Method for producing edible filamentous fungus body

Abstract

Provided is a method for producing an edible filamentous fungus body, the yield of which can be improved.　The present invention provides a method for producing an edible filamentous fungus body, the method including a step for culturing a filamentous fungus in a liquid culture medium containing one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour that pass through a sieve having an opening of 1000 μm. Moreover, the edible filamentous fungus body produced by using the method according to the present invention can be used for producing food.

Description

Method for producing edible filamentous fungal cells

[0001] The present invention relates to a method for producing edible filamentous fungal cells, a liquid medium for culturing edible filamentous fungal cells, and edible filamentous fungal cells, a food composition or a food produced using the same.

[0002] As one of the solutions to the global food problem, a technology of processing microorganisms together with their cells into foods has attracted attention. Among them, filamentous fungi are rich in protein, and are very useful fungal species for food utilization due to their chewy fibrous texture derived from the fibrous morphology.

[0003] Methods for culturing filamentous fungi are divided into two types: solid culture and liquid culture. Compared with solid culture, liquid culture enables strict culture control and facilitates recovery of only fungal cells, so that filamentous fungal culture products with stable quality can be produced. There is a demand for a culture method that can efficiently proliferate fungal cells in such a liquid medium and increase the yield of filamentous fungal cells.

[0004] Japanese Examined Patent Publication No. 3-63350, Japanese Unexamined Patent Publication No. 2024-86167, Japanese Unexamined Patent Publication No. 2007-125002, Japanese Unexamined Patent Publication No. 2014-54245

[0005] An object of the present invention is to provide a method for producing edible filamentous fungal cells capable of improving the yield of filamentous fungal cells in liquid culture.

[0006] As a result of intensive studies, the present inventors found that the above problem can be solved by using cereal flour having a specific particle size in a liquid medium, and completed the following invention.

[0007] The present invention provides the following: [1-1] A method for producing edible filamentous fungal cells, comprising the step of culturing filamentous fungi in a liquid medium containing one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour after passing through a sieve with a mesh size of 1000 μm. [1-2] A method for improving the yield of edible filamentous fungal cells, comprising the step of culturing filamentous fungi in a liquid medium containing one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour after passing through a sieve with a mesh size of 1000 μm. [1-3] A method for improving the amount and / or quality of protein in edible filamentous fungal cells, comprising the step of culturing filamentous fungi in a liquid medium containing one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour after passing through a sieve with a mesh size of 1000 μm. [2] The method according to [1-1] to [1-3], wherein the content of grain flour in the liquid medium is 1 to 10% by mass relative to the total amount of liquid medium. [3] The method according to [1-1] to [1-3] and [2], wherein the viscosity of the liquid medium is 0.15 to 5 dPa·s. [4] The method according to any one of [1-1] to [1-3], [2], and [3], further comprising the step of recovering the microbial cells when the content of grain flour in the liquid medium becomes 0.05% by mass or less relative to the total amount of liquid medium. [5] The method according to any one of [1-1] to [1-3] and [2] to [4], wherein the liquid medium contains 0.5 to 18% by mass of one or more sugars selected from the group consisting of monosaccharides and disaccharides relative to the total amount of liquid medium. [6] A liquid medium for culturing edible filamentous fungal cells, comprising one or more grain flours, wherein the grain flour is selected from the group consisting of corn flour and wheat flour, which pass through a sieve with a mesh size of 1000 μm. [7] The liquid medium according to [6], wherein the content of grain flour in the liquid medium is 1 to 10% by mass relative to the total amount of the liquid medium. [8] The liquid medium according to [6] or [7], wherein the viscosity of the liquid medium is 0.15 to 5 dPa·s. [9] The liquid medium according to any one of [6] to [8], wherein the liquid medium contains 0.5 to 18% by mass of one or more sugars selected from the group consisting of monosaccharides and disaccharides relative to the total amount of the liquid medium.

[10] Edible filamentous fungal cells produced by the method described in any one of items [1] to [5].

[11] A food composition or food containing the edible filamentous fungal cells described in

[10] .

[0008] This invention makes it possible to promote the cell proliferation of filamentous fungi in liquid culture and improve the yield of filamentous fungal cells.

[0009] Figure 1 is a photograph showing the formation of mycelial masses in Example 1 and Comparative Example 1 in Experiment 1 over time.

[0010] One aspect of the present invention relates to a method for producing edible filamentous fungal cells, a method for improving the yield of edible filamentous fungal cells, or a method for improving the amount and / or quality of protein in edible filamentous fungal cells, comprising the step of culturing filamentous fungi in a liquid medium containing one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour that pass through a sieve with a mesh size of 1000 μm. Hereinafter, these methods may be collectively referred to as the methods of the present invention.

[0011] Furthermore, one aspect of the present invention relates to a liquid culture medium comprising one or more grain flours, wherein the grain flours are selected from the group consisting of corn flour and wheat flour, and the grain flours pass through a sieve with a mesh size of 1000 μm. This liquid culture medium may be a liquid culture medium for culturing edible filamentous fungal cells, for improving the yield of edible filamentous fungal cells, for improving the amount and / or quality of protein in edible filamentous fungal cells, or it may be a liquid culture medium used in the method of the present invention. Hereinafter, these culture media may be collectively referred to as the culture medium of the present invention.

[0012] Furthermore, one aspect of the present invention relates to the use of one or more grain flours or the like in a liquid culture medium for producing edible filamentous fungal cells, for improving the yield of edible filamentous fungal cells, and for improving the amount and / or quality of protein in edible filamentous fungal cells, wherein the grain flours are selected from the group consisting of corn flour and wheat flour, and the grain flours pass through a sieve with a mesh size of 1000 μm.

[0013] The filamentous fungi are not limited to those that are edible as food, and examples include filamentous fungi belonging to the genera Aspergillus, Fusarium, Geotrichum, Penicillium, Neurospora, Monascus, Pesilomyces, and Rhizopus. Among these filamentous fungi, microorganisms of the genera Aspergillus and Fusarium are particularly preferred from the standpoint of being edible. In particular, filamentous fungi of the genus Aspergillus are especially preferred due to their long history of use as food. Examples of such filamentous fungi of the genus Aspergillus include Aspergillus oryzae, Aspergillus kawachii, Aspergillus awamori, Aspergillus sojae, Aspergillus glaucus, Aspergillus tamari, Aspergillus luchuensis, and Aspergillus nigar. A single type of filamentous fungus may be used, or a combination of two, three, or more other filamentous fungi may be used, or it may be used in combination with microorganisms other than filamentous fungi.

[0014] Filamentous fungi can be cultured in liquid culture media using various methods depending on the type of fungus, such as aerated culture, stirred culture, shaking culture, and static culture. The culture temperature can be set according to the type of fungus being cultured. For filamentous fungi, the culture temperature can usually be selected from 20 to 50°C, preferably 25 to 40°C, and more preferably 25 to 35°C. From the viewpoint of industrial-scale operations, it is preferable to use a jar fermenter, in which case aerated culture with stirring is usually performed.

[0015] The liquid culture medium may be any liquid culture medium that can be commonly used for culturing filamentous fungi. It may be an experimental culture medium for filamentous fungi or a liquid culture medium for industrial production. The liquid culture medium is usually a solution containing water as the main solvent, with a carbon source, nitrogen source, minerals, and trace elements dissolved in it, and may also be a mixture containing solid components. Possible nitrogen sources include ammonium salts such as ammonium sulfate, amino acids, peptone, and yeast extract. Phosphates, sulfates, potassium, trace metals, vitamins, etc., may be added as minerals and trace elements.

[0016] Starch contained in grain flour is used as the carbon source. The grain flour used in the present invention is a grain flour selected from the group consisting of corn flour and wheat flour that has passed through a sieve with a mesh size of 1000 μm. In one aspect of the present invention, 80% by mass or more, for example, 85% by mass or more, 90% by mass or more, 95% by mass or more, or 100% by mass of the grain flour is a grain flour selected from the group consisting of corn flour and wheat flour that has passed through a sieve with a mesh size of 1000 μm or less, preferably 850 μm or less, for example 500 μm or less, more preferably 300 μm or less, for example 260 μm or less, and even more preferably 150 μm or less, for example 20 μm or less. The corn flour is not limited as long as it is obtained by drying and grinding the endosperm of corn, and corn grits, cornmeal, corn flour, etc. can be used, with corn flour being preferred. In this specification, cornstarch, which is obtained by grinding the endosperm of corn and extracting and purifying the starch, is not included in corn flour. Wheat flour is not limited to wheat flour, and all-purpose flour, cake flour, and bread flour can be used.

[0017] In the present invention, grain flour selected from the group consisting of corn flour and wheat flour that passes through a sieve with a mesh size of 1000 μm or less, preferably 850 μm or less, more preferably 300 μm or less, and even more preferably 150 μm or less is used. As mentioned above, liquid culture media have the advantage of being able to produce filamentous fungal cultures of stable quality, but when filamentous fungi are cultured in liquid culture, there is a problem that the hyphae tend to aggregate and hyphae masses are easily formed. When hyphae masses aggregate and become large, the density of mycelium in the liquid decreases and the yield of the culture decreases. In addition, oxygen and nutrients are not supplied to the center of the hyphae mass, and the center dies, resulting in poor growth. The inventors have discovered that the yield of fungal cells can be improved by using grain flour containing grain flour selected from the group consisting of corn flour and wheat flour of the specific particle size mentioned above. Furthermore, it was confirmed that the formation of hyphae masses was suppressed and the density of mycelium in the culture medium was maintained at a high level compared to cases in which such grain flour is not used. This is thought to be because the formation of mycelial masses was suppressed when corn flour or wheat flour particles of a specific particle size collided with the fungal cells in the liquid culture medium.

[0018] Patent Document 1 addresses the challenge of simultaneously reducing the size of mycelial clumps and promoting the production of a target useful substance. It describes how the addition of plant-derived powders prevents filamentous fungal hyphae from forming large clumps, and lists wood powder, grass and stem powder, and seed coats of soybeans, corn, rice hulls, and buckwheat hulls as examples of plant-derived powders. However, while the examples in Patent Document 1 describe experimental results showing that the production of useful substances by filamentous fungi is promoted, there are no experimental results showing an improvement in the yield of fungal cells. For example, Example 1 shows an increase in the accumulation of mycophenolic acid and L-aspartic acid, but no significant difference in mycelial growth. Furthermore, although the addition of seed coats is described, there is no description of the addition of corn or wheat seeds or their particle size. Patent Document 2 addresses the objective of increasing the content of specific functional components in a protein composition produced by culturing Aspergillus oryzae, and discloses a method for producing a protein composition that includes culturing Aspergillus oryzae in a culture medium containing a sugar-containing nutritional composition to allow the cultured Aspergillus oryzae to produce a protein composition. Examples of the nutritional composition are given as brown rice flour, sake lees, raw sugar, and yeast extract, but there is no mention of corn flour or wheat flour. Patent Document 3 addresses the objective of producing liquid koji with high enzymatic activity, such as starch-degrading enzymes like glucoamylase, and discloses a method for producing liquid koji using grains whose entire or partial surface is covered with at least the husk as a culture material. Examples of grains are given as corn and wheat, but there is no mention that corn flour or wheat flour of a specific particle size improved the yield of Aspergillus oryzae. Patent Document 4 addresses the objective of providing liquid koji that is effective in enhancing saltiness, and discloses that wheat bran, rice, defatted soybeans, corn, etc., can be used as raw materials for a culture medium to produce such liquid koji, but there is no mention of particle size.

[0019] Furthermore, as long as corn flour or wheat flour is present in the liquid culture medium in the following amounts after passing through a sieve with a mesh size of 1000 μm or less, preferably 850 μm or less, more preferably 300 μm or less, and even more preferably 150 μm or less, the particle size or type of other grain flours is not limited. This is because the formation of mycelial masses is suppressed when particles of corn flour or wheat flour of these particle sizes collide with the fungal cells, so even if grain flours of sizes larger than these or other types of grain flours are present in the culture medium, it is thought that there will be no significant impact on the growth or yield of filamentous fungi, and mycelial aggregation will be hindered.

[0020] The inventors have confirmed that as cultivation progresses, the starch derived from grain flour in the liquid medium is consumed and its amount decreases. From the viewpoint of ease of recovering the microbial cells by filtration, etc., the liquid medium may be prepared so that the amount of grain flour recovered is about 0.1% by mass or less, preferably about 0.075% by mass or less, and more preferably about 0.05% by mass or less, relative to the total amount of liquid medium. On the other hand, a certain amount of grain flour of a specific particle size is necessary to obtain a good yield. Examples of lower limits for such grain flour amounts relative to the total amount of liquid medium include about 0.2% by mass or more, about 0.5% by mass or more, about 0.6% by mass or more, about 0.8% by mass or more, about 1.0% by mass or more, about 1.3% by mass, about 2.0% by mass or more, about 2.5% by mass or more, about 3.0% by mass or more, about 3.5% by mass or more, about 4.0% by mass or more, and about 5.0% by mass or more. In light of these circumstances, the grain flour content is preferably in the range of 0.2 to 20.0% by mass, 0.5 to 15.0% by mass, 0.6 to 15.0% by mass, and more preferably 1.0 to 10.0% by mass, relative to the total amount of liquid culture medium. Examples include, but are not limited to, 1.0 to 8.0% by mass, 1.5 to 7.0% by mass, 2.0 to 6.0% by mass, 2.5 to 5.0% by mass, 3.5 to 5.0% by mass, etc.

[0021] Furthermore, in addition to the grain flour described above, the carbon source of the present invention may further contain one or more sugars selected from the group consisting of monosaccharides and disaccharides. Examples of monosaccharides include glucose and fructose. Examples of disaccharides include maltose and sucrose. Although the grain flour used in the present invention alone is sufficient for cultivation as a carbon source, monosaccharides and disaccharides are consumed first immediately after the start of cultivation, so including an appropriate amount of these sugars will increase the rate of increase in the initial stages of cultivation. The content of such sugars depends on the content of the grain flour, but for example, it can be set to about 0.5% by mass or more, about 1.0% by mass or more, about 1.5% by mass or more, about 2.0% by mass or more, about 2.5% by mass or more, about 3.0% by mass or more, about 3.5% by mass or more, about 4.0% by mass or more, about 4.5% by mass or more, etc. On the other hand, adding excessive amounts of sugars can worsen the growth of the fungi due to osmotic pressure, so it is preferable that the upper limit be approximately 18.0% by mass, approximately 19.0% by mass, or approximately 20.0% by mass. Therefore, when adding sugars, the sugar content can be set to 0.5 to 18.0% by mass relative to the total volume of the liquid culture medium, from the viewpoint of good growth of filamentous fungi. Examples of such content, though not limited to them, include 1.0 to 15.0% by mass, 2.0 to 10.0% by mass, and 2.5 to 5.0% by mass.

[0022] From the viewpoint of preventing the aggregation of mycelium during culture and efficiently promoting the growth of filamentous fungi, the culture medium of the present invention may be set to a viscosity of 0.1 to 10 dPa·s, preferably 0.15 to 5 dPa·s, and more preferably 0.3 to 5 dPa·s, as measured by a Type B viscometer (using a No. 3 rotor) at 20°C. If the culture medium contains grain flour in the above-mentioned particle size and amount, the starch contained in the grain flour will achieve a viscosity in this range. However, if necessary, the viscosity of the culture medium may be appropriately adjusted by using a thickening agent or the like.

[0023] Filamentous fungal cultures cultivated in a liquid medium can be easily separated and recovered from the liquid medium by separation treatments such as centrifugation, filtration, and compression. The separated filamentous fungal cells may be subjected to additional treatments such as washing, crushing, and enzymatic treatment. In this invention, edible filamentous fungal cells may refer to the filamentous fungal cells themselves, or they may include culture medium components included in the manufacturing process. When edible filamentous fungal cells include components other than the fungal cells, such as culture medium components, they can be specifically referred to as a composition containing edible filamentous fungal cells. In addition to edible filamentous fungal cells, the composition may include any components, such as culture medium components and additives. The composition may also be a food composition.

[0024] The method of the present invention can improve the quantity or quality of protein in filamentous fungal cells after culture. The amount of protein can be evaluated by any method, such as the combustion method, Dumas method, Kjeldahl method, Biuret method, Lowry method, BCA method, HPLC method, or measuring devices and kits utilizing these methods. Using the method of the present invention, it is possible to obtain protein amounts (after removing water) per filamentous fungal cell after culture, such as 40-80% by mass, 42.5-70% by mass, or 45-60% by mass. Protein quality can be evaluated using various methods, including the amino acid score (AAS), the protein digestibility corrected amino acid score (PDCAAS), the digestible indispensable amino acid score (DIAAS), the biological value (BV), net protein utilization (NPU), the protein efficiency ratio (PER), and measurement devices and kits utilizing these factors. Using the method of the present invention, the protein quality in the filamentous fungal cells after culture can be, for example, expressed as an amino acid score according to the amino acid scoring pattern of international organizations (FAO / WHO / UNU), which is not limited to, but can be, for example, greater than 80, greater than 90, greater than 100, or greater than 110.

[0025] Edible filamentous fungal cells can be used as a food ingredient and can also be added to food. As mentioned above, filamentous fungal cells contain a large amount of high-quality protein in their solid content, so they can be used as a nutritional food ingredient or added to various foods for nutritional enhancement. By adjusting the conditions of additional enzymatic treatment with cell wall-degrading enzymes, the texture can be adjusted as desired, allowing for a melt-in-the-mouth texture while retaining a fibrous texture, or for processing into a paste. Edible filamentous fungal cells possess the high nutritional value of filamentous fungi, especially their high protein content, and can be used in a variety of foods. The edible filamentous fungal cells according to the present invention can be used as a substitute for soybeans, meat products, dairy products, wheat, rice, fish, egg products, etc., but are not intended to be limited to these uses. Edible filamentous fungal cells can also be used in beverages, food, etc. They can also be used as feed for animals such as livestock, pets, and farmed fish.

[0026] One aspect of the present invention relates to a method for producing a food composition or food as described above. This method for producing a food composition or food may include the steps of providing edible filamentous fungal cells produced by the method for producing edible filamentous fungal cells of the present invention, and / or culturing edible filamentous fungi in the culture medium of the present invention. The culture medium and the steps for culturing edible filamentous fungi are as described above.

[0027] A method for producing a food composition or food may include a step of producing a food composition or food using edible filamentous fungal cells obtained by the above step. In the step of producing a food composition or food, the obtained filamentous fungal cells may be collected and processed according to the desired flavor, texture, taste, and use, such as sterilization, cooking, addition of additives, packaging, freezing, or refrigeration. Any method of sterilization can be used, such as heat sterilization, filtration sterilization, drying sterilization, or ultrasonic sterilization. Any method of cooking can be used, such as adding seasonings and other ingredients, kneading, mixing, heating, seasoning, cutting, boiling, blanching, simmering, steaming, baking, roasting, smoking, or fermentation. Examples of additives include any additives such as seasonings, flavorings, flavoring agents, coloring agents, preservatives, pH stabilizers, pH adjusters, sweeteners, coloring agents, thickeners, stabilizers, gelling agents, antioxidants, color fixatives, bleaching agents, acidulants, coagulants, emulsifiers, leavening agents, and nutritional fortifiers. Packaging can be any method, such as retort processing, pouch packaging, canning, or bottling.

[0028] One aspect of the present invention relates to a method for producing a liquid culture medium, comprising the step of mixing one or more types of grain flour in a liquid, wherein the grain flour is selected from the group consisting of corn flour and wheat flour that can pass through a sieve with a mesh size of 1000 μm. The type, particle size, and content of the grain flour, the viscosity of the culture medium, and other components contained in the culture medium are as described above.

[0029] In this specification, "approximately" means that the stated numerical values, concentrations, quantities, times, temperatures, activities, and other values ​​include errors and variations within the range that a person skilled in the art would understand to be acceptable for achieving the effects of the present invention. Typically, this range is within ±10%, more preferably within ±5%, of the stated value, but if the appropriate range of variation recognized by a person skilled in the art differs depending on the measurement item and operating conditions, "approximately" shall be interpreted within the applicable range.

[0030] All references made herein are incorporated herein by citation in their entirety.

[0031] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention.

[0032] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited thereto.

[0033] Experiment 1: Examination of various grain flours 1. Preparation of koji conidia Aspergillus oryzae was spread onto a slant agar medium prepared from potato dextrose agar (manufactured by Becton Dickinson), grown for more than one week, and then suspended in physiological saline containing a surfactant (0.9% sodium chloride aqueous solution) and filtered to obtain conidia. These conidia were sown on polished rice that had been sterilized by pressurized steam, and grown on the rice for a total of 5 days to obtain rice koji. Subsequent cultivation was carried out using a conidial suspension obtained by suspending this rice koji in physiological saline containing a surfactant (0.9% sodium chloride aqueous solution) and filtering it.

[0034] 2. In a pre-culture baffled Erlenmeyer flask, a culture medium containing glucose as a carbon source and ammonium sulfate as a nitrogen source was prepared and sterilized by pressurized steam. Under sterile conditions, the aforementioned conidial suspension was added to the medium until the total number of conidia reached 5.5 × 10⁶. ８ The cells were prepared and inoculated into the aforementioned liquid culture medium. The number of conidia was measured using a TC20 cell counter (BIO-RAD). The liquid culture medium inoculated with conidia was cultured with swirling and shaking for 24 hours in a 30°C incubator to prepare the culture.

[0035] 3. Main Culture The main culture was performed using an aerated and stirred culture apparatus. To a basal medium containing ammonium sulfate as a nitrogen source, each carbon source (glucose, corn flour with a particle size that passes through a 150 μm sieve, wheat flour, corn starch, rice flour, potato starch, tapioca starch) was added to achieve the concentrations shown in Table 1 below, and the viscosity of each medium was measured at 20°C using a B-type viscometer (using a No. 3 rotor). These media were sterilized by pressurized steam, and after sterilization, an amount equivalent to 1% of the volume of the culture medium to be inoculated was inoculated into the next culture vessel from a flask in which pre-culture had been performed under sterile conditions. This was then cultured with aeration and stirring for 48 hours at a culture temperature of 30°C. In addition, the formation of mycelial masses in the culture medium was visually observed at 23, 30, and 47 hours from the start of culture, and the size and number of starch granules in the culture medium were observed over time under a microscope (200x magnification) at 0, 16, 23, and 44 hours.

[0036] 4. Yield Evaluation: The culture solution was filtered by suction, and water was removed by pressing. The weight of the resulting microbial cells was measured to obtain the weight of the Aspergillus oryzae (Koji mold) wet cells. The moisture content of the obtained Aspergillus oryzae was measured using a heat-drying infrared moisture meter ML50 (manufactured by A&D Co., Ltd.), and the weight (g) of Aspergillus oryzae per liter of culture medium was calculated.

[0037] 5. Evaluation of Protein Content The protein content was determined by measuring the total nitrogen content (%) of the sample using a total nitrogen analyzer (Sumigraph: provided by Sumika Analysis Center) with a combustion method, and multiplying that value by 6.25 to obtain the protein content.

[0038] 6. Evaluation of Amino Acid Scores For Examples 1 and 3 and Comparative Example 1, the amino acid scores were calculated using the following method. Hydrolysis was performed at 110°C for 24 hours using 6N hydrochloric acid. After that, the obtained samples were dried while being washed, dissolved in a measuring buffer, and the amount of amino acids was measured using an automated amino acid analyzer (HITACHI Amino Acid Analyzer L-8900). From the measured amounts of each amino acid and protein in the samples, the amino acid score was calculated by referring to the amino acid scoring pattern established by the international organization (FAO / WHO / UNU) in 2007.

[0039] 7. Results The results are shown in Table 1 and Figure 1. As shown in Table 1, compared to Comparative Example 1, which used glucose only as the carbon source, Examples 1 to 3, which used corn flour or wheat flour, showed an increase in yield. On the other hand, no increase in yield was observed in Comparative Examples 2 to 5, which used corn starch, rice flour, potato starch, and tapioca starch, respectively. Furthermore, in Examples 1 to 3, the protein content increased, and the amino acid score also exceeded 100. In addition, the viscosity of the culture medium in Examples 1 to 3 was 0.3 to 3.2, and good yields were observed within this viscosity range. As shown in Figure 1, in Example 1, which used corn flour, the shape of the mycelium was very fine from the beginning of cultivation, and as cultivation progressed, the shape of the mycelium became denser and fibrous, and the formation of mycelial masses was suppressed. Similar suppression of mycelial mass formation was observed in Examples 2 and 3. On the other hand, in Comparative Example 1, which used glucose only, the formation of mycelial masses was observed from the beginning of cultivation, and as cultivation progressed, the mycelial masses became larger, while the density of the mycelium remained low. Furthermore, observation of the size and number of starch granules over time revealed that in all culture solutions, the size decreased and the number decreased.

[0040] Experiment 2: Particle Size Investigation Using the corn flour that showed effectiveness in Experiment 1, the particle size was investigated. 1. In Erlenmeyer flasks with culture baffles containing corn flour of each particle size, 500 mL of culture medium was prepared by adding glucose or corn flour of each particle size that passed through sieves of 5000 μm, 1000 μm, 850 μm, 260 μm, or 150 μm, according to the proportions shown in Table 2, along with a basal medium containing ammonium sulfate as a nitrogen source. The viscosity of each medium was measured at 20°C using a B-type viscometer (using rotor No. 3). These media were sterilized by pressurized steam, and an amount equivalent to 1% of the target medium volume was inoculated into the next culture vessel from a flask that had been pre-cultured under sterile conditions in the same manner as in Experiment 1, 2. This was cultured in a 30°C incubator for 48 hours with swirling and shaking. The formation of mycelial masses in the culture medium was also observed.

[0041] 2. Yield Evaluation The yield of koji alone was calculated by estimating the amount of corn components remaining after decomposition by enzymes produced by koji cultivation, and subtracting the resulting amount. More specifically, the cultured solution was subjected to suction filtration to obtain a mixture of koji cells and corn. Each of these mixtures was freeze-dried for a total of 64 hours to remove water, and its weight was measured to determine the weight of the koji cell mixture (dry). In addition, 100 mL of a culture medium was prepared in a separate baffled Erlenmeyer flask, containing ammonium sulfate as a nitrogen source and glucose or corn of various particle sizes as shown in the table. 100 mL of the above filtrate was added to this medium, and it was shaken at 30°C for the same amount of time as the cultivation (48 hours). After suction filtration, the remaining corn was freeze-dried for a total of 64 hours to remove water, and its weight was measured to assume the weight of corn remaining after cultivation. The weight of the dried Aspergillus oryzae was determined by subtracting this weight from the weight of the above Aspergillus oryzae mixture (dried).

[0042] 3. Results are shown in Table 2. As shown in Table 2, Comparative Example 6, which used glucose only as the carbon source, had a low yield. However, in Examples 4 to 7, which used corn flour of various particle sizes that could pass through a sieve of 1000 μm or less, higher yields were obtained as the sieve opening size decreased from 1000 μm, 850 μm, 260 μm, to 150 μm. In addition, while large mycelial clumps formed by the aggregation of mycelium were observed in Comparative Example 6, the formation of mycelial clumps was less frequent in Examples 4 to 7, which used corn flour of various particle sizes that could pass through a sieve of 1000 μm or less. Even when clumps were formed, their shape was fluffy and the degree of aggregation was low, indicating that the formation of mycelial clumps was suppressed. On the other hand, in Comparative Example 7, which used corn flour with a particle size that could pass through a 5000 μm sieve, the formation of mycelial clumps was suppressed, but no increase in yield was observed. Furthermore, the viscosity of the culture solution increased as the particle size of the corn flour decreased.

[0043] Experiment 3: Examination of Content Using corn flour with a particle size that passed through a 150 µm sieve, which showed effects in Experiment 2, examination was conducted on its content. 1. In Erlenmeyer flasks with baffles for culture using media containing corn at each respective content, 500 mL of a basal medium containing ammonium sulfate as a nitrogen source, and a medium prepared to have the composition shown in Table 3 by adding glucose or corn flour with a particle size that passed through a 150 µm sieve was prepared. The viscosity of each medium was measured at 20°C using a B-type viscometer (using a No. 3 rotor). These media were sterilized by autoclaving. Under aseptic conditions, an amount equivalent to 1% of the volume of the destination culture medium was taken from the flask in which pre-culture had been performed in the same manner as in step 2 of Experiment 1, and inoculated into the next culture vessel. This was subjected to 48 hours of rotary shaking culture in a 30°C incubator.

[0044] 2. Yield Evaluation Aspergillus oryzae was isolated by suction filtration, the moisture content was measured using a heat-drying infrared moisture meter ML50 (manufactured by A&D Company), and the weight (g) of dried Aspergillus oryzae cells per medium volume (500 mL) was calculated.

[0045] 3. Results The results are shown in Table 3. Compared with Comparative Example 8, in which only glucose was used as the carbon source, increased yield was confirmed in Examples 8 to 11, in which the proportion of corn flour with a particle size that passed through a 150 µm sieve was increased. Furthermore, yield and viscosity increased as the proportion of corn flour increased.

[0046] In Experiment 1, good yields were obtained in Examples 1-3, which used corn flour and wheat flour, respectively, and suppression of mycelial mass formation and high density of mycelium in the culture medium were observed. On the other hand, in Comparative Example 1, which used only glucose, and Comparative Examples 1-5, which used other grains, the formation of large, aggregated mycelial masses was observed, and the density of mycelial cells in the culture medium and the yield of mycelial cells were low. Furthermore, Examples 1 and 3, which used corn flour, showed higher yields compared to Comparative Example 2, which used corn starch. Corn starch is extracted and purified from the endosperm of corn, but since mycelial mass formation occurs and the yield of mycelial cells is low with such purified starch, it is possible that components contained in unrefined corn flour that are removed during the purification process are affecting the suppression of mycelial mass formation and the good growth of mycelial cells. In Experiment 2, when corn flour with a particle size that could pass through a sieve of 1000 μm or less was used, it was confirmed that as the mesh size decreased, the formation of mycelial masses decreased, and even when they were formed, the degree of aggregation was low, indicating that mycelial mass formation was suppressed, and a good yield was obtained. It is thought that when mycelial mass formation is small and the degree of aggregation is low, nutrients can easily reach the center of the mycelial mass, promoting fungal growth and thus improving the yield of fungal cells. On the other hand, in Comparative Example 7, which used corn flour with a larger particle size, mycelial mass formation was suppressed, but no increase in yield was observed. This is thought to be because the particle size was too large, so the corn flour was not sufficiently decomposed, and nutrients were depleted. From these results, it is suggested that it is preferable to use grain flour with a particle size that can pass through a sieve with a mesh size of at least 1000 μm or less, preferably 850 μm or less, more preferably 300 μm, for example 260 μm or less, and even more preferably 150 μm or less. With this particle size, the grain flour is moderately decomposed, supplying the nutrients necessary for mycelial growth. At the same time, the collision of grain flour particles with the fungal cells in the liquid culture medium suppresses the formation of mycelial masses, making aggregation of mycelial masses less likely. Therefore, it is possible that filamentous fungi can grow efficiently.In Experiment 3, the results improved as the grain flour content increased from 2.77 g / L (approximately 0.28% by weight) to 27.67 g / L (approximately 2.77% by weight). This suggests that the lower limit is preferably 0.2% by mass or more, preferably 0.5% by mass or more, more preferably 0.8% by mass or more, and even more preferably 1.0% by mass or more, relative to the total volume of the liquid medium. On the other hand, microscopic observation in Experiment 1 confirmed that the amount of starch granules derived from grain flour in the culture medium decreased with cultivation, indicating that the starch granules were utilized for the growth of filamentous fungi. Setting the upper limit of the grain flour content in the medium to 20% by mass or less, more preferably 15% by mass or more, and even more preferably 10% by mass or less, suggests that the amount of grain flour recovered can be adjusted to 0.1% by mass or less, preferably 0.75% by mass or less, and even more preferably 0.05% by mass or less, relative to the total volume of the liquid medium. This range is preferable from the viewpoint of ease of recovering the fungal cells. Furthermore, Experiment 3 shows that sufficient yield can be obtained even with a viscosity of 0 dPa·s, but a viscosity of 0.15 dPa·s or higher is more preferable, and Experiments 1 and 2 show that a viscosity in the range of 0.3 to 5 dPa·s is even more preferable. These results suggest that by using grain flour of a specific particle size, the starch dissolves in the liquid medium, increasing the viscosity of the liquid medium, which may make it more difficult for the fungal cells to aggregate. This suggests that viscosity may contribute to preventing the aggregation of mycelium.

Claims

1. A method for producing edible filamentous fungal cells, comprising the step of culturing filamentous fungi in a liquid medium containing one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour that pass through a sieve with a mesh size of 1000 μm.

2. The method according to claim 1, wherein the content of grain flour in the liquid culture medium is 1 to 10% by mass relative to the total amount of the liquid culture medium.

3. The method according to claim 1, wherein the viscosity of the liquid culture medium is 0.15 to 5 dPa·s.

4. The method according to claim 2, further comprising the step of recovering the microbial cells when the content of grain flour in the liquid medium becomes 0.05% by mass or less relative to the total amount of the liquid medium.

5. The method according to claim 1, wherein the liquid culture medium contains 0.5 to 18% by mass of one or more sugars selected from the group consisting of monosaccharides and disaccharides, relative to the total amount of the liquid culture medium.

6. A liquid culture medium for culturing edible filamentous fungal cells, comprising one or more types of grain flour, wherein the grain flour is selected from the group consisting of corn flour and wheat flour, and passes through a sieve with a mesh size of 1000 μm.

7. The liquid culture medium according to claim 6, wherein the content of grain flour in the liquid culture medium is 1 to 10% by mass relative to the total amount of the liquid culture medium.

8. The liquid culture medium according to claim 6 or 7, wherein the viscosity of the liquid culture medium is 0.15 to 5 dPa·s.

9. The liquid culture medium according to claim 6 or 7, wherein the liquid culture medium contains 0.5 to 18% by mass of one or more sugars selected from the group consisting of monosaccharides and disaccharides, relative to the total amount of the liquid culture medium.

10. Edible filamentous fungal cells produced by the method described in any one of claims 1 to 5.

11. A food composition or food containing the edible filamentous fungal cells described in claim 10.

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