Sugar production method and biofuel production method

Abstract

Provided are: a sugar production method that is capable of producing sugar from a plant or a part thereof and that can be performed, for example, without alkali treatment and high-temperature and high-pressure treatment; and a biofuel production method using the sugar production method. A sugar production method according to the present disclosure comprises: a degradation step for degrading a cell wall of a plant or a part thereof with a cell wall-degrading enzyme in the coexistence of the plant or the part thereof and the cell wall-degrading enzyme; a first separation step for separating cells of the plant or the part thereof from the cell wall or enzymatic degradation products thereof in the plant or the part thereof; a second separation step for separating a solid fraction by subjecting a separated product of the cell wall or the degradation products thereof and the cells of the plant or the part thereof to solid-liquid separation; and a saccharification step for saccharifying the solid fraction in the coexistence of the solid fraction and a saccharifying enzyme.

Description

Sugar production method and biofuel production method

[0001] The present disclosure relates to methods for producing sugars and biofuels.

[0002] The use of fossil fuels increases the concentration of carbon dioxide in the atmosphere and has a significant impact on global warming. On the other hand, biofuels are fuels derived mainly from plant biomass, and the carbon dioxide emitted during their use is absorbed by plants and fixed in the plant biomass. Therefore, the use of biofuels does not substantially increase the concentration of carbon dioxide in the atmosphere (carbon neutral), and therefore has little impact on global warming.

[0003] Patent Document 1 discloses a method for producing biofuel in which alkali-treated plants are heated in the presence of an acid to saccharify them, and the resulting sugars are fermented to synthesize ethanol. Patent Document 2 discloses a method in which cellulose and other substances contained in biomass are saccharified by high-temperature, high-pressure treatment, and the resulting sugars are fermented to synthesize ethanol.

[0004] JP 2012-100594 A JP 2008-182925 A

[0005] However, the above-mentioned method for producing biofuel requires a great deal of cost since the plant is subjected to alkali treatment, high-temperature and high-pressure treatment, etc.

[0006] Therefore, the present disclosure aims to provide a method for producing sugar from plants or parts thereof that can be carried out without, for example, alkaline treatment and high-temperature, high-pressure treatment, and a method for producing biofuel using the sugar production method.

[0007] In order to achieve the above-mentioned object, the method for producing sugar of the present disclosure includes: a decomposition step of decomposing cell walls of a plant or a part thereof with a cell wall-decomposing enzyme in the presence of the plant or a part thereof and the cell wall-decomposing enzyme; a first separation step of separating cells of the plant or part thereof from the cell walls or an enzymatic decomposition product thereof in the plant or part thereof; a second separation step of separating the separated product of the cell walls or the decomposition product thereof and the cells of the plant or part thereof from solid-liquid separation to separate a solid fraction; and a saccharification step of saccharifying the solid fraction in the presence of the solid fraction and a saccharifying enzyme.

[0008] The method for producing biofuel of the present disclosure includes a production step of producing sugar from a plant or part thereof using the method for producing sugar of the present disclosure, and a production step of producing biofuel from the sugar.

[0009] According to the present disclosure, for example, it is possible to provide a method for producing sugar from a plant or a part thereof without the need for alkaline treatment or high-temperature, high-pressure treatment, and a method for producing biofuel using the sugar production method.

[0010] FIG. 1(A) is a schematic diagram of a plant tissue treatment device 1A, and FIG. 1(B) is a schematic diagram of a plant tissue treatment device 1A equipped with a plant tissue supply unit 11 and the like. FIG. 2(A) is a schematic diagram of a plant tissue treatment device 1D, and FIG. 2(B) is a schematic diagram of a plant tissue treatment device 1E. FIG. 3 is a schematic diagram of a plant tissue treatment device equipped with multiple separation tanks 4T. FIG. 4 shows an example of a process flow for producing an extract of a plant or a part thereof. FIG. 5 shows an example of a process flow for producing an extract of a plant or a part thereof. FIG. 6 shows SEM images of tea leaves and used tea leaves before decomposition and separation. FIG. 7 shows SEM images of the slurry-form tea leaves of Example 1, the slurry-form used tea leaves of Example 2, and the tea leaves of Comparative Example 1. FIG. 8 shows SEM images of vein cellulose fibers in the slurry-form tea leaves of Example 1. FIG. 9 shows the soaked tea leaves of Example 3 before (A) and after (B) stirring and crushing. FIG. 10 shows the process of solid-liquid separation in Example 3. FIG. 11 shows soaked tea leaves in Comparative Example 2. FIG. 12 shows the weight results of the wet slurry tea leaves obtained in Example 3. FIG. 13 shows the protein content results obtained in Example 3. FIG. 14 shows the concentrations of each component obtained in Example 3 and Comparative Example 2. FIG. 15 shows soaked tea leaves after stirring and crushing in Example 4. FIG. 16 shows the process of solid-liquid separation in Example 4. FIG. 17 shows soaked tea leaves in Comparative Example 3. FIG. 18 shows the weight results of the wet slurry tea leaves obtained in Example 4. FIG. 19 shows the protein content results obtained in Example 4. FIG. 20 shows the concentrations of each component obtained in Example 4 and Comparative Example 3. FIG. 21 shows soaked coffee beans after stirring and crushing in Example 5. FIG. 22 shows the process of solid-liquid separation in Example 5. FIG. 23 shows soaked coffee beans in Comparative Example 4. Figure 24 shows the weight results for the wet slurry coffee beans obtained in Example 5. Figure 25 shows the protein content results for Example 5. Figure 26 shows soaked strawberry leaves before (A) and after (B) stirring and crushing in Example 6. Figure 27 shows the process of solid-liquid separation treatment in Example 6. Figure 28 shows the weight results for the wet slurry strawberry leaves obtained in Example 6. Figure 29 shows the protein content results for Example 6.Figure 30 shows soaked tomato leaves before (A) and after (B) stirring and crushing in Example 7. Figure 31 shows the process of solid-liquid separation treatment in Example 7. Figure 32 shows the weight results of the wet slurry-like tomato leaves obtained in Example 7. Figure 33 shows the protein content results obtained in Example 7. Figure 34 shows the measurement results of test plots 1 to 6 in Example 11. Figure 35 shows an outline of an example of a method for producing biofuel according to the present disclosure. Figure 36 shows the results of glucose consumption by yeast in the saccharified solution obtained by alkali-treating the solid fraction obtained in Example 12 and then subjecting it to enzymatic saccharification treatment.

[0011] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, and other publications and information referenced herein are incorporated herein by reference in their entirety.

[0012] [Definition] As used herein, the term "plant" refers to any plant. Examples of the plant include tea, coffee, moringa, Brussels sprouts, cabbage, tomato, grape, grasses (e.g., rice, wheat, barley, oats, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, wild rice, sugarcane, and Job's tears) and / or ginkgo. The plant may also be a woody plant (e.g., ginkgo, larch, beech, oak, maple, cherry, dogwood, teak, dipterocarp, cypress, Japanese red pine, camphor tree, Castanopsis cuspidata, and camellia).

[0013] As used herein, "plant parts" refers to any part of a plant. Examples of the plant parts include leaves, stems, flowers, seeds, roots, trunks, skins (e.g., bark, epidermis, bast, pericarp, etc.), sap, and / or fruits, or processed products thereof. The plant parts may be, for example, tea leaves, used tea leaves, tomato leaves, grass leaves, and / or woody leaves. The processed products may be sake lees, shochu lees, beer lees, and / or fruit press residues. Examples of the fruits include grapes and citrus fruits. The fruit press residue refers to the residue remaining after fruit juice is obtained by pressing the fruit, and includes, for example, the peel. The peel may be, for example, the exocarp, mesocarp, and / or endocarp.

[0014] As used herein, "plant tissue" refers to any plant tissue. Examples of the plant tissue include plant tissues of grasses, trees, etc. Examples of the plant tissue include epidermal tissue, conductive tissue, mechanical tissue, parenchyma, etc. Specific examples of the plant tissue include leaves, stems, flowers, seeds, roots, trunks, skin (e.g., bark, epidermis, bast, pericarp, etc.), sap, and / or fruit, or processed products thereof. Specific examples of the plant tissue include tissues rich in nutritional components such as proteins and amino acids. Examples of the plant tissue include tissues of tea plants (tea), coffee plants (coffee), moringa, Brussels sprouts, cabbage, tomatoes, grapes, grasses (e.g., rice, wheat, barley, oats, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, wild rice, sugarcane, Job's tears, etc.), and / or ginkgo. Examples of the tea plant tissue include tea leaves and used tea leaves, and examples of the plant tissue include cellulose fibers.

[0015] In this specification, "cellulose fiber" refers to a fibrous structure composed of cellulose derived from plant tissue. Examples of the cellulose fiber include cellulose fibers contained in leaf veins, i.e., vein cellulose fibers, cellulose fibers contained in mesophyll, and cellulose fibers contained in cell walls. Furthermore, cellulose fibers are not limited to simple cellulose, and may broadly include hemicellulose, lignin, and other fibers.

[0016] As used herein, the term "processed product" refers to any cell aggregate that has been subjected to any treatment. Examples of the processed product include any cell aggregate that has been subjected to physical treatment (cutting, shearing, crushing, etc.), chemical treatment (oxidation-reduction reaction, hydrolysis reaction, enzymatic reaction, etc.), temperature treatment (heating, refrigeration, freezing, etc.), etc.

[0017] As used herein, the term "cell wall-degrading enzyme" refers to any enzyme capable of degrading cell walls. Examples of such enzymes include enzymes that degrade cellulose, hemicellulose, lignin, pectin, cuticle, etc. Examples of such enzymes include cellulase, hemicellulase, lignin peroxidase, and / or pectinase.

[0018] As used herein, "microbial culture solution" refers to a culture solution obtained by culturing a microorganism that expresses a target enzyme. Examples of the microbial culture solution include a culture solution in which the microorganism has grown to an extent that contributes to the decomposition treatment of the target component; a culture solution in which an enzyme has been secreted from the microorganism into the culture solution and accumulated to an extent that contributes to the decomposition treatment; and the like. In the latter case, the microbial culture solution may or may not contain the microorganism. The microorganism is not particularly limited as long as it expresses, for example, the target enzyme. The culture solution is not particularly limited as long as it can culture the microorganism.

[0019] As used herein, "cell" refers to any cell derived from plant tissue. The number of cells may be, for example, one or more. The type of cells may be one or more. When the cells are composed of multiple cells or multiple types, the cells may be either separated from each other or aggregated.

[0020] As used herein, "extraction" refers to the extraction of a specific component from a target object and / or the state in which a specific component has been extracted from a target object. The "extraction" can be performed, for example, by performing at least one extraction process. As used herein, a substance containing a component extracted from a target object is referred to as an extract. Furthermore, a liquid containing a component extracted from a target object is referred to as an extract. The extract or extract may contain cellular components of the plant tissue. Examples of the cellular components include low-molecular-weight compounds, nucleic acids, amino acids, peptides, proteins, and / or cell walls. When the extract or extract contains a cell wall, the extract or extract may contain, for example, cellulose microfibers and / or cellulose nanofibers. The extract may be, for example, cellulose fibers. The cellulose fibers include cellulose microfibers, cellulose nanofibers, and / or hemicellulose. Cellulose fibers can be isolated, for example, by delignification treatment, to obtain cellulose microfibers, cellulose nanofibers, and / or hemicellulose. These can be traded independently as various materials. The cellulose fibers themselves may also be traded. The cellulose fibers may be those contained in the veins of the leaves of any plant, i.e., vein cellulose fibers. The cellulose fibers may also be those contained in the mesophyll or cell walls.

[0021] As used herein, "purified" means identifying and separating, recovering from components in their natural state, being identified and separated, and / or being recovered from components in their natural state. The "purification" can be carried out, for example, by obtaining at least one purification step. The purification can also be referred to as isolation.

[0022] As used herein, the term "low molecular weight compound" refers to a compound having a small molecular weight (less than 900 daltons). Examples of the low molecular weight compound include amino acids, lipids, sugars, and fatty acids. Specific examples of the low molecular weight compound include catechin.

[0023] As used herein, "peptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids.

[0024] As used herein, the term "protein" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids. The protein is, for example, a peptide having a length of 10 amino acids or more.

[0025] As used herein, "biofuel" refers to fuel produced using biomass. Examples of biofuels include ethanol, butanol, jet fuel, and / or diesel fuel.

[0026] As used herein, "biomass" refers to organic resources derived from living organisms, excluding fossil resources. Examples of biomass include edible and inedible parts of plants; plant roots, rhizomes, leaves, stems, flowers, fruits, pericarp, seeds, etc.; plant cell walls; and components contained in plant cell walls, such as cellulose, hemicellulose, lignin, pectin, and cuticle.

[0027] As used herein, "saccharification" refers to converting polysaccharides into sugars with smaller molecular weights by the action of enzymes, acids, etc.

[0028] As used herein, the term "glycating enzyme" refers to an enzyme that exhibits glycating activity.

[0029] As used herein, "fermentation" refers to the decomposition of organic compounds such as sugars by enzymes to produce alcohol, organic acids, carbon dioxide, etc. Microbial fermentation refers to fermentation by enzymes possessed by microorganisms. Examples of the microorganisms include yeast, bacteria, and / or fungi.

[0030] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in the present disclosure and each embodiment can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0031] [Method for Producing Sugar] In one aspect, the present disclosure provides a method for producing sugar from a plant or part thereof. The method for producing sugar of the present disclosure includes: a decomposition step of decomposing cell walls of the plant or part thereof with a cell wall-decomposing enzyme in the presence of the plant or part thereof and the cell wall-decomposing enzyme; a first separation step of separating cells of the plant or part thereof from the cell walls or enzymatic decomposition product thereof in the plant or part thereof; a second separation step of separating the separated product of the cell walls or decomposition product thereof and the cells of the plant or part thereof from a solid-liquid separation to isolate a solid fraction; and a saccharification step of saccharifying the solid fraction in the presence of the solid fraction and a saccharifying enzyme.

[0032] As a result of extensive research, the present inventors discovered that by including the decomposition step, the first and second separation steps, and the saccharification step, cellulose in plants can be saccharified without, for example, alkali treatment or high-temperature, high-pressure treatment, and thus established the present disclosure. Therefore, the sugar production method of the present disclosure can reduce the cost of producing sugar from plants or parts thereof. Furthermore, a liquid fraction derived from the plant or parts thereof can be obtained by solid-liquid separation in the second separation step. The liquid fraction contains, for example, proteins, catechins, and / or caffeine derived from the plant or parts thereof.

[0033] The method for producing sugars according to the present disclosure includes (1) a decomposition step and a first separation step, (2) a second separation step, and (3) a saccharification step. The (1) decomposition step and the first separation step, and the (2) second separation step can also be referred to as pretreatment steps. The (1) decomposition step and the first separation step, and the (2) second separation step can be suitably carried out, for example, using a plant tissue treatment device described below. Each of the steps (1) to (3) is described below.

[0034] (1) Decomposition Step and First Separation Step The sugar production method of the present disclosure includes a decomposition step (decomposition step) in which the cell walls of a plant or a part thereof are decomposed by a cell wall-decomposing enzyme in the presence of the plant or a part thereof, and a first separation step (separation step) in which the cells of the plant or a part thereof are separated from the cell walls or their enzymatic decomposition products in the plant or a part thereof. By including the separation step in addition to the decomposition step, the sugar production method of the present disclosure can effectively separate the cell walls of the plant or a part thereof from other components (e.g., cells or cell components) in the plant or a part thereof. Therefore, the sugar production method of the present disclosure can improve, for example, the extraction efficiency of an extract of the plant or a part thereof. In the following description, the plant or a part thereof may also be referred to as "plant tissue."

[0035] Examples of the plant include tea plant (tea), coffee plant (coffee), moringa, Brussels sprouts, cabbage, tomato, grape, grass plants (e.g., rice, wheat, barley, oats, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, Zizania latifolia, sugarcane, Job's tears, etc.), and / or ginkgo. Examples of the plant part include leaves, stems, flowers, seeds, roots, trunks, skin (bark, epidermis, bast, pericarp, etc.), sap, and / or fruit, or processed products thereof. Examples of the plant part include tea leaves, used tea leaves, tomato leaves, grass plant leaves, and / or woody plant leaves.

[0036] (Decomposition step) The decomposition step is a step in which the cell walls of the plant tissue are decomposed using an enzyme. Therefore, the decomposition step can also be called, for example, an enzyme treatment step. In the decomposition step, the cell walls in the plant tissue are decomposed, and as described above, in the separation step described below, the cell walls in the plant tissue can be effectively separated from other components in the plant tissue (e.g., cells or cell components).

[0037] The decomposition step can be carried out, for example, by contacting the plant tissue with the enzyme. Specifically, the decomposition step involves, for example, mixing the plant tissue with an enzyme solution containing the enzyme to prepare a reaction solution (mixture). The decomposition step is preferably carried out, for example, in a reaction solution system containing the plant tissue, the enzyme, and a solvent. Next, in the decomposition step, for example, the enzyme decomposes the cell wall of the plant tissue in the reaction solution to an extent that the cell wall can be separated from other components in the plant tissue in the separation step described below. In the decomposition step, for example, the enzyme decomposes the cell wall of the plant tissue while stirring the reaction solution. In the decomposition step, for example, part or all of the cell wall of the plant tissue is decomposed.

[0038] In the decomposition step, the enzyme can be appropriately selected depending on, for example, the origin of the plant tissue, i.e., depending on the cell wall constituting the plant tissue. Examples of the enzyme include enzymes that decompose the cell wall (including cellulose, hemicellulose, lignin, pectin, cuticle, etc.). Specific examples of the enzyme include cellulase, hemicellulase, lignin peroxidase, and pectinase.

[0039] When the plant tissue is tissue of the tea plant (tea) or the coffee plant (coffee), the enzyme is preferably, for example, cellulase, hemicellulase, pectinase, amylase, protease, or the like, from the viewpoint of extraction efficiency.

[0040] The enzyme may be, for example, an enzyme capable of decomposing at least one cell wall contained in the plant tissue, and may be a purified enzyme, a crude enzyme, a microorganism expressing the enzyme, a processed product of the microorganism, or a culture solution of the microorganism (microbial culture solution).

[0041] The reaction conditions for the decomposition in the decomposition step are not particularly limited and can be set, for example, under conditions that allow the enzyme to decompose the cell wall of the plant tissue. The pH of the reaction solution can be set appropriately depending on, for example, the type of enzyme, and is, for example, 1 to 10, 2 to 9, 3 to 8, or 4 to 7.

[0042] The temperature of the reaction solution can be appropriately set depending on the type of the enzyme, for example, and is, for example, 20 to 70°C, 25 to 65°C, 35 to 60°C, 40 to 60°C, or 45 to 55°C.

[0043] (Separation Step) The separation step is a step of separating the plant tissue cells and / or cell extracts from the cell walls by separating the plant tissue. Therefore, the separation step can also be referred to as, for example, a cell separation step. The surface area of ​​the plant tissue and plant tissue-derived components on which the enzymes can act is relatively increased in the reaction solution during or after the separation step compared to the reaction solution before the separation step, resulting in more efficient enzymatic decomposition. Furthermore, the stirring effect associated with the separation process can extract nutrients that remain within the cells even after the cell walls are broken down into the solution. Therefore, in the sugar production method disclosed herein, by combining the enzymatic decomposition in the decomposition step with the separation of the plant tissue cells and / or cell extracts from the cell walls in the separation step, the cell walls of the plant tissue can be effectively separated from other components of the plant tissue (e.g., cells or cell components).

[0044] The separation step is preferably carried out on a reaction solution containing the plant tissue and the enzyme. When the separation step is carried out during or after the decomposition step, the separation is carried out on the reaction solution prepared in the decomposition step. When the separation step is carried out before the decomposition step, the separation is carried out on the reaction solution obtained by preparing the reaction solution in advance.

[0045] In the separation step, for example, cells constituting the plant tissue are mechanically separated from the plant tissue. The separation step may include, for example, a process of mechanically shearing the cell walls of the plant tissue (shearing process), which can more efficiently separate the cell walls from the other components. The mechanical shearing can be performed, for example, using a shearing device (shearer). The shearing device preferably includes shearing blades, which can more efficiently separate the cell walls from the other components. Specifically, the separation step includes, for example, a process of mechanically shearing the cell walls of the plant tissue by bringing the plant tissue into contact with an agitator blade having a shearing blade. The separation step includes, for example, a process of agitating and crushing the plant tissue using a homogenizer. This process can effectively separate the cell walls of the plant tissue from other components (e.g., cells or cell components) in the plant tissue.

[0046] In the separation step, the mechanical shearing treatment can be appropriately set depending on, for example, the origin of the plant tissue, i.e., depending on the cell walls that constitute the plant tissue. Examples of the treatment for mechanically shearing the cell walls of the plant tissue include a treatment for stirring and crushing the plant tissue using a homogenizer or the like.

[0047] When the plant tissue is tea or coffee tissue, the mechanical shearing treatment may be performed using any device having blades capable of efficiently shearing tea or coffee tissue.

[0048] The temperature during the treatment of mechanically shearing the cell walls of the plant tissue can be appropriately set depending on, for example, the enzyme used in the decomposition step, and is, for example, a temperature at which the enzyme exhibits enzymatic activity, specifically, 20 to 70°C, 25 to 65°C, 35 to 60°C, 40 to 60°C, or 45 to 55°C.

[0049] The decomposition step and / or the separation step may each be performed once or multiple times. The number of times the decomposition step is performed and the number of times the separation step is performed may be the same or different. The decomposition step and the separation step may be performed separately, or some or all of the decomposition step and the separation step may be performed simultaneously.

[0050] (Order of each step) The decomposition step and the separation step can be performed in any order. For example, the steps can be performed as follows: (1) decomposition step, (2) separation step; (1) separation step, (2) decomposition step.

[0051] Alternatively, one or more decomposition steps and one or more separation steps may be performed any number of times in any order. For example, the steps may be performed as follows: (1) decomposition step, (2) separation step, (3) decomposition step, (4) separation step; (1) separation step, (2) decomposition step, (3) separation step, (4) decomposition step.

[0052] The decomposition step and the separation step can also be performed consecutively any number of times. For example, each step can be performed as follows: (1) decomposition step, (2) decomposition step, (3) separation step; (1) separation step, (2) separation step, (3) decomposition step.

[0053] The decomposition step and the separation step can also be performed simultaneously. Furthermore, the combination of the decomposition step and the separation step can be repeated any number of times. For example, each step can be performed as follows: (1) a decomposition step and a separation step, (2) a decomposition step and a separation step; (1) a decomposition step and a separation step, (2) a decomposition step and a separation step, (3) a decomposition step and a separation step.

[0054] In the sugar production method of the present disclosure, for example, prior to the decomposition step and / or separation step, a pretreatment such as applying physical stimulation (e.g., kneading, crushing, or scratching) to the plant tissue, particularly the cell walls of the plant tissue, may be performed. Examples of the pretreatment include grinding with a food mill, milling, crushing, shearing, heat treatment, kneading (kneading step), squeezing, compressing or pressing, squeezing, crushing, friction welding, etc. Thus, the sugar production method of the present disclosure, for example, including the pretreatment, can improve the efficiency of obtaining an extract (liquid extract) from the plant tissue.

[0055] In the sugar production method of the present disclosure, the obtained extract (extract) contains, for example, an enzyme derived from the plant tissue. In this case, the sugar production method of the present disclosure may include, for example, an enzyme inactivation treatment after the decomposition step and / or the separation step. The enzyme inactivation treatment can be performed, for example, by heating the extract (extract) or changing the pH of the extract (extract) under conditions that inactivate proteins or nucleic acids.

[0056] In the sugar production method of the present disclosure, the conditions for each step may be set to adjust the extraction efficiency of certain components contained in the plant tissue. For example, when the decomposition step and / or separation step are performed at a relatively high temperature, the extraction efficiency of catechins contained in tea leaves or the like from the biological sample into the liquid fraction is improved. On the other hand, when the decomposition step and / or separation step are performed at a relatively low temperature, the extraction efficiency of catechins from the biological sample into the liquid fraction is reduced. Specifically, the extraction efficiency of catechins can be significantly improved by extracting them from plant tissue at a high temperature, such as 80°C, compared to when they are extracted from plant tissue at room temperature, such as 30°C. Thus, components whose extraction efficiency from the biological sample varies depending on temperature can be easily extracted from the solid fraction into the liquid fraction by, for example, exposing the plant tissue to a high-temperature liquid at any timing during the solid-liquid separation process in the separation step.

[0057] (2) Second Separation Step In the second separation step, the separation product of the cell walls or their degradation products and the cells of the plant or its parts is subjected to solid-liquid separation to separate a solid fraction. This allows, for example, the cell walls contained in the treatment liquid after the shearing treatment to be separated from other components. The solid fraction includes, for example, insoluble cellulose fibers. The solid-liquid separation can suitably separate, for example, the cell walls and / or cellulose of the plant tissue from other components derived from the plant tissue. The liquid fraction includes, for example, proteins, catechins, and / or caffeine derived from the plant or its parts. The solid-liquid separation can be performed, for example, using known solid-liquid separation means, and specific examples include filtration means such as filters, membranes, meshes (stainless steel mesh, etc.), and through-nets, as well as cyclones and centrifugal separators. The solid-liquid separation means includes, for example, a mesh. In the solid-liquid separation, it is preferable to use a solid-liquid separation means having an opening of, for example, 1 μm to 10 mm, 10 μm to 9 mm, 25 μm to 8 mm, 50 μm to 7 mm, 100 μm to 5 mm, 1 mm to 3.35 mm, 100 μm to 1 mm, or 1 μm to 100 μm, since this can efficiently separate the solid fraction.

[0058] (3) Saccharification Step In the saccharification step, the solid fraction is saccharified in the presence of the solid fraction and the saccharifying enzyme. The saccharification step is, for example, a step of converting cellulose and / or cellulose fibers contained in the solid fraction into sugars such as glucose using the saccharifying enzyme. The saccharification step can be carried out, for example, in a reaction solution containing the solid fraction and the saccharifying enzyme. For the saccharification step, a known method for saccharifying biomass containing cellulose fibers, such as woody biomass, can be used.

[0059] The saccharifying enzyme can be selected appropriately depending on, for example, the components contained in the solid fraction, i.e., the type of sugar polymer. When the solid fraction contains cellulose and / or hemicellulose, the saccharifying enzyme can be, for example, an enzyme that hydrolyzes cellulose and / or hemicellulose to produce C5 sugars and / or C6 sugars. Specific examples of the enzyme include cellulase, hemicellulase, and β-glucosidase. The hemicellulase is, for example, an enzyme that exhibits hemicellulose decomposition activity, and specific examples include xylanase, xylosidase, mannanase, and pectinase. When the solid fraction contains xyloglucan, the saccharifying enzyme can be, for example, xyloglucanase. The xyloglucanase is, for example, an enzyme that exhibits xyloglucan decomposition activity.

[0060] The saccharifying enzyme may be used in the coexistence of a microorganism that expresses, is capable of expressing, or produces the saccharifying enzyme, such as a microorganism belonging to the genera Trichoderma, Aspergillus, Humicola, Irpex, Acremonium, and / or Penicillium.

[0061] The reaction conditions (temperature, time, pH, etc.) for the saccharification step can be appropriately selected depending on the type and origin (e.g., bacterial species) of the saccharifying enzyme. Specific examples include the reaction temperature of the cellulase, hemicellulase, β-glucosidase, and xyloglucanase, which is, for example, 20 to 60°C, 30 to 60°C, or 40 to 60°C, and preferably about 50°C. The pH during the reaction of the cellulase, hemicellulase, and β-glucosidase is, for example, 2 to 7, 3 to 6, or 3 to 5, and preferably about 4. The pH during the reaction of the xyloglucanase is, for example, 2 to 7, 3 to 6, or 3 to 5, and preferably about 5. For example, the reaction temperature and pH during the reaction of the Trichoderma-derived cellulase and the Aspergillus-derived cellulase are, respectively, 45 to 60°C and 4 to 6. For example, the reaction temperature and pH during the reaction of xyloglucanase derived from the genus Aspergillus are 50° C. and pH 5, respectively.

[0062] In the saccharification step, for example, the enzyme reaction solution (saccharified solution) after the enzyme reaction may be subjected to solid-liquid separation to separate a liquid fraction. The liquid fraction may be supplied to the fermentation step described below. The solid fraction (saccharification residue) can be used as, for example, fuel, fertilizer, etc. The above-mentioned description of solid-liquid separation can be used for the explanation of the solid-liquid separation.

[0063] The method for producing sugar according to the present disclosure may include an alkali treatment step, prior to the saccharification step, in which the solid fraction is contacted with an alkali solution to obtain an alkali-treated solid fraction (alkali-treated product).

[0064] The alkaline solution may be, for example, a solution containing an alkali metal hydroxide (for example, sodium hydroxide, potassium hydroxide, etc.). The concentration of the alkali metal hydroxide may be, for example, 0.1 to 3 mol / L (M), 0.5 to 2.5 M, or 1 to 2 M. The pH of the alkaline solution is not particularly limited, and may be, for example, 12 to 15, or 13 to 14.

[0065] The manner of contacting the solid fraction with the alkaline solution is not particularly limited, but from the viewpoint of processing efficiency, it is preferable to immerse the solid fraction in the alkaline solution. From the viewpoint of processing efficiency, it is preferable that the solid fraction be dried by a drying treatment and then pulverized by a pulverization treatment prior to contacting with the alkaline solution. The drying treatment can be carried out by a known drying method (e.g., hot air drying, vacuum drying, freeze drying, etc.). The pulverization treatment can be carried out by a known pulverization method (e.g., pulverization using a food mill, mill grinding, pulverization using a ball mill, etc.).

[0066] The temperature of the alkaline solution in the alkaline treatment step is not particularly limited, and examples thereof include 60 to 180°C, 80 to 150°C, 100 to 140°C, and 110 to 130°C.

[0067] The treatment time in the alkali treatment step is not particularly limited, and may be, for example, 5 to 100 minutes, 15 to 70 minutes, or 20 to 40 minutes.

[0068] The alkali treatment step may be carried out under pressurized conditions, for example. The pressurized conditions are not particularly limited, and examples thereof include 1 to 4 atmospheres, 1.5 to 3.5 atmospheres, and 2 to 3 atmospheres.

[0069] After contacting the solid fraction with the alkaline solution, for example, a solid-liquid separation treatment (e.g., centrifugation, filtration, standing, etc.) can be performed to obtain an alkali-treated solid fraction.

[0070] Next, the alkali-treated solid fraction is washed with a liquid to obtain a washed solid fraction (washed product) (washing step).

[0071] The liquid used for the washing is not particularly limited, and examples thereof include water (e.g., distilled water, purified water, ultrapure water, etc.) and buffer solutions (e.g., acetate buffer solutions, sodium acetate buffer solutions, phosphate buffer solutions, etc.) from the viewpoint of the efficiency of removing the alkaline solution.

[0072] The method for washing with the liquid is not particularly limited, and can be carried out, for example, by mixing the alkali-treated solid fraction with the liquid, followed by solid-liquid separation (e.g., centrifugation, filtration, standing, etc.), and recovering the washed solid fraction. From the viewpoint of the efficiency of removing the alkaline solution, it is preferable to repeat the washing multiple times. Furthermore, it is preferable to perform the washing until the pH of the washed solid fraction or the liquid fraction after the solid-liquid separation reaches a neutral range (e.g., pH 5 to 8, pH 5.5 to 7.5, pH 6 to 7, etc.). This can reduce the effect of pH on the activity of the saccharifying enzyme in the saccharification step.

[0073] The washed solid fraction can then be subjected to the saccharification step, whereby, for example, the washed solid fraction can be saccharified in the coexistence of the washed solid fraction and the saccharifying enzyme.

[0074] By subjecting the alkali-treated (washed) solid fraction to the saccharification step, some of the components such as cellulose, hemicellulose, and lignin are decomposed and eluted, improving the accessibility of saccharification enzymes, increasing the efficiency of the saccharification reaction, and producing more sugars. Therefore, in the biofuel production method described below, the efficiency of biofuel production can be further improved, and more biofuel can be produced.

[0075] As described above, sugar can be produced from plants or parts thereof by the method for producing sugar disclosed herein.

[0076] [Method for Producing Biofuel] In another aspect, the present disclosure provides a method for producing biofuel from a plant or part thereof, comprising producing sugar from the plant or part thereof using the method for producing sugar of the present disclosure, and producing biofuel from the sugar.

[0077] In the production step, a target biofuel is produced from sugar. The method for producing the target biofuel from sugar can be set, for example, depending on the target biofuel. The target biofuel can be produced, for example, directly or indirectly from the sugar. If the target biofuel cannot be produced directly from the sugar, i.e., if the target biofuel is produced indirectly, the target biofuel may be produced, for example, by producing another biofuel such as ethanol, which is the biofuel, or an intermediate product from the sugar, and using the other biofuel or intermediate product. Specific examples of the production step will be described below, taking the production of ethanol from sugar, the production of butanol from sugar, the production of jet fuel from sugar, and the production of diesel fuel from sugar as examples.

[0078] <Production of Ethanol and Production of Jet Fuel / Diesel Fuel Using the Same> The production of ethanol from sugar can be carried out using, for example, an enzyme or a microorganism. In this case, the production step includes, for example, a step of producing ethanol by fermenting the sugar.

[0079] In the fermentation, for example, a microorganism capable of converting C5 sugars and / or C6 sugars into ethanol is added to the saccharified solution or a liquid fraction thereof obtained in the saccharification step (fermentation solution). Examples of the microorganism include yeast, bacteria, and fungi. As a result, in the fermentation, for example, the C5 sugars and / or C6 sugars in the liquid fraction can be converted into ethanol by the action of the microorganism. From the viewpoint of ethanol production efficiency, the microorganism is preferably yeast. The microorganism may be, for example, a recombinant microorganism that has been genetically engineered to be able to convert C5 sugars and / or C6 sugars into ethanol. For the fermentation, a known method for ethanol fermentation of sugars, such as that described in JP 2013-42727 A, can be used.

[0080] In the production step, after the fermentation, for example, the fermentation liquid may be subjected to solid-liquid separation to separate a liquid fraction containing ethanol. In the solid-liquid separation, for example, a solid fraction (saccharification residue) may also be separated. The solid fraction can be used, for example, as fuel, fertilizer, etc. The above-mentioned description of solid-liquid separation can be used for the explanation of the solid-liquid separation.

[0081] In the production step, ethanol may be distilled from the fermentation liquor after the fermentation or the liquid fraction after the solid-liquid separation. For example, the distillation may involve distilling the fermentation liquor or the liquid fraction to purify ethanol (distilled liquid). For example, a known method for ethanol distillation, such as that described in JP 2013-42727 A, can be used for the distillation. The distilled liquid can be dehydrated using a dehydrating agent such as synthetic zeolite to obtain absolute ethanol.

[0082] The ethanol (bioethanol) can be used, for example, as a substitute for commonly used ethanol, or can be added as an additive to gasoline.

[0083] In the production step, for example, jet fuel (biofuel) and / or diesel fuel (biofuel) may be further produced from the ethanol. The jet fuel and / or diesel fuel can be produced from the ethanol by chemical synthesis using a catalyst, for example, as described in International Publication No. 2019 / 084518.

[0084] The reaction conditions for the chemical synthesis can be appropriately selected depending on the type of catalyst. The chemical synthesis can be carried out in a reactor under predetermined conditions of reaction temperature, pressure, nitrogen, and ethanol. The reaction temperature is, for example, 200 to 500°C, 250 to 450°C, or 300 to 400°C. The pressure is, for example, 500 to 1000 Psi, 550 to 950 Psi, or 600 to 900 Psi. The flow rate of nitrogen is, for example, 20 to 200 cc / min, 30 to 150 cc / min, or 40 to 100 cc / min. The flow rate of ethanol is, for example, 0.1 to 1.0 mL / min, 0.2 to 0.9 mL / min, or 0.3 to 0.8 mL / min.

[0085] The catalyst may be reduced, for example, prior to the reaction, under predetermined reaction temperature, pressure, and hydrogen conditions. The reaction temperature is, for example, 200 to 600°C, 250 to 550°C, or 300 to 500°C. The pressure is, for example, 500 to 900 Psi, 550 to 800 Psi, or 600 to 750 Psi. The hydrogen flow rate is, for example, 20 to 200 cc / min, 30 to 150 cc / min, or 40 to 100 cc / min.

[0086] In the production step, for example, the ethanol (C2) may be converted into ethylene (C2) by dehydration in the presence of a first catalyst, and then polymerized in the presence of a second catalyst to convert it into the C8 to C16 jet fuel and / or the C9 to C22 diesel fuel. Examples of the first catalyst include a 0.5% La-2% H-ZSM-5 catalyst. Examples of the second catalyst include a Ziegler-Natta catalyst, a chromium diphosphine catalyst, and a zeolite. The first and / or second catalyst may be homogeneous or heterogeneous.

[0087] <Production of Butanol and Production of Jet Fuel / Diesel Fuel Using the Same> The production of butanol from sugar can be carried out using, for example, a microorganism. In this case, the production step includes, for example, a step of producing butanol by fermenting the sugar.

[0088] In the fermentation, for example, a microorganism capable of converting C5 sugars and / or C6 sugars into butanol (e.g., isobutanol, n-butanol) is added to the saccharified solution obtained in the saccharification step or a liquid fraction thereof. Examples of the microorganism include butanol-producing bacteria. The butanol-producing bacteria can be prepared, for example, by introducing genes constituting a butanol biosynthetic pathway into the microorganism and transforming it. The transformant can be prepared by referring to, for example, JP 2009-039031 A, JP 2009-183259 A, and WO 2010 / 113832 A.

[0089] In the production step, for example, jet fuel (biofuel) and / or diesel fuel (biofuel) may be further produced from the butanol. The jet fuel and / or diesel fuel can be produced from the butanol by chemical synthesis using a catalyst, for example, with reference to International Publication No. 2019 / 084518. The reaction conditions for the chemical synthesis can be the same as those described above for ethanol.

[0090] In the production step, for example, the butanol (C4) (e.g., isobutanol) is dehydrated in the presence of a first catalyst to produce butenes (C4 olefins), and the butenes are polymerized (oligomerized) in the presence of a second catalyst to produce the C8 to C16 jet fuel and / or the C9 to C22 diesel fuel, which are C4 olefin oligomers. Examples of the first catalyst include γ-alumina catalyst, ZSM-5 zeolite, Y-type zeolite, and Amberlyst acidic resin. Examples of the second catalyst include Group 4 transition metals, Nafion catalyst, and Pd / C-K. ３ P.O. ４ The first and / or second catalyst may be a homogeneous or heterogeneous catalyst. The resulting C4 olefin oligomers may be converted to paraffins, for example, by hydrogenation.

[0091] The jet fuel (biojet fuel) is a mixture of different hydrocarbons, such as linear or branched C8 to C16 alkanes. The jet fuel contains, for example, olefins, substituted or unsubstituted cycloalkanes (e.g., cyclopentane, cyclohexane, etc.), aromatic compounds (e.g., benzene, toluene, naphthalene, etc.), mono-substituted aromatic compounds (e.g., methylbenzene, etc.), di-substituted aromatic compounds (e.g., xylene, etc.), and / or poly-substituted aromatic compounds (e.g., trimethylbenzene, etc.). The jet fuel is also commonly referred to as sustainable aviation fuel (SAF).

[0092] The diesel fuel is a mixture of hydrocarbons, such as C9 to C22 hydrocarbons, aromatic hydrocarbons, and olefinic hydrocarbons. The diesel fuel may contain additives, such as alkyl nitrates (e.g., 2-ethylhexyl nitrate) and di-tert-butyl peroxide, to increase the cetane number. The cetane number is an indicator of the combustion speed of diesel.

[0093] <Production of Diesel Fuel> In addition to the method of producing diesel fuel from sugar using ethanol or butanol as an intermediate product, the production process may be carried out using the sugar and microalgae. In this case, the production process includes, for example, culturing the sugar obtained from the saccharification process and microalgae to produce the diesel fuel (biodiesel fuel). Microalgae are suitable for producing diesel fuel because they have a higher efficiency in producing organic matter through photosynthesis than plants and can be cultured at high density in a short period of time. The microalgae may be, for example, microalgae with a high content of paraffin-based oils and fats. Examples of the microalgae include Euglena and cyanobacteria. The microalgae can be cultured using known methods for microalgae, for example.

[0094] The medium for culturing the microalgae is not particularly limited, and a medium commonly used for culturing the microalgae can be used, and specific examples thereof include various nutrient salts, trace metal salts, vitamins, etc. Examples of the nutrient salts include nitrogen sources such as NaNO3, KNO3, NH4Cl, and urea, and phosphorus sources such as K2HPO4, KH2PO4, and sodium glycerophosphate. Examples of the trace metals include iron, magnesium, manganese, calcium, copper, and zinc. Examples of the vitamins include vitamin B1, vitamin B2, and vitamin B 6、 Vitamin B 12 In addition, the medium is preferably heat sterilized, preferably at 75 to 121°C for 15 to 60 minutes, or filtered through a filter of 0.2 µm or less, for example, in order to prevent growth inhibition of the microalgae by microorganisms present in the medium.

[0095] The microalgae are cultured by, for example, uniformly irradiating the microalgae with light necessary for the photoreaction system in photosynthesis of the microalgae in order to enhance the photosynthetic ability of the microalgae. Furthermore, the microalgae are preferably cultured by, for example, stirring a culture solution containing the microalgae to uniformly irradiate the microalgae with light.

[0096] In the cultivation of the microalgae, for example, sugars produced by the sugar production method of the present disclosure are added to the medium as a carbon source. Examples of the sugars include hexasaccharides and pentasaccharides such as glucose, fructose, xylose, and xylitol, disaccharides or polysaccharides such as sucrose, maltose, trehalose, and inulin, and combinations thereof.

[0097] In the production process, the microalgae cultured in this manner are dried, the cell membranes are broken down, and then the lipids are extracted using a non-polar organic solvent such as hexane or ether, thereby producing the diesel fuel. The diesel fuel thus obtained is preferably further refined. Various separation methods, such as precipitation separation, column separation, liquid-phase separation, gravity separation, and distillation separation, can be used for the purification.

[0098] As described above, the biofuel production method of the present disclosure can produce biofuel from plants or parts thereof.

[0099] The (1) decomposition step, the first separation step, and the (2) second separation step can be suitably carried out using, for example, the following plant tissue treatment device.

[0100] As described above, when extracting (producing) desired cells C and an extract and / or extract solution (hereinafter simply referred to as extract E) containing desired components from plant tissue (subject) T, it is necessary to efficiently separate the cells C, extract E, etc. from the cell walls M and cell membranes of the cells. Below, a description will be given of a plant tissue treatment device 1A that can efficiently separate the cells C and extract E from the cell walls M and cell membranes of the cells to extract the cells, desired extract, etc.

[0101] <Plant Tissue Treatment Apparatus 1A> As shown in FIG. 1(A), the plant tissue treatment apparatus 1A includes a container 2 and a separation apparatus 3.

[0102] (Storage Container 2) The storage container 2 is a container that stores a mixed liquid L (hereinafter sometimes simply referred to as the mixed liquid L) obtained by mixing the enzyme-containing liquid L1 (hereinafter sometimes simply referred to as the liquid L1) with plant tissue T. This storage container 2 is a container formed, for example, from a metal such as stainless steel, steel, or plated copper plate, or a resin such as acrylic, FRP, polypropylene, polyvinyl chloride (PVC), polyethylene, or ABS, and has the property of not affecting the mixed liquid L, the plant tissue T, cells isolated from the plant tissue T, extracts extracted from the plant tissue T, etc. In other words, even when the mixed liquid L or the plant tissue T is stored in the storage space 2h of the storage container 2 and various processes are performed, the storage container 2 is made of a material that does not react with the liquid L1 (e.g., the enzymes contained in the liquid L1), the plant tissue T, cells isolated from the plant tissue T, extracts extracted from the plant tissue T, etc., or is not eroded by the liquid L1 (e.g., the enzymes contained in the liquid L1). The size of the container 2 is not particularly limited, as long as it is large enough to appropriately perform the decomposition process of the plant tissue T contained in the mixed liquid L and the extraction process of the extract, etc.

[0103] Furthermore, the storage container 2 may have a structure that allows the storage space 2h to be kept airtight and liquid-tight from the outside. In other words, the storage container 2 may have a structure that allows the storage space 2h to be kept airtight and liquid-tight from the outside when treating the mixed liquid L described below. For example, the storage container 2 may be a bottomed cylindrical container with an opening at the top, and a lid may be attached to the opening via a packing or the like so that the storage space 2h can be kept airtight and liquid-tight from the outside. Note that, as long as there is no problem with treating the mixed liquid L in an open-to-air state, the storage container 2 does not necessarily have to have a structure that allows the storage space 2h to be kept airtight and liquid-tight from the outside.

[0104] (Separation Device 3) As shown in Figure 1, the separation device 3 includes an agitator 4 that agitates the mixed liquid L in the storage space 2h of the storage container 2 and applies a decomposing force to the plant tissue T to decompose the plant tissue T. The agitator 4 has a drive source 4a such as a motor, a rotating shaft 4b connected to the main shaft of the drive source 4a, and an agitator blade 4c provided at the tip of the rotating shaft 4b. The agitator blade 4c of the agitator 4 is disposed within the storage space 2h of the storage container 2. For example, the agitator blade 4c of the agitator 4 is disposed within the storage space 2h of the storage container 2 so that the central axis CL of the storage space 2h of the storage container 2 and the central axis of the rotating shaft 4b coincide with each other.

[0105] In this specification, the concept that the central axis CL of the storage space 2h and the central axis of the rotation shaft 4b are aligned not only refers to the case where the central axis CL of the storage space 2h and the central axis (rotation axis) of the rotation shaft 4b are coaxial, but also includes a state where the central axis of the rotation shaft 4b is slightly misaligned in a direction intersecting the central axis CL of the storage space 2h, or a state where the central axis of the rotation shaft 4b is slightly tilted with respect to the central axis CL of the storage space 2h.

[0106] The stirring blade 4c of the stirring device 4 has a shape such that its leading edge e, i.e., the edge located at the front of the rotation direction when rotating, can apply a shear force to the plant tissue T, etc. In other words, the stirring device 4 has the function of a shearing device (shearing machine) that can perform a process (shearing process) to mechanically shear the cell walls M, cells, etc. of the plant tissue T. For example, when the rotating stirring blade 4c comes into contact with the cell walls M or cells of the plant tissue T, the leading edge e of the stirring blade 4c is shaped to form a wound S, such as an incision, in the cell walls M or cell membranes of the cells (hereinafter, the cell walls M and the cell membranes may be collectively referred to as the cell walls M, etc.) (see FIG. 1(A)). Furthermore, when the cell walls M, etc. are damaged or when the cell walls M, etc. are decomposed by enzymes contained in the liquid L1, the leading edge e of the stirring blade 4c is shaped to separate the cells C and extract E of the plant tissue T from the cell walls M.

[0107] The tip edge e of the stirring blade 4c corresponds to a shear blade.

[0108] (Processing of plant tissue T using plant tissue processing device 1) In the case of the plant tissue processing device 1 having the structure described above, by intermittently processing the mixed liquid L, it is possible to separate the cell walls M, etc. from the cells C and extract E from the plant tissue T.

[0109] First, a predetermined amount of liquid L1, whose pH and enzyme concentration have been adjusted to predetermined states, is poured into the storage space 2h of the storage container 2, and the drive source 4a of the agitator 4 is operated to agitate the liquid L1 with the agitating blades 4c. In this state, the plant tissue T is poured into the liquid L1 in the storage space 2h of the storage container 2, and a mixed liquid L of the two is prepared in the storage space 2h of the storage container 2.

[0110] Here, when the plant tissue T is mixed with the liquid L1 while the drive source 4a of the agitator 4 is in operation, the following situations 1) to 4) occur simultaneously or in combination in the mixed liquid L, and the cell walls M, etc., the cells C, and the extract E are separated from the plant tissue T, and the cell walls M, etc., the cells C, and the extract E can be suspended in the mixed liquid L. When the liquid extract of the extract E flows out from the cells C, it may be suspended in the mixed liquid L or may be mixed into the mixed liquid L.

[0111] 1) As the mixing of the liquid L1 and the plant tissue T progresses due to the rotation of the stirring blade 4c, the enzymes contained in the liquid L1 come into contact with the plant tissue T, and the enzymes decompose the cell walls M and the like of the plant tissue T. As a result, in some of the plant tissue T, the cells C and extract E of the plant tissue T separate from the cell walls M and the like and flow out from the areas where the cell walls M and the like have been decomposed. In some of the plant tissue T, the cells C and extract E do not separate from the cell walls M and the like, and the plant tissue T in a state where the cells C and extract E are connected to the cell walls M and the like and exposed from the cell walls M and the like (sometimes referred to as unseparated plant tissue T1) floats in the mixed liquid L.

[0112] 2) The rotation of the stirring blades 4c generates a water current in the mixed liquid L, which promotes mixing of the plant tissue T, non-separated plant tissue T1, etc. with the liquid L1. Furthermore, the force of the water current in the mixed liquid L is applied to the biological cells T, non-separated plant tissue T1, etc. When the force from the water current is applied to the non-separated plant tissue T1, cells C and extract E are separated from the cell walls M, etc. of some of the non-separated plant tissue T1. Note that in the non-separated plant tissue T1 where cells C and extract E are not separated from the cell walls M, etc. even when force is applied from the water current, the non-separated plant tissue T1 remains as non-separated plant tissue T1 and floats in the mixed liquid L.

[0113] 3) The rotation of the impeller 4c brings the impeller 4c into contact with the plant tissue T in the mixed liquid L. When the plant tissue T1 comes into contact with the impeller 4c, among the biological cells T that come into contact with the leading edge e of the impeller 4c, incisions S are formed in the cell walls M, etc., of the biological cells T that come into contact with the leading edge e at an appropriate angle. In other words, a shear force is applied from the leading edge e of the impeller 4c to the cell walls M, etc., causing the incisions S. If the incisions S formed at this time are deep, the cells C and extract E that were covered by the cell walls M, etc. may leak out from the cell walls M, etc. at the incisions S, or the cells C and extract E may not be separated from the cell walls M, etc., i.e., the cells C and extract E may be exposed in the state of unseparated plant tissue T1. Furthermore, even if the incisions S are shallow, if an enzyme penetrates the cell walls M, etc. through the incisions S, the enzyme can decompose the cell walls M, etc., which could not be decomposed without the incisions S. In other words, the rotation of the stirring blades 4c can promote the decomposition of cell walls M and the like.

[0114] 4) When non-separated plant tissue T1 is present in the mixed liquid L, the rotation of the impeller 4c causes the impeller 4c to come into contact with the non-separated plant tissue T1 in the mixed liquid L. When the non-separated plant tissue T1 comes into contact with the impeller 4c, a shear force is applied to the non-separated plant tissue T1 with which the leading edge e of the impeller 4c comes into contact at an appropriate angle, separating the cells C and extract E from the cell walls M, etc. As a result, the cell walls M, etc. and the cells C and extract E separated from the cell walls M, etc., float in the mixed liquid L.

[0115] As described above, by using the plant tissue treatment device 1A, it is possible to separate the cell walls M and the like from the cells C and the extract E from the plant tissue T. In other words, it is possible to extract the cells C and the extract E from the plant tissue T.

[0116] (Regarding Operation Control of Plant Tissue Treatment Apparatus 1A) In the plant tissue treatment apparatus 1A, an operator can check the state of the mixed solution L in the storage space 2h of the storage container 2, i.e., the treatment state of the plant tissue T, and adjust the operation or stop of the agitator 4 and the rotation speed of the agitator blades 4c, etc. In this case, the state of the mixed solution L may be visually checked, and the rotation speed of the agitator blades 4c may be adjusted or agitation may be stopped (i.e., treatment may be completed).

[0117] Furthermore, without checking the state of the mixed liquid L, it is also possible to determine that the processing is complete when a certain amount of time has passed since the start of stirring or the addition of the plant tissue T, based on the results of preliminary experiments, etc., and to stop stirring by the stirring device 4 by operating a timer or by the operator, thereby completing the processing.

[0118] Alternatively, a sensor 10s may be provided to monitor the state of the mixed solution L, and the rotation speed of the agitator blades 4c may be adjusted or agitation may be stopped (i.e., processing may be terminated) based on a signal from the sensor 10s. For example, a control unit 10 may be provided in the plant tissue processing device 1A, and the control unit 10 may control the operation of the agitator 4 based on a signal from the sensor 10s (see FIG. 1B). In this case, examples of the sensor 10s that monitors the state of the mixed solution L, i.e., the processing state of the plant tissue T, include a viscometer, an image sensor (using a camera to perform AI analysis of the separation state and color), a pH sensor, and a thermometer. In addition to monitoring the processing state of the plant tissue T, signals obtained from the pH sensor, thermometer, and the like can also be used as information for adjusting the state of the mixed solution L when the control unit 10 controls the operation of the electrolyte supply device 12, the enzyme addition unit 13, the heater for adjusting the temperature of the mixed solution L, and the like, as described below. In addition, in order for the control unit 10 to control the operating state of the agitator 4, it is desirable to provide a load sensor, such as a wattmeter, strain gauge, or rotation speed sensor, for the agitator motor, which is the driving source 4a, as a sensor for grasping the operating state of the agitator 4.

[0119] (Method of Supplying Plant Tissue T) In the above example, a case has been described in which an amount of plant tissue T corresponding to the amount of liquid L1 (in other words, the amount of enzyme) in the storage space 2h of the storage container 2 is supplied all at once to the storage space 2h of the storage container 2. The plant tissue T may be supplied in small amounts to the storage space 2h of the storage container 2. When the plant tissue T is supplied in small amounts to the storage space 2h of the storage container 2, it becomes easier to disperse the plant tissue T in the liquid L1, and it becomes easier to adjust the mixed state of the liquid L1 and the plant tissue T, i.e., the state of the mixed liquid L, to a good state.

[0120] 1(B), a plant tissue supplying unit 11 may be provided that supplies plant tissue T as needed, so that an appropriate amount of plant tissue T is supplied according to the state of the mixed liquid L. The supply of plant tissue T by the plant tissue supplying unit 11 can be controlled by an operator based on the state of the mixed liquid L (for example, by visual inspection or based on a signal from a sensor that grasps the state of the mixed liquid L).

[0121] Furthermore, a control unit 10 may be provided, and based on a signal from a sensor that detects the state of the mixed solution L as described above, the control unit 10 may control the operation of the plant tissue supply unit 11 to supply the plant tissue T. In this case, if the control unit 10 controls the operating state of the stirring device 4 in accordance with the amount of plant tissue T supplied by the plant tissue supply unit 11, the plant tissue T can be processed more appropriately.

[0122] (Method of adjusting liquid L1) In the above example, a case was described in which liquid L1 prepared in advance to a predetermined state was supplied into the storage space 2h of the storage container 2, but it may also be prepared so that it becomes a predetermined state within the storage space 2h of the storage container 2.

[0123] 1(B), by providing an enzyme addition unit 13 that supplies an electrolyte solution adjusted to a predetermined pH to the storage space 2h of the storage container 2 and an electrolyte solution supply device 12 that supplies an enzyme to the storage space 2h of the storage container 2, it is possible to prepare the liquid L1 in the storage space 2h of the storage container 2 so that it is in a predetermined state. Moreover, by supplying the enzyme and electrolyte solution appropriately while the mixed solution L is being treated in the plant tissue treatment device 1A, the state of the mixed solution L can be stabilized. In particular, when the plant tissue T is supplied to the storage space 2h of the storage container 2 in small amounts, the enzyme and electrolyte solution can be supplied according to the supply state and treatment state of the plant tissue T, so that the plant tissue T can be treated appropriately and effectively.

[0124] In this case, it is desirable that the electrolyte solution supply device 12 has an electrolytic water production device 12a that generates an electrolyte solution of a predetermined pH and a reserve tank 12b that stores the electrolyte solution generated by the electrolytic water production device 12a. With this configuration, the electrolyte solution can be stably supplied from the reserve tank 12b to the storage space 2h of the storage container 2. Furthermore, if the reserve tank 12b is provided with a function for adjusting the state of the stored electrolyte solution, the state of the electrolyte solution supplied to the storage space 2h of the storage container 2 can be made more stable. In this case, the reserve tank 12b corresponds to the adjustment unit.

[0125] (Method for Separating Cell Walls M, etc. from Extract E, etc.) The cell walls M, etc., cells C, and extract E (hereinafter, cells C and extract E may be collectively referred to as extract E, etc.) separated by the plant tissue treatment device 1A are recovered from the mixed liquid L. In order to efficiently recover the cell walls M, etc. and the extract E, etc., it is desirable to separate the cell walls M, etc. from the extract E, etc., and then recover them separately. The separation can be carried out by passing the mixed liquid L through a filtration member such as a filter or a mesh member. In other words, the cell walls M, etc. and the extract E, etc. can be separated by passing the mixed liquid L through a filtration member having openings or voids that do not allow the cell walls M, etc. to pass through but that allow the extract E, etc. to pass through together with the mixed liquid L.

[0126] For example, when the plant tissue T is tea leaves, the mixed liquid L processed by the plant tissue treatment device 1A is passed through a mesh with openings (e.g., 1.5 to 3 mm, preferably about 2 mm) that are large enough to allow the cell walls of the tea leaves to pass through but not the extracts (e.g., insoluble proteins, insoluble amino acids, etc.). As a result, the cell walls can be collected on the mesh, and the extract can be separated from the cell walls while still contained in the mixed liquid L. The extract in the mixed liquid L that has been separated from the cell walls can be collected by methods such as concentration separation, freeze concentration spray drying, evaporation, etc.

[0127] The method for passing the mixed liquid L through the filtration member to separate the cell walls M, etc. from the extract E, etc. is not particularly limited, and any method may be employed. For example, the mixed liquid L may be discharged to the outside from the opening of the storage container 2 by tilting the storage container 2, and then the mixed liquid L may be supplied to the filtration member to separate the cell walls M, etc. from the extract E, etc.

[0128] In addition, an outlet 2b equipped with a valve or the like that blocks communication between the inside of the storage space 2h and the outside may be provided near the bottom of the storage container 2, and the mixed liquid L discharged from the outlet 2b may be supplied to a recovery device such as a filtering member, a cyclone, a centrifugal separator, a mesh (a mesh made of stainless steel or the like), or a membrane, to separate the cell walls M, etc. from the extract E, etc.

[0129] When separating cell walls M, etc. from extracts E, etc. using a filtering member, if the filtering member is installed within the storage space 2h of the storage container 2, the task of separating cell walls M, etc. from extracts E, etc. becomes easier.

[0130] For example, as shown in Figure 1, a filter member 6 is provided near the bottom of the storage space 2h of the storage container 2, and an outlet 2b equipped with a valve or the like that blocks communication between the storage space 2h and the outside is provided below the filter member 6. Then, after the treatment of the mixed liquid L is completed, the outlet 2b can be opened to allow the mixed liquid L to flow out through the outlet 2b and be recovered. At this time, the mixed liquid L passes through the filter member 6, so that the cell walls M and the like in the mixed liquid L can be captured by the filter member 6. In other words, the cell walls M and the like can be separated from the extract E.

[0131] 1(B) , if a component recovery section 7 such as activated carbon or montmorillonite is provided in the storage space 2h of the storage container 2, components of the extract E, such as caffeine and catechin, can be adsorbed and recovered by the component recovery section 7 while treating the mixed liquid L in the storage space 2h of the storage container 2. In this way, recovering the caffeine and catechin components by the component recovery section 7 while treating the mixed liquid L has the advantage that caffeine, catechin, etc. can be easily isolated and recovered in the pre-treatment stage of extraction.

[0132] In particular, if caffeine can be removed in the pretreatment stage (i.e., before the extract is recovered), it will be easier to use the extract E, etc. in certain products, etc. For example, when the recovered extract E, etc. is used in soft drinks where excessive caffeine intake is a problem, or when the extract E, etc. (e.g., protein, etc.) is added to livestock feed, it is necessary to remove caffeine from the extract E, etc. before using the extract E, etc. However, if caffeine is recovered by the component recovery unit 7 during processing in the storage space 2h of the storage container 2, it may be unnecessary to separately perform a process to remove caffeine from the extract E, etc. For example, if caffeine can be completely removed (or to an extent that does not affect the quality, etc., of the extract E) in the component recovery unit 7, it may be unnecessary to separately perform a process to remove caffeine from the recovered extract E. Furthermore, even if the caffeine cannot be completely removed by the component recovery section 7 (or to an extent that does not affect the quality of the extract E, etc.), if the caffeine is removed to a certain extent by the component recovery section 7, the efficiency of caffeine removal can be significantly increased when the caffeine removal process is carried out in the post-processing stage (processing after the extract E is recovered), thereby making it easier to use the extract E, etc.

[0133] 2A, the plant tissue processing device 1D is provided with a crushing device 40 such as a crushing pump. Specifically, the supply flow path 21 of the supply unit 20 is connected to the inlet of the crushing device 40, and the return flow path 31 of the return unit 30 is connected to the outlet of the crushing device 40.

[0134] In the case of the plant tissue processing device 1D having the above-described configuration, by passing the mixed liquid L through the crushing device 40 in the following order: storage container 2B → supply flow path 21 of the supply section 20 → crushing device 40 → return flow path 31 of the return section 30 → storage container 2B, the separation process within storage container 2B and the separation process by the crushing device 40 can be carried out continuously.

[0135] Furthermore, in the plant tissue processing device 1D, a small amount of the mixed liquid L can be subjected to separation processing in the crushing device 40, so that shear force can be effectively applied to the plant tissue T and the unseparated plant tissue T1. In other words, processing for separating the extract E and the like from the cell walls M and processing for incising the cell walls M and the like can be effectively performed.

[0136] If the homogenizer 40 is a homogenizer pump or the like having a pumping function, a liquid delivery device need not be provided in the supply flow path 21 of the supply unit 20 or the return flow path 31 of the return unit 30. If a device without a liquid delivery function is used as the homogenizer 40, a liquid delivery device is provided in both or either the supply flow path 21 of the supply unit 20 and the return unit 30. Even if the homogenizer 40 has a liquid delivery function, a liquid delivery device may be provided in both or either the supply flow path 21 of the supply unit 20 and the return unit 30 to stabilize the liquid delivery.

[0137] (Continuous Processing) In the case of the plant tissue treatment device 1D having the above-described configuration, if a plurality of storage containers 2B are provided and the storage container 2B that supplies the mixed liquid L to the crushing device 40 is switched, it becomes possible to continuously treat the mixed liquid L. Hereinafter, an apparatus provided with a plurality of storage containers 2B will be referred to as a plant tissue treatment device 1E.

[0138] As described above, in the plant tissue processing device 1D, the mixed solution L is in one of two states: an enzyme processing state in which only the enzyme processing is performed in the storage container 2, and a separation processing state in which the mixed solution L is continuously subjected to separation processing in the crushing device 40. In the following description, the case in which both the enzyme processing state and the separation processing state are performed, that is, the state in which the processing by the above-described plant tissue processing device 1D is performed, may be simply referred to as "processing."

[0139] 2B, the supply unit 20 includes a plurality of processing vessel-side supply channels 21a each connected to the lower portion of each storage vessel 2B, and a crusher-side supply channel 21d connected to the inlet of the crusher 40. A supply-side switching mechanism 25 is provided between the processing vessel-side supply channels 21a and the crusher-side supply channel 21d, for switching which of the processing vessel-side supply channels 21a communicates with the crusher-side supply channel 21d. For example, a general branch valve having a function of switching the connected channels may be provided as the supply-side switching mechanism 25.

[0140] In addition, the supply side switching mechanism 25 may have the function of blocking the connection between the multiple processing vessel side supply flow paths 21a and the crushing device side supply flow path 21d, that is, the function of completely blocking the connection between the multiple storage vessels 2B and the crushing device 40.

[0141] On the other hand, the return section 30 is provided with a plurality of treatment vessel side return flow paths 31b, each of which is connected to the upper part of each storage vessel 2B. Also, a crusher side return flow path 31d is provided, which is connected to the outlet of the crusher 40. Then, between the plurality of treatment vessel side return flow paths 31b and the crusher side return flow path 31d, a return side switching mechanism 35 is provided for switching the flow path, among the plurality of treatment vessel side return flow paths 31b, that is connected to the crusher side return flow path 31d. For example, a general branch valve having the function of switching the connected flow paths is provided as the return side switching mechanism 35.

[0142] The return-side switching mechanism 35 may have a function of blocking communication between the plurality of processing vessel-side return paths 31b and the crushing device-side return path 31d, that is, a function of completely blocking communication between the plurality of storage vessels 2B and the crushing device 40. The processing vessel-side return path 31b may be provided with a first return path 32b (not shown) and a second return path 33b (not shown).

[0143] With the above-described configuration, by switching the storage container 2B communicating with the crushing device 40 using the supply-side switching mechanism 25 and the return-side switching mechanism 35, the mixed liquid L in the plurality of storage containers 2B can be sequentially switched to treat the mixed liquid L. In other words, the mixed liquid L can be treated in a state in which both the enzyme treatment state and the separation treatment state are realized almost continuously.

[0144] Specifically, the supply flow path 21a on the treatment vessel side of one storage vessel 2B is connected to the supply flow path 21d on the crushing device side by the supply-side switching mechanism 25, and the return flow path 31b on the treatment vessel side of the same storage vessel 2B is connected to the supply flow path 21d on the crushing device side by the return-side switching mechanism 35. Then, the mixed liquid L stored in the one storage vessel 2B can be treated while circulating the mixed liquid L between the one storage vessel 2B and the crushing device 40.

[0145] Then, when the processing of the mixed liquid L contained in one storage vessel 2B is completed, the supply-side switching mechanism 25 switches the processing vessel-side supply flow path 21a, which communicates with the crushing device-side supply flow path 21d of the homogenizer 40, from the processing vessel-side supply flow path 21a of the one storage vessel 2B to the processing vessel-side supply flow path 21a of the other storage vessel 2B. At the same time, the return-side switching mechanism 35 switches the processing vessel-side return flow path 31a, which communicates with the crushing device-side return flow path 31d of the homogenizer 40, from the processing vessel-side return flow path 31a of the one storage vessel 2B to the processing vessel-side return flow path 31a of the other storage vessel 2B. In other words, the return-side switching mechanism 35 connects the processing vessel-side return flow path 31a of the storage vessel 2B, to which the crushing device-side supply flow path 21d and the processing vessel-side supply flow path 21a are connected, with the homogenizer-side return flow path 31d of the homogenizer 40. Then, the mixed liquid L stored in the other storage container 2B can be treated while circulating between the other storage container 2B and the crushing device 40.

[0146] By repeating the above operation and sequentially switching the storage container 2B that is in communication with the crushing device 40, the mixed liquid L stored in each storage container 2B can be processed sequentially.

[0147] On the other hand, if the treatment with the mixed liquid L is to be carried out for a relatively long period of time, the plant tissue treatment device 1E may be operated as follows.

[0148] For example, when the processing of the mixed liquid L contained in one storage container 2B is completed, the first storage container 2B is separated from the homogenizer 40, and then the mixed liquid L is discharged from the discharge port 2b provided in the first storage container 2B and recovered. Next, a new mixed liquid L is supplied to the first storage container 2B from which the processed mixed liquid L has been discharged. Then, when the first storage container 2B is next connected to the homogenizer 40, the new mixed liquid L contained in the first storage container 2B can be processed. In other words, if the processed mixed liquid L is discharged and a new mixed liquid L is supplied from multiple storage containers 2B, the supply-side switching mechanism 25 and the return-side switching mechanism 35 can be used to sequentially switch the storage containers 2B connected to the homogenizer 40, thereby making it possible to continuously process the mixed liquid L for a long period of time even if the number of storage containers 2B is limited.

[0149] In addition, if a filter member 6 is provided in each storage container 2B, when the mixed liquid L is collected, the cell walls M and the like are collected from the filter member 6.

[0150] Furthermore, when a filtration member 6 is provided in each storage vessel 2B, the processing vessel-side supply flow path 21a is connected to a position above the filtration member 6. In other words, the processing vessel-side supply flow path 21a is installed so that the mixed liquid L to be supplied from each storage vessel 2B to the separation tank 4T is collected from a position above the filtration member 6.

[0151] Furthermore, the storage container 2B connected to the crushing device 40 may be switched by an operator after checking the processing state of the mixed liquid L in each storage container 2B, or may be switched by the operator at regular intervals. Furthermore, when the control unit 10 is provided, the processing state in each storage container 2B may be checked by a sensor or the like, or the control unit 10 may switch the storage container 2B connected to the crushing device 40 at regular intervals.

[0152] <Multiple Separation Tanks 4T and Multiple Crushing Devices 40> In the above example, a case has been described in which a single separation tank 4T or a single crushing device 40 is connected to a plurality of storage containers 2B. However, a plurality of separation tanks 4T or multiple crushing devices 40 may be provided, and the plurality of storage containers 2B may be connected to the plurality of separation tanks 4T or multiple crushing devices 40, respectively. That is, each separation tank 4T or each crushing device 40 may be switchably connected to the plurality of storage containers 2B via a supply-side switching mechanism 25 and a return-side switching mechanism 35. For example, as shown in FIG. 3, a plurality of separation tanks 4T (two in FIG. 3) may be provided, and a plurality of storage containers 2B (two in FIG. 3) may be connected to the plurality of separation tanks 4T or multiple crushing devices 40 via the supply-side switching mechanism 25 and the return-side switching mechanism 35. In this case, if the agitation functions of each separation tank 4T and each crushing device 40 are the same, i.e., if each separation tank 4T and each crushing device 40 are the same, it is possible to simultaneously circulate the mixed solution L in each storage container 2B between the separation tank 4T and the crushing device 40, and perform substantially the same treatment on the mixed solution L in each storage container 2B. On the other hand, the agitation functions of each separation tank 4T and each crushing device 40, i.e., the shear force applied to the plant tissue T in each separation tank 4T or each crushing device 40, may be different. For example, by providing multiple separation tanks 4T having agitators 4 with different sizes and shapes of agitator blades 4c, it is possible to apply different shear forces to the plant tissue T in each separation tank 4T. In this way, by changing the separation tank 4T or the crushing device 40, it is possible to apply appropriate shear forces to the cell walls M, etc., depending on the state of decomposition of the cell walls M, etc., and thus the separation process for separating the cell walls M, etc. from the extract E, etc. can be more effectively performed.

[0153] According to the extract production method and plant tissue treatment device and treatment method, for example, waste (e.g., used tea leaves, etc., waste containing moisture) from the production process of various products (e.g., foods, etc.) can be quickly treated at the same production site (e.g., the same factory) to obtain the extract. This reduces waste that would previously be discarded, and extracts obtained from the waste can be used in various products (e.g., foods, etc.). For example, when using used tea leaves containing moisture as products rather than discarding them, the used tea leaves containing moisture had to be temporarily stored or transported outside the factory, which could lead to spoilage. To prevent spoilage, the used tea leaves containing moisture must be dried, but drying the used tea leaves containing moisture requires significant energy and cost. Furthermore, transporting the used tea leaves containing moisture outside the factory is also costly. In contrast, by installing the plant tissue treatment device of the present disclosure at the product production site, the used tea leaves containing moisture generated during the production process can be directly treated, thereby reducing the cost.

[0154] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents and kits were used according to the attached protocols. In the following description, "mol / l" may also be abbreviated as "M."

[0155] [Processing Flow Overview] An exemplary processing flow for the production method of the extract is shown in Figure 4. First, tea leaves or used tea leaves are subjected to decomposition and separation. The resulting product is then subjected to primary solid-liquid separation using a sieve, cyclone, filter, or the like. This separation allows for the production of vein cellulose fibers (including cellulose microfibers and / or cellulose nanofibers). The resulting product is then subjected to secondary solid-liquid separation using a sieve, cyclone, filter, or the like, to separate the resulting product into a slurry of tea leaves or used tea leaves and a solution. The separated slurry of tea leaves or used tea leaves is then subjected to squeezing (squeezing) to separate the residue (solids) and the solution. Cellulose fibers can also be obtained from the slurry of tea leaves or used tea leaves. For example, while vein cellulose fibers are primarily obtained from the primary solid-liquid separation, cellulose fibers other than vein cellulose fibers (i.e., cellulose fibers finer than vein cellulose fibers) are primarily obtained from the secondary solid-liquid separation. The residue (solid matter) can also be further separated.

[0156] The size of the material separated in the primary solid-liquid separation process may differ from the size of the material separated in the secondary solid-liquid separation process. For example, the material separated in the primary solid-liquid separation process (e.g., leaf vein cellulose fiber) may be larger than the material separated in the secondary solid-liquid separation process. Therefore, a mesh or the like having larger openings may be used in the primary solid-liquid separation process than in the secondary solid-liquid separation process.

[0157] The following examples were carried out according to a simplified processing flow of the above processing flow (Figure 5). First, tea leaves or used tea leaves were subjected to decomposition and separation processing. Next, the processed product obtained by the above processing was subjected to solid-liquid separation processing using a mesh screen to separate the processed product into a slurry of tea leaves or used tea leaves and a solution. The separated slurry of tea leaves or used tea leaves was then subjected to a squeezing (squeezing) processing to separate the solution. Cellulose fibers (including leaf vein cellulose fibers) were also obtained from the separated slurry of tea leaves or used tea leaves.

[0158] Example 1 (Decomposition and Separation Treatment of Tea Leaves) 40 L of water was placed in a 50 L container and heated to 45°C using a liquid heater (model number: WPS-110, Kashima Corporation). 120 mL of cellulase (equivalent to 0.3% of the 40 L volume of water) was added to the water and mixed. 2 kg of the tea leaves were then added to the water and soaked for 24 hours (decomposition treatment). The pH of the reaction solution thus prepared was adjusted to 6.64. The tea leaves used were fresh leaves immediately after plucking from tea plants, which were then degreened for 60 seconds by supplying steam at 100°C while stirring.

[0159] The soaked tea leaves were stirred and crushed for 45 minutes at 4,500 rpm using a homogenizer (Automixer 40, Primix Corporation) (separation treatment). The water temperature was maintained at 45°C by heating with a liquid heater. In this way, processed tea leaves were obtained.

[0160] (Obtaining Tea Leaf Extract) The processed tea leaves were poured onto a commercially available through-sieve (mesh opening: approximately 1.5 mm). The tea leaves remained in a slurry state on the screen, and the tea leaf solution passed through the screen and fell into a receiving container. In this way, the tea leaves in a slurry state and the tea leaf solution were separated and obtained. The tea leaves in a slurry state were then squeezed using an oil press and filtered to obtain a tea leaf solution in a slurry state. That is, the tea leaf solution, the tea leaves in a slurry state, and the tea leaf solution in a slurry state were obtained as the tea leaf extract.

[0161] [Example 2] (Decomposition and separation treatment of used tea leaves) Used tea leaves obtained from general tea beverage production were re-dried and subjected to decomposition and separation treatment in the same manner as in Example 1. The only difference from Example 1 is that used used tea leaves instead of tea leaves.

[0162] (Obtaining an extract of used tea leaves) From the processed tea leaves, a solution of used tea leaves, a slurry of used tea leaves, and a squeezed liquid of the slurry of used tea leaves were obtained as extracts of used tea leaves, in the same manner as in Example 1. The only difference from Example 1 is that processed tea leaves were used instead of processed tea leaves.

[0163] [Comparative Example 1] (Decomposition Treatment of Tea Leaves) The tea leaves were subjected to a decomposition treatment in the same manner as in Example 1. The only difference from Example 1 is that no separation treatment was carried out.

[0164] (Obtaining Tea Leaf Extract) The processed tea leaves were poured onto a commercially available through-sieve (mesh opening: approximately 1.5 mm). The tea leaves remained on the screen, and the tea leaf solution passed through the screen and fell into a receiving container. In this way, the tea leaves and the tea leaf solution were separated and obtained. The tea leaves were then squeezed using an oil press and filtered to obtain the tea leaf solution. That is, the tea leaf extract was obtained as the tea leaf solution, the tea leaves, and the tea leaf solution.

[0165] [Measurement of Protein Content in Each Extract] The protein content and amino acid content were measured for each extract obtained in Example 1, Example 2, and Comparative Example 1. The measurements were outsourced to Bureau Veritas FEAC Co., Ltd., a testing organization registered by the Ministry of Health, Labor and Welfare. Specifically, the protein content was measured by the Kjeldahl method, the amino acid (excluding tryptophan) content was measured by PicoTag amino acid analysis, and the tryptophan content was measured by high-performance liquid chromatography (the same applies hereinafter).

[0166] The protein transfer rate (%) was calculated using the following formula: (Protein transfer rate (%)) = (Protein content of each extract) / (Total protein content) × 100. In this formula, "protein content of each extract" refers to a value calculated by multiplying the weight of each extract in Examples 1 and 2 and Comparative Example 1 by the protein concentration (wt%) obtained by the Kjeldahl method. Furthermore, "Total protein content" refers to a value calculated by summing the protein contents of each extract in Examples 1 and 2 and Comparative Example 1. For example, in Example 1, the calculated value refers to a value calculated by summing the protein contents of the tea leaf solution, tea leaf slurry, and squeezed tea leaf slurry.

[0167] As a result, the tea leaf lysate of Example 1 and the used tea leaves lysate of Example 2 generally had higher protein contents, amino acid contents, and protein transfer rates than the tea leaf lysate obtained in Comparative Example 1 (Tables 1 and 2 below). In particular, the tea leaf lysate of Example 1 and the used tea leaves lysate of Example 2 contained large amounts of tryptophan (Table 2 below).

[0168] Furthermore, compared to the squeezed tea leaves obtained from Comparative Example 1, the squeezed tea leaves from Example 1 and the squeezed tea leaves from Example 2 also showed partially similar trends (Tables 1 and 2 below).

[0169] On the other hand, compared to the tea leaves obtained from Comparative Example 1, the slurry tea leaves from Example 1 and the slurry tea leaves from Example 2 generally had lower protein and amino acid contents (Tables 1 and 2 below).

[0170] Therefore, it was found that by carrying out both the decomposition treatment and the separation treatment, large amounts of proteins and amino acids were transferred from the tea leaves or used tea leaves into the water. In particular, it was found that large amounts of tryptophan were transferred from the tea leaves or used tea leaves into the water.

[0171] In addition, the total protein content of Example 1 (the total protein content of the tea leaf solution, the tea leaves, and the squeezed tea leaf slurry) and the total protein content of Example 2 (the total protein content of the used tea leaves solution, the used tea leaves slurry, and the squeezed tea leaf slurry) were smaller than the total protein content of Comparative Example 1 (the total protein content of the tea leaf solution, the tea leaves, and the squeezed tea leaf slurry). This is thought to be caused by the fact that in Examples 1 and 2, when the tea leaves or used tea leaves are transferred from the container to the homogenizer, or when they are transferred from the homogenizer to the mesh, some of the tea leaves or used tea leaves are lost, or some of the tea leaves or used tea leaves remain attached to the homogenizer device.

[0172] Furthermore, the protein content of the used tea leaves slurry of Example 2 was higher than that of the tea leaf slurry of Example 1. This is thought to be because used tea leaves are prepared by extracting an aqueous solution from tea leaves and then drying the tea leaves, and therefore the used tea leaves slurry of Example 2 essentially contains a larger total amount of tea leaves, i.e., more than 2 kg, than the tea leaf slurry of Example 1.

[0173]

[0174]

[0175] From the above, it has been found that by performing both the decomposition treatment and the separation treatment, the protein content and amino acid content of each extract obtained from tea leaves or tea leaves can be improved, and it is possible to provide a method for producing an extract of a plant or its part and a method for treating plant tissue with improved extraction efficiency.

[0176] [SEM observation of each extract] SEM observation was performed on the tea leaves and used tea leaves before being subjected to the decomposition and separation treatments, the slurry-like tea leaves of Example 1, the used tea leaves in the form of a slurry of Example 2, and the tea leaves of Comparative Example 1 using a scanning electron microscope (model number: TM3030Plus, Hitachi High-Tech Corporation).

[0177] The results showed that the pores of both the tea leaves and used tea leaves before the decomposition and separation processes were maintained, and that their surface structures were similar (Figure 6). Furthermore, since the cell walls of the used tea leaves were hardly destroyed, it is believed that most of the intracellular components (proteins, amino acids, catechins, etc.) remained within the cells.

[0178] It was found that the cell walls of the slurried tea leaves of Example 1 were significantly disrupted and intracellular components were released outside the cells through the decomposition and separation processes (Figure 7). It was also found that most of the components that had been adhered to each other in the tea leaves were separated into individual components. In particular, it was found that the vein cellulose fibers that had been adhered to other components such as cells were separated from the mesophyll and veins through the decomposition and separation processes, and separated into individual vein cellulose fibers (Figure 8). The vein cellulose fibers separated in this way can be easily isolated into cellulose and hemicellulose by further separation processes (such as defibration and delignification). The same was true for the slurried used tea leaves of Example 2 (Figure 7).

[0179] On the other hand, for the tea leaves of Comparative Example 1, although the cell walls were destroyed to some extent and intracellular components were released outside the cells through the decomposition treatment, it was found that most of the components that were adhered together in the tea leaves were not separated into individual components (Figure 7). In particular, it was found that in Comparative Example 1, the mesophyll and veins were still adhered together, and the vein cellulose fibers remained adhered to other components such as cells. This is thought to be because the decomposition treatment of the tea leaves alone in Comparative Example 1 was not sufficient to separate the components that were adhered together in the tea leaves into individual components. Furthermore, in Comparative Example 1, the vein cellulose fibers remained adhered to other components such as cells, making it difficult to isolate the vein cellulose fibers.

[0180] The thickness of the leaf vein cellulose fibers in Example 1 is estimated to be 10 μm to 300 μm. The leaf vein cellulose fibers are formed by bundling and branching nano-order cellulose fibers. By subjecting the leaf vein cellulose fibers to a defibration treatment, cellulose microfibers and cellulose nanofibers can be obtained. Cellulose microfibers and cellulose nanofibers have many industrial applications and can be used for a variety of purposes.

[0181] Furthermore, cellulose microfibers and cellulose nanofibers are lightweight and strong, making them suitable for industrial applications such as additives to various organic solvents, thickeners for oils and fats, and reinforcements for resins and rubber. Furthermore, because they are derived from food sources such as tea leaves, vein cellulose fiber itself is also considered suitable for use as a food additive, such as dietary fiber. For example, mixing vein cellulose fiber directly with ingredients for Western and Japanese sweets to create Western and Japanese sweets offers a new texture and provides dietary fiber nutrients. For example, when vein cellulose fiber was mixed into chiffon cake ingredients and cooked, the resulting cake retained its shape and had a firm, fluffy texture. Vein cellulose fiber can also be added to ice cream, bread, and other foods. These applications are expected to have a low environmental impact, as they contribute to the conservation of global resources and the pursuit of a sustainable society in line with the SDGs.

[0182] Below, tea leaves (second-grade green tea leaves and fresh leaves) different from those used in Example 1, coffee beans, strawberry leaves, and tomato leaves were each subjected to the same decomposition and separation processes as in Example 1, and then the results of extracting fiber, protein, solution, etc. are shown.

[0183] Example 3 (Tea Leaf Decomposition and Separation Treatment) Tea leaves (second-grade green tea) were subjected to the same decomposition and separation treatment as in Example 1. The difference from Example 1 is that the tea leaves (25 g) were soaked in 500 mL of water (30°C, pH 4.0) containing an enzyme (3 g of cellulase) for 24 hours, and then stirred and crushed for 1 hour at 4,000 rpm using the plant tissue treatment device 1A shown in Figure 1. Figures 9(A) and (B) show the soaked tea leaves before and after stirring and crushing, respectively. The protein amount and protein content of the tea leaves (25 g) were 6.35 g and 25.40%, respectively. The protein amount was measured using the Kjeldahl method as in Example 1 (the same applies below). The tea leaves used in the treatment were crude tea leaves, which were processed by a crude tea process using fresh leaves picked from tea plants.

[0184] (Solid-liquid separation process (1)) The processed tea leaves were poured onto a commercially available through-sieve (mesh opening: 3.35 mm). The tea leaf slurry remained on the mesh (Fig. 10(A)), and the tea leaf extract passed through the mesh and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 13.35 g, and the weight of the dry tea leaf slurry was calculated to be 4.00 g. The protein content of the wet and dry tea leaf slurry was 0.76% and 2.53%, respectively.

[0185] (Solid-liquid separation process (2)) The tea leaf extract obtained in the solid-liquid separation process (1) was poured onto a commercially available through-sieve (mesh opening: 1.70 mm). The tea leaf slurry remained on the mesh (Figure 10 (B)), and the tea leaf extract passed through the mesh and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 26.35 g, and the weight of the dry tea leaf slurry was calculated to be 7.90 g. The protein content of the wet and dry tea leaf slurry was 0.99% and 3.29%, respectively.

[0186] (Solid-liquid separation process (3)) The tea leaf extract obtained in the solid-liquid separation process (2) was poured onto a commercially available through-sieve (mesh size: 1.00 mm). The tea leaf slurry remained on the mesh (Figure 10(C)), and the tea leaf extract passed through the mesh and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 15.71 g, and the weight of the dry tea leaf slurry was calculated to be 4.71 g. The protein content of the wet and dry tea leaf slurry was 2.69% and 8.96%, respectively.

[0187] (Solid-liquid separation process (4)) The tea leaf extract obtained in the solid-liquid separation process (3) was poured onto a commercially available filter (mesh size: 100 μm). The tea leaf slurry remained on the filter (FIG. 10(D)), and the tea leaf extract passed through the filter and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 14.36 g, and the weight of the dry tea leaf slurry was calculated to be 4.31 g. The protein content of the wet and dry tea leaf slurry was 3.08% and 10.27%, respectively.

[0188] (Solid-liquid separation process (5)) The tea leaf extract obtained in the solid-liquid separation process (4) was poured onto commercially available filter paper (mesh size: 1.00 μm). The tea leaf slurry remained on the filter paper (FIG. 10(E)), and the tea leaf extract passed through the filter paper and fell into a receiving container (FIG. 10(F)). In this way, the wet tea leaf slurry and the tea leaf extract were separated to obtain a tea leaf slurry and a tea leaf extract. The weight of the wet tea leaf slurry was 13.62 g, and the weight of the dry tea leaf slurry was calculated to be 4.08 g. The protein content of the wet and dry tea leaf slurry was 13.64% and 45.50%, respectively. The weight of the tea leaf extract obtained after the solid-liquid separation process (5) was 416.62 g. The amount of protein contained in the tea leaf extract was 3.26 g. The protein content of the tea leaf extract was 0.78%. The amounts of catechin and caffeine contained in the tea leaf extract were 512.66 mg and 1868.62 mg, respectively. High-performance liquid chromatography was used to measure the amounts of catechin, caffeine, and other components contained in the tea leaf extract (the same applies hereinafter).

[0189] Comparative Example 2: Conventional tea extraction was performed on the tea leaves used in Example 3. Specifically, 25 g of the tea leaves were soaked in 500 mL of water (80°C) for 5 minutes. Figure 11 shows the soaked tea leaves. The protein amount, protein content, catechin amount, and caffeine amount contained in 328 g of the tea leaves were 1.23 g, 0.376%, 1523.84 mg, and 377.23 mg, respectively.

[0190] (Results and Discussion) The weight of the wet tea leaf slurry obtained in Example 3 is shown in Figure 12. In Figure 12, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the weight of the wet tea leaf slurry obtained in each treatment (husk recovery weight, bar graph, left axis) and the percentage of that weight to the total (husk recovery rate, line graph, right axis). As shown in Figure 12, the weight of the wet tea leaf slurry obtained in solid-liquid separation treatment (1) was 13.35g. On the other hand, the total weight of the wet tea leaf slurry obtained in all solid-liquid separation treatments (1) to (5) was 83g. Therefore, the weight of the wet tea leaf slurry obtained in solid-liquid separation treatment (1) accounted for 16.01% of the total weight. Similarly, the percentages for solid-liquid separation treatments (2) to (5) are also shown in Figure 12.

[0191] The total amount of slurry tea leaves obtained from the solid-liquid separation processes (1) to (4) was 69.77 g, and the total amount of slurry tea leaves obtained from the solid-liquid separation processes (1) to (5) was 83.39 g. Therefore, the total amount of slurry tea leaves obtained from the solid-liquid separation processes (1) to (4) accounted for 83.67% of the total amount of slurry tea leaves obtained from the solid-liquid separation processes (1) to (5). Therefore, it was found that by performing the solid-liquid separation processes (1) to (4), it was possible to recover most of the slurry tea leaves obtained from the whole tea leaves.

[0192] Next, the results of the protein amount obtained in Example 3 are shown in Figure 13. In Figure 13, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the protein amount obtained in each solid-liquid separation treatment (total recovery amount, bar graph, left axis) and the proportion of the protein amount to the total (total recovery rate, line graph, right axis). As shown in Figure 13, the protein amount obtained in solid-liquid separation treatment (1) was 0.10 g. On the other hand, the total protein amount obtained in solid-liquid separation treatments (1) to (5) was 6.35 g. Therefore, the proportion of the protein amount obtained in solid-liquid separation treatment (1) to the total was 1.60%.

[0193] The total protein content of the tea leaf slurry and tea leaf extract obtained by the solid-liquid separation process (5) was 5.12 g. The protein content of the tea leaves (25 g) used in this example was 6.35 g. Therefore, it was found that by performing the solid-liquid separation process (5), 80.69% of the protein content obtained from the entire tea leaves could be recovered. Furthermore, it was found that most of the protein contained in the tea leaves could be recovered using a filter with a mesh size ranging from approximately 1.00 μm to 100 μm, and that most of the other components such as fiber and dietary fiber could be removed using a filter with a mesh size of 100 μm or more.

[0194] Furthermore, the total amount of protein contained in the slurry tea leaves obtained by the solid-liquid separation processes (1) to (4) was 1.22 g, and the total amount of protein contained in the slurry tea leaves obtained by the solid-liquid separation process (5) was 1.86 g. On the other hand, the amount of protein contained in the tea leaves (25 g) used in this example was 6.35 g. Therefore, the total amount of protein contained in the slurry tea leaves obtained by the solid-liquid separation processes (1) to (4) relative to the amount of protein contained in the tea leaves (25 g) used in this example was 19.31%, and the amount of protein contained in the slurry tea leaves obtained by the solid-liquid separation process (5) was 48.59%. Therefore, it was found that by performing the solid-liquid separation process (5) following the solid-liquid separation processes (1) to (4), an additional 29.28% of the protein amount obtained from the entire tea leaves can be recovered.

[0195] The protein content of the dried tea leaf slurry obtained by the solid-liquid separation process (5) was 45.50%. Furthermore, by drying the dried tea leaf slurry shown in Figure 10(E) in a hot air dryer, a powder-like dry solid with concentrated protein was obtained. Therefore, it was found that by undergoing this solid-liquid separation process, a dry solid with concentrated protein could be obtained.

[0196] The results of the concentration of each component obtained in Example 3 and Comparative Example 2 are shown in Figure 14. In Figure 14, the horizontal axis represents each component, and the vertical axis represents the concentration of each component. The amounts of catechin and caffeine obtained in Example 3 were 1868.62 mg and 512.66 mg, respectively. Furthermore, since the amount of tea leaf extract obtained in Example 3 was 416.62 g, the concentrations of the catechin and caffeine obtained in Example 3 were 4485.20 mg / L and 1230.52 mg / L, respectively.

[0197] On the other hand, the amounts of catechin and caffeine obtained in Comparative Example 2 were 1523.84 mg and 377.23 mg, respectively. Since the amount of tea in Comparative Example 2 was 328 g, the concentrations of catechin and caffeine obtained in Comparative Example 2 were 4650.59 mg / L and 1151.26 mg / L, respectively. Therefore, it was found that the concentrations of caffeine and catechin obtained in Example 3 and Comparative Example 2 were similar.

[0198] From the above, it was found that, when tea leaves (second-grade green tea) different from the tea leaves used in Example 1 were subjected to the degradation and separation treatments in the same manner as in Example 1, solids and extracts containing tea-leaf-derived fiber, dietary fiber, tea-leaf-derived protein, and tea-leaf-derived components could be efficiently extracted. Furthermore, since the extract obtained by the solid-liquid separation treatment (5) contains a much higher concentration of protein than Comparative Example 2, it was found that a tea drink containing a high concentration of protein could be produced by directly processing the extract into a tea drink in the subsequent steps. Furthermore, active ingredients such as proteins and catechins could also be separated and extracted from the extract in subsequent steps using methods such as filtration, concentration separation, freeze-concentration spray drying, and evaporation.

[0199] [Example 4] (Decomposition and Separation Treatment of Tea Leaves) Tea leaves (fresh leaves) were subjected to the decomposition and separation treatments in the same manner as in Example 1. The differences from Example 1 are that the tea leaves (25 g) were soaked in 500 mL of water (30°C, pH 4.0) containing an enzyme (3 g of cellulase) for 24 hours, and then stirred and crushed for 1 hour at 4,000 rpm using the plant tissue treatment device 1A shown in Figure 1 , and that the tea leaves were dried before being subjected to the decomposition treatment. Furthermore, while crude tea was used in Example 3, fresh leaves were used in this example, and therefore the fresh leaves were subjected to pretreatment (physical destruction of cell walls (crushing using a food mill, shearing, etc.)) for the decomposition and separation treatments, which is also different from Example 1. Figure 15 shows tea leaves soaked after stirring and crushing. The amount of protein contained in the tea leaves (25 g) (wet state) was 1.62 g and the protein content was 6.48%, respectively, and the protein content of the tea leaves (6.5 g) (dry state) was 24.92%. The tea leaves used in the treatment were fresh leaves plucked from tea plants.

[0200] (Solid-liquid separation process (1)) The processed tea leaves were poured onto a commercially available through-sieve (mesh opening: 3.35 mm). The tea leaf slurry remained on the mesh (Figure 16 (A)), and the tea leaf extract passed through the mesh and fell into a receiving container. In this way, the tea leaf slurry (wet state) and the tea leaf extract were separated and obtained. The weight of the tea leaf slurry (wet state) was 2.85 g, and the weight of the tea leaf slurry (dry state) was calculated to be 0.44 g. The protein content of the tea leaf slurry was 0 g.

[0201] (Solid-liquid separation process (2)) The tea leaf extract obtained in the solid-liquid separation process (1) was poured onto a commercially available through-sieve (mesh opening: 1.70 mm). The tea leaf slurry remained on the mesh (Figure 16 (B)), and the tea leaf extract passed through the mesh and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 12.99 g, and the weight of the dry tea leaf slurry was calculated to be 2.00 g. The protein content of both the wet and dry tea leaf slurry was 0%.

[0202] (Solid-liquid separation process (3)) The tea leaf extract obtained in the solid-liquid separation process (2) was poured onto a commercially available through-sieve (mesh size: 1.00 mm). The tea leaf slurry remained on the mesh (Figure 16 (C)), and the tea leaf extract passed through the mesh and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 12.93 g, and the weight of the dry tea leaf slurry was calculated to be 1.99 g. The protein content of the wet and dry tea leaf slurry was 1.11% and 7.23%, respectively.

[0203] (Solid-liquid separation process (4)) The tea leaf extract obtained in the solid-liquid separation process (3) was poured onto a commercially available filter (mesh size: 100 μm). The tea leaf slurry remained on the filter (FIG. 16(D)), and the tea leaf extract passed through the filter and fell into a receiving container. In this way, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 4.99 g, and the weight of the dry tea leaf slurry was calculated to be 0.77 g. The protein content of the wet and dry tea leaf slurry was also 0%.

[0204] (Solid-Liquid Separation Process (5)) The tea leaf extract obtained in the solid-liquid separation process (4) was poured onto commercially available filter paper (mesh size: 1.00 μm). The tea leaf slurry remained on the filter paper (FIG. 16(E)), and the tea leaf extract passed through the filter paper and fell into a receiving container (FIG. 16(F)). In this manner, the wet tea leaf slurry and the tea leaf extract were separated and obtained. The weight of the wet tea leaf slurry was 8.49 g, and the weight of the dry tea leaf slurry was calculated to be 1.31 g. The protein content of the wet and dry tea leaf slurries was 9.26% and 60.25%, respectively. The weight of the tea leaf extract obtained after the solid-liquid separation process (5) was 457.75 g. The tea leaf extract contained 0.69 g of protein, 0.15% of protein, and 23.61 mg of catechin and 147.69 mg of caffeine, respectively.

[0205] Comparative Example 3: Conventional tea extraction was performed on the tea leaves used in Example 4. Specifically, 25 g of the tea leaves were soaked in 500 mL of water (80°C) for 5 minutes. Figure 17 shows the soaked tea leaves. The protein amount, protein content, catechin amount, and caffeine amount contained in the tea leaves (439 g) were 0.15 g, 0.034%, 383.90 mg, and 145.83 mg, respectively.

[0206] (Results and Discussion) The weight of the wet slurry tea leaves obtained in Example 4 is shown in Figure 18. In Figure 18, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the weight of the wet slurry tea leaves obtained in each solid-liquid separation treatment (husk recovery weight, bar graph, left axis) and the proportion of that weight to the total (husk recovery rate, line graph, right axis). As shown in Figure 18, the weight of the wet slurry tea leaves obtained in solid-liquid separation treatment (1) was 2.85g. On the other hand, the total weight of the wet slurry tea leaves obtained in all solid-liquid separation treatments (1) to (5) was 42g. Therefore, the proportion of the wet slurry tea leaves obtained in solid-liquid separation treatment (1) to the total weight was 6.75%.

[0207] The results of the protein amount obtained in Example 4 are shown in Figure 19. In Figure 19, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the protein amount obtained in each solid-liquid separation treatment (bar graph) and the percentage of that amount to the total (line graph). As shown in Figure 19, the protein amount obtained in solid-liquid separation treatment (3) was 0.14 g. On the other hand, the total protein amount obtained in solid-liquid separation treatments (1) to (5) was 1.62 g. Therefore, the percentage of the protein amount obtained in solid-liquid separation treatment (3) to the total amount was 8.89%.

[0208] The amount of protein contained in the tea leaf slurry obtained by the solid-liquid separation process (5) was 0.79 g. On the other hand, the amount of protein contained in the tea leaves (25 g) used in this example was 1.62 g. Therefore, the amount of protein contained in the tea leaf slurry obtained by the solid-liquid separation process (5) was 48.77% of the amount of protein contained in the tea leaves (25 g) used in this example.

[0209] The amount of protein contained in the tea leaf extract obtained by the solid-liquid separation process (5) was 0.69 g. Therefore, the amount of protein contained in the tea leaf extract obtained by the solid-liquid separation process (5) was 42.59% of the amount of protein contained in the tea leaves (25 g) used in this example. The total amount of protein contained in the slurry tea leaves and tea leaf extract obtained by the solid-liquid separation process (5) was 91.36% of the amount of protein contained in the tea leaves (25 g) used in this example.

[0210] Furthermore, the protein content of the dried tea leaf slurry obtained by the solid-liquid separation process (5) was 60.25%. Therefore, it was found that by undergoing such solid-liquid separation process, a dry solid with concentrated protein can be obtained.

[0211] The results of the concentrations of each component obtained in Example 4 and Comparative Example 3 are shown in Figure 20. In Figure 20, the horizontal axis represents each component, and the vertical axis represents the concentration (component mass) of each component. In Figure 20, the left bar represents the results of Comparative Example 3, and the right bar represents the results of Example 4. In addition, EGCG, EC, ECG, and EGC in the figure represent epigallocatechin gallate (epigallocatechin gallate), epicatechin, epicatechin gallate, and epigallocatechin, respectively. The catechin and caffeine amounts obtained in Example 4 were 23.61 mg and 147.69 mg, respectively. Since the amount of tea leaf extract obtained in Example 4 was 457.75 g, the catechin and caffeine concentrations obtained in Example 4 were 51.67 mg / L and 323.17 mg / L, respectively.

[0212] On the other hand, the amounts of catechin and caffeine obtained in Comparative Example 3 were 383.90 mg and 145.83 mg, respectively. Since the amount of tea in Comparative Example 3 was 439 g, the concentrations of the amounts of catechin and caffeine obtained in Comparative Example 3 were 874.49 mg / L and 332.19 mg / L, respectively.

[0213] Therefore, it was found that the caffeine concentrations obtained in Example 4 and Comparative Example 3 were comparable. On the other hand, the catechin concentration obtained in Example 4 was less than 1 / 10 of the catechin concentration obtained in Comparative Example 3, and it was found that, unlike caffeine, catechin did not migrate into the solution during the solid-liquid separation process in Example 4, but remained in the solid (slurry tea leaves). In Comparative Example 3, catechin was able to be migrated into the solution by immersing the tea leaves in water at 80°C. Therefore, it is believed that in Example 4, catechin can be migrated into the solution by immersing the solid (slurry tea leaves) in water at 80°C. On the other hand, it is thought that catechin can be left in the solid fraction by maintaining the temperature at or below a certain temperature, for example, below 50°C (30°C in this example).

[0214] From the above, it was found that, when tea leaves (fresh leaves) different from the tea leaves used in Example 1 were subjected to the decomposition treatment and separation treatment in the same manner as in Example 1, solids containing tea leaf-derived fiber, dietary fiber, tea leaf-derived protein, and tea leaf-derived components, extract, etc., could be efficiently extracted. It was also found that the extraction rate of catechins into solids, extract, etc. could be adjusted by adjusting the temperature during the treatment. Furthermore, the extract obtained in the solid-liquid separation treatment (5) can be directly processed into a tea beverage in the subsequent step, as in Example 3, to produce a tea beverage containing a high concentration of protein. Furthermore, active ingredients such as proteins and catechins can be separated and extracted from the extract in the subsequent steps by methods such as filtration, concentration separation, freeze-concentration spray drying, and evaporation.

[0215] Example 5 (Coffee Bean Decomposition Treatment and Separation Treatment) Coffee beans (Brazilian Arabica city roast) were subjected to the decomposition treatment and separation treatment in the same manner as in Example 1. The difference from Example 1 is that the coffee beans (dry weight 36 g) were soaked in 500 mL of water (50°C, pH 4.0) containing an enzyme (3 g of cellulase) for 24 hours, and then stirred and crushed for 1 hour at 4,000 rpm using the plant tissue treatment device 1A shown in FIG. 1 . FIG. 21 shows the soaked coffee beans after stirring and crushing. The amount of protein contained in the coffee beans (36 g) was 4.04 g, and the protein content of the coffee beans (36 g) was 11.22%. The coffee beans subjected to the treatment were milled (espresso ground) and crushed using a coffee mill (Fuji Coffee Machine Co., Ltd., model number: DX R-220).

[0216] (Solid-Liquid Separation Process (1)) The processed coffee beans were poured onto a commercially available through-sieve (mesh opening: 1.00 mm). The slurry coffee beans remained on the screen ( FIG. 22 (A) ), and the coffee bean extract passed through the screen and fell into a receiving container. In this way, the slurry coffee beans (wet state) and the coffee bean extract were separated and obtained. The weight of the slurry coffee beans (wet state) was 55.73 g, and the weight of the slurry coffee beans (dry state) was converted to 17.17 g. The amount of protein contained in the slurry coffee beans was 0 g. The protein content of both the wet and dry slurry coffee beans was 0%.

[0217] (Solid-Liquid Separation Process (2)) The coffee bean extract obtained in the solid-liquid separation process (1) was poured onto a commercially available filter (mesh size: 100 μm). The coffee beans in a slurry state remained on the filter ( FIG. 22 (B) ), and the coffee bean extract passed through the filter and fell into a receiving container. In this manner, the coffee beans in a slurry state (wet state) and the coffee bean extract were separated and obtained. The weight of the coffee beans in a slurry state (wet state) was 55.57 g, and the weight of the coffee beans in a slurry state (dry state) was converted to 17.12 g. The amount of protein contained in the coffee beans in a slurry state was 1.71 g. The protein contents of the wet and dry coffee beans in a slurry state were 3.08% and 10.01%, respectively.

[0218] (Solid-Liquid Separation Process (3)) The coffee bean extract obtained in the solid-liquid separation process (2) was poured onto commercially available filter paper (mesh size: 1.00 μm). The coffee bean slurry remained on the filter paper ( FIG. 22 (C) ), and the coffee bean extract passed through the filter paper and fell into a receiving container ( FIG. 22 (D) ). In this manner, the coffee beans slurry and the coffee bean extract were separated and obtained. The weight of the coffee beans slurry (wet state) was 5.57 g, and the weight of the coffee beans slurry (dry state) was converted to 1.72 g. The protein amount contained in the coffee beans slurry was 0.86 g. The protein contents of the wet and dry coffee beans slurry were 15.45% and 50.16%, respectively. The weight of the coffee bean extract obtained after the solid-liquid separation process (3) was 383.14 g. The amount of protein contained in the coffee bean extract was 1.46 g. The protein content of the coffee bean extract was 0.38%. The amount of caffeine contained in the coffee bean extract was 289.54 mg.

[0219] Comparative Example 4: Conventional coffee extraction was performed on the coffee beans used in Example 5. Specifically, the coffee beans (dry weight 36 g) were soaked in 500 mL of water (80°C) for 5 minutes. Figure 23 shows the soaked coffee beans. The protein amount, protein content, and caffeine amount contained in the coffee beans (366 g) were 1.39 g, 0.38%, and 629.90 mg, respectively.

[0220] (Results and Discussion) The weight results for the wet slurry coffee beans obtained in Example 5 are shown in Figure 24. In Figure 24, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the weight of the wet slurry coffee beans obtained in each solid-liquid separation treatment (shell recovery weight, bar graph, left axis) and the proportion of that weight to the total (shell recovery rate, line graph, right axis). As shown in Figure 24, the weight of the wet slurry coffee beans obtained in solid-liquid separation treatment (1) was 55.73 g. On the other hand, the total weight of the wet slurry coffee beans obtained in all solid-liquid separation treatments (1) to (3) was 117 g. Therefore, the proportion of the weight of the wet slurry coffee beans obtained in solid-liquid separation treatment (1) to the total weight was 47.68%.

[0221] The results of the protein amount obtained in Example 5 are shown in Figure 25. In Figure 25, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the protein amount obtained in each solid-liquid separation treatment (shell recovery weight, bar graph, left axis) and the proportion of that amount to the total (shell recovery rate, line graph, right axis). As shown in Figure 25, the protein amount obtained in solid-liquid separation treatment (2) was 1.71 g. On the other hand, the total protein amount obtained in solid-liquid separation treatments (1) to (3) was 4.04 g. Therefore, the proportion of the protein amount obtained in solid-liquid separation treatment (2) to the total was 42.44%.

[0222] The amount of protein contained in the coffee bean slurry obtained by solid-liquid separation process (3) was 0.86 g. On the other hand, the amount of protein contained in the coffee beans (36 g) used in this example was 4.04 g. Therefore, the amount of protein contained in the coffee bean slurry obtained by solid-liquid separation process (3) was 21.29% of the amount of protein contained in the coffee beans (36 g) used in this example.

[0223] Furthermore, the amount of protein contained in the coffee bean extract obtained by solid-liquid separation process (3) was 1.46 g. Therefore, the amount of protein contained in the coffee bean extract obtained by solid-liquid separation process (3) was 36.14% of the amount of protein contained in the coffee beans (36 g) used in this example. The total amount of protein contained in the slurry coffee beans and coffee bean extract obtained by solid-liquid separation process (3) was 57.43% of the amount of protein contained in the coffee beans (36 g) used in this example.

[0224] The protein content of the dried coffee bean slurry obtained by solid-liquid separation process (3) was 50.16%. Furthermore, by drying the dried coffee bean slurry shown in Figure 22 (C) in a hot air dryer, a powder-like dry solid with concentrated protein was obtained. Therefore, it was found that by undergoing this solid-liquid separation process, a dry solid with concentrated protein could be obtained.

[0225] From the above, it was found that coffee-derived fibers, coffee-derived proteins, a solution containing coffee-derived components, and the like can be efficiently extracted by subjecting coffee beans to the degradation treatment and separation treatment in the same manner as in Example 1. Furthermore, it was found that the extract obtained by the solid-liquid separation treatment (3) contains a higher concentration of protein than in Comparative Example 4, and therefore can be directly processed into a coffee beverage in the subsequent step to produce a coffee beverage with a high concentration of protein.

[0226] Furthermore, Example 5 uses ground coffee beans, but the solid matter recovered through the mesh, filter paper, or filter in the solid-liquid separation processes (1), (2), and (3), and the coffee grounds (which are usually discarded) left after coffee extraction at restaurants, convenience stores, and other establishments, still contain a significant amount of active ingredients such as proteins. Therefore, by using these solid matter, coffee grounds, etc., and subjecting them to the process of Example 5 again, the remaining active ingredients such as proteins can be further extracted. Furthermore, since the concentration of the extract will be reduced in this process, a coffee beverage can be produced by concentrating the extract or adjusting the concentration by mixing it with something more concentrated.

[0227] Example 6 (Strawberry Leaf Decomposition and Separation Treatments) Strawberry leaves were subjected to the decomposition and separation treatments described above, as in Example 1. The difference from Example 1 is that the strawberry leaves (25 g dry weight) were immersed in 500 mL of water (50°C, pH 4.0) containing an enzyme (3 g cellulase) for 24 hours and then agitated and crushed for 1 hour at 4,000 rpm using the plant tissue treatment device 1A shown in Figure 1 . Figures 26(A) and 26(B) show the soaked strawberry leaves before and after agitation and crushing, respectively. The protein content of the strawberry leaves (25 g) was 1.85 g, and the protein content of the strawberry leaves (25 g) was 7.41%. The strawberry leaves used in the treatments were dried fresh leaves and stems remaining after strawberry fruit harvest. These fresh leaves and stems would normally be discarded.

[0228] (Solid-liquid separation process (1)) The processed strawberry leaves were poured onto a commercially available through-sieve (mesh size: 3.35 mm). The strawberry leaf slurry remained on the mesh (Figure 27(A)), and the strawberry leaf extract passed through the mesh and fell into a receiving container. In this way, the strawberry leaf slurry (wet state) and the strawberry leaf extract were separated and obtained. The weight of the strawberry leaf slurry (wet state) was 141.38 g, and the weight of the strawberry leaf slurry (dry state) was calculated to be 12.56 g. The protein content of the strawberry leaf slurry in the wet and dry states was 0.76% and 8.56%, respectively.

[0229] (Solid-Liquid Separation Process (2)) The strawberry leaf extract obtained in the solid-liquid separation process (1) was poured onto a commercially available through-sieve (mesh opening: 1.70 mm). The strawberry leaf slurry remained on the mesh (Figure 27(B)), and the strawberry leaf extract passed through the mesh and fell into a receiving container. In this way, the strawberry leaf slurry and the strawberry leaf extract were separated and obtained. The weight of the strawberry leaf slurry (wet state) was 53.73 g, and the weight of the strawberry leaf slurry (dry state) was calculated to be 4.77 g. The protein content of the strawberry leaf slurry was 0.15 g. The protein contents of the wet and dry strawberry leaf slurries were 0.28% and 3.20%, respectively. The strawberry leaf slurry was also rich in strawberry leaf fiber, dietary fiber, and the like.

[0230] (Solid-liquid separation process (3)) The strawberry leaf extract obtained in the solid-liquid separation process (2) was poured onto a commercially available through-sieve (mesh opening: 1.00 mm). The strawberry leaf slurry remained on the mesh (Figure 27(C)), and the strawberry leaf extract passed through the mesh and fell into a receiving container. In this way, the strawberry leaf slurry (wet state) and the strawberry leaf extract were separated and obtained. The weight of the strawberry leaf slurry (wet state) was 23.77 g, and the weight of the strawberry leaf slurry (dry state) was calculated to be 2.11 g. The protein content of the strawberry leaf slurry was 0.08 g. The protein contents of the wet and dry strawberry leaf slurries were 0.35% and 3.89%, respectively.

[0231] (Solid-liquid separation process (4)) The strawberry leaf extract obtained in the solid-liquid separation process (3) was poured onto a commercially available filter (mesh size: 100 μm). The strawberry leaf slurry remained on the filter (FIG. 27(D)), and the strawberry leaf extract passed through the filter and fell into a receiving container. In this way, the strawberry leaf slurry (wet state) and the strawberry leaf extract were separated and obtained. The weight of the strawberry leaf slurry (wet state) was 55.58 g, and the weight of the strawberry leaf slurry (dry state) was calculated to be 4.94 g. The protein content of the strawberry leaf slurry was 0.17 g. The protein content of the wet and dry strawberry leaf slurries was 0.30% and 3.39%, respectively.

[0232] (Solid-Liquid Separation Process (5)) The strawberry leaf extract obtained in the solid-liquid separation process (4) was poured onto commercially available filter paper (mesh size: 1.00 μm). The strawberry leaf slurry remained on the filter paper (FIG. 27(E)), and the strawberry leaf extract passed through the filter paper and fell into a receiving container (FIG. 27(F)). In this manner, the strawberry leaf slurry (wet state) and the strawberry leaf extract were separated and obtained. The weight of the strawberry leaf slurry (wet state) was 6.98 g, and the weight of the strawberry leaf slurry (dry state) was calculated to be 0.62 g. The protein content of the strawberry leaf slurry was 0.21 g. The protein contents of the wet and dry strawberry leaf slurries were 3.02% and 33.95%, respectively. The weight of the strawberry leaf extract obtained after the solid-liquid separation process (5) was 218.57 g. The amount of protein contained in the strawberry leaf extract was 0.16 g, and the protein content of the strawberry leaf extract was 0.07%.

[0233] (Results and Discussion) Figure 28 shows the weight of the wet strawberry leaf slurry obtained in Example 6. In Figure 28, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the weight of the wet strawberry leaf slurry obtained in each solid-liquid separation treatment (husk recovery weight, bar graph, left axis) and the percentage of that weight to the total (husk recovery rate, line graph, right axis). As shown in Figure 28, the weight of the wet strawberry leaf slurry obtained in solid-liquid separation treatment (1) was 141.38 g. On the other hand, the total weight of the wet strawberry leaf slurry obtained in solid-liquid separation treatments (1) to (5) was 281 g. Therefore, the weight of the wet strawberry leaf slurry obtained in solid-liquid separation treatment (1) accounted for 50.23% of the total weight. Furthermore, the strawberry leaf slurry is rich in strawberry leaf fiber, dietary fiber, and the like.

[0234] The results for the protein amount obtained in Example 6 are shown in Figure 29. As shown in Figure 29, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the protein amount obtained in each solid-liquid separation treatment (shell recovery weight, bar graph, left axis) and the proportion of that amount to the total (shell recovery rate, line graph, right axis). As shown in Figure 29, the protein amount obtained in solid-liquid separation treatment (1) was 1.08 g. On the other hand, the total protein amount obtained in solid-liquid separation treatments (1) to (5) was 1.85 g. Therefore, the proportion of the protein amount obtained in solid-liquid separation treatment (1) to the total was 58.13%.

[0235] The protein content of the strawberry leaf slurry obtained by solid-liquid separation treatment (5) was 0.21 g. On the other hand, the protein content of the strawberry leaves (25 g) used in this example was 1.85 g. Therefore, the protein content of the strawberry leaf slurry obtained by solid-liquid separation treatment (5) was 11.35% of the protein content of the strawberry leaves (25 g) used in this example.

[0236] The amount of protein contained in the strawberry leaf extract obtained by solid-liquid separation treatment (5) was 0.16 g. Therefore, the amount of protein contained in the strawberry leaf extract obtained by solid-liquid separation treatment (5) was 8.65% of the amount of protein contained in the strawberry leaf (25 g) used in this example. The total amount of protein contained in the strawberry leaf slurry and strawberry leaf extract obtained by solid-liquid separation treatment (5) was 20% of the amount of protein contained in the strawberry leaf (25 g) used in this example.

[0237] The protein content of the dried strawberry leaf slurry obtained by the solid-liquid separation process (5) was 33.95%. Furthermore, by drying the dried strawberry leaf slurry in a hot air dryer, a powder-like dry solid with concentrated protein was obtained. Therefore, it was found that a dry solid with concentrated protein could be obtained by undergoing this solid-liquid separation process.

[0238] From the above, it was found that strawberry leaves and the like, which would normally be discarded, can be efficiently extracted with strawberry leaf-derived fiber, dietary fiber, strawberry leaf-derived protein, solids containing strawberry leaf-derived components, and extracts by subjecting them to the decomposition treatment and separation treatment in the same manner as in Example 1. Furthermore, active ingredients such as proteins can be separated and extracted from the extract obtained by the solid-liquid separation treatment (5) in subsequent steps by methods such as filtration, concentration separation, freeze-concentration spray drying, and evaporation treatment.

[0239] Example 7 (Decomposition and Separation Treatment of Tomato Leaves) Tomato leaves were subjected to the decomposition and separation treatments described above in the same manner as in Example 1. The difference from Example 1 was that the tomato leaves (25 g dry weight) were immersed in 500 mL of water (50°C, pH 4.0) containing an enzyme (3 g cellulase) for 24 hours and then crushed by stirring at 4,000 rpm for 1 hour using the plant tissue treatment device 1A shown in Figure 1 . Figures 30(A) and 30(B) show the immersed tomato leaves before and after crushing by stirring, respectively. The protein content of the tomato leaves (25 g) was 5.53 g, and the protein content of the tomato leaves (25 g) was 22.12%. The tomato leaves used in the treatments were dried fresh leaves, stems, etc., remaining after tomato fruit harvesting. These fresh leaves, stems, etc. would normally be discarded.

[0240] (Solid-Liquid Separation Treatment (1)) The processed tomato leaves were poured onto a commercially available through-sieve (mesh opening: 3.35 mm). The tomato leaf slurry remained on the mesh ( FIG. 31(A) ), and the tomato leaf extract passed through the mesh and fell into a receiving container. In this manner, the tomato leaf slurry (wet state) and the tomato leaf extract were separated and obtained. The weight of the tomato leaf slurry (wet state) was 148.22 g, and the weight of the tomato leaf slurry (dry state) was calculated to be 15.01 g. The protein content of the tomato leaf slurry in the wet and dry states was 2.00% and 19.71%, respectively.

[0241] (Solid-Liquid Separation Process (2)) The tomato leaf extract obtained in the solid-liquid separation process (1) was poured onto a commercially available through-sieve (mesh opening: 1.70 mm). The tomato leaf slurry remained on the mesh ( Figure 31 (B) ), and the tomato leaf extract passed through the mesh and fell into a receiving container. In this manner, the tomato leaf slurry (wet state) and the tomato leaf extract were separated and obtained. The weight of the tomato leaf slurry (wet state) was 16.34 g, and the weight of the tomato leaf slurry (dry state) was calculated to be 1.65 g. The protein content of the tomato leaf slurry was 0.42 g. The protein contents of the wet and dry tomato leaf slurries were 2.58% and 25.47%, respectively.

[0242] (Solid-Liquid Separation Process (3)) The tomato leaf extract obtained in the solid-liquid separation process (2) was poured onto a commercially available through-sieve (mesh opening: 1.00 mm). The tomato leaf slurry remained on the mesh ( Figure 31 (C) ), and the tomato leaf extract passed through the mesh and fell into a receiving container. In this manner, the tomato leaf slurry (wet state) and the tomato leaf extract were separated and obtained. The weight of the tomato leaf slurry (wet state) was 16.35 g, and the weight of the tomato leaf slurry (dry state) was calculated to be 1.66 g. The protein content of the tomato leaf slurry in the wet and dry states was 0.35% and 3.44%, respectively.

[0243] (Solid-Liquid Separation Process (4)) The tomato leaf extract obtained in the solid-liquid separation process (3) was poured onto a commercially available filter (mesh size: 100 μm). The tomato leaf slurry remained on the filter ( FIG. 31 (D) ), and the tomato leaf extract passed through the filter and fell into a receiving container. In this manner, the tomato leaf slurry (wet state) and the tomato leaf extract were separated and obtained. The weight of the tomato leaf slurry (wet state) was 58.74 g, and the weight of the tomato leaf slurry (dry state) was calculated to be 5.95 g. The protein content of the tomato leaf slurry was 0.62 g. The protein contents of the wet and dry tomato leaf slurries were 1.06% and 10.51%, respectively.

[0244] (Solid-Liquid Separation Treatment (5)) The tomato leaf extract obtained in the solid-liquid separation treatment (4) was poured onto commercially available filter paper (mesh size: 1.00 μm). The tomato leaf slurry remained on the filter paper ( FIG. 31(E) ), and the tomato leaf extract passed through the filter paper and fell into a receiving container ( FIG. 31(F) ). In this manner, the wet tomato leaf slurry and the tomato leaf extract were separated and obtained. The wet tomato leaf slurry weighed 7.26 g, and the dry tomato leaf slurry weighed 0.74 g. The protein content of the wet and dry tomato leaf slurries was 6.59% and 65.10%, respectively. The weight of the tomato leaf extract obtained after the solid-liquid separation treatment (5) was 253.09 g. The amount of protein contained in the tomato leaf extract was 0.99 g, and the protein content of the tomato leaf extract was 0.39%.

[0245] (Results and Discussion) The weight of the wet tomato leaf slurry obtained in Example 7 is shown in Figure 32. In Figure 32, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the weight of the wet tomato leaf slurry obtained in each solid-liquid separation treatment (husk recovery weight, bar graph, left axis) and the proportion of that weight to the total (husk recovery rate, line graph, right axis). As shown in Figure 32, the weight of the wet tomato leaf slurry obtained in solid-liquid separation treatment (1) was 148.22 g. On the other hand, the total weight of the wet tomato leaf slurry obtained in all solid-liquid separation treatments (1) to (5) was 247 g. Therefore, the proportion of the wet tomato leaf slurry obtained in solid-liquid separation treatment (1) to the total weight was 60.03%.

[0246] The results of the protein amount obtained in Example 7 are shown in Figure 33. In Figure 33, the horizontal axis represents each solid-liquid separation treatment, and the vertical axis represents the protein amount obtained in each solid-liquid separation treatment (shell recovery weight, bar graph, left axis) and the proportion of that amount to the total (shell recovery rate, line graph, right axis). As shown in Figure 33, the protein amount obtained in solid-liquid separation treatment (1) was 2.96 g. On the other hand, the total protein amount obtained in solid-liquid separation treatments (1) to (5) was 5.53 g. Therefore, the proportion of the protein amount obtained in solid-liquid separation treatment (1) to the total was 53.50%.

[0247] The protein amount contained in the tomato leaf slurry obtained by solid-liquid separation treatment (5) was 0.48 g. On the other hand, the protein amount contained in the tomato leaves (25 g) used in this example was 5.53 g. Therefore, the protein amount contained in the tomato leaf slurry obtained by solid-liquid separation treatment (5) was 8.68% of the protein amount contained in the tomato leaves (25 g) used in this example.

[0248] The amount of protein contained in the tomato leaf extract obtained by solid-liquid separation treatment (5) was 0.99 g. Therefore, the amount of protein contained in the tomato leaf extract obtained by solid-liquid separation treatment (5) was 17.90% of the amount of protein contained in the tomato leaves (25 g) used in this example. The total amount of protein contained in the slurry-like tomato leaves and the tomato leaf extract obtained by solid-liquid separation treatment (5) was 26.58% of the amount of protein contained in the tomato leaves (25 g) used in this example.

[0249] The protein content of the dried tomato leaf slurry obtained by the solid-liquid separation treatment (5) was 65.10%. Furthermore, by drying the dried tomato leaf slurry in a hot air dryer, a powder-like dry solid with concentrated protein was obtained. Therefore, it was found that a dry solid with concentrated protein could be obtained by undergoing such solid-liquid separation treatment.

[0250] From the above, it was found that the tomato leaves and the like, which would normally be discarded, can be efficiently extracted with fiber derived from the tomato leaves, dietary fiber derived from the tomato leaves, protein derived from the tomato leaves, an extract containing components derived from the tomato leaves, and the like, by subjecting the tomato leaves and the like to the decomposition treatment and the separation treatment in the same manner as in Example 1. Furthermore, active ingredients such as protein can be separated and extracted from the extract obtained in the solid-liquid separation treatment (5) in subsequent steps by methods such as filtration, concentration separation, freeze-concentration spray drying, and evaporation treatment.

[0251] The various treatment conditions in each of the above examples (liquid volume, water temperature, soaking time, stirring and crushing time, stirring and crushing rotation speed, enzyme type, enzyme amount, etc.) can be appropriately set depending on the subject to be treated, the desired extract, etc.

[0252] The size of the openings of the mesh screen, filter, filter paper, etc., the number of times of solid-liquid separation treatment, etc. can also be appropriately set depending on the object to be treated, the desired extract, etc. For example, the number of times of solid-liquid separation treatment can be reduced from five to two or three, or increased to six or seven. For example, in the example of the protein shown in Example 3, taking into account the size of the protein, it is also possible to perform two solid-liquid separation treatments using filters, filter paper, etc. with openings of 100 μm and 1.00 μm.

[0253] In Examples 4 and 5, fresh leaves and coffee beans were subjected to pretreatment (crushing in a food mill and grinding in a mill, respectively), followed by the decomposition and separation treatments of the present disclosure. Such pretreatments are also effective for strawberry leaves in Example 6 and tomato leaves in Example 7. In the present disclosure, an appropriate pretreatment is selected depending on the type of leaf, and the leaves are subjected to the pretreatment, followed by the decomposition and separation treatments of the present disclosure, thereby improving the extraction rate of active ingredients, etc. This pretreatment is not limited to the above examples, but is also effective for a wide range of plant tissues, including plants. In the present disclosure, an appropriate pretreatment is selected depending on the type of plant tissue, and the plant tissue is subjected to the pretreatment, followed by the decomposition and separation treatments of the present disclosure, thereby improving the extraction rate of active ingredients, etc.

[0254] The pretreatment may be any method that applies a physical stimulus to the plant tissue beforehand (such as kneading, crushing, or wounding). Examples of pretreatment include applying a physical stimulus to the cell walls of leaves, stems, etc., and examples of such pretreatment include grinding with a food mill, milling, crushing, shearing, heat treatment, kneading (kneading process), squeezing, compression / pressing, squeezing, crushing, and friction welding.

[0255] Example 8 (Tea Leaf Decomposition Treatment) 100 L of water was placed in a 200 L container and heated to 35°C using a liquid heater (model number: WPS-110, Kashima Corporation). 600 mL of cellulase (equivalent to 0.6% of the 100 L volume of water) was added to the water and mixed. Next, 5 kg of tea leaves were added to the water and allowed to soak. The mixed solution thus prepared was sampled at the times listed in Table 3 below, and the pH of the mixed solution was measured. The mixed solution was also stirred (4,000 rpm) in the container using a homogenizer (Automixer Type 40, Primix Corporation) at the times listed in Table 3 below. The stirring was performed in the opposite direction to the direction of rotation that would cause stirring and disruption, so as to avoid disruption (i.e., to only stir the mixed solution). The mixed solution was stirred using the homogenizer at the times listed in Table 3 below, and the current consumption of the homogenizer was measured. The steeping (decomposition treatment) was carried out for about 4 hours. The tea leaves used were fresh leaves that had just been picked from tea plants and had been degreened by supplying steam at 100°C for 60 seconds while stirring.

[0256] (Results and Discussion) The measurement results of the pH of the mixed solution and the amount of current used by the homogenizer are shown in Table 3. As shown in Table 3, the pH of the mixed solution was 4.0 at the start of the immersion, but became almost constant at around 4.8 after about 2 to 3 hours from the start of the immersion. Also, as shown in Table 3, the amount of current used by the homogenizer was 1.9 A at the start of the immersion, but became almost constant at around 3.0 A after about 2 to 3 hours from the start of the immersion.

[0257] From the above results, it can be considered that the state of the mixed solution becomes constant after about 2 to 3 hours from the start of the soaking, and the soaking (decomposition treatment) is sufficient. Therefore, by starting the separation treatment using a homogenizer after about 2 to 3 hours from the start of the soaking, it is thought that the plant tissue can be treated efficiently without continuing the soaking for an unnecessarily long time.

[0258] The pH of the mixed solution exceeded 5 for about one hour after the start of the immersion, which is thought to be because the tea leaves decomposed and their components began to mix into the mixed solution. The amount of current used by the homogenizer tended to increase from the start of the immersion and became almost constant after about two to three hours. This is thought to be because the viscosity of the mixed solution increased as the immersion (decomposition treatment) progressed, and the viscosity of the mixed solution became almost constant as the immersion was sufficient, which in turn caused the resistance to the homogenizer due to the viscosity of the mixed solution to become almost constant.

[0259]

[0260] Example 9 (Tea Leaf Separation Treatment) The tea leaf decomposition product obtained in Example 8 was stirred and crushed in the container using a homogenizer (Automixer Type 40, Primix Corporation) at 4,000 rpm for approximately 1 hour (separation treatment). Five minutes after the start of the stirring and crushing, the processed product obtained from the stirring and crushing was circulated through the container, the circulation pipe, and the container again via circulation pipes attached to the top and bottom of the container. A pump was placed inside the circulation pipe. The flow rate of the processed product in the circulation pipe was measured at the times listed in Table 4 below. The current usage of the homogenizer was also measured at the times listed in Table 4 below.

[0261] (Results and Discussion) The measurement results of the flow rate of the material to be treated in the circulation pipe and the amount of current used by the homogenizer are shown in Table 4. As shown in Table 4, the flow rate of the material to be treated in the circulation pipe was 0 L / min at the start of the immersion, but became almost constant at around 55 L / min after about 45 minutes from the start of the immersion. Also, as shown in Table 4, the amount of current used by the homogenizer was 3.7 A at the start of the immersion, but became almost constant at around 2.5 A after about 45 minutes from the start of the immersion.

[0262] From the above results, it can be considered that the state of the processed material becomes constant after about 45 minutes from the start of the stirring and crushing, and the stirring and crushing (separation treatment) is sufficiently performed. Therefore, by ending the separation treatment using the homogenizer after about 45 minutes from the start of the stirring and crushing, it is thought that the plant tissue can be processed efficiently without continuing the stirring and crushing for an unnecessarily long time.

[0263] The flow rate of the processed material in the circulation pipe increased as time passed from the start of the agitation and crushing, which is thought to be because the particle size of the processed material in the circulation pipe, which was large at the start of the agitation and crushing, became smaller as time passed from the agitation and crushing, and thus the circulation became less obstructed.Furthermore, the amount of current used by the homogenizer decreased as time passed from the agitation and crushing, which is thought to be because the particle size of the processed material in the circulation pipe, which was large at the start of the agitation and crushing, became smaller as time passed from the agitation and crushing, and thus the resistance of the processed material to the homogenizer became smaller.

[0264] At the start of the circulation (5 minutes after the stirring and crushing), the flow rate of the treated material in the circulation pipe increases temporarily. This is thought to be because the supernatant liquid in the container preferentially passes through the circulation pipe, and then the sediment in the container passes through the circulation pipe.

[0265]

[0266] [Example 10] (Solid-liquid separation of processed product) The processed tea leaves obtained in Example 9 were subjected to solid-liquid separation using a screw press (44.59 Hz). The screw press was placed inside the circulation pipe. The solid fraction obtained from the solid-liquid separation was collected in a container separate from the container. The rotation speed of the pump placed inside the circulation pipe was set to 7.85 Hz or 4.22 Hz, and the state of the collected solid fraction at each rotation speed was visually confirmed.

[0267] (Results and Discussion) When the rotational speed of the pump placed in the circulation pipe was set to 7.85 Hz, the solid fraction contained a large amount of liquid, whereas when the rotational speed of the pump placed in the circulation pipe was set to 4.22 Hz, the solid fraction contained a small amount of liquid.

[0268] From the above results, it is believed that by lowering the rotation speed of the pump, the amount of liquid contained in the solid fraction can be reduced, and the solid fraction can be recovered efficiently.

[0269] Furthermore, if the rotation speed of the screw press is too high, the amount of the processed material processed by the screw press will be excessive, and the amount of liquid contained in the solid fraction will increase. Also, if the rotation speed of the pump is too high, the amount of the processed material supplied to the screw press by the pump will be excessive, and the amount of liquid contained in the solid fraction will increase. Therefore, in order to reduce the amount of liquid contained in the solid fraction and efficiently recover the solid fraction, it is considered important to adjust the rotation speed of the screw press and / or the pump within an appropriate range.

[0270] Example 11 It was confirmed that sugar can be obtained by saccharifying tea leaves in a slurry state.

[0271] (Decomposition and separation treatment of tea leaves) The tea leaves (25 g dry weight) were immersed in 500 ml of water (35°C, pH 4.0) containing an enzyme (3 g of cellulase) for 24 hours, and then subjected to the decomposition and separation treatment in the same manner as in Example 1, except that the tea leaves were immersed in 500 ml of water (35°C, pH 4.0) containing an enzyme (3 g of cellulase) for 24 hours, and then stirred and crushed at 4,000 rpm for 1 hour using the plant tissue treatment device 1A shown in Figure 1.

[0272] (Solid-liquid separation treatment) The processed tea leaves were poured onto a commercially available through-sieve (mesh opening: 3.35 mm). The tea leaves in a slurry state remained on the screen, and the tea leaf extract passed through the screen and fell into a receiving container. In this way, the tea leaves in a slurry state and the tea leaf extract were separated.

[0273] [Saccharification Treatment of Slurry-State Tea Leaves by Enzymatic Reaction] The slurried tea leaves were subjected to saccharification treatment by enzymatic reaction to obtain glucose.

[0274] (Experimental Method) The enzymes used in the saccharification treatment were divided into test plots 1 to 6, each containing a different enzyme combination as shown in Table 5 below. Test plots 1 to 5 were test plots containing different enzyme combinations, and test plot 6 was a blank containing no enzymes (Table 5 below).

[0275]

[0276] (1) Enzyme Reaction (Test Group 1) 1 g of the slurried tea leaves, 0.3 mL of cellulase SS, and 0.3 mL of cellulase XL-531 were added to 100 mL of 100 mM sodium citrate buffer (pH 4.0) to prepare an enzyme reaction solution. The enzyme reaction solution was then heated and stirred on a hot stirrer at 37°C. 1 mL of the enzyme reaction solution was collected at 0.5, 1, 2, 3, 6, 24, and 48 hours after the start of the reaction. The enzyme reaction solution was centrifuged (4°C, 10,000 x g, 5 minutes), and the supernatant was collected. The supernatant was then heated in a heat block (100°C, 5 minutes) to inactivate the enzyme in the enzyme reaction solution and terminate the enzyme reaction.

[0277] (Test Plot 2) The tea leaves in the slurry form were subjected to saccharification treatment by enzymatic reaction in the same manner as in Test Plot 1. Test Plot 2 was carried out in the same manner as Test Plot 1, except that cellulase SS and cellulase XL-531 were not used, and 10 mg / mL AnBGL (β-glucosidase derived from Aspergillus niger) was used.

[0278] (Test Plot 3) The tea leaves in the slurry form were subjected to saccharification treatment by enzymatic reaction in the same manner as in Test Plot 1. Test Plot 3 was carried out in the same manner as Test Plot 1, except that 10 mg / mL AnBGL was used in addition to cellulase SS and cellulase XL-531.

[0279] (Test Group 4) The tea leaves in the form of a slurry were subjected to saccharification treatment by enzymatic reaction in the same manner as in Test Group 1. Test Group 4 was carried out in the same manner as Test Group 1, except that cellulase SS and cellulase XL-531 were not used, and 10 mg / mL aromatase (β-glucosidase derived from Penicillium sp.) was used.

[0280] (Test Plot 5) The tea leaves in the slurry form were subjected to saccharification treatment by enzymatic reaction in the same manner as in Test Plot 1. Test Plot 5 was carried out in the same manner as Test Plot 1, except that 10 mg / mL aromatase was used in addition to cellulase SS and cellulase XL-531.

[0281] (Test Plot 6 (Blank)) Test Plot 6 was a blank, and was carried out in the same manner as Test Plot 1, except that cellulase SS and cellulase XL-531 were not used.

[0282] (2) Determination of Glucose Concentration: The glucose in the enzyme reaction solution obtained after heating was quantified (glucose oxidase-peroxidase method) using a commercially available kit, Laboassay™ Glucose (Fujifilm Wako Pure Chemical Industries, Ltd.). First, a calibration curve was created based on the relationship between glucose concentration (mM) and absorbance at 505 nm. Absorbance was measured using a spectrophotometer (UV-1800, Shimadzu Corporation). Next, 200 μL of the kit's reagent was added to 50 μL of the enzyme reaction solution obtained after heating, and the mixture was heated and stirred on a hot stirrer at 37°C for 9 minutes. Next, the absorbance of the enzyme reaction solution at 505 nm was measured using a spectrophotometer (UV-1800, Shimadzu Corporation), and the glucose concentration (mM) in the enzyme reaction solution was quantified based on the calibration curve. The measurement results for Tests 1 to 6 are shown in Figure 34.

[0283] (Experimental Results and Discussion) Figure 34 is a graph showing the measurement results for Test Plots 1 to 6. As shown in Figure 34, in Test Plots 1, 3, and 5, which contained cellulase, an increase in the glucose concentration in the reaction solution was confirmed as the reaction time passed. On the other hand, in Test Plots 2 and 4, which did not contain cellulase but contained β-glucosidase, and in Test Plot 6, which was a blank, no increase in the glucose concentration in the reaction solution was confirmed. The amount of glucose produced was greatest in Test Plot 5, 6 hours after the start of the reaction, at approximately 4.0 mg per 1 g of the slurry tea leaves.

[0284] Compared to the glucose concentration at the start of the reaction, the glucose concentration at 6 hours after the start of the reaction increased 1.3-fold in Test Plot 5, while it increased 1.1-fold in Test Plot 3. Therefore, it is estimated that a higher glucose concentration can be obtained by using aromatase as β-glucosidase in combination with cellulase rather than AnBGL.

[0285] From the above, it was confirmed that glucose can be obtained by subjecting the slurry-like tea leaves to saccharification treatment by enzymatic reaction.

[0286] The lignocellulose structure of biomass (woody, herbaceous, etc.) used as a raw material for bioethanol and other products is a complex intertwining of cellulose, hemicellulose, and lignin, making it resistant to the action of enzymes, yeast, etc. Therefore, producing bioethanol by saccharifying and fermenting cellulose and hemicellulose is not easy. When using woody biomass resources, pretreatment is required to weaken the bonds, including lignin, and promote cellulose saccharification. Known pretreatment methods include crushing, hydrothermal treatment (compressed hot water treatment at 100°C or higher), steam explosion, alkali treatment, and dilute sulfuric acid treatment. However, these methods require large-scale equipment and energy, resulting in high costs.

[0287] In contrast, the biofuel production method disclosed herein can use fresh plant leaves, which would normally be treated as waste in various agricultural production processes, and thus can produce bioethanol and the like regardless of food supply and demand. Furthermore, when comparing the amount of lignin in woody biomass such as wood with that in fresh or dried plant leaves, fresh or dried leaves contain approximately 2%, less than one-tenth of the 30-40% lignin content found in woody biomass. Therefore, when using fresh or dried leaves as a biomass resource, the costly pretreatment required for woody biomass is not required.

[0288] Figure 35 shows an outline of an example of a method for producing biofuel according to the present disclosure. In this example, glucose is produced from a plant or a part thereof, and then ethanol is produced from the glucose. As shown in Figure 35, the production of glucose from the plant or a part thereof includes a decomposition step, a first separation step, a second separation step, and a saccharification step. The explanation of each step in the sugar production method described above can be applied to each step.

[0289] In this example, the production of ethanol from glucose includes a fermentation step, a distillation step, a dehydration step, and a solid-liquid separation step, as shown in Figure 35. The fermentation step may be carried out simultaneously with the saccharification step.

[0290] In the fermentation step, yeast is added to the glucose obtained in the saccharification step (fermentation liquid), and the glucose is converted into ethanol by the action of the yeast.

[0291] In the distillation step, ethanol is distilled from the fermentation liquid. In the distillation, the fermentation liquid is distilled to purify ethanol (distillate).

[0292] In the dehydration step, the distillate is dehydrated using a dehydrating agent such as synthetic zeolite to obtain absolute ethanol.

[0293] In the solid-liquid separation step, the fermentation broth is separated into a liquid fraction (stillage) and a solid fraction (residue). The residue can be used as fuel for a biomass boiler or the like.

[0294] The ethanol (bioethanol) can be used, for example, as a substitute for commonly used ethanol, or can be added as an additive to gasoline.

[0295] According to the biofuel production method of the present disclosure, for example, bioethanol can be produced from plants or parts thereof, and useful components derived from the plants or parts thereof, such as proteins, catechins, caffeine, etc., can be simultaneously extracted and used for various purposes.

[0296] The plant part refers to any part of a plant, and examples thereof include tea leaves, tomato leaves, sorghum, grapes, fruit skins, sake lees, beer lees, grasses, pressed fruit residue, and woody leaves. The plant part does not require the above-mentioned pretreatment (crushing, hydrothermal treatment, steam explosion treatment, alkali treatment, dilute sulfuric acid treatment, etc.), but may be subjected to pretreatment such as physical stimulation of the cell walls in the plant tissue (kneading, crushing, wounding, etc.). Examples of the pretreatment include grinding with a food mill, milling, crushing, shearing, heat treatment, kneading (kneading process), squeezing, compression or pressing, squeezing, crushing, friction welding, etc. The inclusion of the pretreatment can improve the efficiency of obtaining an extract (liquid extract) from the plant tissue.

[0297] The biofuel production method of the present disclosure makes it possible to provide alternative energy that contributes to preventing global warming and realizing a sustainable recycling society, for example.

[0298] [Example 12] Used tea leaves obtained from a typical tea beverage production process were subjected to decomposition and separation processes, and the resulting processed product was then subjected to solid-liquid separation to obtain an extract (solid fraction) derived from used tea leaves.

[0299] (Decomposition treatment and separation treatment of used tea leaves) Used tea leaves were subjected to the decomposition treatment and separation treatment in the same manner as in Example 1. The difference from Example 1 is that 200 g of the dried used tea leaves were immersed in 4.0 L of tap water (40°C, pH 4.0) containing an enzyme (24 g of cellulase) for 24 hours, and then stirred and crushed at 4000 rpm for 1 hour using the plant tissue treatment device 1A shown in Figure 1.

[0300] (Solid-liquid separation process) The processed tea leaves were poured onto a commercially available filter (mesh size: 100 μm). The slurry of tea leaves remained on the filter, and the tea leaves extract passed through the filter and fell into a receiving container. In this way, the slurry of tea leaves (solid fraction) was separated from the tea leaves extract and obtained.

[0301] [Alkali Treatment of Solid Fraction] The used tea leaves in a slurry state (solid fraction) was dried, pulverized in a ball mill, and then subjected to an alkali treatment.

[0302] The used tea leaves slurry (solid fraction) of Example 12 was dried at 50°C for 48 hours using a dryer (model WFO-1020, Tokyo Rikakikai Co., Ltd.). The dried solid fraction was then pulverized at 1720 rpm for 0.02 hours using a ball mill (model TI-200, CMT Corporation) (pulverized product).

[0303] 35 g of the ground product was added to 250 mL of 1 M NaOH in a 500 mL polypropylene bottle and heated under pressure using an autoclave (2 atmospheres, 121°C, 20 minutes) (alkali-treated product). The alkali-treated product was then centrifuged (25°C, 8000 x g, 5 minutes) to obtain a precipitated solid fraction.

[0304] The solid fraction was mixed with 250 mL of ultrapure water and then centrifuged again (25°C, 8000 x g, 5 minutes) to obtain a precipitated solid fraction (washing procedure (1)). The washing procedure (1) was repeated until the pH of the supernatant after centrifugation was confirmed to be about 9 using pH test paper.

[0305] The solid fraction obtained after the washing step (1) was mixed with an equal amount of sodium acetate buffer (pH 5.0) and then centrifuged again (25°C, 8000 x g, 5 minutes) to obtain a precipitated solid fraction (washing step (2)). The washing step (2) was repeated twice.

[0306] The solid fraction obtained after the washing step (2) was mixed with an equal amount of ultrapure water and then centrifuged again (25°C, 8000 x g, 5 minutes) to obtain a precipitated solid fraction (washing operation (3)). The washing operation (3) was repeated twice. After the washing operation (3), it was confirmed using pH test paper that the supernatant after centrifugation had a pH of 5 to 7.

[0307] The wet weight and dry weight of the solid fraction after the washing step (3) were 113 g and 11.0 g, respectively. The ratio of the dry weight of the solid fraction (11.0 g) to the dry weight of the solid fraction (ground product) before the alkali treatment (35 g) (recovery rate) was 31.5%.

[0308] [Saccharification and fermentation of solid fraction] The solid fraction after the alkali treatment was subjected to enzymatic saccharification to obtain a saccharified solution, and the saccharified solution was then subjected to fermentation with yeast, whereby it was confirmed that the glucose in the saccharified solution was consumed (assimilated) by the yeast.

[0309] (Experimental Method) (1) Saccharification Treatment: 11.0 g (calculated as bone dry weight) of the solid fraction (wet state) after the alkali treatment, 500 mg of cellulase SS, 100 mg of cellulase XL, and 1 mg of β-glucosidase were added to 50 mM sodium acetate buffer (150 mL, pH 5.0) in a polypropylene bottle (500 mL capacity) to prepare an enzyme reaction solution. The enzyme reaction solution was then subjected to enzymatic saccharification treatment by stirring at 150 rpm for 30 hours under heated conditions of 50°C in a constant temperature shaking incubator. After the saccharification treatment, the enzyme reaction solution was centrifuged (4°C, 10,000 × g, 5 minutes), and the supernatant of the enzyme reaction solution was separated (saccharified solution after alkali treatment). The glucose concentration in the saccharified solution after the alkali treatment was quantified by the same method as in the above-mentioned method for quantifying the glucose concentration, and was found to be 2.3 wt % (22.5 g / L).

[0310] The solid fraction (pulverized product) before the alkali treatment was also subjected to the above-mentioned saccharification treatment (saccharified solution without alkali treatment). The difference from the above saccharification treatment was that 5 g of the solid fraction (pulverized product) before the alkali treatment was used instead of the solid fraction (wet state) after the alkali treatment. The glucose concentration of the saccharified solution without alkali treatment was quantified using the same method as for the glucose concentration quantification described above, and was found to be 0.01 wt % (0.07 g / L). In other words, the solid fraction (pulverized product) before the alkali treatment was hardly saccharified, and the glucose concentration was low. Therefore, the saccharified solution without alkali treatment was not subjected to the fermentation treatment described below.

[0311] (2) Fermentation treatment yeast (Saccharomyces cerevisiae (NBRC100929)) was cultured in 10 mL of YM medium in a polypropylene conical tube (50 mL capacity) at 25°C for 18 hours, and the OD ６００ = 1 (yeast culture solution). The saccharified solution after the alkali treatment was made up to 40 mL by adding YM medium and transferred to a capped flask (250 mL capacity). Next, 250 μL of the yeast culture solution was added to each of the saccharified solution after the alkali treatment for inoculation, and shaking culture was performed under anaerobic conditions (25°C, 150 rpm, 40 hours). The glucose concentrations were quantified at the start of the culture and at 16 and 40 hours after the start of the culture using the same method as for the glucose concentration quantification described above. Note that a control was prepared by adding the same amount of YM medium instead of the yeast culture solution and then performing the shaking culture. The results are shown in Figure 36.

[0312] (Experimental Results and Discussion) Figure 36 is a graph showing the measurement results of the saccharified solution after alkali treatment. As shown in Figure 36, in the saccharified solution to which yeast culture solution was added, a decrease in the glucose concentration in the saccharified solution was confirmed with the passage of culture time ("Saccharomyces cerevisiae (NBRC100929)" in the figure). On the other hand, in the control to which no yeast culture solution was added ("No bacteria" in the figure), no decrease in the glucose concentration in the reaction solution was confirmed.

[0313] Therefore, it was found that in the saccharified solution to which yeast culture medium was added, the glucose in the saccharified solution was consumed (assimilated) by the fermentation reaction by yeast, and the glucose concentration in the saccharified solution decreased.

[0314] Furthermore, yeast performs alcoholic fermentation under anaerobic conditions. Since the fermentation reaction by yeast progressed in the saccharified solution to which the yeast culture solution was added, it can be said that ethanol was produced by the fermentation reaction.

[0315] From the above, it was confirmed that by subjecting the solid fraction after the alkali treatment to enzymatic saccharification treatment to obtain a saccharified solution, and then subjecting the saccharified solution to fermentation treatment with yeast, the glucose in the saccharified solution is consumed (assimilated) by the yeast.

[0316] This application claims priority based on Japanese Patent Application No. 2024-134732, filed August 12, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0317] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0318] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0319] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendixes. <Sugar Production Method> (Appendix 1) A sugar production method comprising: a decomposition step of decomposing cell walls of a plant or a part thereof with a cell wall-decomposing enzyme in the presence of the plant or a part thereof and the cell wall-decomposing enzyme; a first separation step of separating cells of the plant or part thereof from the cell walls or an enzymatic decomposition product thereof in the plant or part thereof; a second separation step of separating the separated product of the cell walls or their decomposition product and the cells of the plant or part thereof from solid-liquid separation to separate a solid fraction; and a saccharification step of saccharifying the solid fraction in the presence of the solid fraction and a saccharifying enzyme. (Appendix 2) The sugar production method according to Appendix 1, wherein the second separation step is performed using solid-liquid separation means. (Appendix 3) The sugar production method according to Appendix 2, wherein the mesh size of the solid-liquid separation means is 1 μm to 10 mm. (Appendix 4) The method for producing sugar according to Appendices 2 or 3, wherein the solid-liquid separation means includes a mesh. (Appendix 5) The method for producing sugar according to any of Appendices 1 to 4, wherein the solid fraction includes insoluble cellulose fibers. (Appendix 6) The method for producing sugar according to any of Appendices 1 to 5, wherein in the first separation step, the cell walls of the plant or parts thereof are mechanically sheared to separate the cells of the plant or parts thereof from the cell walls or enzymatic decomposition products thereof. (Appendix 7) The method for producing sugar according to any of Appendices 1 to 6, wherein in the first separation step, the plant or parts thereof is brought into contact with an agitator blade having a shearing blade to mechanically shear the cell walls of the plant or parts thereof to separate the cells of the plant or parts thereof from the cell walls or enzymatic decomposition products thereof. (Appendix 8) The method for producing sugar according to any of Appendices 1 to 7, wherein at least a part of the decomposition step and the first separation step are carried out simultaneously. (Appendix 9) The method for producing sugar according to any one of Appendices 1 to 8, wherein the decomposition step and / or the first separation step are performed multiple times. (Appendix 10) The method for producing sugar according to any one of Appendices 1 to 9, wherein the cell wall decomposing enzyme is cellulase, hemicellulase, lignin peroxidase and / or pectinase. (Appendix 11) The method for producing sugar according to any one of Appendices 1 to 10, wherein the saccharifying enzyme is cellulase and / or β-glucosidase.(Appendix 12) The method for producing sugar according to any one of Appendices 1 to 11, wherein the plant is Camellia sinensis (tea), Coffea (coffee), Moringa, Brussels sprouts, cabbage, tomato, grape, rice, wheat, barley, oats, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, Zizania latifolia (Willow rice), sugarcane, Job's tears, and / or ginkgo. (Appendix 13) The method for producing sugar according to any one of Appendices 1 to 12, wherein the plant part is a leaf, stem, flower, seed, root, trunk, skin (bark, epidermis, bast, pericarp, etc.), sap, and / or fruit, or a processed product thereof. (Appendix 14) The method for producing sugar according to any one of Appendices 1 to 13, wherein the plant part is tea leaf, used tea leaves, tomato leaf, grass plant leaf, and / or woody plant leaf. (Appendix 15) The method for producing sugar according to any one of Appendices 1 to 14, wherein the liquid fraction separated by solid-liquid separation contains protein, catechin, and / or caffeine derived from the plant or a part thereof. (Appendix 16) The method for producing sugar according to any one of Appendices 1 to 15, comprising, prior to the saccharification step, an alkali treatment step of contacting the solid fraction with an alkali solution to obtain an alkali-treated solid fraction. (Appendix 17) The method for producing sugar according to Appendices 16, comprising a washing step of washing the alkali-treated solid fraction with a liquid to obtain a washed solid fraction. (Appendix 18) The method for producing sugar according to Appendices 17, wherein the saccharification step saccharifies the washed solid fraction in the coexistence of the washed solid fraction and the saccharifying enzyme. <Method for Producing Biofuel> (Appendix 19) A method for producing biofuel, comprising: a production step of producing sugar from a plant or a part thereof using the sugar production method according to any one of Appendices 1 to 18; and a production step of producing biofuel from the sugar. (Appendix 20) The method for producing biofuel according to Appendix 19, wherein the producing step includes fermenting the sugar. (Appendix 21) The method for producing biofuel according to Appendix 20, wherein the fermentation is microbial fermentation. (Appendix 22) The method for producing biofuel according to Appendix 21, wherein the microorganism is yeast, bacteria and / or fungi. (Appendix 23) The method for producing biofuel according to any of Appendices 19 to 22, wherein the biofuel is ethanol, butanol, jet fuel and / or diesel fuel.

[0320] As described above, the present disclosure provides a method for producing sugar from a plant or a part thereof without using alkali treatment or high-temperature, high-pressure treatment, and a method for producing biofuel using the sugar production method. Therefore, the present disclosure is extremely useful in, for example, the energy field.

[0321] REFERENCE SIGNS LIST 1 Plant tissue treatment device 2 Storage container 3 Separation device 4 Stirring device 4c Stirring blade e Tip edge of stirring blade 4c 4T Separation tank 6 Filtering member 7 Component recovery section 10 Control section 11 Plant tissue supply section 12 Electrolyte supply device 12a Electrolyzed water production device 12b Reserve tank 13 Enzyme addition section 20 Supply section 21 Supply flow path 21a Treatment vessel side supply flow path 21b Separation vessel side supply flow path 21d Crushing device side supply flow path 25 Supply side switching mechanism 30 Return section 31 Return flow path 31a Treatment vessel side return flow path 31b Separation vessel side return flow path 31d Crushing device side return flow path 32 First return flow path 33 Second return flow path 35 Return side switching mechanism 40 Crushing device L Mixed liquid L1 Liquid T Plant tissue T1 Non-separated plant tissue M Cell wall C Cell E Extract

Claims

1. A method for producing sugar, comprising: a decomposition step of decomposing cell walls of a plant or part thereof with a cell wall-decomposing enzyme in the presence of the plant or part thereof and the cell wall-decomposing enzyme; a first separation step of separating cells of the plant or part thereof from the cell walls or enzymatic decomposition products thereof in the plant or part thereof; a second separation step of separating the separated product of the cell walls or decomposition products thereof and the cells of the plant or part thereof from solid-liquid separation to separate a solid fraction; and a saccharification step of saccharifying the solid fraction in the presence of the solid fraction and a saccharifying enzyme.

2. The method for producing sugar according to claim 1, wherein the second separation step is carried out using a solid-liquid separation means.

3. The method for producing sugar according to claim 2, wherein the opening of the solid-liquid separation means is 1 μm to 10 mm.

4. The method for producing sugar according to claim 2 or 3, wherein the solid-liquid separation means includes a mesh.

5. A method for producing sugar according to any one of claims 1 to 4, wherein the solid fraction comprises insoluble cellulose fiber.

6. A method for producing sugar described in any one of claims 1 to 5, wherein in the first separation step, the cell walls of the plant or parts thereof are mechanically sheared to separate the cells of the plant or parts thereof from the cell walls or their enzymatic decomposition products.

7. A method for producing sugar described in any one of claims 1 to 6, wherein in the first separation step, the plant or part thereof is brought into contact with an agitator blade having a shearing blade to mechanically shear the cell walls of the plant or part thereof, thereby separating the cells of the plant or part thereof from the cell walls or their enzymatic decomposition products.

8. A method for producing sugars according to any one of claims 1 to 7, wherein at least a portion of the decomposition step and the first separation step are carried out simultaneously.

9. A method for producing sugars according to any one of claims 1 to 8, wherein the decomposition step and / or the first separation step are carried out multiple times.

10. The method for producing sugar according to any one of claims 1 to 9, wherein the cell wall decomposing enzyme is cellulase, hemicellulase, lignin peroxidase and / or pectinase.

11. The method for producing sugar according to any one of claims 1 to 10, wherein the saccharifying enzyme is cellulase and / or β-glucosidase.

12. A method for producing sugar according to any one of claims 1 to 11, wherein the plant is Camellia sinensis (tea), Coffea candida (coffee), Moringa, Brussels sprouts, cabbage, tomato, grape, rice, wheat, barley, oats, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, bamboo, Zizania latifolia (Willow rice), sugarcane, Job's tears, and / or ginkgo.

13. A method for producing sugar according to any one of claims 1 to 12, wherein the plant part is a leaf, stem, flower, seed, root, trunk, skin (bark, epidermis, bast, pericarp, etc.), sap, and / or fruit, or a processed product thereof.

14. A method for producing sugar according to any one of claims 1 to 13, wherein the plant parts are tea leaves, tea leaves, tomato leaves, grass leaves and / or woody leaves.

15. A method for producing sugar according to any one of claims 1 to 14, wherein the liquid fraction separated by the solid-liquid separation contains proteins, catechins and / or caffeine derived from the plant or part thereof.

16. A method for producing sugar according to any one of claims 1 to 15, which comprises, prior to the saccharification step, an alkali treatment step of contacting the solid fraction with an alkali solution to obtain an alkali-treated solid fraction.

17. The method for producing sugar according to claim 16, further comprising a washing step of washing the alkali-treated solid fraction with a liquid to obtain a washed solid fraction.

18. The method for producing sugar according to claim 17, wherein the saccharification step saccharifies the washed solid fraction in the presence of the washed solid fraction and the saccharifying enzyme.

19. A method for producing biofuel, comprising: a production step of producing sugar from a plant or part thereof using the method for producing sugar according to any one of claims 1 to 18; and a production step of producing biofuel from the sugar.

20. The method for producing biofuel of claim 19, wherein the producing step comprises fermenting the sugars.

21. The method for producing biofuel according to claim 20, wherein the fermentation is microbial fermentation.

22. The method for producing biofuel according to claim 21, wherein the microorganism is a yeast, a bacterium and / or a fungus.

23. A method for producing a biofuel according to any one of claims 19 to 22, wherein the biofuel is ethanol, butanol, jet fuel and / or diesel fuel.

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