Method for producing filamentous fungus solid culture, and filamentous fungus solid culture

Abstract

To provide a filamentous fungus solid culture that employs a substrate with a low protein content and a cultivation duration of less than 96 hours, demonstrating an improved ergothioneine content compared to conventional methods, as well as a method for producing the filamentous fungus solid culture.SOLUTION: The present invention provides a method for producing a filamentous fungus solid culture that comprises 20 mg or more of ergothioneine per 100 g of dry mass by inoculating a filamentous fungus onto a substrate and performing solid-state cultivation for a duration of less than 96 hours, wherein the substrate has a protein content of less than 10 mass%. The present invention also provides a filamentous fungus solid culture.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a solid culture of filamentous fungi and a solid culture of filamentous fungi. [Background technology]

[0002] Ergothioneine has been attracting attention as a functional food ingredient. It is a sulfur-containing amino acid with strong antioxidant properties and is known to have elastase and tyrosinase inhibitory properties.

[0003] Patent Document 1 describes a method for producing a fermented product containing ergothioneine by solid-state culture of a substrate, such as defatted soybeans containing 10% or more protein, for 4 days or more. Paragraph 39, Example 1 of Patent Document 1, states that the material is "placed flat on a wooden lid." The wooden lid is presumed to be a small container called a koji lid, which is commonly used for koji production. Since the koji lid does not allow air to pass through, koji production is carried out without ventilation. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2022-135037 A Summary of the Invention [Problem to be solved by the invention]

[0005] Plants and animals cannot synthesize ergothioneine. As described above, Patent Document 1 states that ergothioneine can be detected by culturing a substrate containing 10% or more protein in a solid state without ventilation for a culture period of 4 days or more.

[0006] Patent Document 1 describes, as Comparative Example 2, the use of wheat bran with a protein content of about 16% as a substrate. The protein content of the solid medium after adding hot water and steaming is 9.1%, and the initial water content is 48%. However, under the conditions of Comparative Example 2, only a low concentration of ergothioneine is detected.

[0007] The present invention has been proposed in view of the above, and has an object to provide a solid culture of a filamentous fungus in which a substrate with a low protein content is used and the ergothioneine content is increased to 20 mg or more per 100 g dry mass, even in a culture time of less than 96 hours, as compared to conventional methods, and a method for producing the same. [Means for solving the problem]

[0008] The above-mentioned problems are solved by a method for producing a solid culture of a filamentous fungus, which comprises inoculating a filamentous fungus onto a substrate and culturing the filamentous fungus in a solid state for a culture time of less than 96 hours, thereby producing a solid culture of a filamentous fungus containing 20 mg or more of ergothioneine per 100 g of dry mass, wherein the substrate has a protein content of less than 10 mass%.

[0009] The above-mentioned problems are solved by a solid culture of a filamentous fungus, in which a substrate is solid-state cultured with a filamentous fungus, the substrate contains 20 mg or more of ergothioneine per 100 g of dry mass, and the substrate contains less than 10 mass % of protein.

[0010] The filamentous fungus is preferably a non-mycotoxin producing fungus.

[0011] Preferably, the substrate is wheat bran having a protein content of less than 10% by weight.

[0012] In the above-mentioned production method, it is preferable to ventilate the substrate during solid culture.

[0013] The non-mycotoxin-producing fungus is preferably one or more fungi selected from the group consisting of Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus niger, Aspergillus awamori, and Monascus pilosus.

[0014] It is preferable that the substrate is inoculated with the filamentous fungus and cultured on a solid state for 49 hours or more and less than 96 hours, and water is added during the solid culture so that the water content of the substrate is maintained within the range of 50 to 60% by mass. Effect of the Invention

[0015] According to the present invention, it is possible to provide a solid culture of a filamentous fungus in which the ergothioneine content is improved compared to conventional methods, even when a substrate with a low protein content is used and the culture time is less than 96 hours, and a production method thereof. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a diagram showing an example of a solid culture apparatus used in a method for producing a solid culture of a filamentous fungus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, an embodiment of the present invention will be described, however, the present invention is not limited to the following embodiment.

[0018] <Method for producing solid culture of filamentous fungi>

[0019] The method for producing a solid culture of a filamentous fungus according to the present disclosure involves inoculating a filamentous fungus into a substrate and culturing the substrate in a solid state for a culture time of less than 96 hours to produce a solid culture of a filamentous fungus containing 20 mg or more of ergothioneine per 100 g of dry mass, the substrate having a protein content of less than 10% by mass.

[0020] According to the method for producing a solid culture of a filamentous fungus disclosed herein, a solid culture of a filamentous fungus containing ergothioneine at a higher concentration can be obtained by inoculating a filamentous fungus into a substrate containing less than 10% by mass of protein and culturing the filamentous fungus in a solid state with aeration.

[0021] Since the protein content of the substrate is less than 10% by mass, the content of sulfur-containing amino acids such as histidine, methionine, and cysteine ​​is not high. The sulfur-containing amino acids are involved in the ergothioneine biosynthesis pathway. A substance containing sulfur-containing amino acids may be added to the substrate and solid culture may be performed. The raw material that serves as the source of sulfur-containing amino acids is not particularly limited, but examples thereof include fish meal and soybeans. The substance containing sulfur-containing amino acids may be added during the raw material treatment process, the filamentous fungus inoculation process, the solid culture process, or all of these processes.

[0022] Furthermore, by making the pH of the substrate acidic (pH 2 to 4), the proliferation of bacteria other than the target filamentous fungus can be suppressed. For this reason, a pH adjuster may be added to the substrate before solid culture. The pH can be adjusted, for example, by adding a pH adjuster. There is no particular limitation on the pH adjuster, and examples of the pH adjuster include acidic compounds such as citric acid, calcium carbonate, sodium carbonate, phosphoric acid, succinic acid, tartaric acid, and lactic acid. The pH can be adjusted during the raw material treatment process, during the filamentous fungus inoculation process, during the solid culture process, or during all of these processes.

[0023] The substrate temperature during the culture is preferably 10 to 55°C, more preferably 15 to 50°C. The substrate temperature may be different between a certain time from the start of the culture and after the certain time has elapsed. For example, it is preferable to set the substrate temperature differently before the bacterial cell growth phase, during the bacterial cell growth phase, and after the bacterial cell growth phase. The bacterial cell growth phase refers to a time period during which the number of bacterial cells increases exponentially and heat generation is vigorous. Note that the term "before the bacterial cell growth phase" does not include the start of the bacterial cell growth phase. Furthermore, the term "after the bacterial cell growth phase" does not include the end of the bacterial cell growth phase.

[0024] The substrate temperature is not particularly limited, but for example, before the bacterial cell growth phase, it is preferably adjusted to 25° C. or higher and lower than 35° C. During the bacterial cell growth phase, it is preferably adjusted to 30° C. or higher and 40° C. or lower. After the bacterial cell growth phase, it is preferably adjusted to 25° C. or higher and 40° C. or lower, and more preferably adjusted to 30° C. or higher and 40° C. or lower.

[0025] The culture time in the present method for solid culture of filamentous fungi may be until the surface of the substrate is covered with mycelia of the filamentous fungus. The drying step is not included in the culture time. A specific culture time is less than 96 hours. The culture time is preferably 84 hours or less, and more preferably 72 hours or less. The lower limit of the culture time is, for example, preferably 30 hours or more, more preferably 40 hours or more, and even more preferably 49 hours or more.

[0026] In the production of a solid culture of filamentous fungi, it is preferable to adjust the moisture content of the substrate. Examples of a method for adjusting the moisture content include a method using water spray or drying. Water spray or drying may be performed during solid culture or after the solid culture step. By spraying water during the solid culture step, the water activity suitable for the growth of filamentous fungi can be maintained, and the growth and enzyme production of filamentous fungi can be made more active. A specific moisture content is preferably 40 to 70% by mass, more preferably 45 to 65% by mass, and even more preferably 50 to 60% by mass. From the viewpoint of improving the storage stability of the solid culture of filamentous fungi, the moisture content of the substrate can be adjusted by drying after the solid culture step. This prevents deterioration of the solid culture of filamentous fungi and other mixed materials, and enables stable quality and long-term storage.

[0027] The filamentous fungus used in the present filamentous fungal solid culture is not particularly limited as long as it is not harmful to animals that ingest it and grows by assimilating the substrate. Examples of such filamentous fungi include the Aspergillus and Monascus genera. Of these, from the viewpoint of use as food, it is preferable that the filamentous fungus is a non-mycotoxin-producing fungus. As a non-mycotoxin-producing fungus, for example, a fungus in which a gene related to mycotoxin production is not expressed due to the accumulation of genetic factors such as mutation, deletion, or transcriptional repression of a gene involved in mycotoxin biosynthesis and thus the ability to produce mycotoxin can be preferably used. Specifically, examples of such fungi include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus niger, Aspergillus awamori, and Monascus pilosus. These fungi are commercially available as seed cultures for fermenting fermented foods, and can also be purchased from the National Institute of Technology and Evaluation, National Biotechnology Center (NBRC). The above-mentioned fungi may be wild-type strains that have not been genetically modified, or may be genetically modified by genetic engineering techniques to increase the production of ergothioneine, as described below.

[0028] Examples of mycotoxins include aflatoxin, ochratoxin, zearalenone, and sterigmatocystin.

[0029] The solid culture of filamentous fungi may contain live cells of filamentous fungi, may contain killed cells of filamentous fungi, or may contain both of them.

[0030] For filamentous fungi, a mutant strain that produces more ergothioneine than a wild-type strain may be used by utilizing a known genetic engineering technique. Examples of the method for introducing mutations include self-cloning, ultraviolet (UV) or X-ray irradiation, and treatment with an alkylating agent.

[0031] The substrate may be pretreated before inoculating the filamentous fungus (pretreatment step). The method for pretreatment is not particularly limited, and known methods can be used. For example, water is added to the substrate and the substrate is stirred, followed by steaming and cooling to a temperature at which the substrate can be inoculated with the filamentous fungus.

[0032] The method of inoculating the substrate with the filamentous fungus is not particularly limited, and a known method can be used (filamentous fungus inoculation step). For example, the substrate after the above-mentioned pretreatment step is inoculated by scattering a seed fungus (spores) of the filamentous fungus. The filamentous fungus to be inoculated is not particularly limited, and the above-mentioned filamentous fungus can be used, for example.

[0033] The method for steaming the substrate is not particularly limited, and examples thereof include a method in which water is added in an amount preferably 0.5 to 1.5 times, more preferably 0.7 to 1.3 times the mass of the substrate, followed by stirring and steaming. The steaming temperature is not particularly limited, but is preferably 60 to 160° C., more preferably 90 to 130° C. The steaming time is also not particularly limited, but is preferably 0.5 to 90 minutes, more preferably 2 to 60 minutes.

[0034] The number of spores to be inoculated into the steamed substrate with the filamentous fungus is not particularly limited, but may be, for example, 1.0×10 per 1 g of substrate. 3 ~1.0×10 9 The number of cells is preferably 1.0 × 10 per gram of substrate. 4 ~1.0×10 8 It is more preferable to have one.

[0035] In the solid culture step, when the substrate inoculated with the filamentous fungus is solid cultured, for example, the apparatus shown in FIG. 1 may be used, although it is not essential. The apparatus includes a culture chamber 1 having a culture bed 2 therein on which the substrate 5 is piled up, and an air conditioner 10 capable of blowing air with adjusted temperature and humidity from below the culture bed 2. The culture chamber 1 is divided into an upper chamber 3 and a lower chamber 4 by the culture bed 2. The culture bed 2 is provided with a plurality of openings, and the air supplied from the air conditioner 10 is sent to the lower chamber 4, passes through the openings, passes between the substrates, and escapes to the upper chamber 3. The upper chamber 3 is provided with a product temperature sensor 6 that measures the product temperature of the substrate, and the lower chamber 4 is provided with a humidity sensor 7 that measures the humidity of the air sent to the lower chamber 4.

[0036] When air is supplied to the substrate 5 , a part or all of the air may be exhausted to the outside through an exhaust duct 8 , or a part or all of the air may be circulated through a circulation duct 9 .

[0037] The solid culture apparatus may be provided with a two-fluid nozzle 11 for adjusting humidity and a steam nozzle 12 for adjusting temperature, as in the apparatus of FIG. 1, and may further be provided with a cooling section 13 or a heating section 14. The cooling section may be any section capable of cooling the aspirated gas, for example, a heat exchanger that passes a refrigerant therethrough. The heating section may be any section capable of heating the aspirated gas, for example, a heat exchanger or heater that passes a heating medium therethrough. The heat exchanger may be, for example, a fin-tube type heat exchanger. The refrigerant may be a liquid such as cold water, or a gas such as a refrigerant gas. The heating medium may be a liquid such as hot water, a gas such as hot air, or steam.

[0038] In the process of solid-state culturing a substrate with a filamentous fungus, the temperature of the substrate changes as the culture progresses as the filamentous fungus grows and produces ergothioneine. Therefore, it is preferable to perform solid culture by adjusting at least one of the temperature, humidity, and air volume of the air supplied to the substrate so that the culture progresses at a desired product temperature. This makes it possible to accurately control the temperature and humidity during culture, and allows the filamentous fungus to grow efficiently while suppressing the growth of miscellaneous bacteria. In addition, by controlling the product temperature of the substrate, the concentration of ergothioneine contained in the solid culture of the filamentous fungus can be increased.

[0039] The product temperature of the substrate is preferably within the range of 10 to 55°C, and more preferably within the range of 15 to 50°C, from the start to the end of the culture.

[0040] The air flow rate of the air supplied to the substrate is preferably 1 cm / sec or more during the first 12 to 24 hours of the culture, when the heat generated by the bacteria is high. The air flow rate of the air passing through the substrate is preferably 1 to 25 cm / sec, and more preferably 3 to 20 cm / sec.

[0041] The humidity of the air supplied to the substrate is preferably 50 to 99%, more preferably 70 to 99%, in terms of relative humidity.

[0042] <Solid culture of filamentous fungi> The filamentous fungal solid culture according to this embodiment is produced by the above-mentioned method for solid culture of filamentous fungi, using a substrate containing less than 10% by mass of protein.

[0043] The filamentous fungal solid culture has an ergothioneine content of, for example, 20 mg or more, preferably 20 to 100 mg, more preferably 25 to 90 mg, even more preferably 30 to 90 mg, and even more preferably 30 to 80 mg per 100 g of dry mass of the culture. Thus, the filamentous fungal solid culture according to this embodiment contains a high concentration of ergothioneine.

[0044] The filamentous fungal solid culture can be used as it is, or can be mixed with other materials to form a composition. The other components contained in the composition containing the filamentous fungal culture are not particularly limited, and other raw materials and additives that are usually used in general foods and feeds can be appropriately mixed. The amount of the mixture can be appropriately adjusted depending on the desired effect. In addition, the composition may contain various additives such as antioxidants, pH adjusters, flavorings, various esters, organic acids, organic acid salts, inorganic acids, inorganic acid salts, inorganic salts, pigments, emulsifiers, preservatives, seasonings, or quality stabilizers.

[0045] The solid culture of filamentous fungi or the above-mentioned composition may be fed to animals as it is, or may be included in other foods or animal feeds.Furthermore, the solid culture of filamentous fungi or the above-mentioned composition may be included in food additives or animal feed additives to be mixed with other foods or animal feeds.

[0046] The subjects to which the filamentous fungal solid culture can be given are not particularly limited, and examples thereof include humans, livestock such as cows, pigs, goats, and poultry, crustaceans such as shrimp and crabs, fish (including farmed fish), etc. Poultry includes, for example, chickens, ducks, geese, etc. EXAMPLES

[0047] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. <Example 1> After adding water to wheat bran and stirring, the wheat bran was subjected to steaming treatment under the condition of 0.2 MPa. The steamed wheat bran was cooled until it reached around 30°C, and then a certain amount of inoculum of Aspergillus oryzae (AOK11) that does not produce mycotoxin was inoculated as the koji mold for brewing and mixed uniformly. The moisture content of the wheat bran at the time of inoculation was adjusted to 60%. The Aspergillus oryzae of the inoculum is a non-mycotoxin-producing fungus. The substrate inoculated with this inoculum was placed on the culture bed of the solid culture device, leveled so that the thickness of the deposited substrate was uniform, and then the culture was started. During the culture, air with controlled temperature and humidity was supplied from the lower part of the culture bed. Also, from 12 to 24 hours after the start of the culture when the heat generation of the fungus was large, the temperature of the substrate was strictly controlled by ventilating the deposited substrate at a linear velocity of about 5 to 20 cm / second. The temperature of the air supplied was controlled so that the temperature of the substrate was 30°C from the start of the culture to 8 hours of culture, 38°C from 14 hours of culture to 26 hours of culture, and 30°C after 30 hours of culture. The relative humidity of the air supplied was controlled to be in the range of 90 to 99%. During the culture, the substrate was appropriately stirred using the stirrer equipped in the solid culture device to make the culture state uniform. The culture was carried out for 48 hours, and it was visually confirmed that the surface of the wheat bran granules was sufficiently covered with the mycelia of the filamentous fungus. The moisture content of the substrate during the culture was 30 to 60% by mass. Furthermore, for the purpose of adjusting the moisture content of the substrate, air drying was carried out overnight to obtain a solid culture of filamentous fungus.

[0048] The ergothioneine analysis method was carried out using a well-known method. 2 g of the sample was weighed, extracted by heating and refluxing with water twice at 100°C for 1 hour, centrifuged, the supernatant was recovered, fixed to 200 ml, concentrated to 20 ml, diluted 10-fold with a mixed solution of ethanol and water (7:3), and HPLC analysis was carried out. <HPLC Analysis Conditions> Column: TSKgel Amide-80, inner diameter 4.6 mm × 250 mm, particle size 5 μm Mobile phase: Acetonitrile and 10 mmol / l ammonium acetate solution (8:2) Flow rate: 1.0 ml / min Column temperature: 40°C Measurement wavelength: 258nm

[0049] The ergothioneine content of the solid culture of the filamentous fungus was 34.8 mg per 100 g of dry mass.

[0050] The ergothioneine concentration per 100 g of dry mass was calculated by dividing the filamentous fungal solid culture by the moisture content. Specifically, the following formula was used. X is the ergothioneine content per 100 g of dry mass, Y is the ergothioneine content per 100 g of the filamentous fungal solid culture containing moisture, and A is the moisture content (%) of the filamentous fungal solid culture containing moisture. X = Y ÷ (100 - A) × 100

[0051] The linear velocity during culture was calculated by measuring the flow rate using an anemometer in FIG. 1 and dividing it by the cross-sectional area of ​​the culture bed. The cross-sectional area of ​​the culture bed is the area of ​​the cross section defined by the side wall 15 and the bottom surface of the cylindrical culture bed 2. The following formula was used for the calculation. X is the linear velocity (cm / sec), and Y is the value measured by the area type flow meter (m 3 / min), and A is the cross-sectional area of ​​the culture bed of the solid-state culture device (mm 2 ). X = (Y × 10 8 ÷60)÷A

[0052] The moisture content during cultivation was calculated by drying the sample at 90°C for 17 hours, calculating the moisture content from the difference before and after drying, and dividing the moisture content by the total mass before drying. Specifically, the following formula was used. Moisture content = total mass before drying - total mass after drying Moisture content (%) = water content ÷ total mass before drying × 100

[0053] <Example 2> Wheat bran was steamed at 0.2 MPa after adding water and stirring. The steamed wheat bran was cooled to around 30°C, and a certain amount of Aspergillus oryzae (AOK11), a type of koji mold for sake brewing that does not produce mycotoxins, was inoculated and mixed uniformly. The moisture content of the wheat bran at the time of inoculation was set to 60%. The inoculated Aspergillus oryzae is a type of fungus that does not produce mycotoxins. The substrate inoculated with this inoculation was placed in the culture bed of a solid culture device, and the thickness of the accumulated substrate was evenly distributed before starting the culture. During the culture, air with controlled temperature and humidity was supplied from the bottom of the culture bed. In addition, during the first 12 to 24 hours of culture, when the heat generation of the fungus is high, the temperature of the substrate was strictly controlled by ventilating the accumulated substrate at a linear velocity of about 5 to 18 cm / sec. The temperature and humidity of the supplied air were controlled so that the substrate temperature was 30°C from the start of the culture until 8 hours of culture, 38°C from 14 hours to 26 hours of culture, and 30°C after 30 hours of culture. The relative humidity of the supplied air was controlled to be in the range of 90 to 99%. During the culture, the substrate was appropriately stirred using a stirrer attached to the solid culture device to ensure a uniform culture state. Water was added using a watering nozzle at the timing of stirring, and the moisture content of the substrate was maintained in the range of 50 to 60 mass%. The culture was carried out for 48 hours, and it was visually confirmed that the hyphae of the filamentous fungus were sufficiently covering the surface of the wheat bran granules. Furthermore, in order to adjust the moisture content of the substrate, it was blown dry overnight to obtain a filamentous fungal solid culture product.

[0054] The ergothioneine content of the solid culture of filamentous fungus was 32.0 mg per 100 g of dry mass. The ergothioneine content, linear velocity during culture, water content, and dry mass of the culture were measured by the above-mentioned methods and calculation formulas.

[0055] <Example 3> Wheat bran was steamed at 0.2 MPa after adding water and stirring. The steamed wheat bran was cooled to around 30°C, and a certain amount of Aspergillus oryzae (AOK11), a type of koji mold for sake brewing that does not produce mycotoxins, was inoculated and mixed uniformly. The moisture content of the wheat bran at the time of inoculation was set to 60%. The inoculated Aspergillus oryzae is a type of fungus that does not produce mycotoxins. The substrate inoculated with this inoculation was placed in the culture bed of a solid culture device, and the thickness of the accumulated substrate was evenly distributed before starting the culture. During the culture, air with controlled temperature and humidity was supplied from the bottom of the culture bed. In addition, during the first 12 to 24 hours of culture, when the heat generation of the fungus is large, the product temperature of the substrate was strictly controlled by ventilating the accumulated substrate at a linear velocity of about 5 to 20 cm / sec. The temperature and humidity of the supplied air were controlled so that the substrate temperature was 30°C from the start of the culture until 8 hours of culture, 38°C from 14 hours to 26 hours of culture, and 30°C after 30 hours of culture. The relative humidity of the supplied air was controlled to be in the range of 90 to 99%. During the culture, the substrate was appropriately stirred using a stirrer attached to the solid culture device to ensure a uniform culture state. Water was added using a watering nozzle at the timing of stirring, and the moisture content of the substrate was maintained in the range of 50 to 60 mass%. The culture was carried out for 72 hours, and it was visually confirmed that the hyphae of the filamentous fungus were sufficiently covering the surface of the wheat bran granules. Furthermore, in order to adjust the moisture content of the substrate, it was blown dry overnight to obtain a filamentous fungal solid culture product.

[0056] The ergothioneine content of the solid culture of filamentous fungus was 72.0 mg per 100 g of dry mass. The ergothioneine content, linear velocity during culture, water content, and dry mass of the culture were measured by the above-mentioned methods and calculated by the above-mentioned formulas.

Claims

1. Inoculating the substrate with filamentous fungi; By solid culture for less than 96 hours, A method for producing a solid culture of a filamentous fungus containing ergothioneine at least 20 mg per 100 g of dry mass, The substrate has a protein content of less than 10% by weight. A method for producing a solid culture of filamentous fungi.

2. 2. The method for producing a solid culture of a filamentous fungus according to claim 1, wherein the substrate is wheat bran having a protein content of less than 10% by mass.

3. 2. The method for producing a solid culture of a filamentous fungus according to claim 1, wherein the substrate is aerated during the solid culture.

4. The method for producing a solid culture of a filamentous fungus according to claim 1, wherein the filamentous fungus is a non-mycotoxin producing fungus.

5. 5. The method for producing a solid culture of a filamentous fungus according to claim 4, wherein the non-mycotoxin-producing fungus is one or more fungi selected from the group consisting of Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus niger, Aspergillus awamori, and Monascus pilosus.

6. 2. The method for producing a solid culture of a filamentous fungus according to claim 1, wherein the substrate is inoculated with the filamentous fungus and solid culture is performed for 49 hours or more and less than 96 hours, and water is added during the solid culture so that the water content of the substrate is maintained in the range of 50 to 60 mass%.

7. The substrate is a filamentous fungus cultivated in solid state, Contains 20 mg or more of ergothioneine per 100 g of dry mass, The substrate is less than 10% by weight protein. Solid culture of filamentous fungi.

Images