WO2007039990A1

WO2007039990A1 - Process for producing liquid koji by using aspergillus oryzae

Info

Publication number: WO2007039990A1
Application number: PCT/JP2006/316173
Authority: WO
Inventors: Toshikazu Sugimoto; Hiroshi Shoji
Original assignee: Asahi Breweries, Ltd.
Priority date: 2005-10-04
Filing date: 2006-08-17
Publication date: 2007-04-12
Also published as: TW200745331A

Abstract

It is intended to provide a process for producing a liquid koji having high activities of glucoamylase and α-amylase, which serve as the key enzymes in producing fermented foods and drinks such as sake, by a convenient method without resorting to any troublesome procedures of, for example, such as using a special culture apparatus, pretreating a starting material or strictly controlling. Namely, a process for producing a liquid koji characterized by comprising culturing Aspergillus oryzae in a liquid medium containing at least one starting material for the culture selected from among cereals the which are entirely or partly surface-coated with the seed coat, beans and/or potatoes which are surface-coated with the outer skin, and amaranthus and/or quinua having been subjected to no pretreatment (for example, grinding or milling), a nitric acid salt, a phosphoric acid salt and a sulfuric acid salt; a liquid koji obtained by this process; and a method of producing a fermented food or drink wherein the fermented food or drink is produced by using the liquid koji as described above.

Description

Specification

A method for producing liquid koji using yellow koji mold

Technical field

[0001] 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.

Background art

[0002] In conventional production of fermented foods and beverages 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.

[0003] On the other hand, 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.

However, it is well known that this liquid koji cannot sufficiently obtain the enzyme activity necessary for the production of sake and the like, and so far, there have been few examples used in actual production.

[0004] In particular, regarding the koji mold represented by Aspergillus oryzae used for the production of sake and various brewing seasonings, it is known that the activity of dalcamylase in the liquid culture is extremely low. (See Non-Patent Document 1). Dalcore mirase is one of the key enzymes in the production of fermented foods and beverages that breaks down starch from raw materials into glucose. Therefore, there are almost no reports of liquid koji production methods using jaundice.

[0005] Patent Document 1 discloses a high production method of glucoamylase by culturing yellow koji mold while applying stress to mycelial growth. However, in this method, 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! ,.

[0006] 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. Has found that it is possible to produce a liquid soot with a patent (patent application 2004-35066). 1 specification, Japanese Patent Application No. 2004-352320 specification).

However, the enzyme production behavior when the koji mold is cultured by this production method is unknown, and no high production method of darcoamylase and a-amylase, which are key enzymes in the production of sake, etc. has been found.

[0007] 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

Disclosure of the invention

Problems to be solved by the invention

[0008] 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.

Means for solving the problem

[0009] As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that cereals in which all or part of the surface is covered with at least husks; Amaranthus and Z or Kinuaka without pre-treatment such as comminution or grinding are also selected. Culture of Staphylococcus aureus in a liquid medium containing at least one culture raw material, nitrate, phosphate, and sulfate. Only when this was done, it was revealed that dalcamylase and α-amylase were highly expressed simultaneously.

It was also found that by optimizing the addition amount of inorganic salts and the culture temperature, it is possible to produce a liquid koji having sufficient enzyme activity to produce a fermented food or drink.

Based on these findings, the inventors have completed the present invention.

[0010] That is, 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 method for producing a liquid soot according to claim 1.

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).

[0011] In the present invention according to claim 5, 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 method for producing a liquid soak according to claim 1, wherein

The present invention according to claim 6 is the method for producing a liquid koji according to claim 1, wherein at least darcoamylase and α-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.

[0012] 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.

[0013] 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.

[0014] 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 invention's effect

[0015] According to the present invention, by cultivating yellow koji molds using a liquid medium containing a predetermined culture raw material and inorganic salts, it is possible to perform pretreatment of the raw material, strict control, etc. without using a special culture apparatus. Even without complicated operations, it is possible to produce a liquid koji with high production of dalcamylase and amylase at the same time. Liquid culture allows stricter culture control than solid culture, so that stable liquid koji can be produced inexpensively and efficiently.

[0016] 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. In addition, 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.

Furthermore, 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.

[0017] Since 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.

[0018] Also, when producing fermented foods and drinks using the liquid koji produced according to the present invention, unlike the conventional manufacturing method using solid koji, it is possible to carry out all the steps in the liquid phase. Therefore, it is possible to provide a fermented food and beverage production system that is more efficient and stable than conventional ones.

Brief Description of Drawings

[0019] 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, and (b) is the result of measurement of α-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, and (b) is the result of measurement of α-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, and (b) is the result of measurement of α-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, and (b) is the measurement result of α-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 α-amylase.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] The present invention will be specifically described below.

In the method for producing liquid koji in the present invention, 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. In other words, 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 α-amylase are simultaneously generated and accumulated in a well-balanced manner.

[0021] In the present invention, as 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. In the case of rice, not only brown rice but also rice husks may be attached, or rice husks may be partly attached.

For example, if the raw material for cultivation is barley, 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.

[0022] Here, the milling rate refers to the proportion of cereals that remain after milling the cereals. For example, the milling rate of 90% means that 10% of the skin of the surface layer of the cereals is scraped off. Further, in the present invention, 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.

[0023] In the present invention, 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.

In the present invention, heating or freezing treatment can be carried out while keeping the outer shell of beans or moss as the culture raw material.

[0024] In the present invention, 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, on the other hand, is an annual plant of the Agatha family and is cultivated mainly in the highlands of southern Peru and the Andes Mountains of western Bolivia, 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.

[0025] 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 α-amylase are selectively produced and accumulated during the culture of jaundice.

For example, 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. In addition, when unpolished barley is used, it is more preferable that it is prepared in a liquid medium supplemented with 8 to 10% (w / vol), and when 95% polished barley is used as a raw material. Is prepared in liquid medium supplemented with 1-4% (w / vol). Next, 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).

[0026] 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.

[0027] In addition, for example, when amaranth is 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). On the other hand, 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. Prepared in liquid medium supplemented with 2% (w / vol) to 4% (w / vol).

[0028] As described above, 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.

If 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.

[0029] 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.

[0030] In addition to the above-mentioned culture raw materials, it is necessary to add nitrate, phosphate and sulfate to the liquid medium used in the present invention as nutrient sources. These inorganic salts are not particularly limited as long as they are usually used for cultivation of filamentous fungi.

For example, sodium nitrate or potassium nitrate can be used as the nitrate, and sodium nitrate is particularly preferable. As the phosphate, potassium dihydrogen phosphate, ammonium phosphate and the like can be used, and potassium dihydrogen phosphate is particularly preferable. As the 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 _α- amylase are selectively generated and accumulated during the culture of jaundice. In concrete terms, in the case of nitrate from 0.1 to 2.0%, preferably 0.2 to 1.5%, if ί 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.

[0031] To the liquid medium, organic substances other than the above-mentioned inorganic salts, inorganic salts, and the like can be appropriately added as a nutrient source. These 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 However, it is preferable to add about 0.1 to 5% (wZvol) as an organic substance and about 0.1 to 1% (wZvol) as an inorganic salt.

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.

[0032] After the sterilized liquid medium is cooled to the culture temperature, the koji mold is inoculated into the liquid medium. 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. For example, Aspergillus oryzae Aspergillus sojae ) And the like. Moreover, the form of the yellow koji mold inoculated on the medium is arbitrary, and spores or mycelia can be used.

[0033] 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%.

[0034] 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.

In addition, 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 preferred culture temperature conditions described above can be combined with any aspect of the method of the present invention.

[0035] 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.

[0036] By culturing by the above-mentioned culture method, darcoamylase and (X amylase are simultaneously produced with good tolerance, resulting in a liquid koji having enzyme activity that can be used for the production of fermented foods and drinks such as sake. In addition to the culture itself, the liquid koji includes a culture solution obtained by centrifuging the culture, a concentrate thereof, or a dried product thereof.

[0037] Thus, enzyme production by jaundice can be increased by the culture method described above.

Therefore, the method for producing an enzyme according to claim 17 is the same as the method for producing a liquid koji described above.

[0038] 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. For example, 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. When producing 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.

Further, a part of the obtained liquid soot can be used as a starter in the next liquid soot production. Thus, by continuously producing the liquid soot, stable production can be achieved, and at the same time, production efficiency can be improved.

[0039] Further, when producing fermented foods and drinks such as sake using the above-described liquid koji, all the steps can be performed in a liquid phase. As a method for producing fermented foods and beverages in which the entire process is performed in a liquid phase, for example, when producing sake, a refined rice or its crushed material is used as a raw material, and the raw material is heated at a high temperature of about 80 ° C. A method of producing a mash that has been subjected to alcohol fermentation by adding the above-mentioned liquid cake and yeast after removing the residue by filtration or the like. Can be mentioned. [0040] Due to its high enzyme activity, 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. In this case, 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.

[0041] The liquid koji obtained by the method of the present invention can be used for production of ethanol by fermentation.

As a method for producing ethanol by fermentation using the liquid koji obtained by the method of the present invention, 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.

That is, first, 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.

After charging, after steaming at a low temperature, it can be fermented at a temperature of about 20-30 ° C. After the primary charging, the secondary charging can be performed.

Industrial alcohol (ethanol) can be produced by distilling the mash after fermentation to remove impurities and concentrating.

[0043] In addition, by applying the method of the present invention to a yellow koji mold into which a foreign gene encoding a target heterologous protein has been introduced by a genetic engineering technique, the protein can be produced in a high yield in a koji mold culture. It is possible to make it.

Example

Hereinafter, the present invention will be described more specifically with reference to examples and the like, but the present invention is not limited to these examples and the like.

Example 1 (Effect of carbon source on enzyme productivity in liquid koji production of yellow koji mold)

1. How to make liquid rice cake: Make liquid rice cake by the following method and measure their enzyme activity.

That is, various carbon sources 2.0% (wZvol), sodium nitrate 0.3% (wZvol), salt Hum 0.2% (wZvol), potassium dihydrogen phosphate 0.1% (wZvol), magnesium sulfate 7 hydrate 0.05% (wZvol), iron sulfate heptahydrate 0.02% (wZvol) Then, 100 ml of a liquid medium containing water and water was placed in an Erlenmeyer flask with a 500π baffle and sterilized by autoclaving at 121 ° C for 15 minutes.

As the carbon source, starch (soluble, manufactured by Wako Pure Chemical Industries), dextrin (manufactured by Wako Pure Chemical Industries), 65% refined barley (Australian schooner), and 98% refined barley (Australian stirrer) were used. A test using 98% milled barley crushed with a mill (ground product) was also conducted in the same manner.

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.

[0046] 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).

[0047] 3. Results; The results of enzyme activity measurement are shown in FIG. Dalcoamylase was prominently produced only in the 98% refined barley test area, while production was significantly suppressed in the 98% refined wheat (ground product) test area using the raw material obtained by pulverizing this (Figure 1 (a)). .

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.

a Although the production behavior of 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)).

In this way, by using cereals whose surface is covered with husks such as 98% polished wheat, it is possible to simultaneously produce a high production of enzymes necessary for the production of fermented foods and beverages such as darcoamylase and (X-amylase). It was shown to be possible.

[0048] Example 2 (Effects of various salts added in liquid koji production of yellow koji mold)

1. Liquid rice cake production method: Liquid rice cake is produced by the following method and the enzyme activity is measured. Set.

98% refined barley (Australian schooner) 2.0% (wZvol), sodium nitrate 0.3% (wZvol), potassium chloride 0.2% (wZvol), potassium dihydrogen phosphate 0.1% ( w / vol), magnesium sulfate heptahydrate 0.05% (w / vol), iron sulfate heptahydrate 0.02% (w / vol) and water 100ml liquid medium 500ml Erlenmeyer flask with baffle And autoclaved at 121 ° C for 15 minutes to make a control. Furthermore, as shown in Table 1, except that each salt was removed, a medium was prepared in the same manner as described above and designated Test Groups 1-6. The seven types of media thus prepared were inoculated with Aspergillus oryzae RIB40 at 1 × 10 ⁶ cells / m 1 and cultured with shaking at 30 ° C. for 72 hours at lOOrpm.

[table 1]

[0050] 2. Measurement method: After completion of the culture, darcoamylase activity (GA) in each culture supernatant and

a Measured for amylase activity (AA). 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).

[0051] 3. Results: The results of measuring the enzyme activity in the culture supernatant are shown in FIG.

Dalcore mirase was not produced in Test Zone 1 without sodium nitrate, Test Zone 3 without potassium dihydrogen phosphate, and Test Zone 6 without both magnesium sulfate and iron sulfate ( Figure 2 (a)). On the other hand, almost no amylase was produced in test group 1, and it was confirmed that the production was suppressed in test groups 3, 5, and 6 (Fig. 2 (b)). Thus, it has been clarified that the enzyme productivity of liquid koji molds is greatly influenced by the salts contained in the medium. That is, to produce both enzymes at the same time with high production

It has been shown that cereals with a surface covered with husk, such as 98% polished wheat, sodium nitrate, dibasic hydrogen phosphate, magnesium sulfate or iron sulfate are essential.

[0052] Example 3 (Effect of sulfate on enzyme production in liquid koji production of yellow koji mold)

In 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.

[0053] 1. Method for producing liquid rice cake: Liquid rice cake is produced by the following method, and their enzyme activity is measured.

That is, water, 98% refined barley (Australian schooner) 2.0% (wZvol), potassium nitrate 0.2% (wZvol), potassium dihydrogen phosphate 0.3% (wZvol), magnesium sulfate heptahydrate 0.05% (wZvol) or magnesium chloride hexahydrate 0.041% (wZvol) containing 3 test sections of liquid medium as shown in Table 2 1 After preparing OOml, place it in an Erlenmeyer flask with 500ml baffle, Autoclaved at 121 ° C for 15 minutes. In addition, the amount of magnesium chloride hexahydrate added is calculated as the molar concentration equivalent to 0.05% magnesium sulfate heptahydrate, 0.05 mM, so that the magnesium concentration in each medium becomes equal. Blended into

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.

[0054] [Table 2] Medium composition

Polished barley _{_{_{■ 3 KH 2 P 0 4 MgS0}}} 4 - 7H 2 0 M gCl 2 - 6H 2 0 control group 2.0¾ 0.2% 0.3% - -

2.0? I 0.2% 0.3% 0.05%

Test Zone 2 2.0¾ 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).

[0056] 3. Results; The results of enzyme activity measurement are shown in FIG. Dalcomillase activity was significantly increased only in test group 1 where the medium contained magnesium sulfate. On the other hand, no increase in activity was observed in Test Zone 2, which is a salted and magnesium-added Caro group (Fig. 3 (a)). a Although amylase showed slightly different enzyme production behavior, it was most highly produced in Test Zone 1 containing magnesium sulfate (Fig. 3 (b)).

From the above results, it was suggested that the addition effect of magnesium sulfate lies in the main force of the sulfate radical.

[0057] Example 4 (Influence of added amount of salts)

In Examples 2 and 3, it was confirmed that the inorganic salts added to the medium had a great effect on the enzyme productivity in the production of yellow koji liquid koji. Therefore, we examined whether enzyme productivity could be enhanced by increasing the amount of inorganic salts added.

[0058] 1. 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 500π 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.

[0059] [Table 3] Medium composition

_{_{_{Na N0 3 KCI KH 2 P0 4}}} MgS 0 4 - 7H 2 0 FeS0 4 - 7H 2 0 control group 2.0% 0.3¾ 0.2% 0.1 ¾ 0.05¾ 0.02¾ test group 1 2.0% 0.6¾ 0.4% 0.2% 0.10% 0.04% Test Zone 2 2,0% 1.2¾ 0.8% 0.4¾ 0.20¾ 0.08¾

[0060] 2. Measurement method: After completion of the culture, darcoamylase activity (GA) and a-amylase activity (AA) in each culture supernatant were measured. The measurement of darcoamylase activity (GA) was performed using a sugar squid fraction determination kit (manufactured by Kikkoman), and a monoamylase activity was performed using an ex-amylase measurement kit (manufactured by Kikkoman).

[0061] 3. Results; FIG. 4 shows the measurement results of enzyme activity in each test group.

In 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) ).

Example 5 (Influence of culture temperature)

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.

[0063] 1. Method for producing liquid koji; 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.

98% refined barley (Australian schooner) 2.0% (wZvol), sodium nitrate 1.2% (wZvol), potassium chloride 0.8% (wZvol), potassium dihydrogen phosphate 0.4% ( w / vol), magnesium sulfate heptahydrate 0.2% (w / vol), iron sulphate heptahydrate 0.08% (w / vol), and 100 ml of liquid medium containing water in a 500π baffle Erlenmeyer flask The paste was autoclaved at 121 ° C for 15 minutes. Subsequently, the medium was inoculated with Aspergillus orvzae RIB40 at 1 × 10 ⁶ cells / ml.

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.

[0064] 2. Measurement method: After completion of the culture, darcoamylase activity (GA) and a-amylase activity (AA) in each culture supernatant were measured. The measurement of darcoamylase activity (GA) was performed using a sugar squid fraction determination kit (manufactured by Kikkoman), and a monoamylase activity was performed using an ex-amylase measurement kit (manufactured by Kikkoman).

[0065] 3. Results; FIG. 5 shows the results of enzyme activity measurement in each test group.

In the test group 1 where the culture temperature was 37 ° C—constant, it was confirmed that the enzyme productivity decreased slightly, but in the test group 2 where the culture temperature was shifted from 30 ° C to 37 ° C, the dulcore amylase ( Both enzyme activities of Fig. 5 (a)) and α-amylase (Fig. 5 (b)) were enhanced.

In 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.

In addition, 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.

[0066] Example 6 (Manufacture of dried liquid rice cake)

(I) Manufacture of dried liquid rice cake

After pre-freezing 200 ml of liquid soot produced by the method of Test Zone 2 described in Example 5 at 30 ° C for 2 hours, drying it at 25 ° C and a vacuum degree of 0.5 Torr for 24 hours. 2. Dry product (liquid 麹 vacuum freeze-dried product) 2.5 g was obtained.

[0067] (II) Measurement of enzyme activity

For the liquid koji produced by the method of test section 2 described in Example 5 and not subjected to freeze-drying treatment and the dried liquid koji obtained in the above (I), dalcoamylase (GA) and a— Amylase (AA) activity was measured.

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.

[0068] 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

As a result, enzyme inactivation hardly occurred even when freeze-dried, and it was shown that the dried liquid koji product can be used sufficiently for the production of fermented foods and beverages.

[0069] [Table 4]

Example 7 (Production Method of Shochu)

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.

[0071] [Table 5]

Fermentation proceeded smoothly, and the alcohol content of the final mash (shochu mash) obtained was 17.6%.

As a result of sensory evaluation of shochu raw sake produced by distilling this shochu moromi by vacuum distillation, the expert panel commented that `` clean and nimble '', and that rice shochu with a clean flavor can be obtained. all right.

From the above results, it was shown that shochu can be produced using liquid soot. [0073] Example 8 (Production of sake)

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. Three days later, 750 g of 90% polished rice (Koshihikari from Ibaraki Prefecture) cooked to moromi was added, followed by fermentation at 25 ° C for 15 days to obtain the final moromi. The analysis values of the final moromi are shown in Table 6 below.

[0074] [Table 6]

[0075] The final mash obtained above was filtered to obtain sake. As a result of a sensory evaluation with 10 panelists, the taste was rich and tasted as a fruity aroma.

From the above, it has become clear that it is possible to produce sake using liquid koji by the method of the present invention.

[0076] Example 9 (Production of soy sauce)

Preparation: 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.

[0077] [Table 7] Raw material

Whole soybean 350 (g)

Wheat 350 (g)

210 (g)

Liquid bottle 700 (ml)

[0078] 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.

The analysis values of the final moromi obtained are shown in Table 8 below.

[0079] [Table 8]

* Soluble solids other than salt

[0080] Filtration; The final mash obtained was squeezed with a nylon filter cloth.

Post-treatment: Fired at 80 ° C for 30 minutes, and then pierced with bow I to obtain the final product.

A sensory evaluation of the soy sauce obtained as described above was conducted by six panelists specializing in alcoholic beverages.

From the above, according to the method of the present invention, it has been found that it is possible to produce soy sauce using liquid rice cake.

[0081] Example 10 (Production of miso)

Preparation 'ripening;

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. For 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. To the liquid koji produced by the method of Test Section 2 described in Example 5, Yeast, and Whole soybeans treated as described above were added. Fermentation conditions were 25 ° C-constant, and fermentation and aging were performed for 6 months to obtain miso.

[0082] [Table 9]

[0083] Table 10 below shows the results of component analysis of the miso prepared with the liquid koji obtained as described above.

[0084] [Table 10]

[0085] Further, sensory evaluation of the obtained barley miso was conducted by six specialist panels, and it was determined that the quality was sufficient for use as miso.

From the above, according to the present invention, it became clear that miso can be produced using liquid koji.

[0086] Example 11 (Production of mirin)

Preparation: 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. Steamed glutinous rice, 45% raw alcohol, and liquid rice cake produced by the method of Test Zone 2 described in Example 5 were mixed.

[0087] [Table 11]

[0088] Saccharification and maturation conditions; left at 25 ° C for 2 months. Filtration: Squeezed with a nylon filter cloth.

Post-processing; dragging and final product.

Table 12 shows the results of the moromi composition analysis after the ripening process.

[Table 12]

[0090] When the sensory evaluation of mirin prepared in the above manner was conducted by 6 panelists specializing in alcoholic beverages, the sweetness and umami taste were good, and it was determined that the quality was sufficient for use as mirin.

From the above, it has become clear that it is possible to produce mirin using liquid soot by the method of the present invention.

[0091] Example 12 (production of grain vinegar)

(I) Alcohol fermentation using liquid koji

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.

[0092] [Table 13]

[0093] (II) Using liquid koji alcoholic fermented liquid V, production of cereal vinegar (1) Acetic acid bacteria used: Acetobacter aceti subsp. Aceti (NBRC3284) was used. Acetic acid bacteria were cultured in normal broth + 1% glucose medium.

(2) Preparation: Centrifuge the liquid koji fermented solution obtained in (I) above, add lml of the acetic acid bacteria culture solution obtained in (1) above to the supernatant, and incubate at 30 ° C for 3 months did.

(3) Lowering; After culturing, the fungus on the surface was removed and centrifuged to obtain the final product. 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.

Example 13 (Production of Amazake)

Using the liquid koji produced by the method of Test Zone 2 described in Example 5, 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.

[0095] [Table 14]

[0096] The Brix of the obtained liquor charged with liquid koji was 18.5.

In addition, a sensory evaluation of the resulting liquor prepared with a liquid koji was conducted by six liquor specialist panels.

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

— 1000T special model) was used for continuous distillation to recover industrial ethanol (ethanol).

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.

Claims

The scope of the claims

[I] A method for producing a liquid koji, wherein the whole or part of the surface is covered with at least husk; beans and Z or moss with the hull covered with surface; before pulverization or grinding Untreated ス Amaranthus and Z or quinua force A liquid cocoon characterized by culturing yellow koji molds in a liquid medium containing at least one selected culture raw material, nitrate, phosphate, and sulfate. Manufacturing method.

[2] The method for producing a liquid koji according to claim 1, wherein the liquid medium contains nitrate in a concentration of 0.1 to 2.0% (w / v).

[3] 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% (w / v).

[4] The method for producing a liquid koji according to claim 1, wherein the liquid medium contains a sulfate in a concentration of 0.01 to 0.5% (w / v).

[5] The culture temperature when cultivating yellow koji molds in a liquid medium is 25 to 35 ° C by 12 to 36 hours after the start of cultivation, and 35 to 45 ° C thereafter. Item 2. A method for producing a liquid bag according to Item 1.

[6] The method for producing a liquid koji according to claim 1, wherein at least darcore amylase and a amylase are simultaneously produced and accumulated in the liquid koji.

[7] The method for producing a liquid koji according to claim 1, wherein the enzyme activity of the liquid koji is adjusted by suppressing the release rate of the sugar derived from starch in the culture raw material to the culture system.

[8] A method for producing a fermented food or drink using the liquid koji obtained by the production method according to [1] or [1].

9. 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 amazake.

[10] The method for producing a fermented food or drink according to claim 8 or 9, wherein all steps are performed in a liquid phase.

[II] A liquid koji obtained by the production method according to any one of claims 1 to 7.

[12] A dried liquid koji product obtained by drying the liquid koji according to claim 11.

[13] A concentrated liquid koji product obtained by concentrating the liquid koji according to claim 11.

[14] A method for producing an enzyme preparation using the liquid koji according to claim 11.

[15] A method for producing an enzyme preparation using the dried liquid koji according to claim 12.

[16] A method for producing ethanol using the liquid soot according to [11].

[17] Cereals with at least part of the surface covered with husks; beans and Z or moss with outer husks covered; ； Amaranthus and Z or without pretreatment such as grinding or grinding A method for producing an enzyme, comprising culturing yellow koji molds in a liquid medium containing at least one culture raw material selected from quinua, nitrate, phosphate, and sulfate.