JP7710862B2 - Solid fermentation product of Aspergillus oryzae containing high ergothioneine content and method for producing the same - Google Patents

Description

The present disclosure relates to a method for producing a solid-state fermentation product of koji mold with a high content of ergothioneine (hereinafter sometimes referred to as ERG), and to the technical field of a solid-state fermentation product of koji mold with a high content of ERG produced by the production method.

ERG is a sulfur-containing amino acid with a strong antioxidant effect, and has been found to exist in the living bodies of plants and animals. ERG has been reported to have not only a strong antioxidant effect, but also an elastase inhibitory effect and a tyrosinase inhibitory effect, and has been attracting particular attention in the beauty and food fields, such as skin whitening and wrinkle prevention. It has also been found that ERG is involved in the biological oxidation defense system, and applications in the medical field are also being attempted. Recently, ERG has been attracting attention in relation to longevity and mild dementia disorders, for example, because the ERG content decreases with age and ERG has an effect of mitigating the reduction of telomere length under oxidative stress. 5 mg/day of ERG is required to exert the effect of improving cognitive function in mild dementia disorders (see, for example, Non-Patent Document 1).

Plants and animals cannot synthesize ERG, and it is believed that ERG in the body originates from ERG synthesized by microorganisms such as basidiomycetes. ERG is also found in some edible mushrooms of basidiomycetes, such as enokitake, oyster mushroom, shiitake, maitake, king oyster mushroom, and mushroom, and is known to be found in particularly large amounts in Tamogitake. Foods other than mushrooms do not contain sufficient amounts of ERG to be effective. It is known that Aspergillus oryzae can also produce it, but in order to ingest 5 mg of ERG per day, it is necessary to ingest more than 100 g of commercially available koji per day.

Methods for producing ERG have been attempted, including extraction from basidiomycetes such as Pleurotus cornucopiae, chemical synthesis, and fermentation using microorganisms. Extraction from basidiomycetes such as Pleurotus cornucopiae takes time to obtain the raw materials, and is not suitable for mass production. In order to obtain ERG in large quantities, research has been conducted on fermentation using bacteria or yeast that can assimilate C1 compounds (Patent Document 1: International Publication No. 2016/104437), and fermentation using microorganisms that overexpress ERG biosynthesis genes (Patent Document 2: International Publication No. 2017/150304). Solid culture using Aspergillus oryzae that overexpresses ERG biosynthesis genes has also been attempted, but the production volume was 231 mg/kg (Non-Patent Document 2).

However, the ERG-producing microbial strains identified in this way are not normally used in food, and when gene-introduced microorganisms are used, their use in food is limited. Therefore, considering application to a variety of foods, there is a demand for non-recombinant strains that have been consumed and have high ERG productivity.

International Publication No. 2016/104437 International Publication No. 2017/150304

Pharmacology and Therapy Volume 48, Issue 4, 685-697 (2020) BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, 2019, VOL. 83, NO. 1, 181‐184

The aim is to increase the ERG content in food and provide a method for sufficient intake of ERG from foods other than mushrooms.

The present inventors conducted intensive research in order to provide a food product with a high ERG content, and discovered that the ERG content can be increased by producing a solid fermentation product under specific conditions, thereby arriving at the present invention.
Thus, the present invention relates to:
[1] A solid-fermentation product of Aspergillus oryzae containing 0.35 g or more ergothioneine per kg of solid-fermentation product of Aspergillus oryzae.
[2] A method for producing a solid-fermentation product of koji mold having a high ergothioneine content of 0.35 g/kg of the solid-fermentation product of koji mold, comprising a step of mixing and culturing a solid medium containing 10% or more of protein with koji mold, wherein the method is characterized in that the fermentation is carried out for 4 days or more.
[3]
3. The method according to item 2, wherein water is not added during fermentation and the initial moisture content of the mixture of the solid medium and the koji mold is adjusted to 45% or more.
[4] The method according to item 2 or 3, wherein the moisture content of the mixture during fermentation is maintained at 25% or more.
[5] The method according to any one of items 2 to 4, wherein the fermentation step comprises fermenting at 20 to 40° C.
[6] A solid-fermentation product of an aspergillus oryzae having a high ergothioneine content, produced by the production method according to any one of items 2 to 5.

According to the present disclosure, it is possible to provide foods in which the ERG content has been increased through fermentation.

FIG. 1 is a graph showing the change in ERG content versus the number of days of fermentation when fermentation was performed using Aspergillus oryzae under fermentation conditions that result in a high ERG content in the Aspergillus oryzae solid fermentation product. FIG. 2 is a graph showing the change in water content of the solid fermentation product of koji mold during fermentation. FIG. 3 shows the ERG content in solid fermentation products of koji mold after fermentation for 7 days under specified conditions using koji mold strains NISL2180, RIB326, RIB646, RIB1370, RIB408, RIB40, and NBRC4239. FIG. 4 shows the water content in solid fermentation products of koji mold strains NISL2180, RIB326, RIB646, RIB1370, RIB408, RIB40, and NBRC4239 after fermentation for 7 days under specified conditions.

<Aspergillus oryzae>
As the koji mold, any fungus belonging to the genus Aspergillus can be used. Examples include Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus luchuensis, and Aspergillus tamarii. As the koji mold strain, for example, a publicly available strain such as Aspergillus oryzae RIB326 strain, a strain contained in koji seed commercially available from koji seed makers, or a strain obtained by isolating from a food and beverage production environment such as sake or soy sauce breweries can be used.

As the koji mold, a wild-type strain may be used, or a mutant strain with high ERG production may be used using a general mutation introduction method. Examples of the mutation introduction method include ultraviolet (UV) or X-ray irradiation, which physically damages DNA and introduces mutations, and treatment with alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), which chemically damages DNA and introduces mutations. From the viewpoint of producing food, it is preferable to use a non-recombinant strain. The koji mold may be koji mold cultured in a medium, or koji mold cultured and sufficiently allowed to attach spores and then dried. It may be dried together with the raw material, or only the spores may be collected.

<Method for increasing ERG content>
The method for increasing ERG content according to one embodiment of the present disclosure means a method for solid fermentation of koji mold under fermentation conditions that increase the ERG content in the koji mold solid fermentation product, as described below in detail. The method for increasing ERG content means a method for producing a koji mold solid fermentation product having an increased ERG content compared to a koji mold solid fermentation product produced using raw materials and normal fermentation conditions. In the method for solid fermentation of koji mold with high ERG content, the ERG content is increased by 5 times or more, preferably 10 times or more, more preferably 20 times or more, even more preferably 50 times or more, and even more preferably 100 times or more, compared to normal fermentation conditions, for example, when the koji mold solid fermentation product is fermented with a moisture content of 25% or less. As an example, the ERG-rich koji mold solid fermentation product contains ERG of 0.35 g/kg or more of koji mold solid fermentation product, preferably 0.4 g/kg or more of koji mold solid fermentation product, more preferably 0.5 g/kg or more of koji mold solid fermentation product, even more preferably 0.8 g/kg or more of koji mold solid fermentation product, and even more preferably 1.0 g/kg or more of koji mold solid fermentation product. The ERG contained in the ERG-rich koji mold solid fermentation product of the present disclosure is produced by koji mold and is not added or concentrated. The ERG-rich koji mold solid fermentation product is identified only by its production process, and it is impossible or impractical to identify the produced ERG-rich koji mold solid fermentation product by its ingredients or characteristics.

In order to obtain a high content of ERG in the solid fermented product of koji mold, it is important to control the initial moisture content of the mixture of the solid medium and koji mold and the moisture content during fermentation for 4 days or more. More specifically, it is necessary to maintain the moisture content of the mixture of the solid medium and koji mold during fermentation at 25% or more even after the 4th day. If the moisture content of the mixture of the solid medium and koji mold during fermentation is less than 25%, the ERG content in the solid fermented product of koji mold cannot be high. Therefore, in the method for producing a solid fermented product of koji mold with a high content of ERG according to one embodiment of the present disclosure, the moisture content in the mixture is maintained at 25% or more throughout the fermentation process in the process of mixing the solid medium and koji mold and fermenting it. When producing foods such as soy sauce, miso, and sake, water is usually not added during fermentation, and koji production is not performed for more than 4 days. In addition, heat may be generated at the beginning of fermentation, and moisture may be lost rapidly. Therefore, when the fermentation period and process are taken into consideration, if the initial moisture content of the mixture of solid medium and koji mold at the start of culture is less than 45%, it becomes difficult to maintain the moisture content of the fermented product at 25% or more during fermentation ( FIG. 2 ). Therefore, the method for producing a solid fermented product of koji mold with high ERG content according to one embodiment of the present disclosure may include a step of adjusting the initial moisture content of the mixture of solid medium and koji mold to 45% or more.

The ERG-rich koji mold solid fermentation product is prepared by fermenting a normal solid medium for culturing koji mold. Such a solid medium is preferably a solid medium containing 10% or more protein in the solid content. As raw materials for such a solid medium, beans, fish, meat, algae, and processed products thereof containing 20% or more protein can be used. Examples of processed products containing 20% or more protein include defatted soybeans, fish meal, meat powder, and spirulina powder. It is more preferable that the solid medium of the present invention contains raw materials that have been puffed. The solid medium can be prepared by steaming or boiling the above-mentioned raw materials in boiling water and, if necessary, crushing them.

Other fermentation conditions may be those generally used in the technical field. For example, the initial pH of the medium is adjusted to 5 to 10. The fermentation temperature is set to 20 to 40°C, and the fermentation time is 4 days or more, preferably 4 to 10 days, preferably 5 to 7 days, and more preferably 6 to 7 days. From the viewpoint of maintaining the moisture content of the solid fermentation product of koji mold during fermentation, it is preferable to culture under high humidity, and the humidity can be adjusted to control the moisture value of the solid fermentation product of koji mold. In addition, since the fermentation is a long-term fermentation of 4 days or more and involves a high moisture content, it is preferable to carry out the fermentation in an environment that prevents the proliferation of miscellaneous bacteria. A container that allows for aseptic culture, such as a Yamazaki-type koji-making device or a drum koji-making device, may be used.

As an example, when using Aspergillus oryzae RIB326, a representative strain of koji mold, and producing an ERG-rich koji mold solid fermentation product using the production method of the present invention, it is possible to produce an ERG-rich koji mold solid fermentation product containing 0.35 g/kg or more of ERG per kg of koji mold solid fermentation product, preferably 0.4 g/kg or more of ERG per kg of koji mold solid fermentation product, more preferably 0.5 g/kg or more of ERG per kg of koji mold solid fermentation product, even more preferably 0.8 g/kg or more of ERG per kg of koji mold solid fermentation product, and even more preferably 1.0 g/kg or more of ERG per kg of koji mold solid fermentation product (FIG. 1).

ERG is known to have various effects such as an antioxidant effect, an elastase inhibitory effect, a tyrosinase inhibitory effect, and a polyphenol oxidase (PPO) inhibitory effect. Therefore, the solid fermentation product of ERG-rich koji mold can be made into a functional food. Such functional claims can display functions such as an anti-wrinkle effect based on the elastase inhibitory effect of ERG, a skin-beautifying or whitening effect based on tyrosinase inhibitory activity, a lifestyle-related disease prevention effect based on the generation of lipid peroxides, and a dementia or Alzheimer's disease prevention effect based on the removal of active oxygen. In yet another embodiment, a composition for anti-wrinkle or whitening, or a composition for preventing lifestyle-related diseases, or dementia or Alzheimer's disease, containing the solid fermentation product of ERG-rich koji mold is provided. In addition, the solid fermentation product of ERG-rich koji mold may be used as a raw material or a fermenting raw material, and further used in the production of seasonings that are fermented using lactic acid bacteria and yeast. Furthermore, the solid fermentation product of ERG-rich koji mold can be used as a raw material for pet food, ornamental fish feed, and as a raw material for meat substitutes.

<Method of ERG analysis>
The method of extracting ERG from the ERG-rich koji mold solid fermentation product after fermentation can be carried out using a method well known in the art. The extraction solvent is not particularly limited as long as it dissolves ERG, and examples of the extraction solvent include organic solvents such as methanol, ethanol, isopropanol, and acetone; aqueous organic solvents obtained by mixing these organic solvents with water; water, warm water, and hot water. After adding the solvent, ERG can be extracted while performing a crushing treatment as appropriate. The extraction solvent temperature can be set to room temperature to 100°C.

One embodiment of the method for extracting ERG is, for example, a method in which a solid fermentation product of Aspergillus oryzae with a high ERG content is added to water to prepare a suspension, and the resulting suspension is then heated at 98 to 100°C for 15 minutes, for example, and then centrifuged to recover the supernatant, which is then filtered to remove insoluble matter. The heat-treated suspension may also be filtered without being centrifuged.

In place of the above-mentioned heating treatment, the cells may be subjected to a disruption treatment such as a method of disrupting the cells using a disrupting means such as an ultrasonic disrupter, French press, Dynomill, or mortar; a method of dissolving the cell walls of the cells using a cell wall-dissolving enzyme such as Yatalase; or a method of dissolving the cells using a surfactant such as SDS or Triton X-100. These methods may be used alone or in combination.

The resulting extract can be subjected to purification processes such as centrifugation, filter filtration, ultrafiltration, gel filtration, separation by solubility difference, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography, reverse phase chromatography, etc.), crystallization, activated carbon treatment, membrane treatment, etc. to purify ERG.

Qualitative or quantitative analysis of ERG is not particularly limited and can be performed, for example, by HPLC. HPLC separation conditions can be appropriately selected by a person skilled in the art, and can be performed, for example, under the conditions described in the examples described below.

All references cited in this disclosure are incorporated herein by reference in their entirety. In this disclosure, % means % by weight unless otherwise specified.

The following examples of the present invention are for illustrative purposes only and are not intended to limit the scope of the present invention. The scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of the constituent elements of the present invention, may be made without departing from the spirit of the present invention.

Example 1: Production of ERG-rich koji mold solid fermentation product using defatted soybeans as a medium 640 g of puffed defatted soybeans (protein content about 48%) were placed in a vinyl bag, and 480 ml of boiling water was added and steamed (protein content of solid medium after adding hot water and steaming about 27%). After cooling to room temperature, 2.4 g of Aspergillus oryzae RIB326 strain seed culture (wheat bran culture) was added and thoroughly stirred, and the mixture was placed evenly on a plate cover. Fermentation was performed at 95% humidity and 32°C. When the temperature of the koji mold solid fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was performed for 7 days to obtain an ERG-rich koji mold solid fermentation product. After the start of fermentation, the koji mold solid fermentation product was weighed on the 1st, 3rd, 4th, and 7th days, and the moisture content was calculated (Figure 2). In addition, portions of the solid fermentation product of the koji mold were collected on the 1st, 2nd, 3rd, and 6th days after the start of fermentation.

Example 2: ERG Analysis Method (1) Extraction of ERG 5 g of ERG-rich koji mold solid fermentation product was weighed out, 75% ethanol was added, the ERG-rich fermentation product was crushed, and left at room temperature for one day to extract ERG from the ERG-rich koji mold solid fermentation product. The ERG content was measured under the following analysis conditions, and the ERG content in 1 kg of ERG-rich koji mold solid fermentation product was 1.1 g. Similarly, the ERG content was measured for the fermentation products on the first, second, third, and sixth days after the start of fermentation, and the results are shown in a graph (Figure 1).
As a reference example, ERG was similarly extracted from Miyako Koji Square Type (manufactured by Isesosha Co., Ltd.), Kome Koji H (manufactured by Kose Foods Co., Ltd.), and Kome Koji S (manufactured by Kose Foods Co., Ltd.), and the ERG content was measured under the following analytical conditions. The ergothioneine content of each was as follows:

(2) LCMS analysis conditions analyzer: UPLC CQ micro; Waters
UPLC
Column: 2.5 HILIC 3.0 mm L.D. x 150 mm
Solvent A: Acetonitrile Solvent B: 5 mM ammonium acetate/ _H2O
Flow rate: 0.5 ml/min 80% solvent A
Inject: 2μL
Mass spectrometer ESI: ES+
Cone V: 21V
Capillary V: 4.5kV
Source temperature: 120℃
Desolvation temperature: 400℃
MS Scan Mode

(3) Ergothioneine analysis conditions by LC-MS/MS Analytical equipment: UPLC CQ micro; Waters
UPLC
Column: 2.5 HILIC 3.0 mml. D. x 150 mm
Solvent A: 0.1% formic acid/acetonitrile Solvent B: 0.1% formic acid/ _H2O
Flow rate: 0.5ml/min 80%A
Inject: 2μL
Mass spectrometer ESI: ES+
Cone V: 21V
Capillary V: 4.5kV
Source temperature: 120℃
Desolvation temperature: 400℃
Collision energy: 11V
Trace: m/z 230.1>186.1
Collision energy: 20V
Trace: m/z 230.1>127.0

Example 3: Comparison of ERG production of each koji mold 640g of defatted soybeans (protein content about 48%) that had been expanded were placed in a plastic bag, and 480ml of boiling water was added and steamed (protein content of solid medium after adding hot water and steaming was about 27%, initial water content was 47%). After cooling to room temperature, 2.4g of seed culture (wheat bran culture) of Aspergillus oryzae NISL2180 strain, RIB326 strain, RIB646 strain, RIB1370 strain, RIB408 strain, RIB40 strain, and Aspergillus sojae NBRC4239 strain was added and stirred well, and the mixture was placed evenly on a plate cover. Fermentation was performed at 95% humidity and 32°C, and when the temperature of the koji mold solid fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was performed for 7 days to obtain a koji mold solid fermentation product with high ERG content. The ERG content of the fermented products after 7 days of fermentation was measured by the same analytical method as in Example 2, and the ERG production amount for each strain was compared (Figure 3). In addition, the moisture content of the culture after 7 days of fermentation was also measured, and the moisture content of the fermented products for each strain was compared (Figure 4).

Example 4: Production of ERG-rich koji mold solid fermentation product in a medium containing a mixture of defatted soybeans and wheat. 640 g of a mixture of equal amounts of expanded defatted soybeans and crushed wheat (protein content: 25.2%) was placed in a plastic bag, to which 480 ml of boiling water was added and steamed (protein content of the solid medium after adding hot water and steaming: 14.4%, initial water content: 47%). After cooling to room temperature, 2.4 g of a seed culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added, stirred thoroughly, and spread evenly on a wooden lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji mold solid fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was continued for 7 days to obtain a koji mold solid fermentation product with a high ERG content. Extraction and ERG analysis were carried out in the same manner as in Example 2. The ERG content was 0.42 g/kg of koji mold solid fermentation product.

Example 5: Production of ERG-rich koji mold solid fermentation product using fish meal as a raw material 27.8 g of 60% fish meal (Izukawa Feed Co., Ltd., protein content 60% or more), 30.4 g of crushed wheat, and 41.7 g of water were mixed thoroughly in a vinyl bag, and then 10 g was dispensed into 150 ml Erlenmeyer flasks and autoclaved at 121 ° C for 50 minutes (protein content of solid medium after autoclaving is about 19%, initial water content is 46%), and 100 μL of RIB326 strain conidia (1 × 10 ⁷ / ml) were inoculated into each of three tubes and solid culture was performed. After 16 hours and 24 hours, the culture was performed for 6 days. After the end of the culture, ERG was analyzed in the same manner as in Example 1. The amount of ergothioneine produced was 0.9 g / kg of koji mold solid fermentation product.

Example 6: Production of ERG-rich koji mold solid fermentation product using soybean pulp powder as a medium 640 g of soybean pulp powder (protein content 23.1%) manufactured by Kikkoman Soy Foods Corporation was placed in a vinyl bag, and 480 ml of boiling water was added and steamed (protein content of solid medium after adding hot water and steaming: 13.2%, initial water content: 45%). After cooling to room temperature, 2.4 g of seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain was added and stirred well, and the mixture was placed evenly on a plate cover. Fermentation was performed at 95% humidity and 32°C. When the temperature of the koji mold solid fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was performed for 7 days to obtain an ERG-rich koji mold solid fermentation product. ERG was analyzed and found to be 0.35 g/kg of koji mold solid fermentation product.

Example 7: Production of ERG-rich koji mold solid fermentation product using peas as a medium 640 g of crushed peas (protein content 21.7%) was placed in a vinyl bag, and 480 ml of boiling water was added and steamed (protein content of solid medium after adding hot water and steaming was 12.4%, initial water content was 49%). After cooling to room temperature, 2.4 g of seed culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added and stirred well, and the mixture was placed evenly on a plate cover. Fermentation was performed at 95% humidity and 32°C. When the temperature of the koji mold solid fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was performed for 7 days to obtain an ERG-rich koji mold solid fermentation product. ERG was analyzed and found to be 0.59 g/kg of koji mold solid fermentation product.

Example 8: Production of ERG-rich koji mold solid fermentation product using spirulina as a medium 340g of spirulina powder (protein content about 60%) and wheat bran (protein content 16%) were placed in a vinyl bag, and 480ml of boiling water was added and steamed (protein content of solid medium after adding hot water and steaming is about 21%, initial water content is 47%). After cooling to room temperature, 2.4g of seed culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added and stirred well, and the mixture was placed evenly on a plate cover. Koji was made at 95% humidity and 32°C. When the temperature of the koji mold solid fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was continued for 7 days to obtain an ERG-rich koji mold solid fermentation product. ERG was analyzed and found to be 0.36g/kg of koji mold solid fermentation product.

Example 9: Production of ERG-rich koji mold solid fermentation product using dried yeast powder as a medium 3.4 g of dried yeast powder (protein content 54.7%) and 3.0 g of wheat bran were dispensed into a 150 ml Erlenmeyer flask, 4.8 g of water was added, and the mixture was autoclaved at 121 ° C for 50 minutes (protein content of solid medium after autoclaving 20.9%, initial water content 47%), and 100 μL of conidia (1 × 10 ⁷ / ml) of RIB326 strain were inoculated and fermented. After 16 hours and 24 hours, the mixture was cleaned and fermented for 6 days. After the end of fermentation, ERG was analyzed in the same manner as in Example 2. The amount of ERG produced was 0.42 g / kg of koji mold solid fermentation product.

Comparative Example 1: ERG Production by the Method of Making Soy Sauce Koji 640 g of an equal mixture of expanded defatted soybeans and crushed wheat (protein content 25.2%) was placed in a plastic bag, and 480 ml of boiling water was added and steamed (protein content of solid medium after adding hot water and steaming: 14.4%, initial water content: 47%). After cooling to room temperature, 2.4 g of seed culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added and stirred well, and the mixture was placed evenly on a wooden lid. Fermentation was performed at 95% humidity and 32°C, and when the koji temperature reached 40°C, the room temperature was changed to 25°C, and koji was made for 3 days and the ERG content was measured. The ERG production was 0.15 g/kg of solid koji fermentation product.

Comparative Example 2: ERG production using low-protein raw materials Solid culture was carried out using wheat bran (protein content: about 16%) as the raw material in the same manner as in Example 1 (protein content of the solid medium after adding hot water and steaming: 9.1%, initial water content: 48%). The ERG production was 0.034 g/kg of koji mold solid fermentation product.

Claims (8)

A solid fermentation product of koji mold containing ergothioneine at a rate of 0.35 g/kg or more. A solid fermentation product of Aspergillus oryzae containing ergothioneine of 0.8 g/kg or more. 3. The koji mold solid-fermentation product according to claim 1 or 2, which is a fermentation product of koji mold and a solid medium made from wheat and a raw material selected from the group consisting of defatted soybeans, fish meal, meat powder, and spirulina powder. 1. A method for producing a solid-fermented koji mold product with a high ergothioneine content of 0.35 g/kg of solid-fermented koji mold product, the method comprising a step of mixing and culturing a solid medium containing 10% or more of protein with koji mold, the method being characterized in that the fermentation is carried out for 4 days or more, no water is added during fermentation, the initial moisture content of the mixture of the solid medium and the koji mold is adjusted to 45% or more, and the moisture content of the mixture during fermentation is maintained at 25% or more . The method according to claim 4, wherein the solid medium is prepared by subjecting at least one raw material selected from the group consisting of defatted soybeans, fish meal, meat meal, and spirulina powder to a swelling treatment together with a wheat raw material. The method according to claim 4 or 5, wherein the high-ergothioneine content koji mold solid fermentation product contains 0.8 g or more of ergothioneine per kg of koji mold solid fermentation product. The method according to any one of claims 4 to 6 , wherein the fermentation step comprises fermenting at 20 to 40°C. A solid fermentation product of Aspergillus oryzae with a high ergothioneine content, produced by the production method according to any one of claims 4 to 7 .