Matsutake primordia and method for producing the same

Abstract

To provide a method for artificially producing a fruiting body primordium of Tricholoma matsutake under a non-symbiotic environment with plants, and a fruiting body primordium of Tricholoma matsutake produced by the method, and a novel Tricholoma matsutake strain used for producing the fruiting body primordium of Tricholoma matsutake.SOLUTION: A cell having primordium-forming ability separated from a Tricholoma matsutake fruiting body is cultured in culture medium that contains a protein decomposition product and has specific free amino acid concentration.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel Matsutake strain, as well as the primordia of said Matsutake and a method for producing the same. [Background technology]

[0002] Although there have been reports of the formation of Matsutake fruit body primordia of about several millimeters in size by fully artificial culture (e.g., Non-Patent Documents 1 and 2), there have been no reports of the growth of fruit body primordia. In addition, there are few reports of the reproduction of fruit body primordium formation, and currently, there is no established method for the formation and growth of Matsutake fruit body primordia (e.g., Non-Patent Document 3).

[0003] Research has also been conducted for many years on the so-called "shiro," which is an aggregate of Matsutake mycelium and red pine roots in the soil, and it is estimated that in nature, it takes about seven years for shiro to form from the time of spore fall to the time of the first fruit body appearing (e.g., Non-Patent Document 4). In addition, Matsutake fruit body primordia appear from vegetatively growing mycelium, and it is said that about 100 g of mycelium is required in dry weight to produce one Matsutake fruit body (e.g., Non-Patent Document 5). Therefore, it is thought that even in artificial cultivation of Matsutake, a large amount of mature Matsutake mycelium that functions as a colony that grows toward the Matsutake fruit body primordia is required (e.g., Non-Patent Documents 4 and 6). Furthermore, since the growth of Matsutake mycelium is extremely slow (approximately 20 mm / month) and requires a long time for growth (e.g., non-patent literature 7), studies on culture conditions and growth-promoting substances have been conducted in order to mass-cultivate Matsutake mycelium (e.g., non-patent literature 3, 8, and 9). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Journal of the Mycological Society of Japan 16(4), p406-415, 1976-01 [Non-Patent Document 2] “Forestry and Forest Products Research Institute Research Report” (Bulletin of FFPRI) Vol.19-No.2 (No.454)153-157 August 2020 [Non-Patent Document 3] 2009 Doctoral thesis, University of Tokyo (Katsumi Shindo) [Non-Patent Document 4] Hiroshima Prefectural Forestry Research Institute Bulletin 28:49-54,1993 [Non-Patent Document 5] Journal of Forestry 87:90-102 [Non-Patent Document 6] Biology of "Matsutake" P326,1978 [Non-Patent Document 7] Mycobiology 29(4):183-189(2001) [Non-Patent Document 8] Mycol Prog 2,37-44,2003 [Non-Patent Document 9] Okayama Prefectural Agriculture, Forestry and Fisheries Research Center Forest Research Institute Research Report 32:19-23(2016) Summary of the Invention [Problem to be solved by the invention]

[0005] However, there have been no reports to date of artificially inducing differentiation of Matsutake into fruiting body primordia and then causing them to further differentiate into fruiting bodies, and conventional techniques have only been limited in their effectiveness as a method for stably obtaining Matsutake fruiting body primordia and growing said fruiting body primordia.

[0006] Therefore, the present invention relates to a method for artificially producing matsutake fruiting body primordia in an environment that does not coexist with plants, matsutake fruiting body primordia produced by said method, and a novel matsutake strain that can be used to produce the matsutake fruiting body primordia. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that the above-mentioned problems can be solved by culturing cells isolated from the fruiting body of Tricholoma matsutake under specific conditions. The present invention is based on this finding. That is, the gist of the present invention is as follows.

[0008] [1] A method for producing an elongated Matsutake primordium, comprising the steps of: (a) culturing cells capable of forming primordia separated from a fruiting body of Matsutake in a first medium containing a first protein hydrolysate or amino acids and having a free amino acid concentration of 0.1 to 2.5 (w / v) % to form Matsutake primordia; (b) culturing the Matsutake primordia in a culture support containing a highly absorbent material infiltrated with a second medium containing a second protein hydrolysate or amino acids and having a free amino acid concentration of 0.05 to 2.5 (w / v)%, thereby elongating the Matsutake primordia vertically upward to obtain elongated Matsutake primordia; Including, The first protein hydrolysate and the second protein hydrolysate each contain at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone and tryptone, or contain a combination of casamino acid and at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone and tryptone; The method. [2] The method according to [1], wherein the culture temperature in the step (a) is 18 to 25°C. [3] The method according to [1] or [2], wherein the culture temperature in the step (b) is 18 to 30°C. [4] The method according to any one of [1] to [3], wherein the highly absorbent material comprises at least one selected from the group consisting of plant fibers, highly absorbent fibers, highly absorbent resins, agar, and artificial mineral fibers. [5] The method according to any one of [1] to [4], wherein both or one of the first medium and the second medium further contain 0.2 to 5 (w / v)% of sugars. [6] The method according to any one of [1] to [5], wherein both or one of the first medium and the second medium further contains at least one selected from the group consisting of inorganic salts, vitamins, lipids and amino acids. [7] The method according to any of [1] to [6], wherein the fruiting body is identified as Tricholoma matsutake by molecular phylogenetic analysis based on the base sequence of ITS-5.8S rDNA. [8] The method according to [7], wherein the fruiting body is a fruiting body of Tricholoma matsutake, which is identified by accession number NITE P-03769. [9] A method for producing a fruiting body-like Matsutake primordium, further comprising a step of growing the elongated Matsutake primordium obtained by the method according to any one of [1] to [8].

[10] An elongated Matsutake primordium produced by the method according to any one of [1] to [8].

[11] [9] A fruiting body-like Matsutake primordium produced by the method described in

[11] [9].

[12] Cells isolated from the fruiting body of Tricholoma matsutake, identified by accession number NITE P-03769. Effect of the Invention

[0009] According to the present invention, it is possible to provide a method for artificially producing matsutake fruiting body primordia in an environment that does not coexist with plants, matsutake fruiting body primordia produced by said method, and a novel matsutake strain that can be used to produce the matsutake fruiting body primordia. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a photograph showing an elongated Matsutake primordium obtained by the production method of the present invention. [Diagram 2] FIG. 2 is a photograph showing the appearance of Matsutake primordia or colonies obtained from each of the Matsutake cells of NITE P-03769 and NBRC#109050. [Diagram 3]FIG. 3 is a photograph showing the extension of hyphae in Matsutake primordia or colonies obtained from each of the Matsutake cells of NITE P-03769 and NBRC#109050. [Figure 4] FIG. 4 is a photograph showing the appearance of Matsutake primordia or colonies obtained from each of the Matsutake cells of NITE P-03769, NBRC#33136, and NBRC#109050. [Diagram 5] Figure 5 is a photograph showing the time-dependent growth of Matsutake primordia obtained from Matsutake cells of NITE P-03769 when they were cultured in a highly absorbent medium-containing material and then further cultured in another highly absorbent medium-containing material. [Figure 6] FIG. 6 is a photograph showing the appearance of Matsutake primordia or colonies obtained from Matsutake cells of NITE P-03769, NBRC#109050, NBRC#108259, and NBRC#108261. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [Method of manufacturing Matsutake fruit body primordia] According to one aspect of the present invention, there is provided a method for producing an elongated Matsutake fruiting body primordium (hereinafter also simply referred to as "Matsutake primordium") (hereinafter also simply referred to as "the production method of the present invention").

[0012] In this specification, "matsutake fruiting body primordium" and "matsutake primordium" refer to a mass of hyphae (hyphal mass) formed by the proliferation of dikaryotic hyphae (secondary hyphae) and assembly in a certain direction, a part of which (particularly the central part) is raised vertically upward, and is, so to speak, the "base" of the matsutake fruiting body. In general, secondary hyphae assembled in a certain direction are considered to be more differentiated than secondary hyphae that do not have such directionality, and are sometimes called tertiary hyphae to distinguish them from secondary hyphae. In addition, "elongated matsutake primordium" refers to a matsutake primordium in a state in which the raised part of the above-mentioned matsutake primordium is further raised, and as a result, the entire or almost entire matsutake primordium is raised.

[0013] In this specification, with regard to "cells having the ability to form primordia isolated from a Matsutake fruiting body", "having the ability to form primordia" means that the cells isolated from a Matsutake fruiting body have a property in which the mycelium grows (elongates) vertically upward on a plate agar medium depending on the concentration of free amino acids, and when the free amino acids reach a certain concentration, a raised mycelium mass is formed. Typical examples of such cells are: Pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (hereinafter referred to simply as the "Budapest Treaty"), The material was deposited domestically at the National Institute of Technology and Evaluation (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan) under the accession number NITE P-03769. It was subsequently transferred to international depository status and deposited internationally under the accession number NITE BP-03769 (domestic deposit date: October 18, 2022; date of receipt of request for transfer to international depository: October 16, 2023). Examples of cells that do not have the ability to form primordia include cells isolated from the fruiting body of Tricholoma matsutake. As shown in Example 9 described later, cells isolated from the fruiting body of Tricholoma matsutake, identified by the accession number NITE P-03769, have the characteristic that, on a plate agar medium with a free amino acid concentration of 0.05 (w / v)% or more and less than 0.1 (w / v)%, mycelia are densely packed in the center of the colony, and the densely packed mycelia further rise vertically upward. Furthermore, as shown in Example 7 described later, on a plate agar medium with a free amino acid concentration of 0.1 to 2.5 (w / v)%, mycelia densely packed and raised in the center of the colony further rise and extend vertically upward to form a mycelium mass (i.e., Matsutake primordia). On the other hand, examples of cells that do not have the ability to form primordia include cells isolated from the fruiting body of Tricholoma matsutake, which is typically deposited in NPMD under NBRC#109050. As shown in Example 9 below, in cells isolated from the fruiting body of Tricholoma matsutake deposited under NBRC#109050, on agar plate media with a free amino acid concentration of 0.05% (w / v) or more and less than 0.1% (w / v), mycelia mainly extend horizontally, and mycelia are hardly clustered in the center of the colony or protrude vertically upward. Furthermore, as shown in Example 7 below, on agar plate media with a free amino acid concentration of 0.1 to 2.5% (w / v), mycelia are clustered in the center of the colony, but they do not form a mycelium mass.

[0014] The production method of the present invention includes a step of culturing cells separated from a Matsutake fruiting body under specific conditions to form a Matsutake primordium (step (a)), and a step of culturing the formed Matsutake primordium under specific conditions to obtain an elongated Matsutake primordium (step (b)). According to the production method of the present invention, elongated Matsutake primordium can be artificially produced in an environment that does not coexist with plants. Steps (a) and (b) of the production method of the present invention are described in detail below.

[0015] <Process (a)> In step (a), cells capable of forming primordia separated from a Matsutake fruiting body are cultured under specific conditions to form Matsutake primordia.

[0016] As the Matsutake fruiting body, any fruiting body of Tricholoma matsutake can be used without any particular limitation. As the Matsutake fruiting body, a fruiting body identified as Tricholoma matsutake by molecular phylogenetic analysis based on the base sequence of ITS-5.8S rDNA, particularly preferably a fruiting body of Tricholoma matsutake identified by the accession number NITE P-03769, is used.

[0017] (Culture medium) The first medium used in step (a) is not particularly limited as long as it is a medium that is usually used for culturing fungal, particularly mushroom, cells. As the first medium, either a solid medium or a liquid medium can be used, but a solid medium is preferably used. As the solid medium, for example, a medium in which a liquid medium has been solidified with a solidifying agent, preferably a gel-like solid medium having no fluidity, can be used. As the solid medium, any of a plate medium, a slant medium, a semi-slant medium, and a high-layer medium can be used, but a plate medium is preferably used.

[0018] The liquid medium can be any liquid medium that is commonly used for culturing fungi, particularly mushroom cells, without any particular limitations. In addition, examples of solidifying agents that solidify the liquid medium include agar, mannan, gellan, gelatin, and Kelcogel. Agar is preferably used as the solidifying agent, that is, an agar medium is preferably used as the first medium.

[0019] When an agar medium is used as the first medium, the concentration of agar in the medium is not particularly limited as long as the effects of the present invention are achieved, but is, for example, 0.5 to 3.0 (w / v)%, 1.0 to 2.0 (w / v)%, 1.2 to 1.8 (w / v)%, and preferably about 1.5 (w / v)%.

[0020] In one embodiment, the first medium contains the first protein hydrolysate. The first medium may contain, in addition to the above-mentioned first protein hydrolysate, additional components such as sugars, minerals (e.g., inorganic salts, etc.), vitamins, lipids, amino acids, etc. These additional components may be used alone or in combination of two or more.

[0021] The first protein hydrolysate is not particularly limited as long as it is a collection of amino acids or peptides obtained by hydrolyzing a protein using an enzyme, acid, etc., and examples of the first protein hydrolysate include protein hydrolysates and enzymatic digests of proteins.

[0022] The first protein hydrolysate is not limited as long as the effects of the present invention are achieved, and examples thereof include yeast extract, beef extract, peptone, tryptone, casamino acid, etc. The first protein hydrolysate may be used alone or in combination of two or more kinds.

[0023] In one embodiment, the first protein hydrolysate is at least one selected from the group consisting of yeast extract, beef extract, peptone and tryptone.

[0024] In another embodiment, the first protein hydrolysate is a combination of casamino acids and at least one selected from the group consisting of yeast extract, beef extract, peptone and tryptone.

[0025] In yet another embodiment, when casamino acids are used alone as the first protein hydrolysate, inorganic salts such as magnesium salts are further added to the culture medium as necessary, and preferably amino acids such as tryptophan, vitamins, lipids, etc., as described below, are further added.

[0026] In one embodiment, the first medium contains amino acids instead of or in addition to the above-mentioned first protein hydrolysate. In this embodiment, the "amino acids" refer to amino acids other than those derived from the above-mentioned "first protein hydrolysate". The amino acids may be synthesized amino acids (synthetic amino acids) or naturally occurring amino acids (natural amino acids), and any amino acids can be used. The amino acids can be obtained by methods commonly used for producing amino acids, such as fermentation, enzymatic, extraction, and synthetic methods.

[0027] The type of amino acid is not particularly limited as long as the effects of the present invention are achieved, and examples include proteinogenic amino acids (i.e., α-amino acids), non-proteinogenic amino acids, and derivatives thereof.

[0028] Examples of amino acids that make up proteins include essential amino acids for humans (isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, histidine) and non-essential amino acids (tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, arginine).

[0029] An example of a derivative is cystine (3,3'-dithiobis(2-aminopropionic acid)), in which two cysteine ​​molecules are linked via a disulfide bond formed by oxidation of the hydrosulfide groups (-SH).

[0030] The first medium may contain at least one selected from the above-mentioned amino acids and derivatives, preferably two or more, more preferably five or more, even more preferably ten or more, and particularly preferably fifteen or more amino acids and / or derivatives. In a preferred embodiment, the first medium contains at least one amino acid and / or derivative selected from the group consisting of valine, leucine, isoleucine, threonine, methionine, tryptophan, lysine, histidine, glycine, arginine, glutamic acid, alanine, aspartic acid, proline, serine, cystine, and tyrosine, more preferably two or more, even more preferably five or more, even more preferably ten or more, even more preferably fifteen or more amino acids and / or derivatives, and particularly preferably all eighteen amino acids and derivatives.

[0031] The total concentration of amino acids and derivatives in the first medium is not particularly limited as long as the first medium satisfies the range of free amino acid concentration described below, and can be appropriately set according to the type of amino acid used, the type and concentration of other components of the first medium, the total nitrogen amount described below, etc. For example, the total concentration of amino acids and derivatives in the first medium can be 0.1 to 3.0 (w / v)%, 0.5 to 2.0 (w / v)%, 0.7 to 1.8 (w / v)%, etc. In addition, when the first medium contains amino acids and / or derivatives other than the amino acids derived from the above-mentioned first protein hydrolysate, the concentrations of each amino acid and derivative in the first medium can be set as follows, for example. Valine: 0.001 to 2.0 (w / v)% (preferably 0.01 to 0.2 (w / v)%) Leucine: 0.001 to 1.0 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Isoleucine: 0.001 to 1.0 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Threonine: 0.001 to 1.0 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Methionine: 0.001 to 1.0 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Tryptophan: 0.0001 to 1.0 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Lysine: 0.001 to 1.0 (w / v)% (preferably 0.01 to 0.5 (w / v)%) Histidine: 0.001 to 0.5 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Glycine: 0.001 to 0.5 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Arginine: 0.001 to 0.2 (w / v)% (preferably 0.01 to 0.1 (w / v)%) Glutamic acid: 0.01 to 1.0 (w / v)% (preferably 0.03 to 0.5 (w / v)%) Alanine: 0.01 to 0.5 (w / v)% (preferably 0.05 to 0.2 (w / v)%) Aspartic acid: 0.001 to 0.5 (w / v)% (preferably 0.01 to 0.2 (w / v)%) Asparagine: 0.0001 to 0.1 (w / v)% (preferably 0.01 to 0.05 (w / v)%) Proline: 0.001 to 2.0 (w / v)% (preferably 0.01 to 0.5 (w / v)%) Serine: 0.001 to 2.0 (w / v)% (preferably 0.01 to 0.5 (w / v)%) Cystine: 0.0001 to 0.05 (w / v)% (preferably 0.001 to 0.01 (w / v)%) Tyrosine: 0.001 to 2.0 (w / v)% (preferably 0.01 to 0.5 (w / v)%)

[0032] The sugars contained in the first medium are not particularly limited as long as they are sugars that are usually used in the culture of fungi, particularly mushroom cells, and may be monosaccharides or sugars of disaccharides or more. Examples of monosaccharides include glucose, fructose, xylose, mannose, galactose, rhamnose, arabinose, and fucose. Examples of sugars of disaccharides or more include lactose, maltose, isomaltose, cellobiose, maltotriose, maltotetraose, and maltopentaose. The sugars may be used alone or in combination of two or more, and preferably glucose is used alone.

[0033] The concentration of the saccharide in the first medium is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type of saccharide used, the types and concentrations of other components of the first medium, etc. Specifically, the concentration of the saccharide in the first medium can be 0.2 to 5.0 (w / v)%, 0.4 to 4.0 (w / v)%, 0.5 to 2.0 (w / v)%, etc.

[0034] The minerals contained in the first medium are not particularly limited as long as they are minerals that are commonly used in culturing fungi, particularly mushroom cells, and examples of such minerals include various inorganic salts. Examples of inorganic salts include various calcium salts (e.g., calcium carbonate, calcium chloride, calcium hydroxide, etc.), various potassium salts (e.g., potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc.), various magnesium salts (e.g., magnesium sulfate, magnesium hydrochloride, etc.), various copper salts (e.g., copper citrate, copper phthalate, copper tartrate, copper sulfate, copper chloride, etc.), various iron salts (e.g., iron fumarate, iron citrate, iron chloride, iron gluconate, iron lactate, iron sulfate, iron phosphate, etc.), various manganese salts (e.g., manganese chloride, manganese sulfate, etc.), and various ferric salts (e.g., iron sulfate, iron sulfate, etc.). Examples of the mineral include manganese acetate, manganese gluconate, etc.), various zinc salts (e.g., zinc lactate, zinc citrate, zinc gluconate, zinc propionate, zinc sulfate, zinc chloride, etc.), various ammonium salts (e.g., ammonium acetate, ammonium chloride, etc.), various nitrites (e.g., ammonium nitrite, sodium nitrite, etc.), various nitrates (e.g., ammonium nitrate, sodium nitrate, potassium nitrate, etc.), selenium salts (e.g., sodium selenate, sodium selenite, etc.), various chromium salts, various molybdenum salts, etc. Minerals may be used alone or in combination of two or more.

[0035] The concentration of the mineral in the first medium is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type of mineral used, the types and concentrations of other components of the first medium, etc. Specifically, the concentration of the mineral in the first medium can be 0.05 to 0.5 (w / v)%, 0.075 to 0.3 (w / v)%, 0.1 to 0.2 (w / v)%, etc.

[0036] In one preferred embodiment, potassium dihydrogen phosphate is used alone as the mineral. In this embodiment, the concentration of potassium dihydrogen phosphate in the first medium can be 0.05 to 0.5 (w / v)%, 0.075 to 0.3 (w / v)%, 0.1 to 0.2 (w / v)%, etc.

[0037] In another preferred embodiment, at least one of calcium, magnesium, iron, manganese, copper, zinc, selenium, chromium, and molybdenum is used alone, preferably two or more, more preferably four or more, even more preferably six or more, and particularly preferably all eight are used in combination with the potassium dihydrogen phosphate. In this embodiment, the total concentration of minerals other than potassium dihydrogen phosphate in the first medium can be appropriately set within a range in which the effects of the present invention are achieved.

[0038] The vitamins contained in the first medium are not particularly limited as long as they are vitamins that are usually used for culturing fungi, particularly mushroom cells, and may be fat-soluble or water-soluble vitamins. Examples of fat-soluble vitamins include vitamin A, vitamin D, vitamin E, vitamin K, etc. Examples of water-soluble vitamins include vitamin B group (e.g., vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, folic acid, biotin, etc.), vitamin C, etc. Vitamins may be used alone or in combination of two or more.

[0039] The vitamin concentration in the first medium is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type of vitamin used, the types and concentrations of other components of the first medium, etc. Specifically, the vitamin concentration in the first medium can be 0.001 to 0.05 (w / v)%, 0.005 to 0.03 (w / v)%, 0.01 to 0.02 (w / v)%, etc.

[0040] In one preferred embodiment, the vitamins are at least one of vitamin B1, vitamin E, vitamin A, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, folic acid, biotin, vitamin C, and vitamin D, preferably two or more, more preferably four or more, even more preferably six or more, even more preferably eight or more, even more preferably ten or more, and particularly preferably all twelve. In this embodiment, the total vitamin concentration in the first medium can be appropriately set within the range in which the effects of the present invention are achieved.

[0041] The lipids contained in the first medium are not particularly limited as long as they are commonly used in culturing fungal, particularly mushroom, cells, and examples thereof include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, polyketides, etc. The lipids may be used alone or in combination of two or more kinds.

[0042] The lipid concentration in the first medium is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type of lipid used, the types and concentrations of other components of the first medium, etc. Specifically, the lipid concentration in the first medium can be 0.01 to 0.2 (w / v)%, 0.03 to 0.1 (w / v)%, 0.04 to 0.08 (w / v)%, etc.

[0043] The first medium contains free amino acids at a concentration of 0.1 to 2.5 (w / v)%, preferably 0.12 to 2.4 (w / v)%, more preferably 0.14 to 2.3 (w / v)%, and even more preferably 0.145 to 2.2 (w / v)%. In this specification, the "concentration" of free amino acids means the total concentration of free amino acids derived from all components contained in the medium. When the free amino acid concentration in the first medium is within the above-mentioned range, cells separated from the Matsutake fruiting body grow and form a mycelium aggregate (mycelium mass), and the mycelium mass rises vertically upward to form a Matsutake primordium. The concentration of free amino acids in the first medium can be adjusted mainly by the type and concentration of the protein hydrolysate described above, and can also be further adjusted by adding the above-mentioned additional components. In this specification, the free amino acid concentration in the medium can be calculated based on the data in the Difco & BBL Manual (Second Edition).

[0044] The first medium preferably contains a nitrogen source. The total nitrogen content in the first medium is not particularly limited as long as the effect of the present invention is exhibited, but can be, for example, 0.01 to 5 (w / v)%, 0.05 to 3 (w / v)%, 0.1 to 2 (w / v)%, etc. The nitrogen source in the first medium is mainly contained in the above-mentioned protein hydrolysate, and can be further adjusted as necessary by adding the above-mentioned additional components, particularly nitrogen-containing inorganic salts such as ammonium salts, nitrites, and nitrates, and nitrogen-containing amino acids such as tryptophan and histidine. In this specification, the nitrogen content of the medium can be calculated based on the data in the Difco & BBL Manual (Second Edition), and can also be measured by the Kjeldahl method, combustion method, etc.

[0045] In step (a), the temperature at which the cells separated from the Matsutake fruiting body are cultured is preferably 15 to 30° C., more preferably 18 to 29° C., even more preferably 19 to 28° C., and particularly preferably 20 to 25° C. When the culture temperature in step (a) is within the above-mentioned range, the formation of Matsutake primordia is further promoted.

[0046] In step (a), the time for culturing the cells separated from the Matsutake fruiting body is not particularly limited as long as it is a time sufficient for the formation of Matsutake primordia, and can be appropriately set depending on the composition of the medium, the number of cells at the start of the culture, the culture temperature, etc. The time for culturing the cells separated from the Matsutake fruiting body can be, for example, 2 to 12 weeks, 3 to 10 weeks, 4 to 7 weeks, etc.

[0047] In this step (a), the humidity at which the cells separated from the Matsutake fruiting body are cultured is not particularly limited as long as the humidity is such that cell growth is not inhibited due to excessive dryness or wetness, and can be, for example, 50 to 100%, 60 to 90%, 70 to 100%, etc.

[0048] <Process (b)> In step (b), the Matsutake primordia formed in the above-mentioned step (a) are cultured under specific conditions to elongate the Matsutake primordia vertically upward, thereby obtaining elongated Matsutake primordia.

[0049] (Culture medium) The second medium used in step (b) is not particularly limited as long as it is a medium that is commonly used for culturing fungal, particularly mushroom, cells, and can be appropriately selected from those described above as the first medium.

[0050] In one embodiment, the second medium contains a second protein hydrolysate, which may be appropriately selected from those described above as the first protein hydrolysate.

[0051] The second medium may contain, in addition to the second protein hydrolysate described above, further components such as sugars, minerals such as inorganic salts, vitamins, lipids, and amino acids. These further components may be used alone or in combination of two or more. The types of sugars, minerals (inorganic salts), vitamins, lipids, and amino acids may be appropriately selected from those described above for the first medium. The respective concentrations of sugars, minerals (inorganic salts), vitamins, lipids, and amino acids may be the respective concentrations of sugars, minerals (inorganic salts), vitamins, lipids, and amino acids described above for the first medium.

[0052] The second medium contains free amino acids at a concentration of 0.05 to 2.5 (w / v)%, preferably 0.05 to 2.0 (w / v)%, more preferably 0.055 to 1.8 (w / v)%, even more preferably 0.06 to 1.5 (w / v)%, even more preferably 0.065 to 1.4 (w / v)%. When the free amino acid concentration in the second medium is within the above-mentioned range, the elongation of Matsutake primordia is further promoted. The concentration of free amino acids in the second medium can be adjusted mainly by the type and concentration of the protein hydrolysate described above, and can also be further adjusted by adding the above-mentioned further components.

[0053] The second medium preferably contains a nitrogen source. The total nitrogen content in the second medium is not particularly limited as long as the effects of the present invention are achieved, and may be, for example, 0.01 to 2 (w / v)%, 0.02 to 1.5 (w / v)%, 0.05 to 1 (w / v)%, etc. The nitrogen source in the second medium is mainly contained in the above-mentioned protein hydrolysate, and can be further adjusted as necessary by adding the above-mentioned additional components, particularly nitrogen-containing inorganic salts such as ammonium salts, nitrites, and nitrates, and nitrogen-containing amino acids such as tryptophan and histidine.

[0054] In step (b), the temperature for culturing the Matsutake primordia is preferably 15 to 30° C., more preferably 16 to 29° C., even more preferably 17 to 28° C., and particularly preferably 18 to 25° C. When the culture temperature in step (b) is within the above-mentioned range, the elongation of the Matsutake primordia is further promoted.

[0055] In step (b), the time for culturing the Matsutake primordia is not particularly limited as long as it is a time sufficient for the Matsutake primordia to grow to a desired size, and can be appropriately set depending on the composition of the medium, the culture temperature, etc. The time for culturing the Matsutake primordia can be, for example, 2 to 30 weeks, 2 to 20 weeks, 2 to 15 weeks, 2 to 12 weeks, 4 to 10 weeks, 5 to 8 weeks, etc.

[0056] In this step (b), the humidity when culturing the Matsutake primordia is not particularly limited as long as it is a humidity that does not inhibit the elongation of the Matsutake primordia due to excessive dryness or wetness, and can be, for example, 50 to 100%, 60 to 90%, 70 to 100%, etc.

[0057] The cultivation of Matsutake primordia in step (b) is carried out using a culture support containing a highly absorbent material infiltrated with the second medium containing a specific concentration of free amino acids as described above. The culture support is in contact with the Matsutake primordia to supply the components of the second medium to the Matsutake primordia and support the elongating Matsutake primordia.

[0058] The highly absorbent material used in the culture support is not particularly limited as long as it can be sufficiently infiltrated with the second culture medium (i.e., can be highly absorbed) and can be used for culturing fungal, particularly mushroom, cells. Examples of highly absorbent materials include vegetable fiber, highly absorbent fiber (SAF), highly absorbent resin (SAP), agar, artificial mineral fiber, etc. One type of highly absorbent material may be used alone, or two or more types may be used in combination.

[0059] <Process (c)> In addition to the above-mentioned steps (a) and (b), the production method of the present invention may further include a step (c) of growing the elongated Matsutake primordia obtained by steps (a) and (b) to obtain fruiting body-like Matsutake primordia.

[0060] As a method for growing the elongated Matsutake primordium, for example, there is a method of supplying the elongated Matsutake primordium with nutritional components (free amino acids, sugars, vitamins, minerals such as inorganic salts, lipids, water, etc.) necessary for the growth of the Matsutake primordium and fruiting body during or after the above-mentioned step (b). As a method for supplying nutritional components, for example, a method of adding nutritional components to a medium, etc. can be mentioned. When supplying nutritional components during the above-mentioned step (b), for example, a culture solution to which nutritional components have been added is added to the medium (i.e., the culture support) based on the type and amount of nutritional components consumed for the elongation of the primordium. Also, when supplying nutritional components after the above-mentioned step (b), for example, the elongated Matsutake primordium is further cultured in a medium to which necessary nutritional components have been added. The type and amount of nutritional components to be supplied can be appropriately set according to the type and amount of nutritional components (necessary nutritional components) that are insufficient.

[0061] According to another aspect of the present invention, there is provided a strain which is a Tricholoma matsutake strain identified by accession number NITE P-03769, which can be used in the above-mentioned production method of the present invention.

[0062] The strain identified by accession number NITE P-03769 exhibits good formation of Matsutake primordia in the production method of the present invention, and can be suitably used for producing elongated Matsutake primordia. EXAMPLES

[0063] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In each example, the free amino acid concentration in the medium was calculated based on the data in the Difco & BBL Manual (Second Edition).

[0064] Example 1: Study on the formation of primordia of Matsutake mushroom using a medium containing protein hydrolysate 1 The formation of primordia of Matsutake mushrooms using a medium containing a protein hydrolysate was investigated according to the following procedure. This example corresponds to the above-mentioned step (a).

[0065] Matsutake Cells New Matsutake cells (strains) were obtained from tissues isolated from naturally occurring Matsutake fruiting bodies by screening and other isolation processes. The obtained Matsutake cells have been deposited at the National Institute of Technology and Evaluation (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-03769 on October 18, 2022.

[0066] Preparation of slant medium for preserving Matsutake cells A medium for culturing the above-mentioned Matsutake cells was prepared according to the following procedure. First, a culture medium containing 0.2 (w / v)% yeast extract (Gibco), 1.5 (w / v)% agar (Bacto Agar, Becton Dickinson), and 2 (w / v)% glucose was prepared. All ingredients in the culture medium except for yeast extract, agar, and glucose were distilled water. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and after heating in an autoclave, 8 ml of the culture medium was dispensed into test tubes and sealed with a silicone stopper. Next, the test tubes were sterilized at high pressure at 121°C for 20 minutes, tilted, and left at room temperature to solidify the culture medium, preparing a slant medium.

[0067] Cultivation and storage of Tricholoma matsutake cells on slant medium The above-mentioned Matsutake cells were inoculated into the slant medium obtained according to the above-mentioned procedure. Then, the medium was left to stand at 20°C for 2 months to allow Matsutake mycelium to fully spread in the test tube, and the medium was stored at a low temperature of 4°C until use.

[0068] Preparation of pre-culture plates A medium for culturing the Matsutake mycelium obtained according to the above-mentioned procedure was prepared according to the following procedure. First, a culture medium containing 0.2 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco), 0.1 (w / v)% potassium dihydrogen phosphate, and 1.5 (w / v)% agar (Bacto Agar, manufactured by Becton Dickinson) was prepared. In addition, all the ingredients in the culture medium except for yeast extract, potassium dihydrogen phosphate, and agar were distilled water. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C using an autoclave. Next, the culture medium was cooled to about 60-70°C, and a glucose solution that had been sterilized in advance was added to a final concentration of 2%, and the mixture was thoroughly mixed. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and left to stand at room temperature to solidify the culture medium, and a plate medium for pre-culture was prepared.

[0069] Pre-culture of Matsutake mycelium Matsutake mycelia were pre-cultured according to the following procedure using the flat medium for pre-culture obtained according to the procedure described above. First, a part of the Matsutake mycelium obtained by culturing on a slant medium was cut out with a needle with a handle and inoculated onto a plate medium for pre-cultivation. The plate medium was then left to stand at 20°C for two months to obtain radially spreading mycelium. The sides of the plate medium were covered with film to seal and prevent drying during the culturing.

[0070] Preparation of plates for investigating primordium formation in Tricholoma matsutake Plate media for test plots 1-1 to 1-12 for investigating primordium formation of Matsutake were prepared according to the following procedure. A culture medium containing 0.1 (w / v)% potassium dihydrogen phosphate, 1.5 (w / v)% agar (Bacto Agar, Becton Dickinson), and protein hydrolysates such as yeast extract (Bacto Yeast Extract, Gibco), peptone (Bacto Peptone, Gibco), tryptone (Bacto Tryptone, Gibco) or beef extract (Beef Extract Powder, Gibco) was prepared. All ingredients in the culture medium were distilled water except for potassium dihydrogen phosphate, agar, and protein hydrolysates. The pH of the culture medium was then adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C in an autoclave. The culture medium was then cooled to about 60-70°C, and a pre-sterilized glucose solution was added and mixed thoroughly. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and left to stand at room temperature to solidify the culture medium, preparing plate media for examining the formation of primordia of Matsutake. The final concentrations of protein hydrolysates and glucose in the media for each test were as shown in Table 1.

[0071] [Table 1]

[0072] Primordium formation in Matsutake The radially spreading mycelium obtained in the pre-culture described above was punched out together with the agar using a sterilized straw with a diameter of 3.5 mm, the agar portion was removed as much as possible, and the bacteria was inoculated onto a plate medium for examining the formation of Matsutake primordia prepared by the procedure described above. The medium was then left to stand at 20°C or 25°C for 4 to 7 weeks of culture. The sides of the plate medium were covered with film to seal it, and culture was performed in a dark place to prevent drying. The culture temperature and culture period for each test group are shown in Table 1.

[0073] Studies on medium components and primordium formation of Tricholoma matsutake The presence or absence of primordia formation in the case where Matsutake mycelium was cultured using each of the media in test plots 1-1 to 1-13 is shown in Table 1. Regarding the formation of primordia in Matsutake, a case in which a mycelium mass was formed and a part of it rose vertically upward was considered to have "primordia formation", and a case in which a mycelium mass was not formed and vegetative mycelium extended radially around the inoculated mycelium was considered to have "no primordia formation".

[0074] From the results shown in Table 1, the formation of Matsutake primordia was confirmed in test plots 1-2 to 1-13, where the free amino acid concentration was 0.148 to 0.984 (w / v)%, while the formation of Matsutake primordia was not confirmed in test plot 1-1, where the free amino acid concentration was 0.066 (w / v)% (protein hydrolysate added amount 0.2 (w / v)%).

[0075] Example 2: Study on the formation of primordia of Matsutake using a medium containing protein hydrolysate 2 The formation of primordia of Matsutake mushrooms using a medium containing a protein hydrolysate was investigated according to the following procedure. This example corresponds to the above-mentioned step (a).

[0076] Pre-culture of Matsutake mycelium The Matsutake mycelium was pre-cultured according to the following procedure using the flat medium for pre-culture obtained according to the procedure described above. Note that the Matsutake mycelium used was obtained from cells derived from the fruit body of the Matsutake mushroom identified by the accession number NITE P-03769 described in Example 1. First, a part of the Matsutake mycelium obtained by culturing on a slant medium was cut out with a needle and inoculated onto a plate medium for pre-cultivation. The plate medium was then left to stand at 20°C for two months for cultivation. The sides of the plate medium were covered with film to seal and prevent drying during cultivation.

[0077] Preparation of plates for investigating primordium formation in Tricholoma matsutake Plate media for test plots 2-1 to 2-5 for investigating primordium formation of Matsutake were prepared according to the following procedure. A culture medium was prepared containing 0.1 (w / v)% potassium dihydrogen phosphate, 1.5 (w / v)% agar (Bacto Agar, Becton Dickinson), and L-tryptophan (Fujifilm Wako Pure Chemical Industries, Ltd.), yeast extract (Bacto Yeast Extract, Gibco), which is a protein hydrolyzate, and casamino acids (Bacto Casamino Acids, Gibco), either alone or in combination. All ingredients in the culture medium were distilled water, except for potassium dihydrogen phosphate, agar, L-tryptophan, and various protein hydrolyzates. The pH of the culture medium was then adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C in an autoclave. The culture medium was then cooled to about 60-70°C, and a pre-sterilized glucose solution was added and mixed thoroughly. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and left to stand at room temperature to solidify the culture medium, preparing plate media for examining the formation of primordia of Matsutake. The final total concentrations of L-tryptophan and various protein hydrolysates and the final concentration of glucose in the media for each test were as shown in Table 2.

[0078] In addition, plate media for test plots 2-6 and 2-7 were prepared in the same manner as the plate media for test plots 2-1 to 2-5, except that 2 (w / v)% casamino acids (protein hydrolysates) and amino acids (added at various concentrations as an amino acid-vitamin-mineral supplement with the amino acid composition shown below) were added to 0.1 (w / v)% potassium dihydrogen phosphate and 1.5 (w / v)% agar (Bacto Agar, Becton Dickinson). Valine 21.65mg / g Leucine 30.30mg / g Isoleucine 21.65mg / g Threonine 15.15mg / g Methionine 28.14mg / g Phenylalanine 30.30mg / g Tryptophan 7.58mg / g Lysine 25.97mg / g Histidine 17.32mg / g Glycine 34.34mg / g Arginine 25.40mg / g Glutamic acid 20.85mg / g Alanine 19.91mg / g Aspartic acid 12.19mg / g Proline 10.61mg / g Serine 7.07mg / g Cystine 3.25mg / g Tyrosine 1.15mg / g Total 332.828mg / g The final concentrations of the total components of the protein hydrolysate and amino acid-vitamin-mineral supplement in the medium for each test group, as well as the final concentration of glucose, were as shown in Table 2.

[0079] [Table 2]

[0080] Primordium formation in Matsutake The radially spreading mycelium obtained in the pre-culture described above was punched out together with the agar using a sterilized straw with a diameter of 3.5 mm, the agar portion was removed as much as possible, and the mycelium was inoculated onto the plate medium for examining the formation of Matsutake primordia prepared by the procedure described above. The plate medium was then left to stand at 20°C or 25°C for 5 weeks for culture. The sides of the plate medium were covered with film to seal it, and the culture was carried out in a dark place to prevent drying.

[0081] Studies on medium components and primordium formation of Tricholoma matsutake The presence or absence of primordia of Matsutake when Matsutake mycelium was cultured using the media of each of the test plots 2-1 to 2-7 is shown in Table 2. The presence or absence of primordia of Matsutake was determined by visual inspection.

[0082] From the results shown in Table 2, the formation of Matsutake primordia was confirmed in test area 2-1, which had a free amino acid concentration of 0.164 (w / v)% and contained yeast extract as a protein hydrolysate. The formation of Matsutake primordia was also confirmed in test areas 2-2 to 2-5, which had a free amino acid concentration of 0.501 to 2.186 (w / v)% and contained a combination of yeast extract and casamino acids as a protein hydrolysate. The formation of Matsutake primordia was also confirmed in test areas 2-6 and 2-7, which had free amino acid concentrations of 1.331 and 1.361 (w / v), respectively, and contained an amino acid-vitamin-mineral supplement. Although not shown in Table 2, the formation of Matsutake primordia was confirmed to be higher in test areas 2-2 to 2-5, which used a medium containing yeast extract, casamino acids, and tryptophan, compared to test area 2-1, which used a medium containing only yeast extract, especially when the temperature was 20°C.

[0083] Example 3: Study on the elongation of Matsutake primordia using highly absorbent materials as culture supports According to the following procedure, the elongation of Matsutake primordia was investigated when the Matsutake primordia were cultured using a highly absorbent material as a culture support. This example corresponds to the above-mentioned step (b).

[0084] Primordium formation in Matsutake Using the flat medium for investigating the formation of Matsutake primordia obtained according to the above-mentioned procedure, Matsutake primordia were formed from Matsutake mycelia according to the following procedure. As the Matsutake mycelia, Matsutake mycelia obtained from cells derived from the Matsutake fruit body identified by the accession number NITE P-03769 described in Example 1 were used. First, a portion of the Matsutake mycelium obtained by culturing on a slant medium was cut out using a needle with a handle, and inoculated onto a plate medium (0.5 (w / v)% yeast extract, 0.1 (w / v)% potassium dihydrogen phosphate, 2 (w / v)% glucose, 1.5 (w / v)% agar, the remainder being distilled water) for examining the formation of Matsutake primordia, and cultured at 25°C for 6 weeks to form Matsutake primordia. The sides of the plate medium were covered with film to seal and cultured in a dark place to prevent drying.

[0085] Preparation of medium-containing highly absorbent material (culture support) According to the following procedure, medium-containing highly absorbent materials (culture supports) shown in Test Plots 3-1 to 3-12 were prepared. First, a culture solution containing yeast extract (Bacto Yeast Extract, manufactured by Gibco) and potassium dihydrogen phosphate, which are protein hydrolysates, was prepared. In addition, distilled water was used in the culture solution except for yeast extract and potassium dihydrogen phosphate. Next, the pH of the culture solution was adjusted to 5.0 using HCl, and the culture solution was sterilized at high pressure at 121°C for 20 minutes using an autoclave. Next, the culture solution was cooled to room temperature, and a glucose solution that had been sterilized in advance was added at the concentration shown in Table 3, and the mixture was thoroughly mixed to prepare a liquid medium. The final concentrations of yeast extract and glucose in the medium for each test group were as shown in Table 3.

[0086] On the other hand, 1 g of the superabsorbent polymer was weighed out into a 125 ml plant box (for plant culture, manufactured by AS ONE Corporation) and sterilized in an autoclave at high pressure for 20 minutes at 121° C. 50 ml of the liquid medium for each test group obtained according to the above-mentioned procedure was added to the resulting plant box, and the liquid medium was absorbed into the superabsorbent polymer to produce a medium-containing superabsorbent material that would serve as a culture support.

[0087] Cultivation using medium-containing highly absorbent material The Matsutake primordia formed according to the above-mentioned procedure were cut out from the agar to a diameter of about 0.5 cm and a height of about 0.5 cm, and the Matsutake primordia were inoculated into each medium-containing highly absorbent material of test plots 3-1 to 3-12. The medium-containing highly absorbent material was then left to stand in the dark at 18°C, 20°C, 22°C, or 25°C for 7 weeks for culture. The final concentrations of free amino acids and glucose in the medium contained in the highly absorbent material in each test plot, as well as the culture temperature and culture period, were as shown in Table 3.

[0088] [Table 3]

[0089] A study on highly water-absorbent medium and the elongation of Tricholoma matsutake primordia The height of Matsutake primordia when the highly absorbent material-containing medium was used in each of the test plots 3-1 to 3-12 is shown in Table 3. Photographs of the Matsutake primordia grown in each test plot are shown in FIG.

[0090] From the results shown in Table 3, the elongation of Matsutake primordia was confirmed in all of the test plots 3-1 to 3-12, which were cultured using a highly absorbent material (culture support) containing a medium with a free amino acid concentration of 0.066 to 0.328 (w / v)%. In addition, a comparison of the culture temperatures showed that the higher the free amino acid concentration, the greater the degree of elongation of Matsutake primordia, and in particular, the elongation of Matsutake primordia was remarkable in test plots 3, 6, 9, and 12. In addition, a tendency was observed in which the degree of elongation of Matsutake primordia was greater as the culture temperature was higher.

[0091] Furthermore, as shown in Figure 1, when the free amino acid concentration was relatively high, such as in test plots 3, 6, 9 and 12, no pleated hyphae were observed around the Matsutake primordia, and the Matsutake primordia had a good shape.

[0092] Example 4: Examination of Matsutake primordium elongation using various highly absorbent materials as culture supports According to the following procedure, the growth of primordia of Matsutake mushroom was investigated when various highly absorbent materials were used as culture supports. This example corresponds to the above-mentioned step (b).

[0093] Primordium formation in Matsutake Using the flat medium for investigating the formation of Matsutake primordia obtained according to the above-mentioned procedure, Matsutake primordia were formed from Matsutake mycelia according to the following procedure. As the Matsutake mycelia, Matsutake mycelia obtained from cells derived from the Matsutake fruit body identified by the accession number NITE P-03769 described in Example 1 were used. First, a portion of the Matsutake mycelium obtained by culturing on a slant medium was cut using a needle with a handle and inoculated onto a plate medium (0.5 (w / v)% yeast extract, 0.1 (w / v)% potassium dihydrogen phosphate, 2 (w / v)% glucose, 1.5 (w / v)% agar, the remainder being distilled water) for examining the formation of Matsutake primordia, and left to stand at 25°C for six weeks of culture to form Matsutake primordia. The sides of the plate medium were covered with film to seal and prevent drying during the culture.

[0094] Preparation of medium-containing highly absorbent material (culture scaffold) According to the procedure described below, medium-containing highly absorbent materials (culture supports) shown in test plots 4-1 to 4-7 and 4-9 to 4-10 were prepared. First, a culture solution containing 1 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco) and 0.1 (w / v)% potassium dihydrogen phosphate, which are protein hydrolysates, was prepared. In addition, in the culture solution, distilled water was used except for the yeast extract and potassium dihydrogen phosphate, and the free amino acid concentration of the culture solution was 0.328 (w / v)%. Next, the pH of the culture solution was adjusted to 5.0 using HCl, and high-pressure sterilization was performed at 121 ° C. for 20 minutes by autoclaving. Next, the culture solution was cooled to room temperature, and a glucose solution (final concentration 2 (w / v)%) that had been sterilized in advance was added and thoroughly mixed to prepare a liquid medium.

[0095] In addition, the liquid medium for test plot 4-8 was prepared in the same manner as the liquid media for test plots 4-1 to 4-7 and 4-9 to 4-10, except that the culture solution was prepared by adding 2 (w / v)% casamino acids and amino acids (added at a concentration of 0.1 (w / v)% as the same amino acid-vitamin-mineral blend supplement used in Example 2) to 0.1 (w / v)% potassium dihydrogen phosphate instead of 1 (w / v)% yeast extract.

[0096] On the other hand, each highly absorbent material shown in Table 4 was placed in a 125 ml plant box (for plant culture, manufactured by AS ONE Corporation) and sterilized at high pressure for 20 minutes at 121°C using an autoclave. The liquid medium obtained according to the above-mentioned procedure was added to the resulting plant box to prepare a highly absorbent material containing the medium (culture support). The amount of liquid medium added was as shown in Table 4 depending on the type of highly absorbent material.

[0097] [Table 4]

[0098] Culture using various media containing highly absorbent materials The Matsutake primordia formed according to the above-mentioned procedure were cut out from the agar to have a diameter of about 0.5 cm and a height of about 0.5 cm, and the Matsutake primordia were inoculated into each medium-containing highly absorbent material of test plots 4-1 to 4-10. Next, the primordia were left to stand in a dark place at each temperature (18 to 25°C) listed in Table 4 and cultured for 5 to 14 weeks.

[0099] The height of Matsutake primordia when they were cultured using the highly absorbent materials containing the culture media of test plots 4-1 to 4-10 is shown in Table 4. From the results shown in Table 4, the elongation of Matsutake primordia was confirmed in all test plots 4-1 to 4-10, which were cultured using various highly absorbent materials (culture supports) containing media with a free amino acid concentration of 0.328 (w / v)% or 1.361 (w / v)%.

[0100] Example 5: Comparison of primordium shapes derived from various Matsutake cells using a confocal laser microscope Using different strains of Matsutake cells, Matsutake primordia were formed according to the procedure corresponding to the above-mentioned step (a), and their shapes were observed using a confocal laser microscope. The Matsutake cells used were those deposited at the National Institute of Technology and Evaluation, Japan Patent Microorganism Depositary Center (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-03769 and those deposited under the accession number NBRC#109050.

[0101] Preparation of plates for primordium formation of Tricholoma matsutake A plate medium for the formation of primordia of Matsutake was prepared according to the following procedure. First, a culture medium containing 0.5 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco), 0.1 (w / v)% potassium dihydrogen phosphate, and 1.5 (w / v)% agar (Bacto Agar, manufactured by Becton Dickinson) was prepared. All ingredients in the culture medium except for yeast extract, potassium dihydrogen phosphate, and agar were distilled water. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C using an autoclave. Next, the culture medium was cooled to about 60-70°C, and a pre-sterilized glucose solution was added and thoroughly mixed. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and left to stand at room temperature to solidify the culture medium, and a plate medium for the formation of Matsutake primordia was prepared.

[0102] Primordium formation in Matsutake Using the flat medium for forming Matsutake primordia obtained according to the procedure described above, Matsutake primordia were formed from Matsutake mycelia of each line according to the procedure described below. First, a part of the Matsutake mycelium obtained by culturing on a slant medium was cut out with a needle with a handle, inoculated onto a plate medium for the formation of Matsutake primordia, and cultured at 20°C for 10 weeks to form Matsutake primordia. The sides of the plate medium were covered with film to seal and prevent drying during the culture.

[0103] Observation of Matsutake mycelium The appearance of colonies obtained from NITE P-03769 and NBRC#109050 Matsutake cells is shown in Figure 2. As shown in Figure 2, when NITE P-03769 Matsutake cells were used, the mycelium was tightly raised and extended vertically, confirming the formation of Matsutake primordia. On the other hand, when NBRC#109050 Matsutake cells were used, the pleated vegetative mycelium spread and extended horizontally, and no Matsutake primordium was formed.

[0104] Example 6: Comparison of hyphal elongation derived from various Matsutake cells using a confocal laser microscope Hyphal elongation in Matsutake primordia derived from different lineages of Matsutake cells was observed using a confocal laser microscope according to the following procedure. First, hyphae in Matsutake primordia derived from each Matsutake cell of NITE P-03769 and NBRC#109050 were collected, and their cell walls were fluorescently stained with Calcofluor white (Merck), and observed with a confocal laser microscope (Olympus FV1000). Calcofluor white was detected at an excitation wavelength of 405 nm and a fluorescence wavelength of 425 to 475 nm using a 100x oil immersion objective lens (NA 1.4). For the hyphae in the primordia derived from NITE P-03769 that extended vertically upward, images were taken while shifting by 0.5 μm in the Z-axis direction to observe the three-dimensional extension of the hyphae, and finally a three-dimensional stack image was obtained by superimposing 20 images onto one sheet. For the hyphae in the primordia derived from NBRC#109050, where the hyphae extended horizontally, a two-dimensional image was taken by adjusting the focus to the point where the hyphae were most clearly visible. The results are shown in Figure 3.

[0105] As shown in Figure 3, in the primordia derived from NITE P-03769, the hyphae were branched and extended in all directions, both horizontally and vertically, whereas in the primordia derived from NBRC#109050, the hyphae did not branch and extended only horizontally.

[0106] Example 7: Comparison of morphological characteristics of Matsutake primordia derived from various Matsutake cells The morphological characteristics of Matsutake primordia derived from the cells of NITE P-03769, NBRC#33136 and NBRC#109050 were compared.

[0107] Pre-culture of Matsutake mycelium Each Matsutake cell was cultured on a slant medium to obtain mycelium, and a portion of the mycelium was cut out using a needle with a handle and inoculated onto a plate medium for pre-culture. The medium was then left to stand at 20°C for two months to obtain radially spreading mycelium. The sides of the plate medium were covered with film to seal and prevent drying during the culture.

[0108] Preparation of plates for primordium formation of Tricholoma matsutake A medium for the formation of primordia of Matsutake was prepared according to the following procedure. First, a culture medium containing 0.5 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco), 0.1 (w / v)% potassium dihydrogen phosphate, and 1.5 (w / v)% Bacto Agar (manufactured by Becton Dickinson) was prepared, which are protein hydrolysates. All ingredients in the culture medium except for yeast extract, potassium dihydrogen phosphate, and agar were distilled water. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C using an autoclave. Next, the culture medium was cooled to about 60-70°C, and a pre-sterilized glucose solution (final concentration 2 (w / v)%) was added and thoroughly mixed. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and left to stand at room temperature to solidify the culture medium, and a plate medium for the formation of Matsutake primordia was prepared.

[0109] Primordium formation in Matsutake The mycelium derived from each Matsutake cell that spread radially in the pre-culture described above was punched out together with the agar using a sterilized straw with a diameter of 3.5 mm, the agar portion was removed as much as possible, and the mycelium was inoculated onto the plate medium for primordium formation prepared by the procedure described above. The plate medium was then left to stand in a dark place at 25°C for 6 weeks of culture. The sides of the plate medium were covered with film to seal and prevent drying during the culture.

[0110] Observation of Matsutake primordia The appearance of colonies obtained from Matsutake cells of NITE P-03769, NBRC#33136, and NBRC#109050 is shown in Figure 4. As shown in Figure 4, when Matsutake cells of NITE P-03769 were used, the mycelium was firm and raised, elongating vertically, and the formation of Matsutake primordia was confirmed. On the other hand, when Matsutake cells of NBRC#33136 and Matsutake cells of NBRC#109050 were used, the pleated vegetative mycelium spread widely and the mycelium elongated mainly horizontally, and the formation of Matsutake primordia was not confirmed.

[0111] Table 5 shows the external characteristics and morphology of each colony, quantified.

[0112] [Table 5]

[0113] From the results shown in Table 5, when NITE P-03769 Matsutake cells were used, the "mycelium height / colony diameter" was 0.37, whereas when NBRC#33136 and NBRC#109050 Matsutake cells were used, the values ​​were 0.09 and 0.07, respectively (both P<0.01), confirming a significant difference in morphology.

[0114] Example 8: Morphological characteristics of Matsutake primordia obtained by the production method of the present invention The morphology of Matsutake primordia derived from NITE P-03769 Matsutake cells obtained by the production method of the present invention was observed according to the following procedure.

[0115] Primordium formation in Matsutake NITE P-03769 Matsutake cells were cultured on a slant medium to obtain mycelium, and a portion of the mycelium was excised using a needle with a handle and inoculated onto a plate medium for primordial culture (0.5 (w / v)% yeast extract, 0.1 (w / v)% potassium dihydrogen phosphate, 2 (w / v)% glucose, 1.5 (w / v)% agar, the remainder being distilled water), and cultured at 20°C for 5 weeks to form Matsutake primordia. The sides of the plate medium were covered with film to seal and prevent drying during the culture.

[0116] Preparation of medium-containing highly absorbent material (culture support) A medium-containing highly absorbent material (culture support) was prepared according to the following procedure. First, a culture medium containing 1 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco) and 0.1 (w / v)% potassium dihydrogen phosphate, which are protein hydrolysates, was prepared. In addition, distilled water was used in the culture medium except for the yeast extract and potassium dihydrogen phosphate. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C using an autoclave. Next, the culture medium was cooled to room temperature, and a glucose solution (final concentration 4 (w / v)%) that had been sterilized in advance was added and thoroughly mixed to prepare a liquid medium.

[0117] On the other hand, 1 g of the superabsorbent polymer was weighed into a 125 ml plant box (for plant culture, manufactured by AS ONE Corporation) and sterilized by autoclave at high pressure for 20 minutes at 121° C. 50 ml of the liquid medium obtained according to the above-mentioned procedure was added to the resulting plant box, and the liquid medium was absorbed into the superabsorbent polymer to produce a medium-containing highly absorbent material that would serve as a culture support.

[0118] Culture using medium-containing highly absorbent material The Matsutake primordia formed according to the above-mentioned procedure were cut out from the agar to a diameter of about 0.3 cm and a height of about 0.3 cm, and the Matsutake primordia were inoculated into the medium-containing highly absorbent material prepared according to the above-mentioned procedure. The Matsutake primordia were then left to stand at 20°C for 11 weeks for cultivation. As a result, it was confirmed that the Matsutake primordia had grown to a diameter of 0.6 cm and a height of 0.7 cm.

[0119] Transplantation of Matsutake primordia cultured in highly absorbent medium containing culture medium Further medium-containing highly absorbent materials (culture supports) were prepared according to the following procedure. First, 4 g of pulp (Kimtowel, Nippon Paper Crecia Co., Ltd.) was weighed into a plant box and sterilized at high pressure for 20 minutes at 121° C. in an autoclave. 20 ml of the liquid medium obtained according to the above-mentioned procedure was added to the resulting plant box, and the liquid medium was absorbed into the pulp to produce a medium-containing highly absorbent material that would serve as a culture support.

[0120] The Matsutake primordia grown as described above were transplanted into the highly absorbent medium-containing material obtained according to the procedure described above. They were then left to stand at 20°C for 12 weeks for cultivation. As a result, it was confirmed that the Matsutake primordia had grown to a diameter of 1.8 cm and a height of 1.3 cm. Observation results (photographs) of the Matsutake primordia at each time point during cultivation are shown in Figure 5.

[0121] As shown in Figure 5, when the cross section of the Matsutake primordium was observed after 12 weeks of culture, it was observed that the primordium had differentiated into a mushroom cap. Furthermore, when the part that had differentiated into a cap was cut vertically and the cross section was observed, it was found that there were cracks (arrows) symmetrically on the left and right sides, and that these parts had turned brown. From these findings, it was confirmed that the Matsutake primordium would gradually differentiate and grow if it was cultured and grown in a highly absorbent polymer that further promotes the growth of the Matsutake primordium, and then transferred to a fibrous culture support with a slightly lower water absorption rate for the culture solution in order to reduce osmotic stress and further cultured.

[0122] Example 9: Confirmation of the primordium formation ability of a new species of Matsutake The following four types of Matsutake cells were examined for their ability to form primordia in the step of forming Matsutake primordia in the production method of the present invention (ie, "step (a)" of the present invention).

[0123] Matsutake Cells As the Matsutake cells, four types of Matsutake cells were used: NITE P-03769 and NBRC#109050 used in the above-mentioned examples, and different NBRC#108259 and NBRC#108261.

[0124] Preparation of slant medium for preserving Matsutake cells Media for culturing each of the above-mentioned Matsutake cells were prepared according to the following procedure. First, a culture medium was prepared containing 0.2 (w / v)% yeast extract (Gibco), 1.5 (w / v)% agar (Bacto Agar, Becton Dickinson), and 2 (w / v)% glucose, which are protein hydrolysates. All ingredients in the culture medium were distilled water except for yeast extract, agar, and glucose. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and after heating in an autoclave, 8 ml of the culture medium was dispensed into test tubes and sealed with a silicone stopper. Next, the test tubes were sterilized at high pressure at 121°C for 20 minutes, tilted, and left at room temperature to solidify the culture medium, and a slant medium was prepared.

[0125] Cultivation and storage of Tricholoma matsutake cells on slant medium The above-mentioned Matsutake cells were inoculated into the slant medium prepared according to the above-mentioned procedure. Then, the medium was left to stand at 20°C for 2 months to allow Matsutake mycelium to fully spread in the test tube, and then the medium was stored at a low temperature of 4°C until use.

[0126] Preparation of pre-culture plates A medium for culturing the Matsutake mycelium prepared according to the above-mentioned procedure was prepared according to the following procedure. First, a culture medium containing 0.2 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco), 0.1 (w / v)% potassium dihydrogen phosphate, and 1.5 (w / v)% agar (Bacto Agar, manufactured by Becton Dickinson) was prepared. In addition, distilled water was used in the culture medium except for yeast extract, potassium dihydrogen phosphate, and agar. Next, the pH of the culture medium was adjusted to 5.0 using HCl, and the medium was sterilized at high pressure for 20 minutes at 121°C using an autoclave. Next, the culture medium was cooled to about 60-70°C, and a glucose solution that had been sterilized in advance was added to a final concentration of 1%, and the mixture was thoroughly mixed. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and left to stand at room temperature to solidify the culture medium, and a plate medium for pre-culture was prepared.

[0127] Cultivation of Matsutake mycelium Matsutake mycelia were pre-cultured according to the following procedure using the flat medium for pre-culture obtained according to the procedure described above. First, a portion of the Matsutake mycelium obtained by culturing on a slant medium was cut out with a needle with a handle and inoculated onto a plate medium for pre-cultivation. The plate medium was then left to stand at 25°C for 13 weeks to obtain Matsutake primordia derived from each Matsutake cell. The sides of the plate medium were covered with film to seal and prevent drying during the culturing process. The observation results (photos) of each colony after the end of the culturing process are shown in Figure 6.

[0128] As shown in Figure 6, after 13 weeks of cultivation, the mycelia obtained from all of the Matsutake cells had formed colonies with densely packed mycelia in the center (i.e., a Matsutake primordium-like morphology). Table 6 shows the external characteristics of each colony after cultivation and the diameter of the densely packed central part.

[0129] [Table 6]

[0130] From the results shown in Table 6 and Figure 6, when NITE P-03769 Matsutake cells were used, many hyphae were densely packed in the center of the colony and extended vertically upwards, while there were few hyphae extending horizontally, confirming that they had the ability to form Matsutake primordia. On the other hand, when the other three Matsutake cells were used, there were few hyphae densely packed in the center and extended vertically upwards, while there were many hyphae extending horizontally. Therefore, it was confirmed that these cells did not have the ability to form Matsutake primordia.

Claims

1. A method for producing an elongated Matsutake primordium, comprising the steps of: (a) culturing cells capable of forming primordia separated from a fruiting body of Matsutake in a first medium containing a first protein hydrolysate or amino acids and having a free amino acid concentration of 0.1 to 2.5 (w / v) % to form Matsutake primordia; and (b) culturing the Matsutake primordia on a culture support containing a highly absorbent material infiltrated with a second medium containing a second protein hydrolysate or amino acids and having a free amino acid concentration of 0.05 to 2.5 (w / v)%, thereby elongating the Matsutake primordia vertically upward to obtain elongated Matsutake primordia; Including, The first protein hydrolysate and the second protein hydrolysate each contain at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone and tryptone, or contain a combination of casamino acid and at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone and tryptone; The method.

2. The method according to claim 1, wherein the culture temperature in the step (a) is 18 to 25°C.

3. The method according to claim 1, wherein the culture temperature in the step (b) is 18 to 30°C.

4. The method of claim 1, wherein the highly absorbent material comprises at least one selected from the group consisting of vegetable fibers, highly absorbent fibers, highly absorbent resins, agar, and man-made mineral fibers.

5. The method according to claim 1, wherein either or both of the first medium and the second medium further comprise 0.2 to 5 (w / v) % sugar.

6. The method of claim 1, wherein both or one of the first and second culture media further comprises at least one selected from the group consisting of inorganic salts, vitamins, lipids and amino acids.

7. The method according to claim 1, wherein the fruiting body is identified as Tricholoma matsutake by molecular phylogenetic analysis based on the base sequence of ITS-5.8S rDNA.

8. The method according to claim 7, wherein the fruiting body is a fruiting body of Tricholoma matsutake, identified by accession number NITE P-03769.

9. A method for producing fruiting body-like Matsutake primordia, further comprising a step of growing the elongated Matsutake primordia obtained by the method according to any one of claims 1 to 8.

10. Cells isolated from the fruiting body of Tricholoma matsutake, identified by accession number NITE P-03769.

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