Method for producing protein-rich composition

Abstract

The purpose of the present invention is to provide a technique for safely and efficiently producing a protein that is usable as a food material. The present invention provides: a method for producing a protein-rich composition, the method including a step for subjecting a koji fungus to solid culture in a culture medium prepared by adding a cereal and a nitrogen compound, preferably a culture medium prepared by further adding an inorganic salt component comprising at least one component selected from the group consisting of a magnesium salt and an iron salt to the above-mentioned culture medium to produce a protein, wherein the inorganic salt component preferably comprises a magnesium salt and an iron salt, and the amount of the magnesium salt in terms of magnesium atom content is preferably 0.001% by weight or more with respect to the dry weight of the cereal; and a food product or a feed composition each using a composition produced by the method.

Description

Method for producing a protein-rich composition

[0001] The present invention relates to a method for producing a protein-rich composition.

[0002] With the rapid increase in the world population and the economic growth of emerging countries, demand for meat and other proteins is expected to increase dramatically. The main sources of protein are grains and legumes, with 500 million tons of rice, 700 million tons of wheat, 1.1 billion tons of corn, and 300 million tons of soybeans produced annually, some of which are also used for meat production. Because the arable land area required for the production of these agricultural products cannot be expanded in the short term, there are concerns about the occurrence of a "global protein crisis," in which it becomes difficult internationally to supply protein to meet demand.

[0003] In recent years, growing interest in the environmental impact of meat production and animal welfare has led to interest in edible insects and cultured meat as alternative protein sources. Producing edible insects requires large-scale insect breeding facilities and many days of rearing. Furthermore, cultured meat requires large bioreactors for cell culture, and the cost of the components required for cell culture is known to be high. To solve the social issue of global protein shortages, a technology that can produce protein more simply and efficiently than conventional technologies is essential.

[0004] Methods that use yeast bioproteins called single cell proteins and bioproteins from filamentous fungi called mycoproteins have been developed as cheaper and more efficient protein production methods compared to insect food and cultured meat (Non-Patent Documents 1 and 2).

[0005] Manda, H. (1967) IV. Microbial protein production, Nutrition and Food, No. 20, 92-97. Marilyn G. Wiebe (2004), Quorn Myco-protein - Overview of successful fungal product, 18, 17-20.

[0006] Microbial protein production that has been put into practical use to date has basically been achieved using large-scale culture equipment. Culture equipment capable of stirring and aeration requires significant installation costs, making it difficult to develop enough equipment to expand protein production on a global scale. In order to significantly increase protein production, there is a need for a method to produce high-protein food ingredients easily and quickly, anytime, anywhere, without the need for large equipment.

[0007] An object of the present invention is to provide a technique for safely and efficiently producing proteins that can be used as food ingredients.

[0008] Therefore, the present inventors investigated a process for producing proteins by cultivating koji mold by adding nitrogen compounds to grains, applying the traditional koji-making method that has been practiced in Japan since ancient times. As a result, they found that proteins can be efficiently produced by cultivating koji mold by adding nitrogen compounds to grains, and preferably by further adding inorganic salts.

[0009] That is, the present invention provides the following [1] to

[13] . [1] A method for producing a protein-rich composition, comprising the step of solid-state culturing koji mold in a medium prepared by adding grains and a nitrogenous compound to produce a protein. [2] A method for producing a protein-rich composition, comprising the step of solid-state culturing koji mold in a medium prepared by adding grains, a nitrogenous compound, and inorganic salts containing one or more selected from the group consisting of magnesium salts and iron salts to produce a protein. [3] A method for producing a protein-rich composition, comprising the step of solid-state culturing koji mold in a medium prepared by adding grains, a nitrogenous compound, and inorganic salts containing magnesium salts and iron salts to produce a protein. [4] The method according to [2] or [3], wherein the amount of magnesium salt in magnesium atom equivalent relative to the dry weight of grains in the medium is 0.001% by weight or more. [5] The method according to any one of [2] to [4], wherein the amount of iron salt in iron atom equivalent relative to the dry weight of grains in the medium is 0.00003% by weight or more. [6] The method according to any one of [1] to [5], wherein the medium is prepared by further adding water in an amount of 400% by weight or less based on the dry weight of the grain. [7] The method according to any one of [1] to [6], wherein the nitrogen compound is one or more selected from the group consisting of ammonium salts, nitrates, ammonia, urea, and amino acids. [8] The method according to any one of [1] to [7], wherein the content of nitrogen compounds is 0.5% by weight or more based on the dry weight of the grain. [9] The method according to any one of [2] to [8], wherein the inorganic salt further comprises one or more selected from the group consisting of calcium salts and phosphates.

[10] The method according to any one of [1] to [9], wherein the medium is prepared by further adding an oil or fat.

[11] The method according to any one of [1] to

[10] , wherein the grain includes at least one of rice and corn.

[12] A method for producing a food or feed composition, comprising producing a protein-rich composition by the method according to any one of [1] to

[11] , and using the composition to produce food or feed.

[13] A method for producing a food or feed composition for preventing or reducing the risk of hyperuricemia or gout, comprising producing a protein-rich composition by the method according to any one of [1] to

[11] , and using the composition to produce a food or feed composition for preventing or reducing the risk of hyperuricemia or gout.

[0010] According to the present invention, proteins can be produced easily and efficiently in a short period of time using koji mold. Because koji mold has been traditionally used in food production since ancient times, the protein compositions produced by the koji mold are guaranteed to be safe and can be widely used as substitutes for conventional protein sources such as meat, as additives for foods, feeds, and raw materials for these.

[0011] FIG. 1 is a graph showing A: protein production amount from ammonium salt, B: nucleic acid concentration, C: ammonia concentration, and D: changes in pH over time in the medium shown in Table 1, and E: protein production amount in a control experiment in the medium shown in Table 1 in Example 2. FIG. 2 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH in the medium shown in Table 2 under various koji mold solid culture conditions for producing protein from ammonium salt in the medium shown in Table 2 in Example 3. FIG. 3 is a graph comparing the ratio of protein to nucleic acid produced by koji mold from ammonium salt under various koji mold solid culture conditions in the medium shown in Table 2 in Example 3. FIG. 4 is a graph showing A: improvement in protein productivity and B: changes in nucleic acid concentration, C: ammonia concentration, and D: pH in the medium shown in Table 3 by addition of magnesium in Example 4. FIG. 5 is a graph showing A: improvement in protein productivity and B: changes in nucleic acid concentration, C: ammonia concentration, and D: pH in the medium shown in Table 4 by addition of iron in Example 5. Figure 6 is a graph showing the changes over time in A: protein production amount from ammonium salt and urea, B: nucleic acid concentration, C: ammonia concentration, and D: pH in the medium shown in Table 5 in Example 6. Figure 7 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH in the medium shown in Table 6 for each koji mold solid culture condition for producing protein from ammonium salt and urea in Example 7. Figure 8 is a graph comparing the ratio of protein to nucleic acid produced by koji mold from ammonium salt and urea for each koji mold solid culture condition in the medium shown in Table 6 in Example 7. Figure 9 is a graph showing the changes over time in A: protein production amount from nitrate, B: nucleic acid concentration, C: ammonia concentration, and D: pH in the medium shown in Table 7 in Example 8. FIG. 10 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH for each of the solid culture conditions for koji mold for producing protein from nitrate in the medium shown in Table 8 in Example 9.In the graph, "p<0.05" indicates that the p-value was below the significance level of 0.05 as a result of comparing the protein production amounts between the two groups under conditions 1 and 2, and conditions 1 and 3, and that there was a statistically significant difference. Figure 11 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH by adding fat or oil to the medium shown in Table 9 in Example 10, for each koji mold solid culture condition. Figure 12 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH in the medium using corn shown in Table 10, for each koji mold solid culture condition in Example 11.

[0012] [1. Method for Producing a High-Protein Composition] The method of the present invention includes a step of producing a protein by solid-state culture of koji mold in a medium prepared by adding grains and nitrogenous compounds, preferably an inorganic salt, and more preferably an oil or fat, thereby enabling efficient protein production.

[0013] [1.1 Koji mold (seed koji)] The koji mold is preferably one that has been used in the production of foods (e.g., fermented foods such as miso, soy sauce, mirin, vinegar, sake, awamori, shochu, and pickles), which can produce safe proteins and are useful for food and feed. Examples of such koji molds include Aspergillus oryzae, Aspergillus niger, Aspergillus luchuensis, Aspergillus luchuensis var kawachii, Aspergillus kawachii, Aspergillus sojae, Aspergillus tamari, and Aspergillus awamori. Examples of fungi that can be used include fungi of the genus Aspergillus, such as Aspergillus awamori and Aspergillus glaucus. Of these, the genus Aspergillus is preferred, and Aspergillus oryzae (e.g., Aspergillus oryzae RIB40 strain) is preferred.

[0014] The koji mold is preferably added to the system as a culture containing spores (seed koji). The seed koji can be prepared by a conventional method, for example, by culturing the koji mold in a medium containing yeast extract or the like.

[0015] [1.2 Grains] Grains may be any grain material that can be used in the production of so-called koji, and may be processed products such as ground or dried grains. Examples of grains include rice, barley, wheat, oats, corn, rye, pearl barley, sorghum, foxtail millet, barnyard millet, buckwheat, sorghum, triticale, and other cellulosic biomass, of which rice and corn are preferred. Rice is classified into, for example, non-glutinous rice, glutinous rice, japonica, indica, and javanica (japanica), and any of these may be used. Examples of corn include dent, hard grain, soft grain, explosive, glutinous, and sweet varieties, and any of these may be used, but dent is preferred.

[0016] The grain is usually added to the system as a heated (steamed) grain, but heat treatment is not essential, and unheated grain can also be used. In this specification, steamed grain refers to grain that has been steamed (usually treated at high temperature and / or high pressure in a water-absorbed state). When the grain is rice, steamed rice can be obtained by appropriately soaking the rice and then cooking it (for example, by heating it to a temperature above the gelatinization temperature (e.g., 80 to 120°C) and maintaining the temperature for 10 to 60 minutes). The steamed grain may be so-called gelatinized grain obtained by rapid drying such steamed grain, or waste such as leftover food or starch-containing surplus food ingredients.

[0017] [1.3 Nitrogen Compounds] In this specification, the term "nitrogen compound" refers to a compound containing a nitrogen atom. The nitrogen compound may be any compound capable of supplying a nitrogen source during the cultivation of koji mold, and may be either an inorganic nitrogen compound or an organic nitrogen compound. Examples of inorganic nitrogen compounds include ammonium salts, nitrates, and ammonia, with ammonium salts and nitrates being more preferred. Examples of ammonium salts include ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium bicarbonate, and ammonium chloride, with ammonium sulfate being preferred. Examples of nitrates include sodium nitrate and potassium nitrate, with sodium nitrate being preferred. Examples of organic nitrogen compounds include urea and amino acids, with urea being preferred. The nitrogen compounds may be used alone or in combination of two or more.

[0018] [1.4 Inorganic Salt] The inorganic salt is one or more selected from the group consisting of magnesium salts and iron salts. For example, a magnesium salt or an iron salt may be added alone, or both a magnesium salt and an iron salt may be added. Adding both a magnesium salt and an iron salt is preferred. This increases the amount of protein produced by the koji mold. In this specification, the inorganic salt may be any salt composed of an inorganic element and is distinguished from nitrogen compounds. Examples of other inorganic salts that may be used as needed include calcium salts, phosphate salts, sodium salts, potassium salts, and aluminum salts. Calcium salts and phosphate salts are preferred, and calcium salts are more preferred. The inorganic salt may contain multiple inorganic elements in one compound, and the counter ion is not particularly limited.

[0019] Examples of magnesium salts include magnesium sulfate, magnesium chloride, and magnesium carbonate, with magnesium sulfate being preferred.

[0020] As the iron salt, for example, divalent iron salts such as ferrous citrate, ferrous gluconate, ferrous sulfate, and ferrous chloride, and trivalent iron salts such as ferric pyrophosphate and ferric oxide are preferred, with divalent iron salts being more preferred and ferrous sulfate being even more preferred.

[0021] Examples of calcium salts include calcium carbonate, calcium sulfate, and calcium chloride, with calcium carbonate being preferred.

[0022] Examples of phosphates include potassium phosphate, sodium phosphate, and magnesium phosphate, and may also include monohydrogen phosphate, dihydrogen phosphate, metaphosphate, and polyphosphate. As the phosphate, potassium phosphate is preferred, and potassium dihydrogen phosphate is more preferred.

[0023] The inorganic salts may be used alone or in combination of two or more, preferably in combination of two or more or three or more. Preferred combinations of two or more include a combination of a magnesium salt and an iron salt, a combination of a magnesium salt, an iron salt and a calcium salt, and a combination of a magnesium salt, an iron salt, a calcium salt and a phosphate.

[0024] [1.5 Fats and Oils] The fats and oils may be either vegetable fats or animal fats. The vegetable fats and oils may be derived from plants, and the production method thereof is not particularly limited. Examples of vegetable fats and oils include rapeseed oil (canola oil, salad oil), soybean oil, sunflower seed oil, cottonseed oil, peanut oil, safflower oil, rice bran oil, corn oil, safflower oil, olive oil, kapok oil, sesame oil, evening primrose oil, coconut oil, palm oil, palm kernel oil, shea oil, monkey fat, cacao butter, coconut oil, perilla oil, lavender oil, mango kernel oil, and processed fats and oils made from at least one of these. The animal fats and oils may be derived from animals, and the production method thereof is not particularly limited. Examples of animal fats and oils include beef tallow, lard, fish oil, whale oil, milk fat derived from dairy ingredients, and processed fats and oils made from at least one of these. Examples of processed oils include hardened oils, interesterified oils, and fractionated oils. Among these, vegetable oils are preferred, and soybean oil is more preferred. By including oils, the amount of protein produced by koji mold can be further increased.

[0025] [1.6 Culture Conditions] Solid-state culture of koji mold may be carried out in a medium prepared by adding grains and nitrogenous compounds, preferably inorganic salts, and more preferably fats and oils. During the culture, other components (e.g., water, vitamins) may be added to the system as necessary.

[0026] The grain content (dry weight) is usually 20% by weight or more, preferably 30% by weight or more, and more preferably 35% by weight or more, relative to the weight of the medium. The upper limit is usually 70% by weight or less, preferably 60% by weight or less, and more preferably 55% by weight or less. Therefore, the grain content (dry weight) is usually 20 to 70% by weight, preferably 30 to 60% by weight, and more preferably 35 to 55% by weight, relative to the weight of the medium.

[0027] The content of the nitrogenous compounds is preferably 0.5% by weight or more, more preferably 1% by weight or more, and even more preferably 2% by weight or more, based on the dry weight of the grain. The upper limit is preferably 15% by weight or less, more preferably 10% by weight or less, and even more preferably 8% by weight or less. Therefore, the content of the nitrogenous compounds is preferably 0.5 to 15% by weight, more preferably 1 to 10% by weight, and even more preferably 2 to 8% by weight, based on the dry weight of the grain. When the content of the nitrogenous compounds satisfies the above numerical range, the effects of the present invention can be more suitably exhibited.

[0028] When magnesium salt is contained, its content is preferably 0.01 wt% or more, more preferably 0.02 wt% or more, and even more preferably 0.04 wt% or more, based on the dry weight of the grain. There is no particular upper limit, but it is usually 2 wt% or less, preferably 1 wt% or less, and more preferably 0.5 wt% or less. Therefore, the content of magnesium salt is preferably 0.01 wt% or more, more preferably 0.01 to 2 wt%, even more preferably 0.02 to 1 wt%, and even more preferably 0.04 to 0.5 wt% based on the dry weight of the grain.

[0029] The amount of magnesium salt in the medium in terms of magnesium atoms relative to the dry weight of the grain is preferably 0.001% by weight or more, more preferably 0.002% by weight or more, and even more preferably 0.004% by weight or more. There is no particular upper limit, but it is usually 0.2% by weight or less, preferably 0.1% by weight or less, and more preferably 0.05% by weight or less. Therefore, the amount of magnesium salt in the medium in terms of magnesium atoms relative to the dry weight of the grain is preferably 0.001% by weight or more, more preferably 0.001 to 0.2% by weight, even more preferably 0.002 to 0.1% by weight, and even more preferably 0.004 to 0.05% by weight.

[0030] When iron salt is contained, its content is preferably 0.0002 wt% or more, more preferably 0.0003 wt% or more, and even more preferably 0.0005 wt% or more, based on the dry weight of the grain. There is no particular upper limit, but it is usually 0.1 wt% or less, preferably 0.01 wt% or less, and more preferably 0.005 wt% or less. Therefore, the content of iron salt is preferably 0.0002 wt% or more, more preferably 0.0002 to 0.1 wt%, even more preferably 0.0003 to 0.01 wt%, and even more preferably 0.0005 to 0.005 wt%, based on the dry weight of the grain.

[0031] The amount of iron salt in the medium in terms of iron atoms relative to the dry weight of the grain is preferably 0.00003% by weight or more, more preferably 0.00004% by weight or more, and even more preferably 0.00008% by weight or more. There is no particular upper limit, but it is usually 0.02% by weight or less, preferably 0.002% by weight or less, and more preferably 0.001% by weight or less. Therefore, the amount of iron salt in the medium in terms of iron atoms relative to the dry weight of the grain is preferably 0.00003% by weight or more, more preferably 0.00003 to 0.02% by weight, even more preferably 0.00004 to 0.002% by weight, and even more preferably 0.00008 to 0.001% by weight.

[0032] When the inorganic salt includes a calcium salt, the content thereof is preferably 0.5% by weight or more, more preferably 0.7% by weight or more, and even more preferably 0.8% by weight or more, based on the dry weight of the grain. The upper limit is not particularly limited, but is preferably 5% by weight or less, and even more preferably 4% by weight or less. Therefore, the content of the calcium salt is preferably 0.5 to 5% by weight, more preferably 0.7 to 5% by weight, and even more preferably 0.8 to 4% by weight, based on the dry weight of the grain.

[0033] When the inorganic salt contains phosphate, the content thereof is preferably 4 wt% or less, more preferably 3.5 wt% or less, based on the dry weight of the grain. This can increase the protein to nucleic acid concentration ratio in the koji mold culture, making it suitable for use as a food or feed. The lower limit of the phosphate content is not particularly limited, and may be, for example, 0 wt% or more, or 0.1 wt% or more.

[0034] When fats and oils are contained, the content thereof is preferably 0.1% by weight or more, more preferably 1% by weight or more, and even more preferably 3% by weight or more, based on the dry weight of the grain. The upper limit is preferably 20% by weight or less, more preferably 10% by weight or less, and even more preferably 7% by weight or less. Therefore, the content of fats and oils is preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight, and even more preferably 3 to 7% by weight, based on the dry weight of the grain.

[0035] In solid culture, the medium may contain other components in addition to the above-mentioned components. An example of such other components is water. When the medium contains water as an additional component, the medium is prepared by adding water in an amount of preferably 400% by weight or less, or 300% by weight or less, more preferably 200% by weight or less, even more preferably 170% by weight or less, or 150% by weight or less, based on the dry weight of the grain. This increases the accessibility of koji mold to oxygen, thereby promoting protein production. The lower limit is preferably 10% by weight or more, more preferably 50% by weight or more, and even more preferably 100% by weight or more. This allows the effects of the present invention to be more suitably exhibited. Therefore, the medium is prepared by adding water in an amount of preferably 400% by weight or less, or 300% by weight or less, more preferably 10 to 200% by weight, even more preferably 50 to 170% by weight, and even more preferably 100 to 170% by weight or 100 to 150% by weight, based on the dry weight of the grain.

[0036] Cultivation is preferably carried out with the culture exposed to air. The culture temperature is usually room temperature, for example, 10 to 40°C, preferably 20 to 37°C. The culture period is usually 1 day or more, preferably 2 days or more, and more preferably 3 days or more. The upper limit is not particularly limited as long as the culture is continued until no nitrogen source remains, but may be, for example, 10 days or less, 8 days or less, 7 days or less, or 6 days or less. The pH during culture is usually near neutral (e.g., 4 to 10) and tends to decrease as the culture progresses. The culture may be terminated when the pH reaches less than 4. The culture period can be adjusted by adding a nitrogen compound. Specifically, the culture period can be shortened by adding a smaller amount of nitrogen compound while satisfying the above-mentioned numerical range of the amount of nitrogen compound added during koji mold culture. Alternatively, protein production can be continued during koji mold culture when no nitrogen compound remains or when the pH during culture falls below 4 by adding a new nitrogen compound within the above-mentioned numerical range of the amount of nitrogen compound added.

[0037] After the cultivation, a culture product with a high protein content can be obtained. The proteins contained in the culture product include proteins produced by the koji mold from nitrogen compounds and proteins derived from the koji mold. The culture product containing the proteins can be recovered as an extract or purified product either directly or by subjecting it to extraction and / or purification treatment (e.g., filtration, centrifugation).

[0038] [2. Protein-rich composition] The culture and purified product obtained by the above-mentioned method are protein-rich compositions. That is, since Aspergillus oryzae can produce proteins using nitrogen compounds as nitrogen sources in the presence of inorganic salts, the culture obtained contains a larger amount of protein than that obtained by a conventional culture method.

[0039] The high-protein composition may have 100% protein purity or may contain other components. Examples of other components include nucleic acids. A relatively small amount of nucleic acid is preferred. For example, the ratio of protein to nucleic acid in the culture (protein / nucleic acid) may be 2.7 or more, 2.8 or more, 3 or more, 4 or more, or 5 or more. This makes it possible to anticipate the use of the koji mold culture as a functional food, such as a food or feed for preventing or alleviating hyperuricemia and gout.

[0040] The dosage form of the high-protein composition is not particularly limited, and may be any of a solid (e.g., powder, pellet, granule, lump, mince), a liquid, etc. The molding method for preparing these dosage forms can be a conventional method.

[0041] [3. Uses] The protein-rich composition can be used as a raw material or additive for food or feed. For example, it can be used as a substitute (alternative meat) for meat (livestock meat or fish meat). Specifically, it can be added when preparing processed meat products (e.g., ham, sausages, and minced meat). It can also be used together with meat during cooking or as a meat substitute, by appropriately seasoning and shaping it to resemble meat. Furthermore, as described above, cultures with low nucleic acid content are lower in calories than meat, and are expected to be used as so-called diet foods and foods for treating and preventing lifestyle-related diseases such as obesity and metabolic syndrome. Furthermore, the protein-rich composition of the present invention can be consumed as a so-called purine-reduced food without worrying about gout. Therefore, it is expected to be used as a food or feed for preventing or reducing the risk of hyperuricemia or gout, suitable for people at risk of hyperuricemia or gout.

[0042] The present invention will be described below with reference to examples, which are not intended to limit the scope of the present invention.

[0043] Example 1: Preparation of Koji Mold Spore Suspension 20 g of glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 g of yeast extract (Thermo Fisher Scientific), 30 g of sodium chloride (Nacalai Tesque), and 20 g of agar (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to 1 liter of pure water. After sterilization by autoclave, the mixture was placed in a plastic petri dish and solidified to form GYN agar medium. Aspergillus oryzae RIB40 strain (NBRC100959 strain), a representative strain of Koji mold, was cultured on GYN agar medium for 4 to 6 days. Spores formed on the medium were collected with a sterile cotton swab and suspended in a solution containing 1 g of Tween 80 and 4 g of sodium chloride per liter to form a spore suspension. The spore suspension was diluted appropriately and placed in a plastic cuvette, and the optical density (OD) at a wavelength of 600 nm was measured using a spectrophotometer. Based on the results, the spore concentration was adjusted so that the OD of the spore suspension was approximately 10.

[0044] Example 2: Protein Production from Ammonium Salts by Solid Culture of Aspergillus oryzae. 100 mg of dried cooked rice (Alpha Foods) was placed in a 2 mL plastic tube and sterilized at 100°C for at least 1 hour. Nitrogen compounds, various inorganic salts, and a spore suspension were added to the dried cooked rice in the plastic tube as shown in Table 1, mixed thoroughly, and then cultured at 30°C. Culture was terminated after 0, 3, 4, and 5 days (two replicates per day). One stainless steel bead for homogenization was added to the plastic tube, and 500 μL of water was added. The sample was then left to stand for a while to allow the sample to absorb water. The sample was then homogenized using a bead homogenizer (FastPrep FP100A, MP-Biomedical) by repeating homogenization treatment at level 4 for 30 seconds four times. The mixture was then diluted with 1000 μL of water and vortex-mixed to obtain a homogenized sample solution.

[0045]

[0046] The protein concentration of the sample disruption solution was measured using the following method. Bovine serum albumin solutions at 1, 2, 5, and 10 mg / mL were used as standard solutions for protein concentration measurement. Water was used as a blank. 50 μL of the sample disruption solution, standard solution, or blank water was collected in a plastic tube, 400 μL of 1N NaOH was added, and the tube was mixed by vortexing. After heating at 100°C for 3 minutes, the tube was neutralized by adding 200 μL of 2N HCl, vortexed, and centrifuged at 15,000 rpm for 40 seconds. A 96-well plate was prepared, and 10 μL of the centrifuged supernatant was added to each well of the 96-well plate. Then, 200 μL of Quickstart Bradford reagent (Bio-Rad) was added. After allowing to stand for 5 minutes, the absorbance at a wavelength of 595 nm was measured using a microplate reader (Multiskan® GO, Thermo Fisher Scientific). The protein concentration was calculated from a calibration curve prepared using the standard solution and blank water.

[0047] The nucleic acid concentration of the sample disruption solution was measured as follows. 50 μL of the disrupted bacterial cell solution was placed in a plastic tube, and 500 μL of 0.5 N aqueous perchloric acid solution was added. After vortexing, the tube was heated at 100°C for 15 minutes and vortexed again. The tube was centrifuged at 15,000 rpm for 40 seconds. The absorbance of the centrifuged supernatant at a wavelength of 260 nm was measured using a NanoDrop (registered trademark, Thermo Fisher Scientific). The results are shown in Figure 1.

[0048] The ammonia concentration of the sample disruption solution was measured as follows. The sample disruption solution was centrifuged at 15,000 rpm for 40 seconds to obtain the sample disruption solution supernatant. In addition to the sample disruption solution supernatant, a 0.1% aqueous solution of ammonium carbonate was used as a standard. Water was also used as a blank. A 96-well plate was prepared, and 80 μL of E-kit Liquid Ammonia Reagent 1 (JK International) was added to each well. 4 μL of the sample disruption solution supernatant, standard solution, or blank water was added to each well and mixed with a pipette. After leaving the plate at room temperature for 1 minute, the absorbance at 340 nm was measured, and the result was designated Measurement 1. Next, 20 μL of E-kit Liquid Ammonia Reagent 2 was added to each solution in the 96-well plate and mixed with a pipette. After leaving the plate at room temperature for 15 minutes or more, the absorbance at 340 nm was measured, and the result was designated Measurement 2. The difference between the results of Measurement 1 and Measurement 2 was calculated for the supernatant of the sample disruption solution, the standard solution, and the blank water. A calibration curve was then created from the results of the standard solution and the blank water, and the ammonia concentration in the sample disruption solution was calculated. The results are shown in Figure 1.

[0049] The pH of the sample disruption solution was determined by adding 10 μL of the sample disruption solution to a pH test paper and visually checking the pH. The results are shown in Figure 1.

[0050] The measurement results for protein, nucleic acid, ammonia, and pH (average values ​​for duplicate samples) are shown in Figures 1A, 1B, 1C, and 1D, respectively. Figure 1A demonstrates that solid-state cultivation of koji mold with the addition of ammonium salts, phosphate salts, calcium salts, magnesium salts, and iron salts to rice allows for rapid and easy production of protein in excess of the amount originally contained in rice (protein amount at day 0 of cultivation). Koji mold cultures on grains have been safely used as food. Therefore, the resulting koji mold cultures are expected to be used as high-protein food and feed ingredients. Furthermore, Figures 1B and 1C demonstrate that solid-state cultivation of koji mold simultaneously produces not only protein but also nucleic acids, consuming the ammonia necessary for protein and nucleic acid synthesis. Furthermore, Figure 1D demonstrates that the ammonia from the added ammonium sulfate is consumed, leaving sulfate ions, resulting in a decrease in pH. These results are scientifically valid, demonstrating that koji mold synthesizes protein from ammonium salts.

[0051] As a control experiment, cultivation was carried out under the same conditions as in Table 1, except that ammonium sulfate, potassium dihydrogen phosphate, calcium carbonate dihydrate, magnesium sulfate heptahydrate, and ferrous sulfate heptahydrate were omitted, i.e., 250 mg of dried cooked rice, koji mold spore suspension, and pure water were used. The resulting protein concentration was as shown in Figure 1E. The results in Figure 1E demonstrate that protein does not increase when koji mold is cultivated using only grains without the addition of nitrogen compounds and inorganic salts.

[0052] Example 3 Comparison of solid culture conditions for koji mold for protein production from ammonium salts. 100 mg of dried cooked rice (Alpha Foods) was collected in 2 mL plastic tubes and subjected to dry heat sterilization at 100°C for at least 1 hour. As shown in Table 2, nitrogen compounds, various inorganic salts, and koji mold spores were added to the dried cooked rice, mixed thoroughly, and then cultured at 30°C (triplicates were performed for each condition). After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein concentration, nucleic acid concentration, ammonia concentration, and pH were measured.

[0053]

[0054] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 2A, B, C, and D, respectively. Comparing conditions 1 and 2, 1 and 3, 1 and 4, and 1 and 5 in Figure 2A shows that adding ammonium salts, phosphate salts, calcium salts, magnesium salts, and iron salts to rice increases protein production. This demonstrates that adding inorganic salts selected from phosphate salts, calcium salts, magnesium salts, and iron salts, as well as nitrogen compounds, in solid-state cultivation of koji mold enables efficient protein production.

[0055] Furthermore, based on the data in Figures 2A and 2B, the ratio of protein to nucleic acid was calculated as shown in Figure 3. The results revealed that the ratio of protein to nucleic acid was higher when nitrogen compounds, calcium salts, magnesium salts, and iron salts were added (Condition 2) compared to other conditions. It is known that excessive intake of nucleic acids as food can cause diseases such as hyperuricemia and gout. Therefore, when using koji mold cultures as food, those with high protein content and low nucleic acid content are desirable. Therefore, it was suggested that the protein obtained by cultivating koji mold in rice with the addition of nitrogen compounds and inorganic salts other than phosphates could be used as a functional food.

[0056] Example 4: Improvement of protein productivity by adding magnesium salt 100 mg of dried steamed rice was collected in 2 mL plastic tubes and subjected to dry heat sterilization at 100°C for at least 1 hour. As shown in Table 3, nitrogen compounds, various inorganic salts, and koji mold spores were added to the dried steamed rice, mixed well, and then cultured at 30°C (triplicates were performed for each condition). After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein, nucleic acid, ammonia, and pH levels were measured.

[0057]

[0058] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 4A, 4B, 4C, and 4D, respectively. When the amount of magnesium added was 0.0048% based on the dry weight of rice, protein production increased significantly. The results in Figures 2 and 4 demonstrate that in solid-state cultivation of koji mold, adding 0.0048% magnesium based on the dry weight of rice results in efficient protein production.

[0059] Example 5: Improvement of protein productivity by adding iron salt 100 mg of dried steamed rice was collected in a 2 ml plastic tube and subjected to dry heat sterilization at 100°C for at least 1 hour. As shown in Table 4, various components and koji mold spores were added to the dried steamed rice, mixed well, and then cultured at 30°C (triplicate runs for each condition). After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein, nucleic acid, ammonia, and pH levels were measured.

[0060]

[0061] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 5A, 5B, 5C, and 5D. When the amount of iron added was 0.0001% based on the dry weight of rice, protein production increased significantly. The results in Figures 2 and 5 demonstrate that in solid-state cultivation of koji mold, adding 0.0001% iron based on the dry weight of rice leads to efficient protein production.

[0062] Example 6: Protein production from ammonium salt and urea by solid culture of koji mold. 100 mg of dried steamed rice was placed in a 2 ml plastic tube and sterilized by dry heat at 100°C for at least 1 hour. Nitrogen compounds, various inorganic salts, and koji mold spores were added to the dried steamed rice in the plastic tube according to Table 5, mixed well, and then cultured at 30°C. Culture was terminated after 0, 2, and 4 days (two replicates were performed for each day). Sample disruption solutions were prepared in the same manner as in Example 2, and protein, nucleic acid, ammonia, and pH were measured.

[0063]

[0064] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 6A, 6B, 6C, and 6D, respectively. As shown in Figure 6A, it was revealed that protein can be efficiently produced by solid-state cultivation of Aspergillus oryzae with the addition of ammonium salt and urea as nitrogen compounds.

[0065] Furthermore, the results in Figure 6B revealed that solid-state cultivation of koji mold simultaneously produced not only proteins but also nucleic acids. koji mold is known to produce urease, which breaks down urea. Because urease breaks down urea into ammonia, the ammonia concentration temporarily increased, as shown in Figure 6C, and the ammonia was then consumed for the synthesis of proteins and nucleic acids. Furthermore, as shown in Figure 6D, the pH temporarily increased due to the ammonia supplied from urea, but then the ammonia was consumed and the pH decreased due to the remaining sulfate ions from ammonium sulfate. These results are scientifically valid, indicating that koji mold synthesized proteins from ammonium salts and urea.

[0066] Example 7 Comparison of solid culture conditions for koji mold for protein production from ammonium salts and urea 100 mg of dried cooked rice (Alpha Foods) was collected in a 2 mL plastic tube and subjected to dry heat sterilization at 100°C for at least 1 hour. As shown in Table 6, nitrogen compounds, various inorganic salts, and koji mold spores were added to the dried cooked rice, mixed well, and then cultured at 30°C. After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein concentration, nucleic acid concentration, ammonia concentration, and pH were measured.

[0067]

[0068] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 7A, 7B, 7C, and 7D, respectively. Comparing Conditions 1 and 2, 1 and 3, and 1 and 4 in Figure 7A reveals that adding not only urea and ammonium salt but also phosphate, magnesium salt, and iron salt to rice increases protein production. Considering the results of the previous examples, particularly the results of Examples 2 and 3, this example revealed that even when urea and ammonium salt are used in combination as nitrogen compounds in solid culture of koji mold, protein can be efficiently produced in combination with each inorganic salt, just as in the case of ammonia salt alone.

[0069] Furthermore, based on this data, the ratio of protein to nucleic acid was calculated as shown in Figure 8. As a result, it was revealed that when ammonium salt, urea, magnesium salt, and iron salt were added (Condition 2), the ratio of protein to nucleic acid was higher than under other conditions. Therefore, taking into account the results of Example 3 (Figure 3), it was suggested that the protein obtained by combining urea and ammonium sulfate as nitrogen compounds with inorganic salts other than phosphates could be used as a functional food.

[0070] Example 8: Protein production from nitrate by solid culture of koji mold 100 mg of dried steamed rice was placed in a 2 ml plastic tube and subjected to dry heat sterilization at 100°C for at least 1 hour. As shown in Table 7, nitrogen compounds, various inorganic salts, and koji mold spores were added to the dried steamed rice, mixed well, and then cultured at 30°C. After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein, nucleic acid, ammonia, and pH were measured.

[0071]

[0072] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 9A, 9B, 9C, and 9D. It was revealed that protein can be efficiently produced by solid-state cultivation of koji mold with the addition of nitrate as a nitrogen compound.

[0073] Example 9 Comparison of Solid Culture Conditions of Koji Mold for Protein Production from Nitrate 100 mg of dried cooked rice (Alpha Foods) was collected in a 2 mL plastic tube and subjected to dry heat sterilization at 100°C for at least 1 hour. As shown in Table 8, nitrogen compounds, various inorganic salts, and Koji mold spores were added to the dried cooked rice, mixed well, and then cultured at 30°C. After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein concentration, nucleic acid concentration, ammonia concentration, and pH were measured.

[0074]

[0075] The results of measuring protein, nucleic acid, ammonia, and pH are shown in Figures 10A, 10B, 10C, and 10D, respectively. Protein was efficiently produced under all conditions. Comparing Conditions 1 and 2, and Conditions 1 and 3 in Figure 10A, it can be seen that the addition of magnesium sulfate and iron sulfate significantly increases protein production. Considering the results of the previous examples, particularly Examples 2, 3, and 7, this example revealed that even when nitrate is used in combination as a nitrogen compound, protein is produced more efficiently by combining it with each inorganic salt, just as when ammonium salt or urea is used. The results of these examples demonstrate that the present invention enables efficient protein production using koji mold from various nitrogen sources.

[0076] Example 10: Evaluation of the effect of adding oils and fats on protein production by solid culture of koji mold. 100 mg of dried cooked rice (Alpha Foods) was collected in a 2 mL plastic tube and sterilized at 100°C for at least 1 hour. As shown in Table 9, soybean oil, nitrogen compounds, various inorganic salts, and koji mold spores were added to the dried cooked rice, mixed thoroughly, and then cultured at 30°C (Condition 1). A control culture was also performed under the same conditions without adding soybean oil (Condition 2). After 4 days of culture, a sample disruption solution was prepared in the same manner as in Example 2, and the protein concentration, nucleic acid concentration, ammonia concentration, and pH were measured.

[0077]

[0078] The results of the measurements of protein, nucleic acid, ammonia, and pH are shown in Figures 11A, 11B, 11C, and 11D, respectively. The results of Figure 11A confirmed that the addition of oil increased protein production. The results of Figure 11B also confirmed that the amount of nucleic acid increased under oil-added conditions, and the results of Figure 11C also confirmed that the amount of ammonia consumed by koji mold increased under oil-added conditions. Furthermore, the results of Figure 11D confirmed that the pH decreased under oil-added conditions. These results suggest that the addition of oil promoted protein synthesis from ammonia by koji mold. As described above, it was revealed that protein production was further increased by culturing koji mold in a mixture of grains with not only nitrogen compounds and various inorganic salts but also oil.

[0079] Example 11: Protein production from corn and ammonium salts by solid culture of koji mold. Dent kernels were purchased from a domestic feed company as a representative corn sample. These kernels were ground in a food mill to prepare crushed corn kernels. 10 g of crushed corn kernels were placed in a 500 mL Erlenmeyer flask. Water was added to the crushed corn kernels and autoclaved at 121°C for 20 minutes to gelatinize the starch. Ammonium sulfate, various inorganic salts, and koji mold spores were then added to the crushed kernels as shown in Table 10, mixed thoroughly, and cultured at 30°C. After 4 days of culture, a crushed sample solution was prepared in the same manner as in Example 2, and the protein concentration, nucleic acid concentration, ammonia concentration, and pH were measured.

[0080]

[0081] The measurement results for protein, nucleic acid, ammonia concentrations, and pH are shown in Figures 12A, 12B, 12C, and 12D, respectively. The results in Figure 12A confirmed that solid-state cultivation of koji mold with the addition of nitrogen compounds and inorganic salts increased protein production, not only in rice but also in corn. Furthermore, a comparison of conditions 1 to 3 with condition 4 suggested that the addition of magnesium salts and iron salts increased protein production. In Figure 12C, a comparison of conditions 1 to 3 with condition 4 showed that more ammonia was consumed under the former. These results suggest that the addition of magnesium salts and iron salts promotes protein synthesis from ammonium salts by koji mold. Furthermore, the dent variety used in the medium in this example is corn, which is primarily used as animal feed. This suggests that the present invention enables efficient protein synthesis using less expensive raw materials.

Claims

1. A method for producing a high-protein composition, comprising the step of solid-state culturing koji mold in a medium prepared by adding grains and a nitrogenous compound to produce protein.

2. A method for producing a high-protein composition, comprising the step of solid-state culturing koji mold in a medium prepared by adding grains, a nitrogenous compound, and inorganic salts including one or more selected from the group consisting of magnesium salts and iron salts, to produce protein.

3. A method for producing a high-protein composition, comprising the step of solid-state culturing koji mold in a medium prepared by adding grains, nitrogen compounds, and inorganic salts including magnesium salts and iron salts, to produce protein.

4. The method according to claim 2 or 3, wherein the amount of magnesium atom of the magnesium salt in the medium is 0.001% by weight or more relative to the dry weight of the grain.

5. The method according to claim 2 or 3, wherein the amount of iron atom of the iron salt in the medium is 0.00003% by weight or more relative to the dry weight of the grain.

6. The method according to any one of claims 1 to 3, wherein the medium is prepared by further adding water in an amount of 400% by weight or less based on the dry weight of the grain.

7. The method according to any one of claims 1 to 3, wherein the nitrogen compound is one or more selected from the group consisting of ammonium salts, nitrates, ammonia, urea and amino acids.

8. The method according to any one of claims 1 to 3, wherein the content of nitrogen compounds is 0.5% by weight or more based on the dry weight of the grain.

9. The method according to claim 2 or 3, wherein the inorganic salt further comprises one or more selected from the group consisting of calcium salts and phosphate salts.

10. The method according to any one of claims 1 to 3, wherein the medium is prepared by further adding an oil or fat.

11. The method according to any one of claims 1 to 3, wherein the grain comprises at least one of rice and corn.

12. A method for producing a food or feed composition, comprising producing a protein-rich composition by the method according to any one of claims 1 to 3, and using the composition to produce food or feed.

13. A method for producing a food or feed composition for preventing or reducing the risk of antiuricemia or gout, comprising producing a protein-rich composition by the method according to any one of claims 1 to 3 and using the composition to produce a food or feed composition for preventing or reducing the risk of antiuricemia or gout.

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