three molecules of randomly coiled β-1,3-glucan forming a triple helix structure in water, which then self-assemble into bundles, with a thickness of approximately several to several tens of nanometers. In the network structure according to this embodiment, the nanofibers further aggregate to form submicron fibrils that are thicker than conventional nanofibers, and these fibrils are further connected to each other so as to extend in the length direction, thereby forming a network structure.

[0020] The fibrous paramylon produced by the conventional top-down method described in Patent Document 1 is obtained by mechanically shearing paramylon particles with a diameter of several micrometers, and the length of each fibrous object does not exceed that of the natural fibrous object that constitutes the paramylon particle. The inventors observed single-stranded nanofibers obtained by immersing paramylon particles in a solvent (solvent name: dimethyl sulfoxide (DMSO)) and taking scanning probe micrographs. The nanofibers were approximately 3 μm long and 40 nm wide. Furthermore, because the fibrous paramylon produced by the top-down method is fibrous due to shear force, it does not undergo the process of individual fibrous objects sharing parts with each other, as occurs with the bottom-up method. In other words, the fibrous paramylon produced by the top-down method cannot form a network structure by connecting fibrils to each other, extending linearly. Fibrous paramylon produced using conventional top-down methods such as those described in Patent Document 1 is simply a collection of multiple fibrous materials that have become entangled with each other, so it has characteristics such as the fibrous materials not being connected to each other, the presence of many gaps (at the ends of the fibrous materials) in the mesh structure when observed under a microscope, and few gaps between each fibrous material.

[0021] In one embodiment, the fibrils preferably extend in a straight or curved line while being connected in a continuous manner to define a mesh. In one embodiment, the fibrils preferably form a continuous mesh structure. "Formed in a continuous mesh structure" means that the fibrils are arranged continuously to form one mesh structure.

[0022] In one embodiment, it is preferred that no boundaries between individual fibrils are observed when viewed under a scanning electron microscope at 500x magnification (preferably 2000x magnification).

[0023] In one embodiment, the fibrils preferably extend linearly while branching to define a network. In this case, the network structure has two or more branches. By extending linearly while branching, it is easy to define two or more adjacent networks. In this embodiment, fibrils are preferably separated from other fibrils between branches. When fibrils are separated from other fibrils between branches, the fibrils are not entangled with each other. The linear distance between adjacent branches is not limited, but is preferably 0.5 to 100 μm, more preferably 0.5 to 100 μm, more preferably 0.5 to 50 μm, and even more preferably 2 to 20 μm.

[0024] In one embodiment, the fibrils are preferably not entangled with one another. In one embodiment, the fibrils are preferably observed as a single fibrous object between the branches when observed under a scanning electron microscope at 500x magnification (more preferably 2000x magnification). When observed as a single fibrous object, the fibrils are not entangled with one another. In one embodiment, fibrils may extend with a space between them in the width direction. When a space is formed between them in the width direction, the fibrils are not entangled. The space may be a circular region, an irregular region, or a rectangular region. In one embodiment, the fibrils are preferably connected seamlessly to form a network structure.

[0025] (mesh structure) The mesh structure is formed by two or more meshes separated by fibrils. The mesh structure is preferably a state in which a large number of meshes separated by fibrils are gathered together. When a large number of meshes are gathered together, a mesh structure having a stronger network can be obtained. Examples of a mesh structure in which a large number of meshes separated by fibrils are gathered together include the following: That is, in one embodiment, the network structure is preferably formed by two or more connected networks partitioned by fibrils. "Two or more connected networks" means that two or more adjacent networks are continuously arranged to form one network structure. In one embodiment, the network structure may be formed by two or more networks of different sizes arranged adjacent to each other. In one embodiment, the mesh structure preferably has three or more meshes in contact with each other. In one embodiment, the mesh structure preferably has a densely packed structure, where "densely packed" means that there are many meshes (pores) packed together. In one embodiment, the meshes are preferably arranged to extend in the planar direction and one or more directions intersecting the planar direction at a certain angle. A mesh structure formed by meshes arranged to extend in the planar direction and one or more directions intersecting the planar direction at a certain angle can be said to be a mesh structure in which a large number of meshes are gathered in all directions.

[0026] Fiberized paramylon produced by conventional top-down methods such as those described in Patent Document 1 is in a state where multiple fibrous materials are entangled with each other and gathered together, and does not have a mesh structure with two or more adjacent meshes. Nanofibers produced by conventional bottom-up methods such as those described in Non-Patent Document 1 are composed of individual nanofibers that are aggregated or entangled in bundles, but do not have a network structure with two or more adjacent networks.

[0027] In one embodiment, the ratio of the mesh pores in the mesh structure is preferably 90% or more, more preferably 95% or more, as a percentage of the area obtained by binarizing an image in a 64 micrometer × 48 micrometer field of view under a scanning electron microscope using image processing software. On the other hand, in a network structure that is aggregated into a spherical shape, such as β-1,3-glucan beads described below, the pores may be crushed in the region near the surface. In one embodiment, the proportion of the pores in the network structure may be 50% or less as the proportion of the area obtained by binarizing an image using image processing software in a 64 micrometer × 48 micrometer field of view under a scanning electron microscope.

[0028] In one embodiment, the size of the mesh pores, as the maximum distance of the inner diameter, is preferably 0.5 to 100 μm, more preferably 0.5 to 50 μm, and even more preferably 0.5 to 20 μm. In one embodiment, it is even more preferable that the proportion of small pores of 1 μm or less, more preferably about 0.5 μm to 1 μm, is high. By having a mesh structure with many small pores, it is possible to obtain a mesh structure with a stronger network (mesh structure).

[0029] In one embodiment, the number of fibril ends in the mesh structure, in one field of view observed at 2000x magnification with a scanning electron microscope, is preferably 10 or less, more preferably 8 or less, even more preferably 5 or less, and particularly preferably 0. When the number of fibril ends is 10 or less, the number of breaks between fibrils is reduced, and a mesh structure having a stronger network (mesh structure) can be obtained.

[0030] (network structure) A network structure is an article having, at least on the surface and / or part of the interior thereof, a mesh structure that can be observed with a scanning electron microscope at a magnification of at least 100 to 10,000 times. The network structure includes one or more of the above-described mesh structures. In addition to the above-described mesh structures, the network structure may have regions without meshes, regions where fibrils are densely packed with no gaps, regions where fibrils are entangled with each other, etc. The size of the network structure is not limited and can be determined according to the intended use. In one embodiment, the network structure may take various shapes, such as beads, powder, sheet (film), or a mixture thereof. For example, the network structure may contain powder, spherical particles (beads) and / or broken pieces thereof, with an average particle size of 1 to 3 mm or 0.5 to 5 mm. The average particle size is determined by measuring the diameters (maximum linear distance) of any 10 network structures in a 2000x field of view using a scanning electron microscope, and averaging the measured diameters. In one embodiment, the network structure may be, but is not limited to, a film-like shape measuring 100 to 1000 mm x 100 to 1000 mm.

[0031] (Application) The network structure according to this embodiment is resistant to aggregation and has excellent dispersibility in water or resin, and therefore can be preferably used, for example, as a filler (reinforcing material) for resin. In the network structure according to this embodiment, the fibrils are connected to each other, and the fibrils are bonded to each other by sharing parts of their cores, forming a high-density, highly cross-linked network with high mechanical strength. Because of its excellent mechanical strength, it can be preferably used as a functional separation membrane or other membrane material. The network structure according to this embodiment is a submicron fibril network with a 100% natural component ratio, and therefore can be used as a biodegradable filler. Furthermore, since it can maintain a strong network even in water, it is also water-resistant. Therefore, it can be used as a water-resistant network structure.

[0032] [Method for manufacturing network structure] In the method for manufacturing a network structure according to this embodiment, natural β-1,3-glucan nanofibers that constitute raw material β-1,3-glucan such as paramylon particles are not used as a constituent component of the fiber as they are, but are first dissolved in a good solvent to produce a β-1,3-glucan solution with a random coil conformation, which is then dropped into a poor solvent to prepare beads in which a high concentration of random coil-shaped β-1,3-glucan is contained in a spherical space, and a network structure is formed within the beads.

[0033] That is, the method for producing a network structure according to this embodiment is as follows: (Step 1) A β-1,3-glucan solution (hereinafter simply referred to as "β-1,3-glucan solution") containing raw material β-1,3-glucan and one or more solvents selected from aprotic solvents and ionic liquids is dropped into a poor solvent to obtain β-1,3-glucan beads. Includes:

[0034] (melting process) The method for producing a network structure according to this embodiment may include, as necessary, a step of preparing a β-1,3-glucan solution prior to step 1. The β-1,3-glucan solution can be obtained by dissolving the raw material β-1,3-glucan in a good solvent. The "good solvent" refers to a solvent that can dissolve the raw material β-1,3-glucan. Examples of good solvents include aprotic solvents, ionic liquids, deep eutectic solvents, etc. The good solvent preferably contains one or more selected from these, more preferably contains one or more solvents selected from aprotic solvents and ionic liquids, and even more preferably contains an aprotic solvent. Aprotic solvents, ionic liquids, and deep eutectic solvents can dissolve and defibrate the raw material β-1,3-glucan into random-coil molecular chains, and then dropwise addition to a poor solvent can form a strong network of submicron fibrils. In contrast, when an alkaline solution such as an aqueous sodium hydroxide solution is used, the raw material β-1,3-glucan can be dissolved, but as shown in the comparative example described below, a strong network of submicron fibrils cannot be formed even when the raw material β-1,3-glucan is then dropwise addition to a poor solvent.

[0035] In one embodiment, the source β-1,3-glucan has the following formula (1): It is preferable that the structure be as shown in TIFF0007789401000002.tif26170. In chemical formula (1), n ​​represents an integer of 60 to 3000. n is preferably 500 to 2800, more preferably 700 to 2500, still more preferably 800 to 2200, and particularly preferably 1000 to 2000.

[0036] From the viewpoint of reducing the environmental load, the raw material β-1,3-glucan is preferably derived from a living organism, and more preferably from a plant. Among these, it is preferable to use β-1,3-glucan isolated from microalgae that synthesize β-1,3-glucan intracellularly as the raw material. Euglena (a microalgae belonging to the Euglenophyta phylum) is preferred as the microalgae. Euglena is easy to culture, has a rapid growth cycle, and accumulates large amounts of paramylon particles intracellularly. The paramylon synthesized and accumulated by Euglena is typically a β-1,3-glucan composed of 1,500 to 2,000 glucose units linked together in a β-1,3 bond. β-1,3-glucans such as paramylon can be isolated from microalgae by conventional methods. In one embodiment, the β-1,3-glucan preferably contains paramylon. In one embodiment, the β-1,3-glucan is paramylon. In one embodiment, the β-1,3-glucan may contain paramylon and a β-1,3-glucan other than paramylon.

[0037] Examples of aprotic solvents include acetone, N,N-dimethylacetamide, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). Examples of ionic liquids include known ionic liquids that are a combination of a positively charged compound and a negatively charged compound, such as 1-allyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium acetate, and N,N-diethyl-N-(2-methoxyethyl)-N-methylammonium 2-methoxyacetate. An example of a deep eutectic solvent is choline chloride-zinc chloride. In one embodiment, the good solvent preferably includes dimethyl sulfoxide (DMSO), and a mixed solvent of dimethyl sulfoxide (DMSO) and other aprotic solvents can also be used, such as a mixed solvent of dimethyl sulfoxide (DMSO) and methanol, or a mixed solvent of dimethyl sulfoxide (DMSO) and water. In one embodiment, when the good solvent contains N,N-dimethylacetamide, it is preferable to use a mixed solvent of N,N-dimethylacetamide and lithium chloride (inorganic salt).

[0038] The method for dissolving the raw material β-1,3-glucan in a good solvent is not limited, and for example, the raw material β-1,3-glucan can be dissolved by placing it in a good solvent and stirring it, if necessary using a magnetic stirrer or the like, at 10 to 60°C, preferably 20 to 30°C, for 1 to 72 hours, preferably 10 to 24 hours. The concentration of the raw material β-1,3-glucan in the β-1,3-glucan solution is preferably 1 to 12% by weight, more preferably 4 to 10% by weight.

[0039] (Step 1: β-1,3-glucan bead production step) In step 1, a β-1,3-glucan solution containing β-1,3-glucan and one or more selected from an aprotic solvent and an ionic liquid is added dropwise to a poor solvent to obtain β-1,3-glucan beads. When the β-1,3-glucan solution is added dropwise to the poor solvent, spherical particles containing β-1,3-glucan (also referred to as "β-1,3-glucan beads" in this specification) are rapidly formed.

[0040] The term "poor solvent" as used herein refers to a solvent that does not dissolve β-1,3-glucan. Examples of poor solvents include water; alcohols such as methanol, ethanol, n-propanol, and 2-propanol; halogenated solvents such as chloroform, dichloromethane, carbon tetrachloride, and 1,2-dichloroethane; ethyl acetate, acetonitrile, tetrahydrofuran, pyridine, acetone, DMF, petroleum ether, and toluene. In one embodiment, the poor solvent preferably contains an alcohol, and more preferably contains ethanol. In one embodiment, the poor solvent can be a mixed solvent of water and an alcohol. In one embodiment, the poor solvent can be a mixed solvent of different types of alcohols (for example, a mixed solvent of ethanol and methanol).

[0041] The step of producing β-1,3-glucan beads is preferably carried out at 4 to 60°C, more preferably at 10 to 40°C, and even more preferably at 20 to 30°C, while stirring the poor solvent. The total amount of β-1,3-glucan solution to be dropped is not limited, and can be, for example, 0.01 to 10 g per 50 mL of poor solvent. The size of the droplets is not limited, and can be determined by the size of the opening of the pipette, nozzle, or other device used, depending on the desired size of the resulting β-1,3-glucan beads.

[0042] In one embodiment, the method preferably includes immersing the β-1,3-glucan beads in a poor solvent. For example, after adding a β-1,3-glucan solution dropwise to the poor solvent, the resulting β-1,3-glucan beads are preferably left immersed in the poor solvent, preferably while stirring the poor solvent. Immersing the β-1,3-glucan beads in the poor solvent allows the poor solvent to easily penetrate deeper into the β-1,3-glucan beads, forming a stronger network.

[0043] The time for immersing the β-1,3-glucan beads in the poor solvent is preferably 0.1 to 72 hours, more preferably 0.5 to 10 hours, and even more preferably 1 to 3 hours.

[0044] Figure 1 is a schematic diagram showing the formation of a network of submicron fibrils when paramylon particles are used as the raw β-1,3-glucan. As shown in Figure 1, random-coiled paramylon is formed in the β-1,3-glucan solution. By dropping this β-1,3-glucan solution into a poor solvent, spherical particles (beads) are formed. The beads contain a high concentration of random-coiled β-1,3-glucan. The good solvent in the beads is gradually replaced by the poor solvent, and the encapsulated random-coiled β-1,3-glucan gradually comes into contact with the poor solvent. This allows the β-1,3-glucan to self-organize and become fibers. The beads contain a high concentration of randomly coiled β-1,3-glucan, which results in the formation of submicron fibrils that are thicker than the conventional nanofibers with a diameter of 20 nanometers described in Non-Patent Document 1. Furthermore, multiple submicron fibrils share structural components and form a series of spherical networks (mesh structures) with few breaks, high density, and highly cross-linked.

[0045] The size of the β-1,3-glucan beads formed in step 1 can be adjusted to various sizes depending on the size of the droplets when a solution of β-1,3-glucan dissolved in a good solvent is dropped into a poor solvent. For example, the size of the β-1,3-glucan beads can have an average particle size of 10 μm to 10 mm, 0.5 to 5 mm, or 1 to 3 mm, depending on the size of the nozzle or pipette. By using an instrument with a larger nozzle, beads with diameters in the centimeter range can be produced. The size of the resulting network structure can be adjusted by adjusting the size of the beads. The average particle size is determined by measuring the diameters (maximum linear distance) of 10 random beads in a 2000x field of view using a scanning electron microscope, and averaging the measured diameters.

[0046] Figure 2 shows a scanning electron microscope photograph of β-1,3-glucan beads obtained using paramylon particles. Figure 2(b) is an enlarged photograph of one particle in Figure 2(a). As shown in Figures 2(a) and (b), the β-1,3-glucan beads are rounded and roughly circular in plan view. The diameter of the β-1,3-glucan beads shown in Figure 2 is about 1 mm, which is roughly 10 times smaller than the diameter of paramylon particles, which are several microns in diameter. 7 This allows the size of the high-density, high-crosslinked network (mesh structure) to be on the order of centimeters.

[0047] Figures 3 and 4 show scanning electron microscope photographs of the cross section of β-1,3-glucan beads obtained using paramylon particles. Figures 3 and 4 show photographs of β-1,3-glucan beads obtained using paramylon particles that were cut with a knife, quickly frozen in liquid nitrogen, and then observed under a scanning electron microscope. Figures 3(a) to (e) are photographs of the same particles observed at different magnifications. Figure 4 is a photograph observing the formation of a submicron fibril network in a different region of the β-1,3-glucan beads than that shown in Figure 3.

[0048] As shown in Figures 3(a) to 3(e), fibrils are formed inside the β-1,3-glucan beads, and a network is formed in which the fibrils are bonded to each other. In other words, the β-1,3-glucan beads are a network structure in which the beads are aggregated into a spherical shape. As shown in Figures 3(b) to (d), a fibril network is also formed on the surface of the particles. These results indicate that fibrillation progresses throughout the entire area of ​​the β-1,3-glucan beads, including the interior and surface, forming a high-density, highly cross-linked network.

[0049] As shown in Figure 4, the β-1,3-glucan beads contain densely packed meshes separated by fibrils, forming a high-density, highly cross-linked network.

[0050] (Process 2: Fiber defibration process) If necessary, the β-1,3-glucan beads can be defibrated following step 1. Defibration defibrates the high-density submicron fiber network constructed in the β-1,3-glucan beads, yielding a network structure with relatively large meshes exposed on the surface.

[0051] The defibration treatment may be, for example, defibrating β-1,3-glucan beads by shearing, and is preferably carried out by shearing in a poor solvent. The poor solvent is as described above. When step 2 is carried out subsequent to step 1, the poor solvent used in step 1 can be used as is. The shearing treatment can be carried out by any known method using known equipment, and can be, for example, mechanical shearing treatment such as homogenization treatment; or pulverization or powdering treatment using a water jet method, a ball mill method, or a grinder method. In one embodiment, the defibration treatment preferably includes homogenizing the β-1,3-glucan beads in water, which can be performed using a known device such as a homogenizer.

[0052] The defibration temperature is preferably 4 to 40° C. The defibration time is preferably 1 to 60 minutes, more preferably 3 to 30 minutes, and even more preferably 5 to 20 minutes.

[0053] Figure 5 shows a scanning electron microscope photograph of the network structure obtained after defibration. This network structure was obtained by dropping a β-1,3-glucan solution into a poor solvent, immersing it for 3 hours with stirring (step 1), and then homogenizing it in water for 20 minutes. Figures 5(a) to 5(c) are photographs at magnifications of 100x, 1000x, and 2000x, respectively. The scales shown in the lower right corner of Figure 5 are 500 μm for Figure 5(a), 50.0 μm for Figure 5(b), and 20.0 μm for Figure 5(c).

[0054] The network structure shown in Figure 5 is made up of fibrils (submicron fibrils) with an average fiber diameter of more than 100 nm but less than 1000 nm that are connected to each other and extend linearly to separate two or more adjacent networks, forming a network structure. Each fibril is connected to another fibril in a series. The fibrils are not entangled with each other, nor do they appear to be gathered in bundles. The fibrils are connected by sharing parts with other fibrils. Almost all fibrils are connected to other fibrils. Networks constructed by the sharing of fibril constituent parts can be seen everywhere. The mesh structure shown in Figure 5 has two or more adjacent meshes, each partitioned by one or more fibrils. The mesh structure spreads out like a honeycomb. The mesh structure is formed by a series of numerous mesh holes, large and small. The mesh structure contains many small meshes with pore sizes of approximately 0.5 μm to 20 μm. The proportion of mesh holes in the mesh structure shown in Figure 5(c) is 90% or more as determined by the area ratio obtained by binarizing the image using image processing software in a 64 μm × 48 μm field of view under a scanning electron microscope. The number of discontinuities in the mesh structure shown in Figure 5(c) is 10 or less per field of view observed at 2000x magnification under a scanning electron microscope. These structural features contribute to improving the mechanical strength of the network, a feature not seen in fibrous materials obtained by mechanical shearing of paramylon particles, a conventional technology. Furthermore, the construction of a strong network prevents aggregation of submicron fibrils.

[0055] (Other processes) The method for producing a network structure according to this embodiment may, if necessary, include a washing step, a drying step, a chemical modification step, or the like, after step 2, using known methods.

[0056] [β-1,3-glucan beads] The β-1,3-glucan beads according to this embodiment comprise a network structure in which fibrils containing β-1,3-glucan and having an average fiber diameter of more than 100 nm but less than 1000 nm are interconnected and extend linearly to define the network. "β-1,3-glucan beads" refer to spherical particles containing β-1,3-glucan. "Spherical" means rounded in plan view. In one embodiment, it is preferable that a surface obtained by cutting a single β-1,3-glucan bead through its center is substantially circular regardless of the angle from which the bead is cut. In one embodiment, the β-1,3-glucan beads are a network structure aggregated into a spherical shape.

[0057] As described above, the β-1,3-glucan beads shown in FIGS. 2(a) and 2(b) are rounded and have a substantially circular shape when viewed from above. The internal mesh structure of β-1,3-glucan beads is as shown in Figures 3 and 4 and described above. The description of β-1,3-glucan and other descriptions of β-1,3-glucan beads are also as described above.

[0058] The average particle size of β-1,3-glucan beads is not limited and can be adjusted by the size of the opening of tools such as pipettes and nozzles used during production. For example, the average particle size of β-1,3-glucan beads can be 1 to 3 mm, or 0.5 to 5 mm. In one embodiment, the average particle size of β-1,3-glucan beads can be 10 μm to 10 mm. The average particle size is determined by measuring the diameters (maximum linear distance) of 10 random beads in a 2000x field of view using a scanning electron microscope, and averaging the measured diameters.

[0059] [Method of manufacturing β-1,3-glucan beads] The method for producing β-1,3-glucan beads according to this embodiment comprises adding dropwise a β-1,3-glucan solution containing β-1,3-glucan and one or more solvents selected from aprotic solvents and ionic liquids to a poor solvent, as described in step 1 (β-1,3-glucan bead production step) of the method for producing a network structure.

[0060] The method for producing β-1,3-glucan beads according to this embodiment may optionally include a step of dissolving the raw material β-1,3-glucan in a solvent containing at least one selected from an aprotic solvent and an ionic liquid prior to dropping the β-1,3-glucan solution into the poor solvent, as described in detail in the dissolving step in the method for producing the network structure. [Example]

[0061] The present invention will be explained in more detail below by showing examples, but the interpretation of the present invention is not limited to these examples.

[0062] [Example 1] Paramylon dope (1) (1.0 g / DMSO 10 mL) Euglena gracilis (NIES-48, provided by the National Institute for Environmental Studies) was cultured in Koren-Hutner medium (pH 3.5) at 28°C for 4 days, and the resulting Euglena cells were centrifuged and freeze-dried. The resulting dry powder was dispersed in a 0.25 mol / L aqueous solution of sodium hydroxide and stirred at room temperature for 2.5 hours. The precipitated white solid was washed repeatedly with water to obtain paramylon particles. The paramylon particles were freeze-dried to produce a dry powder. 13 C-NMR measurement (Bruker AVANCE 500) confirmed that the product was high-purity paramylon.

[0063] 1.00 g of the raw paramylon particles obtained above was dispersed in 10 mL of DMSO and dissolved by stirring with a stirrer bar to prepare paramylon dope (1) (a homogeneous solution containing random-coiled paramylon, paramylon concentration 10 wt%) (dissolution process). Paramylon dope (1) (1.38 g) was added dropwise to 50 mL of ethanol to produce paramylon beads (Step 1). The mixture was stirred, and after 1 hour and 3 hours, approximately 80 mg of paramylon beads (wet weight) were removed and dispersed in 10 mL of Milli-Q water. The beads were then homogenized at room temperature using a homogenizer (model number AHG-160A, shift generator HT1008, rotation speed 13,500 rpm, AS ONE) (Step 2). The paramylon beads removed after 1 hour were homogenized for 20 minutes, and the paramylon beads removed after 3 hours were homogenized for 10 minutes.

[0064] After the homogenization process, 100 μL of each dispersion was sampled and the shape of the solids contained in the dispersion was observed using a scanning electron microscope (TM4000, Hitachi High-Technologies). Scanning electron microscope photographs (500x magnification) are shown in Figure 6. Figure 6(a) is a scanning electron microscope photograph of the solid obtained by dropping paramylon dope (1) into ethanol, stirring for 1 hour, and defibrating the resulting paramylon beads for 20 minutes. Figure 6(b) is a scanning electron microscope photograph of the solid obtained by dropping paramylon dope (1) into ethanol, stirring for 3 hours, and defibrating the resulting paramylon beads for 10 minutes. As shown in Figure 6, a network composed of submicron fibrils with an average fiber diameter of 500 nm was confirmed in both samples, and a network-like structure was obtained.

[0065] [Example 2] Paramylon dope (2) (0.5 g / DMSO 10 mL) 0.5 g of raw paramylon particles prepared in the same manner as in Example 1 was dispersed in 10 mL of DMSO and dissolved by stirring with a stirrer bar to prepare paramylon dope (2) (a homogeneous solution containing random-coil paramylon, paramylon concentration 5 wt%). Paramylon dope (2) (1.07 g) was added dropwise to 50 mL of ethanol, and the resulting paramylon beads were stirred. After 4 hours, they were removed and homogenized for 20 minutes. The shape of the beads was confirmed using a scanning electron microscope (500x magnification). A scanning electron microscope photograph is shown in Figure 7. As shown in Figure 7, a network composed of submicron fibrils with an average fiber diameter of 500 nm was confirmed, and a network structure was obtained.

[0066] [Example 3] Paramylon dope (3) (1.2 g / DMSO 10 mL) 1.2 g of raw paramylon particles prepared in the same manner as in Example 1 was dispersed in 10 mL of DMSO and dissolved by stirring with a stirrer bar to prepare paramylon dope (3) (a homogeneous solution containing random-coiled paramylon, paramylon concentration 12 wt%). Paramylon dope (3) (1.36 g) was added dropwise to 50 mL of ethanol, and the resulting paramylon beads were stirred. After 3 hours, they were removed and homogenized for 20 minutes. The shape of the beads was confirmed using a scanning electron microscope (500x magnification). A scanning electron microscope photograph is shown in Figure 8. As shown in Figure 8, a network composed of submicron fibrils with an average fiber diameter of 500 nm was confirmed, and a network structure was obtained.

[0067] [Example 4] Dry beads Paramylon beads prepared from paramylon dope (1) (1.0 g / DMSO 10 mL) prepared in the same manner as in Example 1 were air-dried to remove all DMSO contained in the beads, yielding dried beads. 49 mg of dried beads were dispersed in 10 mL of Milli-Q water and homogenized. After 10 minutes, the beads were removed and their shape was confirmed using a scanning electron microscope (500x magnification). A scanning electron microscope photograph is shown in Figure 9. As shown in Figure 9, a network composed of submicron fibrils with an average fiber diameter of 500 nm was confirmed, and a network structure was obtained.

[0068] [Comparative Example 1] Homogenization of natural paramylon particles 12 mg of raw paramylon particles, prepared using the same method as in Example 1, were dispersed in 10 mL of Milli-Q water and homogenized for 20 minutes. The shape of the particles was confirmed using a scanning electron microscope. A scanning electron microscope photograph (2000x magnification) is shown in Figure 10. As shown in Figure 10, no fibrous material was observed; only paramylon particles were confirmed. This result demonstrates that the dissolution of paramylon and immersion in a poor solvent (ethanol), which are included in each step of Examples 1 to 4, are essential for constructing a submicron fibril network.

[0069] [Comparative Example 2] Fiberization from sodium hydroxide solution 503 mg of raw paramylon particles prepared using the same method as in Example 1 was dispersed in 10 mL of 1 mol / L aqueous sodium hydroxide solution and stirred at room temperature until homogenous. The resulting homogenous solution was added dropwise to 100 mL of ethanol with stirring, yielding a white precipitate. This was separated by decantation, washed with 100 mL of ethanol (80 minutes) and then with methanol (100 mL, approximately 60 minutes x 4), then dried on a filter paper (by sandwiching the particles between filter paper to allow the water to penetrate the filter paper) and air-dried. 340 mg of the resulting solid was placed in a test tube with 10 mL of Milli-Q water and left for 2 days (pH 7.54). The dispersion was homogenized, and 100 μL of the dispersion was withdrawn after 10 minutes. A scanning electron microscope photograph (500x magnification) is shown in Figure 11. As shown in Figure 11, the majority of the dispersion consisted of irregularly arranged aggregates of fibrils approximately 10 μm in length, with no network formation. Therefore, the use of aqueous sodium hydroxide solution is not suitable for constructing a submicron fibril network.

[0070] [Comparative Example 3] Fiber formation from heated DMSO solution 996 mg of raw paramylon particles, prepared using the same method as in Example 1, were immersed in 10 mL of DMSO for 30 minutes, followed by heating and stirring at 90°C for 3 hours to obtain a homogeneous solution. The resulting homogeneous solution was then placed in a Teflon® dish and cooled to room temperature, yielding 5.44 g of a gel-like solid containing DMSO. 113 mg of the sheet-like gel solid was dispersed in 10 mL of Milli-Q water and homogenized at room temperature. 100 μL of the dispersion was removed after 10 minutes. A scanning electron microscope photograph (JSM-6060, JEOL, 500x magnification) is shown in Figure 12. As shown in Figure 12, only amorphous aggregates were observed. Therefore, in order to construct a submicron fibril network, it is essential to drop the solution into a poor solvent such as ethanol, which is included in the paramylon bead preparation process.

[0071] [Example 5] Composite of polylactic acid and paramylon submicron fibril network (polylactic acid-paramylon submicron fibril network composite) 8.6 g of polylactic acid (NatureWorks Ingeo Biopolymer 10361D) and 0.96 g of a paramylon submicron fibril network prepared by vacuum heating and drying (100°C, 8 hours) the network structure obtained by the same method as in Example 1 were mixed (185°C, 10 minutes) in a Labo Plastomill (manufactured by Toyo Seiki Co., Ltd.) equipped with a small segment mixer KF6 to obtain a composite of polylactic acid and a paramylon submicron fibril network. The composite of polylactic acid and a paramylon submicron fibril network is a resin composition containing polylactic acid and a paramylon submicron fibril network.

[0072] The elastic modulus (storage modulus and loss modulus) of the obtained kneaded sample was measured using a viscoelasticity measuring device (MCR302, Anton Paar) while changing the measurement frequency from 0.01 Hz to 100 Hz. The storage modulus and loss modulus of polylactic acid were measured in the same manner. The data are shown in Figure 13. In Figure 13, "polylactic acid + fiber" represents a composite of polylactic acid and a paramylon submicron fibril network.

[0073] The storage modulus of the composite of polylactic acid and paramylon submicron fibril network (shown by the open circle in Figure 13) shows a plateau region at around 0.1 Hz. In this region, the following relationship exists between the storage modulus G and the entanglement transition molecular weight Me of the material: Me = ρRT / G (where ρ is the specific gravity, R is the gas constant, and T is the temperature) is known to hold. where ρ=1000kg / m 3 Substituting the difference (88.3 - 17.2 = 71.1 Pa) between the "storage modulus of polylactic acid + fiber" and the "loss modulus of polylactic acid + fiber" at R = 8.31, T = 300 K, and 0.1 Hz gives Me = 35,063. Since the molecular weight of the paramylon monomer unit (glucose) is 162, this formula suggests that 35,063 / 162 = 216, or one crossover occurs for every 216 degrees of polymerization. Since paramylon has approximately 2000 polymerizations, roughly 10 times the 216, this indicates that paramylon forms a dispersed structure with enough crossovers to exert a reinforcing effect on polylactic acid. [Industrial Applicability]

[0074] The network structure according to this embodiment has industrial applicability as, for example, a filler (reinforcing material) for resins, a functional separation membrane or other membrane material, a biodegradable filler, a water-resistant network structure, and the like.

Claims

1. The fiber has a mesh structure in which fibrils containing β-1,3-glucan and having an average fiber diameter of more than 100 nm but less than 1000 nm are connected to each other and extend linearly to define a mesh, The fibrils include the following (1) to (2): (1) The fibrils are not entangled with each other and are not gathered in bundles. (2) The fibrils share parts with each other and extend linearly. A network structure that satisfies either or both of the above.

2. The network structure according to claim 1 , wherein the fibrils are connected in a continuous manner and extend in straight or curved lines to define meshes.

3. The network structure according to claim 1 , wherein the fibrils extend linearly while branching to define meshes.

4. The network structure of claim 3 , wherein the fibrils are separated from each other between the branches.

5. The network structure according to claim 1 , wherein the network structure is formed by two or more of the networks being connected together.

6. The network structure according to claim 1 , wherein the network structure is formed by three or more of the networks being in contact with each other.

7. 5. The network structure according to claim 1, wherein the number of ends of the fibrils in the network structure is 10 or less in one field of view observed at 2000x magnification using a scanning electron microscope.

8. A method for producing the network structure according to any one of claims 1 to 4, The production method includes dropping a β-1,3-glucan solution containing raw material β-1,3-glucan and a good solvent into a poor solvent to obtain β-1,3-glucan beads.

9. The method according to claim 8, further comprising defibrating the β-1,3-glucan beads.

10. The method according to claim 8 , wherein the good solvent comprises one or more solvents selected from aprotic solvents and ionic liquids.

11. The method according to claim 8 , wherein the anti-solvent comprises an alcohol.

12. The manufacturing method according to claim 8 , wherein the defibrating treatment comprises a shearing treatment in a poor solvent.

13. The fiber has a mesh structure in which fibrils containing β-1,3-glucan and having an average fiber diameter of more than 100 nm but less than 1000 nm are connected to each other and extend linearly to define a mesh, The fibrils include the following (1) to (2): (1) The fibrils are not entangled with each other and are not gathered in bundles. (2) The fibrils share parts with each other and extend linearly. β-1,3-glucan beads that satisfy either or both of the above.

14. A method for producing β-1,3-glucan beads, comprising dropping a β-1,3-glucan solution containing raw material β-1,3-glucan and a good solvent into a poor solvent.

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