Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
  • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
  • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

• the present invention relates to a method for producing a high-protein composition.
• Non-Patent Documents 1-2 Compared to insect food and cultured meat, methods of protein production that are cheaper and more efficient have been developed, such as using a living protein from yeast called single cell protein and a living protein from the cells of filamentous fungi called mycoprotein (Non-Patent Documents 1-2).
• the inventors therefore applied the traditional koji-making method that has been practiced in Japan since ancient times to investigate a process for producing protein by adding nitrogenous compounds to grains and cultivating koji mold. As a result, they discovered that protein can be produced efficiently by cultivating koji mold by adding nitrogenous compounds to grains, and preferably by further adding inorganic salts.
• the present invention provides the following [1] to [13].
• [1] A method for producing a high-protein composition, comprising a 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 a step of solid-state culturing koji mold in a medium prepared by adding grains, nitrogen compounds, and inorganic salts including one or more selected from the group consisting of magnesium salts and iron salts, to produce protein.
• FIG. 4 is a graph showing A: improvement in protein productivity, B: changes in nucleic acid concentration, C: ammonia concentration, and D: changes in pH by adding magnesium to the medium shown in Table 3 in Example 4.
• FIG. 5 is a graph showing A: improvement in protein productivity, B: changes in nucleic acid concentration, C: ammonia concentration, and D: changes in pH by addition of iron in the medium shown in Table 4 in Example 5.
• FIG. 6 is a graph showing the time-dependent changes in A: protein production 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.
• 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 media shown in Table 8 in Example 9.
• “p ⁇ 0.05" indicates that the p-value was below the significance level of 0.05 as a result of comparing the protein production amount between the two groups under conditions 1 and 2, and conditions 1 and 3, and that there was a statistically significant difference.
• FIG. 11 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH by adding fats and oils to the medium shown in Table 9 in Example 10, for each of the solid culture conditions for koji mold.
• FIG. 12 is a graph comparing A: protein production amount, B: nucleic acid concentration, C: ammonia concentration, and D: pH in media using corn shown in Table 10 for each of the solid culture conditions for koji mold in Example 11.
• the koji mold is preferably added to the system as a culture containing spores (seed koji).
• seed koji can be prepared according to standard methods, for example, by culturing koji mold in a medium containing yeast extract, etc.
• Grains are usually added to the system as heated (steamed) grains, but heat treatment is not essential and unheated grains can also be used.
• steamed grains refer to grains that have been steamed (usually treated at high temperature and/or high pressure while in a water-absorbed state).
• steamed rice can be obtained by appropriately soaking the grain and then cooking it (for example, heating it to a temperature above the gelatinization temperature (e.g., 80-120°C) and maintaining the temperature for 10-60 minutes).
• Steamed grains can also be so-called gelatinized grains, which are obtained by rapid drying such steamed grains, or waste materials such as leftover food and starch-containing surplus food ingredients.
• a nitrogen compound refers to a compound containing a nitrogen atom.
• the nitrogen compound may be either an inorganic nitrogen compound or an organic nitrogen compound, as long as it can supply a nitrogen source during the cultivation of koji mold.
• inorganic nitrogen compounds include ammonium salts, nitrates, and ammonia, with ammonium salts and nitrates being more preferred.
• ammonium salts include ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium bicarbonate, and ammonium chloride, with ammonium sulfate being preferred.
• the inorganic salt is one or more selected from the group consisting of magnesium salts and iron salts.
• 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.
• the term "inorganic salt” refers to 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 a single compound, and the counter ion is not particularly limited.
• iron salts 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.
• Examples of calcium salts include calcium carbonate, calcium sulfate, and calcium chloride, with calcium carbonate being preferred.
• the inorganic salts may be used alone or in combination of two or more, with combinations of two or more or three or more being preferred.
• Preferred combinations of two or more include a combination of magnesium salt and iron salt, a combination of magnesium salt, iron salt and calcium salt, and a combination of magnesium salt, iron salt, calcium salt and phosphate.
• Solid-state cultivation 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 cultivation, other components (e.g., water, vitamins) may be added to the system as needed.
• nutrients e.g., water, vitamins
• magnesium salt its content is preferably 0.01% by weight or more, more preferably 0.02% by weight or more, and even more preferably 0.04% by weight or more, based on the dry weight of the grain.
• the magnesium salt content is preferably 0.01% by weight or more, more preferably 0.01 to 2% by weight, even more preferably 0.02 to 1% by weight, and even more preferably 0.04 to 0.5% by weight, based on the dry weight of the grain.
• the content 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 fat and oil content 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.
• the medium may contain other components in addition to those listed above.
• An example of such a component is water.
• 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 koji mold's access 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 effectively achieved.
• the high-protein composition may be 100% pure protein, or may contain other components.
• examples of other components include nucleic acids.
• a relatively small amount of nucleic acid is preferred.
• the ratio of protein to nucleic acid in the culture 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 koji mold cultures as functional foods, such as foods or feed for the prevention or alleviation of hyperuricemia and gout.
• 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 ⁇ L 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 ⁇ L of water and vortex-mixed to obtain a homogenized sample solution.
• the pH of the sample disruption solution was determined by adding 10 ⁇ L of the sample disruption solution to pH test paper and visually determining the pH. The results are shown in Figure 1.
• Figure 1A The measurement results for protein, nucleic acid, ammonia, and pH (average values for duplicate samples) are shown in Figures 1A, B, C, and D, respectively.
• Figure 1A demonstrates that adding ammonium salts, phosphate salts, calcium salts, magnesium salts, and iron salts to rice and culturing koji mold in solid state allows for the rapid and easy production of more protein than the amount originally contained in rice (protein amount on day 0 of cultivation). Koji mold cultures on grains have been safely used as food to date. Therefore, the resulting koji mold cultures are expected to be used as high-protein food and feed ingredients.
• Figures 1B and 1C also demonstrate that solid-state koji mold cultivation simultaneously produces not only protein but also nucleic acids, consuming the ammonia necessary for protein and nucleic acid synthesis.
• Figure 1D also demonstrates that the ammonia from the added ammonium sulfate is consumed, leaving sulfate ions, which lowers the pH.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• Example 11 Protein production from corn and ammonium salts by solid culture of Aspergillus oryzae Dent kernels were purchased from a domestic feed company as a representative corn sample and were crushed in a food mill to prepare crushed corn kernels. 10 g of crushed corn kernels were placed in a 500 mL Erlenmeyer flask, and water was added to the mixture 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 well, and cultured at 30°C. After 4 days of culture, a sample crushed solution was prepared in the same manner as in Example 2, and the protein concentration, nucleic acid concentration, ammonia concentration, and pH were measured.

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