Method for producing koji mold mycelium, and meat-like mycelium

Abstract

To provide a novel technique which enables efficient production of mycelium in a short time.SOLUTION: The present invention provides a method for producing mycelium of koji mold, the method including a culture step involving liquid-culture of koji mold in a culture medium containing grains, and a recovery step involving recovery of the mycelium obtained in the culturing step.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a mycelium of Aspergillus oryzae and a meat-like mycelium produced by the production method.

Background Art

[0002] In recent years, technologies for processing mycelia obtained by culturing filamentous fungi into meat substitute foods have been developed. The obtained mycelia are also called "mycoprotein" and are mainly produced and sold actively in Europe and the like. For example, Quorn, a pioneer in mycoprotein production, produces mycoprotein using bacteria of the genus Fusarium.

[0003] In addition, production of mycoprotein using Aspergillus oryzae known for food use has also been carried out. For example, Patent Document 1 describes culturing Aspergillus oryzae in a liquid culture medium, collecting the grown cells, and using them as mycella for meat substitutes. Patent Document 2 describes a method for producing mycella for meat, in which a seed culture of Aspergillus oryzae is inoculated into cooked grains, Aspergillus oryzae is solid-cultured, and the obtained solid culture is mixed with water to collect mycella.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the method for producing mycella according to the above prior art, it was necessary to continue culturing for a long time of 3 days or more in order to obtain an amount of cells that could be processed into food. In view of the above circumstances, an object of the present invention is to provide a novel technique capable of efficiently producing mycelia in a short period of time.

Means for Solving the Problem

[0006] The present invention for solving the above problems and its preferred forms are as follows. [1] A culturing step of liquid-culturing Aspergillus oryzae in a culture solution containing grains, and A recovering step of recovering the mycelium obtained in the culturing step, A method for producing a mycelium of Aspergillus oryzae, comprising the above.

[0007] By liquid-culturing Aspergillus oryzae in a culture solution containing grains, the present invention can efficiently produce mycelium.

[0008] [2] The culturing method according to [1], wherein the culturing period of Aspergillus oryzae in the culturing step is 48 hours or less. In the present invention of the above form, even if the culturing period is 48 hours or less, a sufficient amount of mycelium can be produced, and the production efficiency is good.

[0009] [3] The manufacturing method according to [1] or [2], wherein the grains are one or more selected from seeds of Gramineae crops, seeds of Leguminosae crops, and processed products thereof. By adopting such a form, the production efficiency of the mycelium can be improved.

[0010] [4] The manufacturing method according to any one of [1] to [3], wherein the grains include one or more selected from rice, soybeans, wheat, barley, millet, adzuki beans, corn, and processed products thereof. By adopting such a form, the production efficiency of the mycelium can be improved.

[0011] [5] The manufacturing method according to any one of [1] to [4], wherein the grains include one or more selected from okara, rice bran, brown rice, wheat flour, defatted rice germ, defatted soybeans, sake lees, and beer lees. By adopting such a form, the production efficiency of the mycelium can be improved.

[0012] [6] The culture solution contains 6 w / v% or more of the grains in terms of dry mass, and is the production method according to any one of [1] to [5]. By adopting such a form, the production efficiency of the mycelium can be increased, and a sufficient amount of mycelium can be produced in a short time.

[0013] [7] The culture solution contains raw materials other than the grains, The raw materials other than the grains are saccharides and / or nitrogen sources, and are the production method according to any one of [1] to [6]. By adopting such a form, the production amount of the mycelium can be further increased.

[0014] [8] The saccharides are one or more selected from monosaccharides, disaccharides, and oligosaccharides, and are the production method according to [7]. By adopting such a form, the production amount of the mycelium can be further improved.

[0015] [9] The nitrogen source is one or more selected from proteins and their hydrolysates, and extracts derived from animals, and is the production method according to [7] or [8]. By adopting such a form, the production amount of the mycelium can be further improved.

[0016]

[10] The production method according to any one of [1] to [9] includes a washing step of washing and dehydrating the mycelium obtained in the recovery step. By adopting such a form, the processability of the mycelium into foods and the like can be improved.

[0017]

[11] After the culturing step, the titer of total protease (pH 7) in the culture solution measured by the following measurement method A is 50 U / g or less, and is the production method according to any one of [1] to

[10] . [Measurement method A] (1) Mix 0.5 ml of the culture solution with 10 ml of 66.7 mmol / L phosphate buffer, filter with filter paper, and obtain a filtrate. (2) After culturing 5 ml of the casein solution (pH 7.0) at 37 °C for 5 minutes, add 1 ml of the filtrate to the casein solution and culture at 37 °C for 10 minutes. (3) After culturing, add 5 ml of trichloroacetic acid precipitation reagent and culture at 37 °C for 30 minutes. Filter the resulting solution to obtain a reaction solution. (4) Add 5 ml of 0.55 mol / L sodium carbonate test solution to 2 ml of the reaction solution and stir. Add 1 ml of the three-fold diluted Folin test solution to the resulting reaction solution, stir, and culture at 37 °C for 30 minutes. (5) For the reaction solution obtained in (4) above, use a spectrophotometer and measure the absorbance (Abs S1 ) at a wavelength of 660 nm by ultraviolet-visible absorbance measurement method. (6) Separately, after adding 5 ml of trichloroacetic acid precipitation reagent to 1 ml of the filtrate, further add 5 ml of the casein solution and culture at 37 °C for 30 minutes to obtain a control solution. (7) Add 5 ml of 0.55 mol / L sodium carbonate test solution to 2 ml of the control solution and stir. Add 1 ml of the three-fold diluted Folin test solution to the resulting reaction solution, stir, and culture at 37 °C for 30 minutes. (8) For the control solution obtained in (7) above, use a spectrophotometer and measure the absorbance (Abs B1 ) at a wavelength of 660 nm by ultraviolet-visible absorbance measurement method. (9) Calculate the activity of total protease (pH 7) based on the following formula 1. (Formula 1) Activity of total protease (pH 7) (U / g) = (Abs S1 - Abs B1 ) × Σ × 11 / 2 × 1 / 10 × 1 / 0.5 (However, the coefficient Σ represents the amount of tyrosine (μg) when the absorbance difference obtained from the tyrosine calibration curve is 1.) In the present invention of the above-described form, since the protease titer in the culture solution is low, the production amount of enzymes such as protease is very small. That is, in the present invention of such a form, since the mycelium is preferentially produced rather than the enzyme, the productivity of the mycelium is high. Further, since the obtained mycelium has a low content of enzymes such as protease, according to the present invention of such a form, the odor derived from the enzyme reaction in the mycelium can be suppressed.

[0018]

[12] A meat-like mycelium produced by the production method according to any one of [1] to

[11] .

Effect of the Invention

[0019] According to the present invention, mycelium can be efficiently produced in a short period of time.

Mode for Carrying Out the Invention

[0020] In this specification, "%" means "mass%". Unless otherwise specified, "w / v%" refers to the mass in the volume of water used for culture.

[0021] The method for producing the mycelium of Aspergillus oryzae of the present invention (hereinafter, also referred to as the production method of the present invention) includes a culturing step of liquid-culturing Aspergillus oryzae in a culture solution containing grains, and a recovering step of recovering the mycelium obtained in the culturing step. Further, in a preferred form of the present invention, the production method of the present invention includes a washing step of washing and dehydrating the mycelium obtained in the recovering step. Hereinafter, preferred forms of each step will be described.

[0022] (1) Culturing step The culturing step is a step of liquid-culturing Aspergillus oryzae in a culture solution containing grains. In the present invention, unlike the solid culture method in which Aspergillus oryzae is propagated in a state of being attached to grains or the like, Aspergillus oryzae is cultured in liquid culture. By performing liquid culture, it is possible to completely sterilize the medium (culture solution) for culturing Aspergillus oryzae with a medium sterilizer such as an autoclave sterilizer, so that the generation of putrid odor derived from miscellaneous bacteria can be prevented.

[0023] Aspergillus spp. are used as the koji molds in the present invention. Aspergillus spp. used for producing fermented foods and fermented seasonings such as sake, miso, vinegar, pickles, soy sauce, shochu, awamori, and dried bonito include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis mut. Kawachii (former name: Aspergillus kawachii), Aspergillus luchuensis (former name: Aspergillus awamori), Aspergillus saitoi, and Aspergillus glaucus. Either wild-type strains or mutant strains may be used. Also, one strain of these koji molds may be inoculated, or two, three, four, or five or more types of strains may be inoculated and mixed cultured. In the present invention, one or more selected from Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, and Aspergillus luchuensis mut. Kawachii can be preferably used.

[0024] The koji mold to be inoculated may be spores or pre-cultured mycelia, but it is desirable to inoculate mycelia in that the time required for the logarithmic growth phase is shortened.

[0025] For example, water is added to wheat bran (the part of the wheat husk removed in the wheat flour milling process), koji is inoculated and cultured, and the resulting koji is used as a solid seed. The solid seed is diluted and inoculated as a starter. The solid seed refers to grains such as wheat bran and rice made into koji. The solid seed can be made according to the following procedure.

[0026] When koji is scattered on grains and left standing at room temperature and high humidity, white hyphae grow and penetrate into the grains, starting enzyme production and heat generation. If this is stirred to dissipate heat and ventilate, sporulation occurs, and further culturing results in coloring and good solid koji. The culturing of Aspergillus oryzae can be carried out for 1 to 7 days, preferably 3 to 5 days. Since solid koji forms spores, it can be stored for 2 to 3 months in the refrigerator, but it is preferably used within 1 month.

[0027] The inoculation amount into the culture solution of Aspergillus oryzae is not limited, but in the case of spores, it is 1×10 2 ~10 6 / mL, and from the viewpoint of the growth rate of Aspergillus oryzae, it is more preferably inoculated into the liquid medium so as to have a density of 1×10 4 ~10 6 / mL. In the case of mycelia obtained by preculturing, the preculture solution may be added to the liquid medium at 0.1 to 10 w / v%.

[0028] The culture solution used for culturing Aspergillus oryzae contains grains. The grains contained in the culture solution are preferably one or more selected from seeds of Gramineae crops, seeds of Leguminosae crops, and processed products thereof. Here, in the present invention, the processed product of grains includes pulverized products obtained by pulverizing seeds of Gramineae crops or Leguminosae crops, fermented products obtained by fermenting the pulverized products, and by-products (end materials) such as husks and bran generated during the production of grains.

[0029] In a preferred embodiment of the present invention, the grains added to the culture solution include one or more selected from rice, soybeans, wheat, barley, millet, adzuki beans, corn, and processed products thereof. Among them, it is more preferable that the grains added to the culture solution are one or more selected from rice, soybeans, wheat, barley, and processed products thereof.

[0030] When using rice as the grain, brown rice, rice bran, rice germ, defatted rice germ, sake lees, etc. can be preferably used. Polished rice bran is the bran generated during the polishing of rice, and there are red bran on the bran side and mid flour on the endosperm side. In the present invention, either red bran or mid flour can be preferably used. Defatted rice germ is the residue obtained by extracting rice oil from the rice germ removed during the polishing of rice. Sake lees is the residue remaining after squeezing sake from the moromi obtained by fermenting rice and koji in the production of sake.

[0031] When using barley as the grain, barley, malt, beer lees, etc. can be preferably used. Beer lees is the residue remaining after extracting saccharides from malt during the production of beer.

[0032] When using wheat as the grain, wheat flour (flour milled excluding the epidermis and germ), whole grain flour, etc. can be preferably used. The wheat flour to be used is not particularly limited, and any of weak flour, medium flour, and strong flour can be used, but weak flour is particularly preferably used.

[0033] When using soybeans as the grain, okara, defatted soybeans, soybean flour, soy milk, etc. can be preferably used. Defatted soybeans are the residue obtained by extracting soybean oil from soybeans and are also called soybean meal. As defatted soybeans, commercially available products such as "ZFS Soya" of Nisshin Shokai Co., Ltd. can be used, for example.

[0034] In one embodiment, the grain added to the culture solution is preferably a defatted product. Examples of defatted products include those obtained by defatting the above-mentioned grains, and for example, defatted soybeans, defatted rice germ, etc. can be preferably used.

[0035] In the present invention, the grain contained in the culture solution is preferably one or more selected from okara, polished rice bran, brown rice, wheat flour, defatted rice germ, defatted soybeans, sake lees, and beer lees.

[0036] The lower limit of the content of grains in the culture medium is preferably 1 w / v% or more, more preferably 3 w / v% or more, more preferably 5 w / v% or more, more preferably 6 w / v% or more, more preferably 8 w / v% or more, more preferably 10 w / v% or more in terms of dry mass. The upper limit of the content of grains is not particularly limited, but is preferably 25 w / v% or less, more preferably 20 w / v% or less, more preferably 18 w / v% or less, more preferably 15 w / v% or less in terms of dry mass. By setting the content of grains in the culture medium within the above range, mycelium can be efficiently produced.

[0037] As a result of intensive research, the inventors have found that when the content of grains in the culture medium is particularly 6 w / v% or more in terms of dry mass, the production amount of mycelium further increases. That is, in the present invention, the content of grains in the culture medium is preferably 6 w / v% or more, more preferably 7 w / v% or more, more preferably 8 w / v% or more, more preferably 9 w / v% or more, more preferably 10 w / v% or more in terms of dry mass. By setting the lower limit of the content of grains in the culture medium within the above range, the production efficiency of mycelium can be further improved, and a sufficient amount of mycelium can be obtained in a short time.

[0038] Also, the range of the content of grains can be preferably 1 to 25 w / v%, more preferably 2 to 20 w / v%, still more preferably 3 to 18 w / v%, still more preferably 4 to 18 w / v%, still more preferably 5 to 15 w / v%, still more preferably 6 to 15 w / v%, still more preferably 6 to 10 w / v% in terms of dry mass. In the present invention, powdery grains can be preferably used. Also, grains in a form containing moisture (for example, undried raw grains, okara, soy milk, etc.) instead of dried ones can be used. When using grains containing a large amount of moisture, the above content of grains can be calculated by converting it to the mass of the dried product after subtracting the moisture content. Also, when using soy milk, it is only necessary that the solid content (in terms of mass) is included in the range of the above content of grains (dry mass).

[0039] In a preferred embodiment of the present invention, the culture medium contains raw materials other than grains. The raw materials other than grains are preferably saccharides and / or nitrogen sources. Hereinafter, in this specification, "sugars other than grains" and "nitrogen sources other than grains" are simply referred to as "sugars" and "nitrogen sources", respectively.

[0040] As the sugars contained in the culture medium, refined sugars generally used for culturing Aspergillus oryzae can be used, and monosaccharides, disaccharides, and polysaccharides such as oligosaccharides and starch can be used. In one embodiment of the present invention, the sugars to be used are preferably one or more selected from monosaccharides, disaccharides, oligosaccharides, and polysaccharides, more preferably one or more selected from monosaccharides, disaccharides, and oligosaccharides, and even more preferably one or more selected from monosaccharides and disaccharides. Preferred examples of such sugars include monosaccharides such as glucose and fructose, disaccharides such as maltose, sucrose, and laminaribiose, oligosaccharides such as fructooligosaccharide, galactooligosaccharide, and isomaltooligosaccharide, and polysaccharides such as starch, glycogen, cellulose, and pectin. In the present invention, it is preferable to use one or more selected from glucose, maltose, fructose, and sucrose as the sugars.

[0041] Also, in the present invention, a mixture of a plurality of sugars may be added. For example, isomerized sugars such as fructose-glucose syrup and glucose-fructose syrup can be used.

[0042] The nitrogen sources contained in the culture medium include proteins or their hydrolysates (HVP), animal-derived extracts, fungus-derived extracts, inorganic nitrogen compounds, etc. Among them, one or more selected from proteins and their hydrolysates, animal-derived extracts, and fungus-derived extracts can be preferably used.

[0043] Examples of the protein or its hydrolyzate include peptones, casein, casein hydrolyzate, etc. In the present invention, it is preferable to use peptones as the protein or its hydrolyzate.

[0044] Examples of the peptones used in the present invention preferably include casein peptone, meat peptone, myocardial peptone, gelatin peptone, and soy peptone. Examples of the digestive enzymes in the peptones used in the present invention include trypsin, pancreatin, pepsin, papain, etc., and they may be enzymes derived from bacteria, animals, or plants. In the present invention, it is preferable to use casein peptone as the nitrogen source.

[0045] Examples of the animal-derived extract preferably include meat extract. The meat extract refers to a component extracted as a water-soluble component from mammalian meat or seafood, and includes a concentrated extract obtained by extracting meat with hot water, or a concentrated juice obtained by decomposing meat with an enzyme and / or an acid. The meat extract contains many medium- to low-molecular-weight proteins that have been peptidized. In the present invention, it is preferable to use beef extract and / or bonito extract.

[0046] Examples of the fungus-derived extract preferably include yeast extract.

[0047] Commercially available products can be used for peptones, animal-derived extracts, and fungus-derived extracts.

[0048] In the present invention, when using a protein or its hydrolyzate (HVP), animal-derived extract, fungus-derived extract, etc. as the nitrogen source, the component used as the nitrogen source preferably has a total nitrogen content of 7% by mass or more, more preferably 9% by mass or more, still more preferably 10% by mass or more, even more preferably 10.5% by mass or more, and still more preferably 11% by mass or more.

[0049] Examples of the inorganic nitrogen compound preferably include ammonium sulfate, ammonium phosphate, ammonium carbonate, ammonium acetate, etc.

[0050] In the present invention, as raw materials other than grains, either of the above-mentioned saccharides or nitrogen sources may be added, or a plurality of combinations of the above-mentioned saccharides and nitrogen sources may be added. In one embodiment of the present invention, the culture solution can contain one or more selected from saccharides and one or more selected from nitrogen sources as raw materials other than grains.

[0051] The content of saccharides in the culture solution is preferably 0.5 w / v% or more, more preferably 0.8 w / v% or more, and still more preferably 1.0 w / v% or more in terms of dry mass. The content of saccharides in the culture solution is preferably 5.0 w / v% or less, more preferably 4.0 w / v% or less, and still more preferably 3.0 w / v% or less in terms of dry mass. The content of saccharides in the culture solution is preferably 0.5 - 5.0 w / v%, more preferably 0.8 - 4.0 w / v%, and still more preferably 1.0 - 3.0 w / v% in terms of dry mass. By using a medium containing saccharides in the above content, the production amount of mycelia can be further increased.

[0052] The content of the nitrogen source in the culture solution is preferably 0.5 w / v% or more, more preferably 0.8 w / v% or more, and still more preferably 1.0 w / v% or more in terms of dry mass. The content of the nitrogen source in the culture solution is preferably 5.0 w / v% or less, more preferably 4.0 w / v% or less, and still more preferably 3.0 w / v% or less in terms of dry mass. The content of the nitrogen source in the culture solution is preferably 0.5 - 5.0 w / v%, more preferably 0.8 - 4.0 w / v%, and still more preferably 1.0 - 3.0 w / v% in terms of dry mass. By using a medium containing the nitrogen source in the above content, the production amount of mycelia can be further increased.

[0053] The total content of saccharides and nitrogen sources in the culture solution is preferably 0.5 w / v% or more, more preferably 0.8 w / v% or more, and still more preferably 1.0 w / v% or more in terms of dry mass. The total content of saccharides and nitrogen sources in the culture solution is preferably 6.0 w / v% or less, more preferably 5.0 w / v% or less, and even more preferably 4.5 w / v% or less in terms of dry mass. The total content of saccharides and nitrogen sources is preferably 0.5 - 6.0 w / v%, more preferably 0.8 - 5.0 w / v%, and even more preferably 1.0 - 4.5 w / v% in terms of dry mass. By using the medium containing the saccharides and / or nitrogen sources with the above content, the production amount of mycelia can be further increased.

[0054] In addition, the total content of the above saccharides and nitrogen sources relative to the content of grains in the culture solution is preferably 0.05 times or more, more preferably 0.08 times or more, and even more preferably 0.1 times or more in terms of dry mass conversion. The total content of the above saccharides and nitrogen sources relative to the content of grains in the culture solution is preferably 1 time or less, more preferably 0.9 times or less, and even more preferably 0.8 times or less in terms of dry mass conversion. The content of the above saccharides and nitrogen sources relative to the content of grains in the culture solution can also be 0.5 times or less, more preferably 0.45 times or less. The total content of the above saccharides and nitrogen sources relative to the content of grains in the culture solution is preferably 0.05 - 1 time, more preferably 0.08 - 0.9 times, and even more preferably 0.1 - 0.8 times in terms of dry mass conversion. The content of the above saccharides and nitrogen sources relative to the content of grains in the culture solution can also be preferably 0.05 - 0.5 times, more preferably 0.08 - 0.5 times, and even more preferably 0.1 - 0.45 times in terms of dry mass conversion. When only one type of sugar source or nitrogen source is contained in the culture solution, the total content of the above saccharides and nitrogen sources refers to the content of one type of saccharide or nitrogen source.

[0055] The culture solution (liquid medium) used may contain components suitable for culturing mycelia. For example, the culture solution can contain inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphate salts, manganese salts, iron salts, zinc salts, etc., and vitamins. In one embodiment of the present invention, the culture medium may be in a form that does not contain additives other than the above-mentioned grains, sugars, and nitrogen sources. For example, in one embodiment, the culture medium to be used may be one to which five or fewer, four or fewer, or three or fewer additives are added to water, and the number of the additives can be the number including the above-mentioned grains, sugars, and nitrogen sources.

[0056] The culturing method in the culturing step is not particularly limited, and a method used in a general liquid culturing method can be adopted. Specifically, it can be cultured by shaking culture using an Erlenmeyer flask with a baffle or aerobic culture using a jar fermenter. In the present invention, either batch culture or continuous culture can be adopted.

[0057] In one embodiment, in the culturing step of the present invention, Aspergillus oryzae can be cultured by batch culture. That is, in the culturing step, after the start of the liquid culture of Aspergillus oryzae, it may be in a form in which culture medium components such as the above-mentioned grains, sugars, or nitrogen sources are not newly added. In such a form, since operations such as replenishment of the culture medium components are unnecessary, the culturing step becomes simpler.

[0058] In the culturing step, it is preferable to perform the culture while shaking the culture vessel. The shaking conditions of the culture vessel are not particularly limited, and the rotation speed of the stirring blades of the culture vessel can be 100 to 1000 rpm.

[0059] The culturing temperature in the culturing step is not particularly limited as long as it is a temperature suitable for culturing Aspergillus oryzae, but it is preferably 25 to 40 °C, more preferably 30 to 35 °C.

[0060] In addition, the aeration rate to the culture medium in the culturing step can be appropriately adjusted according to the capacity of the culture device such as the jar fermenter to be used, and can be, for example, 0.1 to 20 L / min. Taking specific examples, in a 10 L mini-jar fermenter, the aeration rate can be 2.0 L / min. In a 90 L jar fermenter, the aeration rate can be 18 L / min.

[0061] In a preferred embodiment of the present invention, the culture period of Aspergillus oryzae in the culturing step is 48 hours or less. That is, in such an embodiment, after completing the culturing period of 48 hours or less, a recovery step of immediately recovering the mycelia is carried out. In the present invention of the above-described embodiment, a sufficient amount of mycelia can be produced without culturing Aspergillus oryzae for longer than 48 hours in the culturing step. Also, when the culture period extends over a long period of 3 days to 10 days or more, Aspergillus oryzae starts to produce metabolites such as enzymes in addition to mycelia. Metabolites and enzyme degradation products derived from koji produce an unfavorable aroma (i.e., off-flavor). In the present invention, as shown in the examples described later, when the culture period is 48 hours or less, the titer of total protease and / or α-amylase in the culture solution is low, so that mycelia in which off-flavors derived from enzyme degradation products and metabolites by Aspergillus oryzae are suppressed can be obtained.

[0062] Also, the culture period of Aspergillus oryzae in the culturing step may be less than 48 hours, preferably 45 hours or less, more preferably 42 hours or less, more preferably 40 hours or less, more preferably 38 hours or less, more preferably 35 hours or less, more preferably 32 hours or less, more preferably 28 hours or less, more preferably 25 hours or less, and still more preferably 24 hours or less. By setting the culture period of Aspergillus oryzae within the above range, off-flavors of the mycelia derived from enzyme degradation products and metabolites by Aspergillus oryzae can be more effectively suppressed.

[0063] Also, the culture period of Aspergillus oryzae in the culturing step may be 16 hours or less.

[0064] In a preferred embodiment of the present invention, the titer of total protease (pH 7) in the culture solution after the culturing step is preferably 50 U / g or less, more preferably 40 U / g or less, still more preferably 30 U / g or less, still more preferably 20 U / g or less, still more preferably 10 U / g or less, still more preferably 8 U / g or less, still more preferably 6 U / g or less, still more preferably 5 U / g or less, still more preferably 4 U / g or less, and even more preferably 3 U / g or less. Further, the titer of the total protease (pH 7) activity in the culture solution after the culturing step may be 2 U / g or less, may be 1 U / g or less, or may be below the measurement limit (i.e., 0 U / g). When the total protease (pH 7) activity in the culture solution is within the above range, since the mycelium is preferentially produced rather than the enzyme, the mycelium can be efficiently produced. Further, since the obtained mycelium has a low content of enzymes such as protease, the odor derived from the enzyme reaction in the mycelium can be suppressed. That is, by setting the total protease (pH 7) activity in the culture solution within the above range, a mycelium with suppressed Aspergillus oryzae-derived off-flavors can be obtained.

[0065] The total protease activity can be measured according to the 2. Protein digestion power test method of the 4.03 Digestive power test method of the 18th revised Japanese Pharmacopoeia. More specifically, the titer of total protease (pH 7) in the culture solution after the culturing step can be measured by the procedure described in the following Measurement method A. [Measurement method A] (1) Mix 0.5 ml of the culture solution with 10 ml of 66.7 mmol / L phosphate buffer, filter with filter paper, and obtain a filtrate. (2) After culturing 5 ml of the casein solution (pH 7.0) at 37°C for 5 minutes, add 1 ml of the filtrate to the casein solution and culture at 37°C for 10 minutes. (3) After culturing, add 5 ml of trichloroacetic acid precipitation reagent and culture at 37°C for 30 minutes, filter the obtained solution, and use it as a reaction solution. (4) Add 5 ml of 0.55 mol / L sodium carbonate test solution to 2 ml of the reaction solution and stir. To the obtained reaction solution, add 1 ml of the Folin test solution diluted 3 times, stir, and incubate at 37 °C for 30 minutes. (5) For the reaction solution obtained in (4) above, using a spectrophotometer, by the ultraviolet-visible absorbance measurement method, measure the absorbance (Abs S1 ) at a wavelength of 660 nm. (6) Separately, after adding 5 ml of trichloroacetic acid precipitation reagent to 1 ml of the filtrate, further add 5 ml of the casein solution, incubate at 37 °C for 30 minutes to obtain a control solution. (7) Add 5 ml of 0.55 mol / L sodium carbonate test solution to 2 ml of the control solution obtained in (7) above and stir. To the obtained reaction solution, add 1 ml of the Folin test solution diluted 3 times, stir, and incubate at 37 °C for 30 minutes. (8) For the control solution obtained in (7) above, using a spectrophotometer, by the ultraviolet-visible absorbance measurement method, measure the absorbance (Abs B1 ) at a wavelength of 660 nm. (9) Calculate the activity of total protease (pH 7) based on the following formula 1. (Formula 1) Activity of total protease (pH 7) (U / g) = (Abs S1 - Abs B1 ) × Σ × 11 / 2 × 1 / 10 × 1 / 0.5 (However, the coefficient Σ represents the amount of tyrosine (μg) when the absorbance difference obtained from the tyrosine calibration curve is 1.)

[0066] In a preferred embodiment of the present invention, the titer of α-amylase in the culture broth after the culturing step is preferably 50 U / g or less, more preferably 40 U / g or less, still more preferably 30 U / g or less, even more preferably 20 U / g or less, even more preferably 10 U / g or less, even more preferably 8 U / g or less, even more preferably 6 U / g or less, even more preferably 5 U / g or less, and still more preferably 4.5 U / g or less. Further, the titer of α-amylase in the culture broth after the culturing step may be 3 U / g or less, may be 2.5 U / g or less, may be 2 U / g or less, may be 1.5 U / g or less, may be 1 U / g or less, and may even be below the measurement limit (i.e., 0 U / g). By setting the titer of α-amylase in the culture broth within the above range, it is possible to preferentially produce mycelia rather than the enzyme, and it is possible to efficiently produce mycelia. Further, it is possible to obtain mycelia with suppressed off-flavors derived from Aspergillus.

[0067] The titer of α-amylase can be measured according to the 1.2 starch dextrinization power measurement method of the 4.03 digestion power test method in the 18th revised Japanese Pharmacopoeia. More specifically, the titer of α-amylase in the culture broth after the culturing step can be measured by the procedure described in the following Measurement Method B. [Measurement Method B] (1) Mix 0.5 ml of the culture broth and 10 ml of 50 mmol / L acetate buffer, and filter through filter paper to obtain a filtrate. (2) After heating 10 ml of a potato starch test solution at 37°C for 10 minutes, add 1 ml of the filtrate and culture at 37°C for 10 minutes to obtain a reaction solution. (3) To 1 ml of the obtained reaction solution, add 10 ml of 0.1 mol / L hydrochloric acid test solution and stir. (4) To 0.5 ml of the reaction solution obtained in (3) above, add 10 ml of 0.0002 mol / L iodine test solution and stir. (5) For the reaction solution obtained in (4) above, using a spectrophotometer, by the ultraviolet-visible absorbance measurement method, measure the absorbance (Abs S2 ) at a wavelength of 660 nm with water as a control. (6) Separately, add 10 ml of the potato starch test solution to 1 ml of 50 mmol / L acetic acid buffer, and incubate at 37 °C for 10 minutes to obtain a control solution. (7) To 1 ml of the obtained control solution, add 10 ml of 0.1 mol / L hydrochloric acid test solution and stir. (8) For the control solution obtained in (7) above, using a spectrophotometer, by the ultraviolet-visible absorbance measurement method, measure the absorbance (Abs B2 ) at a wavelength of 660 nm with water as a control. (9) Calculate the activity of α-amylase based on the following formula 2. (Formula 2) Activity of α-amylase (U / g) = (Abs B2 - Abs S2 ) / Abs B2 × 1 / 0.5

[0068] In a preferred form of the present invention, it is preferable that both the activity of total protease (pH 7) and the activity of α-amylase are below the above upper limit values. In one embodiment, the activity of protease and the activity of α-amylase can both preferably be 10 U / g or less, more preferably 8 U / g or less, still more preferably 6 U / g or less, and even more preferably 5 U / g or less. Also, in one embodiment, the activity of protease and the activity of α-amylase can both be 4 U / g or less, 3 U / g or less, 2 U / g or less, 1 U / g or less, or even below the measurement limit (i.e., 0 U / g). By adopting such a form, the mycelium is preferentially produced rather than the enzyme, and the efficiency of mycelium production can be further improved. Also, off-flavors derived from Aspergillus oryzae can be more effectively suppressed.

[0069] (2) Recovery step The recovery step is a step of recovering the mycelium from the culture solution. The method for recovering the mycelium is not particularly limited, and known methods such as centrifugal separation, filtration separation, pressing separation, and slide culture separation can be adopted.

[0070] The mycelium obtained in the recovery process can be used as food as it is, but preferably, the washing process described below is carried out.

[0071] (3) Washing process The washing process is a process of washing and dehydrating the mycelium obtained in the recovery process. By going through the washing process, it is possible to remove the medium components attached to the mycelium and suppress the deterioration of the mycelium due to spoilage or the like. In addition, the generation of off-flavors can be suppressed for the obtained mycelium.

[0072] In the washing process, the obtained mycelium is washed with water. The water to be used is not particularly limited, and distilled water or the like can be used. The number of washing times can be appropriately adjusted according to the production amount of the mycelium, but it is preferably carried out 2 times or more.

[0073] The washed mycelium is dehydrated by an arbitrary method. As the dehydration method, existing methods can be adopted. For example, pressing by a press, vacuum filtration drying, or the like can be used.

[0074] In the washing process, it is preferable to perform heat treatment on the mycelium before washing. By the heat treatment, the growth of the mycelium can be stopped. The conditions of the heat treatment are not particularly limited, but it is preferable to heat at a temperature of 60 °C or higher for 20 minutes or more.

[0075] (4) Mycelium and its processed products The mycelium of Aspergillus oryzae obtained by the production method of the present invention is preferably in a pulp form rather than in a pellet form. The pellet-shaped mycelium forms spherical mycelium masses, and the pulp-shaped mycelium forms small amorphous mycelium masses and forms a uniformly dispersed fibrous mycelium group. The fibrous pulp-shaped mycelium has a texture closer to that of meat and is easier to homogenize and more stable than the pellet-shaped mycelium. Therefore, the mycelium can be suitably used as a meat substitute for producing a more palatable meat-like food.

[0076] The mycelium that has undergone the washing process can be processed into food. In a preferred form, the obtained mycelium can be used as a meat substitute. When using the mycelium as a meat substitute, the obtained mycelium can be processed such as shaping and baking to produce a meat-like food.

[0077] The mycelium obtained by the production method of the present invention may be processed into a meat-like food by mixing with one or more selected from vegetables such as soybeans and lotus roots, grains such as breadcrumbs, vegetable protein materials such as hydrolyzates of vegetable proteins (HVP), eggs (whole eggs), egg whites, egg yolks, etc. The meat-like food containing the mycelium according to the present invention is not particularly limited, and various processed meat foods such as hamburgers, meatballs, gyoza, shumai, and fillings for meat buns are preferably mentioned.

Example

[0078] Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0079] [Test Example 1] In Test Example 1, according to the following procedure, the production of mycelium using a culture solution containing grains was carried out.

[0080] (1) Preparation of starter Deionized water in an equal amount in terms of mass was added to wheat bran and autoclaved. The obtained wheat bran was inoculated with the mycelium or spores of Aspergillus oryzae and cultured in a thermo-hygrostat at 80% RH and 30 °C for 3 days to obtain solid koji bran. Next, the obtained solid koji bran was diluted in a buffer solution to obtain a starter.

[0081] (2) Preparation of culture solution In Examples 1 to 8, 6 g of grains and 100 ml of ion-exchanged water were added to a 500-ml Erlenmeyer flask with a baffle, and sterilized using an autoclave to prepare a culture solution. The obtained culture solution contained 6 w / v% of grains in terms of dry mass. As the grains in the culture solution, one type of defatted soybeans, brown rice, defatted rice germ, polished rice bran (red bran), polished rice bran (medium flour), wheat flour (weak flour), sake lees, or beer lees was added.

[0082] In Comparative Examples 1 to 4, the following general media were used. The media used in Comparative Examples 1 to 4 were all media that did not contain grain raw materials. · Comparative Example 1: YM medium (composition: yeast extract 0.3 w / v%, malt extract 0.3 w / v%, peptone 0.5 w / v%, dextrose 1.0 w / v%) · Comparative Example 2: CZAPEK medium (composition: sucrose 3.0 w / v%, sodium nitrate 0.2 w / v%, DL-α-glycerophosphate magnesium 0.05 w / v%, potassium chloride 0.05 w / v%, potassium sulfide 0.035 w / v%, iron sulfide 0.001 w / v%) · Comparative Example 3: MRS medium (MRS BROTH, Thermo Fisher Sciemtific) · Comparative Example 4: YPD medium (composition: polypeptone (HypoPolypeptone, Shioya MS Co., Ltd.) 2.0 w / v%, yeast extract (powdered yeast extract D-3H, Shioya MS Co., Ltd.) 1.0 w / v%, glucose 2.0 w / v%)

[0083] (3) Cultivation of mycelia The starter in (1) was inoculated into the culture solution obtained in (2), and cultured for 24 hours while shaking at a speed of 200 rpm on a constant temperature shaking incubator BR-43FL (TAITEC) under the condition of a culture temperature of 30°C.

[0084] After cultivation, the mycelia together with the Erlenmeyer flask were heat-treated at 60°C for 20 minutes, and then heat-treated at 95°C for 20 minutes. After heating, the mycelia were washed twice with distilled water and dehydrated by vacuum filtration using a circulating aspirator WJ-20 (Shibata Scientific Technology) to obtain wet bacterial cells.

[0085] The mass and moisture content of the obtained wet bacterial cells were measured. The moisture content of the wet bacterial cells was measured using atmospheric pressure heating and drying. That is, the wet bacterial cells were dried under the conditions of 105 °C for 5 hours, and the moisture content was calculated from the mass before and after drying. From the measured mass, the mass of the wet bacterial cells after vacuum filtration with respect to the culture solution volume (yield of the wet bacterial cells after vacuum filtration) was determined. Further, from the yield of the wet bacterial cells after vacuum filtration, the mass of the mycelia was calculated assuming the moisture content of the wet bacterial cells was 85% by mass, and this was taken as the yield (w / v%) of the wet bacterial cells.

[0086] (4) Results As shown in Table 1, in Examples 1 to 8 where Aspergillus oryzae was cultured using a culture solution containing grains, mycelia could be obtained in high yields. On the other hand, in the comparative examples cultured in a general medium without grains, the yield of mycelia was lower than that in the examples. Further, when the odor of the obtained mycelia (wet bacterial cells) was smelled and confirmed, the mycelia in Examples 1 to 8 did not have an off-flavor derived from Aspergillus oryzae. From the above, it became clear that mycelia derived from Aspergillus oryzae can be efficiently produced by liquid culture in a culture solution containing grains.

[0087]

Table 1

[0088] [Test Example 2] In Test Example 2, an experiment was conducted to produce mycelia by changing the amount of grains added to the culture solution. Specifically, it was carried out in the same procedure as in Test Example 1 except that brown rice in the amounts shown in Table 2 below was added to ion-exchanged water to obtain a culture solution.

[0089]

Table 2

[0090] As shown in Table 2, when the addition amount of brown rice in the culture solution was 6 to 10 w / v%, the yield of the wet bacterial cells became a high value exceeding 10 w / v%. Also, when the addition amount of brown rice was increased, the yield of the wet bacterial cells increased.

[0091] [Test Example 3] In Test Example 3, raw materials other than grains added to the culture solution were examined. Specifically, it was carried out in the same procedure as Test Example 1, except that a culture solution in which 6 w / v% of defatted rice bran (middlings), sake lees, or beer lees in dry mass and one of the auxiliary raw materials in the amounts shown below were added to ion-exchanged water was used. [Auxiliary raw materials used] Sugars: glucose, maltose, sucrose, fructose glucose syrup (Kato Chemical Co., Ltd.) Nitrogen sources: polypeptone (casein peptone) (Hypopeptone, Shioya MS Co., Ltd.), beef extract (LAB LEMCO POWDER, Kanto Chemical Co., Inc.), bonito extract (Yotsukaido Fisheries Co., Ltd.), yeast extract (Shioya MS Co., Ltd.)

[0092] As shown in Table 3, when sugars or nitrogen sources were added as raw materials other than grains, the yield of wet mycelia increased compared to the case where these raw materials were not added. Also, when the smell of the obtained mycelia (wet mycelia) was smelled and confirmed, the mycelia did not have an off-flavor derived from Aspergillus. From the above, it became clear that by further adding sugars and nitrogen sources such as peptones and meat extracts together with grains, mycelia can be produced more efficiently.

[0093] [Table 3]

[0094] [Test Example 4] In Test Example 4, mycelia were produced using multiple Aspergillus bacteria. The bacterial cells used in Test Example 4 are as follows. ·Aspergillus oryzae (Numbers (1) to (3) in Table 4 indicate that they are different strains.) ·Aspergillus sojae ·Aspergillus luchuensis · Aspergillus luchuensis mut. Kawachii

[0095] In Test Example 4, koji mold was cultured in the same procedure as in Test Example 1 using a culture solution containing 6 w / v% polished rice bran (middlings) as the grain, 1 w / v% glucose as the auxiliary raw material, and 1 w / v% polypeptone to obtain mycelia. After culturing for 24 hours and before recovering the mycelia, the titer of total protease (pH 7) and α-amylase in the culture solution was measured according to the following measurement method.

[0096] The titer of total protease (pH 7) was measured according to the following procedure. [Measurement Method A] (1) 0.5 ml of the said culture solution was mixed with 10 ml of 66.7 mmol / L phosphate buffer, and filtered through filter paper (Advantec No. 131 filter paper) to obtain a filtrate. (2) After culturing 5 ml of casein solution (pH 7.0) in a constant temperature water bath at 37 °C for 5 minutes, 1 ml of the said filtrate was added to the casein solution, and cultured at 37 °C for 10 minutes. (3) After culturing, 5 ml of trichloroacetic acid precipitation reagent was added and cultured in a constant temperature water bath at 37 °C for 30 minutes, and the obtained solution was filtered to obtain a reaction solution. (4) To 2 ml of the said reaction solution, 5 ml of 0.55 mol / L sodium carbonate test solution was added and stirred. To the obtained said reaction solution, 1 ml of 3-fold diluted Folin test solution was added and stirred, and cultured in a constant temperature water bath at 37 °C for 30 minutes. (5) Regarding the reaction solution obtained in (4) above, using a spectrophotometer (manufactured by Shimadzu Corporation), by the ultraviolet-visible absorbance measurement method, the absorbance (Abs S1 ) at a wavelength of 660 nm was measured. (6) Separately, after adding 5 ml of trichloroacetic acid precipitation reagent to 1 ml of the said filtrate, 5 ml of the said casein solution was further added, and cultured in a constant temperature water bath at 37 °C for 30 minutes to obtain a control solution. (7) 5 ml of 0.55 mol / L sodium carbonate test solution was added to 2 ml of the control solution and stirred. 1 ml of the three-fold diluted Folin test solution was added to the obtained reaction solution, stirred, and cultured in a constant temperature water bath at 37 °C for 30 minutes. (8) Regarding the control solution obtained in (7) above, using a spectrophotometer (manufactured by Shimadzu Corporation), by the ultraviolet-visible absorbance measurement method, the absorbance (Abs B1 ) at a wavelength of 660 nm was measured. (9) Based on the following formula 1, the titer of total protease (pH 7) was calculated. (Formula 1) Titer of total protease (pH 7) (U / g) = (Abs S1 - Abs B1 ) × Σ × 11 / 2 × 1 / 10 × 1 / 0.5 (However, the coefficient Σ indicates the amount of tyrosine (μg) when the absorbance difference obtained from the tyrosine calibration curve is 1.)

[0097] The reagents used in Measurement Method A were adjusted as follows. (i) Phosphate buffer 0.2 mol / L KH2PO4 solution was added to 0.2 mol / L Na2HPO4 solution and adjusted to pH 7.0. 60 ml of the obtained 0.2 mol / L phosphate buffer was diluted three-fold with distilled water to obtain 180 ml of 66.7 mmol / L phosphate buffer (pH 7.0).

[0098] (ii) Casein solution 160 ml of 50 mmol / L Na2HPO4 test solution was added to 1 g of dry casein powder, boiled in a water bath for 4 minutes to dissolve, cooled, and then adjusted to pH 7.0 with KH2PO4. The obtained solution was filled up to 200 ml with distilled water to obtain a casein solution.

[0099] (iii) Trichloroacetic acid precipitation reagent Trichloroacetic acid reagent B in the 2. Protein digestion test method of the 4.03 Digestive power test method of the Japanese Pharmacopoeia was used. That is, 5.5 mL of 6 mol / L acetic acid test solution and water were added to 1.80 g of trichloroacetic acid and 1.80 g of anhydrous sodium acetate, dissolved, and made up to 100 ml.

[0100] In addition, the coefficient Σ in Formula 1 was determined by the following procedure. That is, 5 ml of 0.55 mol / L sodium carbonate reagent and 1 ml of Folin reagent were added to 1 ml of a tyrosine standard solution (containing 0.01, 0.02, 0.03 or 0.04 mg / ml of tyrosine), and color development was carried out at 37 °C for 30 minutes. The obtained tyrosine standard solution was used with a solution containing no tyrosine (0.2 mol / L hydrochloric acid test solution) as a control, and the absorbance at 660 nm was measured with an absorbance cell having an optical path length of 10 mm to prepare a calibration curve. Based on the calibration curve, the amount of tyrosine (μg) corresponding to an absorbance difference of 1 was determined and used as the coefficient Σ.

[0101] In addition, the activity of α-amylase was measured according to the following procedure. [Measurement Method B] (1) 0.5 ml of the above-mentioned culture solution was mixed with 10 ml of 50 mmol / L acetate buffer, and filtered through filter paper (Advantec No. 131 filter paper) to obtain a filtrate. (2) After heating 10 ml of a potato starch test solution at 37 °C for 10 minutes, 1 ml of the above-mentioned filtrate was added, and the mixture was cultured at 37 °C for 10 minutes in a constant temperature water bath to obtain a reaction solution. (3) 10 ml of 0.1 mol / L hydrochloric acid test solution was added to 1 ml of the obtained reaction solution and stirred. (4) 10 ml of 0.0002 mol / L iodine test solution was added to 0.5 ml of the reaction solution obtained in (3) above and stirred. (5) For the reaction solution obtained in (4) above, using a spectrophotometer (manufactured by Shimadzu Corporation), by the ultraviolet-visible absorbance measurement method, the absorbance (Abs S2 ) at a wavelength of 660 nm with water as a control was measured. (6) Separately, 10 ml of the potato starch test solution was added to 1 ml of 50 mmol / L acetate buffer, and the mixture was cultured at 37 °C for 10 minutes in a constant temperature water bath to obtain a control solution. (7) 10 ml of 0.1 mol / L hydrochloric acid test solution was added to 1 ml of the obtained control solution and stirred. (8) For the control solution obtained in (7) above, using a spectrophotometer (Shimadzu Corporation), by the ultraviolet-visible absorbance measurement method, the absorbance (Abs B2 ) at a wavelength of 660 nm with water as a control was measured. (9) Based on the following formula 2, the activity of α-amylase was calculated. (Formula 2) Activity of α-amylase (U / g) = (Abs B2 - Abs S2 ) / Abs B2 × 1 / 0.5

[0102] The reagents used in Measurement Method B were adjusted as follows. (i) Acetate buffer After preparing 1 mol / L acetic acid solution and 1 mol / L sodium acetate solution respectively, the two solutions were mixed so as to have a pH of 5.0 to prepare 1 mol / L acetic acid - sodium acetate buffer (pH 5.0). This 1 mol / L acetic acid - sodium acetate buffer was diluted 20-fold to obtain 50 mmol / L acetate buffer (pH 5.0).

[0103] (ii) Potato starch test solution To 1 g of potato starch (dry matter), 25 ml of water was added and mixed, and then 2 mol / L sodium hydroxide solution was gradually added to make it viscous. Then, after heating in a water bath for 3 minutes, it was cooled and neutralized with 2 mol / L hydrochloric acid reagent. To the obtained solution, 10 ml of 1 mol / L acetic acid - sodium acetate buffer (pH 5.0) was added, and further water was added to obtain 100 ml of potato starch test reagent.

[0104]

Table 4

[0105] As shown in Table 4, it was confirmed that mycelia can be produced in the same manner even when using Aspergillus species other than Aspergillus oryzae. Moreover, regardless of the Aspergillus bacterium used, the titer of total protease (pH 7) was 3 U / g or less. Furthermore, regardless of the Aspergillus bacterium used, the titer of α-amylase was 5 U / g or less. This result indicates that in the production method of the present invention, enzymes such as total protease (pH 7) and α-amylase are hardly produced, and mainly mycelia are produced. Also, when the smell of the obtained mycelia (wet cells) was confirmed by smelling, the mycelia did not have an off-flavor derived from Aspergillus koji.

[0106] [Test Example 5] In Test Example 5, the scale-up of the production of mycelia was carried out using a jar fermenter. The specific production procedure is as follows.

[0107] Similar to Example 1, koji bran of Aspergillus oryzae was prepared. Next, 4 L of a culture solution (containing 30 w / v% of liquid okara (equivalent to 3.16 w / v% in dry mass), 2 w / v% of fructose glucose syrup, and 0.5 w / v% of yeast extract) was placed in a 10 L jar fermenter and sterilized by autoclaving. To the sterilized culture solution, 2 g of koji bran suspended in an inoculation medium was inoculated. After inoculation, stirring culture was carried out for 24 hours under the conditions of a stirring speed of 100 rpm of the stirring blade of the jar fermenter, a culture temperature of 30 °C, and an aeration rate of 1 L / min.

[0108] After the culture, heating was carried out at 70 °C for 20 minutes, and further sterilization was carried out at 121 °C for 15 minutes. The sterilized mycelia were recovered, frozen and thawed once, washed twice with distilled water, and dehydrated by the vacuum filtration method in the same manner as in Example 1 to obtain wet cells.

[0109] The obtained wet cells had a yield of wet cells after vacuum filtration of 11.87 w / v% and a yield of wet cells at 85 mass% moisture of 11.52 w / v%. From the above, it was found that even under the scaled-up conditions, mycelia can be produced in high yield by carrying out stirring culture in a culture solution containing grains.

[0110] [Test Example 6] In Test Example 6, mycelia were produced in the same procedure as in Test Example 5, except that the following points of the culture conditions and the culture time were changed. · Culture medium: Contains 6 w / v% polished rice bran (medium powder), 2 w / v% fructose / glucose liquid sugar, and 2 w / v% hy-polypeptone. · Stirring speed of the stirring blades of the jar fermenter: 700 rpm, culture temperature: 30 °C, aeration rate: 2 L / min

[0111] Mycelia were collected at 16, 20, 24, 36, and 42 hours from the start of the culture, and wet cells were obtained in the same procedure as in Test Example 5. The yields of the obtained wet cells (when the water content was 85% by mass) are shown in Table 5. Also, in the same procedure as in Test Example 4, the activity of total protease (pH 7) in the culture broth after the culture was measured.

[0112] [Table 5]

[0113] As shown in Table 5, when the culture time of Aspergillus oryzae was 16 to 42 hours, the yields of the wet cells were all as high as 10 w / v% or more. From this result, it was found that the production method of the present invention can produce mycelia in a high yield even when the culture time is 48 hours or less. No unpleasant odor such as off-flavor was felt in the obtained wet cells.

[0114] Also, it was confirmed that the total protease activity increased as the culture time increased. In particular, when the culture time was 36 hours and 42 hours, the protease activity was higher than that at 24 hours. From this result, it can be said that it is more preferable to set the culture time of Aspergillus oryzae to 24 hours or less from the viewpoint of further suppressing off-flavors derived from metabolites of Aspergillus oryzae such as enzymes and degradation products of the enzymes. [Industrial Applicability]

[0115] The present invention can be applied to the production of foods and the like using mycelia.

Claims

1. A culturing step of liquid-culturing Aspergillus oryzae in a culture solution containing grains, and A recovering step of recovering the mycelia obtained in the culturing step, A method for producing mycelia of Aspergillus oryzae, comprising the above steps.

2. The method for production according to claim 1, wherein the culturing period of Aspergillus oryzae in the culturing step is 48 hours or less.

3. The method for production according to claim 1, wherein the grains are one or more selected from seeds of Gramineae crops, seeds of Leguminosae crops, and processed products thereof.

4. The method for production according to claim 1, wherein the grains include one or more selected from rice, soybeans, wheat, barley, millet, adzuki beans, corn, and processed products thereof.

5. The method for production according to claim 1, wherein the grains include one or more selected from okara, polished rice bran, brown rice, wheat flour, defatted rice germ, defatted soybeans, sake lees, and beer lees.

6. The method for production according to claim 1, wherein the culture solution contains the grains at 6 w / v% or more in terms of dry mass.

7. The culture solution contains raw materials other than the grains, and The raw materials other than the grains are saccharides and / or a nitrogen source. The method for production according to claim 1.

8. The method for production according to claim 7, wherein the saccharides are one or more selected from monosaccharides, disaccharides, and oligosaccharides.

9. The method for production according to claim 7, wherein the nitrogen source is one or more selected from proteins and their hydrolysates, and extracts derived from animals.

10. The method for production according to claim 1, further comprising a washing step of washing and dehydrating the mycelia obtained in the recovering step.

11. The method for production according to claim 1, wherein the titer of total protease (pH 7) in the culture solution measured by the following measurement method after the culturing step is 50 U / g or less. [Measurement Method A] (1) Mix 0.5 ml of the culture solution with 10 ml of 66.7 mmol / L phosphate buffer, filter with filter paper to obtain a filtrate. (2) After culturing 5 ml of casein solution (pH 7.0) at 37°C for 5 minutes, add 1 ml of the filtrate to the casein solution and culture at 37°C for 10 minutes. (3) After culturing, add 5 ml of trichloroacetic acid precipitation reagent and culture at 37°C for 30 minutes, filter the obtained solution to obtain a reaction solution. (4) Add 5 ml of 0.55 mol / L sodium carbonate test solution to 2 ml of the reaction solution and stir. To the obtained reaction solution, add 1 ml of 3-fold diluted Folin test solution, stir, and culture at 37°C for 30 minutes. (5) With respect to the reaction solution obtained in (4) above, using a spectrophotometer, the absorbance (Abs Ｓ１ ) at a wavelength of 660 nm is measured by ultraviolet-visible absorbance measurement method. (6) Separately, after adding 5 ml of trichloroacetic acid precipitation reagent to 1 ml of the filtrate, 5 ml of the casein solution is further added, and the mixture is cultured at 37°C for 30 minutes to obtain a control solution. (7) To 2 ml of the control solution, 5 ml of 0.55 mol / L sodium carbonate test solution is added and stirred. To the resulting reaction solution, 1 ml of 3-fold diluted Folin test solution is added and stirred, and the mixture is cultured at 37°C for 30 minutes. (8) For the control solution obtained in (7) above, using a spectrophotometer, the absorbance (Abs Ｂ１ ) at a wavelength of 660 nm is measured by ultraviolet-visible absorbance measurement method. (9) Based on the following formula 1, the titer of total protease (pH 7) is calculated. (Formula 1) Activity (U / g) of total protease (pH 7) = (Abs Ｓ１ - Abs Ｂ１ ) × Σ × 11 / 2 × 1 / 10 × 1 / 0.5 (However, the coefficient Σ indicates the amount of tyrosine (μg) when the absorbance difference determined from the tyrosine calibration curve is 1.) (12) According to claim (5) A meat-like mycelium produced by the production method according to any one of claims 1 to 11.

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