Methods for inducing primordia of Tricholoma matsutake fruiting bodies, methods for manufacturing primordia of Tricholoma matsutake fruiting bodies, culture medium, and culture solution.

Abstract

The purpose of this invention is to provide a new technique for inducing primordia of *Tricholoma matsutake* fruiting bodies. A method for inducing primordia of *Tricholoma matsutake* fruiting bodies is characterized by culturing *Tricholoma matsutake* strains in a fungal bed culture medium, wherein the fungal bed culture medium comprises a culture solution and a culture medium substrate permeated with the culture solution, the culture solution comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + It is one or more of the following primary metabolites: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate (G6P), pyruvate, oxaloacetic acid, succinic acid, and malic acid.

Description

Technical Field

[0001] This invention relates to a method for inducing primordia of fruiting bodies of matsutake mushrooms, etc. Background Technology

[0002] Tricholoma matsutake is a basidiomycete belonging to the family Tricholomataceae. It is a mycorrhizal fungus that forms symbiotic relationships with the roots of red pine trees. Because the fruiting bodies of Tricholoma matsutake, which grow in symbiosis with red pine trees, have a unique aroma, it is popular and consumed in Japan.

[0003] If various conditions, such as the specific growth requirements, are not met, matsutake mushrooms cannot form fruiting bodies. Therefore, the harvest of matsutake fruiting bodies that naturally reproduce in places like mountains is limited and traded at high prices. Given this situation, there is a need for techniques for matsutake mushroom cultivation (artificial cultivation). However, compared to saprophytic fungi (such as shiitake mushrooms) that grow by decomposing dead trees or fallen leaves, matsutake mushrooms are more difficult to cultivate in mushroom beds, and commercially viable methods for matsutake cultivation have not yet been established.

[0004] The fruiting body of *Tricholoma matsutake* develops from primordia formed by the aggregation of mycelia. Therefore, when cultivating *Tricholoma matsutake* in a spawn bed, it is necessary to induce primordia from the *Tricholoma matsutake* spawn. The following are some reports on techniques for inducing primordia from *Tricholoma matsutake* spawn.

[0005] Non-Patent Document 1 reports the formation of fruiting body primordia by culturing *Tricholoma matsutake* spores in a culture medium containing a prescribed composition of nutrient solution from red pine forest soil (paleozoic soil). Non-Patent Document 2 reports the formation of fruiting body primordia by culturing *Tricholoma matsutake* spores in a culture medium containing a prescribed composition of nutrient solution from vermiculite. Furthermore, Patent Document 1 describes obtaining fruiting body primordia of mycorrhizal fungi by culturing mycorrhizal fungi spores in a culture medium containing water-moistened wheat at a temperature below 30°C. *Tricholoma matsutake* is an example of a mycorrhizal fungi.

[0006] Prior art literature

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 07-115844.

[0009] Non-patent literature

[0010] Non-patent literature 1: Makoto Ogawa and Minoru Hamada, “Formation of primordia of pure cultured matsutake fruiting bodies”, Nihon Mycology Journal, Vol. 16, pp. 406-415, 1975;

[0011] Non-Patent Literature 2: Masayoshi Kawai and Makoto Ogawa, Research on the Cultivation of *Tricholoma matsutake*, "Research on the Cultivation of the 4th Strain and Attempts at Bed Cultivation", *Nikko Kogyo*, Vol. 17, pp. 499-505.

[0012] 1976. Summary of the Invention

[0013] The problem the invention aims to solve

[0014] The purpose of this invention is to provide a new technology for inducing primordia of matsutake fruiting bodies.

[0015] Problem-solving methods

[0016] The inventors attempted to cultivate *Tricholoma matsutake* in a culture bed using the culture solutions described in Non-Patent Literature 1 and Non-Patent Literature 2. However, even when using any culture solution, it was impossible to induce the fruiting body primordia of *Tricholoma matsutake*.

[0017] Based on the experimental results, the inventors conducted in-depth research. They discovered that by adding a specific primary metabolite to the culture medium added to the mushroom bed, the fruiting body primordia of *Tricholoma matsutake* became more easily induced compared to the case without the specific primary metabolite, thus completing this invention.

[0018] The key points of this invention are as follows.

[0019] [1] A method for inducing primordia of matsutake fruiting bodies, characterized in that it includes:

[0020] Tricholoma species are cultivated using a substrate culture medium comprising a culture solution and a substrate impregnated with the culture solution.

[0021] The culture medium includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + It is one or more of the following primary metabolites: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate (G6P), pyruvate, oxaloacetic acid, succinic acid, and malic acid.

[0022] [2] The induction method according to [1] is characterized in that,

[0023] The primary metabolites are pyruvate and reduced nicotinamide adenine dinucleotide (NADH).

[0024] [3] The induction method according to [2] is characterized in that,

[0025] The concentration of pyruvate in the culture medium is 0.01–1 g / L.

[0026] The concentration of reduced nicotinamide adenine dinucleotide (NADH) in the culture medium is 0.1–15 mg / L.

[0027] [4] A method for manufacturing primordia of matsutake fruiting bodies, characterized in that it includes:

[0028] Tricholoma species are cultivated using a substrate culture medium comprising a culture solution and a substrate impregnated with the culture solution.

[0029] The culture medium includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + It is one or more of the following primary metabolites: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate (G6P), pyruvate, oxaloacetic acid, succinic acid, and malic acid.

[0030] [5] A culture medium for culturing matsutake mushrooms, characterized in that it comprises:

[0031] The culture medium and the culture medium substrate impregnated by the culture medium,

[0032] The culture medium includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + It is one or more of the following primary metabolites: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate (G6P), pyruvate, oxaloacetic acid, succinic acid, and malic acid.

[0033] [6] A culture medium, contained in a substrate culture medium for culturing *Tricholoma matsutake* species, characterized in that it comprises:

[0034] The free radicals selected are adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). +It is one or more of the following primary metabolites: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate (G6P), pyruvate, oxaloacetic acid, succinic acid, and malic acid.

[0035] Invention Effects

[0036] According to the present invention, a new technique for inducing primordia of matsutake fruiting bodies can be provided. Attached Figure Description

[0037] Figure 1 This is a graph showing the average number of primary cells for each test tube (test numbers 1-27).

[0038] Figure 2 This is a graph showing the average number of primary fruiting bodies in each test tube (test numbers 28–48).

[0039] Figure 3 It is a photograph of a culture medium in which mushroom primordia have formed. Detailed Implementation

[0040] Hereinafter, one embodiment of the present invention will be described.

[0041] This embodiment relates to a method for inducing the fruiting body primordia of *Tricholoma matsutake* (hereinafter referred to as "Tricholoma fruiting body primordia"). The induction method of this embodiment includes culturing a *Tricholoma* spawn in a prescribed culture medium (hereinafter also referred to as the "culturing process"). Furthermore, in this specification, *Tricholoma* refers to four species: *Tricholoma matsutake*, *Tricholoma bakamatsutake*, *Tricholoma fulvocastaneum*, and *Tricholoma robustum*. In the culturing process, at least one spawn of these four species is cultured.

[0042] The culture medium used in the cultivation process contains a culture solution and a culture medium substrate soaked in the culture solution. The culture solution includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). +The primary metabolites are selected from one or more of the following groups (hereinafter also referred to as "specific primary metabolites"): reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate (G6P), pyruvate, oxaloacetic acid, succinic acid, and malic acid. Furthermore, in this specification, primary metabolites refer to the collective term for substances required to maintain an organism.

[0043] Of the aforementioned primary metabolites, substances other than cyclic AMP (c-AMP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are primary metabolites involved in carbohydrate metabolism (the reaction of breaking down sugars to generate energy). They are intermediate and final products generated during the period leading up to the breakdown of sugars to generate energy (hereinafter also referred to as "carbohydrate metabolites"), as well as enzymes and coenzymes required to carry out (or promote) the various reactions leading up to the breakdown of sugars to generate energy (hereinafter also referred to as "catalysts of carbohydrate metabolism").

[0044] In the aforementioned primary metabolite group, the carbohydrate metabolites are adenosine triphosphate (ATP), guanosine triphosphate (GTP), glucose-6-phosphate (G6P), pyruvate, oxaloacetate, succinate, and malic acid. On the other hand, in the aforementioned specific primary metabolite group, the catalytic substances (more specifically, coenzymes) of carbohydrate metabolism are flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + ) and reduced nicotinamide adenine dinucleotide (NADH).

[0045] The culture medium may contain any primary metabolite selected from one or more of the aforementioned primary metabolite groups. However, among these primary metabolite groups, those selected from adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD) are preferred. + It is one or more of the following primary metabolites: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), pyruvate, oxaloacetic acid, succinic acid, and malic acid.

[0046] The culture medium may contain a specific primary metabolite, which may be selected from one or more substances from the primary metabolite group. When using two or more substances from the primary metabolite group, the combination is not particularly limited; for example, pyruvate (an intermediate product of sugar metabolism) and reduced nicotinamide adenine dinucleotide (NADH) (a catalyst in sugar metabolism) may be used in combination.

[0047] The concentration of a specific primary metabolite in the culture medium is not particularly limited, but the preferred concentrations are as follows.

[0048] From the perspective of more easily inducing primordia of matsutake fruiting bodies, the culture medium contains adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + ), oxidized nicotinamide adenine dinucleotide (NAD) + The concentrations of reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are preferably 0.02–50.0 mg / L, more preferably 0.1–15.0 mg / L, and particularly preferably 0.2–5.0 mg / L, respectively.

[0049] From the viewpoint that it is easier to induce primordia of matsutake fruiting bodies, the concentrations of glucose 6-phosphate (G6P), pyruvic acid, oxaloacetic acid, succinic acid and malic acid in the culture medium are preferably 0.001-5.0 g / L, more preferably 0.005-3.0 g / L, and particularly preferably 0.01-1.0 g / L.

[0050] The culture medium containing the aforementioned specific primary metabolites is a liquid containing nutrients required for the growth of fungi (not limited to matsutake mushrooms), and in addition to the aforementioned specific primary metabolites, it also contains at least a nutrient source and water.

[0051] Nutrient sources are the nutrients required for the growth of fungi (not limited to matsutake mushrooms), such as carbon sources, nitrogen sources, inorganic salts, vitamins, and plant hormones.

[0052] Examples of carbohydrates that can serve as carbon sources include glucose, maltose, molasses, dextrin, glycerol, and starch.

[0053] Examples of nitrogen sources include amino acids such as aspartic acid, arginine, citrulline, ornithine, serine, glutamine, alanine, glycine, methionine, L-isoleucine, and DL-isoleucine. Nitrogen sources are not limited to amino acid monomers; they can also be nitrogen sources containing amino acids and other components. Examples of such nitrogen sources include peptone, meat extract, cottonseed meal, soybean meal, yeast extract, dried yeast, casein, corn steep liquor, and NZ-amine (registered trademark). Alternatively, nitrogen sources that do not contain amino acids can also be used. Examples of such nitrogen sources include ammonium sulfate, ammonium nitrate, and ammonium chloride.

[0054] Examples of inorganic salts include potassium dihydrogen phosphate, sodium phosphate, table salt, calcium carbonate, calcium chloride, magnesium sulfate, manganese chloride, ferric chloride (III), ferrous citrate, ferric citrate, zinc sulfate, manganese sulfate (II), copper sulfate (II), cobalt sulfate (II), and nickel sulfate (II).

[0055] Vitamins refer to vitamins and substances that have the same function as them, such as thiamine hydrochloride (vitamin B1), niacin (vitamin B3), folic acid, biotin (vitamin B7), carnitine chloride, adenine sulfate, etc.

[0056] Plant hormones refer to plant hormones and substances with the same function, such as benzyladenine and gibberellin (GA3).

[0057] The nutrient source in the culture medium can be a water-soluble substance or an insoluble substance. Examples of insoluble substances include dried yeast.

[0058] The concentrations of the above-mentioned components (nutrient sources) in the culture medium can be set based on the concentrations of the components (nutrient sources) in a known substrate culture medium (or culture medium) capable of growing fungi (not limited to matsutake mushrooms), and are not subject to any particular limitation.

[0059] Examples of water contained in a culture medium include pure water, ion-exchange water, filtered water, tap water, and well water.

[0060] In addition to specific primary metabolites, nutrients, and water, culture media may contain other components. Examples of other components that may be included in culture media include pH adjusters such as hydrochloric acid, citric acid, and potassium hydroxide; ribonucleotides such as adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP), and guanosine monophosphate (UMP); and deoxyribonucleotides such as thymidine monophosphate (TMP).

[0061] The pH of the culture medium is not particularly limited; for example, it can be 4.0 to 6.0.

[0062] Regarding the amount of culture solution in the substrate, it should be sufficient to ensure that the substrate is adequately moistened by the culture solution, but without leakage. The amount can be determined by considering the absorbency and water retention of the substrate. For example, in a substrate using a mixture of barley and sawdust (e.g., a mass ratio of 2.5:1 (barley:sawdust)) as the substrate, the culture solution content can be 60-75% by mass relative to 100% of the substrate. From the viewpoint of more easily inducing primordia of *Tricholoma matsutake* fruiting bodies, 65-70% by mass is preferred. Furthermore, in a substrate using soil as the substrate, the culture solution content can be 10-20% by mass relative to 100% of the substrate. From the viewpoint of more easily inducing primordia of *Tricholoma matsutake* fruiting bodies, 15-20% by mass is preferred. Additionally, for example, in a mushroom bed culture medium using vermiculite as the culture medium substrate, the content of the culture medium can be 75-85% by mass relative to 100% by mass of the mushroom bed culture medium, and preferably 80-85% by mass from the viewpoint that it is easier to induce the primordia of matsutake fruiting bodies.

[0063] In the induction method of this embodiment, the culture medium containing a specific primary metabolite may also contain the specific primary metabolite relative to a known culture medium containing a nutrient source and water (a culture medium without the specific primary metabolite). Examples of known culture media containing a nutrient source and water (a culture medium without the specific primary metabolite) include the culture medium described in Non-Patent Document 1 (Hamada medium) and the culture medium described in Non-Patent Document 2 (Kawai / Ogawa medium).

[0064] The culture medium substrate, saturated with nutrient solution, is a solid component used to retain the moisture required for the mycelial elongation of mushrooms (not limited to matsutake mushrooms) and to provide the space necessary for mycelial elongation. The culture medium substrate can be any substrate that can be saturated with nutrient solution; for example, sawdust, barley, chip dust, sphagnum moss, sphagnum moss, corncob, cottonseed hulls, bagasse, alfalfa, beet pulp, rice hulls, timothy, tofu residue, soybean hulls, silica gel, superabsorbent polymers, soil collected from the natural environment, and artificially prepared soil (e.g., vermiculite). Furthermore, two or more of these components (culture medium substrate) can be mixed, and the particle size can be adjusted (e.g., 2–5 mm).

[0065] The method for manufacturing a culture medium is not particularly limited as long as it involves impregnating a culture medium substrate with a culture solution containing a specific primary metabolite. As an example, a culture medium can be manufactured by pre-preparing a culture solution by mixing a specific primary metabolite, a nutrient source, and water, and then adding this culture solution to the culture medium substrate to impregnate it.

[0066] Alternatively, a culture medium can be prepared by sequentially adding a culture medium without the specific primary metabolite and a culture medium containing the specific primary metabolite to a culture medium substrate and allowing them to saturate. Furthermore, in a culture medium prepared using this method, the concentration of the specific primary metabolite in the culture medium is determined as the ratio of the mass of the primary metabolite contained in the culture medium to the total volume of the culture medium in the culture medium (i.e., the total volume of the culture medium without the specific primary metabolite and the culture medium containing the specific primary metabolite).

[0067] In the manufacturing process of the bacterial culture medium, a sterilization process is preferred, which includes killing (or removing) the culture medium substrate and the microorganisms (containing bacteria) contained in the culture medium. The sterilization method used in the sterilization process is not particularly limited; heat sterilization to kill microorganisms by heating or filtration sterilization to remove microorganisms by filtration can be used. Regarding the heating conditions for heat sterilization, if it is heat sterilization under normal pressure, it can be at 98–100°C for 4–12 hours; if it is heat sterilization under high pressure, it can be at 110–125°C for 30–180 minutes. Filtration sterilization can use, for example, membrane filters A020A025A manufactured by ADVANTEC Toyo Co., Ltd.

[0068] Regarding the sterilization treatment to kill (or remove) microorganisms contained in the culture medium substrate and culture medium, this can be performed on the prepared culture medium after the culture medium substrate has been impregnated with the culture medium. However, some primary metabolites may be modified (inactivated) by heating. Therefore, when the sterilization process is included in the preparation of the culture medium, it is preferable to pre-sterilize the culture medium substrate (or the culture medium substrate impregnated with a culture medium that does not contain primary metabolites) by heating, and then add a culture medium containing the specific primary metabolites after filtration and sterilization to the culture medium substrate and impregnate it.

[0069] When cultivating matsutake mushroom strains in a substrate culture medium, at least one strain selected from the group consisting of *Tricholoma matsutake*, *Tricholoma bakamatsutake*, *Tricholoma fulvocastaneum*, and *Tricholoma robustum* can be used. However, from the viewpoint of easily inducing fruiting body primordia, *Tricholoma matsutake* strains are preferred.

[0070] In the cultivation of *Tricholoma matsutake* strains in the culture medium, known strains of *Tricholoma* can be used, and there is no limitation on the strains. Examples of known *Tricholoma matsutake* strains include those with accession numbers NITEBP-03662, NITE BP-03663, NITE BP-03664, NITE BP-03665, NITE BP-03666, NITE BP-03667, and NITE BP-03668. These *Tricholoma matsutake* strains were internationally deposited on August 16, 2022, at the Patent and Microbial Collection Center of the Technical Base for Evaluation of Products, an independent administrative agency, as stipulated by the Budapest Treaty (Tel: 292-0818; Room 122, 2-5-8 Kamezuma-zu, Kisarazu City, Chiba Prefecture, Japan). In addition to these strains, other well-known *Tricholoma matsutake* strains include NBRC 33136 (acceptance date: June 2, 2000) and NBRC 108713 (acceptance date: July 29, 2011). These *Tricholoma matsutake* strains are deposited at the National Institute for Product Evaluation and Technical Support (NITE) (Tel: 292-0818; 2-5-8 Kamezuma-zutari, Kisarazu City, Chiba Prefecture, Japan), and their transfer is permitted through the prescribed procedures. Furthermore, the inoculum of *Tricholoma matsutake* cultured in the culture medium is not limited to the strains mentioned above; the strains used in the examples described later may also be used. Among the *Tricholoma matsutake* strains used in the examples described later, those numbered starting with "FFPRI" are deposited at the National Research and Development Agency for Forestry and Forest Management (1 Matsunosato, Tsukuba City, Ibaraki Prefecture, Japan), and their transfer is permitted through the prescribed procedures.

[0071] Furthermore, the matsutake mushrooms used as inoculum are culture media for the proliferation or maintenance of mycorrhizal fungi (symbiotic fungi). For example, Ohta medium (Ohta, A. (1990), Trans. Mycol. Soc. Japan 31: 323-334), MMN medium (Marx, DH (1969) Phytopathology 59: 153-163), Hamada medium (Hamada (1964) Matsutake, 97-100), etc., can be used to proliferate them. The matsutake mushrooms proliferated (or maintained) in the above-mentioned known culture media can be cultured in the culture medium of the fungal bed involved in this embodiment.

[0072] The inoculum size of *Tricholoma matsutake* species inoculated into the culture medium (hereinafter referred to as "inoculation amount") is not particularly limited; for each 1 cm³ of culture medium... 2 The surface area of ​​the upper surface of the substrate culture medium can be, for example, 0.04 to 3.6 mg by dry weight, but from the viewpoint that it is easier to induce the fruiting body primordia of Tricholoma matsutake, it is preferably 0.4 to 0.9 mg by dry weight.

[0073] The cultivation method for the spawn of matsutake mushrooms in the cultivation process is not particularly limited as long as it enables the growth of mushrooms (not limited to matsutake mushrooms). For example, shaking culture method or static culture method can be used.

[0074] The cultivation conditions for the spawn of *Tricholoma matsutake* in the cultivation process are not particularly limited, as long as the spawn can grow mushrooms (not limited to *Tricholoma matsutake*). For example, the cultivation temperature can be 10–28°C, and from the viewpoint of more easily inducing *Tricholoma matsutake* fruiting body primordia, 15–23°C is preferred. The cultivation humidity can be, for example, 50–95%, and from the viewpoint of more easily inducing *Tricholoma matsutake* fruiting body primordia, 70–90% is preferred. The cultivation time varies, for example, depending on the strain used, and can be, for example, 30–200 days. Sunlight conditions do not affect the induction of *Tricholoma matsutake* fruiting body primordia, but can be, for example, set to 8–16 hours / day, or can be set to dark conditions. The air pressure inside and outside the cultivation container and the composition of the gas phase can be, for example, ordinary air.

[0075] The induction method of this embodiment, including the cultivation steps described above, is more effective at inducing primordia of *Tricholoma matsutake* than culturing *Tricholoma matsutake* strains in a culture medium containing a culture solution that does not contain specific primary metabolites. Furthermore, in this specification, *Tricholoma matsutake* primordia refer to a hairball-like structure (an aggregate of hyphae) formed on the surface of the culture medium; when inducing *Tricholoma matsutake* primordia, these hairball-like protrusions protruding from the culture medium can be visually confirmed.

[0076] Furthermore, according to the induction method of this embodiment that includes the above-described cultivation process, since the fruiting body primordia of *Tricholoma matsutake* are induced, as an embodiment of the present invention, a method for manufacturing fruiting body primordia of *Tricholoma matsutake* including the cultivation process can also be provided.

[0077] Example

[0078] Next, examples will be provided to illustrate the invention in more detail. However, the invention is not limited to these examples.

[0079] <Evaluation 1: Efficacy of a specific primary metabolite>

[0080] The following evaluations 1-1 to 1-2 were conducted to evaluate the effectiveness of specific primary metabolites in inducing the fruiting body primordia of *Tricholoma matsutake*.

[0081] [Evaluation 1-1: Efficacy of using a specific primary metabolite of NITE BP-03663]

[0082] Mix the components (nutrient source, pH adjuster) shown in Table 1 below with water to obtain a culture medium with a pH of 5.1. The content of each component per 1000 ml of culture medium is shown in Table 1 below.

[0083] [Table 1]

[0084]

[0085] The obtained culture medium was divided into multiple portions of 23.5 ml each. The primary metabolites shown in Table 2 below were each mixed with 8 ml of culture medium from each 23.5 ml portion to obtain a culture medium containing the primary metabolites (hereinafter referred to as "culture medium"). 代谢 The obtained culture medium 代谢 The concentrations of the primary metabolites contained therein are shown in Table 2, which will be described later. On the other hand, the remaining culture medium (hereinafter referred to as "culture medium")... 无代谢 The 15.5ml sample does not contain any primary metabolites.

[0086] Prepare barley and sawdust as culture medium substrates. Furthermore, these culture medium substrates should not contain primary metabolites. Add 13.5 ml of culture medium to 7.5 g of the prepared barley. 无代谢 Let the barley stand for 4–16 hours to allow it to swell. Mix 3g of the prepared sawdust with the swelled barley, and then add 2ml of culture medium. 无代谢 Mixing is then performed. Through this operation, the culture medium is cultured in a culture medium substrate composed of barley (7.5g) and sawdust (3g) (hereinafter referred to as "barley culture medium substrate"). 无代谢 Soak a total of 15.5 ml.

[0087] The culture medium 无代谢 Barley culture medium was filled into test tubes with an inner diameter of 30 mm and plugged with a Shin-Etsu SILICOSEN T-32 (trade name) plug manufactured by Shin-Etsu Polymer Co., Ltd. The tubes were sterilized at 121°C for 60 minutes and then cooled. After filtration and sterilization using a membrane filter A020A025A manufactured by ADVANTEC Toyo Co., Ltd., 8 ml of the culture medium was... 代谢 Add it to the cooled barley culture medium substrate and let it soak in to obtain the mycelium culture medium (hereinafter, the mycelium culture medium is referred to as "barley culture medium").

[0088] In addition, the obtained barley culture medium contained 15.5 ml of culture medium. 无代谢 and 8ml of culture medium 代谢 It contains a total of 23.5 ml of culture medium (hereinafter referred to as "culture medium"). 总计 The culture medium contained in the barley culture medium. 总计 The concentrations of the primary metabolites are shown in Table 2, which will be described later.

[0089] NITE BP-03663 was prepared as the spawn for *Tricholoma matsutake*. The prepared spawn was inoculated into barley medium containing primary metabolites and cultured. Specifically, 5.4 mg (dry weight) of liquid-cultured *Tricholoma matsutake* mycelium was inoculated into the aforementioned barley medium filled in test tubes, which were then plugged with a plug made of Shin-Etsu SILICOSEN T-32 manufactured by Shin-Etsu Polymer Co., Ltd. This operation was repeated for each of the test numbers (No.) shown in Table 2 below, so that the spawn for *Tricholoma matsutake* could be inoculated into the barley medium in 5 test tubes (hereinafter referred to as "test tubes 1 to 5"). The tubes were then cultured for 90 days under the following conditions. Additionally, a control medium, prepared under the same conditions as the aforementioned barley medium (barley medium containing primary metabolites), was used as the control medium, and the spawn for *Tricholoma matsutake* was inoculated and cultured using the same method as described above.

[0090] <Cultivation Conditions>

[0091] Temperature: 22℃ for 30 days, then 17℃ for 60 days

[0092] Atmosphere: Same as indoors (air)

[0093] Lighting: None

[0094] Culture method: static culture

[0095] After inoculating *Tricholoma matsutake* with mycelium and culturing for 90 days, the number of hairy, bulbous protuberances (fruiting body primordia) with a diameter of 1 mm or more formed on the surface of the barley culture medium was counted. For each test number (No.) shown in Table 2 below, the number of fruiting body primordia for each test tube was determined (hereinafter referred to as the "average number of fruiting body primordia"). The results are shown in Table 2 and... Figure 1 .

[0096] [Table 2]

[0097]

[0098]

[0099] like Figure 1 As shown in Table 2, the number of fruiting body primordia increased when the barley culture medium contained a specific primary metabolite selected from the primary metabolite group, compared to the case where no primary metabolite was present. This result indicates that the presence of a specific primary metabolite in the barley culture medium can induce the formation of fruiting body primordia.

[0100] In addition, Figures 1-2 In this context, PiAc represents pyruvate, OxAc represents oxaloacetic acid, ScAc represents succinic acid, and MaAc represents malic acid.

[0101] [Evaluation 1-2: Efficacy of using specific primary metabolites of NITE BP-03662]

[0102] In this evaluation, a subset of the primary metabolites used in Evaluation 1-1 were employed. Additionally, NITE BP-03662 was used as the inoculum for *Tricholoma matsutake* ingested in barley medium. Furthermore, under the same conditions as in Evaluation 1-1, the average number of fruiting body primordia per test tube as shown in (1) above was determined. The results are presented in Table 3 and... Figure 2 .

[0103] [Table 3]

[0104]

[0105]

[0106] like Figure 2 As shown in Table 3, the barley culture medium contained adenosine triphosphate (ATP), guanosine triphosphate (GTP), cyclic AMP (c-AMP), flavin mononucleotide (FMN), and oxidized nicotinamide adenine dinucleotide (NAD). + When NITE BP-03662 was used, the number of fruiting body primordia increased compared to the absence of these specific primary metabolites. This result indicates that even when using NITE BP-03662, the presence of specific primary metabolites in the barley medium induces the formation of fruiting body primordia.

[0107] <Evaluation 2: The Influence of the Composition of the Mycobacterial Bed Culture Medium>

[0108] The following evaluations 2-1 to 2-3 were conducted to evaluate the effect of the composition of the culture medium on the induction performance of Tricholoma matsutake fruiting body primordia.

[0109] [Evaluation 2-1: Induction Performance in Soil Culture Medium]

[0110] The components (nutrient source, pH adjuster) shown in Table 4 below were mixed with water to obtain a culture medium with a pH of 5.1. The concentration of each component per 1000 ml of culture medium is shown in Table 4 below. Furthermore, this culture medium is equivalent to the culture medium (Hamada medium) described in Non-Patent Document 1.

[0111] [Table 4]

[0112]

[0113] The obtained culture medium was divided into multiple portions of 12.5 ml each. Reduced nicotinamide adenine dinucleotide (NADH) and pyruvate were each mixed with 1.25 ml of culture medium from each 12.5 ml culture medium to obtain a culture medium containing these primary metabolites (hereinafter, "culture medium"). 代谢 In the obtained culture medium 代谢 In this culture medium, the concentration of reduced nicotinamide adenine dinucleotide (NADH) was 25 mg / L, and the concentration of pyruvate was 2.5 g / L. On the other hand, the remaining 11.25 ml of culture medium (hereinafter referred to as "culture medium")... 无代谢 It does not contain primary metabolites.

[0114] Soil (collected from Shiga Prefecture, Japan) using granite as the parent material was prepared as the culture medium substrate. Furthermore, this culture medium substrate did not contain primary metabolites. 62 g (55 mL) of the prepared soil was filled into 30 mm inner diameter test tubes, sterilized at 120°C for 50 minutes, and then cooled to obtain the soil-based culture medium substrate (hereinafter referred to as "soil culture medium substrate"). Additionally, 11.25 mL of culture medium... 无代谢 Sterilize at 120°C for 15 minutes, then cool. Add it to the soil culture medium substrate to prepare the culture solution. 无代谢 Soaking. Soaking in culture medium. 无代谢 1.25 ml of culture medium, sterilized by filtration using an ADVANTEC membrane filter (A020A025A) manufactured by Toyo Co., Ltd., was added to the soil culture medium substrate. 代谢 The soil is then soaked in the soil to obtain the culture medium for the mushroom bed (hereinafter referred to as the "soil culture medium").

[0115] In addition, the obtained soil culture medium contained 11.25 ml of culture medium. 无代谢 and 1.25 ml of culture medium 代谢 The total volume contains 12.5 ml of culture medium (hereinafter referred to as "culture medium"). 总计 The culture medium contained in the soil culture medium. 总计The concentration of reduced nicotinamide adenine dinucleotide (NADH) was 2.5 mg / L, and the concentration of pyruvate was 0.25 g / L.

[0116] NITE BP-03665, NITE BP-03666, NITE BP-03667, and NITE BP-03668 were prepared as inoculum for *Tricholoma matsutake*. Additionally, FFPRI 460553, FFPRI 460555, FFPRI 460556, FFPRI 460558, FFPRI 460564, FFPRI460565, FFPRI460566, FFPRI 460567, and FFPRI 460568 were prepared as inoculum for *Tricholoma matsutake* (these were isolated from different fruiting bodies collected in Ueda City, Nagano Prefecture, Japan, and are all preserved by the National Forestry and Grassland Administration).

[0117] The prepared *Tricholoma matsutake* spawn was inoculated into soil culture medium containing primary metabolites and cultured. Specifically, 4.5 mg (dry weight) of liquid-cultured *Tricholoma matsutake* spawn was inoculated into the aforementioned soil culture medium containing primary metabolites and cultured for 150 days under the following conditions. Additionally, a soil culture medium prepared under the same conditions as the aforementioned soil culture medium (containing primary metabolites) was used as a control medium, except that it did not contain primary metabolites, and the *Tricholoma matsutake* spawn was cultured using the same method as described above.

[0118] <Cultivation Conditions>

[0119] Temperature: 22℃ for 30 days, then 17℃ for 120 days

[0120] Atmosphere: Same as indoors (air)

[0121] Sunshine duration: None

[0122] Culture method: static culture

[0123] After culturing *Tricholoma matsutake* spawn for 150 days, the presence and diameter of hairy, bulbous protrusions (fruiting body primordia) on the surface of the soil culture medium were visually confirmed. Hairy, bulbous protrusions with a diameter of 1 mm or more were considered to have formed fruiting body primordia. The number of soil culture media from which fruiting body primordia could be confirmed was counted. The counting results are shown in Table 5 below. Furthermore, Figure 3(a) shows a photograph of a soil culture medium in which fruiting body primordia have formed. Furthermore, regarding the fractions shown in Table 5 below, the numerator represents the number of test tubes (soil culture medium) in which fruiting body primordia could be identified, and the denominator represents the number of test tubes (soil culture medium) ingested with Tricholoma spores.

[0124] [Table 5]

[0125]

[0126]

[0127] As shown in Table 5, when the soil culture medium contained reduced nicotinamide adenine dinucleotide (NADH) and pyruvate as primary metabolites, fruiting body primordia were induced in at least one of the test tubes (soil culture medium) containing *Tricholoma matsutake* spores. Conversely, when the soil culture medium did not contain primary metabolites, fruiting body primordia were not induced in any of the test tubes (soil culture medium) containing *Tricholoma matsutake* spores.

[0128] [Evaluation 2-2: Induction performance in vermiculite medium]

[0129] The components shown in Table 6 below were mixed with water to obtain a first solution with a pH of 5.0–5.2. The concentration of each component in the first solution per 1000 ml is shown in Table 6 below. Furthermore, the components shown in Table 7 below were mixed with water to obtain a second solution with a pH of 5.0–5.2. The concentration of each component in the second solution per 1000 ml is shown in Table 7 below. These culture media are equivalent to the culture media (Kawai / Ogawa medium) described in Non-Patent Document 2.

[0130] [Table 6]

[0131]

[0132] [Table 7]

[0133] Element Concentration ( / 1000ml) Thiamine 1mg Nicotinic acid 1mg folic acid 1mg Biotin 0.1mg AMP 1mg TMP 1mg GMP 1mg CMP 1mg UMP 1mg

[0134] Further, reduced nicotinamide adenine dinucleotide (NADH) and pyruvate were mixed with the obtained second solution to obtain a culture medium containing primary metabolites (hereinafter also referred to as "culture medium"). 代谢 In the obtained culture medium 代谢 The concentration of reduced nicotinamide adenine dinucleotide (NADH) was 25 mg / L, and the concentration of pyruvate was 2.5 g / L.

[0135] Prepare vermiculite as the culture medium substrate. Furthermore, this culture medium substrate should not contain primary metabolites. Fill 27g (200mL) of the prepared vermiculite into a 500mL flask to prepare the culture medium substrate composed of vermiculite (hereinafter referred to as "vermiculite culture medium substrate"). Add 120mL of the first solution to each flask, seal, sterilize at 120°C for 50 minutes, and then cool. Filter 12mL of culture medium sterilized using a membrane filter (A020A025A) manufactured by ADVANTEC Toyo Co., Ltd. 代谢 The culture medium is added to the cooled vermiculite culture medium substrate and allowed to saturate it to obtain the culture medium (hereinafter referred to as "vermiculite culture medium").

[0136] In addition, the obtained vermiculite culture medium contained 120 ml of the first solution and 12 ml of culture medium. 代谢 The total volume contains 132 ml of culture medium (hereinafter referred to as "culture medium"). 总计 The culture medium contained in vermiculite medium contains this culture solution. 总计 The concentration of reduced nicotinamide adenine dinucleotide (NADH) was 2.3 mg / L, and the concentration of pyruvate was 0.23 g / L.

[0137] As inoculum for *Tricholoma matsutake*, NITE BP-03665, NITE BP-03666, NITE BP-03667, and NITE BP-03668 were prepared. Additionally, as inoculum for *Tricholoma matsutake*, FFPRI 460552, FFPRI 460558, FFPRI 460559, FFPRI 460560, FFPRI 460561, FFPRI460563, FFPRI460564, and FFPRI 460567 were prepared (these were isolated from different fruiting bodies collected in Ueda City, Nagano Prefecture, Japan, and are all preserved by the National Forestry and Grassland Administration).

[0138] The prepared spawn of *Tricholoma matsutake* was inoculated into the vermiculite medium containing primary metabolites and cultured. Specifically, 43.2 mg (dry weight) of liquid-cultured *Tricholoma matsutake* spawn was inoculated into the vermiculite medium containing primary metabolites and cultured for 150 days under the following conditions.

[0139] <Cultivation Conditions>

[0140] Temperature: 17℃

[0141] Atmosphere: Same as indoors (air)

[0142] Lighting: None

[0143] Culture method: static culture

[0144] After culturing *Tricholoma matsutake* spawn for 150 days, the presence and diameter of hairy, bulbous protrusions (fruiting body primordia) forming on the surface of the soil culture medium were visually confirmed. Hairy, bulbous protrusions with a diameter of 1 mm or more were considered to indicate the formation of *Tricholoma matsutake* fruiting body primordia. The number of test tubes (soil culture medium) from which fruiting body primordia could be confirmed was counted. The counting results are shown in Table 8 below. Additionally, Figure 3 (b) shows a photograph of the vermiculite medium on which the fruiting body primordia of *Tricholoma matsutake* formed. Furthermore, regarding the fractions shown in Table 8 below, the numerator represents the number of test tubes (vermiculite medium) on which the fruiting body primordia could be identified, and the denominator represents the number of test tubes (vermiculite medium) on which *Tricholoma matsutake* spores were ingested.

[0145] [Table 8]

[0146]

[0147]

[0148] As shown in Table 8, when the vermiculite medium contained reduced nicotinamide adenine dinucleotide (NADH) and pyruvate as primary metabolites, fruiting body primordia were induced in at least one of the test tubes (vermiculite medium) containing *Tricholoma matsutake* spores. Conversely, when the vermiculite medium did not contain primary metabolites, fruiting body primordia were not induced in any of the test tubes (soil medium) containing *Tricholoma matsutake* spores.

[0149] [Evaluation 2-3: Induction performance in barley medium]

[0150] Except that the raw materials used were doubled (i.e., barley culture medium was prepared in a 300ml flask with 15g barley, 6g sawdust, and 47ml culture medium) and reduced nicotinamide adenine dinucleotide (NADH) and pyruvate were used as primary metabolites in the culture medium, the barley culture medium was prepared using the same method as in Evaluation 1-1. The culture medium contained in the prepared barley culture medium... 总计 In 47 ml, the concentration of reduced nicotinamide adenine dinucleotide (NADH) was 1.7 mg / L and the concentration of pyruvate was 0.17 g / L.

[0151] As inoculum for *Tricholoma matsutake*, NITE BP-03663, NITE BP-03664, NITE BP-03666, NITE BP-03667, and NITE BP-03668 were prepared. Additionally, as inoculum for *Tricholoma matsutake*, FFPRI 460561, FFPRI460564, FFPRI 460565, FFPRI 460566, and FFPRI460567 were prepared (these were isolated from different fruiting bodies collected in Ueda City, Nagano Prefecture, Japan, and are all preserved by the National Forestry and Grassland Administration).

[0152] The prepared *Tricholoma matsutake* spawn was inoculated into the aforementioned barley culture medium containing primary metabolites and cultured. Specifically, 36 mg (dry weight) of liquid-cultured *Tricholoma matsutake* spawn was inoculated into the aforementioned barley culture medium containing primary metabolites and cultured for 150 days under the following conditions. Additionally, a control medium, prepared under the same conditions as the aforementioned barley culture medium (containing primary metabolites), was used, and the *Tricholoma matsutake* spawn was cultured using the same method as described above.

[0153] <Cultivation Conditions>

[0154] Temperature: 22℃ for 30 days, then 17℃ for 120 days

[0155] Atmosphere: Same as indoors (air)

[0156] Sunshine duration: None

[0157] Culture method: static culture

[0158] After culturing *Tricholoma matsutake* mycelium for 120 days, the presence and diameter of hairy, bulbous protrusions (fruiting body primordia) forming on the surface of barley culture medium were visually confirmed. Hairy, bulbous protrusions with a diameter of 1 mm or more were considered to indicate the formation of *Tricholoma matsutake* fruiting body primordia. The number of test tubes (barley culture medium) from which fruiting body primordia could be confirmed was counted. The counting results are shown in Table 9 below. Additionally, Figure 3 (c) shows a photograph of barley culture medium in which fruiting body primordia of *Tricholoma matsutake* were formed. Furthermore, regarding the fractions shown in Table 9 below, the numerator represents the number of test tubes (barley culture medium) in which fruiting body primordia could be identified, and the denominator represents the number of test tubes (barley culture medium) in which *Tricholoma matsutake* inoculum was ingested.

[0159] [Table 9]

[0160]

[0161]

[0162]

[0163] As shown in Table 9, when the barley culture medium contained reduced nicotinamide adenine dinucleotide (NADH) and pyruvate as primary metabolites, fruiting body primordia were induced in at least one test tube (barley culture medium) containing *Tricholoma matsutake* spores. Conversely, when the barley culture medium did not contain primary metabolites, fruiting body primordia were not induced in any of the test tubes (barley culture medium) containing *Tricholoma matsutake* spores.

[0164] As demonstrated in Evaluations 2-1 to 2-3 above, even if the composition of the substrate (i.e., the type of substrate and the composition of the culture medium) changes, the fruiting body primordia of *Tricholoma matsutake* will be induced as long as a specific primary metabolite is present in the culture medium. Furthermore, as shown in Evaluations 2-1 to 2-3, it is evident that if the culture medium contains a primary metabolite, the fruiting body primordia will be induced for various strains of *Tricholoma matsutake*.

Claims

1. A method for inducing primordia of matsutake fruiting bodies, characterized in that, include: Tricholoma species are cultivated using a substrate culture medium comprising a culture solution and a substrate impregnated with the culture solution. The culture medium comprises, selected from 0.02–50.0 mg / L of adenosine triphosphate (ATP), 0.02–50.0 mg / L of guanosine triphosphate (GTP), 0.02–50.0 mg / L of flavin adenine dinucleotide (FAD), 0.02–50.0 mg / L of flavin mononucleotide (FMN), and 0.02–50.0 mg / L of oxidized nicotinamide adenine dinucleotide (NAD). + It consists of one or more primary metabolites from the group consisting of 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide (NADH), 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide phosphate (NADPH), 0.001–5.0 g / L of glucose-6-phosphate (G6P), 0.001–5.0 g / L of pyruvate, 0.001–5.0 g / L of oxaloacetic acid, 0.001–5.0 g / L of succinic acid, and 0.001–5.0 g / L of malic acid.

2. The induction method according to claim 1, characterized in that, The primary metabolites are pyruvate and reduced nicotinamide adenine dinucleotide (NADH).

3. The induction method according to claim 2, characterized in that, The concentration of pyruvate in the culture medium is 0.01–1 g / L. The concentration of reduced nicotinamide adenine dinucleotide (NADH) in the culture medium is 0.1–15 mg / L.

4. A method for manufacturing primordia of matsutake fruiting bodies, characterized in that, include: Tricholoma species are cultivated using a substrate culture medium comprising a culture solution and a substrate impregnated with the culture solution. The culture medium comprises, selected from 0.02–50.0 mg / L of adenosine triphosphate (ATP), 0.02–50.0 mg / L of guanosine triphosphate (GTP), 0.02–50.0 mg / L of flavin adenine dinucleotide (FAD), 0.02–50.0 mg / L of flavin mononucleotide (FMN), and 0.02–50.0 mg / L of oxidized nicotinamide adenine dinucleotide (NAD). + It consists of one or more primary metabolites from the group consisting of 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide (NADH), 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide phosphate (NADPH), 0.001–5.0 g / L of glucose-6-phosphate (G6P), 0.001–5.0 g / L of pyruvate, 0.001–5.0 g / L of oxaloacetic acid, 0.001–5.0 g / L of succinic acid, and 0.001–5.0 g / L of malic acid.

5. A substrate culture medium for culturing matsutake mushrooms, characterized in that, include: The culture medium and the culture medium substrate impregnated by the culture medium, The culture medium comprises, selected from 0.02–50.0 mg / L of adenosine triphosphate (ATP), 0.02–50.0 mg / L of guanosine triphosphate (GTP), 0.02–50.0 mg / L of flavin adenine dinucleotide (FAD), 0.02–50.0 mg / L of flavin mononucleotide (FMN), and 0.02–50.0 mg / L of oxidized nicotinamide adenine dinucleotide (NAD). + It consists of one or more primary metabolites from the group consisting of 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide (NADH), 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide phosphate (NADPH), 0.001–5.0 g / L of glucose-6-phosphate (G6P), 0.001–5.0 g / L of pyruvate, 0.001–5.0 g / L of oxaloacetic acid, 0.001–5.0 g / L of succinic acid, and 0.001–5.0 g / L of malic acid.

6. A culture medium contained in a substrate culture medium for culturing matsutake mushrooms, characterized in that, include: Selected from 0.02–50.0 mg / L adenosine triphosphate (ATP), 0.02–50.0 mg / L guanosine triphosphate (GTP), 0.02–50.0 mg / L flavin adenine dinucleotide (FAD), 0.02–50.0 mg / L flavin mononucleotide (FMN), and 0.02–50.0 mg / L oxidized nicotinamide adenine dinucleotide (NAD) + It consists of one or more primary metabolites from the group consisting of 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide (NADH), 0.02–50.0 mg / L of reduced nicotinamide adenine dinucleotide phosphate (NADPH), 0.001–5.0 g / L of glucose-6-phosphate (G6P), 0.001–5.0 g / L of pyruvate, 0.001–5.0 g / L of oxaloacetic acid, 0.001–5.0 g / L of succinic acid, and 0.001–5.0 g / L of malic acid.

Images