Matsutake primordium and production method thereof

Abstract

[Problem] To provide a matsutake primordium having a high methyl cinnamate content and a method capable of obtaining such a matsutake primordium having a high methyl cinnamate content at an artificially high yield. [Solution] Cells having the ability to form a primordium that have been isolated from matsutake fruiting bodies are cultured in a medium containing a protein degradation product and having a specific phenylalanine concentration.

Description

Matsutake primordia and method for producing the same

[0001] The present invention relates to a novel Matsutake primordium and a method for producing the same.

[0002] Although there have been reports of the formation of Matsutake fruiting body primordia measuring several millimeters in size through fully artificial culture (e.g., Non-Patent Documents 1 and 2), there have been no reports of fruiting body primordia growing. Furthermore, there are few reports of the reproduction of fruiting body primordium formation, and currently, no established method has been established for the formation and growth of Matsutake fruiting body primordia (e.g., Non-Patent Document 3).

[0003] It has been reported that three major aroma components of matsutake mushrooms are the alcohol compounds 1-octen-3-ol and cis-2-octen-1-ol, and the aromatic alkyl ester compound methyl cinnamate (see, for example, Non-Patent Documents 4-9). Among these, methyl cinnamate has a refreshing resinous and balsamic aroma. Because mushrooms rich in methyl cinnamate have not been reported other than those of matsutake and related species, it is believed to be the most important component contributing to matsutake's distinctive aroma (see, for example, Non-Patent Documents 9-11). Furthermore, it is known that methyl cinnamate, which most characterizes the matsutake mushroom aroma, tends to be found in the cap of mature fruiting bodies, followed by the upper and lower stalks. It has been reported that methyl cinnamate is particularly abundant in the cap gills and spores within the cap (see, for example, Non-Patent Documents 8 and 12). However, the methyl cinnamate content in the stalk is extremely low compared to the cap, and is known to be below the detection limit, or even if detected, only in small amounts, particularly in the lower stalk. Specifically, it is known that the methyl cinnamate content in the lower part of the fruiting body stalk is approximately 0.51% of that in the cap (e.g., Non-Patent Document 8). Because even in mature fruiting bodies, some tissues have low methyl cinnamate content, it has been commonly accepted that the methyl cinnamate content in the primordia before differentiation into fruiting bodies is extremely low, similar to the lower part of the stalk described above. Furthermore, it has been shown that the methyl cinnamate content is also very low in mycelia obtained by artificial culture (e.g., Non-Patent Document 13). Therefore, it is speculated that the methyl cinnamate content in primordia obtained by either natural or artificial cultivation methods is extremely low compared to fruiting bodies, or is undetectable.

[0004] Regarding the biosynthetic pathway of methyl cinnamate, it is known that cinnamic acid is produced from L-phenylalanine by phenylalanine ammonia-lyase (PAL), and methyl cinnamate is produced by cinnamic acid carboxyl methyltransferase (CCMT) (e.g., Non-Patent Documents 14-15). Therefore, studies have been conducted to grow Matsutake mycelia and produce methyl cinnamate by adding L-phenylalanine, a precursor of methyl cinnamate, to liquid media. Specifically, it has been shown that when the concentration of L-phenylalanine is 0.5 to 6 mM, the amount of methyl cinnamate per amount of soluble protein in the mycelium increases in a concentration-dependent manner (Non-Patent Document 13).

[0005] On the other hand, it has been shown that mycelial yield decreases depending on the L-phenylalanine concentration in the medium, and it has been reported that adding L-phenylalanine to the medium at concentrations of even 1 mM tends to inhibit the growth of Matsutake mycelia. Furthermore, it has been shown that adding 64 mM L-phenylalanine reduces mycelial yield by 68% (Non-Patent Document 13). Thus, in artificial culture, the methyl cinnamate content of mycelia is very low, and the addition of L-phenylalanine, a precursor of methyl cinnamate, increases the amount of methyl cinnamate in the mycelia, but inhibits growth. Furthermore, there have been very few reports on the culture and reproducibility of primordia, and no reports have been published on the addition of L-phenylalanine to the medium.

[0006] In addition, it is known that the content of 1-octen-3-ol, one of the main aroma components of Matsutake, similar to methyl cinnamate, is lower in mycelia obtained by liquid culture than in natural fruiting bodies (Non-patent Document 16).

[0007] Bulletin of the Mycological Society of Japan, 16 (4), pp. 406-415, 1976-01. Bulletin of the Forestry and Forest Products Research Institute (FFPRI), Vol. 19-No. 2 (No. 454), 153-157, August 2020. Doctoral thesis, University of Tokyo, 2009 (Katsumi Shindo). Journal of the Japanese Forestry Society, 18, pp. 528-536 (1936). Journal of the Japanese Forestry Society, 19, pp. 414-420 (1937). Bulletin of the Institute of Physical and Chemical Research, 15, pp. 1186-1196 (1936). Bulletin of the Institute of Physical and Chemical Research, 16, pp. 548-561 (1937). Hokkaido Research Organization, Forest Products Experiment Station News, January 2020 issue. Journal of the Japanese Society of Food Technology, 31, pp. 14-18 (1984) Microorganisms and Fragrances - The Power of Aroma in the Microscopic World, Fragrance Journal, Tokyo (2002) Journal of the Society for Odor and Fragrance Environment, 44, pp. 315-322 (2013) Abstracts of the 18th Annual Meeting of the Japanese Society of Mushroom Science, 2P-22, pp. 136 (2014) Mycoscience, 59 (2018) 8-11 Mycoscience, 56 (2015) 503-511 Mycoscience, 57 (2016) 181-186 Bulletin of the Mycological Society of Japan, Vol. 32, No. 4, pp. 477-484 (1991-12)

[0008] Under these circumstances, there is a demand for Matsutake primordia with a high content of methyl cinnamate, and a method for artificially obtaining Matsutake primordia with such a high content of methyl cinnamate in high yield.

[0009] Therefore, the present invention relates to Matsutake primordia having a high content of methyl cinnamate, and a method for artificially obtaining Matsutake primordia having such a high content of methyl cinnamate in high yield.

[0010] As a result of extensive research, the present inventors have discovered that the above-mentioned problems can be solved by culturing cells isolated from the fruiting bodies of Tricholoma matsutake under specific conditions. The present invention is based on this discovery. Specifically, the gist of the present invention is as follows.

[0011] [1] A matsutake primordium containing 4 μg or more of methyl cinnamate per unit mass of the matsutake primordium. [2] A matsutake primordium according to [1], which is identified as Tricholoma matsutake by molecular phylogenetic analysis based on the base sequence of ITS-5.8S rDNA. [3] The matsutake primordium according to [1] or [2], which is a matsutake primordium of Tricholoma matsutake identified by accession number NITE BP-03769. [4] A method for producing matsutake primordia, comprising: (a) culturing cells capable of forming primordia separated from a matsutake fruiting body in a first medium containing a first protein hydrolysate or amino acids to form matsutake primordia; and (b) culturing the matsutake primordia on a culture support comprising a highly absorbent material infiltrated with a second medium containing a second protein hydrolysate or amino acids and having a phenylalanine concentration of 10 to 100 mM to allow the matsutake primordia to elongate vertically upward, thereby obtaining matsutake primordia, wherein the first protein hydrolysate and the second protein hydrolysate each comprise at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone, and tryptone, or comprise a combination of casamino acids and at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone, and tryptone. [5] The method according to [4], wherein the culture temperature in step (a) is 18 to 25°C. [6] The method according to [4] or [5], wherein the culture temperature in step (b) is 18 to 30°C. [7] The method according to any one of [4] to [6], wherein the highly absorbent material comprises at least one selected from the group consisting of plant fiber, highly absorbent fiber, highly absorbent resin, agar, and artificial mineral fiber. [8] The method according to any one of [4] to [7], wherein the fruiting body is identified as Tricholoma matsutake by molecular phylogenetic analysis based on the nucleotide sequence of ITS-5.8S rDNA. [9] The method according to any one of [4] to [8], wherein the fruiting body is a fruiting body of Tricholoma matsutake identified by accession number NITE BP-03769.

[10] A method for producing fruiting body-like matsutake primordia, further comprising a step of growing the matsutake primordia obtained by the method according to any one of [4] to [9].

[11] A matsutake primordium produced by the method according to any one of [4] to [9].

[12] A fruiting body-like matsutake primordium produced by the method according to

[10] .

[0012] According to the present invention, it is possible to provide Matsutake primordia having a high content of methyl cinnamate, and a method for artificially obtaining Matsutake primordia having such a high content of methyl cinnamate in high yield.

[0013] FIG. 1 is a photograph showing matsutake primordia obtained by the production method of the present invention. FIG. 2 is a graph showing the yield increase rate of matsutake primordia cultured in media supplemented with different concentrations of phenylalanine at different temperatures. FIG. 3 is a graph showing the time-dependent change in the yield increase rate (weight increase rate) (%) of matsutake primordia (cultured primordia) cultured at 20°C for 30, 50, 70, and 92 days with a phenylalanine concentration of 50 mM. FIG. 4 is a graph showing the methyl cinnamate content in matsutake primordia cultured in media supplemented with different concentrations of phenylalanine at different temperatures. FIG. 5 is a graph showing the time-dependent change in the methyl cinnamate content in matsutake primordia cultured at 20°C for 30, 50, 70, and 92 days with a phenylalanine concentration of 50 mM. FIG. 6 is a graph showing the methyl cinnamate content in the cap and stalk of domestically grown wild matsutake. Figure 7 is a graph showing the 1-octen-3-ol content in matsutake primordia cultured at different temperatures in media supplemented with different concentrations of phenylalanine, and Figure 8 is a graph showing the 1-octen-3-ol content in the cap and stalk of domestically grown wild matsutake.

[0014] [Method for producing matsutake fruiting body primordia] According to one aspect of the present invention, there is provided a method for producing matsutake fruiting body primordia (also simply referred to as "matsutake primordia" in this specification) (also simply referred to as "the production method of the present invention" in this specification).

[0015] In this specification, "matsutake fruiting body primordium" and "matsutake primordium" refer to a mass (primordium) of hyphae (secondary hyphae) that grows and assembles in a certain direction, with a portion (particularly the central portion) of the primordium rising vertically upward, which serves as the "base" of the matsutake fruiting body. Generally, secondary hyphae that assemble in a certain direction are considered to be more differentiated than secondary hyphae that do not have such directionality, and are sometimes referred to as tertiary hyphae to distinguish them from secondary hyphae. Furthermore, "matsutake fruiting body primordium" and "matsutake primordium" are concepts that encompass elongated matsutake primordium. An "elongated matsutake primordium" refers to a matsutake primordium in which the raised portion of the above-mentioned matsutake primordium has further raised, resulting in the entire or nearly entire matsutake primordium being raised.

[0016] In this specification, with regard to "cells having the ability to form primordia isolated from the fruiting body of Matsutake," "having the ability to form primordia" means that the cells isolated from the fruiting body of Matsutake grow (elongate) their hyphae vertically upward on agar plate medium in a free amino acid concentration-dependent manner, and when the free amino acids reach a certain concentration, they form raised primordia. Such cells are typically deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (hereinafter also simply referred to as the "Budapest Treaty") at the National Institute of Technology and Evaluation, Japan, Patent Microorganism Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE BP-03769 (domestic deposit date: October 18, 2022, date of receipt of request for transfer to international deposit: October 16, 2023), and include cells isolated from the fruiting body of Tricholoma matsutake.

[0017] The production method of the present invention includes a step (step (a)) of culturing cells isolated from matsutake fruiting bodies under specific conditions to form matsutake primordia, and a step (step (b)) of culturing the formed matsutake primordia under specific conditions to obtain matsutake primordia. According to the production method of the present invention, matsutake primordia can be artificially produced in an environment that is not symbiotic with plants. Steps (a) and (b) of the production method of the present invention are described in detail below.

[0018] <Step (a)> In step (a), cells capable of forming primordia separated from a Matsutake fruiting body are cultured under specific conditions to form Matsutake primordia.

[0019] As the matsutake fruiting body, any fruiting body of Tricholoma matsutake can be used without 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 BP-03769, is used.

[0020] (Culture Medium) The first culture medium used in step (a) is not particularly limited as long as it is a medium commonly used for culturing fungal, particularly mushroom, cells. As the first culture medium, either a solid or liquid culture medium can be used, with a solid culture medium being preferred. Examples of solid culture media include a liquid culture medium solidified with a solidifying agent, preferably a gel-like solid culture medium with no fluidity. As the solid culture medium, any of a plate culture medium, a slant culture medium, a semi-slant culture medium, and a high-rise culture medium may be used, with a plate culture medium being preferred.

[0021] The liquid medium can be any liquid medium commonly used for culturing fungal, particularly mushroom, cells, without any particular limitations. Examples of solidifying agents for solidifying the liquid medium include agar, mannan, gellan, gelatin, and Kelcogel. Agar is preferably used as the solidifying agent, and thus an agar medium is preferably used as the first medium.

[0022] 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)%.

[0023] In one embodiment, the first culture medium contains a first protein hydrolysate. The first culture medium may further contain, in addition to the first protein hydrolysate, additional components such as sugars, minerals (e.g., inorganic salts), vitamins, lipids, and amino acids. These additional components may be used singly or in combination of two or more.

[0024] The first protein hydrolysate is not particularly limited as long as it is a collection of amino acids or peptides obtained by decomposing a protein using an enzyme, acid, etc., and examples thereof include protein hydrolysates and enzymatic digests of proteins.

[0025] 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 acids, etc. One type of first protein hydrolysate may be used alone, or two or more types may be used in combination.

[0026] In one embodiment, the first protein hydrolysate is at least one selected from the group consisting of yeast extract, beef extract, peptone, and tryptone.

[0027] In another embodiment, the first protein hydrolysate is a combination of casamino acid and at least one selected from the group consisting of yeast extract, beef extract, peptone, and tryptone.

[0028] 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.

[0029] In one embodiment, the first medium contains amino acids instead of or in addition to the first protein hydrolysate. In this embodiment, "amino acids" refer to amino acids other than those derived from the "first protein hydrolysate." The amino acids may be synthesized amino acids (synthetic amino acids) or naturally occurring amino acids (natural amino acids), and either amino acids can be used. The amino acids can be obtained by methods commonly used for producing amino acids, such as fermentation, enzymatic methods, extraction methods, and synthesis methods.

[0030] The type of amino acid is not particularly limited as long as the effects of the present invention are achieved, but examples include proteinogenic amino acids (ie, α-amino acids), non-proteinogenic amino acids, and derivatives thereof.

[0031] Examples of amino acids that make up proteins include essential amino acids for humans (isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, histidine), non-essential amino acids (tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, arginine), etc.

[0032] An example of a derivative is cystine (3,3'-dithiobis(2-aminopropionic acid)), in which two cysteine ​​molecules are bonded via a disulfide bond formed by oxidation of the hydrosulfide groups (-SH).

[0033] The first medium may contain at least one amino acid and / or derivative 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. 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, and even more preferably fifteen or more amino acids and / or derivatives, and particularly preferably all 18 amino acids and derivatives.

[0034] The total concentration of amino acids and derivatives in the first medium is not particularly limited as long as the first medium satisfies the free amino acid concentration range described below, and can be appropriately set depending on the type of amino acid used, the type and concentration of other components of the first medium, the total nitrogen content 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. Furthermore, when the first medium contains amino acids and / or derivatives other than the amino acids derived from the first protein hydrolysate described above, the concentrations of each amino acid and derivative in the first medium can be set, for example, as follows: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)%).

[0035] The sugars contained in the first medium are not particularly limited as long as they are sugars commonly used in culturing fungal, particularly mushroom, cells, and may be monosaccharides or sugars of disaccharides or higher. Examples of monosaccharides include glucose, fructose, xylose, mannose, galactose, rhamnose, arabinose, and fucose. Examples of sugars of disaccharides or higher include lactose, maltose, isomaltose, cellobiose, maltotriose, maltotetraose, and maltopentaose. One type of sugar may be used alone, or two or more types may be used in combination, with glucose being preferred.

[0036] The concentration of the sugar 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 sugar used, the types and concentrations of other components of the first medium, etc. Specifically, the concentration of the sugar 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.

[0037] The minerals contained in the first culture medium are not particularly limited as long as they are minerals commonly used in culturing fungal, particularly mushroom, cells, and examples thereof 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 Examples of minerals 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 nitrate salts (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 singly or in combination of two or more.

[0038] The concentration of minerals 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 minerals used, the types and concentrations of other components of the first medium, etc. Specifically, the concentration of minerals 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.

[0039] 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 culture 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.

[0040] 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, as minerals 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.

[0041] The vitamins contained in the first culture medium are not particularly limited as long as they are vitamins commonly used in culturing fungal, particularly mushroom, cells, and may be fat-soluble or water-soluble vitamins. Fat-soluble vitamins include, for example, vitamin A, vitamin D, vitamin E, vitamin K, etc. Water-soluble vitamins include, for example, B vitamins (e.g., vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, folic acid, biotin, etc.) and vitamin C, etc. Vitamins may be used alone or in combination of two or more.

[0042] The concentration of the vitamin 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 concentration of the vitamin 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.

[0043] In one preferred embodiment, the vitamin is 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, used alone, or 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 12 types are used in combination. In this embodiment, the total concentration of vitamins in the first medium can be appropriately set within a range in which the effects of the present invention are achieved.

[0044] The lipid contained in the first medium is not particularly limited as long as it is a vitamin that is commonly used in culturing fungal, particularly mushroom, cells, and examples thereof include fatty acyl, glycerolipid, glycerophospholipid, sphingolipid, sterol lipid, prenol lipid, glycolipid, polyketide, etc. One type of lipid may be used alone, or two or more types may be used in combination.

[0045] 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.

[0046] In one embodiment, the first medium may contain free amino acids. The concentration of free amino acids in the first medium is, for example, 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 refers to 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 mycelial aggregates (primordia), and Matsutake primordia are formed, with the primordia protruding vertically upward. 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 additional components described above. 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).

[0047] The first medium preferably contains a nitrogen source. The total nitrogen content in the first medium is not particularly limited as long as the effects of the present invention are achieved, 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 protein hydrolysate described above, and can be further adjusted as needed by adding the additional components described above, 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), or can be measured by the Kjeldahl method, combustion method, etc.

[0048] 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.

[0049] 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.

[0050] 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 by excessive dryness or wetness, and can be, for example, 50 to 100%, 60 to 90%, 70 to 100%, etc.

[0051] <Step (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 Matsutake primordia.

[0052] (Culture medium) The second culture medium used in step (b) is not particularly limited as long as it is a culture medium that is commonly used for culturing fungal, particularly mushroom, cells, and can be appropriately selected from those described above as the first culture medium.

[0053] The second medium contains phenylalanine at a concentration of 10 to 100 mM, preferably 15 to 90 mM, more preferably 20 to 75 mM, even more preferably 25 to 60 mM, and particularly preferably 25 to 50 mM. In this specification, the phenylalanine concentration in the medium can be measured by post-column derivatization with a ninhydrin reagent using an automatic amino acid analyzer.

[0054] In one embodiment, the second medium contains a second protein hydrolysate, which may be selected from the first protein hydrolysates described above.

[0055] In addition to the second protein hydrolysate described above, the second culture medium may contain additional components such as sugars, minerals such as inorganic salts, vitamins, lipids, and amino acids. These additional components may be used singly 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 culture medium. Furthermore, the respective concentrations of sugars, minerals (inorganic salts), vitamins, lipids, and amino acids may be the same as those described above for the first culture medium.

[0056] In one embodiment, the first medium may contain free amino acids. The concentration of free amino acids in the second medium is, for example, 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)%, and particularly 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 additional components described above.

[0057] 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, but can 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 protein hydrolysate described above, and can be further adjusted as needed by adding the additional components described above, particularly nitrogen-containing inorganic salts such as ammonium salts, nitrites, and nitrates, and nitrogen-containing amino acids such as tryptophan and histidine.

[0058] In step (b), the temperature at which the Matsutake primordia are cultured 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.

[0059] 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.

[0060] In this step (b), the humidity at which the Matsutake primordia are cultured is not particularly limited as long as the humidity is such that the elongation of the Matsutake primordia is not inhibited by excessive dryness or wetness, and can be, for example, 50 to 100%, 60 to 90%, 70 to 100%, etc.

[0061] The cultivation of Matsutake primordia in step (b) is carried out using a culture support comprising a highly absorbent material infiltrated with the second medium containing the specific concentration of free amino acids as described above. The culture support is in contact with the Matsutake primordia to supply them with the components of the second medium and to support the elongating Matsutake primordia.

[0062] The highly absorbent material used for 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 plant fiber, superabsorbent fiber (SAF), superabsorbent resin (superabsorbent polymer, superabsorbent polymer, 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.

[0063] <Step (c)> In addition to the above-described steps (a) and (b), the production method of the present invention may further include, as step (c), a step of growing the Matsutake primordia obtained by steps (a) and (b) to obtain fruiting body-like Matsutake primordia.

[0064] One method for growing Matsutake primordia is to supply the Matsutake primordia with nutrients (free amino acids, sugars, vitamins, minerals such as inorganic salts, lipids, water, etc.) necessary for the growth of Matsutake primordia and fruiting bodies during or after the above-mentioned step (b). Examples of methods for supplying nutrients include adding the nutrients to the medium. When supplying nutrients during the above-mentioned step (b), for example, a culture solution containing the nutrients is added to the medium (i.e., the culture support) based on the type and amount of the nutrients consumed for primordial elongation. Furthermore, when supplying nutrients after the above-mentioned step (b), for example, the Matsutake primordia are further cultured in a medium supplemented with the necessary nutrients. The type and amount of the nutrients supplied can be appropriately determined depending on the type and amount of the missing nutrients (necessary nutrients).

[0065] The Matsutake primordia obtained by the production method of the present invention may contain 4 μg or more, preferably 5 μg, more preferably 10 μg, and particularly preferably 15 μg or more of methyl cinnamate per unit mass of the Matsutake primordium. Meanwhile, the upper limit of the methyl cinnamate content per unit mass of the Matsutake primordium is not particularly limited, and may be, for example, 100 μg, 90 μg, 80 μg, 70 μg, 60 μg, 50 μg, etc. Therefore, the Matsutake primordium obtained by the production method of the present invention may contain, for example, 4 to 100 μg, 5 to 100 μg, 10 to 100 μg, 15 to 100 μg, 4 to 90 μg, 5 to 90 μg, 10 to 90 μg, 15 to 90 μg, 4 to 80 μg, 5 to 80 μg, 10 to 80 μg, 15 to 80 μg, 4 to 70 μg, 5 to 70 μg, 10 to 70 μg, 15 to 70 μg, 4 to 60 μg, 5 to 60 μg, 10 to 60 μg, 15 to 60 μg, 4 to 50 μg, 5 to 50 μg, 10 to 50 μg, or 15 to 50 μg of methyl cinnamate per unit mass of the Matsutake primordium. In this specification, the "unit mass" of a matsutake primordium refers to the unit mass of a matsutake primordium in a wet (raw) state, unless otherwise specified. Furthermore, according to the production method of the present invention, matsutake primordium having such a high content of methyl cinnamate can be artificially obtained in high yield. In this specification, the methyl cinnamate content in matsutake primordium and fruiting bodies can be measured by gas chromatography-mass spectrometry (GC / MS) under the conditions shown below. Apparatus: 8890 GC, 5977 GC / MSD (manufactured by AGILENT), MPS sampler (manufactured by GERSTEL) Column: DB-5, 60M, inner diameter 0.32 mm, film thickness 0.25 μm (manufactured by AGILENT) Injection mode: splitless Injection port temperature: 260°C Oven temperature: 40°C (3 min) -15°C / min -200°C (0 min) -4°C / min -250°C (3 min) Column flow rate: helium flow rate 1.2 mL / min Heater: transfer 250°C, MS 230°C, quadrupole 150°C Sample volume: 10 mL SPME fiber: polydimethylsiloxane / divinylbenzene (PDMS / DVB) 65 μm, needle size 23GA (manufactured by SUPELCO) Vapor phase extraction: 60°C, 10 min Vial penetration: 38 mm

[0066] The matsutake primordia obtained by the production method of the present invention contain, for example, 1 / 100 or more, 1 / 90 or more, 1 / 80 or more, 1 / 70 or more, 1 / 60 or more, or 1 / 50 or more of the 1-octen-3-ol per unit mass in a wet state compared to naturally obtained matsutake fruiting bodies, while also containing, for example, 5 times, 4 times, 3 times, 2 times, 1.5 times, 1.2 times, or 1 times (similar to naturally obtained matsutake fruiting bodies) of 1-octen-3-ol. More specifically, the matsutake primordia obtained by the production method of the present invention contain, for example, 6 μg or more, 7 μg or more, or 10 μg or more of 1-octen-3-ol per unit mass in a wet state of the matsutake primordium. On the other hand, the matsutake primordia obtained by the production method of the present invention contain, for example, 200 μg or less, 100 μg or less, or 50 μg or less of 1-octen-3-ol per unit mass of the matsutake primordium in a wet state. Furthermore, the matsutake primordium obtained by the production method of the present invention preferably contains 6 to 200 μg, more preferably 7 to 100 μg, and even more preferably 10 to 50 μg of 1-octen-3-ol per unit mass of the matsutake primordium in a wet state. In this specification, the content of 1-octen-3-ol in matsutake primordia and fruiting bodies can be measured by gas chromatography-mass spectrometry (GC / MS). The GC / MS conditions can be the same as those used to measure the content of methyl cinnamate.

[0067] The mass ratio of methyl cinnamate to 1-octen-3-ol (mass of methyl cinnamate:mass of 1-octen-3-ol) in the Matsutake primordia obtained by the production method of the present invention can be, for example, 1:0.01 or more, 1:0.05 or more, 1:0.06 or more, 1:0.1 or more, etc. Note that "1:X or more" means that the mass of 1-octen-3-ol is X or more relative to 1 mass of methyl cinnamate. For example, a mass of methyl cinnamate:mass of 1-octen-3-ol of 1:0.01 or more means that the mass of 1-octen-3-ol is 0.1 or more relative to 1 mass of methyl cinnamate. On the other hand, the mass ratio of methyl cinnamate to 1-octen-3-ol (mass of methyl cinnamate:mass of 1-octen-3-ol) in the Matsutake primordia obtained by the production method of the present invention can be, for example, 1:100 or less, 1:80 or less, 1:50 or less, 1:30 or less, etc. Note that "1:Y or less" means that the mass of 1-octen-3-ol is Y or less per 1 mass of methyl cinnamate. For example, a ratio of 1:100 or less of methyl cinnamate to 1 mass of 1-octen-3-ol means that the mass of 1-octen-3-ol is 100 or less per 1 mass of methyl cinnamate. The mass ratio of methyl cinnamate to 1-octen-3-ol (mass of methyl cinnamate:mass of 1-octen-3-ol) in the Matsutake primordia obtained by the production method of the present invention can be, for example, 1:0.01 to 1:100, 1:0.05 to 1:80, 1:0.06 to 1:50, 1:0.1 to 1:30, etc.

[0068] The present invention will be described in more detail with reference to the following examples, but 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).

[0069] Example 1: Examination of the relationship between phenylalanine concentration and matsutake primordium formation The relationship between phenylalanine concentration and matsutake primordium formation was examined according to the following procedure.

[0070] As the Matsutake cells, Matsutake cells internationally deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan) under accession number NITE BP-03769 were used.

[0071] Preparation of Slant Medium for Preserving Matsutake Cells The 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 (Bacto Yeast Extract, manufactured by Gibco), 1.5 (w / v)% agar (Bacto Agar, manufactured by Becton Dickinson), and 2 (w / v)% glucose was prepared. Note that all components in the culture medium, except for the 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 silicone stoppers. The test tubes were then sterilized at high pressure at 121°C for 20 minutes, tilted, and left at room temperature to solidify the culture medium, preparing slant medium.

[0072] Cultivation and storage of Matsutake cells in slant medium The Matsutake cells were inoculated into the slant medium obtained according to the procedure described above. The slant medium was then left to stand in a dark place at 20°C for 2 months to allow Matsutake mycelia to fully spread in the test tube, and the tube was then stored at a low temperature of 4°C until use.

[0073] Preparation of Plate Medium for Cultivating Matsutake Mycelia A medium for further culturing the Matsutake mycelia obtained according to the above procedure was prepared according to the following procedure. This medium corresponds to the first medium described above. 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. Distilled water was used for all components of the culture medium except for the yeast extract, potassium dihydrogen phosphate, and agar. The pH of the culture medium was then adjusted to 5.0 using HCl and autoclaved at 121°C for 20 minutes. The culture medium was then cooled to approximately 60-70°C, and a pre-sterilized glucose solution was added to a final concentration of 2 (w / v)% and thoroughly mixed. Next, 18 ml of the culture medium was dispensed into sterilized plastic petri dishes (diameter 90 mm) and allowed to stand at room temperature to solidify the culture medium, thereby preparing plate media for culturing Matsutake mycelia.

[0074] Cultivation of Matsutake Mycelia on Plates Matsutake mycelia were cultured using the plate medium for culturing Matsutake mycelia obtained according to the procedure described above, according to the following procedure. First, a portion of the mycelia was excised from the Matsutake mycelia obtained by culturing on the slant medium using a needle with a handle, and inoculated onto the plate medium for culturing Matsutake mycelia. Next, the plate was left to stand in a dark place at 20°C for two months, and radially spreading mycelia were obtained. The sides of the plate medium were covered with film to seal and prevent drying during the culturing.

[0075] Preparation of Plate Medium for Tricholoma matsutake Primordium Culture. Medium for Tricholoma matsutake primordium culture was prepared according to the following procedure. First, a culture medium containing 1 (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. Distilled water was used for the culture medium except for the yeast extract, potassium dihydrogen phosphate, and agar. The pH of the culture medium was then adjusted to 5.0 with HCl and autoclaved at 121°C for 20 minutes. The culture medium was then cooled to approximately 60-70°C, and a pre-sterilized glucose solution was added to a final concentration of 2 (w / v)% and thoroughly mixed. Next, 18 ml of the culture medium was dispensed into sterilized plastic dishes (diameter 90 mm) and allowed to stand at room temperature to solidify, thereby preparing plate media for culturing primordial buds of Matsutake.

[0076]

[0043] The radially spreading Matsutake mycelia obtained in the above-described culture were punched out together with the agar using a sterilized straw with a diameter of 3.5 mm, and the agar portion was removed as much as possible. The mycelia were then inoculated onto the plate medium for primordial culture prepared by the above-described procedure. The plate was then left to stand in the dark at 25°C for 3 weeks, yielding primordia measuring 0.6-0.8 cm in diameter and 0.2-0.25 cm in height. The sides of the plate medium were covered with film to seal and prevent drying during the culture.

[0077] Preparation of a medium-containing highly absorbent material (culture support) for culturing Tricholoma matsutake primordia. 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 was prepared. Distilled water was used for all ingredients in the culture medium except for the yeast extract and potassium dihydrogen phosphate. The pH of the culture medium was then adjusted to 5.0 using HCl and autoclaved at 121°C for 20 minutes. The culture medium was then cooled to room temperature, and a pre-sterilized glucose solution was added to a final concentration of 2 (w / v)%. The mixture was thoroughly mixed to prepare a liquid medium.

[0078] Meanwhile, 1 g of superabsorbent polymer was weighed into a 125 ml plant box (for plant culture, manufactured by AS ONE Corporation) and sterilized in an autoclave at 121° C. for 20 minutes under high pressure. 50 ml of the liquid medium obtained according to the procedure described above 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.

[0079]

[0046] The primordia of Matsutake formed according to the above-mentioned procedure were excised from the agar and inoculated into the medium-containing highly absorbent material (culture support) prepared according to the above-mentioned procedure. The primordia were then left to stand in a dark place at 25°C for 9 weeks.

[0080] Preparation of a highly absorbent material (culture support) containing synthetic phenylalanine-supplemented medium for culturing Tricholoma matsutake primordia. The medium-containing highly absorbent materials (culture supports) shown in Table 1 for test plots 1-1 to 5-4 were prepared according to the following procedure. First, a culture medium containing 1 (w / v)% yeast extract (Bacto Yeast Extract, manufactured by Gibco), 0.1 (w / v)% potassium dihydrogen phosphate, and 0-100 mM synthetic phenylalanine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared. Distilled water was used for all ingredients in the culture medium except for the yeast extract and potassium dihydrogen phosphate. The pH of the culture medium was then adjusted to 5.0 using HCl and autoclaved at 121°C for 20 minutes. The culture medium was then cooled to room temperature, and a pre-sterilized glucose solution was added to a final concentration of 2 (w / v)%. The mixture was thoroughly mixed to prepare a liquid medium. The media in test plots 1-2 to 1-4, 2-2 to 2-4, 3-2 to 3-4, 4-2 to 4-4, and 5-2 to 5-4 correspond to the second medium described above. The amount (final concentration) of synthetic phenylalanine (synthetic L-Phe) added to each test plot was as shown in Table 1.

[0081]

[0082] On the other hand, for test plots 1-1 to 4-4, 4 g of water-absorbent polyester fiber was placed in a 125 ml plant box (for plant culture, manufactured by AS ONE Corporation) and sterilized at 121 ° C. for 20 minutes under high pressure in an autoclave. 40 ml of the liquid medium obtained according to the procedure described above was added to the resulting plant box, and a medium-containing superabsorbent material (culture support) was prepared. Furthermore, for test plots 5-1 to 5-4, 1 g of superabsorbent polymer (superabsorbent polymer) was weighed into a 125 ml plant box (for plant culture, manufactured by AS ONE Corporation) and sterilized at 121 ° C. for 20 minutes under high pressure in an autoclave. 50 ml of the liquid medium obtained according to the procedure described above was added to the resulting plant box, and the liquid medium was absorbed into the superabsorbent polymer to prepare a medium-containing superabsorbent material that would serve as a culture support.

[0083] Cultivation of Primordium in a Highly Absorbent Material Containing a Synthetic Phenylalanine-Supplemented Medium (Main Culture) Primordium of Matsutake grown according to the procedure described above were excised from the culture support and inoculated into each medium-containing highly absorbent material shown in Test Areas 1-1 to 5-4 in Table 1. Culture was performed for 13 weeks in a dark place at culture temperatures of 18°C, 20°C, 25°C, and 30°C as shown in Table 2 below. Furthermore, for the test areas cultured at a culture temperature of 20°C, culture was performed separately for 30, 50, 70, and 92 days to examine the culture progress over time. On day 0 of culture, various measurements were performed using the primordium obtained in the preculture described above. The weight of the tare containing the medium was measured before inoculation, and the weight of each inoculated primordium was calculated.

[0084] Harvesting of Primordium Cultures Grown on Superabsorbent Materials Containing Synthetic Phenylalanine-Supplemented Medium As described above, for the main cultures of primordia obtained by culturing on each superabsorbent material containing synthetic phenylalanine-supplemented medium, the grown primordia were removed from the plant box along with a portion of the culture support, and photographs of their appearance are shown in Figure 1 . Note that for primordia cultured on a medium-containing superabsorbent material (culture support) prepared using a superabsorbent polymer (superabsorbent polymer), photographs were taken with the superabsorbent polymer attached to the primordium (test plots 5-1 to 5-4). Furthermore, the culture primordium was excised from the culture support and weighed individually. Because the yield of primordia obtained in the above-mentioned precultures varied due to the influence of the inoculation volume of the second preculture, the weight of the primordium was measured immediately after inoculation, and the growth rate (%) of the main culture relative to the primordium preculture was calculated and compared. The results are shown in Table 2 and Figure 2 .

[0085]

[0086] From Table 2 and Figure 2, it can be seen that the yield increase rate (weight increase rate) of matsutake primordia was greater in test plots 1-2 to 1-4 compared to test plot 1-1. Similarly, it can be seen that the yield increase rate (weight increase rate) of matsutake primordia was greater in test plots 2-2 to 2-4 compared to test plot 2-1. Similarly, it can be seen that the yield increase rate (weight increase rate) of matsutake primordia was greater in test plots 3-2 to 3-4 compared to test plot 3-1. Similarly, it can be seen that the yield increase rate (weight increase rate) of matsutake primordia was greater in test plots 4-2 to 4-4 compared to test plot 4-1. Therefore, when culturing in a medium-containing highly water-absorbent material (culture support) prepared using water-absorbent polyester fiber, the yield increase rate (weight increase rate) of Matsutake primordia is greater when the phenylalanine concentration is 25 mM, 50 mM, or 100 mM compared to when the phenylalanine concentration is 0 mM, at culture temperatures of 18° C., 20° C., 25° C., and 30° C. Furthermore, Table 2 and Figure 2 show that the yield increase rate (weight increase rate) of Matsutake primordia is greater in test plots 5-2 to 5-4 compared to test plot 5-1. Therefore, when cultured in a medium-containing superabsorbent material (culture support) prepared using a superabsorbent polymer (superabsorbent polymer), the yield increase rate of Matsutake primordia is greater when the phenylalanine concentration is 25 mM, 50 mM, or 100 mM compared to when the phenylalanine concentration is 0 mM, at culture temperatures of 20° C., 20° C., 25° C., and 30° C. Furthermore, Figure 3, which shows the results of examining the time-course of culture, shows that when cultured at a culture temperature of 20° C. for 30, 50, 70, and 92 days, the yield increase rate of Matsutake primordia stagnated at 50 days, but tended to increase over time.

[0087] Example 2: Investigation of the content of methyl cinnamate in matsutake primordia The content of methyl cinnamate in matsutake primordia was investigated according to the following procedure.

[0088] Preparation of Aroma Component Extract from Primordium Cultured in a Highly Absorbent Material Containing a Synthetic Phenylalanine-Supplemented Medium Primordium obtained by culturing in a highly absorbent material containing a synthetic phenylalanine-supplemented medium as described in Example 1 above was harvested, and 1 g of primordium was collected in a conical tube. For primordium yields of less than 1 g, the entire amount was used. Next, 7 ml of ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added per 1 g of primordium, and the tissue was pulverized using an electric homogenizer (manufactured by IKA Japan Co., Ltd.) until the tissue was finely divided. The mixture was then centrifuged at 3,000 rpm for 10 minutes, and the supernatant of the extract was recovered. Next, pure water was added to the supernatant to make the supernatant 20% (v / v). 10 ml of the obtained diluted solution was collected in a 20 ml headspace vial (manufactured by GERSTEL), and methyl laurate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added as an internal standard substance, followed by headspace-solid phase microextraction (HS-SPME) gas chromatography mass spectrometry (GC / MS).

[0089] Preparation of Aroma Extract from Domestic Wild Matsutake Fruiting Bodies Domestic wild matsutake mushrooms from Iwate Prefecture were used, purchased from a department store in Tokyo. On the day of purchase, the caps (including gills) and stalks of the matsutake mushrooms were separated and finely diced, and 1 g of each was collected in a conical tube. Next, 7 ml of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was added per 1 g of each sample, and the tissue was pulverized using an electric homogenizer (IKA Japan Co., Ltd.) until finely divided. The mixture was then centrifuged at 3,000 rpm for 10 minutes to collect the supernatant. Pure water was then added to the supernatant to make a 20% (v / v) concentration. 10 ml of the resulting diluted solution was placed in a 20 ml headspace vial, and methyl laurate was added as an internal standard. The solution was then subjected to HS-SPME GC / MS.

[0090] Preparation of standard solutions of methyl cinnamate and 1-octen-3-ol To prepare standard solutions for HS-SPME GC / MS, methyl cinnamate and 1-octen-3-ol (both manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were each dissolved in ethanol and diluted with pure water to a final ethanol concentration of 20%. 10 ml of the resulting diluted solution was placed in a 20 ml headspace vial, and methyl laurate was added as an internal standard substance before being subjected to HS-SPME GC / MS.

[0091] Quantitative determination of methyl cinnamate by headspace-solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC / MS) Methyl cinnamate was determined by headspace-solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC / MS). Specifically, the amount of methyl cinnamate in the aroma component extract obtained by the above procedure was determined under the following conditions. Apparatus: 8890 GC, 5977 GC / MSD (manufactured by AGILENT), MPS sampler (manufactured by GERSTEL) Column: DB-5, 60M, inner diameter 0.32 mm, film thickness 0.25 μm (manufactured by AGILENT) Injection mode: splitless Injection port temperature: 260°C Oven temperature: 40°C (3 min) -15°C / min -200°C (0 min) -4°C / min -250°C (3 min) Column flow rate: helium flow rate 1.2 mL / min Heater: transfer 250°C, MS 230°C, quadrupole 150°C Sample volume: 10 mL SPME fiber: polydimethylsiloxane / divinylbenzene (PDMS / DVB) 65 μm, needle size 23GA (manufactured by SUPELCO) Vapor phase extraction: 60°C, 10 min Vial penetration: 38 mm

[0092] Quantitative Determination of 1-Octen-3-ol by Gas Chromatography Mass Spectrometry (GC / MS) Quantitative determination of 1-octen-3-ol was carried out by gas chromatography mass spectrometry (GC / MS). Specifically, the aroma component extract obtained by the above procedure was subjected to quantitative determination of 1-octen-3-ol by GC / MS under the same conditions as those for the above-mentioned determination of methyl cinnamate.

[0093] Amounts of methyl cinnamate and 1-octen-3-ol produced by Matsutake mushroom primordia were analyzed by HS-SPME GC / MS under the conditions described above for an aroma component extract from Matsutake mushroom primordia grown in a synthetic phenylalanine-supplemented medium. The results of quantification of the amount of methyl cinnamate produced using the internal standard method are shown in Figure 4. When cultured on a medium-containing highly absorbent material (culture support) made from absorbent polyester fiber, at a culture temperature of 18°C, the amount of methyl cinnamate per gram of primordia was 0.12 μg when the phenylalanine concentration was 0 mM, but increased to 24.03 μg when the phenylalanine concentration was 25 mM, further increasing to 39.08 μg at 50 mM, and further increasing to 52.50 μg at 100 mM. Furthermore, at an incubation temperature of 20°C, the amount of methyl cinnamate per gram of primordia was 1.14 μg at a phenylalanine concentration of 0 mM, increased to 33.43 μg at a phenylalanine concentration of 25 mM, and further increased to 48.83 μg at a phenylalanine concentration of 50 mM. Even at a phenylalanine concentration of 100 mM, the amount of methyl cinnamate was 18.11 μg, indicating an increase compared to when the phenylalanine concentration was 0 mM. Furthermore, at an incubation temperature of 25°C, the amount of methyl cinnamate per gram of primordia was 1.49 μg at a phenylalanine concentration of 0 mM, increased to 16.54 μg at a phenylalanine concentration of 25 mM, and further increased to 40.60 μg at a phenylalanine concentration of 50 mM. Furthermore, even when the phenylalanine concentration was 100 mM, the amount of methyl cinnamate was 19.25 μg, which is an increase compared to when the phenylalanine concentration was 0 mM. On the other hand, at a culture temperature of 30°C, no increase in the amount of methyl cinnamate produced was observed with the addition of phenylalanine. Furthermore, when cultured in a medium-containing highly absorbent material (culture support) prepared using a highly absorbent polymer (highly water-absorbent polymer), at a culture temperature of 18°C, the amount of methyl cinnamate per gram of primordium yield was 0.35 μg when the phenylalanine concentration was 0 mM, but increased to 32.67 μg when the phenylalanine concentration was 25 mM, further increased to 42.00 μg at 50 mM, and further increased to 48.42 μg at 100 mM.Furthermore, Figure 5, which shows the time-dependent culture progress, indicates that when cultured at a culture temperature of 20°C for 30, 50, 70, and 92 days, the amount of methyl cinnamate produced increases over time up to the 70th day of culture but decreases after 92 days. Figure 6 also shows the quantitative results of the amount of methyl cinnamate produced in domestically grown wild matsutake fruiting bodies. The amount of methyl cinnamate per gram of fruiting body was 43-70 μg in the cap, where methyl cinnamate is reported to be localized, and 1-3 μg in the stalk. These results indicate that the matsutake primordia obtained by the production method of the present invention produce the same amount of methyl cinnamate, a characteristic matsutake aroma component, as the cap of domestically grown wild matsutake fruiting bodies.

[0094] The amount of 1-octen-3-ol produced was quantified by the internal standard method and shown in Figure 7. When cultured on a medium-containing highly absorbent material (culture support) prepared using absorbent polyester fiber, the amount of 1-octen-3-ol was 42.35 μg per gram of primordium yield at a culture temperature of 18°C ​​when the phenylalanine concentration was 0 mM, but this decreased to 13.65 μg at a phenylalanine concentration of 25 mM and further decreased to 8.52 μg at a phenylalanine concentration of 50 mM. Even at a phenylalanine concentration of 100 mM, the amount of 1-octen-3-ol was 17.97 μg, indicating a decrease compared to when the phenylalanine concentration was 0 mM. At a culture temperature of 20°C, the amount of 1-octen-3-ol per gram of primordia was 26.25 μg at a phenylalanine concentration of 0 mM, increased to 30.68 μg at a phenylalanine concentration of 25 mM, decreased to 19.48 μg at a phenylalanine concentration of 50 mM, and further decreased to 6.53 μg at a phenylalanine concentration of 100 mM. At a culture temperature of 25°C, the amount of 1-octen-3-ol per gram of primordia was 33.60 μg at a phenylalanine concentration of 0 mM, decreased to 14.23 μg at a phenylalanine concentration of 25 mM, decreased further to 12.25 μg at a phenylalanine concentration of 50 mM, and further decreased to 9.33 μg at a phenylalanine concentration of 100 mM. Furthermore, at a culture temperature of 30°C, the amount of 1-octen-3-ol per gram of primordia was 6.3 μg at a phenylalanine concentration of 0 mM, increased to 7.88 μg at a phenylalanine concentration of 25 mM, and remained almost unchanged at 50 mM and 100 mM. The quantitative results of the amount of 1-octen-3-ol produced in domestically grown wild matsutake fruiting bodies are shown in Figure 8. The amount of 1-octen-3-ol per gram of fruiting body ranged from 291.78 to 443.68 μg in the cap and from 231.12 to 469.00 μg in the stalk. These results indicate that the amount of 1-octen-3-ol produced in matsutake primordia obtained by the production method of the present invention is approximately 1 / 10 lower than that of wild matsutake fruiting bodies, and is further reduced by approximately 1 / 10 to 1 / 50 when phenylalanine is added.

Claims

1. Matsutake primordia containing 4 μg or more of methyl cinnamate per unit mass of the primordium.

2. The Matsutake primordium according to claim 1, which is identified as Tricholoma matsutake by molecular phylogenetic analysis based on the base sequence of ITS-5.8S rDNA.

3. The matsutake primordium according to claim 1, which is a matsutake primordium of Tricholoma matsutake identified by accession number NITE BP-03769.

4. A method for producing matsutake primordia, comprising: (a) culturing cells capable of forming primordia separated from matsutake fruiting bodies in a first medium containing a first protein hydrolysate or amino acids to form matsutake primordia; and (b) culturing the matsutake primordia on a culture support comprising a highly absorbent material infiltrated with a second medium containing a second protein hydrolysate or amino acids and having a phenylalanine concentration of 10 to 100 mM to allow the matsutake primordia to elongate vertically upward, thereby obtaining matsutake primordia, wherein the first protein hydrolysate and the second protein hydrolysate each comprise at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone, and tryptone, or comprise a combination of casamino acids and at least one protein hydrolysate selected from the group consisting of yeast extract, beef extract, peptone, and tryptone.

5. The method according to claim 4, wherein the culture temperature in step (a) is 18 to 25°C.

6. The method according to claim 4, wherein the culture temperature in step (b) is 18 to 30°C.

7. The method of claim 4, 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.

8. The method according to claim 4, wherein the fruiting body is identified as Tricholoma matsutake by molecular phylogenetic analysis based on the base sequence of ITS-5.8S rDNA.

9. The method according to claim 4, wherein the fruiting body is a fruiting body of Tricholoma matsutake, identified by accession number NITE BP-03769.

10. A method for producing fruiting body-like Matsutake primordia, further comprising the step of growing the Matsutake primordia obtained by the method according to any one of claims 4 to 9.

11. A Matsutake primordium produced by the method according to any one of claims 4 to 9.

12. A fruiting body-like Matsutake primordium produced by the method according to claim 10.

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