Solid Fermentation Product of Aspergillus oryzae with High Content of Ergothioneine and Method for Producing the Same

Abstract

To provide a food having a high content of ergothioneine.SOLUTION: A fermented solid koji mold having a high content of ergothioneine contains ergothioneine of 0.35 g relative to fermented solid koji mold 1 kg. The fermented solid koji mold having a high content of ergothioneine is made by a process of mixing a solid medium and koji mold for culturing, in which the fermentation is performed for four days or longer while the moisture content of the mixture under fermentation is kept 25% or higher.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a method for producing an ergothioneine (hereinafter sometimes referred to as ERG) - rich Aspergillus solid fermented product, and to the technical field of an ERG - rich Aspergillus solid fermented product produced by the production method.

Background Art

[0002] ERG is a sulfur - containing amino acid having a strong antioxidant action and has been found to be present in the living bodies of plants and animals. ERG not only has a strong antioxidant action, but also has been reported to have an elastase inhibitory action and a tyrosinase inhibitory action, and has attracted particular attention in the fields of beauty and food such as whitening and wrinkle prevention. In addition, it has been found that ERG is involved in the body's antioxidant defense system, and its application in the medical field has also been attempted. Recently, it has also been attracting attention in relation to longevity and mild cognitive impairment, etc., such as the fact that the ERG content decreases with aging and that ERG has a mitigating effect on telomere length reduction under oxidative stress. In order to exert the cognitive function improvement effect of mild cognitive impairment, 5 mg / day of ERG is required (see, for example, Non - Patent Document 1).

[0003] Plants and animals cannot synthesize ERG, and the ERG in the living body is considered to be derived from ERG synthesized by microorganisms such as basidiomycetes. ERG is also contained in some edible mushrooms of basidiomycetes, such as enoki mushrooms, oyster mushrooms, shiitake mushrooms, maitake mushrooms, eryngii mushrooms, and champignon mushrooms, and is known to be particularly abundant in tremella mushrooms. Foods other than mushrooms do not contain a sufficient amount of ERG to exert an effect. Although it is known that Aspergillus produces ERG, in order to ingest 5 mg / day of ERG, it is necessary to ingest 100 g / day or more of generally sold Aspergillus.

[0004] As methods for producing ergosterol (ERG), extraction from basidiomycetes such as Ganoderma tsugae, chemical synthesis, and fermentation using microorganisms have been attempted. Extraction from basidiomycetes such as Ganoderma tsugae takes time to obtain raw materials and is not suitable for mass production. In order to obtain a large amount of ERG, fermentation using C1 compound-assimilating bacteria or yeast (Patent Document 1: International Publication No. 2016 / 104437), and further fermentation using microorganisms overexpressing the ERG biosynthetic gene (Patent Document 2: International Publication No. 2017 / 150304) have been studied. Solid culture using Aspergillus overexpressing the ERG biosynthetic gene has also been attempted, but the production amount is 231 mg / kg (Non-Patent Document 2).

[0005] However, the ERG-producing microbial strains thus identified are microorganisms not normally used in foods, and when using genetically engineered microorganisms, their use in foods is restricted. Therefore, considering applications to various foods, a non-recombinant strain with food experience and high ERG productivity is desired.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for increasing the ergothioneine (ERG) content in foods and enabling sufficient intake of ERG from foods other than mushrooms.

Means for Solving the Problems

[0009] When the inventors of the present invention conducted intensive research to provide foods with a high ERG content, they found that the ERG content increases by producing a solid fermented product under predetermined conditions, leading to the present invention. Therefore, the present invention relates to the following: [1] Aspergillus solid fermented product containing 0.35 g or more of ergothioneine per 1 kg of Aspergillus solid fermented product. [2] A method for producing an Aspergillus solid fermented product with a high ergothioneine content of 0.35 g or more of ergothioneine per 1 kg of Aspergillus solid fermented product, comprising a step of mixing and culturing a solid medium containing 10% or more of protein and Aspergillus, characterized in that fermentation is carried out for 4 days or more. [3] The production method according to item 2, characterized in that water is not added during fermentation and the initial water content of the mixture of the solid medium and Aspergillus is adjusted to 45% or more. [4] The production method according to item 2 or 3, characterized in that the water content of the mixture during fermentation is maintained at 25% or more. [5] The production method according to any one of items 2 to 4, characterized in that the fermentation step includes fermentation at 20 to 40°C. [6] An Aspergillus solid fermented product with a high ergothioneine content produced by the production method according to any one of items 2 to 5.

Effects of the Invention

[0010] According to the present disclosure, it is possible to provide a food in which the ERG content in the food is increased by fermentation.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0012] <Aspergillus> As any Aspergillus belonging to the genus Aspergillus can be used as the Aspergillus. As an example, Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus luchuensis, Aspergillus tamarii can be mentioned. As the Aspergillus strain, for example, publicly available strains such as Aspergillus oryzae RIB326 strain, strains contained in commercially available seed koji from seed koji makers, etc., or strains obtained by isolation from food and beverage manufacturing environments such as sake breweries, soy sauce brewing cellars, etc. can be used.

[0013] In addition, as the Aspergillus oryzae, a wild strain may be used, or a mutant strain with high ERG production may be further used by using a general mutagenesis method. Examples of the mutagenesis method include treatment with alkylating agents such as ultraviolet (UV) or X-ray irradiation that physically damages DNA and introduces mutations, and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) that chemically damages DNA and introduces mutations. From the perspective of food production, it is preferable to use non-genetically modified strains. The Aspergillus oryzae may be one obtained by culturing Aspergillus oryzae in a medium, or one obtained by culturing Aspergillus oryzae, allowing spores to sufficiently grow and then drying. It may be dried together with the raw material, or only the spores may be recovered.

[0014] <Method for increasing ergosterol content> The method for increasing ergosterol content according to an embodiment of the present disclosure means a method of solid-state fermentation of Aspergillus oryzae under fermentation conditions that highly contain ergosterol in the solid-state fermented product of Aspergillus oryzae, which will be described in detail below. Such a method for increasing ergosterol content means a method for producing a solid-state fermented product of Aspergillus oryzae with an increased ergosterol content as compared with a solid-state fermented product of Aspergillus oryzae produced under raw materials and normal fermentation conditions. In the method for solid-state fermentation of Aspergillus oryzae with high ergosterol content, the ergosterol content increases by 5 times or more, preferably 10 times or more, more preferably 20 times or more, still more preferably 50 times or more, and even more preferably 100 times or more as compared with the case where the moisture content of the solid-state fermented product of Aspergillus oryzae is fermented at 25% or less under normal fermentation conditions. As an example, the solid-state fermented product of Aspergillus oryzae with high ergosterol content contains 0.35 g or more per 1 kg of the solid-state fermented product of Aspergillus oryzae, preferably 0.4 g or more per 1 kg of the solid-state fermented product of Aspergillus oryzae, more preferably 0.5 g or more per 1 kg of the solid-state fermented product of Aspergillus oryzae, still more preferably 0.8 g or more per 1 kg of the solid-state fermented product of Aspergillus oryzae, and even more preferably 1.0 g or more per 1 kg of the solid-state fermented product of Aspergillus oryzae. The ergosterol contained in the solid-state fermented product of Aspergillus oryzae with high ergosterol content of the present disclosure is produced by Aspergillus oryzae and is not added or concentrated. The solid-state fermented product of Aspergillus oryzae with high ergosterol content is identified only by its manufacturing process, and it is impossible or impractical to identify it by the components and characteristics of the produced solid-state fermented product of Aspergillus oryzae with high ergosterol content.

[0015] For the culture conditions to enrich ERG in the Aspergillus solid fermented product, it is important to control the initial water content of the mixture of the solid medium and Aspergillus and the water content during fermentation for at least 4 days. More specifically, it is necessary to maintain the water content of the mixture of the solid medium and Aspergillus during fermentation at 25% or more even after the 4th day. If the water content of the mixture of the solid medium and Aspergillus during fermentation is less than 25%, it is not possible to enrich ERG in the Aspergillus solid fermented product. Therefore, in the method for producing an ERG-rich Aspergillus solid fermented product according to an embodiment of the present disclosure, in the step of mixing and fermenting the solid medium and Aspergillus, it is characterized in that the water content in the mixture is maintained at 25% or more throughout the fermentation process. In the production of foods such as soy sauce, miso, and sake, water is usually not added during fermentation, and koji making is not carried out for more than 4 days. Also, heat is generated at the initial stage of fermentation, and water can be rapidly lost. Therefore, considering the fermentation period and process, when the initial water content of the mixture of the solid medium and Aspergillus at the start of culture is less than 45%, it becomes difficult to maintain the water content of the fermented product during fermentation at 25% or more (Figure 2). Therefore, the method for producing an ERG-rich Aspergillus solid fermented product according to an embodiment of the present disclosure may include a step of adjusting the initial water content of the mixture of the solid medium and Aspergillus to 45% or more.

[0016] The ERG-rich Aspergillus solid fermented product is prepared by fermenting a normal solid medium for culturing Aspergillus. 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. The solid medium of the present invention preferably further contains an expanded raw material. The solid medium can be prepared by steaming or boiling the above-mentioned raw materials in boiling water and optionally crushing them.

[0017] For other fermentation conditions, normal conditions used in the relevant technical field can be employed. For example, the initial pH of the medium is adjusted to 5 - 10. The fermentation temperature is set at 20 - 40°C, the fermentation time is 4 days or more, preferably 4 - 10 days, more preferably 5 - 7 days, and even more preferably 6 - 7 days. From the perspective of maintaining the moisture content of the Aspergillus solid ferment during fermentation, it is preferable to culture under high humidity, and the humidity can be adjusted to control the moisture value of the Aspergillus solid ferment. Also, since the fermentation is a long-term fermentation of 4 days or more with a high moisture content, it is desirable to conduct the fermentation in an environment that prevents the growth of contaminants. A container that can be aseptically cultured, a Yamazaki koji-making device, or a drum koji-making device may be used.

[0018] As an example, when using the Aspergillus oryzae RIB326 strain, a typical strain of Aspergillus, to produce an ergosterol (ERG)-rich Aspergillus solid ferment by the production method according to the present invention, an ERG-rich Aspergillus solid ferment containing 0.35 g or more, preferably 0.4 g or more, more preferably 0.5 g or more, even more preferably 0.8 g or more, and even more preferably 1.0 g or more of ERG per 1 kg of the Aspergillus solid ferment can be produced (Figure 1).

[0019] ERG is known to have various functions, including antioxidant activity, elastase inhibitory activity, tyrosinase inhibitory activity, polyphenol oxidase (PPO) inhibitory activity, etc. Therefore, Aspergillus solid fermented product with high ERG content can also be used as a food with functional claims. As such functional claims, anti-wrinkle effects based on the elastase inhibitory activity of ERG, skin beautifying or whitening effects based on tyrosinase inhibitory activity, lifestyle disease prevention effects based on the production of lipid peroxides, and prevention effects of dementia and Alzheimer's disease based on the removal of reactive oxygen species can be indicated. In yet another embodiment, there is provided a composition for anti-wrinkle and whitening, or a composition for preventing lifestyle diseases, or preventing dementia and Alzheimer's disease, which contains Aspergillus solid fermented product with high ERG content. In addition, it may be used in the production of seasonings by using Aspergillus solid fermented product with high ERG content as a raw material or an auxiliary raw material, and further performing fermentation using lactic acid bacteria, yeast, etc. Furthermore, Aspergillus solid fermented product with high ERG content can also be used in pet food, feed for ornamental fish, raw materials for alternative meat, etc.

[0020] <Analysis method of ERG> The method for extracting ERG from the Aspergillus solid fermented product with high ERG content after fermentation can be carried out using methods well-known in the art. The extraction solvent is not particularly limited as long as ERG can dissolve in it. For example, 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, etc. can be mentioned. After adding the solvent, ERG can be extracted while appropriately performing crushing treatment. The temperature of the extraction solvent can be set from room temperature to 100°C.

[0021] As an embodiment of the method for extracting ERG, for example, a suspension prepared by adding Aspergillus solid fermented product with high ERG content to water is subjected to heating treatment such as 98 - 100°C for 15 minutes, and then the supernatant is recovered by centrifugation. Next, the recovered supernatant is filtered to remove insoluble matters. Also, the heated suspension may be filtered without being subjected to centrifugation.

[0022] Alternatively, instead of the above heat treatment, for example, methods of disrupting the cells using destruction means such as ultrasonic disruptors, French presses, dynomills, mortars, etc.; methods of lysing the cell walls of the cells using cell wall-lysing enzymes such as lyticase; methods of lysing the cells using surfactants such as SDS and Triton X-100, etc. may be used for the cell disruption treatment. These methods can be used alone or in combination.

[0023] The obtained extract can be purified for ERG by subjecting it to purification treatments 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.

[0024] The qualitative or quantitative analysis of ERG is not particularly limited and can be performed, for example, by HPLC. The HPLC separation conditions can be appropriately selected by those skilled in the art and can be carried out, for example, under the conditions described in the examples below.

[0025] All documents mentioned in this disclosure are hereby incorporated by reference in their entirety into this specification. In this disclosure, % means mass % unless otherwise specified.

[0026] The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description in the claims. Changes to the present invention, for example, addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Examples

[0027] Example 1: Production of Aspergillus oryzae solid fermented product with high ergosterol content using defatted soybeans as the medium Put 640 g of puffed defatted soybeans (protein content about 48%) into a plastic bag, add 480 ml of boiling water and steam (the protein content of the solid medium after adding water and steaming is about 27%). After cooling to room temperature, add 2.4 g of the seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain, stir well, and spread evenly on a plate lid. Ferment at 95% humidity and 32 °C. When the temperature of the Aspergillus solid fermented product reaches 40 °C, change the room temperature to 25 °C, stop humidity control, and ferment for 7 days to obtain an Aspergillus solid fermented product with high ergothioneine content. After the start of fermentation, the Aspergillus solid fermented product was weighed on the 1st, 3rd, 4th, and 7th days, and the moisture content was calculated (Figure 2). In addition, a part of the Aspergillus solid fermented product was obtained on the 1st, 2nd, 3rd, and 6th days after the start of fermentation.

[0028] Example 2: Analysis method of ergosterol (1) Extraction of ergothioneine Weigh 5 g of the Aspergillus solid fermented product with high ergothioneine content, add 75% ethanol, crush the fermented product with high ergothioneine content, leave it at room temperature for 1 day, and extract ergothioneine from the Aspergillus solid fermented product with high ergothioneine content. When the ergothioneine content was measured under the following analysis conditions, the content of ergothioneine contained in 1 kg of the Aspergillus solid fermented product with high ergothioneine content was 1.1 g. Similarly, the ergothioneine content of the fermented products on the 1st, 2nd, 3rd, and 6th days after the start of fermentation was also measured and shown in a graph (Figure 1). As a reference example, for Miyako Koji Square (manufactured by Ise Souza Co., Ltd.), Rice Koji H (manufactured by Kose Foods Co., Ltd.), and Rice Koji S (manufactured by Kose Foods Co., Ltd.), ergothioneine was extracted in the same way, and the ergothioneine content was measured under the following analysis conditions. The ergothioneine content of each was as follows.

Table 1

[0029] (2) LCMS analysis conditions Analytical instrument: UPLC CQ micro; Waters UPLC Column: 2.5 HILIC 3.0 mm l.D.×150 mm Solvent A: Acetonitrile Solvent B: 5 mM ammonium acetate / H2O Flow rate: 0.5 ml / min with 80% Solvent A Inject: 2 μL Mass spectrometer ESI: ES+ Cone V: 21 V Capillary V: 4.5 kV Source temperature: 120 °C Desolvation temperature: 400 °C MS Scan mode

[0030] (3) Ergothioneine analysis conditions in LC-MS / MS Analytical instrument: UPLC CQ micro; Waters UPLC Column: 2.5 HILIC 3.0 mm I.D. × 150 mm Solvent A: 0.1% formic acid / acetonitrile Solvent B: 0.1% formic acid / H2O Flow rate: 0.5 ml / min with 80% A Inject: 2 μL Mass spectrometer ESI: ES+ Cone V: 21 V Capillary V: 4.5 kV Source temperature: 120 °C Desolvation temperature: 400 °C Collision energy: 11 V Trace: m / z 230.1 > 186.1 Collision energy: 20 V Trace: m / z 230.1 > 127.0

[0031] Example 3: Comparison of ergosterol production amounts of each Aspergillus oryzae Put 640 g of puffed defatted soybeans (protein content: about 48%) into a plastic bag, add 480 ml of boiling water, and steam (the protein content of the solid medium after adding water and steaming is about 27%, and the initial moisture content is 47%). After cooling to room temperature, add 2.4 g of seed culture (wheat bran culture) of each of Aspergillus oryzae NISL2180 strain, RIB326 strain, RIB646 strain, RIB1370 strain, RIB408 strain, RIB40 strain, and Aspergillus sojae NBRC4239 strain, stir well, and spread evenly on a plate lid. Ferment at 95% humidity and 32 °C. When the temperature of the koji solid ferment reaches 40 °C, change the room temperature to 25 °C, stop humidity control, and ferment for 7 days to obtain a koji solid ferment rich in ergosterol (ERG). For the ferment after 7 days of fermentation, measure the ERG content based on the same analysis method as in Example 2, and compare the ERG production of each strain (Figure 3). Also, measure the moisture content of the culture after 7 days of fermentation, and compare the moisture content of the ferment for each strain (Figure 4).

[0032] Example 4: Production of Aspergillus oryzae solid fermented product with high ergosterol content using a medium mixed with defatted soybeans and wheat Put 480 ml of boiling water into 640 g of an equal mixture (protein content: 25.2%) of puffed defatted soybeans and crushed wheat in a plastic bag and steam (the protein content of the solid medium after adding water and steaming is 14.4%, and the initial moisture content is 47%). After cooling to room temperature, add 2.4 g of seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain, stir well, and spread evenly on a plate lid. Ferment at 95% humidity and 32 °C. When the temperature of the koji solid ferment reaches 40 °C, change the room temperature to 25 °C, stop humidity control, and ferment for 7 days to obtain a koji solid ferment rich in ergosterol (ERG). Extract and analyze ERG in the same manner as in Example 2. The ERG content was 0.42 g / kg of koji solid ferment.

[0033] Example 5: Production of Aspergillus oryzae solid fermented product with high ergosterol content using fish meal as the raw material Into a plastic bag, 27.8 g of 60% fish meal (manufactured by Izukawa Feed Co., protein content 60% or more), 30.4 g of crushed wheat, and 41.7 g of water were well mixed. After that, 10 g portions were dispensed into 150 ml Erlenmeyer flasks, and autoclaved at 121°C for 50 minutes (the protein content of the solid medium after autoclaving was about 19%, and the initial moisture content was 46%). Conidia of the RIB326 strain (1×10 7 cells / ml) were inoculated at 100 μL each, three replicates per flask, and solid culture was carried out. Maintenance was performed at 16 hours and 24 hours, and the culture was carried out for 6 days. After the culture was completed, ERG analysis was performed in the same manner as in Example 1. The production amount of ergothioneine was 0.9 g per 1 kg of koji solid fermented product.

[0034] Example 6: Production of Aspergillus oryzae solid fermented product with high ergosterol content using okara powder as the medium 640 g of Kikkoman Soyfoods Co., Ltd. okara powder (protein content 23.1%) was placed in a plastic bag, and 480 ml of boiling water was added and steamed (the protein content of the solid medium after adding boiling water and steaming was 13.2%, and the initial moisture content was 45%). After cooling to room temperature, 2.4 g of a seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain was added and stirred well, and then evenly spread on a plate lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji solid fermented product reached 40°C, the room temperature was changed to 25°C, and humidity control was stopped. Fermentation was carried out for 7 days to obtain an ERG-rich koji solid fermented product. ERG was analyzed and it was 0.35 g per 1 kg of koji solid fermented product.

[0035] Example 7: Production of Aspergillus oryzae solid fermented product with high ergosterol content using peas as the medium 640 g of crushed broad beans (protein content 21.7%) was placed in a plastic bag, and 480 ml of boiling water was added and steamed (the protein content of the solid medium after adding boiling water and steaming was 12.4%, and the initial moisture content was 49%). After cooling to room temperature, 2.4 g of a seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain was added and stirred well, and then evenly spread on a plate lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji solid fermented product reached 40°C, the room temperature was changed to 25°C, and humidity control was stopped. Fermentation was carried out for 7 days to obtain an ERG-rich koji solid fermented product. ERG was analyzed and it was 0.59 g per 1 kg of koji solid fermented product.

[0036] Example 8: Production of Aspergillus oryzae solid fermented product with high ergosterol content using Spirulina as the medium Put 340 g of Spirulina powder (protein content about 60%) and wheat bran (protein content 16%) into a plastic bag, add 480 ml of boiling water and steam (the protein content of the solid medium after adding water and steaming is about 21%, and the initial moisture content is 47%). After cooling to room temperature, add 2.4 g of the seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain, stir well, and spread evenly on a plate lid. Ferment at 95% humidity and 32 °C. When the temperature of the koji mold solid ferment reaches 40 °C, change the room temperature to 25 °C, stop humidity control, and ferment for 7 days to obtain a koji mold solid ferment rich in ERG. The analysis of ERG showed that it was 0.36 g / kg of koji mold solid ferment.

[0037] Example 9: Production of Aspergillus oryzae solid fermented product with high ergosterol content using dried yeast powder as the medium Dispense 3.4 g of dry yeast powder (protein content 54.7%) and 3.0 g of wheat bran into a 150 ml Erlenmeyer flask, add 4.8 g of water, and autoclave at 121 °C for 50 minutes (the protein content of the solid medium after autoclaving is 20.9%, and the initial moisture content is 47%). Inoculate 100 μL of conidia of RIB326 strain (1×10 7 cells / ml) and carry out fermentation. Perform maintenance at 16 hours and 24 hours, and ferment for 6 days. After the fermentation is completed, analyze ERG in the same manner as in Example 2. The production amount of ERG was 0.42 g / kg of koji mold solid ferment.

[0038] Comparative Example 1: Ergosterol production by the soy sauce koji making method Put 640 g of an equal amount mixture of puffed defatted soybeans and crushed wheat (protein content 25.2%) into a plastic bag, add 480 ml of boiling water and steam (the protein content of the solid medium after adding water and steaming is 14.4%, and the initial moisture content is 47%). After cooling to room temperature, add 2.4 g of the seed culture (wheat bran culture) of Aspergillus oryzae RIB326 strain, stir well, and spread evenly on a plate lid. Ferment at 95% humidity and 32 °C. When the koji temperature reaches 40 °C, change the room temperature to 25 °C, carry out koji making for 3 days, and measure the ERG content. The ERG production amount was 0.15 g / kg of koji mold solid ferment.

[0039] Comparative Example 2: Ergosterol production with low protein raw materials Solid culture was carried out in the same manner as in Example 1 using wheat bran (protein content: approximately 16%) as a raw material (protein content of the solid medium after adding hot water and steaming: 9.1%, initial moisture content: 48%). The ERG production amount was 0.034 g per 1 kg of the Aspergillus solid fermented product.

Claims

1. A solid koji fermentation product containing 1 kg or more of ergothioneine per 0.35 g of solid koji fermentation product of Aspergillus oryzae.

2. A solid koji fermentation product containing 1 kg or more of ergothioneine per 0.8 g of solid koji fermentation product of Aspergillus oryzae.

3. The solid koji fermentation product according to Claim 1 or 2, wherein the solid koji fermentation product is a fermentation product of Aspergillus oryzae and a solid medium made from raw materials selected from the group consisting of defatted soybeans, fish meal, meat powder, and spirulina powder and wheat.

4. A method for producing a solid koji fermentation product with a high ergothioneine content of 1 kg or more of ergothioneine per 0.35 g of solid koji fermentation product of Aspergillus oryzae, comprising the step of mixing and culturing a solid medium containing 10% or more of protein and Aspergillus oryzae, characterized in that it ferments for 4 days or more, does not add water during fermentation, adjusts the initial moisture content of the mixture of the solid medium and Aspergillus oryzae to 45% or more, and maintains the moisture content of the mixture during fermentation at 25% or more.

5. The production method according to Claim 4, wherein the solid medium is prepared by puffing at least one raw material selected from defatted soybeans, fish meal, meat powder, and spirulina powder and together with a wheat raw material.

6. The production method according to Claim 4 or 5, wherein the solid koji fermentation product with a high ergothioneine content contains 1 kg or more of ergothioneine per 0.8 g of solid koji fermentation product of Aspergillus oryzae.

7.

8. The production method according to any one of Claims 4 to 6, wherein the fermentation step includes fermenting at 20 to 40°C.

9. A solid koji fermentation product with a high ergothioneine content produced by the production method according to any one of Claims 4 to 7.

Images