Method for producing aspergillus tissue material

Abstract

To provide a method for producing an Aspergillus tissue material with a high content of γ-aminobutyric acid.SOLUTION: A method for producing an Aspergillus tissue material enriched with γ-aminobutyric acid, including subjecting a mycelium of Aspergillus to anaerobic treatment, and a method for producing an Aspergillus tissue material enriched with γ-aminobutyric acid, including adding acetic acid into a liquid medium of Aspergillus 1 to 24 hours prior to completion of the culture of Aspergillus in the liquid medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a koji mold tissue product, and particularly to a method for producing a koji mold tissue product enriched with γ-aminobutyric acid. [Background technology]

[0002] Research into alternative protein sources is progressing to solve food shortages caused by a growing global population and the environmental impact of livestock production. Alternative protein sources include plant-based meat made from soybeans and peas, cultured meat produced artificially through cell culture, edible insects such as crickets, and fermentation-derived proteins.

[0003] Fungi used as a source of fermentation-derived protein have the advantages of being fast growing and capable of multiplying in a short period of time, requiring few resources (water and land) and therefore having a low environmental impact, and their mycelium has a fibrous structure that makes it easy to create a fibrous texture, and being rich in nutrients. Technologies using koji mold, a fungus that has been used for many years for various purposes in traditional Japanese cuisine, are also being developed. This makes it a viable alternative protein source from the perspectives of safety and consumer sentiment.

[0004] Patent Document 1 discloses a method for producing koji fungus cells for use as a meat substitute, which includes a step of initiating cultivation of koji fungus in a liquid medium, and then recovering and washing the grown fungus cells.

[0005] γ-Aminobutyric acid is known as an inhibitory neurotransmitter. Its known effects include lowering blood pressure, promoting brain metabolism, tranquilizing the mind, and promoting growth hormone secretion, and the development of foods enriched with this substance has been intensified. Fermentation is a method for producing large amounts of γ-aminobutyric acid. Patent Document 2 discloses a method for producing γ-aminobutyric acid by culturing the koji mold Aspergillus oryzae. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2021-023172 [Patent Document 2] Japanese Patent Application Publication No. 10-165191 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for producing a koji mold tissue product with a high content of γ-aminobutyric acid. Patent Document 1 does not disclose any adjustment of the content of specific nutritional components in the koji mold cells produced as a meat substitute. Furthermore, Patent Document 2 only discloses a method for producing γ-aminobutyric acid as a compound using koji mold. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have been studying the culture conditions of koji mold in the production of koji mold tissue products, and have found a method for increasing the content of γ-aminobutyric acid (referred to herein as "GABA") in the resulting koji mold tissue product. Based on this finding, the present inventors have conducted further studies and have completed the present invention. Representative aspects of the present invention include the following.

[0009] <1> A method for producing a koji mold tissue product enriched in γ-aminobutyric acid, comprising anaerobic treatment of koji mold mycelium. <2> The anaerobic treatment is carried out at 20°C to 45°C for 1 hour to 36 hours. <1> The manufacturing method described in <3> The anaerobic treatment is carried out by wrapping the mycelium in a film at 20°C to 45°C for 1 hour to 36 hours. <1> The manufacturing method described in <4> The anaerobic treatment is followed by sterilization. <1> ~ <3> 1. The manufacturing method according to any one of the preceding claims. <5> obtaining the mycelium by culturing koji mold in a liquid medium; <1> ~ <4> 1. The manufacturing method according to any one of the preceding claims. <6> The method includes obtaining the mycelium by culturing the following (1) to (3) in this order: <1> ~ <4> a manufacturing method according to any one of the methods; (1) Initiating the cultivation of a starter koji mold in a liquid medium containing at least a carbon source; (2) adding a first nitrogen source to the liquid medium after the koji mold starts to grow in the liquid medium; and (3) After the addition of the first nitrogen source, the culture is continued for the time required for the production of a protein using the first nitrogen source as a raw material. <7> The method includes culturing koji mold in a liquid medium to obtain mycelium, and adding acetic acid to the liquid medium 1 hour to 24 hours before the end of the culture. A method for producing a koji mold tissue product enriched with γ-aminobutyric acid. <8> A koji mold tissue product containing koji mold mycelium, The koji mold tissue product contains 6.0 mg / g or more of γ-aminobutyric acid relative to the total dry mass of the koji mold tissue product, The koji mold tissue, wherein the gamma-aminobutyric acid is present inside the mycelium. <9> The koji mold tissue product contains 40% by mass to 70% by mass of protein based on the total dry mass of the koji mold tissue product. <8> The koji mold tissue product according to claim 1. <10> edible <8> or <9> The koji mold tissue product according to claim 1. [Effects of the Invention]

[0010] The present invention provides a method for producing a koji mold tissue product having a high GABA content. The production method of the present invention makes it possible to produce a koji mold tissue product having a high GABA content by a simple procedure at low cost. [Brief explanation of the drawings]

[0011] [Figure 1] The figures show the GABA content (1) and glutamic acid content (2) of koji mold tissue obtained by leaving it to stand under aerobic or anaerobic conditions (wrapped in plastic wrap). [Figure 2] The GABA content of koji mold tissue obtained after anaerobic treatment (wrap film) for different periods of time is shown. [Figure 3]The GABA content of koji mold tissue obtained after anaerobic treatment (wrap film) at different temperatures is shown. [Figure 4] The GABA content of koji mold tissue obtained by anaerobic treatment using a de-enzyme agent is shown. [Figure 5] The GABA content of koji mold tissue obtained by anaerobic treatment (wrap film) and koji mold tissue obtained by adding GABA is shown before and after washing with water. [Figure 6] The GABA content of koji mold tissue samples obtained by adding various acids at the start of culture or 4 hours before the end of culture (1) and the glutamic acid content of koji mold tissue samples obtained by adding various acids 4 hours before the end of culture (2) are shown. [Figure 7] The GABA content of koji mold tissue samples obtained by adding acetic acid at different times before the completion of the culture is shown. [Figure 8] The GABA content of Aspergillus oryzae tissue samples obtained by adding different amounts of acetic acid before the completion of the incubation is shown. [Figure 9] The protein content of mycelia obtained by culturing in a liquid medium containing 2% sake lees and 2% raw sugar or in a liquid medium containing 2% brown rice flour with yeast extract added is shown. [Figure 10] The protein content of mycelia obtained by culturing in a liquid medium containing 2% sake lees and 2% raw sugar with different amounts of yeast extract added is shown. [Figure 11] The protein content of mycelia obtained by culturing in liquid medium containing 2% or 4% raw sugar with different amounts of yeast extract added is shown. [Figure 12] The protein content of mycelia obtained by culturing in a liquid medium containing 2% sake lees and 2% raw sugar, to which casamino acids or peptone were added, is shown. [Figure 13] The wet weight and protein content of the mycelium obtained by culturing in a liquid medium containing 2% sake lees and 2% raw sugar to which yeast extract or casamino acids were added 24 hours or 4 hours before harvesting the koji mold tissue are shown. [Figure 14] The wet weight and protein content of mycelia obtained by culturing in a liquid medium containing 2% sake lees and 2% raw sugar with the addition of yeast extract are shown. [Figure 15]The wet weight and protein content of mycelia obtained by culturing in a liquid medium containing 2% sake lees and 2% raw sugar with yeast extract added at different times are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. Hereinafter, when "%" is used to describe a component in a liquid medium, it means a mass volume percent concentration relative to the volume of the liquid medium (w / v % concentration) unless otherwise specified.

[0013] <Anaerobic treatment> The present invention relates to a method for producing a koji mold tissue product, which comprises anaerobic treatment of koji mold mycelium. The mycelium of koji mold may contain functional components such as amino acids, vitamins, and hydrocarbon compounds. The present inventors have found that the GABA content in the mycelium increases through anaerobic treatment. Herein, koji mold mycelium with a high GABA content (e.g., koji mold mycelium in which the GABA content has been increased by a specific treatment compared to that without the specific treatment) may be referred to as a GABA-enriched koji mold tissue product or simply as a koji mold tissue product. That is, a koji mold tissue product may contain or consist of mycelium.

[0014] GABA is produced from L-glutamic acid by the action of glutamic acid decarboxylase. For example, Japanese Patent Publication No. 2009-39082 discloses that raw mushroom fruiting bodies are placed under anaerobic conditions to convert a portion of the glutamic acid contained in the mushrooms to GABA, thereby increasing the GABA content of the mushrooms. However, Japanese Patent Publication No. 2009-39082 does not disclose the GABA content in mushroom mycelium. It is believed that at least a portion of the glutamic acid is converted to GABA in the mycelium, as shown in the examples below. The present invention is the first to discover that the GABA content in mycelium increases through anaerobic treatment. Because GABA is produced within the mycelium of koji mold, washing the product after anaerobic treatment is unique in that, unlike compositions obtained by adding GABA to the mycelium later, the GABA content does not essentially decrease.

[0015] Anaerobic treatment is carried out by placing mycelia under conditions of lower oxygen concentration than under air conditions.

[0016] The method for obtaining the mycelia is not particularly limited, but it is preferably mycelia of koji mold that can be used as an alternative protein source. As described below, it is particularly preferred that the mycelia be obtained by culturing koji mold in a liquid medium containing a carbon source and a nitrogen source. The mycelia to be anaerobically treated may be in the medium in which the culture was performed or may be recovered from the medium. In this case, the mycelia may be mycelia immediately after recovery from the medium or may be mycelia that have been statically cultured for a certain period of time after recovery. For example, the mycelia may be mycelia that have formed in the medium during koji mold culture. The recovery method for the cultured mycelia is not particularly limited, and any known separation method may be used. For example, centrifugation, filtration separation, compression separation, or the like may be appropriately used. The mycelia may be in a state in which they have been separated from the medium and moisture and the like have been removed to a certain extent. The mycelia to be subjected to the anaerobic treatment may be washed with purified water or the like after recovery, or may be suspended in water or an aqueous solution containing other components.

[0017] The method for placing mycelia under conditions of lower oxygen concentration than under air conditions is not particularly limited, but examples include a method in which mycelia separated from the culture medium and with a certain amount of moisture and the like removed are stored in an airtight container or bag, a method in which mycelia are stored in an airtight container or bag with an oxygen absorber, and a method in which mycelia are placed under a nitrogen or carbon dioxide atmosphere. Furthermore, since the mycelia can be prepared in a shape such that the surface easily adheres to a wrap, wrapping them in food wrap film also consumes oxygen within the wrap film, thereby creating a low oxygen concentration condition. While the wrap film is not particularly limited, a wrap film with a high gas barrier property is preferred, and for example, polyvinylidene chloride film is preferably used. Anaerobic treatment of mycelia occurring in the culture medium can be carried out, for example, by stopping the oxygen supply, such as aeration. After the completion of the culture, the oxygen supply may be stopped and the mycelia may be allowed to stand in the culture medium.

[0018] The anaerobic treatment is preferably carried out at a temperature at which glutamic acid decarboxylase is active, specifically, at 5°C to 60°C, preferably 10°C to 50°C, and more preferably 20°C to 45°C.

[0019] Anaerobic treatment may be carried out for, for example, 30 minutes to 40 hours, preferably 1 hour to 36 hours. Anaerobic treatment for 30 minutes or longer facilitates the effect of increasing the amount of GABA. Furthermore, limiting the anaerobic treatment time to 40 hours or less can prevent a decrease in the GABA content. Since the amount of GABA may decrease due to the action of enzymes if the mycelium is placed under aerobic conditions after anaerobic treatment, it is preferable to inactivate the enzymes immediately after anaerobic treatment. Enzyme inactivation can be carried out by a drying process such as freeze-drying or a sterilization process. Examples of sterilization processes include high-temperature treatment and UV irradiation.

[0020] <Acetic acid added> In another aspect of the present invention, the present inventors have found that in a method for producing a koji mold tissue product, which comprises culturing koji mold to obtain mycelium, the GABA content can also be increased by adding acetic acid to the liquid medium 24 hours to 1 hour before the end of the culture. Here, the end of the culture refers to, for example, the time when recovery of the cultured mycelium begins (the time when the mycelium is separated from the medium) or the time when the koji mold is inactivated. Acetic acid is added preferably at least 18 hours, more preferably at least 12 hours, and even more preferably at least 6 hours before the end of the culture, and preferably at least 2 hours, and even more preferably at least 3 hours before the end of the culture. Acetic acid may be added in an amount such that the concentration in the liquid medium after addition is 1 mM to 100 mM, preferably 2 mM to 50 mM, more preferably 3 mM to 40 mM, and even more preferably 4 mM to 30 mM. The pH of the liquid medium should be adjusted to about 4.0 to 5.5 by adding acetic acid. The mycelia obtained by culturing with the addition of acetic acid can be provided as a koji mold tissue product by sterilization, washing, recovery, etc., in the same manner as the mycelia obtained by culturing koji mold described in this specification. The mycelium obtained by the culture with the addition of acetic acid as described above may be further subjected to the above-mentioned anaerobic treatment to obtain a koji mold tissue product enriched in GABA.

[0021] <Koji mold> As for koji mold, any koji mold that has traditionally been used in the production of sake, miso, soy sauce, mirin, shochu, etc. can be used. Specifically, strains such as Aspergillus oryzae, Aspergillus sojae, Aspergillus awamori, Aspergillus kawachii, Aspergillus glaucus, Aspergillus tamari, Aspergillus luchuensis, and Aspergillus niger can be used. Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachii, and Aspergillus leuconoensis are preferred, with Aspergillus oryzae being more preferred, as they are safe microorganisms that have been used in the Japanese industrial sector for brewing soy sauce, miso, and other products. Commercially available koji molds can be used for cultivation, and can be purchased, for example, from the Biotechnology Center of the National Institute of Technology and Evaluation.

[0022] <Cultivation: Aspergillus oryzae> The following describes how mycelia are obtained by culturing. The koji mold used as a starter may be a mycelium tissue obtained by liquid culture of koji mold, or a collection of spores on which koji mold grows by solid culture, or koji obtained by growing and propagating koji mold on grains such as rice, beans, or wheat, or a processed product thereof. The koji mold used as a starter is preferably one that contains at least spores that are normally used as "seed koji."

[0023] In the culture, for example, commercially available koji mold cultured by a fungal culture method known to those skilled in the art can be used as a starter. To produce koji mold in a large-capacity culture tank, mycelium is formed in a small culture tank and then subcultured in stages. Culture to obtain a starter can be carried out, for example, on a plate medium for fungal culture (such as PDA agar medium) under heating. For example, the culture temperature is 20 to 40°C, and the culture time is approximately 72 to 192 hours. The koji mold after culture or spores obtained after culture can be used as a starter. When using spores, they can be used as a suspension at a desired concentration. For example, the spores can be used as a suspension in sterilized water containing a surfactant such as Tween 20 or Tween 80.

[0024] The number of spores can be measured by dropping the spore suspension onto a hemocytometer (such as a Thoma counting chamber) and counting them under a microscope using standard methods. Alternatively, an automatic particle counting device used for the same purpose may be used.

[0025] In the culture, the starter koji mold can be added to the liquid medium to start the culture. When spores are used, the amount of koji mold added to the liquid medium at the start of the culture is, for example, 10 3 pieces / ml~10 10 / ml, preferably 10 4 pieces / ml~10 7 / ml, more preferably 10 4 pieces / ml~10 6 It is sufficient if it is 1 / ml.

[0026] <Culture: Medium> The culture is preferably carried out in a liquid medium. The liquid medium is not particularly limited as long as it contains components that serve as carbon and nitrogen sources necessary for culturing the koji mold. Examples of carbon sources include sugars such as glucose, maltose, sucrose, lactose, starch, and blackstrap molasses. Unrefined raw sugar is particularly preferred as a sugar. Carbon sources may also be sources other than sugars, and may also contain proteins, vitamins, and the like. For example, fermentation residues such as sake lees, soy sauce lees, beer lees, and shochu lees, as well as rice bran, wheat bran, and residues of other grain (rice, barley, beans, etc.) powders or paste-processed grains, can also be used. These are grain derivatives with relatively limited uses, and are preferred from the perspective of effective resource utilization. Of these, fermentation residues or raw sugars can be preferably added to the liquid medium. Fermentation residues and raw sugars may be added alone, or two or more of them may be added. When two or more of them are used, for example, sake lees and raw sugars are preferred.

[0027] Examples of nitrogen sources that can be used include amino acids, peptides, inorganic nitrogen compounds (e.g., ammonia, ammonium sulfate, ammonium chloride, etc.), and mixtures thereof. It is preferable to use a component containing amino acids or low-molecular-weight peptides as the nitrogen source, and particularly preferred is an amino acid-containing component containing a high concentration of amino acids or low-molecular-weight peptides. Examples of amino acid-containing components include yeast extract, malt extract, barley fermentation extract, meat extract, casamino acids, and peptone. Protein-containing fermentation residues, such as those listed in the previous section, can also be used as nitrogen sources. Preferred nitrogen sources include yeast extract, malt extract, barley fermentation extract, fermentation residue, or a mixture of two or more of these, with yeast extract being more preferred. Extracts are extracted components; for example, yeast extract is an extracted component of the chemical or physical hydrolysis of yeast cells. Casamino acids are casein acid hydrolysates, and peptones are enzymatic casein hydrolysates, enzymatic soy protein hydrolysates, etc. According to the document (Basic Bioengineering Course, Bio Trivia "Do You Know the Ingredients of Culture Media?", 2011, No. 4, p. 195), yeast extract and meat extract contain 68% and 80% peptides and amino acids, respectively, while the peptones Bacto Tryptone (derived from milk casein), Bacto peptone (derived from meat), and Bacto Soytone contain approximately 81%, 97%, and 59% peptides and amino acids, respectively.

[0028] In the production of koji mold mycelium using sake lees or brown rice flour as ingredients in a liquid medium, the protein content of the resulting mycelium can be increased by adding about 1% of yeast extract, casamino acids, peptone, or a mixture of two or more of these to the liquid medium, thereby achieving a protein content of, for example, more than 40% by mass. Because ingredients such as yeast extract, which contain high concentrations of amino acids and low-molecular-weight peptides, are expensive, obtaining koji mold tissue products with a high protein content in a medium containing these ingredients at 1.5% or less makes it possible to produce high-protein products at low cost.

[0029] In liquid media, medium components such as grain derivatives and fermentation residues may be present in water as solids without being completely dissolved. After mixing the medium components in water, it is preferable to sterilize the mixture in an autoclave or the like before use as a liquid medium.

[0030] If necessary, inorganic salts may be added to the liquid medium, such as phosphate salts, magnesium salts, iron salts, sodium salts, potassium salts, calcium salts, zinc salts, copper salts, manganese salts, and cobalt salts.

[0031] The liquid medium may be PDB (Potato Detoxulose Broth), which is used for culturing various microorganisms. Alternatively, the liquid medium or medium components may be solutions, suspensions (such as broth), residues, or solids containing carbon and nitrogen sources generated during food production, or nutritional components derived from non-standard agricultural products.

[0032] <Culture: Post addition> By adding the first nitrogen source to the liquid medium after the starter koji mold has begun to grow following the initiation of culture, the protein content in the resulting mycelium (koji mold tissue) can be increased. In this specification, the addition of a nitrogen source after the initiation of culture as described above is sometimes referred to as "late addition," and the point in time at which it is added is sometimes referred to as "time of late addition."

[0033] As used herein, the term "first nitrogen source" refers to the component in a liquid medium that has the highest nitrogen content. The first nitrogen source may consist of one component or two or more components. For example, the term "first nitrogen source" does not refer to a carbon source or other components that contain trace amounts of nitrogen. The "first nitrogen source" is preferably one of the preferred examples of the "nitrogen source," and particularly preferably yeast extract, casamino acids, peptone, or a mixture of two or more of these.

[0034] When the first nitrogen source is added later, the protein content of the resulting mycelium can be higher than when the entire amount is included in the liquid medium at the start of cultivation. Without being bound by any theory, this is thought to be because, as the mycelium grows during cultivation, the protein amount also increases. As the mycelium grows further, the accumulated protein is consumed by the koji mold. By adding the first nitrogen source later, the protein accumulation can be efficiently maintained. This is presumably because the first nitrogen source, which is primarily used as a raw material for protein production, is efficiently consumed by adding it after the koji mold has reached a state in the liquid medium where it can produce protein. Therefore, the amount of the first nitrogen source added to the liquid medium at the start and the amount of the first nitrogen source added later can be adjusted appropriately depending on the type of first nitrogen source and the culture conditions. Based on the above-presumed principle, the amount of the first nitrogen source added later is preferably more than 50% by mass of the total amount of the first nitrogen source added to the liquid medium, more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more. From the viewpoint of simplifying the procedure, it is preferable that the first nitrogen source is not added to the liquid medium at the start of culture, but is added only at a later time after the start of culture.

[0035] Whether the time has passed since the starter koji mold began to grow can be confirmed, for example, by visually observing the amount of bacterial mass in the liquid medium, measuring the pH of the liquid medium, or measuring the dissolved oxygen concentration in the liquid medium. For example, post-addition can be performed after an increase in the amount of bacterial mass, a decrease in the pH of the liquid medium, or a decrease in the dissolved oxygen concentration in the medium is confirmed. In particular, post-addition is preferably performed after sufficient growth of koji mold in the liquid medium, for example, when the increase in the amount of bacterial mass, the decrease in the pH of the liquid medium, or the decrease in the dissolved oxygen concentration in the medium have almost ceased. Under the conditions described herein, at least a portion of koji mold is considered to have begun to grow at least 8 hours after the start of culture. However, from the perspective of performing post-addition after sufficient growth of koji mold, post-addition is usually performed 18 hours or later after the start of culture, more preferably 24 hours or later, and even more preferably 36 hours or later. The start of culture typically refers to the time when the starter koji mold is added to the liquid medium.

[0036] The later addition is carried out at a point by the end of the culture when there is sufficient time to produce protein using the later-added first nitrogen source as a raw material. In particular, it is preferable that the later addition be carried out at a point when there is sufficient time to digest most of the later-added first nitrogen source. Based on the experience of the present inventors, taking into account the time it takes for a decrease in the amount of protein, which is thought to be due to decomposition within the koji mold cells, to begin, the later addition is preferably carried out at least 4 hours, more preferably 8 hours, and even more preferably 16 hours before the end of the culture.

[0037] The later addition may be carried out once or twice or more times, as long as it is within a suitable time for the later addition (for example, at least 18 hours after the start of culture and at least 4 hours before the end of culture). It is preferable to add the component multiple times sequentially in accordance with the consumption rate of the component in the liquid medium, or it is also preferable to add it once in consideration of the labor involved in the operation. It may also be added in multiple divided portions within a short period of time.

[0038] The post-addition of the first nitrogen source is preferably carried out so that the first nitrogen source is present at a concentration of 0.1 to 2%, more preferably 0.25 to 1%, relative to the volume of the liquid medium. The first nitrogen source may be added as a solution or suspension in an appropriate solvent such as sterile water, or as a sterilized, dried powder. As shown in the examples (Example R5 and Figure 13), post-addition allows the production of mycelia with a protein content of over 40% by mass, even when the amino acid-containing component in the liquid medium is only 0.25 to 0.5%.

[0039] <Cultivation: temperature conditions, procedures, etc.> The culture temperature is not particularly limited as long as it is within a temperature range in which koji mold can grow, and can be appropriately selected depending on the purpose, but is preferably 20°C to 40°C, more preferably 25°C to 37°C, and even more preferably 28°C to 32°C. By setting the culture temperature to 20°C or higher, it is possible to promote the growth of koji mold and the production of proteins. Furthermore, by setting the temperature to 40°C or lower, it is possible to prevent the koji mold from becoming unable to grow.

[0040] The culture is not particularly limited as long as it is carried out at a pH that is optimal for the growth of koji mold or allows cultivation. The pH may vary during the culture process. It may also vary depending on components added later. Unless specifically controlled, the pH of the liquid medium gradually decreases from the start to the end of the culture, but the pH is preferably about 3 to 8, and more preferably about 4 to 7, throughout the culture period.

[0041] Cultivation can be carried out by a general aerobic culture method. From the viewpoint of supplying the oxygen necessary for growth, for example, shaking culture or stirring culture using a jar fermenter can be used. Furthermore, gas supply to the culture vessel may be performed in addition to shaking and stirring. A method of achieving oxygen supply by gas supply only without shaking or stirring can also be used. In this case, gas supply may be performed so that the dissolved oxygen concentration is, for example, 1.0 ppm to 8.0 ppm.

[0042] Anaerobic treatment can be performed during the culture period by intentionally stopping the gas supply. In this case, the gas supply is preferably stopped 36 hours to 1 hour before the completion of the culture and continued until the completion of the culture. However, depending on the duration of the anaerobic treatment after the completion of the culture, the time for stopping the gas supply can be adjusted so that the anaerobic treatment time falls within the above-mentioned preferred range.

[0043] The cultivation of koji mold may be a batch culture in which the cultivation is started using an initially prepared liquid medium and terminated upon recovery of the resulting cultured mycelium, or a continuous culture in which new liquid medium is added as the cultured mycelium is recovered and the cultivation is continued continuously. When post-addition is performed at each cultivation period, continuous cultivation may be performed by repeating the following steps in this order: addition of new liquid medium and koji mold, cultivation, post-addition, continuation of cultivation, and recovery of cultured mycelium. Anaerobic treatment may be performed on the harvested cultured mycelium each time it is harvested.

[0044] The culture time is not particularly limited and can be selected appropriately depending on the purpose. For example, the culture time can be adjusted depending on the amount of koji mold at the start of culture. In the case of batch culture, in order to generally recover the cultured mycelium before the nutrients in the medium are depleted, 38 to 240 hours is preferred, 42 to 120 hours is more preferred, and 48 to 100 hours is particularly preferred. Protein can be produced by culture for 38 hours or more, and in consideration of production efficiency, it is preferable that the culture time not exceed 240 hours. In the case of continuous culture, the culture period used in batch culture can be repeated and continued.

[0045] The cultured mycelia may be used as is after cultivation, or may be recovered and then used. The recovery method is as described above and is not particularly limited. When producing a koji mold tissue product in which components contained in the liquid medium are attached separately from and not mixed with components in the mycelia, the mycelia may be washed with purified water or the like after recovery.

[0046] <Koji mold tissue product> As described above, koji mold tissue products contain or consist of mycelia. When koji mold is cultured in a liquid medium, the koji mold aggregates and may take the form of pellets, such as spheres, roughly spheres, or tablets, or may take the form of fibers (filaments), such as lines or fibers. The form of the mycelia is not particularly limited, and may be pellet-shaped, fibrous, or non-aggregated. It may also be a mixture of two or more of these forms. Note that a fibrous form refers to mycelia that have been dehydrated using a Nutsche or similar device and become completely sheet-like. A pellet-shaped form refers to a form in which the mycelium remains partially or completely intact even after dehydration using a Nutsche or similar device.

[0047] The koji mold tissue product obtained by the production method of the present invention contains GABA as an intracellular component (intracellular component). Furthermore, the koji mold tissue product obtained by the production method of the present invention preferably contains protein. Protein is contained in the koji mold tissue product as an intracellular component (intracellular component).

[0048] The GABA content can be measured after drying (e.g., freeze-drying) the koji mold tissue product. Measurement can be performed using any amino acid analysis method known or known to those skilled in the art. The GABA content of the koji mold tissue product obtained by the production method of the present invention is preferably 4.0 mg / g or more, more preferably 5.0 mg / g or more, and even more preferably 6.0 mg / g or more, based on the total dry mass. There is no upper limit, and a high value can be obtained depending on the glutamic acid content in the mycelium, etc. Even under conditions similar to those of the examples, it is believed that a GABA content of, for example, about 25 mg / g or about 20 mg / g can be obtained.

[0049] As described above, GABA is produced in koji mold by an enzymatic reaction from glutamic acid. Therefore, koji mold tissue products obtained by the production method of the present invention are characterized by a higher GABA content relative to the glutamic acid content compared to koji mold tissue products (koji mold mycelium) produced by production methods that do not involve anaerobic treatment or the addition of acetic acid. The glutamic acid and GABA contents and their ratios vary depending on the medium components used during koji mold culture, and therefore the GABA content relative to the glutamic acid content in koji mold tissue products obtained by anaerobic treatment and the addition of acetic acid also varies depending on the mycelium used and the culture method. The GABA content relative to the glutamic acid content in koji mold tissue products is, for example, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 100% by mass or more, or 150% by mass or more. In the examples described below, koji mold tissue products were obtained that had approximately twice the GABA content relative to the glutamic acid content. There is no particular upper limit, but depending on the mycelium used, the GABA content may be as high as 300% by mass relative to the glutamic acid content.

[0050] The protein content can be measured after drying (lyophilization, etc.) of the koji mold tissue product by the Kjeldahl method, which is well known to those skilled in the art. The koji mold tissue product preferably contains 15% to 70% by mass, more preferably 20% to 60% by mass, and even more preferably 25% to 55% by mass of protein relative to the total dry mass. As described above, by appropriately selecting the medium components or by performing culture with subsequent addition of the first nitrogen source, it is possible to obtain a koji mold tissue product containing 30% or more by mass, 35% or more by mass, or even 40% or more by mass of protein. A koji mold tissue product containing 30% or more by mass of protein can be used as an alternative protein source.

[0051] The koji mold tissue product obtained by the production method of the present invention may contain components derived from the medium components used in culturing koji mold. The content of these components usually decreases when the mycelium is washed with purified water or the like after culturing, compared to the mycelium immediately after culturing. For example, by adding GABA or a functional component other than GABA to the medium components used in culturing and providing the final product without a washing step, a koji mold tissue product with enhanced functionality can be provided.

[0052] The koji mold tissue product obtained by the production method of the present invention can be used as a functional food. Furthermore, the koji mold tissue product, which is rich in protein, can be used as an alternative protein source. The koji mold tissue product may be adjusted to a water content (for example, 60% to 95% by mass) according to the intended use. The koji mold tissue product can be distributed as a fresh food product, and may also be frozen or dried (lyophilized, etc.) for storage or distribution. Furthermore, the koji mold tissue obtained after anaerobic treatment or addition of acetic acid may be sterilized. Sterilization can prevent changes in the components caused by enzyme reactions in the koji mold. When washing the mycelium after anaerobic treatment or addition of acetic acid, sterilization may be performed either before or after washing.

[0053] The koji mold tissue product obtained by the production method of the present invention can be used for food. For example, koji mold tissue products with a high protein content can be cooked as a meat substitute in the same way as regular meat, or can be mixed with meat, eggs, egg whites, etc. and cooked. Furthermore, koji mold tissue products can be added to various foods and beverages, for example, as a dried powder. Koji mold tissue products provided as foods may further contain seasonings such as salt, sugar, soy sauce, miso, and alcoholic beverages, spices, sweeteners, preservatives, emulsifiers, flavorings, etc. These ingredients can be mixed, for example, with the koji mold tissue product obtained after anaerobic treatment or acetic acid addition treatment, or with a dried powder obtained by drying the koji mold tissue product.

[0054] Furthermore, the koji mold tissue product can also be used as a feed additive to be incorporated into feed for livestock, poultry, and seafood. [Example]

[0055] The present invention will be described in more detail below with reference to examples. The materials, reagents, amounts and proportions of substances, procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the following examples.

[0056] In the examples, the koji mold used as a culture starter was Aspergillus oryzae RIB40 strain, which was obtained from the NITE Biological Resource Center. The bacteria were inoculated onto PDA medium and cultured at 30°C for one week to allow spores to adhere to the plate surface. Sterilized 0.05% Tween 20 solution was poured onto the plate, and the surface was rubbed to remove the spores, yielding a spore suspension. The spore concentration of the spore suspension was measured using a hemocytometer, and the suspension was diluted appropriately for use.

[0057] In the examples, the protein content of koji mold tissue or mycelium was measured using the Kjeldahl method, a common method for measuring the protein content of foods, as follows: First, to prepare the sample, koji mold tissue (mycelium) was thoroughly freeze-dried (FDS-2000: Tokyo Rikakiki Co., Ltd.). The amount to be measured was weighed out, and the measurement was carried out according to the procedure described in the manual for the measuring device (Superkel 1600: Actac Co., Ltd.). To convert the measured total nitrogen amount to protein content, a conversion factor of 6.25 was used. In the examples, the "%" indicated for protein content is % by mass.

[0058] <Production method including anaerobic treatment or acetic acid treatment> (1) Preparation of liquid medium Both the yeast extract and glucose were commercially available products used as medium components for microbial culture. For example, a predetermined amount of the medium components shown in each example was weighed into a 2.8 L Erlenmeyer flask, 1500 mL of distilled water was added, the flask was fitted with a lid fitted with a breathable filter, and the flask was sterilized in an autoclave (121°C, 15 minutes). Alternatively, a predetermined amount was weighed into a 200 mL Erlenmeyer flask, 50 mL of distilled water was added, the flask was fitted with a cork stopper, and the flask was sterilized in an autoclave (121°C, 15 minutes).

[0059] (2) Cultivation in liquid medium After the liquid medium was sterilized in an autoclave and cooled thoroughly, it was inoculated with bacteria in a clean bench. 7 ~1.0×10 8 / mL spore suspension was used, resulting in a final spore concentration of 1.0 × 10 4 The spore suspension was added at a volume of 1 / mL. After addition, the flask was placed in an incubator shaker (Innova S44i, manufactured by Eppendorf), and the incubator was set to 30°C and cultured by rotation at 120 or 180 rpm for 2 or 3 days. When adding acetic acid or other substances during the culture, the flask was removed from the culture and, in a sterile space such as a clean bench, the lid was opened and a predetermined amount of acetic acid solution was added. After the addition, the lid was replaced, and the culture was continued in the incubator.

[0060] (3) Method for collecting mycelium After the cultivation was completed, the cultured bacterial solids were separated from the culture solution. The culture solution containing the bacterial cells was poured into a Buchner funnel equipped with a filter paper or a filter, and the water was efficiently removed using a suction pump. The collected mycelium was washed with an appropriate amount of pure water to avoid affecting the bacterial component values.

[0061] (4) Anaerobic treatment of mycelium The wet weight of the obtained mycelium was recorded, and the mycelium was cut into several pieces with a knife or the like so that each piece was approximately the same shape and weight, between 5 and 10 g, and then subjected to anaerobically treatment. In the anaerobic treatment using plastic wrap, the mycelium was wrapped in food wrap with a high oxygen barrier property (made of polyvinylidene chloride, Asahi Kasei, Saran Wrap) to remove as much air as possible, and then left to stand in an incubator for a specified time to reach the specified temperature. After standing, the sample was transferred to a freezer at -20°C or below without removing the plastic wrap, frozen, and stored until measurement. For anaerobic treatment using an oxygen scavenger, the sample was placed in a sealed storage container together with the oxygen scavenger and left to stand for a specified period of time. After standing, the sample was immediately transferred to a freezer at -20°C or below, frozen, and stored until measurement. For koji mold tissues that were not subjected to oxygen blocking treatment, the samples cut as described above were immediately transferred to a freezer at -20°C or below, frozen, and stored until the time of measurement.

[0062] (5) Method for measuring GABA content in koji mold tissue To prepare the measurement sample, frozen koji mold tissue was first freeze-dried to obtain dried mycelia. Purified water was added to the tube to a concentration of 10 mg / ml. Glass beads were added to the tube, and the mycelia were disrupted using a bead-type disrupter (FastPrep24: MP Biomedicals). The tube was then centrifuged at 12,000 rpm for 3 minutes, and the supernatant was transferred to a separate tube as the measurement sample. GABA levels were measured using a GABA measurement kit (Enzyme Sensor, Inc.) according to the protocol.

[0063] [Example 1] Aspergillus oryzae was cultured in YG liquid medium containing 1% yeast extract and 2% glucose at 30°C for 3 days, washed with pure water, and then dehydrated to obtain mycelium. The mycelium was cut as described above and stored at 30°C for 3, 6, and 24 hours, respectively, to obtain koji mold tissues. After storage, the samples were frozen and lyophilized, and the GABA and glutamic acid contents were measured. The results are shown in Figure 1. At all treatment times, the GABA content of the oxygen-deprived samples was significantly higher and the glutamic acid content significantly lower than the samples without anaerobic treatment. In Figure 1, "No treatment" indicates the results for the samples without the above storage.

[0064] [Example 2] Aspergillus oryzae was cultured in YG liquid medium (1.5 L) containing 1% yeast extract and 2% glucose at 30°C for 2 days (Figure 2(b) and (c)) or 3 days (Figure 2(a)). The mycelium was then washed with pure water and dehydrated to obtain mycelia. The effects of anaerobic treatment using plastic wrap for different periods of time on the mycelia were examined. Cut samples were placed in plastic wrap to block oxygen and allowed to stand at 30°C for 1, 2, and 3 hours (a); 3, 24, 48, and 72 hours (b); or 3, 24, 30, and 36 hours (c). The samples were freeze-dried and the GABA content was measured. The content of the same mycelia without oxygen blocking treatment was also measured. The results are shown in Figure 2. Compared to untreated samples, the GABA content was significantly higher in samples that had been oxygen-deprived for 1 to 36 hours.

[0065] Furthermore, the protein content was measured for each of the samples (c) above and an untreated sample. The results are shown in Table 1. In all cases, the protein content exceeded 40% by mass.

[0066] [Table 1]

[0067] [Example 3] Aspergillus oryzae was cultured in YG liquid medium containing 1% yeast extract and 2% glucose at 30°C for two days, washed with pure water, and then dehydrated to obtain mycelium. The effects of anaerobic treatment at different temperatures using plastic wrap were examined. Cut samples were placed in plastic wrap to block oxygen and allowed to stand for three hours at 4°C, 30°C, 37°C, and 42°C. After standing, the samples were freeze-dried and their GABA content was measured. The results are shown in Figure 3. Compared to the untreated sample, there was no difference in GABA content in the sample treated at 4°C, but the GABA content was significantly higher in the samples treated with oxygen at 37°C and 42°C.

[0068] [Example 4] Aspergillus oryzae was cultured in YG liquid medium containing 1% yeast extract and 2% glucose at 30°C for two days, washed with pure water, and then dehydrated to obtain mycelium. The effect of anaerobic treatment using an oxygen scavenger on this mycelium was examined. Cut samples were placed in a sealed storage container with an oxygen scavenger and left to stand at 30°C for three hours. After standing, the samples were freeze-dried and the GABA content was measured. The results are shown in Figure 4. Compared to the untreated sample, the GABA content was significantly higher in the sample that had been oxygen-blocked using an oxygen scavenger.

[0069] [Example 5] Aspergillus oryzae was cultured in YG liquid medium containing 1% yeast extract and 2% glucose at 30°C for 2 days, washed with pure water, and then dehydrated to obtain mycelia. The effects of water washing on the GABA content of these mycelia were examined when anaerobic treatment or the addition of GABA solution was used to increase the GABA content. Cut samples were blocked with plastic wrap and allowed to stand at 30°C for 3 hours. Alternatively, 10 mg of GABA solution per gram of mycelial tissue was added dropwise to the cut samples and allowed to stand at 30°C for 3 hours. After each incubation, the samples were suspended in pure water in an amount 50 times the weight of the mycelial tissue, dehydrated, and the mycelia were re-collected. The mycelia were freeze-dried, and the GABA content was measured. The results are shown in Figure 5. No effect of water washing was observed in samples with increased GABA content due to anaerobic treatment. The GABA content of samples with added GABA solution, which was higher than that of samples without added GABA solution, decreased after water washing.

[0070] [Example 6] Aspergillus oryzae was cultured in YG liquid medium (50 mL) containing 1% yeast extract and 2% glucose at 30°C for 2 days, washed with pure water, and then dehydrated to obtain mycelia. To confirm the effect of medium acidification, citric acid, acetic acid, and hydrochloric acid were added at the beginning of the culture to adjust the medium pH to 4.5–5.0. The concentrations and dilutions of the solutions added were 200-fold for a 10% citric acid solution, approximately 110-fold for a 1M acetic acid solution, and 220-fold for a 1M hydrochloric acid solution. Furthermore, the same amounts of citric acid, acetic acid, and hydrochloric acid were added during the culture, starting from the start of the culture and 4 hours before the end of the culture. The koji mold tissues obtained from these cultures were lyophilized and the GABA content was measured. The results are shown in Figure 6(1). Samples to which citric acid, acetic acid, or hydrochloric acid had been added at the beginning of the culture all had similar GABA content to samples without added acid. When the acidic solution was added 4 hours before the end of the incubation, the GABA content in the sample with acetic acid was significantly higher than in the sample without it. The samples with citric acid or hydrochloric acid added showed the same level of GABA content as the sample without it, even 4 hours before the end of the incubation.

[0071] Furthermore, the glutamic acid content was also measured for samples to which the same amounts of citric acid, acetic acid, and hydrochloric acid were added 4 hours before the end of the culture. The results are shown in Figure 6(2). It can be seen that the glutamic acid content was significantly lower in the sample to which acetic acid was added than in the sample to which no addition was made.

[0072] Furthermore, the protein content was measured for samples obtained by adding citric acid, acetic acid, or hydrochloric acid 4 hours before the end of the culture, as well as for a sample to which no acid was added, using the same procedure as above, except that the culture was carried out for 3 days instead of 2 days. The results are shown in Table 1. In all cases, the protein content exceeded approximately 50% by mass.

[0073] [Table 2]

[0074] [Example 7] Aspergillus oryzae was cultured in YG liquid medium containing 1% yeast extract and 2% glucose at 30°C for 3 days, washed with pure water, and then dehydrated to obtain mycelia. The effect of the incubation time from adding acetic acid to the medium until harvesting the mycelia on GABA content was examined. 1M acetic acid was added 24 hours, 4 hours, and 1 hour before the end of incubation to a 110-fold dilution. The koji mold tissue obtained from these incubations was freeze-dried and the GABA content was measured. The results are shown in Figure 7. The samples with acetic acid added 24 hours, 4 hours, and 1 hour before the end of incubation all had higher GABA content than the untreated samples. The sample with acetic acid added 4 hours before the end of incubation had the highest GABA content, followed by the sample with acetic acid added 1 hour before the end of incubation.

[0075] [Example 8] Aspergillus oryzae was cultured in YG liquid medium containing 1% yeast extract and 2% glucose at 30°C for 3 days, washed with pure water, and then dehydrated to obtain mycelia. The effect of the amount of acetic acid added 4 hours before the end of culture on GABA content was examined. Four hours before the end of culture, 1M acetic acid was added to the medium at 220-, 110-, and 55-fold dilutions. The koji mold tissues obtained from these cultures were freeze-dried and the GABA content was measured. The results are shown in Figure 8. The samples diluted 220-, 110-, and 55-fold with acetic acid four hours before the end of culture all had higher GABA content than the undiluted samples. The sample with 110-fold acetic acid added had the highest GABA content, followed by the sample with 55-fold acetic acid added.

[0076] <Other culture methods> The production method of the present invention is not limited to the mycelia obtained by the culture in the above example, but can be carried out using mycelia of various koji molds. Below, an example is shown in which a method for producing mycelia with a higher protein content was investigated.

[0077] (1) Preparation of liquid medium The yeast extract, casamino acids, and peptones used were all commercially available products used as medium components for microbial culture. The sake lees used were from Kikuya Oita Co., Ltd., and the raw sugar used was low-refined sugar (raw sugar) made from 100% sugarcane. Brown rice flour was from Ishizaka Farm. The medium components shown in each example (components added from the start of culture) were weighed in predetermined amounts into, for example, a 200 mL Erlenmeyer flask, and 50 mL of distilled water was added. The flask was then sealed with a silicone stopper and sterilized in an autoclave (121°C, 15 minutes). When a first nitrogen source was added from the start of culture, the first nitrogen source was added to the concentration described in the description of each example (0.25%, 0.5%, 1%, 2%) before the autoclave sterilization. When adding the first nitrogen source later, 2.5%, 5%, and 10% solutions of the first nitrogen source were prepared in advance, sterilized in an autoclave, and cooled. At the designated time points, 5.5 mL of this concentrated solution was added to the original medium to adjust the final concentrations to approximately 0.25%, 0.5%, and 1%, respectively, and the culture was continued.

[0078] (2) Cultivation in liquid medium After the liquid medium was sterilized in an autoclave and cooled thoroughly, it was inoculated with bacteria in a clean bench. 7 Use 50 µL of the spore suspension at 100 µL / mL, resulting in a final spore concentration of 5.0 x 10 4 The spore suspension was added at a volume of 1 / mL. After addition, the flask was placed in a shaker (MMS-1020, manufactured by Tokyo Rikakikai Co., Ltd.) installed in an incubator (FMC-1000, manufactured by Tokyo Rikakikai Co., Ltd.), and the incubator was set to 30°C and cultured at 120 rpm for 3 days. When the first nitrogen source was to be added later, the flask was removed from the culture and, in a sterile space such as a clean bench, the lid was opened and a solution of the first nitrogen source at a predetermined concentration was added. After the addition, the lid was replaced, and the culture was continued in the incubator.

[0079] (3) Method for collecting cultured mycelium After the cultivation was completed, the obtained mycelium (solid mycelium) was separated from the culture solution. The culture solution containing the cultivated mycelium was poured into a funnel equipped with filter paper, and the water was efficiently removed using a suction pump. The collected mycelium was washed with an appropriate amount of pure water so as not to affect the values ​​of the mycelium components. The wet weight of the cultivated mycelium obtained was recorded, and the mycelium was stored frozen until it was used for measurement.

[0080] [Example 1R] At the start of cultivation, yeast extract was added to a liquid medium containing 2% sake lees and 2% raw sugar, or a liquid medium containing 2% brown rice flour, and the protein content of the resulting mycelium was confirmed. Yeast extract was added to the liquid medium from the start of cultivation. The results are shown in Figure 9. In both cases, the addition of 1% yeast extract significantly increased the protein content.

[0081] [Example 2R] We investigated the differences in protein content of the mycelium obtained by adding different amounts of yeast extract to a liquid medium containing 2% sake lees and 2% raw sugar at the start of cultivation. Yeast extract was added to the liquid medium from the start of cultivation. The results are shown in Figure 10. When the yeast extract was increased from 1% to 2%, no significant increase in protein content was observed, indicating that the threshold protein content could be achieved by adding about 1% yeast extract.

[0082] [Example 3R] We investigated the difference in protein content of mycelia obtained by adding 1% or 2% yeast extract to liquid medium containing 2% or 4% raw sugar at the start of cultivation. Yeast extract was added to the liquid medium from the start of cultivation. The results are shown in Figure 11. No significant increase in protein content was observed when the yeast extract concentration was increased from 1% to 2%, indicating that even in liquid medium containing only raw sugar as a carbon source, the threshold protein content can be roughly achieved by adding approximately 1% yeast extract. Furthermore, no significant increase in protein content was observed when the raw sugar concentration was increased from 2% to 4%.

[0083] [Example 4R] We investigated the difference in protein content of the resulting mycelium when casamino acids or peptone was added to a liquid medium containing 2% sake lees and 2% raw sugar at the start of cultivation. Casamino acids or peptone were added to the liquid medium from the start of cultivation. The results are shown in Figure 12. The addition of 1% casamino acids or peptone significantly increased the protein content.

[0084] [Example 5R] The wet weight and protein content of the mycelia obtained when 1% yeast extract or 1% casamino acids was added to a liquid medium containing 2% sake lees and 2% raw sugar at the start of cultivation, 24 hours or 4 hours before harvesting the mycelia, were examined. The results (average values ​​of two samples) are shown in Figure 13. In either case, the protein content was significantly higher in the mycelia obtained from the cultivation in which yeast extract or casamino acids was added 24 hours before than in the cultivation in which it was added 4 hours before.

[0085] [Example 6R] The wet weight and protein content of mycelia were measured when yeast extract was added to a liquid medium containing 2% sake lees and 2% raw sugar at the start of cultivation. The results are shown in Figure 14. In Figure 14, YE represents yeast extract.

[0086] The wet weight of the mycelia obtained from the culture without the addition of yeast extract was 1.036 g, and the protein content was 25.9%. On the other hand, the wet weight of the mycelia obtained from the culture with the addition of yeast extract at 0.25%, 0.5%, and 1% also increased, and the protein contents were 33.1%, 41.8%, and 46.1%, respectively. All of the protein contents were higher than those of the mycelia obtained from the culture without the addition of yeast extract.

[0087] As an example of post-addition, culture was initiated in a liquid medium containing 2% sake lees and 2% raw sugar. Two days after the start of culture (24 hours before harvesting), 5.5 mL of water or 5.5 mL of yeast extract solution was added to adjust the yeast extract concentration in the liquid medium to 0%, 0.25%, 0.5%, or 1%, and culture was continued. The protein contents of the resulting mycelia were 26.4%, 40.6%, 45.5%, and 48.9%, respectively. Compared to the mycelia obtained from the culture with water, the protein content of the mycelia obtained from the culture with yeast extract was significantly higher.

[0088] When the same amount of yeast extract was used, the protein content of the mycelia obtained from the culture in which the yeast extract was added from the start of the culture was compared with that obtained from the culture in which the yeast extract was added 24 hours before harvesting, as shown below. With 0.25% yeast extract: 33.1% for initial addition and 40.6% for later addition; With 0.5% yeast extract: 41.8% for initial addition and 45.5% for later addition; With 1% yeast extract: 46.1% for initial addition and 48.9% for later addition. These results show that the effect of increasing the protein content by post-addition is more pronounced in mycelia obtained by culturing in a liquid medium to which yeast extract has been added at a concentration of 0.5% or less.

[0089] [Example 7R] The liquid medium at the start of cultivation was 2% sake lees and 2% raw sugar, and the wet weight and protein content of the mycelium obtained by adding yeast extract at different times were compared. The results are shown in Figure 15. In Figure 15, YE represents yeast extract. When yeast extract was added to a concentration of 0.25% or 0.5%, adding it on the second day after the start of cultivation (24 hours before harvesting) had the greatest effect on increasing protein content, while adding it between the first day (48 hours before harvesting) and 16 hours before harvesting was most effective. [Industrial Applicability]

[0090] The method for producing a koji mold tissue product of the present invention can provide a koji mold tissue product with a high content of GABA, a functional component.

Claims

1. A method for producing a koji mold tissue product enriched in γ-aminobutyric acid, which comprises anaerobic treatment of koji mold mycelium.

2. The method according to claim 1, wherein the anaerobic treatment is carried out at 20°C to 45°C for 1 hour to 36 hours.

3. 2. The method according to claim 1, wherein the anaerobic treatment is carried out by wrapping the mycelium in a film at 20°C to 45°C for 1 hour to 36 hours.

4. The method according to claim 1 , further comprising a sterilization treatment after the anaerobic treatment.

5. The method according to any one of claims 1 to 4, comprising culturing koji mold in a liquid medium to obtain the mycelium.

6. The method according to any one of claims 1 to 4, wherein the mycelium is obtained by culturing the mycelium containing the following (1) to (3) in this order: (1) Initiating the cultivation of a starter koji mold in a liquid medium containing at least a carbon source; (2) adding a first nitrogen source to the liquid medium after the koji mold starts to grow in the liquid medium; and (3) After the addition of the first nitrogen source, the culture is continued for the time required for the production of a protein using the first nitrogen source as a raw material.

7. The method includes culturing koji mold in a liquid medium to obtain mycelium, and adding acetic acid to the liquid medium 1 hour to 24 hours before the end of the culture. A method for producing a koji mold tissue product enriched with γ-aminobutyric acid.

8. A koji mold tissue product containing koji mold mycelium, The koji mold tissue product contains 6.0 mg / g or more of γ-aminobutyric acid relative to the total dry mass of the koji mold tissue product, The koji mold tissue, wherein the γ-aminobutyric acid is present inside the mycelium.

9. The koji mold tissue product according to claim 8, which contains 40% by mass to 70% by mass of protein relative to the total dry mass of the koji mold tissue product.

10. The koji mold tissue product according to claim 8 or 9, which is edible.

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