Ergothioneine-high content aspergillus solid fermented product and method for producing the same

Abstract

To provide an ergothioneine-high content food.SOLUTION: In a step of culturing by mixing a solid medium and Aspergillus, the water content of the mixture during fermentation is kept at 25% or more and fermentation is continued for 4 days or more. This results in an ergothioneine-high content Aspergillus solid fermented product with an ergothioneine content of 0.35 g or more per 1 kg of Aspergillus solid fermented product.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] 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 abbreviated 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. [Background technology]

[0002] ERG is a sulfur-containing amino acid with potent antioxidant properties and has been found to exist in the living bodies of plants and animals. ERG not only has potent antioxidant properties, but has also been reported to have elastase and tyrosinase inhibitory properties, drawing particular attention in the beauty and food industries for its role in skin whitening and wrinkle prevention. Furthermore, ERG has been shown to be involved in the body's oxidation defense system, and attempts are being made to apply it in the medical field. Recently, ERG has been attracting attention in relation to longevity and mild cognitive impairment, due to the fact that ERG content decreases with age and that ERG has the effect of mitigating telomere length reduction under oxidative stress. A daily dose of 5 mg of ERG is required to exert the cognitive function improvement effect in mild cognitive impairment (see, for example, Non-Patent Document 1).

[0003] 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 basidiomycete mushrooms, such as enokitake, oyster mushroom, shiitake, maitake, king oyster mushroom, and mushroom, with Tamogi mushroom being particularly abundant. Foods other than mushrooms do not contain sufficient amounts of ERG to exert an effect. Aspergillus oryzae is also known to produce ERG, but to obtain 5 mg of ERG per day, one would need to consume more than 100 g of commercially available koji per day.

[0004] Methods for producing ERG have been attempted, including extraction from basidiomycetes such as Pleurotus cornucopius, chemical synthesis, and fermentation using microorganisms. Extraction from basidiomycetes such as Pleurotus cornucopius requires time to obtain raw materials, making it unsuitable for mass production. To obtain large amounts of ERG, research has been conducted on fermentation using C1 compound-utilizing bacteria or yeast (Patent Document 1: International Publication No. 2016 / 104437) and fermentation using microorganisms overexpressing ERG biosynthetic genes (Patent Document 2: International Publication No. 2017 / 150304). Solid-state culture using koji mold overexpressing ERG biosynthetic genes has also been attempted, but the production yield was only 231 mg / kg (Non-Patent Document 2).

[0005] However, the ERG-producing microbial strains identified in this way are not commonly used in food, and their use in food is limited when gene-transfected microorganisms are used. Therefore, considering the application to various foods, a non-recombinant strain with high ERG productivity that has been used in food is desired. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 104437 [Patent Document 2] International Publication No. 2017 / 150304 [Non-patent literature]

[0007] [Non-Patent Document 1] Pharmacology and Therapeutics Volume 48, Issue 4, 685-697 (2020) [Non-patent document 2] BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, 2019, VOL. 83, NO. 1, 181‐184 Summary of the Invention [Problem to be solved by the invention]

[0008] The purpose of the present invention is to provide a method for increasing the ERG content in food and enabling sufficient intake of ERG from foods other than mushrooms. [Means for solving the problem]

[0009] The present inventors conducted extensive research to provide a food product with a high ERG content, and discovered that the ERG content can be increased by producing a solid fermented product under specific conditions, leading to the present invention. Thus, the present invention relates to: [1] A koji mold solid-fermentation product containing 0.35g or more ergothioneine per kg of koji mold solid-fermentation product. [2] A method for producing a solid-fermentation product of koji mold with a high content of ergothioneine of 0.35 g / kg or more of the koji mold solid-fermentation product, the method comprising the step of mixing koji mold with a solid medium containing 10% or more protein and culturing the mixture, wherein the fermentation is carried out for 4 days or more. [3] 3. The production 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 production 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 koji mold with a high ergothioneine content, produced by the production method described in any one of items 2 to 5. [Effects of the Invention]

[0010] According to the present disclosure, it is possible to provide foods in which the ERG content has been increased by fermentation. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the change in ERG content with respect to the number of days of fermentation when fermentation was carried out using koji mold under fermentation conditions that resulted in a high ERG content in the koji mold solid-fermentation product. [Figure 2] FIG. 2 is a graph showing the change in water content of the solid fermentation product of koji mold during fermentation. [Figure 3] FIG. 3 shows the ERG content in solid fermentation products of koji mold after 7 days of fermentation under specified conditions using koji mold strains NISL2180, RIB326, RIB646, RIB1370, RIB408, RIB40, and NBRC4239. [Figure 4] FIG. 4 shows the water content in the solid fermentation products of koji mold strains NISL2180, RIB326, RIB646, RIB1370, RIB408, RIB40, and NBRC4239 after fermentation for 7 days under specified conditions. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Koji mold> Any fungus belonging to the genus Aspergillus can be used as the koji mold. Examples include Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus luchuensis, and Aspergillus tamarii. Examples of koji mold strains that can be used include publicly available strains such as Aspergillus oryzae RIB326, strains contained in koji starters commercially available from koji makers, and strains isolated from food and beverage production environments such as sake and soy sauce breweries.

[0013] Also, as the Aspergillus oryzae, a wild strain may be used, or a mutant strain with higher ERG production may be used by using a general mutagenesis method. Examples of the mutagenesis method include treatment with alkylating agents such as ultraviolet (UV) rays or X-rays that physically damage DNA to introduce mutations, and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) that chemically damage DNA to introduce mutations. From the perspective of food production, it is preferable to use non-genetically modified strains. The Aspergillus oryzae may be one cultured in a medium, or one cultured, with spores sufficiently grown and dried. It may be dried together with the raw material, or only the spores may be recovered.

[0014] <Method for increasing ERG content> The method for increasing ERG 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 ERG in the solid-state fermented product of Aspergillus oryzae, which will be described in detail below. Such a method for increasing ERG content means a method for producing a solid-state fermented product of Aspergillus oryzae with an increased ERG content as compared to a solid-state fermented product of Aspergillus oryzae produced under raw materials and normal fermentation conditions. In the method for highly containing ERG in the solid-state fermentation of Aspergillus oryzae, the content of ERG 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 to the case where the moisture content of the solid-state fermented product of Aspergillus oryzae is 25% or less under normal fermentation conditions. As an example, the solid-state fermented product of Aspergillus oryzae with high ERG 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 ERG contained in the solid-state fermented product of Aspergillus oryzae with high ERG content according to the present disclosure is produced by Aspergillus oryzae and is not added or concentrated. The solid-state fermented product of Aspergillus oryzae with high ERG content is identified only by its manufacturing process, and it is impossible or impractical to identify it by the components and properties of the produced solid-state fermented product of Aspergillus oryzae with high ERG content.

[0015] To achieve a high ERG content in a koji mold solid-fermentation product, it is important to control the initial moisture content of the mixture of solid medium and koji mold and the moisture content during fermentation for at least four days. More specifically, it is necessary to maintain the moisture content of the mixture of solid medium and koji mold at 25% or higher even after the fourth day of fermentation. If the moisture content of the mixture of solid medium and koji mold during fermentation is less than 25%, the ERG content in the koji mold solid-fermentation product cannot be increased. Therefore, in one embodiment of the present disclosure, a method for producing a koji mold solid-fermentation product with a high ERG content is characterized by maintaining the moisture content of the mixture at 25% or higher throughout the fermentation process during 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 carried out for more than four days. Furthermore, heat may be generated in the early stages of fermentation, causing rapid loss of moisture. 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, a method for producing a solid fermented product of koji mold with a 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.

[0016] The ERG-rich koji mold solid fermentation product is prepared by fermenting a conventional solid medium for culturing koji mold. Such a solid medium preferably contains 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 meal, 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 simmering the above-mentioned raw materials in boiling water, and optionally crushing them.

[0017] Other fermentation conditions may be those typically used in this 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 longer, 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 koji mold solid-fermentation product during fermentation, it is preferable to culture under high humidity, and humidity can be adjusted to control the moisture content of the koji mold solid-fermentation product. Furthermore, since the fermentation is a long-term fermentation of 4 days or longer and involves a high moisture content, it is desirable to carry out the fermentation in an environment that prevents the growth of unwanted bacteria. Containers that allow for aseptic culture, such as a Yamazaki-type koji-making apparatus or a drum koji-making apparatus, may also be used.

[0018] As an example, when Aspergillus oryzae RIB326, a representative strain of koji mold, is used, and when an ERG-rich koji mold solid fermentation product is produced by the production method of the present invention, it is possible to produce an ERG-rich koji mold solid fermentation product containing 0.35 g ERG / 1 kg or more of koji mold solid fermentation product, preferably 0.4 g ERG / 1 kg or more of koji mold solid fermentation product, more preferably 0.5 g ERG / 1 kg or more of koji mold solid fermentation product, even more preferably 0.8 g ERG / 1 kg or more of koji mold solid fermentation product, and even more preferably 1.0 g ERG / 1 kg or more of koji mold solid fermentation product ( FIG. 1 ).

[0019] ERG is known to have various functions, including antioxidant activity, elastase inhibitory activity, tyrosinase inhibitory activity, polyphenol oxidase (PPO) inhibitory activity, and others. Therefore, a solid fermented product of Aspergillus containing a high content of ERG can also be used as a food with functional claims. As such functional claims, it is possible to indicate functions such as 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. 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 a solid fermented product of Aspergillus containing a high content of ERG. In addition, it may be used in the production of seasonings by using a solid fermented product of Aspergillus containing a high content of ERG as a raw material or an auxiliary raw material, and further performing fermentation using lactic acid bacteria, yeast, and the like. Furthermore, the solid fermented product of Aspergillus containing a high content of ERG can also be used for pet food, feed for ornamental fish, and raw materials for alternative meat.

[0020] <Analysis method of ERG> The method for extracting ERG from the solid fermented product of Aspergillus containing a high content of ERG 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 be dissolved therein, and examples include organic solvents such as methanol, ethanol, isopropanol, and acetone; water-containing 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 appropriately performing crushing treatment. The temperature of the extraction solvent can be set from room temperature to 100°C.

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

[0022] Instead of the above-mentioned heating treatment, the cells may be subjected to disruption treatment such as disrupting the cells using disruption means such as an ultrasonic disrupter, French press, Dynomill, or mortar; dissolving the cell walls of the cells using a cell wall-lytic enzyme such as Yatalase; or dissolving the cells using a surfactant such as SDS or Triton X-100. These methods may be used alone or in combination.

[0023] The resulting extract can be subjected to purification processes such as centrifugation, filter filtration, ultrafiltration, gel filtration, separation based on solubility differences, 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.

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

[0025] All documents referred to in this disclosure are incorporated herein by reference in their entirety. In this disclosure, % means % by weight unless otherwise specified.

[0026] The following examples of the present invention 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 claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0027] 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: approximately 48%) were placed in a plastic bag, and 480 ml of boiling water was added and steamed (protein content of the solid medium after steaming with hot water: approximately 27%). After cooling to room temperature, 2.4 g of Aspergillus oryzae RIB326 seed culture (wheat bran culture) was added and mixed thoroughly. The mixture was then flattened onto a lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji mold solid-fermentation mixture reached 40°C, the room temperature was changed to 25°C, humidity control was discontinued, and fermentation continued for 7 days to obtain a koji mold solid-fermentation mixture with a high ERG content. The koji mold solid-fermentation mixture was weighed on days 1, 3, 4, and 7, and the moisture content was calculated (Figure 2). In addition, aliquots of the koji mold solid-fermentation mixture were taken on days 1, 2, 3, and 6.

[0028] Example 2: ERG analysis method (1) Extraction of ERG Five grams of ERG-rich koji mold solid-state fermentation product was weighed out, 75% ethanol was added, and the product was then crushed and left at room temperature for one day to extract ERG from the ERG-rich koji mold solid-state fermentation product. The ERG content was measured under the analytical conditions described below, and the ERG content per kg of ERG-rich koji mold solid-state fermentation product was found to be 1.1 g. Similarly, the ERG content was measured for the fermentation product on days 1, 2, 3, and 6 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 (manufactured by Isesosha Co., Ltd.), Rice Koji H (manufactured by Kose Foods Co., Ltd.), and Rice 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: [Table 1]

[0029] (2)LCMS analysis conditions Analyzer: UPLC CQ micro;Waters UPLC Column: 2.5 HILIC 3.0mm l.D. x 150mm Solvent A: Acetonitrile Solvent B: 5mM 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

[0030] (3) Ergothioneine analysis conditions by LC-MS / MS Analyzer: UPLC CQ micro;Waters UPLC Column: 2.5 HILIC 3.0 mm l.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

[0031] Example 3: Comparison of ERG production by each koji mold 640 g of expanded defatted soybeans (protein content: approximately 48%) were placed in a plastic bag, and 480 ml of boiling water was added and steamed (protein content of the solid medium after steaming was approximately 27%, initial moisture content: 47%). After cooling to room temperature, 2.4 g of seed culture (wheat bran culture) of Aspergillus oryzae strains NISL2180, RIB326, RIB646, RIB1370, RIB408, and RIB40, and Aspergillus sojae strain NBRC4239 was added and mixed well. The mixture was then spread evenly on a wooden lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji mold solid-state fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was discontinued, and fermentation continued for 7 days to obtain a koji mold solid-state fermentation product with a high ERG content. The ERG content of the fermented products after 7 days of fermentation was measured using the same analytical method as in Example 2, and the ERG production amount for each strain was compared (Figure 3). In addition, the water content of the culture after 7 days of fermentation was measured, and the water content of the fermented products for each strain was compared (Figure 4).

[0032] Example 4: Production of ERG-rich koji mold solid-state fermentation product in a medium containing a mixture of defatted soybeans and wheat 480 ml of boiling water was added to 640 g of a mixture of equal parts expanded defatted soybeans and crushed wheat (protein content: 25.2%) in a plastic bag and steamed (protein content of the solid medium after adding hot water: 14.4%, initial moisture content: 47%). After cooling to room temperature, 2.4 g of a seed culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added and mixed well, and the mixture was 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, resulting in a koji mold solid-fermentation product with a high ERG content. Extraction and ERG analysis were carried out as in Example 2. The ERG content was 0.42 g / kg of koji mold solid-fermentation product.

[0033] Example 5: Production of a high-ERG content 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 well in a plastic bag, and then 10 g of each was dispensed into 150 ml Erlenmeyer flasks and autoclaved at 121°C for 50 minutes (protein content of the solid medium after autoclaving was approximately 19%, initial water content was 46%). Conidia (1 × 10 7 Three tubes were inoculated with 100 μL of a solution of 100 μL of the fermented soybean extract (cells / ml) and subjected to solid culture. The culture was continued for 6 days, with the cells being cleaned after 16 and 24 hours. After the culture was completed, ERG analysis was performed as in Example 1. The amount of ergothioneine produced was 0.9 g / kg of koji mold solid fermentation product.

[0034] Example 6: Production of a solid fermentation product of koji mold with a high ERG content using okara powder as a medium 640 g of Kikkoman Soyfoods okara powder (protein content: 23.1%) was placed in a plastic bag, and 480 ml of boiling water was added and steamed (protein content of the solid medium after adding hot water and steaming: 13.2%, initial moisture content: 45%). After cooling to room temperature, 2.4 g of Aspergillus oryzae RIB326 seed culture (wheat bran culture) was added and mixed well, then the mixture was 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 discontinued, and fermentation continued for 7 days, resulting in a koji mold solid-fermentation product with a high ERG content. ERG was analyzed and found to be 0.35 g / kg of koji mold solid-fermentation product.

[0035] Example 7: Production of ERG-rich koji mold solid-state fermentation product using peas as a medium 640 g of crushed peas (protein content 21.7%) were placed in a plastic bag, and 480 ml of boiling water was added and steamed (protein content of the solid medium after adding hot water and steaming was 12.4%, initial moisture content 49%). After cooling to room temperature, 2.4 g of a starter culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added and mixed well, and the mixture was spread evenly on a wooden lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji mold solid-state fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was discontinued, and fermentation continued for 7 days, resulting in a koji mold solid-state fermentation product with a high ERG content. ERG was analyzed and found to be 0.59 g / kg of koji mold solid-state fermentation product.

[0036] Example 8: Production of ERG-rich koji mold solid fermentation product using spirulina as a medium 340 g of spirulina powder (approximately 60% protein content) and wheat bran (16% protein content) were placed in a plastic bag, and 480 ml of boiling water was added and steamed (the protein content of the solid medium after adding hot water and steaming was approximately 21%, with an initial moisture content of 47%). After cooling to room temperature, 2.4 g of Aspergillus oryzae RIB326 seed culture (wheat bran culture) was added and mixed well, then the mixture was spread evenly on a wooden lid. Koji was made at 95% humidity and 32°C. When the temperature of the koji mold solid-state fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was discontinued, and fermentation continued for 7 days, resulting in a koji mold solid-state fermentation product with a high ERG content. ERG was analyzed and found to be 0.36 g / kg of koji mold solid-state fermentation product.

[0037] Example 9: Production of ERG-rich koji mold solid-fermentation product using dry yeast powder as a medium 3.4 g of dry 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 the solid medium after autoclaving was 20.9%, initial water content was 47%). Conidia (1 × 10 7 100 μL of a solution of koji mold (cells / ml) was inoculated and fermentation was carried out. The mixture was cleaned after 16 and 24 hours and fermentation was continued for 6 days. After fermentation was completed, 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.

[0038] Comparative Example 1: ERG production using the soy sauce koji method A 640g mixture of equal parts expanded defatted soybeans and crushed wheat (protein content 25.2%) was placed in a plastic bag, and 480ml of boiling water was added and steamed (protein content of the solid medium after adding hot water and steaming was 14.4%, initial moisture content 47%). After cooling to room temperature, 2.4g of a starter culture of Aspergillus oryzae RIB326 strain (wheat bran culture) was added and mixed well, and the mixture was spread evenly on a wooden lid. Fermentation was carried out at 95% humidity and 32°C. When the koji temperature reached 40°C, the room temperature was changed to 25°C. Koji production was continued for 3 days, and the ERG content was measured. ERG production was 0.15g / kg of solid koji fermentation product.

[0039] Comparative Example 2: ERG production with low protein ingredients Solid culture was carried out using wheat bran (protein content: approximately 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%). ERG production was 0.034 g / kg of koji mold solid fermentation product.

Claims

1. A koji mold solid-fermentation product containing ergothioneine at 0.35 g / kg or more.

2. 1. A method for producing a solid-fermented product of koji mold having a high content of ergothioneine of 0.35 g / kg or more of the koji mold solid-fermented product, the method comprising a step of mixing koji mold with a solid medium containing 10% or more of protein and culturing the mixture, wherein the fermentation is carried out for 4 days or more.

3. 3. The method according to claim 2, wherein the initial moisture content of the mixture of the solid medium and the koji mold is adjusted to 45% or more without adding water during fermentation.

4. 4. The method according to claim 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 claims 2 to 4, wherein the fermentation step comprises fermenting at 20 to 40°C.

6. A solid-state fermentation product of koji mold with a high ergothioneine content, produced by the production method according to any one of claims 2 to 5.

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