WO2012049737A1 - Procédé de fabrication de koji liquide dont l'activité enzymique d'amilolyse et l'activité enzymique de dégradation des fibres alimentaires sont augmentées - Google Patents

Procédé de fabrication de koji liquide dont l'activité enzymique d'amilolyse et l'activité enzymique de dégradation des fibres alimentaires sont augmentées Download PDF

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WO2012049737A1
WO2012049737A1 PCT/JP2010/067894 JP2010067894W WO2012049737A1 WO 2012049737 A1 WO2012049737 A1 WO 2012049737A1 JP 2010067894 W JP2010067894 W JP 2010067894W WO 2012049737 A1 WO2012049737 A1 WO 2012049737A1
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liquid
fiber
degrading enzyme
koji
enzyme activity
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PCT/JP2010/067894
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English (en)
Japanese (ja)
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晋 舛田
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アサヒグループホールディングス株式会社
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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention relates to a method for producing liquid koji with enhanced enzyme activity, and more particularly, to a method for producing liquid koji with enhanced amylolytic enzyme activity and dietary fiber degrading enzyme activity.
  • the starch When producing alcoholic beverages from grains such as wheat, rice and rice bran, since the carbohydrates in the grains exist in the form of starch, the starch is first decomposed into sugar (ie, saccharified). In order to saccharify starch, an amylolytic enzyme such as amylase is required. As the enzyme supply source, straw and malt are used.
  • Non-Patent Document 1 enzyme activity is measured for various rice cakes in which wheat bran, rice bran, corn husk and the like are combined with filamentous fungi, Aspergillus niger and Aspergillus awamori as rice bran media. The measurement results show that these wrinkles mainly have amylolytic enzyme activity.
  • a solid koji that inoculates and inoculates filamentous fungal spores on the raw material after the treatment such as steaming, and a liquid medium is prepared by adding the raw materials and other nutrients to the water, to which the koji mold spores or There are liquid rice cakes that are inoculated with pre-cultured mycelia.
  • the liquid culture method is easy to control culture and quality control, and is a culture format suitable for efficiently producing koji.
  • liquid koji is actually used as an enzyme supply source for producing alcoholic beverages and the like because saccharification power cannot be sufficiently obtained.
  • Patent Document 1 culturing white koji mold or black koji mold using a liquid medium containing a nitrogen source such as cereal with all or part of the grain surface covered with husk and potassium nitrate, It is described that the amylolytic enzyme activity of liquid koji is enhanced.
  • the raw material for saccharification contains a large amount of fiber
  • a problem has been revealed that saccharification does not proceed easily with conventional liquid koji.
  • the reason why saccharification is difficult to proceed in a saccharified raw material containing a large amount of fiber is considered to be because the fiber remains as a solid content in the process of saccharifying starch. That is, in order to obtain a practical level of saccharification even for grains containing a large amount of fiber, it is necessary to enhance not only the starch degrading enzyme activity of liquid koji but also the dietary fiber degrading enzyme activity.
  • the present inventor solves the above-mentioned conventional problems, and the object is to enhance not only the starch degrading enzyme activity of liquid koji but also the dietary fiber degrading enzyme activity in liquid koji. .
  • the present invention includes a step of cultivating white koji mold or black koji mold using a liquid medium containing a carbon source containing a substrate containing cereal and fiber containing all or part of the grain surface covered with husk.
  • a method for producing a liquid koji with enhanced starch degrading enzyme activity and dietary fiber degrading enzyme activity is provided.
  • the starch degrading enzyme is at least acid-resistant ⁇ -amylase and glucoamylase, and the dietary fiber degrading enzyme is at least ⁇ -glucanase and xylanase.
  • the substrate containing the fiber is at least one selected from the group consisting of beet fiber and radish.
  • the substrate containing the fiber is beet fiber.
  • the white gonococcus is Aspergillus kawachi and the black gonococcus is Aspergillus awamori.
  • the grain in which the whole or part of the grain surface is covered with husks is brown rice, rice with all or part of rice husks, or barley or wheat having an unpolished to polished ratio of 92% or more. It is.
  • the present invention also provides a liquid bottle produced by any one of the methods described above.
  • the present invention also includes a step of causing the starch-degrading enzyme and the dietary fiber-degrading enzyme contained in the liquid koji to act on the fiber cereal or the fiber koji.
  • a method for saccharifying glycation is provided.
  • the fibrous cereals are wheat, barley, corn, straw, and the like, and the fibrous cereals are cassava, sweet potato, potato, and the like.
  • the amylolytic enzyme activity and the dietary fiber degrading enzyme activity are enhanced in the liquid koji. Therefore, even a saccharification raw material containing a large amount of fiber can be efficiently saccharified using a liquid koji. That is, the object of practical saccharification by liquid koji is expanded to cereals or koji that contain a large amount of fiber.
  • 3 is a graph showing ⁇ -glucanase activity of a liquid koji of the present invention produced using a substrate containing various fibers and Aspergillus kawachi. It is a graph which shows the xylanase activity about the liquid rice cake of this invention manufactured using the substrate and various Aspergillus kawachi containing various fiber. 3 is a graph showing ⁇ -glucanase activity of a liquid koji of the present invention produced using a substrate containing various fibers and Aspergillus awamori. It is a graph which shows the xylanase activity about the liquid rice cake of this invention manufactured using the substrate and various Aspergillus awamori containing various fiber.
  • 3 is a graph showing acid-resistant ⁇ -amylase activity of a liquid koji of the present invention produced using a substrate containing various fibers and Aspergillus kawachi. It is a graph which shows the glucoamylase activity about the liquid koji of this invention manufactured using the substrate and Aspergillus kawachi containing various fiber. 3 is a graph showing acid-resistant ⁇ -amylase activity of a liquid koji of the present invention produced using a substrate containing various fibers and Aspergillus awamori. It is a graph which shows the glucoamylase activity about the liquid rice cake of this invention manufactured using the board
  • the liquid medium used in the method of the present invention is a liquid in which nutrients necessary for growth and growth of white koji mold or black koji mold are dissolved or suspended in water.
  • nutrients include, for example, carbon sources, nitrogen sources, inorganic salts and the like.
  • Cereal is used as one of the carbon sources. Then, an amylolytic enzyme that degrades starch is produced. Examples of cereals include barley, wheat, rice, buckwheat, millet, millet, millet, cucumber, and corn. Cereal particles, or grains, need to be entirely or partially covered, preferably all, of the surface. The husk is a film covering the surface of the grain.
  • the surface of the grain is covered with husk, it takes time to saccharify the starch in the grain, the rate of sugar release to the culture system is suppressed, and the enzyme activity of the liquid koji is enhanced.
  • unpolished products such as brown rice, brown barley, and brown wheat, or refined products that have been refined to such an extent that the grain skin is left on the surface of the grain
  • the rice husk may be entirely or partially attached.
  • the milling ratio used in the method of the present invention is that the milling ratio is unmilled milling ratio (100% ) Or more than the ratio obtained by subtracting the grain percentage of the grain.
  • barley having a grain ratio of 7-8% and a milling ratio of 92-93% or more can be used.
  • Cereals have a content of 1 to 20% (w / vo1) in the liquid medium, preferably 8 to 10% (w / vol) when the grains are unmilled, and preferably 95%. Is used in an amount of 1 to 4% (w / vo1). If the cereal content is less than 1%, the koji mold does not grow or proliferate sufficiently, and the enzyme activity becomes insufficient. If it exceeds 20%, the viscosity of the culture solution becomes high, and the koji mold is aerobically cultured. The supply of necessary oxygen and air becomes insufficient, the oxygen concentration in the culture decreases, and the culture becomes difficult to proceed.
  • Starch contained in cereals may be gelatinized in advance before culturing.
  • the starch gelatinization method is not particularly limited, and may be performed according to a conventional method such as a steaming method or a roasting method.
  • a steaming method or a roasting method.
  • gelatinization of starch is simultaneously performed by this treatment.
  • a substrate containing fiber is also used in combination with cereals. Then, not only starch degrading enzymes but also dietary fiber degrading enzymes are produced.
  • the substrate containing fiber is preferably at least one selected from the group consisting of beet fiber and radish. The radish is preferably cut and dried. In order to produce a high amount of dietary fiber degrading enzyme, beet fiber is preferable.
  • the substrate containing fibrous material accounts for 1 to 40% (w / v), preferably 5 to 30% (w / v), more preferably 10 to 25% (w / v) of the carbon source in the liquid medium. ), And if it is less than 1%, there is no effect, and if it exceeds 40%, the activity decreases.
  • the nitrogen source is not particularly limited as long as it is a nitrogen supply source necessary for the growth and growth of Neisseria gonorrhoeae.
  • organic substances include yeast cells or processed products thereof (for example, yeast cell decomposition products, yeast extracts, etc.), and examples of inorganic substances include nitrates.
  • nitrate potassium nitrate, sodium nitrate or the like can be used, and potassium nitrate is particularly preferable.
  • the nitrogen source may be used alone or in combination of two or more organic substances and / or inorganic substances.
  • the amount of nitrogen source added is not particularly limited as long as it promotes the growth of Aspergillus, but it is 0.1 to 2% (w / vol), preferably 0.5 to 1.0% (w / Vol).
  • the amount of nitrate added as an inorganic substance is 0.05 to 2.0% (w / vol), preferably 0.1 to 2.0% (w / vol), most preferably 0.2 to 1.5%. % (W / vol).
  • the nitrogen source is added beyond the upper limit, it is not preferable because it inhibits the growth of Aspergillus. Moreover, when the addition amount is less than the lower limit, enzyme production is not promoted, which is also not preferable.
  • Yeasts used as a kind of nitrogen source in the present invention include beer yeasts, wine yeasts, whiskey yeasts, shochu yeasts, sake yeasts, baker's yeasts used in brewing processes and food production, genus Saccharomyces , Candida ( Candida) genus, Torulopsis (Torulopsis) genus, Han Zegna Supora (Hanseniaspora) genera, Hansenula (Hansenula) spp., Debaryomyces (Debaryomyces) genus Saccharomyces Maiko-flops cis (Saccharomycopsis) genus Saccharomyces Maiko death (Saccharomycodes) genus Pichia ( Pichia ) and yeasts such as Pachysolen can be mentioned.
  • yeasts can be used as a nitrogen source, but can also be used as a yeast cell decomposition product or yeast extract.
  • Yeast cell degradation products or yeast extracts are produced by self-digestion of yeast cells (method of solubilizing cells using the proteolytic enzyme inherent in yeast cells), enzyme decomposition methods (from microorganisms and plants) (Methods of solubilization by adding enzyme preparations, etc.), hot water extract method (method of soaking yeast cells in hot water for a certain period of time), acid or alkali decomposition method (various acids or alkalis) Additive solubilization method), physical crushing method (sonication, high-pressure homogenization method, crushing method by mixing and stirring solid materials such as glass beads), freeze-thawing method (freezing / thawing) For example, by crushing once or more).
  • liquid medium used in the present invention may contain sulfate and phosphate in addition to the carbon source or nitrogen source. Enzyme activity is enhanced by using these inorganic salts in combination.
  • magnesium sulfate heptahydrate for example, calcium sulfate, magnesium sulfate heptahydrate, iron sulfate heptahydrate, ammonium sulfate and the like can be used, and magnesium sulfate heptahydrate is particularly preferable.
  • phosphate potassium dihydrogen phosphate, ammonium phosphate or the like can be used, and potassium dihydrogen phosphate is 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 enzymes such as amylolytic enzymes, dietary fiber degrading enzymes, and proteolytic enzymes are selectively generated and accumulated in the koji mold culture.
  • enzymes such as amylolytic enzymes, dietary fiber degrading enzymes, and proteolytic enzymes are selectively generated and accumulated in the koji mold culture.
  • sulfate 0.01 to 0.5% (w / vo1), preferably 0.02 to 0.2% (w / vo1)
  • phosphate 0.05 to 1.0. % (W / vo1), preferably 0.1 to 0.8% (w / vol).
  • organic substances and inorganic salts other than the aforementioned nitrogen sources and inorganic salts can be added as nutrient sources as appropriate.
  • These additives are not particularly limited as long as they are generally used for culturing koji molds, but organic substances such as wheat koji, corn steep liquor, soybean koji, defatted soybeans, etc., and inorganic salts such as ammonium salt and potassium Water-soluble compounds such as salts, calcium salts and magnesium salts can be mentioned, and two or more kinds of organic substances and / or inorganic salts may be used simultaneously.
  • the amount of these additives is not particularly limited as long as it promotes the growth of Neisseria gonorrhoeae, but is about 0.1 to 5% (w / vo1) for organic substances, and 0.1 to 1% (w / vo1) for inorganic salts. It is preferable to add about vo1).
  • the liquid medium of Aspergillus obtained by mixing the above-mentioned culture raw material and nitrogen source with water may be sterilized as necessary, and the treatment method is not particularly limited.
  • a high-temperature and high-pressure sterilization method can be mentioned, which may be performed at 121 ° C. for 15 minutes.
  • Manufacture of liquid koji After cooling the sterilized liquid medium to the culture temperature, inoculate white koji mold and / or black koji mold on the liquid medium.
  • the form of the koji mold inoculated into the medium is arbitrary, and spores or hyphae can be used.
  • the amount of koji mold inoculated into the liquid medium there is no particular limitation on the amount of koji mold inoculated into the liquid medium, but about 1 ⁇ 10 4 to 1 ⁇ 10 6 spores per 1 ml of the liquid medium, and 0.1 to 10 of the preculture solution for mycelia. It is preferable to inoculate about 1%.
  • the culture temperature of Aspergillus 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 is delayed, and contamination with various bacteria is likely to occur.
  • the culture time is suitably 24 to 120 hours.
  • koji mold having an ability to produce starch-degrading enzymes such as glucoamylase, acid-resistant ⁇ -amylase and ⁇ -amylase, and dietary fiber degrading enzymes such as cellulase, ⁇ -glucosidase and xylanase are preferable.
  • starch-degrading enzymes such as glucoamylase, acid-resistant ⁇ -amylase and ⁇ -amylase
  • dietary fiber degrading enzymes such as cellulase, ⁇ -glucosidase and xylanase are preferable.
  • Aspergillus kawachii and the like are Aspergillus or aspergillus niger and Aspergillus niger and the like are Aspergillus awachi and Aspergillus niger .
  • Aspergillus oryzae Aspergillus kawachi is preferable. Aspergillus niger is preferably Aspergillus awamori. This is because by using these, starch-degrading enzymes and dietary fiber-degrading enzymes are highly produced.
  • koji molds can be used either by culturing with one type of strain or by mixed culturing with two or more types of strains of the same or different types. There is no problem with using any form of spores or hyphae obtained by preculture, but it is preferable to use hyphae because the time required for the logarithmic growth phase is shortened.
  • the culture apparatus may be any apparatus that can perform liquid culture. However, since Neisseria gonorrhoeae needs to perform aerobic culture, it needs to be performed under aerobic conditions in which oxygen and air can be supplied into the medium. Moreover, it is preferable to stir so that the raw material, oxygen, and koji mold in the medium are uniformly distributed in the apparatus during the culture.
  • the stirring conditions and the aeration amount may be any conditions as long as the culture environment can be maintained aerobically, and may be appropriately selected depending on the culture apparatus, the viscosity of the medium, and the like.
  • a liquid koji having enzyme activity such as starch-degrading enzyme that degrades starch and dietary fiber-degrading enzyme that degrades dietary fiber and hemicellulose can be obtained.
  • the liquid koji includes a liquid culture itself, a culture supernatant, a clarified liquid obtained by filtering or centrifuging the culture, a concentrate thereof, and the like. Also, dried liquid koji is equivalent to liquid koji and can be used as an enzyme source as well.
  • the liquid cake of the present invention preferably exhibits, for example, the following enzyme activity in an unconcentrated state. Each enzyme activity is measured according to the method described in the examples.
  • the acid-resistant ⁇ -amylase (ASAA) activity is 16 U / ml or more, preferably 18 U / ml or more, more preferably 20 U / ml or more, and further preferably 23 U / ml or more.
  • About glucoamylase (GA) activity it is 45 U / ml or more, Preferably it is 50 U / ml or more, More preferably, it is 60 U / ml or more, More preferably, it is 70 U / ml or more.
  • the ⁇ -glucanase (BG) activity is 0.15 U / ml or more, preferably 0.20 U / ml or more, more preferably 0.80 U / ml or more, and further preferably 1.0 U / ml or more.
  • the xylanase (XY) activity is 0.20 U / ml or more, preferably 0.28 U / ml or more, more preferably 0.30 U / ml or more, and further preferably 0.40 U / ml or more.
  • the liquid koji obtained by the production method of the present invention can be used in the same manner as solid koji as an enzyme source for producing fermented foods and drinks such as shochu, sake, soy sauce, miso, mirin and amazake.
  • liquid soot a part of the obtained liquid soot can be used as a starter in the next liquid soot production.
  • stable production is possible, and at the same time, production efficiency can be improved.
  • the liquid koji of the present invention can be used as a pharmaceutical preparation such as an enzyme preparation and a digestive agent because of its high enzyme activity.
  • 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.
  • Saccharification of saccharification raw material When the saccharification raw material is saccharified using the liquid koji of the present invention, the amylolytic enzyme and dietary fiber degrading enzyme contained in the liquid koji of the present invention are allowed to act on the saccharification raw material.
  • the saccharified raw material is pretreated as necessary, and then poured into water containing a sufficient amount of liquid soot for the saccharified raw material to be immersed, and statically maintained at a temperature suitable for the enzyme to act. Or shaken or agitated as necessary.
  • pretreatment of the saccharification raw material operations such as washing, pulverization, heating, and alkali treatment are generally performed.
  • the liquid koji of the present invention is rich in starch degrading enzymes and dietary fiber degrading enzymes. That is, this liquid koji is excellent in the decomposing power of starch contained in the saccharification raw material and also in the decomposing power of the fiber. Therefore, even when the saccharification raw material contains a large amount of fiber, the fiber does not remain as a solid content during the saccharification process, and the progress of saccharification is promoted.
  • the saccharification raw material rich in fiber which has been difficult to saccharify with conventional liquid koji, or has low saccharification efficiency, includes cereals rich in fiber or potatoes rich in fiber, specifically Are wheat, barley, corn, and straw as cereals, and cassava, sweet potato, and potato as potatoes.
  • a saccharified solution obtained by saccharifying a saccharified raw material containing a large amount of fiber is used to produce alcoholic beverages such as shochu or bioethanol by, for example, fermenting alcohol using yeast and, if necessary, distilling it. Is done.
  • Example 1 Manufacture of liquid cake Aspergillus oryzae strains, Aspergillus kawachi (NBRC4308 strain), which is a standard strain related to white mold, and Aspergillus awamori (NBRC4388 strain), which is a standard strain related to Aspergillus niger, were prepared.
  • Preculture method 100 ml of a preculture medium having a composition of 65% refined barley 8% (w / v), KNO 3 0.2% (w / v), KH 2 PO 4 0.3% (w / v), It put into the conical flask with a capacity
  • Substrate containing fiber As a substrate containing fiber, beet fiber (derived from sugar beet: manufactured by Nippon Sugar Sugar Co., Ltd.) and Japanese radish (cut dried radish) were prepared. The radish was pulverized before use.
  • starch soluble starch
  • Enzyme activity was measured using a culture supernatant obtained by centrifugation from the culture medium in which main culture was performed, as a liquid sputum sample.
  • the measuring method is as follows.
  • Acid-resistant ⁇ -amylase (ASAA) activity After 9 ml of 100 mM acetate buffer (pH 3) was added to 1 ml of the culture supernatant and acid treatment was performed at 37 ° C. for 1 hour, the measurement was performed using an ⁇ -amylase measurement kit (manufactured by Kikkoman).
  • Glucoamylase (GA) activity Measured according to the National Tax Agency prescribed analysis method. Specifically, 0.2 ml of 0.2 M acetate buffer was added to 1 ml of starch solution and preheated at 40 ° C. for 5 minutes. To this, 0.1 ml of the culture supernatant was added and reacted at 40 ° C. for 20 minutes, and 0.1 ml of 1N sodium hydroxide solution was added to stop the reaction. Thereafter, the mixture was allowed to stand for 30 minutes and neutralized by adding 0.1 ml of 1N hydrochloric acid solution. As a control, add 0.2 ml of 0.2 M acetate buffer to 1 ml of starch solution, preheat at 40 ° C.
  • the glucoamylase activity was expressed with 1 unit of activity to produce 1 mg of glucose from soluble starch at 40 ° C. for 60 minutes.
  • ⁇ -glucanase activity was determined by measuring the absorbance of a stained fragment produced by enzymatic degradation using ⁇ -glucan labeled with a dye, using a ⁇ -glucanase measurement kit manufactured by Megazyme. Specifically, 0.5 ml of the culture supernatant was added to 0.5 ml of the azo barley glucan substrate solution, and the enzyme reaction was carried out at 40 ° C. for exactly 10 minutes, and then the stop solution [4% sodium acetate, 0% .4% zinc acetate and 80% methyl cellosolve (pH 5)] 3.0 ml was added and left for 5 minutes to stop the reaction. Subsequently, after centrifugation, the absorbance of the supernatant was measured at 590 nm.
  • 1 unit of ⁇ -glucanase activity was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 ⁇ mol of glucose per minute under the reaction conditions of 40 ° C. and 10 minutes.
  • Xylanase (XY) activity The xylanase activity was determined by measuring the absorbance of a stained fragment produced by enzymatic degradation using azo-xylan as a substrate, using a xylanase measurement kit manufactured by Megazyme. More specifically, 0.5 ml of the culture supernatant was added to 0.5 ml of 1% azo-xylan substrate solution (manufactured by Megazyme), and the enzyme reaction was performed at 40 ° C. for exactly 10 minutes. [Ethanol (95% v / v)] 2.5 ml was added and mixed well to stop the reaction. Subsequently, after centrifugation, the absorbance of the supernatant was measured at 590 nm.
  • 1 unit of xylanase activity was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 ⁇ mol of glucose per minute under the reaction conditions of 40 ° C. and 10 minutes.
  • FIG. 1 and FIG. 2 show BG activity (U / ml) and XY activity (U / ml) for a liquid sputum sample using Aspergillus kawachi.
  • FIG. 3 and FIG. 4 show the BG activity (U / ml) and XY activity (U / ml) of the liquid sputum sample using Aspergillus awamori.
  • BG activity and XY activity which are dietary fiber degrading enzyme activities, were increased in both liquid aspergillus samples using Aspergillus kawachi and Aspergillus awamori.
  • ASAA activity (U / ml) and GA activity (U / ml) for the liquid sputum sample using Aspergillus kawachi are shown in FIG. 5 and FIG.
  • ASAA activity (U / ml) and GA activity (U / ml) about the liquid sputum sample using Aspergillus awamori are shown in FIG. 7 and FIG.
  • ASAA activity and GA activity which are amylolytic enzyme activities, were increased in both liquid aspergillus samples using Aspergillus kawachi and Aspergillus awamori.
  • FIG. 9 shows the results for a liquid koji sample using Aspergillus kawachi as a saccharification ratio, and the results for a liquid koji sample using Aspergillus awamori. As shown in FIG.
  • FIG. 11 shows the results for the liquid koji sample using Aspergillus kawachi as the saccharification rate, and the results for the liquid koji sample using Aspergillus awamori as the saccharification rate with respect to the total starch amount in 1.0 g of cassava. As shown in FIG.

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Abstract

L'objectif de l'invention est non seulement d'augmenter l'activité enzymique d'amilolyse mais aussi l'activité enzymique de dégradation des fibres alimentaires, dans un koji liquide. Dans cet objectif, l'invention fournit un procédé de fabrication de koji liquide dont l'activité enzymique d'amilolyse et l'activité enzymique de dégradation des fibres alimentaires sont augmentées, et qui comporte un processus de culture de moisissures de koji blanc et de koji noir, à l'aide d'un milieu de culture liquide comprenant une source de carbone qui comprend un substrat contenant des fibres alimentaires et des céréales dont l'ensemble ou une partie des grains est recouvert d'une enveloppe.
PCT/JP2010/067894 2010-10-12 2010-10-12 Procédé de fabrication de koji liquide dont l'activité enzymique d'amilolyse et l'activité enzymique de dégradation des fibres alimentaires sont augmentées WO2012049737A1 (fr)

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CN103773648A (zh) * 2012-10-18 2014-05-07 柳州市京阳节能科技研发有限公司 液态生料速效免蒸馏酿酒

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FR2302336A1 (fr) * 1975-02-26 1976-09-24 Baxter Laboratories Inc Procede pour la fabrication d'un melange d'enzymes
JPS5739773A (en) * 1980-08-21 1982-03-05 Naganoken Miso Kogyo Kyodo Kumiai Preparation of "koji"
JPS60237988A (ja) * 1984-05-10 1985-11-26 Yonemi Tanaka 製麹方法
JP2002543773A (ja) * 1999-04-14 2002-12-24 アンスティテュ ナショナル ド ラ ルシェルシュ アグロノミク（イーエヌエルアー） アスペルギルス・ニガー培養物を得るための方法およびフェルラ酸とバニリン酸を生産するためのそれらの使用
JP2003265165A (ja) * 2002-03-13 2003-09-24 Asahi Kyowa Shurui Seizo Kk 麹菌の培養方法
JP2005318886A (ja) * 2004-04-09 2005-11-17 Asahi Breweries Ltd 液体麹の製造方法
WO2007034670A1 (fr) * 2005-09-20 2007-03-29 Asahi Breweries, Ltd. Procédé pour la production de koji liquide ayant une enzyme digestive de fibre végétale amelioree, koji liquide produit par le procédé et utilisation du koji liquide
JP2007125002A (ja) * 2005-07-22 2007-05-24 Asahi Breweries Ltd 液体麹の製造方法
JP2009273472A (ja) * 2005-10-05 2009-11-26 Asahi Breweries Ltd 糸状菌培養物の製造方法
WO2010070146A1 (fr) * 2008-12-19 2010-06-24 Danisco A/S Procédé pour la production d'un produit de type enzyme

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Publication number Priority date Publication date Assignee Title
FR2302336A1 (fr) * 1975-02-26 1976-09-24 Baxter Laboratories Inc Procede pour la fabrication d'un melange d'enzymes
JPS5739773A (en) * 1980-08-21 1982-03-05 Naganoken Miso Kogyo Kyodo Kumiai Preparation of "koji"
JPS60237988A (ja) * 1984-05-10 1985-11-26 Yonemi Tanaka 製麹方法
JP2002543773A (ja) * 1999-04-14 2002-12-24 アンスティテュ ナショナル ド ラ ルシェルシュ アグロノミク（イーエヌエルアー） アスペルギルス・ニガー培養物を得るための方法およびフェルラ酸とバニリン酸を生産するためのそれらの使用
JP2003265165A (ja) * 2002-03-13 2003-09-24 Asahi Kyowa Shurui Seizo Kk 麹菌の培養方法
JP2005318886A (ja) * 2004-04-09 2005-11-17 Asahi Breweries Ltd 液体麹の製造方法
JP2007125002A (ja) * 2005-07-22 2007-05-24 Asahi Breweries Ltd 液体麹の製造方法
WO2007034670A1 (fr) * 2005-09-20 2007-03-29 Asahi Breweries, Ltd. Procédé pour la production de koji liquide ayant une enzyme digestive de fibre végétale amelioree, koji liquide produit par le procédé et utilisation du koji liquide
JP2009273472A (ja) * 2005-10-05 2009-11-26 Asahi Breweries Ltd 糸状菌培養物の製造方法
WO2010070146A1 (fr) * 2008-12-19 2010-06-24 Danisco A/S Procédé pour la production d'un produit de type enzyme

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CAI J.M. ET AL.: "Production, properties, and application of xylanase from Aspergillus niger A3", ANN. NY ACAD. SCI., vol. 864, 1998, pages 214 - 218 *
LEVASSUR A. ET AL.: "Overproduction and characterization of xylanase B from Aspergillus niger", CAN. J. MICROBIOL., vol. 51, no. 2, 2005, pages 177 - 183 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773648A (zh) * 2012-10-18 2014-05-07 柳州市京阳节能科技研发有限公司 液态生料速效免蒸馏酿酒

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