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WO2007039990A1 - Procédé servant à produire du koji liquide en utilisant aspergillus oryzae - Google Patents

Procédé servant à produire du koji liquide en utilisant aspergillus oryzae Download PDF

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Publication number
WO2007039990A1
WO2007039990A1 PCT/JP2006/316173 JP2006316173W WO2007039990A1 WO 2007039990 A1 WO2007039990 A1 WO 2007039990A1 JP 2006316173 W JP2006316173 W JP 2006316173W WO 2007039990 A1 WO2007039990 A1 WO 2007039990A1
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Prior art keywords
liquid
koji
producing
culture
liquid koji
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Japanese (ja)
Inventor
Toshikazu Sugimoto
Hiroshi Shoji
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Asahi Breweries Ltd
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Asahi Breweries Ltd
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    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/023—Preparation of other alcoholic beverages by fermentation of botanical family Solanaceae, e.g. potato
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

Definitions

  • the present invention relates to a method for producing liquid koji using yellow koji mold, in particular, liquid koji having enzyme activity necessary for producing fermented foods and drinks such as sake.
  • Solid koji made by growing koji molds on the surface of raw materials such as cereals has been used.
  • Solid koji is obtained by a traditional production method, but is not suitable for large-scale production because it is a special culture form called solid culture.
  • liquid koji which is a koji mold culture obtained by liquid culture of koji mold, can be said to be a culture form suitable for efficient production because of easy culture control.
  • Patent Document 1 discloses a high production method of glucoamylase by culturing yellow koji mold while applying stress to mycelial growth.
  • gonococci are cultured on a porous membrane or in a entrapping immobilization agent having voids, and a novel gene glaB encoding darcoamylase is expressed to increase the enzyme activity.
  • Control or special culture equipment is required, and it is difficult to say a simple method! ,.
  • the present inventors sufficiently cultivate birch or black koji molds in a liquid medium containing cereals and the like whose surfaces are covered with husks as a culture raw material, thereby obtaining sufficient enzyme activity for the production of shochu and the like.
  • a patent patent application 2004-35066. 1 specification, Japanese Patent Application No. 2004-352320 specification).
  • Non-patent literature l Ishida H. et al: J. Ferment. Bioeng., 86, 301-307 (1998)
  • Patent Document 1 Japanese Patent Laid-Open No. 11-225746
  • An object of the present invention is to cultivate yellow koji molds by a simple method that does not require special operations such as a special culture apparatus, pretreatment of raw materials, and strict control, so that sake or the like can be obtained. It is to provide a method for producing a liquid koji with high activity of darcoamylase and OC amylase, which are key enzymes for the production of fermented foods and drinks.
  • the present invention according to claim 1 is a method for producing a liquid koji, wherein the whole or a part of the surface is covered with at least a husk; Or agar; pretreatment such as crushing and crushing, amaranthus and koji or quinuaka are also selected.
  • At least one culture material, nitrate, phosphate, and sulfate in a liquid medium containing sulfate Is a method for producing a liquid koji.
  • the present invention according to claim 2 comprises liquid medium strength nitrate at a concentration of 0.1 to 2.0% (wZv). 2.
  • the present invention according to claim 3 is the method for producing a liquid koji according to claim 1, wherein the liquid medium contains phosphate at a concentration of 0.05 to L 0% (wZv).
  • the present invention according to claim 4 is the method for producing a liquid koji according to claim 1, wherein the liquid medium contains sulfate in a concentration of 0.01 to 0.5% (wZv).
  • the culture temperature when cultivating yellow koji molds in a liquid medium is 25 to 35 ° C until 12 to 36 hours from the start of the culture, and thereafter 35 to 45 ° C. 2.
  • the present invention according to claim 6 is the method for producing a liquid koji according to claim 1, wherein at least darcoamylase and ⁇ -amylase are simultaneously generated and accumulated in the liquid koji.
  • the present invention according to claim 7 is characterized in that the enzyme activity of liquid koji is adjusted by suppressing the release rate of the sugar derived from starch in the culture raw material to the culture system. It is a manufacturing method of a liquid cake.
  • the present invention according to claim 8 is a method for producing a fermented food or drink using the liquid koji obtained by the production method according to any one of claims 1 to 7.
  • the present invention according to claim 9 is the method for producing a fermented food or drink according to claim 8, wherein the fermented food or drink is at least one selected from sake, shochu, soy sauce, miso, mirin, brewed vinegar and sweet sake power. .
  • the present invention according to claim 10 is the method for producing a fermented food or drink according to claim 8 or 9, wherein all steps are performed in a liquid phase.
  • the present invention according to claim 11 is a liquid bottle obtained by the manufacturing method according to any one of claims 1 to 7.
  • the present invention according to claim 12 is a dried product of the liquid cake obtained by drying the liquid cake of claim 11.
  • the present invention according to claim 13 is a concentrated product of the liquid cake obtained by concentrating the liquid cake according to claim 11.
  • the present invention according to claim 14 is a method for producing an enzyme preparation using the liquid koji according to claim 11.
  • the present invention according to claim 15 is a method for producing an enzyme preparation using the dried liquid koji according to claim 12.
  • the present invention according to claim 16 is a method for producing ethanol using the liquid soot according to claim 11.
  • the present invention according to claim 17 is a cereal whose surface is entirely or partially covered with husks; beans and Z or moss whose surface is covered with husks; A maranthus and Z or quinua force
  • a method for producing an enzyme comprising culturing yellow koji molds in a liquid medium containing at least one selected culture raw material, nitrate, phosphate, and sulfate.
  • the liquid koji produced according to the present invention has sufficient enzyme activity to produce fermented foods and drinks such as sake, so that it is possible to produce fermented foods and drinks using the liquid koji. Become.
  • the liquid koji having high enzyme activity can be used for Chiji IJ as a pharmaceutical product such as an enzyme preparation and a digestive agent.
  • yellow koji molds are often used in the field of genetic engineering as transformation hosts, and according to the present invention, mass production of high-value-added heterogeneous proteins used for pharmaceuticals and the like is facilitated.
  • the culture raw material used in the present invention is unpolished, unprocessed, or at least refined to the extent that the outer skin is left on the surface, the raw material utilization rate and the yield are high. Improvement can be expected.
  • Fig. 1 is a graph showing the enzyme activity in Example 1 when the koji mold was cultured in a liquid medium containing various carbon sources.
  • A is the result of measurement of darcoamylase
  • b is the result of measurement of ⁇ -amylase.
  • FIG. 2 is a graph showing the enzyme activity of Example 2 in which various salts are removed and yellow koji molds are cultured in a liquid medium.
  • A is the result of measurement of darcoamylase
  • b is the result of measurement of ⁇ -amylase.
  • FIG. 3 is a graph showing the enzyme activity in Example 3 when the koji mold was cultured in a liquid medium excluding various salts.
  • A is the result of measurement of darcoamylase
  • b is the result of measurement of ⁇ -amylase.
  • FIG. 4 is a graph showing the enzyme activity in Example 4 when the koji mold was cultured in a liquid medium in which the added amount of inorganic salts was changed.
  • A is the measurement result of darcoamylase
  • b is the measurement result of ⁇ -amylase.
  • FIG. 5 is a graph showing the enzyme activity in Example 5 when liquid culture of yellow koji molds was performed at different culture temperatures.
  • (A) shows the measurement results for dalcore mirase, and
  • (b) shows the measurement results for ⁇ -amylase.
  • koji molds are cultured in a liquid medium prepared by adding raw materials such as the above-mentioned cereals, beans, koji, specific millet grains, koji, darcoamylase, and OC amylase.
  • a process for producing a liquid koji with enhanced enzyme activity since the koji molds are cultured using the various raw materials described above, it takes time for the starch in the raw materials to take time, and the release rate of nutrients including sugar into the culture system is suppressed, so that Enzyme activity is enhanced.
  • Dalcoylase and ⁇ -amylase are simultaneously generated and accumulated in a well-balanced manner.
  • cereals used as a raw material for culturing liquid koji, barley, rice, wheat examples include buckwheat, shrimp, yam, millet, culyan, and corn.
  • the shape of these culture raw materials requires that all or a part of the surface is covered with at least the husk, and the unmilled product or at least the husk remains on the surface of the grain.
  • Those that are higher than the degree of whitening that have been polished to the extent can be used, and brown rice, brown wheat, etc. can also be used.
  • rice not only brown rice but also rice husks may be attached, or rice husks may be partly attached.
  • the unpolished milling rate is 100%, or the unpolished milling rate is 100%, and the unmilled milling rate (100%) is used to determine the barley grain ratio (general The ratio is less than 7-8%), that is, more than 92 to 93% of the milling rate.
  • the milling rate refers to the proportion of cereals that remain after milling the cereals.
  • the milling rate of 90% means that 10% of the skin of the surface layer of the cereals is scraped off.
  • the unpolished barley is one that has been refined to such an extent that the unmilled wheat power husk remains on the surface of the grain, and includes those having a milling ratio of 90% or more.
  • the husk is the outer part of the grain that covers the surface of the grain.
  • examples of beans and moss used as a raw material for culturing liquid koji include soybeans, red beans, and sweet potatoes. These culture materials are used for the preparation of a liquid medium in a state where they are completely covered with the outer skin without any processing such as cutting and crushing, only by washing away the dirt on the outer skin.
  • heating or freezing treatment can be carried out while keeping the outer shell of beans or moss as the culture raw material.
  • amaranth used as a raw material for culturing liquid koji is a general term for a plant belonging to the genus Huaceae, and the content of lysine, which is one of amino acids having a high protein content in cereals, is soybean. Comparable to In addition, it is a highly nutritious grain that contains more calcium, iron and fiber than milled rice, and the country of origin is a specific region in Latin America, India, Himalayas, and Nepal.
  • Quinua is an annual plant of the Agatha family and is cultivated mainly in the highlands of southern Peru and the Andes Mountains of western Peru, and is rich in minerals, vitamins, proteins and dietary fiber.
  • the culture materials amaranth and quinua may be used alone or in combination. These are used for the preparation of liquid media without pretreatment such as comminution or grinding.
  • the above culture raw materials are used alone or in combination of two or more for the preparation of the following liquid medium. That is, the above culture raw material is mixed with water to prepare a liquid medium. The mixing ratio of the raw materials is adjusted to such a level that darcoamylase and ⁇ -amylase are selectively produced and accumulated during the culture of jaundice.
  • barley when barley is used as a culture raw material, it is prepared in a liquid medium supplemented with 1-20% (wZvol) of barley with respect to water.
  • wZvol liquid medium supplemented with 8 to 10% (w / vol)
  • 95% polished barley Is prepared in liquid medium supplemented with 1-4% (w / vol).
  • brown rice excluding rice husks when brown rice excluding rice husks is used as a culture raw material, the brown rice is 1% (WZV ol) to 20% (w / vol), preferably 5% (w / vol) to 13% of water. (w / vol), more preferably 8% (w / vol) to 10% (w / vol).
  • beans When beans are used as a culture raw material, beans are 1 to 10% (w / vol), preferably 8 to 10% (w / vol) for soybeans, 1 for beans. Prepared in liquid medium supplemented with ⁇ 2% (w / vol). In addition, when moss is used as a culture raw material, it is prepared in a liquid medium with moss 1 to 10% (w / vol) added to water.
  • amaranth when used as a culture raw material, it is 1.5% (w / vol) to 15% (w / vol), preferably 2% (w / vol) to 10% with respect to water. (w / vol), more preferably 2% (wZvol) force is also prepared in a liquid medium supplemented with 8% (wZvol).
  • it in the case of quinua, it is 1.5% (w / vol) to 7% (w / vol), preferably 2% (w / vol) to 6% (w / vol), more preferably relative to water.
  • liquid medium supplemented with 2% (w / vol) to 4% (w / vol).
  • the optimum blending amount varies depending on the degree of milling of the culture raw material to be used, the koji mold used, the type of the culture raw material, and the like, and may be appropriately selected.
  • the amount of culture raw material used exceeds the upper limit, the viscosity of the culture solution becomes high, the supply of oxygen and air necessary for aerobic culture of Staphylococcus aureus becomes insufficient, and the oxygen concentration in the culture becomes low. It is not preferable because it decreases and culture becomes difficult to proceed. On the other hand, if the amount of the raw material used is less than the lower limit, the target enzyme will not be produced at a high rate.
  • the starch contained in the culture raw material may be gelatinized before culturing.
  • the gelatinization method of starch is not particularly limited and may be carried out according to conventional methods such as the steaming method and roasting method. In the liquid medium sterilization process described later, when the starch is heated to a temperature higher than the gelatinization temperature by high-temperature high-pressure sterilization or the like, the starch paste is simultaneously performed by this treatment.
  • nitrate, phosphate and sulfate are not particularly limited as long as they are usually used for cultivation of filamentous fungi.
  • sodium nitrate or potassium nitrate can be used as the nitrate, and sodium nitrate is particularly preferable.
  • phosphate potassium dihydrogen phosphate, ammonium phosphate and the like can be used, and potassium dihydrogen phosphate is particularly preferable.
  • sulfate magnesium sulfate heptahydrate, iron sulfate heptahydrate, ammonium sulfate and the like can be used, and magnesium sulfate heptahydrate and iron sulfate heptahydrate are particularly preferable.
  • These inorganic salts can be used alone or in combination of two or more.
  • the concentration of the above-mentioned inorganic salts in the liquid medium is adjusted to such a level that darcoamylase and ⁇ - amylase are selectively generated and accumulated during the culture of jaundice.
  • concentration of the above-mentioned inorganic salts in the liquid medium is adjusted to such a level that darcoamylase and ⁇ - amylase are selectively generated and accumulated during the culture of jaundice.
  • nitrate from 0.1 to 2.0%, preferably 0.2 to 1.5%, if ⁇ or 0. 05-1 phosphate. 0 0/0, preferably I or 0. 1 to 0. 5 0/0, I or 0. 01-0 when sulfate. 5 0/0, the good Mashiku 0.02 to 0.1% 0, the deviation also /) to .
  • the preferred concentration conditions for the inorganic salts described above can be used in combination with each other, and can be combined with any aspect of the method of the present invention.
  • organic substances other than the above-mentioned inorganic salts, inorganic salts, and the like can be appropriately added as a nutrient source.
  • additives are not particularly limited as long as they are generally used for culturing filamentous fungi, but organic substances include rice bran, wheat straw, corn steep liquor, soybean meal, defatted soybean, etc.
  • Water-soluble compounds such as ammonium salt, potassium salt, calcium salt and magnesium salt can be mentioned, and two or more kinds of organic substances and salt or inorganic salt may be used simultaneously.
  • These additions promote the growth of jaundice
  • the liquid culture medium thus obtained is not particularly limited in the treatment method in which sterilization treatment may be performed as necessary.
  • An example is the high-temperature and high-pressure sterilization method, which can be performed at 121 ° C for 15 minutes.
  • the yellow koji mold used in the present invention is a koji mold having a saccharide-degrading enzyme-producing ability, preferably a darcoamylase-producing ability and an a-amylase-producing ability.
  • a saccharide-degrading enzyme-producing ability preferably a darcoamylase-producing ability and an a-amylase-producing ability.
  • Aspergillus oryzae Aspergillus sojae Aspergillus oryzae Aspergillus sojae
  • the form of the yellow koji mold inoculated on the medium is arbitrary, and spores or mycelia can be used.
  • These yellow koji molds can be used either by culturing with one kind of strain or by mixed culturing with two or more kinds of the same or different kinds of strains. There is no problem whether these are used in the form of spores or mycelia obtained by preculture, but it is preferable to use mycelia because the time required for the logarithmic growth phase is shortened. There is no particular limitation on the inoculum of jaundice in liquid medium, but about 1 X 10 4 to 1 X 10 6 spore per ml of liquid medium, and 0.1 to It is preferable to inoculate about 10%.
  • the culture temperature of the yellow koji mold is not particularly limited as long as it does not affect the growth, but it is preferably 25 to 45 ° C, more preferably 30 to 40 ° C. When the culture temperature is low, the growth of Aspergillus oryzae slows down and contamination with various bacteria is likely to occur.
  • the enzyme activity can be enhanced by controlling the culture temperature according to the growth phase of the yellow koji mold. Specifically, the cell growth period until 12-36 hours after the start of cultivation is 25-35 ° C, preferably 28-33 ° C, and the subsequent enzyme production period is 35-45 ° C, preferably 37 What is necessary is just to maintain at -42 degreeC.
  • the total culture time is preferably 24 to 72 hours.
  • the culture apparatus is not limited as long as it can perform liquid culture. However, it is necessary to perform aerobic culture for jaundice, so it is performed under aerobic conditions in which oxygen and air can be supplied into the medium. There is a need. Further, during the culture, it is preferable to stir so that the raw materials, oxygen, and jaundice in the medium are uniformly distributed in the apparatus.
  • the stirring conditions and aeration amount may be appropriately selected depending on the culture apparatus, the viscosity of the medium, etc., as long as the culture environment can be maintained aerobically.
  • the liquid koji includes a culture solution obtained by centrifuging the culture, a concentrate thereof, or a dried product thereof.
  • the method for producing an enzyme according to claim 17 is the same as the method for producing a liquid koji described above.
  • the liquid koji obtained by the production method of the present invention can be used for the production of fermented foods and drinks such as sake.
  • sake in the case of producing sake, in the sake mother and each mash preparation stage, in the case of producing shochu, in the case of making soy sauce in the mash preparation stage, in the case of producing miso in the preparation stage.
  • brewed vinegar at the preparation stage when preparing mirin at the preparation stage, when manufacturing sweet sake at the preparation stage, Can be used instead.
  • liquid soot a part of the obtained liquid soot can be used as a starter in the next liquid soot production.
  • stable production can be achieved, and at the same time, production efficiency can be improved.
  • the liquid koji obtained by the method of the present invention can also be used as a pharmaceutical preparation such as an enzyme preparation and a disinfectant.
  • the obtained koji mold culture may be concentrated and purified to a desired degree, and an appropriate excipient, thickener, sweetener and the like may be added to prepare a preparation by a conventional method.
  • the liquid koji obtained by the method of the present invention can be used for production of ethanol by fermentation.
  • a known industrial alcohol (ethanol) production method is used except that the liquid koji is used instead of the solid koji. Can be manufactured according to.
  • the liquid koji is charged by adding yeast having ethanol-producing ability, such as shochu yeast, raw materials, and water. Lactic acid can also be used as necessary.
  • the above raw materials may be any starchy raw material, such as barley, bare barley, rice, wheat, buckwheat, rice, wheat, millet, corn, corn, etc .; sweet potatoes, sweet potatoes, etc .; etc. Can be mentioned.
  • the secondary charging can be performed.
  • Industrial alcohol can be produced by distilling the mash after fermentation to remove impurities and concentrating.
  • the protein can be produced in a high yield in a koji mold culture. It is possible to make it.
  • Example 1 Effect of carbon source on enzyme productivity in liquid koji production of yellow koji mold
  • the medium thus prepared was inoculated with Aspergillus oryzae RIB40 at 1 ⁇ 10 6 cells / ml, and cultured with shaking at 30 ° C. for 72 hours at lOOrpm.
  • 98% refined wheat is equivalent to cereals whose surface is covered with husk disclosed in the specification of Japanese Patent Application No. 2004-350661 by the applicant of the present invention, and when this was ground, enzyme production was suppressed. Therefore, it was inferred that it was important to have a structure in which barley starch was physically covered with husk.
  • amylase was slightly different from that of darcoamylase, it produced a large amount of enzyme in the 98% polished wheat test plot (Fig. 1 (b)).
  • Example 2 (Effects of various salts added in liquid koji production of yellow koji mold)
  • Liquid rice cake production method Liquid rice cake is produced by the following method and the enzyme activity is measured. Set.
  • AA a Measured for amylase activity
  • Measurement of darcoamylase activity (GA) was performed using a sugar squid fraction determination kit (manufactured by Kikkoman), and a-amylase activity was performed using a amylase measurement kit (manufactured by Kikkoman).
  • cereals with a surface covered with husk such as 98% polished wheat, sodium nitrate, dibasic hydrogen phosphate, magnesium sulfate or iron sulfate are essential.
  • Example 3 (Effect of sulfate on enzyme production in liquid koji production of yellow koji mold)
  • Example 2 the magnesium sulfate-deficient medium and the sulfuric acid sulfate in the test group 6 were not affected by the magnesium sulfate-deficient medium in the test group 4 and the iron sulfate-deficient medium in the test group 5 although there was no tendency to decrease the enzyme productivity. It was confirmed that there was a marked decrease in the medium lacking both iron. Therefore, in order to search for the essential factors of enzyme productivity in liquid koji mold of yellow koji mold, a test to confirm the effect of sulfate was conducted.
  • Liquid rice cake is produced by the following method, and their enzyme activity is measured.
  • the medium was inoculated with Aspergillus oryzae RIB40 at 1 ⁇ 10 6 cells / ml and cultured with shaking at 30 ° C. for 72 hours at lOOrpm.
  • Polished barley ⁇ 3 KH 2 P 0 4 MgS0 4 - 7H 2 0 M gCl 2 - 6H 2 0 control group 2.03 ⁇ 4 0.2% 0.3% - -
  • Test Zone 2 2.03 ⁇ 4 0.2% 0.3% 0.041% [0055] 2. Measurement method: After completion of the culture, darcoamylase activity (GA) and a-amylase activity (AA) in each culture supernatant were measured. Measurement of darcoamylase activity (GA) was performed using a sugar squid fraction determination kit (manufactured by Kikkoman), and ex-amylase activity was performed using an ex-amylase measurement kit (manufactured by Kikkoman).
  • Method for producing liquid rice cake Manufacture liquid rice cake by the following method and measure their enzyme activity.
  • Table 3 shows the composition of 98% refined barley (Australian schooner), sodium nitrate, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron sulfate heptahydrate and water.
  • 100 ml of the liquid culture medium formulated in the above was applied to a triangular flask with 500 ⁇ baffle and autoclaved at 121 ° C for 15 minutes.
  • the medium was inoculated with Aspergillus oryzae RIB40 at 1 ⁇ 10 6 cells / ml and cultured with shaking at 30 ° C. for 72 hours at lOOrpm.
  • FIG. 4 shows the measurement results of enzyme activity in each test group.
  • test group 1 the amount of inorganic salts added was doubled compared to the control group, and test group 2 was increased four times. Darko amylase activity at that time, an inorganic salt concentration amount highest immediately inorganic salts added in 4-fold enhanced test group 2 were shown to contribute to the enzyme productivity improvement (FIG. 4 (a)) 0 a Amylase also showed the highest activity in test group 1 where inorganic salts were enhanced by a factor of 2 compared to the control group, confirming that the amount of inorganic salts added affects enzyme productivity (Fig. 4 (b) ).
  • Tests were conducted to confirm the effect of culture temperature on enzyme production in liquid koji production of yellow koji mold, and the possibility of further high enzyme production was examined.
  • liquid koji mold of yellow koji mold was produced under the same culture medium conditions as in test group 2 of Example 4 at different culture temperatures, and the enzyme activity was measured.
  • the culture conditions are as follows: control group, 30 ° C—constant, 72 hours; test group 1, 37 ° C—constant, 72 hours; In test group 2, the cells were cultured at 30 ° C. from the start of culture to 24 hours and at 37 ° C. from 24 to 72 hours of culture.
  • the stirring conditions were shaking culture at lOOrpm in all test sections.
  • FIG. 5 shows the results of enzyme activity measurement in each test group.
  • Test Zone 2 it is assumed that cell growth occurs at a culture temperature of 30 ° C, and that enzyme production occurs at a culture temperature of 37 ° C. It was suggested that control of the culture temperature was effective in enhancing enzyme activity.
  • Test Zone 2 the enzyme activity in Test Zone 2 is 106.5 U / ml for dalcore amylase and 563.5 U / ml for a-amylase, which is sufficient for producing fermented foods and drinks such as rice shochu and sake. available.
  • Example 6 Manufacture of dried liquid rice cake
  • the measurement of darcoamylase activity was performed using a sugar squid fraction determination kit (manufactured by Kikkoman), and the a-amylase activity was performed using an a-amylase measurement kit (manufactured by Kikkoman). Furthermore, enzyme activity measurement of the liquid koji dry product, the liquid koji dry product 250mg was measured using a solution in 10mM acetate buffer (P H5) 20ml.
  • Table 4 shows the enzyme activity measurement results of the liquid koji and the dried liquid koji product obtained in the above (I) when the freeze-drying process produced by the method of test section 2 described in Example 5 was not performed. Shown in
  • the charging composition is as shown in Table 5.
  • the rice was washed with 90% polished rice (Koshihikari from Ibaraki Prefecture), soaked for 15 minutes, drained for 10 minutes, and steamed for 30 minutes.
  • the liquid rice cake produced by the method of Test Zone 2 described in Example 5 was used.
  • Lactic acid and water This was inoculated with 50 1 shochu yeast (Kagoshima yeast) that was statically cultured in YPD medium at 30 ° C for 48 hours. Fermentation conditions were 25 ° C-constant, and fermentation was performed for 18 days.
  • the charging composition was as follows. That is, after washing 580 g of 90% polished rice (Koshihikari from Ibaraki Prefecture), soaked for 15 minutes, drained for 10 minutes and boiled for 30 minutes, 1630 ml of water, manufactured by the method of Test Zone 2 described in Example 5 500 ml of the liquid koji and 1.6 ml of 90% lactic acid were mixed. To this was added 100 1 sake yeast (Association No. 7) that had been shaken and cultured in YPD medium at 30 ° C for 24 hours, and fermentation was performed at 25 ° C.
  • the preparation composition was as shown in Table 7 below. Whole soybeans were washed, dipped in water, drained for 60 minutes, and steamed for 4 hours, and then crushed with a food processor. Wheat (Agriculture No. 61) was roasted and then ground. Salt was added to the liquid koji produced by the method of test section 2 described in Example 5, and 10 ml of the Zygosaccharomyces rouxii (NBRC0510) culture solution and the whole soybeans and wheat treated as described above were added thereto. The Z. rouxii culture solution used was a shaking culture in YPD medium at 30 ° C. for 24 hours.
  • Fermentation conditions After fermentation at 15 ° C for 30 days, the temperature was increased to 2 ° CZ days to 30 ° C. After reaching the temperature, fermentation was performed at 30 ° C for another 3 months. Up to this point, the mixture was properly stirred. Thereafter, the mixture was aged at 25 ° C for 2 months without stirring, and the final mash was obtained.
  • Post-treatment Fired at 80 ° C for 30 minutes, and then pierced with bow I to obtain the final product.
  • the charging composition was as shown in Table 9.
  • the whole soybeans were washed, dipped in water, drained for 60 minutes, boiled for 4 hours, and crushed (crushed) with a food processor.
  • yeast Zygosaccharomyces rouxii (NBRC0510) in 30 ml of 10 ml YPD medium.
  • C after 24 hours of shaking culture, collected by centrifugation, and the resulting cells were washed twice with sterilized water.
  • Table 10 below shows the results of component analysis of the miso prepared with the liquid koji obtained as described above.
  • the preparation composition was as shown in Table 11.
  • the glutinous rice (Hyakumochi, 90% whitened) was washed, soaked in water for 60 minutes, drained for 30 minutes, boiled for 1 hour, and allowed to cool.
  • Table 12 shows the results of the moromi composition analysis after the ripening process.
  • Example 12 production of grain vinegar
  • the charging composition was as shown in Table 13. As the round barley (domestic 2 barley barley, 70% koji ratio), it was soaked for 60 minutes, drained for 30 minutes, and steamed for 60 minutes.
  • the yeast was shochu yeast (Kagoshima yeast), and the liquid koji produced by the method of test section 2 described in Example 5 was inoculated with 50 1 that was cultured in YPD medium at 30 ° C for 48 hours.
  • the yeast and the barley treated as described above, 90% lactic acid and water were added.
  • the fermentation conditions were 25 ° C-constant, and alcohol fermentation was performed for 10 days to obtain a liquid koji alcoholic fermentation broth.
  • the grain vinegar obtained as described above had an acidity of 6.1% and a pH of 3.1. A sensory evaluation of this grain vinegar was conducted by six specialist panels, and it was judged that the quality was sufficient for use as grain vinegar.
  • amazake was produced as follows.
  • the charging recipe is as shown in Table 14.
  • the rice was 90% polished rice (Koshihikari from Ibaraki Prefecture), washed, soaked in water for 60 minutes, drained for 30 minutes, cooked for 1 hour, and allowed to cool. Using the raw materials of the following blend, sugar washes were performed at 55 ° C for 16 hours to produce amazake.
  • Example 14 (Method for producing ethanol)
  • the charging composition is as shown in Table 15.
  • the rice was prepared by the method of Test Zone 2 described in Example 5, using 90% polished rice (Koshihikari from Ibaraki Prefecture), washed with water for 15 minutes, drained for 10 minutes, and cooked for 30 minutes. Liquid koji, lactic acid and water were added. This was inoculated with 50 1 shochu yeast (Kagoshima yeast) that was statically cultured in YPD medium at 30 ° C for 48 hours. Fermentation conditions were 25 ° C-constant, and fermentation was performed for 16 days. [Table 15]
  • the fermentation proceeded smoothly, and the alcohol content of the final mash obtained was 17.8%.
  • the mash after the fermentation obtained above is purified using a precision distillation machine (Shibata Kagaku Co., Ltd., HP
  • the alcohol content of the obtained industrial alcohol (ethanol) was 95.5%. From the above, according to the present invention, it has become apparent that industrial alcohol (ethanol) can be produced using liquid koji mold.

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Abstract

L'invention a pour objet un procédé servant à produire un koji liquide ayant des activités de glucoamylase et d'α-amylase élevées, lesquelles servent d'enzymes clés lors de la production d'aliments et boissons fermentés tels que le saké, par un procédé commode sans avoir recours à des procédures pénibles, telles que, par exemple, celles consistant à utiliser un appareil de culture spécial, à prétraiter une matière de départ ou à réguler strictement le procédé. L'invention concerne en l'occurrence un procédé servant à produire un koji liquide caractérisé en ce qu'il comprend d'effectuer la culture d'Aspergillus oryzae dans un milieu liquide contenant au moins une matière de départ pour la culture sélectionnée parmi des céréales, lesquelles sont entièrement ou partiellement recouvertes en surface du tégument, des graines et/ou des pommes de terre qui sont recouvertes en surface de la peau externe et l'amarante et/ou le quinoa n'ayant été soumis à aucun prétraitement (par exemple de broyage ou de mouture), un sel de l'acide nitrique, un sel de l'acide phosphorique et un sel de l'acide sulfurique ; un koji liquide obtenu par ce procédé ; et un procédé de production d'un aliment fermenté ou d'une boisson fermentée comprenant de produire l'aliment fermenté ou la boisson fermentée en utilisant le koji liquide tel que décrit ci-dessus.
PCT/JP2006/316173 2005-10-04 2006-08-17 Procédé servant à produire du koji liquide en utilisant aspergillus oryzae Ceased WO2007039990A1 (fr)

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US8124374B2 (en) 2005-10-12 2012-02-28 Asahi Breweries, Ltd. Method of producing recombinant protein
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US8802170B2 (en) 2004-04-09 2014-08-12 Asahi Breweries, Ltd. Method of manufacturing liquid koji
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US8715979B2 (en) 2005-10-05 2014-05-06 Asahi Breweries, Ltd. Method of producing filamentous fungus culture product
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WO2025205715A1 (fr) * 2024-03-28 2025-10-02 キッコーマン株式会社 Assaisonnement contenant du shio kôji lyophilisé et du shio kôji

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