JP7705287B2 - Additives for mushroom cultivation - Google Patents

Description

The present invention relates to a medium additive for mushroom cultivation and a method for increasing mushroom components.

(1) Mushrooms Mushrooms are a common name for relatively large fruiting bodies or basidiomycetes of certain fungi. In Japan, more than 300 kinds are eaten, and several dozen of them are cultivated artificially. Shiitake mushrooms, enokitake mushrooms, shimeji mushrooms (bunashimeji, honshimeji, hatakeshimeji, etc.), oyster mushrooms, himematsutake mushrooms, mushrooms, porcini mushrooms, king oyster mushrooms, matsutake mushrooms, and nameko mushrooms are very commonly eaten and are also cultivated.

The reported efficacy of mushrooms includes antibacterial, antiviral, cholesterol-lowering, blood sugar-lowering, blood pressure-lowering, antithrombotic, lymphocyte blastogenesis-inhibiting, and antitumor properties.
Mushrooms contain water, protein, fiber, minerals, vitamins, etc. as major components. Trace components include amino acids, nucleic acids, organic acids, sugars, β-glucans, etc., and these trace components also have a significant impact on the efficacy and taste of mushrooms.

Ornithine is an amino acid and one of the components of the urea cycle (also called the ornithine cycle), which converts harmful ammonia into urea. It is produced by the decomposition of arginine (Figure 1). Although ornithine is not an amino acid that constitutes protein, it plays an important role in the body as a free amino acid and is known as a functional food. It has been reported that ingesting ornithine activates the urea cycle and enhances the detoxification of ammonia. In addition, it has been reported that ornithine has many physiological effects, such as improving hepatic encephalopathy, promoting growth hormone secretion, stimulating the immune system, reducing fatigue, improving hepatic encephalopathy, promoting growth hormone secretion, stimulating the immune system, and reducing fatigue.

Ornithine is known to be found in foods such as clams, yellowfin tuna, cheese, flounder, dadachamame beans, and also in shimeji mushrooms such as Bunashimeji. Furthermore, its use as a health functional food, including supplements containing ornithine, is also on the rise.

JP 2014-100112 A (Patent Document 1) discloses a method for producing a food composition containing ornithine, which includes culturing an edible basidiomycete in a culture medium containing dried barley shochu lees, harvesting the fruiting bodies or culture medium containing the produced free arginine, and seeding and culturing lactic acid bacteria. In the method described in this document, the use of dried barley shochu lees causes a large amount of free arginine to accumulate in the cultured edible basidiomycete. By subjecting this to lactic acid bacteria culture, the free arginine is converted to ornithine. In the method of Patent Document 1, lactic acid bacteria culture is essential to increase ornithine.

Prior to the present invention, no method was known for selectively increasing the amino acids, nucleic acids and other components of mushrooms, in particular ornithine and other components with particularly high nutritional value.
(2) Yeast Yeast cells contain various nutritionally important components such as proteins, vitamins, minerals, nucleic acids, glutathione, and dietary fiber. Therefore, extracts and nutritional components extracted from yeast cells are used as seasonings, etc. The yeast cells after extracting yeast extract are also rich in various nutritional components, and it is necessary to consider how to utilize them.

Yeast cells are often used as nutritional supplements because they contain many nutritional components. They are also known to be used as a culture material for mushrooms. For example, JP 5-284852 (Patent Document 2) describes that by using yeast after yeast extract extraction as a cultivation medium, the cultivation period can be shortened and the mycelium concentration can be increased. In addition, JP 2014-121288 (Patent Document 3) describes a flavorful food obtained by cultivating or fermenting a basidiomycete using yeast residue as a culture medium.

Patent Publication 2014-100112 JP 5-284852 A Patent Publication 2014-121288

Japan Patent Office "2005 Standard Technology Collection: Mushroom Cultivation Methods"

The inventors of the present invention conducted extensive research to find a method for selectively increasing the amino acids, nucleic acids, polyamines, and other components of mushrooms, particularly ornithine and other highly nutritious components, and discovered that adding yeast cells to a mushroom cultivation medium increases specific amino acids, nucleic acids, polyamines, and other components of mushrooms, leading to the invention.

The present invention provides a medium additive for mushroom cultivation.
The present invention also provides a method for increasing a specific component of a mushroom.

Although not limited thereto, the present invention includes the following aspects [Aspect 1]
A medium additive for mushroom cultivation comprising yeast cells, the medium additive comprising the following components of the mushroom:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
A medium additive for mushroom cultivation, which increases the content of at least one component of (4) choline; and/or (5) trigonelline.
[Aspect 2]
The medium additive for mushroom cultivation according to aspect 1, which increases the content of ornithine, arginine, glutamic acid, aspartic acid, glutamine and/or ergothioneine.
[Aspect 3]
The medium additive for mushroom cultivation according to embodiment 1, which increases the content of guanylic acid (GMP) and/or adenylic acid (AMP).
[Aspect 4]
The medium additive for mushroom cultivation according to any one of Aspects 1 to 3, which increases the content of components from two or more groups of substances (1) to (5).
[Aspect 5]
The medium additive for mushroom cultivation according to any one of aspects 1 to 4, wherein the content of the at least one component is increased to 120% or more compared to when the additive is not added.
[Aspect 6]
The medium additive for mushroom cultivation according to any one of Aspects 1 to 5, which further increases the umami taste of mushrooms.
[Aspect 7]
The medium additive for mushroom cultivation according to any one of Aspects 1 to 6, wherein the yeast cells are yeast cells in which components have been released outside the yeast cells by heat treatment and/or enzyme treatment of the yeast cells.
[Aspect 8]
The medium additive for mushroom cultivation according to any one of aspects 1 to 7, wherein the mushroom is selected from edible mushrooms consisting of Bunashimeji, Enokitake, Honshimeji, Hatakeshimeji, Oyster Mushroom, Himematsutake, Shiitake, mushroom, porcini, king oyster mushroom, matsutake, truffle, nameko, Tamogitake, and Bakamatsutake.
[Aspect 9]
The medium additive for mushroom cultivation according to any one of Aspects 1 to 7, wherein the mushroom is Bunashimeji or Enokitake.
[Aspect 10]
In addition, the following:
(i) increasing the umami flavor of mushrooms;
(ii) increasing the content of polyamines in mushrooms;
(iii) increasing the protein content in mushrooms; and (iv) not increasing mushroom yield;
The medium additive for mushroom cultivation according to any one of aspects 1 to 9, which satisfies one or more requirements selected from the group consisting of:
[Aspect 11]
The following components of mushrooms:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) choline; and/or (5) trigonelline, comprising:
The method further comprises adding yeast cells to a medium for growing mushrooms.
[Aspect 12]
12. The method according to aspect 11, wherein the content of ornithine, arginine, glutamic acid, aspartic acid, glutamine and/or ergothioneine is increased.
[Aspect 13]
12. The method according to aspect 11, wherein the content of guanylic acid (GMP) and/or adenylic acid (AMP) is increased.
[Aspect 14]
14. The method according to any one of aspects 11-13, wherein the content of components of two or more groups of substances (1)-(5) is increased.
[Aspect 15]
15. The method of any one of aspects 11-14, wherein the content of the at least one component is increased by 120% or more compared to when the additive is not added.
[Aspect 16]
Aspect 16. The method according to any one of aspects 11 to 15, further comprising increasing the umami taste of the mushroom.
[Aspect 17]
The method according to any one of Aspects 11 to 16, wherein the yeast cells are yeast cells from which components have been released outside the yeast cells by heat treatment and/or enzyme treatment of the yeast cells.
[Aspect 18]
A method according to any one of aspects 11-17, wherein the mushroom is selected from edible mushrooms consisting of Bunashimeji, Enokitake, Honshimeji, Hatakeshimeji, Pleurotus oyster, Himematsutake, Shiitake, Bacillus subtilis, Porcini, Eringi, Matsutake, Truffle, Nameko, Tamogitake and Bakamatsutake.
[Aspect 19]
Aspects 19. The method according to any one of aspects 11-18, wherein the mushroom is Bunashimeji or Enokitake.
[Aspect 20]
In addition, the following:
(i) increasing the umami flavor of mushrooms;
(ii) increasing the content of polyamines in mushrooms;
(iii) increasing the protein content in mushrooms; and (iv) not increasing mushroom yield;
20. The method according to any one of aspects 11-19, wherein the method achieves one or more advantages selected from the group consisting of:
[Aspect 21]
The method according to any one of aspects 11 to 20, further comprising carrying out stalk length cultivation during fruit body growth.
[Aspect 22]
22. The method according to aspect 21, wherein the ornithine and/or arginine content is increased.

By using the medium additive for mushroom cultivation of the present invention, specific components of mushrooms, specifically the following components:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
The effect of increasing the content of at least one of the components: (4) choline; and/or (5) trigonelline can be obtained.

By using the medium additive for mushroom cultivation of the present invention, it is now possible to selectively increase specific components with high nutritional value without the need for additional culture processes such as lactic acid bacteria culture.

FIG. 1 is a schematic diagram of the urea cycle (ornithine cycle).

1. Medium additive for mushroom cultivation In one aspect, the present invention relates to a medium additive for mushroom cultivation. The medium additive for mushroom cultivation contains yeast cells and the following components of mushrooms:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) increasing the content of at least one component of choline; and/or (5) trigonelline.

The present invention is based on the discovery that the addition of yeast cells to a medium for growing mushrooms increases the content of components such as amino acids, nucleic acids and polyamines in the mushrooms.
The type of yeast cells contained in the additive is not particularly limited. In one embodiment (i.e., not limited to), yeast cells include cells of torula yeast, baker's yeast, brewer's yeast, sake yeast, etc. In one embodiment, torula yeast (scientific name: Candida utilis), which includes torula yeast and baker's yeast, is rich in cysteine peptides (glutathione) and is used as a protein source in livestock feed and as an additive for processed foods. Baker's yeast (scientific name: Saccharomyces cerevisiae) is widely known as a yeast mainly used in the production of bread.

The yeast cells may also be in various forms, such as compressed yeast, dried yeast, active dry yeast, killed yeast, pasteurized dry yeast, and the like.
In one embodiment, the yeast cells may be residual yeast cells in which the content components have been released outside the yeast cells by subjecting the yeast cells to heat treatment and/or enzyme treatment. "Content components released outside the yeast cells" refers to a state in which the content components of the yeast cells have been released outside the yeast cells and some of the content components remain. "Heat treatment" refers, but is not limited to, for example, to treatment at about 40°C to about 100°C for about 30 minutes to 20 hours. In one embodiment, the heat treatment is performed under alkaline conditions. "Enzyme treatment" refers, for example, to protease treatment. Yeast cells in which the protein content and/or cell wall content have been increased by heat treatment and/or enzyme treatment may be used. "Content components" refer to components leaked from the yeast cells by various treatments. They contain amino acids, nucleic acid-related substances, minerals, and vitamins as main components, and are used in "seasonings,""culture media for microbial culture,""livestockfeed,""dietarysupplements,""fertilizers," and the like. In one embodiment, dried yeast cells in which the content components have been released outside the yeast cells may be used.

In one embodiment, yeast cells that have not been subjected to heat treatment or enzyme treatment may be used. Dried yeast cells that have not been subjected to heat treatment or enzyme treatment may be used.
The yeast cells may be of one type or a mixture of two or more types.

The concentration of yeast cells in the mushroom cultivation medium when the above mushroom cultivation medium additive is used is not particularly limited. Non-limitingly, the concentration of yeast cells in the mushroom cultivation medium may be 0.1% by weight or more, 0.3% by weight or more, 0.5% by weight or more, 0.7% by weight or more, 0.8% by weight or more, 0.9% by weight or more, 1.0% by weight or more, 1.1% by weight or more, 1.2% by weight or more, 1.3% by weight or more, 1.4% by weight or more, 1.5% by weight or more, 1.6% by weight or more, 1.7% by weight or more, 1.8% by weight or more, or 1.9% by weight or more. In one embodiment, the concentration of yeast cells in the mushroom cultivation medium is 0.5% by weight or more, 0.8% by weight or more, 1.0% by weight or more, or 1.5% by weight. Without limitation, the concentration of yeast cells in the mushroom cultivation medium may be 10.0% by weight or less, 8.0% by weight or less, 6.0% by weight or less, 5.0% by weight or less, 3.0% by weight or less, 2.0% by weight or less, 1.5% by weight or less, 1.2% by weight or less, or 1.1% by weight or less. In one embodiment, the concentration of yeast cells in the mushroom cultivation medium is 0.3% by weight to 2.0% by weight, 0.5% by weight to 2.0% by weight, 1.0% by weight to 2.0% by weight, or 1.0% by weight to 1.5% by weight.

The type of mushroom that can provide the medium additive for mushroom cultivation is not particularly limited. It is preferably an edible mushroom. Without limitation, the mushroom is selected from edible mushrooms consisting of Bunashimeji, Enokitake, Honshimeji, Hatakeshimeji, Pleurotus oyster, Himematsutake, Shiitake, mushroom, Porcini, Eringi, Matsutake, truffle, Nameko, Tamogitake, and Bakamatsutake. In one embodiment, the mushroom is Bunashimeji or Enokitake.

The "mushroom cultivation medium" is not particularly limited. Those skilled in the art can use an appropriate medium according to the type of mushroom. It is preferable that the medium contains, but is not limited to, a medium base material and any optional components.
Examples of medium substrates include sawdust, sawdust, chips, chip dust, and corn cob meal.

Optional components may include nutrients, additives, and other components. Nutrients, additives, and other components may be freely combined from known components within the scope of the present invention. Examples of nutrients and additives include rice bran, corn cob, corn bran, grain sorghum, corn stalk, cotton husk, bean husk, bran, chitosan, okara, milo, beet pulp, vitamins, minerals, proteins (soybean meal, soybean meal, corn gluten feed, potato protein, cottonseed meal, rapeseed meal, peanut meal, tofu meal, sake lees, brewer's lees, dried brewer's yeast, etc.), sugar, Takara Clean (Takara Bio Inc.), Kinoko Lime (Maruei Co., Ltd.), etc. The nutrients and additives may be commercially available products or may be produced by known methods.

Other ingredients include, for example, organic materials such as rice straw, wheat straw, the central axis of the female corn ear, bran, rice husks, and crushed or pulverized dead grass; inorganic materials such as diatomaceous earth, white earth, and volcanic ash soil; soil suitable for cultivating matsutake mushrooms, such as mountain soil and soil from pine forests, mixed with the required amount of white earth and leaf mold; hydrated lime, shell fossils, nitrogen sources, vitamins, minerals, sugars, etc.

For example, as a culture medium for Bunashimeji, YKB medium and YK2 medium (composition: corn cob meal, rice bran, powdered beet, cotton husk, wheat bran, dried bean curd refuse, shell fossils) described in the examples of this specification can be used. As a culture medium for Enokitake mushrooms, YK3 medium described in the examples of this specification can be used.

Mushrooms can also be cultured using known methods appropriate to the type of mushroom. For example, but not limited to, the following cultivation processes are known for Bunashimeji, Enokitake, Shiitake, Nameko, Oyster Mushroom, Maitake, Yanagimatsutake, Hatakeshimeji, King Oyster Mushroom, Armillaria arborescens, Sugitake, Usuhirake, Yamabushitake, and Honshimeji.

Bunashimeji cultivation process [Cultivation process]
Sterilization of medium → Inoculation of seed bacteria → Cultivation → Surrounding of fungi (when mycelium has spread throughout the medium) → Cultivation (cultivation period) → Scrape (to stimulate fruiting body formation) → Fruiting body growth → Harvest Enoki mushroom cultivation process [Cultivation process]
Sterilization of medium → Inoculation of seed bacteria → Cultivation (cultivation period) → Scratching (stimulation to form fruit bodies) → Sprouting → Inhibition → Fruit body growth → Harvesting Nameko cultivation process [Cultivation process]
Culture medium sterilization → seed culture inoculation → cultivation (cultivation period) → scratching (stimulation to form fruit bodies) → germination → fruit body growth → harvest Among the above cultivation steps, the above-mentioned medium additive for mushroom cultivation is added to the cultivation medium used at least in the "cultivation (cultivation period)" step.

Other details about the mushroom cultivation process are listed in the Japan Patent Office's "2005 Collection of Standard Techniques: Mushroom Cultivation Methods" (National Diet Library Digital Collection: https://dl.ndl.go.jp/info:ndljp/pid/1249443).

The mushrooms cultivated in the medium containing the above-mentioned medium additive for mushroom cultivation contain the following components:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
The content of at least one of the components, (4) choline; and/or (5) trigonelline, is increased compared to when the additive is not used. The mushroom cultivation medium additive has the effect of increasing the mushroom components (compared to when the additive is not used). Preferably, each mushroom component is increased by 101% or more, 102% or more, 103% or more, 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, 160% or more, 180% or more, or 200% or more compared to when the additive is not used.

The increase rate of each mushroom component due to the use of the medium additive for mushroom cultivation may vary depending on the type of mushroom and the type of yeast cell used.
In one embodiment, the medium additive for mushroom cultivation increases the content of components in two or more groups among the substances (1)-(5) in a mushroom cultivated in a medium containing the additive. In one embodiment, the medium additive for mushroom cultivation increases the content of components in three or more groups, components in four or more groups, or components in all groups among the substances (1)-(5) in a mushroom cultivated in a medium containing the additive.

In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increase in 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more amino acid components. In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increase in all of the amino acid components described in (1).

In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have increased contents of two or more nucleic acid components. In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have increased contents of guanylic acid (GMP) and/or adenylic acid (AMP).

In one aspect, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased content of ornithine, arginine, glutamic acid, aspartic acid, glutamine and/or ergothioneine. In one aspect, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased content of two or more components selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, glutamine and/or ergothioneine. In one aspect, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased content of all components selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, glutamine and/or ergothioneine.

Ornithine is a free amino acid produced from arginine in the urea cycle. Preferably, the ornithine content is increased by 102% or more, 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, or 160% or more compared to when no additive is used.

The arginine content increases by 102% or more, 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, 160% or more, 170% or more, 180% or more, or 190% or more compared to when no additive is used.

Pyroglutamic acid is an amino acid formed by intramolecular condensation reaction between the carboxyl group and amino group of glutamic acid to form a lactam. In one embodiment, the content of pyroglutamic acid is increased in mushrooms cultivated in a medium containing the medium additive for mushroom cultivation. Preferably, the pyroglutamic acid content is increased by 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, 160% or more, 180% or more, or 200% or more compared to when no additive is used.

Preferably, the glutamic acid content increases by 101% or more, 102% or more, 104% or more, 105% or more, 108% or more, 110% or more, 115% or more, or 120% or more compared to when no additive is used.

Preferably, the aspartic acid content increases by 101% or more, 103% or more, 105% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, 160% or more, 180% or more, or 190% or more compared to when no additive is used.

Preferably, the alanine content increases by 101% or more, 103% or more, 105% or more, 108% or more, 110% or more, 112% or more, or 115% or more compared to when no additive is used.

Preferably, the tyrosine content is increased by 101% or more, 105% or more, 108% or more, 110% or more, 112% or more, 115% or more, 118% or more, 120% or more, 125% or more, or 130% or more compared to when no additive is used.

Preferably, the lysine content is increased by 101% or more, 105% or more, 110% or more, 115% or more, 118% or more, 120% or more, 125% or more, 130% or more, 140% or more, 150% or more, or 170% or more compared to when no additive is used.

Preferably, the glutamine content increases by 101% or more, 105% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, 170% or more, 200% or more, or 250% or more compared to when no additive is used.

Preferably, the citrulline content increases by 101% or more, 105% or more, 110% or more, 115% or more, 120% or more, 125% or more, 130% or more, 135% or more, or 140% or more compared to when no additive is used.

Preferably, S-adenosylmethionine (SAM) is increased by 101% or more, 105% or more, 110% or more, 120% or more, 125% or more, 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, or 160% or more compared to when no additive is used.

Preferably, the hydroxyproline is increased by 101% or more, 105% or more, 108% or more, 110% or more, 112% or more, 115% or more, or 118% or more compared to when no additive is used.

Preferably, the amount of β-alanine increases by 101% or more, 105% or more, 108% or more, 110% or more, 112% or more, 115% or more, or 118% or more compared to when no additive is used.

Ergothioneine is a rare amino acid derivative and a substance with strong antioxidant properties. Preferably, ergothioneine is increased by 101% or more, 105% or more, 108% or more, 110% or more, 112% or more, 115% or more, 118% or more, or 120% or more compared to when no additive is used.

Gamma-aminobutyric acid (GABA) is a type of amino acid that functions primarily as an inhibitory neurotransmitter. Preferably, GABA is increased by 101% or more, 105% or more, 108% or more, 110% or more, 112% or more, 115% or more, 118% or more, or 120% or more compared to when no additive is used.

In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased content of guanylic acid (GMP). Preferably, the GMP content is increased by 102% or more, 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, 160% or more, 180% or more, 200% or more, or 250% or more compared to when no additive is used. Guanylic acid is known as a nucleic acid involved in the umami taste of mushrooms.

In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased content of adenylic acid (AMP). Preferably, the AMP content increases by 101% or more, 105% or more, 110% or more, 115% or more, 118% or more, 120% or more, 130% or more, 140% or more, 160% or more, 180% or more, or 200% or more compared to when no additive is used.

In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased content of uridylic acid (UMP). Preferably, the UMP content increases by 101% or more, 105% or more, 110% or more, 115% or more, 118% or more, 120% or more, 130% or more, or 140% or more compared to when no additive is used.

In one embodiment, mushrooms grown in a medium containing the mushroom culture medium additive have increased contents of both guanylic acid (GMP) and adenylic acid (AMP).
In one aspect, the medium additive for mushroom cultivation can increase the umami of mushrooms cultivated using a medium containing the additive. "Increasing the umami of mushrooms" means that the umami of mushrooms is felt stronger compared to when the medium additive for mushroom cultivation is not used. Non-limitingly, for example, when a sensory test is performed as in the examples of this specification, when a control without additive is evaluated on a scale of ±4 (total of 9 scales) with the control without additive as the standard (0), the evaluation is preferably 0.05 or more, 0.1 or more, or 0.2 or more. In one aspect, the mushrooms cultivated in a medium containing the medium additive for mushroom cultivation have an increased content of glutamic acid, which is one of the umami components of mushrooms. Preferably, the glutamic acid content increases by 102% or more, 105% or more, 108% or more, 110% or more, 115% or more, or 120% or more compared to when the additive is not used.

In one aspect, the medium additive for mushroom cultivation can increase the content of polyamines in mushrooms cultivated using a medium containing the additive. "Polyamine" is a general term for straight-chain aliphatic hydrocarbons to which three or more primary amino groups are bonded, and may also include diamines to which only two primary amino groups are bonded. Polyamines are found in all living organisms, from viruses to humans, and are growth factors involved in activities such as cell division and protein synthesis. In the case of human organisms, it is believed that more than 20 types of polyamines are contained, the main ones being putrescine (PUT), spermidine (SPD), and spermine (SPM). Polyamines that increase in mushrooms cultivated in a medium containing the medium additive for mushroom cultivation include, but are not limited to, spermidine, spermine, and/or putrescine. In one embodiment, the polyamine content increases by 102% or more, 103% or more, 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, 130% or more, or 140% or more compared to when no additive is used.

Preferably, the spermidine content is increased by 101% or more, 105% or more, 110% or more, 120% or more, 125% or more, 130% or more, 135% or more, 140% or more, 145% or more, or 150% or more compared to when no additive is used.

Preferably, the spermine content is increased by 101% or more, 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 170% or more, 180% or more, 200% or more, 250% or more, or 300% or more compared to when no additive is used.

Preferably, putrescine is increased by 101% or more, 105% or more, 110% or more, 120% or more, 125% or more, or 130% or more compared to when no additive is used.
Preferably, two or more of the polyamines spermidine, spermine and putrescine, or all of the polyamines are increased.

Preferably, the choline is increased by 102% or more, 103% or more, 105% or more, 108% or more, 110% or more, 115% or more, 120% or more, or 130% or more compared to when no additive is used.

Preferably, trigonelline is increased by 101% or more, 105% or more, 110% or more, 120% or more, 125% or more, 130% or more, 135% or more, 140% or more, 145% or more, or 150% or more compared to when no additive is used.

In one embodiment, mushrooms grown in a medium containing the medium additive for mushroom cultivation have an increased protein content. In one embodiment, the protein content is increased by 102% or more, 105% or more, 110% or more, 115% or more, or 120% or more compared to when no additive is used.

In one embodiment, the yield of mushrooms grown in a medium containing the medium additive for mushroom cultivation does not change substantially. Without limitation, "not change substantially" means that the change (increase or decrease) in the yield of mushrooms is within 10%, within 9%, or within 8% compared to when the additive is not used.

The medium additive for mushroom cultivation increases the content of specific amino acids and/or nucleic acids in mushrooms, and in addition, has the following properties:
(i) increasing the umami flavor of mushrooms;
(ii) increasing the content of polyamines in mushrooms;
(iii) increasing the protein content in mushrooms; and (iv) not increasing mushroom yield;
In one embodiment, the medium additive for mushroom cultivation satisfies two or more of the above requirements (i)-(ii).

2. Method for Increasing Mushroom Components In one aspect, the present invention also relates to a method for increasing mushroom components. The method of the present invention comprises increasing the following components of mushrooms:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) choline; and/or (5) trigonelline, comprising:
The method involves adding yeast cells to a medium for growing mushrooms.

In one embodiment, the method increases the content of components from two or more groups of substances (1)-(5). In one embodiment, the method increases the content of components from three or more groups, four or more groups, or all groups of substances (1)-(5).

In one embodiment, the method increases the content of the at least one component by 120% or more compared to the content without the additive.
In one embodiment, the method increases the amount of amino acid components in the mushroom by 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more. In one embodiment, the method increases all of the amino acid components described in (1).

In one embodiment (i.e., without limitation), the method increases the content of ornithine, arginine, glutamic acid, aspartic acid, glutamine, and/or ergothioneine. In one embodiment, the method increases the content of two or more components selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, glutamine, and/or ergothioneine. In one embodiment, the method increases all of the components selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, glutamine, and/or ergothioneine.

In one embodiment, the method increases the content of guanylate (GMP) and/or adenylate (AMP).
In one embodiment, it may further increase the umami flavor of the mushrooms.

In one embodiment, the yeast cells are yeast cells from which components have been released outside the yeast cells by subjecting the yeast cells to heat treatment and/or enzyme treatment.
In one embodiment, the mushroom is selected from edible mushrooms consisting of Bunashimeji, Enokitake, Honshimeji, Hatakeshimeji, Pleurotus oyster, Himematsutake, Shiitake, mushroom, Porcini, Honshimeji, Eringi, Matsutake, truffle, Nameko, Tamogitake, and Bakamatsutake. In one embodiment, the mushroom is Bunashimeji or Enokitake.

In one embodiment, the use of yeast cells in the method does not substantially alter mushroom yield.
In one embodiment, the method described above increases the protein content in mushrooms through the use of yeast cells.

Unless otherwise specified, the definitions and explanations regarding "yeast cells," "cultivation medium," "cultivation process," "mushrooms," and the increase rate of each component are as described in "1. Additives for mushroom cultivation medium."

The method, in addition to increasing the specific amino acid and/or nucleic acid content of mushrooms by adding yeast cells, also comprises the steps of:
(i) increasing the umami flavor of mushrooms;
(ii) increasing the content of polyamines in mushrooms;
(iii) increasing the protein content in mushrooms; and (iv) not increasing mushroom yield;
In one embodiment, the method has two or more of the above effects (i)-(ii).

The method of the present invention for increasing mushroom components may further include carrying out stalk length cultivation during fruit body growth.
"Long stem cultivation" is a method in which the part of the plant that will become the stem is wrapped in paper during the growth period of the fruit, so that the stem grows long. In the case of mushrooms, the fruit body is wrapped in paper during the period during which the fruit grows or part of that period, preventing the fruit body from spreading out laterally and allowing the stem to grow long. Long stem cultivation is generally used for cultivation of Enokitake mushrooms. In the present invention, when the free amino acid content of the cap and stalk of Bunashimeji was compared, it was found that the amino acid content of ornithine, arginine, aspartic acid, etc., which are involved in the ornithine cycle (Figure 1), was high in the stalk, and it was considered that the stalk should be allowed to grow long in order to increase the amino acid content. In the present invention, long stem cultivation was also performed on Bunashimeji, and the effect on the components in the resulting mushroom was examined.

Carrying out stalk-length cultivation during fruit body growth increases various mushroom components, especially when combined with the addition of yeast cells. In particular, the ornithine and arginine contents are increased compared to when yeast cells are not added and stalk-length cultivation is not performed. Preferably, the ornithine content is increased by 110% or more, 115% or more, 120% or more, 130% or more, 150% or more, or 160% or more compared to when yeast cells are not added and stalk-length cultivation is not performed. Preferably, the arginine content is increased by 115% or more, 120% or more, 130% or more, 150% or more, 160% or more, 170% or more, 180% or more, or 190% or more compared to when no additive is used.

The present invention also relates to a method for the preparation of yeast cells comprising administering to a subject the following components of a mushroom:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) choline; and/or (5) trigonelline in a method for increasing the content of at least one component.

The present invention also provides the following components of mushrooms:
(1) an amino acid selected from the group consisting of ornithine, arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
(2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP) and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) choline; and/or (5) trigonelline,

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are included in the technical scope of the present invention.

Materials and Methods In the examples herein, the following materials and methods were used unless otherwise specified.
(1) Method for Producing Yeast Cells In this specification, unless otherwise specified, yeast cells produced as follows were used.

DYP: Baker's yeast cells that have been treated to increase the protein content and then dried (product name: DYP-SY-02, Fuji Food Industry Co., Ltd.)
TFD: yeast cells obtained by treating torula yeast so that the cell wall content is high and then drying FD: yeast cells obtained by treating baker's yeast so that the cell wall content is high and then drying (product name: Yeast Powder FD, Fuji Food Industry Co., Ltd.)
BY: yeast cells obtained by drying baker's yeast without heat treatment/enzyme treatment (2) Cultivation of Bunashimeji [Test variety] "Nagano Agricultural and Industrial Research Institute NN12" 1 variety [Cultivation container] Capacity: 850 ml Diameter: 58 mm PP bottle cap [Cultivation process]
Sterilization of medium → Inoculation of seed bacteria → Cultivation → Surrounding of fungi (when mycelium has spread throughout the medium) → Cultivation (cultivation period) → Scrape (to stimulate fruiting body formation) → Fruiting body growth → Harvest [Cultivation period and number of specimens]

(3) Cultivation of Enoki mushrooms [Test varieties] Three varieties: "Variety A", "Variety B", and "Variety C".

[Cultivation container] Capacity: 850ml Diameter: 58mm PP bottle with cap [Cultivation process]
Media sterilization → seed inoculation → culture (cultivation period) → scratching (stimulation for fruit body formation) → germination → inhibition → fruit body growth → harvest During the fruit body growth period, paper wrapping (stem-length cultivation) was performed. In the examples of this specification, in the case of cultivation of Enoki mushroom, stem-length cultivation was performed unless otherwise specified.

[Cultivation period and number of specimens]

Example 1: Effect of adding yeast cells to Bunashimeji cultivation medium (first time)
In this example, component analysis and sensory evaluation were carried out when yeast cells were added to a Bunashimeji cultivation medium (first time).

1-1. Cultivation of Bunashimeji The composition of the YKB-1 medium used as the cultivation medium for Bunashimeji is shown in Table 2-1.

Sawdust: Wood powder produced when wood is cut with a saw (large saw) and torn off with a blade Corn cob: Crushed corn cob Cottonseed husk: A mixture of husk and short fiber obtained after cottonseeds are crushed and the kernels are removed Bean husk: The skin of soybeans and other beans. Includes crushed and powdered materials.

Yeast cells DYP or FD were added to YKB-1 medium at the concentrations shown in Table 2-2.

"Takaraclean" (manufactured by Takara Bio Inc.) is an activator for Bunashimeji mushrooms.
In this example, the cultivation period (from seed ingestion to scratching) was 83 days. The number of days for inoculation, the number of days for growth, the uniformity and yield of the plants, and other fruit body characteristics are shown in Tables 2-3 to 2-5.

The yield was almost the same as the control.

1-2. Component analysis <Test specimen>
Bunashimeji mushrooms cultivated in YKB-1 medium supplemented with the control (no yeast cells added), 2.5g DYP, 5g DYP, 2.5g FD, and 5g FD shown in Table 2-2. <Analysis items and analytical methods>

<Pretreatment>
1) Nutritional analysis and β-glucan The edible portion of a Bunashimeji mushroom stalk (approximately 1 cm from the base) was cut and then shredded (finely chopped) with a knife to prepare a sample for analysis.

2) Analysis of components other than β-glucan (1) Cut the edible part of the Bunashimeji mushroom (about 1 cm from the base) and then shred (finely chop) it with a knife.

(2) Add 35 g of distilled water to 25 g of the shredded material from (1) and homogenize.
(3) (2) is heated and extracted for 5 minutes until boiling, and then boiled for 6 minutes.
(4) (3) is centrifuged (3000 rpm x 10 minutes).

The supernatant obtained by the above pretreatment was used as a sample for component analysis.
<Moisture>

<General analysis>

An increase in the protein content of Bunashimeji was observed by adding yeast cells of DYP and FD (DYP 5g 115%, FD 5g 126%).
<5'-ribonucleic acid (as anhydrous disodium salt)>

The mushroom's umami component guanylic acid (GMP) increased significantly (approximately 160% to 290% higher than the control). Adenylic acid also increased by approximately 120% to 230% higher than the control.

<Organic acid>

The content of pyroglutamic acid increased (approximately 140% to 190% compared to the control). Pyroglutamic acid is an amino acid formed by an intramolecular condensation reaction between the carboxyl group and amino group of glutamic acid to form a lactam, and is known to have anticancer effects.

<Sugars and sugar alcohols>

The addition of yeast cells did not result in significant differences from the control.
<β-glucan>

The addition of yeast cells did not result in significant differences from the control.
<Free amino acids>

The total free amino acid content increased by more than 10% in the yeast cell addition group, and increased with increasing amounts of DYP and FD. The content of ornithine, the main free amino acid in Bunashimeji, was about 140% of the control in the DYP 5g and FD 5g addition group. The arginine content, which is a precursor of ornithine in the urea cycle, also increased by about 130% to about 160% compared to the control. Similarly, the aspartic acid content, which forms argininosuccinic acid together with citrulline in the urea cycle, also increased by about 130% to about 160% compared to the control. Furthermore, alanine and glutamic acid, which has a strong umami taste, also increased.

1-3. Sensory evaluation <Test specimens>
Bunashimeji mushrooms cultivated in YKB-1 medium supplemented with the control (no yeast cells added), 2.5 g of DYP, 5 g of DYP, 2.5 g of FD, and 5 g of FD, as shown in Table 2-2. <Pretreatment>
(1) Cut the Bunashimeji mushrooms about 1 cm from the base.

(2) (1) Weigh 80 g of the test specimen into a dish and cover it with plastic wrap.
(3) Heat in a microwave oven (500 W, 3 minutes).
(4) Leave the product wrapped for 30 minutes and then subject it to a sensory evaluation test.

<Sensory evaluation test>
Testers: 9-14 people (men and women in their 20s to 60s, trained panelists)
Evaluation items: sweetness, umami, bitterness, texture (crunchiness), overall evaluation (taste)
Evaluation index: Evaluation is made on a scale of ±4 levels (total of 9 levels) with the control as the standard (0). Evaluation items: Only the control is indicated, and the test areas are blinded (A, B, C, D). <Sensory evaluation results>

The yeast cells other than 2.5 g of DYP were evaluated as having a higher umami taste than the control, and the crispy texture was also felt. The overall evaluation was also high.
Example 2 Effect of adding yeast cells to Bunashimeji cultivation medium (second time)
In this example, component analysis and sensory evaluation were carried out when yeast cells were added to a Bunashimeji cultivation medium (second time).

2-1. Cultivation of Bunashimeji The composition of the YKB-1 medium used as the cultivation medium for Bunashimeji is as shown in Table 2-1, the same as in the first experiment.

<Test specimen>
The cultivation periods for Bunashimeji mushrooms were three types, 69 days, 83 days, and 97 days, as shown in Table 1-1.
The first control (no yeast cells added) and the yeast cell addition group were prepared by adding 5 g of yeast TFD and 6.7 g of BY in addition to 5 g of DYP and 5 g of FD. The amount of BY added was large because yeast cells that were not heat-treated/enzyme-treated (yeast extract was not extracted) were used.

<Cultivation process>
The results of the number of days for the fungus to grow and the yield are shown in Table 3-1.

As shown in Table 3-1, the yield of Bunashimeji grown using cultivation medium with added yeast cells was almost the same as the control, but only slightly higher. The thickness of the cap tended to be slightly thinner than the control.

2-2. Component analysis (whole Bunashimeji)
<Pretreatment> and <Analysis items and analysis method> are the same as in Example 1.
<Nutritional Information>

Regardless of the cultivation period or the type of yeast cells added, the protein content of Bunashimeji increased with the addition of yeast cells. There was a tendency for the protein content to increase slightly as the cultivation period became longer.

<Free amino acids>

The total free amino acid content increased by approximately 10% or more in the yeast cell-added group. The ornithine content and arginine content also increased by approximately 120% to approximately 160%. The aspartic acid content also increased by approximately 120% to approximately 140%. Furthermore, glutamic acid also increased by approximately 100% or more to approximately 120% compared to the control.

<5'-ribonucleic acid (as anhydrous disodium)>

At 83 and 96 days of the cultivation period, guanylic acid (GMP) and other nucleic acids also increased.
<Organic acid>

The pyroglutamic acid content increased during all cultivation periods (approximately 120% to 200% compared to the control). In particular, the acetic acid content increased on the 69th day of cultivation (approximately 120% to 200% compared to the control). In particular, the succinic acid content increased on the 97th day of cultivation (approximately 120% compared to the control).

2-3. Component analysis (umbrella/shaft analysis)
Below, component analysis was performed on the cap and stalk of Bunashimeji mushrooms grown for 97 days after addition of 5.0 g DYP or 5.0 g FD.

The "cap" is the hat-like part at the top of the mushroom, and the "stalk" is the cylindrical part attached under the mushroom cap.
<Nutrition analysis>

The addition of yeast cells increased the protein content. The protein content of DYP increased in the cap, and that of FD increased in the stalk.
<Free amino acids>

Most of the free amino acids examined were found to be more abundant in the cap than in the stalk, but only ornithine and aspartic acid were found to be more abundant in the stalk.

By adding yeast, the ornithine and arginine contents increased in both the cap and stalk. The aspartic acid content also increased in both the cap and stalk, but the DYP-added area showed an increase especially in the cap, and the FD-added area showed an increase especially in the stalk.

It was suggested that the amino acids related to the urea cycle (ornithine cycle, Figure 1), such as ornithine, arginine, and aspartic acid, were increased in the stalk compared to the cap.
<5'-ribonucleic acid (as anhydrous disodium salt)>

The cap has a higher nucleic acid content than the stalk.

In the umbrella, guanylic acid (GMP) and uridylic acid (UMP) were increased compared to controls.
Adenylic acid (AMP) increased in both the calyx and stalk.

<Organic acid>

Organic acids found in greater amounts in the cap than in the stalk: pyroglutamic acid, acetic acid, citric acid, phosphoric acid Organic acids found in greater amounts in the cap than in the stalk: lactic acid, malic acid Succinic acid

In the group in which FD was added to the stalk, the pyroglutamic acid content was increased compared to the control.
In the plot where DYP was added to the stalk, the acetic acid content increased compared to the control.
2-4. Sensory Evaluation As in "1-3. Sensory Evaluation" of Example 1, sensory evaluation was performed by 9 to 14 testers (men and women in their 20s to 60s, trained panelists) on sweetness, umami, bitterness, texture (crunchiness), and overall evaluation (taste).

The yeast-added group tended to have a stronger umami flavor. Although only slightly, the yeast-added group tended to have a more favorable overall evaluation.
Example 3: Effect of adding yeast cells to Enokitake mushroom cultivation medium (first time)
In this example, component analysis and sensory evaluation were performed when yeast cells were added to a medium for cultivating Enoki mushroom (first time).

3-1. Cultivation of Enokitake Mushrooms The composition of the YK3 medium used as the cultivation medium for Enokitake Mushrooms is shown in Table 4-1.

Cotton hulls: A mixture of husks and short fibers remaining after cotton seeds are crushed and the kernels removed. Grain sorghum: Scientific name: Sorghum bicolor. An annual plant of the Sorghum genus of the Poaceae family used as an food grain.

Corn cob: crushed corn cores Vaccine: sugar cane cake Yeast cells DYP or FD were added to the YK3 medium at the concentrations shown in Table 4-2.

"Kinoko Lime" (Shinshu Seikaken Co., Ltd.) is a mushroom culture medium conditioner.
3-2. Component analysis <Test specimen>
Enoki mushrooms were grown in YK3 medium supplemented with a control (no yeast cells added), 2.5 g of DYP, 5 g of DYP, 2.5 g of FD, or 5 g of FD, as shown in Table 4-2.

The types of Enoki mushroom used, the cultivation period, and the number of specimens are as shown in Table 1-3.
<Analysis items and analysis methods>
The same as in Example 1, <Analysis items and analysis method> for Bunashimeji.

<Pretreatment>
1) Nutritional analysis and β-glucan Enoki mushrooms were cut 2 cm above the line at the top of their base, the base of the mushroom was removed, and the cut into 1-1.5 cm pieces was used as samples for analysis.

2) Analysis of components other than β-glucan (1) Cut the enoki mushroom 2 cm above the line at the top of its base, remove the base, and cut into 1-1.5 cm pieces.

(2) Add 70 g of distilled water to 50 g of (1) and homogenize.
(3) (2) is heated and extracted for 5 minutes until it boils, and then boiled for 5 minutes.
(4) (3) is centrifuged (3000 rpm x 10 minutes).

The supernatant obtained by the above pretreatment was used as a sample for component analysis.
<Moisture>

<5'-ribonucleic acid (as anhydrous disodium)>

The content of guanylic acid (GMP), a savory component of mushrooms, increased significantly (about 120% to about 160% compared to the control).
<Organic acid>

The pyroglutamic acid content increased. In particular, in the FD-added group, it was about 250% to about 290% compared to the control. Succinic acid also increased (about 110% to about 170% compared to the control).
<Sugars and sugar alcohols>

The addition of yeast cells did not result in significant differences from the control.
<β-glucan>

The addition of yeast cells did not result in significant differences from the control.
<Free amino acids>

The content of ornithine, the main free amino acid in Enoki mushroom, increased in all yeast cell-added groups (approximately 110% to 130% compared to the control). In the urea cycle, the content of arginine, a precursor of ornithine, also increased by approximately 130% in the case of DYP5g compared to the control.

3-3. Sensory evaluation <Test specimens>
Enoki mushrooms grown in YK3 medium supplemented with the control (no yeast cells added), 2.5 g of DYP, 5 g of DYP, 2.5 g of FD, or 5 g of FD, as shown in Table 4-2. <Pretreatment>
(1) Cut the enoki mushroom 2 cm above the line at the top of its base.

(2) (1) Weigh 80 g of the test specimen into a dish and cover it with plastic wrap.
(3) Heat treatment in a microwave oven (500 W, 1 minute 20 seconds).
(4) Leave the product wrapped for 30 minutes and then subject it to a sensory evaluation test.

<Sensory evaluation test>
Testers: 12 people (men and women in their 20s to 60s, trained panelists)
Evaluation items: sweetness, umami, bitterness, texture (crunchiness), overall evaluation (taste)
Evaluation index: Evaluation is made on a scale of ±4 levels (total of 9 levels) with the control as the standard (0). Evaluation items: Only the control is indicated, and the test areas are blinded (A, B, C, D). <Sensory evaluation results>

The yeast cell-added area received positive ratings (superior to the control) for umami, bitterness (astringency), texture (crunchiness), and overall evaluation (tastiness) other than sweetness. The FD-added area tended to receive higher positive scores than the DYP-added area, and the FD 5g-added area received the highest score, including the overall evaluation.

Example 4 Effect of adding yeast cells to Enokitake mushroom cultivation medium (2nd time)
In this example, three varieties of Enoki mushroom ( variety A, variety B, variety C ) were used and component analysis and sensory evaluation were performed when yeast cells were added to the Enoki mushroom cultivation medium (second time).

4-1. Cultivation of Enokitake Mushrooms The composition of the YK3 medium used as the cultivation medium for Enokitake Mushrooms is as shown in Table 4-1, the same as in the first experiment.

<Test specimen>
Enoki mushrooms were grown in YK3 medium supplemented with a control (no yeast cells added), 5 g of DYP, or 5 g of FD, as shown in Table 4-2.

The types of Enoki mushroom used, the cultivation period, and the number of specimens are as shown in Table 1-3.
4-2. Component analysis <Pretreatment> and <Analysis items and analysis method> are the same as in Example 3.

<Nutritional ingredients>

Regardless of the cultivation period and the type of yeast cells added, the protein content of Enoki mushroom increased with the addition of yeast cells. <Free amino acids>

The total free amino acid content increased by more than 10% in the yeast cell-added areas. The ornithine content increased in all yeast cell-added areas. The free amino acids glutamic acid, arginine, alanine, tyrosine, and lysine also increased.

<5'-ribonucleic acid (as anhydrous disodium)>

In cultivars A and B , guanylic acid (GMP), adenylic acid (AMP), uridylic acid (UMP), and cytidylic acid (CMP) (i.e., nucleic acids other than inosinic acid (IMP)) were increased (about 110% to about 150% compared to the control). Cultivar C had increased CMP and UMP.

<Organic acid>

In all varieties, the pyroglutamic acid content increased (about 125% to about 160% compared to the control). In addition, several other organic acids increased in the yeast cell-added group.
Similar to "3-3. Sensory evaluation" in Example 3, sensory evaluation was performed by 9 to 13 testers (men and women in their 20s to 60s, trained panelists) on sweetness, umami, bitterness, texture (crunchiness), and overall evaluation (taste).

The yeast-added group tended to have a stronger umami and sweetness. The yeast-added group tended to have a slightly more favorable overall evaluation.
Example 5 Polyamine Analysis
In Example 1-4, the ornithine content in the yeast cell-added group of Bunashimeji increased. This suggests that the addition of yeast cells to the mushroom cultivation medium has an effect on the polyamine content. In this Example, the polyamine content of Bunashimeji was analyzed.

<Test specimen>
Bunashimeji variety: Nagano Agricultural and Industrial Research Institute NN12
Culture medium: YKB-1 medium (moisture 65%, 505g per bottle)
Control: no DYP added, DYP group: DYP 5g (1% in medium)
Culture: 89 days <Analysis method>
The edible parts, with the base of the plant removed, were analyzed using the Japan Food Research Center's HPLC method.

Analysis items: spermidine, spermine, putrescine <Analysis results>
Polyamine analysis results of Bunashimeji

Putrescine and spermidine were detected in both the control and DYP-added areas. The DYP area had higher contents than the control, with the total amount of putrescine and spermidine being approximately 145% of that in the control. This suggests that the addition of DYP increases polyamines.

Example 6 Effect of adding yeast cells to Bunashimeji cultivation medium (3rd time)
In this example, a component analysis was performed when yeast cells were added to a Bunashimeji cultivation medium (third time). In this example, the amount of yeast cells added was increased from Examples 1 and 2 to 1 wt%, 1.5 wt%, and 2 wt% in the medium.

6-1. Cultivation of Bunashimeji The composition of the YKB-1 medium used as the cultivation medium for Bunashimeji is as shown in Table 2-1, the same as in the first experiment.

The Bunashimeji cultivation period was 97 days. The cultivation was carried out under conditions of a temperature of 20° C., humidity of 50-80%, and CO ₂ of 3000 ppm, after which the following steps were carried out: scratching → germination → inhibition → growth (21 days).

The composition of the cultivation medium is as shown in Table 7-1. A control (no yeast cells added) and yeast cell-added plots containing 5 g DYP (1% by weight in the medium), 7.5 g DYP (1.5% by weight), 10 g DYP (2% by weight), 5 g FD (1% by weight in the medium), 7.5 g FD (1.5% by weight), and 10 g FD (2% by weight) were prepared.

The nutritional analysis values of the control, DYP 5g, and DYP 10g cultivation media are shown in Table 7-2.

The more yeast cells were added, the slower the mycelium growth, i.e., the rate at which the mycelium spread throughout the medium during cultivation. The number of days the mycelium remained around was 27 days for the control, 29.5 days for 5 g DYP, 33 days for 7.5 g DYP, 35 days for 10 g DYP, 28.5 days for 5 g FD, 30 days for 7.5 g FD, and 31.5 days for 10 g FD. There was no significant difference in the growth rate of the fruiting bodies.

The results, including yield, are shown in Table 7-3.

As shown in Table 7-3, the yield of Bunashimeji cultivated using the cultivation medium to which yeast cells had been added was almost the same as that of the control, but only slightly higher.
6-2. Component analysis (whole Bunashimeji)
<Pretreatment> and <Analysis items and analysis method> are the same as in Example 1.

<Nutritional ingredients>

The protein content in Shimeji increased with increasing amounts of yeast cells added. The increase in protein content in Shimeji correlates with the increase in protein content in the culture medium.
<Free amino acids>

By increasing the amount of yeast cells added, the total content of the 20 major free amino acids increased by approximately 10% or more. When 7.5g and 10g of DYP were added, the content increased to approximately 140%, but reached a plateau at 10g of DYP. When 5g, 7.5g, and 10g of FD were added, the content increased according to the amount added, reaching approximately 130% at 10g of FD.

By increasing the amount of DYP and FD added, many amino acids increased. The changes tended to be greater for amino acids that were originally present in large amounts, such as glutamine, glutamic acid, ornithine, and arginine. In terms of relative values, with the control set at 100, the following amino acids in particular increased.

Aspartic acid: about 95 to about 194
Glutamine: about 137 to about 294
Threonine: about 124 to about 212
Arginine: about 126 to about 190
Ornithine: about 120 to about 143
Ornithine was highest with DYP 7.5g, and tended to decrease with DYP 10g (same trend in both cultivation tests). With FD added, it was highest with FD 10g. The ornithine content of commercially available Bunashimeji is about 70-150mg/100g (listed on the packaging of Shimeji mushrooms made by Hokuto Co.), but it was confirmed that the ornithine content of Bunashimeji mushrooms containing DYP and FD greatly exceeded this, reaching 200mg/100g or more.

<Sugar>

As for sugars, trehalose was the most abundant, with the control containing about 1%, but this content decreased as the amount of DYP and FD added increased.
<Organic acid>

With the addition of DYP or FD, the pyroglutamic acid content increased (from about 254% to about 925% compared to the control).
<5'-ribonucleic acid (as anhydrous disodium salt)>

Example 7 Effect of adding yeast cells to the Enokitake mushroom cultivation medium (3rd time)
In this example, a component analysis was performed when yeast cells were added to the Enokitake mushroom cultivation medium (third time). In this example, the amount of yeast cells added was increased from Examples 3 and 4 to 0.9 wt%, 1.3 wt%, and 1.8 wt% of the medium.

7-1. Cultivation of Enokitake Mushrooms The composition of the YK-3 medium used as the cultivation medium for Enokitake Mushrooms is as shown in Table 4-1, the same as in the first experiment.

The cultivation period for Enoki mushroom was 20 days. The cultivation was carried out under the conditions of a temperature of 17 to 21°C and a humidity of 70±5%, and then the following steps were carried out: scratching → germination → inhibition → fruit body growth.
The composition of the culture medium is as shown in Table 8-1. A control (no yeast cells added) and yeast cell-added plots containing 5 g DYP (0.9% by weight in the medium), 7.5 g DYP (1.3% by weight), 10 g DYP (1.8% by weight), 5 g FD (0.9% by weight in the medium), 7.5 g FD (1.3% by weight), and 10 g FD (1.8% by weight) were prepared.

The nutritional analysis values of the control, DYP 5g, and DYP 10g cultivation media are shown in Table 8-2.

The results, including yield, are shown in Table 8-3.

As shown in Table 8-3, the yield of Enoki mushrooms grown using the culture medium containing added yeast cells was almost the same as that of the control, but only slightly higher.
7-2. Component analysis (whole Enoki mushroom)
<Pretreatment> and <Analysis items and analysis method> were the same as in Example 3.

<Nutritional ingredients>

The protein content in the Enoki mushroom increased with increasing amounts of DYP added. In the case of FD, no increase in protein content was observed depending on the amount added.
<Free amino acids>

As the amount of yeast cells added increased, the total content of the 20 major free amino acids increased. When DYP was added, the total increased from about 110% to about 117%. When FD was added, the total increased from about 105% to about 112%.

By increasing the amount of DYP and FD added, many amino acids increased. There was a tendency for the changes to be greater for amino acids that were originally present in large amounts, such as glutamine, alanine, phenylalanine, and lysine. The relative values, with the control set at 100, are as follows.

Glutamine: about 109 to about 137
Alanine: about 101 to about 118
Phenylalanine: about 113 to about 148
Lysine: about 116 to about 134
Valine, isoleucine, proline, arginine, etc. showed little change. Ornithine and aspartic acid, which are involved in the ornithine cycle, decreased with increasing amounts of yeast cells.

GABA (gamma-aminobutyric acid) stagnated at DYP 5g and FD 5g, then rose at DYP 7.5g, DYP 10g, and FD 7.5g. It then rose again at FD 10g. It was highest at DYP 10g, with a relative value of 124 when the control was taken as 100.

<Sugar>

As for sugars, trehalose was the most abundant, with the control containing about 0.61%, but this content decreased as the amount of DYP and FD added increased.
<Organic acid>

The addition of DY increased the pyroglutamic acid content (about 106% to about 170% compared to the control). The addition of FD decreased the pyroglutamic acid content.
<5'-ribonucleic acid (as anhydrous disodium salt)>

The addition of DYP and FD increased adenylic acid (AMP), the most abundant ribonucleic acid (RNA), (approximately 114% to approximately 130% compared to the control).
Example 8 Effect of adding yeast cells to Bunashimeji cultivation medium: Capillary column electrophoresis mass spectrometry (CE-MS analysis)
In this example, DYP was added to the Bunashimeji cultivation medium at 1% by weight of the medium in the same manner as in Examples 1 and 2. Capillary column electrophoresis mass spectrometry (CE-MS analysis) was performed on a liquid obtained by extracting cultivated Bunashimeji with hot water, and the detected functional components were examined.

The composition of the YKB-1 medium used as the cultivation medium for Bunashimeji is as shown in Table 2-1, the same as in the first experiment. The composition of the cultivation medium is the same as in the case of DYP5g in Table 2-2 (505g per bottle).

Bunashimeji mushrooms were cultivated under three conditions of 69 days, 83 days, and 97 days as shown in Table 1-1. Cultivation was carried out under conditions of a temperature of 20°C, humidity of 50-80%, and _CO2 of 3000 ppm. After that, the mushrooms were scratched, sprouted, inhibited, and then grown.

8-2. Capillary column electrophoresis mass spectrometry (CE-MS analysis)
The edible parts of the harvested Bunashimeji were cut, hydrated (25 g of shimeji, 35 g of water), homogenized, and then heated in a hot water bath at a temperature of 95°C or higher for 6 minutes for extraction. The centrifuged liquid was then subjected to analysis.

The CE-MS analysis was outsourced to Professor Akira Oikawa of the Faculty of Agriculture, Yamagata University. The following equipment was used:
Agilent G7100A CE Instrument (Agilent Technologies, Sacramento, CA),
Agilent G6224A TOF LC/MS system,
Agilent 1200 Infinity series G1311C Quad Pump VL,
G1603A Agilent CE-MS adapter, and G1607A Agilent CE-ESI-MS sprayer kit
In addition, G1601BA 3D-CE ChemStation software for CE and G3335-64002 MH Workstation were used.

Separation was performed using a fused silica capillary (50 μm inner diameter x 100 cm total length) with 1 M formic acid for cation analysis or 20 mM ammonium formate (pH 10.0) for anion analysis as the electrolyte. The capillary temperature was maintained at 20°C. Sample solutions were injected at 50 mbar for 15 seconds (15 nl). ESI-TOFMS analysis of cation ions and anion ions was performed with an applied voltage of 30 kV. Substances that tend to increase with the addition of DYP are summarized in Table 9-1 based on the relative ratio of the peak areas of the detected and identified functional components between the DYP-added and control.

Glutamine: 30-50% of muscle-forming amino acids, involved in muscle protein synthesis. Prevents muscle breakdown after strenuous exercise, speeds up recovery from muscle fatigue. Enhances immunity, protects gastric mucosa.
Arginine: Strengthens muscles by promoting the secretion of growth hormone, improves blood flow, reduces the risk of lifestyle-related diseases, and strengthens the immune system;
Ornithine: Promotes ammonia metabolism, promotes growth hormone secretion to build muscle, maintains muscle mass, strengthens the immune system, and improves sleep quality;
Citrulline: Vasodilator, reduces fatigue, improves athletic performance, reduces oxidative stress;
Spermidine: polyamine, activates new metabolism, improves colon barrier function, has anti-inflammatory and anti-allergic effects, stabilizes DNA and inhibits mutations, and acts as an anti-aging agent;
Spermine: polyamine, activates new metabolism, improves colon barrier function, has anti-inflammatory and anti-allergic effects, stabilizes DNA and inhibits mutations, and acts as an anti-aging agent;
S-Adenosylmethionine (SAM): Supports neurological function and liver detoxification;
Pyroglutamic acid: Improves memory and thinking ability, treats brain damage caused by alcohol, a functional ingredient in agaric;
Choline: raw material for acetylcholine (converted into acetylcholine in the brain), acetylcholine is a neurotransmitter that plays a role in memory and information transmission;
Trigonelline: Promotes nitric oxide production in endothelial cells to keep blood vessels flexible;
Hydroxyproline: contributes to the stabilization of collagen structure in the skin (it is abundant in the skin);
β-alanine: A raw material for carnosine, reduces neuromuscular fatigue, improves endurance, and is a raw material for pantothenic acid.

Detailed data on each substance is provided below.
Effects of DYP on the amino acid metabolism in the ornithine and polyamine cycles

In particular, the increases in arginine, citrulline, spermidine, spermine, and SAM were significant. Other increases included aspartic acid.
Effect of DYP on amino acid metabolism in the citric acid cycle

In particular, there was a significant increase in glutamine, arginine, ornithine, citrulline, and pyroglutamic acid. Aspartic acid and other substances also increased.
Effect of DYP on substances involved in nucleic acid synthesis

As substances related to the nucleic acid synthesis system, nucleic acids and their decomposition products were analyzed. In particular, there was a significant increase in β-alanine and glutamine, which are decomposition products of nucleic acids. In addition, allantoic acid, AMP, GMP, etc. also increased.

Example 9 Effect of Adding Yeast Cells to Enokitake Mushroom Cultivation Medium: Analysis of Ergothioneine by Capillary Column Electrophoresis-Mass Spectrometry (CE-MS Analysis) In this example, capillary column electrophoresis-mass spectrometry (CE-MS analysis) was carried out to examine ergothioneine for a hot water extract of Enokitake mushrooms cultivated in the same manner as in Example 7. The amount of yeast cells added was 1 wt %, 1.5 wt %, and 2 wt % of the medium, as in Example 7.

The CE-MS analysis was outsourced to Professor Akira Oikawa of the Faculty of Agriculture, Yamagata University, as in Example 8. The instruments used were an Agilent G7100A CE Instrument (Agilent Technologies, Sacramento, CA), an Agilent G6224A TOF LC/MS system, an Agilent 1200 Infinity series G1311C Quad Pump VL, an Agilent CE-MS adapter G1603A, and an Agilent CE-ESI-MS sprayer kit G1607A, and the analysis was performed in the same manner as in Example 8.

The results are shown in Table 10-1.

The addition of DYP and FD increased the ergothioneine content in Enoki mushrooms. When DYP was added, the content increased depending on the amount added, ranging from about 111% to about 129% compared to the control. When FD was added, the content increased to about 110% compared to the control, regardless of the amount added.

Example 10: Effect of stalk length cultivation on organic acid content In this example, a cultivation method (stalk length cultivation) was attempted in which the stalk of Bunashimeji is lengthened by wrapping it in paper during fruit body growth, similar to that of Enokitake. From the results of Example 2, the amino acid content related to the urea cycle (ornithine cycle, Figure 1), such as ornithine and arginine, in the stalk was increased. Based on this, this example was carried out on the hypothesis that these amino acids would increase by lengthening the growth of the stalk in the same way as Enokitake. In this example, the amount of yeast cells added was 1% or 2% by weight of the medium. The composition of the YKB-1 medium used as the cultivation medium for Bunashimeji is as shown in Table 2-1, the same as in the first experiment.

The composition of the cultivation medium was the same as in Table 7-1, and a control (no yeast cells added) and yeast cell-added areas containing 5 g of DYP (1% by weight in the medium) and 10 g of DYP (2% by weight) were prepared.

The Bunashimeji cultivation period was 97 days. Cultivation was carried out under conditions of 20°C temperature, 50-80% humidity, and 3000 _ppm CO2, after which the following steps were carried out: scratching → germination → inhibition → growth (21 days). During this growth period, the fruiting bodies of the Bunashimeji were wrapped in paper. This allows the Bunashimeji stalks to grow longer than when they are not wrapped in paper.

The results of yield etc. for Control 1 (normal cultivation, no DYP added), Control 2 (long-stem cultivation, no DYP added), DYP5g (long-stem cultivation, 5g DYP added), and DYP10 (long-stem cultivation, 10g DYP added) are shown in Table 11-1.

By cultivating with long stalks, the ratio of stalks increases, i.e. the fruiting bodies grow longer. On the other hand, as shown in the results of Example 7, etc., the weight of one plant increases with the addition of DYP. Even with long stalk cultivation, the ratio of stalks decreased when the amount of DYP added was increased. When the ratio of stalks was low, the fruiting bodies had a strong ability to spread, and the wrapping paper tended to come off.

Table 11-2 shows the nutritional analysis values for each of the following: Control 1 (normal cultivation, no DYP added), Control 2 (long-stem cultivation, no DYP added), DYP5g (long-stem cultivation, 5g DYP added), and DYP10 (long-stem cultivation, 10g DYP added).

The results in Table 11-2 and Table 7-4 are considered together. Comparing Control 1 and Control 2, protein decreases with stalk length cultivation. Protein increases depending on the amount of DYP added, but when combined with stalk length cultivation, the degree of protein increase decreases with the addition of DYP.

The results of investigating the content of free amino acids (18 types) are shown in Table 11-3.

With only elongated cultivation, the total content of the 18 major free amino acids was almost the same as the control (normal cultivation). On the other hand, as the amount of DYP added increased, the total content of the 18 major free amino acids increased. When the addition of DYP was combined with elongated cultivation, the free amino acid content increased further. With the combination of elongated cultivation and DYP, the major free amino acid content was approximately 109% to 143% compared to normal cultivation and elongated cultivation alone.

The ornithine content increased with long-stem cultivation. It also increased with the addition of DYP. It increased further with the combination of long-stem cultivation and DYP addition. The combination of DYP10 (addition of 2% by weight) and long-stem cultivation resulted in 240 mg/100 g, the highest value among the examples in this specification.

The arginine content in the case of only the long-span cultivation was almost the same as in the control (normal cultivation). On the other hand, as the amount of DYP added increased, the arginine content increased. When the addition of DYP was combined with the long-span cultivation, the arginine content increased further.

Claims (22)

A medium additive for mushroom cultivation comprising yeast cells,
Ornithine content of mushrooms; and
(1) the content of at least one amino acid selected from the group consisting of arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
and,
A content of at least one component selected from the group consisting of the following (2) to (5): (2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP), and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) choline; and/or (5) increasing the content of at least one component of trigonelline;
Culture medium additive for mushroom cultivation. The medium additive for mushroom cultivation according to claim 1, which increases the content of γ-aminobutyric acid and/or ergothioneine. 1. The method according to claim 1, which increases the content of guanylic acid (GMP) and/or adenylic acid (AMP).
The medium additive for mushroom cultivation described in claim 1. A medium additive for mushroom cultivation according to any one of claims 1 to 3, which increases the content of components from two or more groups of substances (2) to (5). The medium additive for mushroom cultivation according to any one of claims 1 to 4, which increases the content of at least one of the ornithine, the amino acid (1), or (2) to (5) by 120% or more compared to the case where the additive is not added. The medium additive for mushroom cultivation according to any one of claims 1 to 5 further increases the umami taste of mushrooms. The medium additive for mushroom cultivation according to any one of claims 1 to 6, wherein the yeast cells are yeast cells whose components have been released outside the yeast cells by subjecting the yeast cells to heat treatment and/or enzyme treatment. The medium additive for mushroom cultivation according to any one of claims 1 to 7, wherein the mushroom is selected from edible mushrooms consisting of Bunashimeji, Enokitake, Honshimeji, Hatakeshimeji, Pleurotus oyster, Himematsutake, Shiitake, mushroom, Porcini, King Oyster Mushroom, Matsutake, truffle, Nameko, Tamogitake and Bakamatsutake. The medium additive for mushroom cultivation according to any one of claims 1 to 7, wherein the mushroom is Bunashimeji or Enokitake. In addition, the following:
(i) increasing the umami flavor of mushrooms;
(ii) increasing the content of polyamines in mushrooms;
(iii) increasing the protein content in mushrooms; and (iv) not increasing mushroom yield;
The medium additive for mushroom cultivation according to any one of claims 1 to 9, which satisfies one or more requirements selected from the group consisting of: Ornithine content of mushrooms; and
(1) the content of at least one amino acid selected from the group consisting of arginine, glutamic acid, aspartic acid, alanine, tyrosine, lysine, pyroglutamic acid, glutamine, citrulline, S-adenosylmethionine, hydroxyproline, γ-aminobutyric acid, and ergothioneine;
and,
A content of at least one component selected from the group consisting of the following (2) to (5): (2) a nucleic acid selected from the group consisting of guanylic acid (GMP), adenylic acid (AMP), and uridylic acid (UMP);
(3) a polyamine selected from the group consisting of spermidine, spermine and putrescine;
(4) choline; and/or (5) trigonelline,
The method further comprises adding yeast cells to a medium for growing mushrooms. The method according to claim 11, wherein the content of gamma-aminobutyric acid and/or ergothioneine is increased. The method according to claim 11, which increases the content of guanylic acid (GMP) and/or adenylic acid (AMP). The method according to any one of claims 11-13, in which the content of components from two or more groups of substances (2)-(5) is increased. The method according to any one of claims 11 to 14, in which the content of at least one of the ornithine, the amino acid (1), or (2) to (5) is increased to 120% or more compared to the case in which no additive is added. The method according to any one of claims 11 to 15, further comprising increasing the umami taste of mushrooms. The method according to any one of claims 11 to 16, wherein the yeast cells are yeast cells whose components have been released outside the yeast cells by subjecting the yeast cells to heat treatment and/or enzyme treatment. The method according to any one of claims 11 to 17, wherein the mushroom is selected from edible mushrooms consisting of Bunashimeji, Enokitake, Honshimeji, Hatakeshimeji, Pleurotus oyster, Himematsutake, Shiitake, mushroom, Porcini, King Oyster Mushroom, Matsutake, truffle, Nameko, Tamogitake and Bakamatsutake. The method according to any one of claims 11 to 18, wherein the mushroom is Bunashimeji or Enokitake. In addition, the following:
(i) increasing the umami flavor of mushrooms;
(ii) increasing the content of polyamines in mushrooms;
(iii) increasing the protein content in mushrooms; and (iv) not increasing mushroom yield;
The method according to any one of claims 11 to 19, further comprising one or more effects selected from the group consisting of: The method according to any one of claims 11 to 20, further comprising carrying out stalk length cultivation during fruit body growth. The method according to claim 21, wherein the content of ornithine and/or arginine is increased.