JP4068649B2 - Method for producing liquid koji using yellow koji mold - Google Patents

Description

  The present invention relates to a method for producing a liquid koji using yellow koji mold, particularly a liquid koji having an enzyme activity necessary for producing a fermented food or drink such as sake.

  In the conventional production of fermented foods and drinks such as sake, solid koji made by growing koji molds on the surface of raw materials such as cereals has been used. Solid koji is obtained by a traditional production method, but is not suitable for large-scale production because it is a special culture form called solid culture.

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.

  In particular, regarding yellow koji molds represented by Aspergillus oryzae used for the production of sake and various brewing seasonings, it is known that the activity of glucoamylase in the liquid culture is extremely low (non- Patent Document 1). Glucoamylase is one of the key enzymes in the production of fermented foods and beverages that decomposes starch derived from raw materials into glucose. Therefore, there are almost no reports on the method for producing liquid koji using jaundice.

  Patent Document 1 discloses a high production method of glucoamylase by culturing yellow koji mold while applying stress to the growth of mycelia. However, in this method, gonococci are cultured on a porous membrane or in a entrapping immobilization agent having voids to express a novel gene glaB encoding glucoamylase to enhance the enzyme activity. A simple culture apparatus is necessary, and it is difficult to say that it is a simple method.

The present inventors have cultivated white koji mold or black koji mold in a liquid medium containing cereals and the like whose surface is covered with husk as a culture raw material, thereby having a liquid koji having sufficient enzyme activity necessary for the production of shochu and the like. Has already been filed (see Japanese Patent Application Nos. 2004-350661 and 2004-352320).
However, the enzyme production behavior when yellow koji molds are cultured by this production method is unknown, and no high production method of glucoamylase or α-amylase, which are key enzymes in the production of sake or the like, has been found.

Ishida H. et al: J. Ferment. Bioeng., 86, 301-307 (1998) Japanese Patent Laid-Open No. 11-225746

  The purpose of the present invention is to produce fermented foods and beverages such as sake by liquid culture of yellow koji molds by a simple method that does not require complicated operations such as special culture equipment, raw material pretreatment, and strict control. It is to provide a method for producing a liquid koji having high activities of glucoamylase and α-amylase, which are key enzymes of the present invention.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the whole or a part of the surface is covered with at least husks; beans and / or moss with the husks covered with hulls; Only when the koji mold is cultured in a liquid medium containing at least one culture raw material selected from amaranthus and / or quinoa without pretreatment such as crushing and crushing, nitrate, phosphate, and sulfate, It was revealed that glucoamylase and α-amylase were highly expressed simultaneously.
It has also been 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.

That is, the present invention according to claim 1 is a method for producing a liquid koji, wherein the whole surface is covered with cereals (excluding pulverized matter) , sodium nitrate and potassium nitrate, or 1 type selected from 2 types of nitrates, 1 type or 2 types of phosphates selected from potassium dihydrogen phosphate and ammonium phosphate, and magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate A method for producing liquid koji characterized by culturing yellow koji molds in a liquid medium containing sulfates of seeds or three kinds .
In the present invention according to claim 2, the liquid medium contains one or two kinds of nitrates selected from sodium nitrate and potassium nitrate at a concentration of 0.1 to 2.0% (w / v). The manufacturing method of the liquid soot described in 1.
The present invention according to claim 3 is characterized in that the liquid medium contains 0.05 to 1.0% (w / v) of phosphate that is one or two selected from potassium dihydrogen phosphate and ammonium phosphate. It is a manufacturing method of the liquid soot of Claim 1 contained in a density | concentration.
The present invention according to claim 4 is characterized in that the liquid medium contains 0.01 to 0.5% ( 1 to 3 % ) sulfate selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate. It is a manufacturing method of the liquid soot of Claim 1 contained in the density | concentration of w / v).

The present invention according to claim 5 is characterized in that the culture temperature when culturing jaundice 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. The method for producing a liquid soak according to claim 1.
The present invention according to claim 6 is the method for producing a liquid koji according to claim 1, wherein at least glucoamylase and α-amylase are simultaneously produced and accumulated in the liquid koji .

  In the present invention according to claim 7, the cereals whose surfaces are entirely covered with husks are brown rice, rice with all or part of rice husks, and wheat with an unpolished to polished ratio of 92% or more. The manufacturing method of the liquid soot described in 1.

  The present invention according to claim 8 is the method for producing a liquid koji according to claim 1, wherein the koji mold is Aspergillus oryzae.

The present invention according to claim 9 is a cereal whose surface is entirely covered with husk (excluding pulverized matter), sodium nitrate and potassium nitrate, which are one or two kinds of nitrates, potassium dihydrogen phosphate And one or two phosphates selected from ammonium phosphate, and one or three sulfates selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate A method for producing glucoamylase and / or α-amylase , comprising culturing yellow koji molds in a liquid medium.

  According to the present invention, by culturing yellow koji mold using a liquid medium containing a predetermined culture raw material and inorganic salts, complicated operations such as raw material pretreatment and strict control can be performed without using a special culture apparatus. Even without this, a liquid koji with high production of glucoamylase and α-amylase can be produced. Since liquid culture allows stricter culture control than solid culture, a liquid koji with stable quality can be produced inexpensively and efficiently.

Since the liquid koji produced by the present invention has sufficient enzyme activity to produce fermented food and drink such as sake, fermented food and drink using the liquid koji can be produced.
In addition, the liquid koji having high enzyme activity can be used 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.

  In addition, since the culture raw material used in the present invention is unwhitened, unprocessed, or at least refined to the extent that the outer skin is left on the surface, it is expected to improve the raw material utilization rate and the yield.

  In addition, 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 steps in the liquid phase, so that It is possible to provide a fermented food and beverage production system that is more efficient and stable.

Hereinafter, the present invention will be specifically described.
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, etc., and glucoamylase and α-amylase It includes a step of producing a liquid koji with enhanced enzyme activity. That is, since the koji mold is cultured using the various raw materials described above, it takes time to saccharify the starch in the raw materials, the release rate of nutrients including sugars into the culture system is suppressed, and the enzyme activity of liquid koji Is strengthened. Moreover, glucoamylase and α-amylase are simultaneously generated and accumulated in a balanced manner.

In the present invention, examples of cereals used as a raw material for culturing liquid koji include barley, rice, wheat, buckwheat, millet, millet, millet, cucumber, and corn. As the shape of these culture raw materials, it is necessary that all or a part of the surface is covered with at least the husk, and the degree to which the unmilled product or at least the husk is left on the surface of the grain More than the whitening ratio that has been refined until now can be used, and brown rice, brown wheat and the like 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, when the cultivation raw material is barley, the unpolished milling rate is 100%, or the unmilled milling rate is 100%, and the unmilled milling rate (100%) to the barley grain ratio (generally Is a ratio obtained by subtracting 7 to 8%), that is, a polishing rate of about 92 to 93%.

  Here, the milling ratio refers to the ratio of cereals left after cerealing, and for example, the milling ratio of 90% means that 10% of the skin of the surface layer of the cereal is scraped off. Moreover, in the present invention, the unpolished barley is one that has been whitened from unmilled wheat to the extent that the grain skin is left on the surface of the grain, and includes one with a fineness ratio of 90% or more. Moreover, a grain skin means the outer side part which has covered the surface of the grain of cereals.

In the present invention, examples of beans and pods used as a raw material for culturing liquid koji include soybeans, red beans, and sweet potatoes. These culture raw materials are used for the preparation of a liquid medium in a state in which they are completely covered with the outer skin without being subjected to any processing such as cutting and pulverization, only by washing away the dirt on the outer skin.
In the present invention, heating or freezing treatment can also be performed while keeping the outer shell of beans or moss as the culture raw material.

In the present invention, amaranth used as a raw material for culturing liquid koji is a general term for an Amaranthaceae plant, and has a high protein content in cereals, and the content of lysine, which is one of the amino acids, is comparable to that of soybean. In addition, it is a highly nutritious grain that contains more calcium, iron and fiber than milled rice, and its 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 Agasaceae family and is cultivated mainly in the highlands of southern Peru and the Andes in western Bolivia and is rich in minerals, vitamins, proteins and dietary fiber.
The culture raw materials amaranth and quinoa may be used alone or in combination. These are used for the preparation of a liquid medium without pretreatment such as comminution or pulverization.

The above culture raw materials are used alone or in combination of two or more for preparing the following liquid medium. That is, the above culture raw material is mixed with water to prepare a liquid medium. The blending ratio of the raw materials is adjusted to such a level that glucoamylase and α-amylase are selectively generated and accumulated during the cultivation of yellow koji mold.
For example, when barley is used as a culture raw material, it is prepared in a liquid medium in which 1 to 20% (w / vol) of barley is added to water. Further, when non-refined barley is used, it is more preferably prepared in a liquid medium supplemented with 8 to 10% (w / vol), and when 95% polished barley is used as a raw material, 1 is more preferable. Prepared in liquid medium supplemented with ~ 4% (w / vol).
Next, when brown rice from which rice husks are removed is used as a culture raw material, the brown rice is 1% (w / vol) to 20% (w / vol), preferably 5% (w / vol) to 13 with respect to water. % (W / vol), more preferably 8% (w / vol) to 10% (w / vol).

  When beans are used as culture raw materials, the beans are 1 to 10% (w / vol) with respect to water, preferably 8 to 10% (w / vol) for soybeans, and 1 to 2% for red beans. (W / vol) Prepared in added liquid medium. Moreover, when moss is used as a culture raw material, it is prepared in a liquid medium to which moss is added in an amount of 1 to 10% (w / vol) with respect to water.

  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% (w / w) with respect to water. vol), more preferably 2% (w / vol) to 8% (w / vol). On the other hand, in the case of quinoa, 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).

As described above, the optimum blending amount varies depending on the degree of whitening of the culture raw material used, the jaundice strain used, the type of the culture raw material, and the like.
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 yellow koji mold becomes insufficient, and the oxygen concentration in the culture decreases. Therefore, the culture is difficult to proceed, which is not preferable. On the other hand, if the amount of the raw material used is less than the lower limit, the target enzyme will not be produced at a high rate.

  The starch contained in the culture raw material may be gelatinized before culturing. The 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. In the sterilization step of the liquid medium described later, when the starch is heated to a starch gelatinization temperature or higher by high-temperature high-pressure sterilization or the like, the starch gelatinization is simultaneously performed by this treatment.

It is necessary to add nitrate, phosphate and sulfate to the liquid medium used in the present invention as a nutrient source in addition to the above-mentioned culture raw materials. These inorganic salts are not particularly limited as long as they are usually used for culturing 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 or 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.
In addition, the concentration of the inorganic salts in the liquid medium is adjusted to such a level that glucoamylase and α-amylase are selectively generated and accumulated during the culture of jaundice. Specifically, in the case of nitrate, it is 0.1 to 2.0%, preferably 0.2 to 1.5%, and in the case of phosphate, 0.05 to 1.0%, preferably 0.1 to 0.1%. In the case of 0.5% and sulfate, 0.01 to 0.5%, preferably 0.02 to 0.1% (both are w / v).
In addition, the preferable concentration conditions of the above-mentioned inorganic salts can be employed in combination with each other, and can be combined with any aspect of the method of the present invention.

In the liquid medium, organic substances other than the above-mentioned inorganic salts, inorganic salts, and the like can be appropriately added as nutrient sources. 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., and inorganic salts such as ammonium. Water-soluble compounds such as salts, potassium salts, calcium salts, and magnesium salts can be mentioned, and two or more 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 Staphylococcus aureus, but is about 0.1 to 5% (w / vol) as an organic substance, and 0.1 to 1% (w as an inorganic salt) / Vol) is preferably added.
The liquid medium thus obtained may be sterilized as necessary, and the treatment method is not particularly limited. As an example, a high-temperature and high-pressure sterilization method can be mentioned, which may be performed at 121 ° C. for 15 minutes.

  The sterilized liquid medium is cooled to the culture temperature and then inoculated with jaundice. The yellow koji mold used in the present invention is a yellow koji mold having saccharide-degrading enzyme-producing ability, preferably glucoamylase-producing ability and α-amylase-producing ability. For example, Aspergillus oryzae and Aspergillus sojae ) And the like. Moreover, the form of the yellow koji mold inoculated into the medium is arbitrary, and spores or hyphae can be used.

These yellow koji molds can be used either by culturing with one kind of strain or mixed culturing with two or more kinds of strains of the same kind or different kinds. There is no problem whether these are used in the 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. There is no particular limitation on the inoculation amount of jaundice to 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 10%.

The culture temperature of the yellow koji mold is not particularly limited as long as it does not affect the growth, but it is preferably 25 to 45 ° C, more preferably 30 to 40 ° C. If the culture temperature is low, the growth of Aspergillus oryzae slows down, and contamination with various bacteria tends to occur.
In addition, the enzyme activity can be enhanced by controlling the culture temperature in accordance with the growth phase of jaundice. Specifically, the cell growth period from 12 to 36 hours after the start of culture is 25 to 35 ° C, preferably 28 to 33 ° C, and the subsequent enzyme production period is 35 to 45 ° C, preferably 37 to 42 ° C. Should be maintained. Further, the total culture time is preferably 24 to 72 hours.
In addition, said preferable culture | cultivation temperature conditions can be combined with any aspect of the method of this invention.

  Any culture apparatus may be used as long as it can perform liquid culture. However, since yellow koji molds need to be subjected to aerobic culture, it is necessary to perform them 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 jaundice 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.

  By culturing by the above culture method, glucoamylase and α-amylase are simultaneously produced in a well-balanced state, and a liquid koji having enzyme activity that can be used for producing fermented foods and drinks such as sake is obtained. In addition, in this invention, the liquid koji shall also include the culture solution obtained by centrifuging a culture, those concentrates, those dry matters, etc. besides the culture itself.

Thus, the enzyme production by the yellow koji mold can be increased by the above culture method.
Therefore, the method for producing an enzyme according to claim 17 is the same as the method for producing a liquid koji described above.

The liquid koji obtained by the production method of the present invention can be used for the production of fermented foods and beverages such as sake. For example, when producing sake, when producing shochu at the mash mother and each mash preparation stage, when producing soy sauce at the mash preparation stage, when producing miso at the preparation stage When producing brewed vinegar in the preparation stage, in the preparation stage, when producing mirin, in the preparation stage, when producing amazake, in the preparation stage, liquid rice cake is replaced with solid rice cake. Can be used.
Moreover, a part of the obtained liquid soot can also be used as a starter in the next liquid soot production. Thus, by continuously producing the liquid soot, stable production is possible, and at the same time, production efficiency can be improved.

  Moreover, when manufacturing fermented foods and drinks, such as sake, using the above-mentioned liquid koji, all processes can be performed in a liquid phase. As a method for producing fermented foods and beverages in which the entire process is carried out in a liquid phase, for example, when producing sake, a heat-resistant enzyme agent is used at a high temperature of about 80 ° C. by using polished rice or its crushed material as a raw material. There is a method of producing a mash that has been subjected to alcohol fermentation by adding the above-described liquid koji and yeast, after removing the residue by filtration or the like.

  Due to its high enzyme activity, the liquid koji obtained by the method of the present invention can also be used as an enzyme preparation and a pharmaceutical product such as a digestive agent. 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 and formulated in a conventional manner.

Moreover, the liquid koji obtained by the method of the present invention can be used for production of ethanol by fermentation.
As a method of producing ethanol by fermentation using the liquid koji obtained by the method of the present invention, except for using the liquid koji instead of the solid koji, in accordance with a known method for producing industrial alcohol (ethanol) Can be manufactured.

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 raw material may be any starchy raw material, such as barley, bare barley, rice, wheat, buckwheat, millet, millet, millet, cucumber, corn, etc .; sweet potato (sweet potato), cassava, etc. be able to.
After the preparation, the mixture is cooked at a low temperature, fermented at a temperature of about 20 to 30 ° C., and the secondary preparation can be performed after the primary preparation.
Industrial-use alcohol (ethanol) can be manufactured by distilling the mash after completion | finish of fermentation, removing an impurity, and concentrating.

  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 at a high yield in a koji mold culture. .

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention more concretely, this invention is not limited to these Examples etc.

Example 1 (Effect of carbon source on enzyme productivity in liquid koji production of yellow koji mold)
1. Liquid soot production method: Liquid soot was produced by the following method, and the enzyme activity was measured.
That is, various carbon sources 2.0% (w / vol), sodium nitrate 0.3% (w / vol), potassium chloride 0.2% (w / vol), potassium dihydrogen phosphate 0.1% (w / Vol), magnesium sulfate heptahydrate 0.05% (w / vol), iron sulfate heptahydrate 0.02% (w / vol) and 100 ml of liquid medium containing water in a 500 ml baffled Erlenmeyer flask It was applied and autoclaved at 121 ° C. for 15 minutes.
As a 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 schooner) were used. A test using 98% milled barley pulverized 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 100 rpm.

2. Measurement method: After completion of the culture, glucoamylase activity (GA) and α-amylase activity (AA) in each culture supernatant were measured. The glucoamylase activity (GA) was measured using a saccharification power fractionation kit (manufactured by Kikkoman), and the α-amylase activity was measured using an α-amylase measurement kit (manufactured by Kikkoman).

3. Results; The enzyme activity measurement results are shown in FIG. Glucoamylase was prominently produced only in the 98% refined wheat test section, while production was significantly suppressed in the 98% refined wheat (ground product) test section using the raw material obtained by pulverizing the glucoamylase (FIG. 1 (a)). .
98% refined barley corresponds to the cereal 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.
Although α-amylase produced a little different from glucoamylase, it produced a large amount of enzyme in the 98% polished wheat test zone (FIG. 1 (b)).
In this way, by using cereals with a surface covered with husks such as 98% refined wheat, it is possible to simultaneously produce a large amount of enzymes necessary for the production of fermented foods and beverages such as glucoamylase and α-amylase. It was shown.

Example 2 (Effects of various salts added in liquid koji production of yellow koji mold)
1. Liquid soot production method: Liquid soot was produced by the following method, and the enzyme activity was measured.
That is, 98% refined barley (Australian schooner) 2.0% (w / vol), sodium nitrate 0.3% (w / vol), potassium chloride 0.2% (w / vol), potassium dihydrogen phosphate 100 ml of liquid medium containing 0.1% (w / vol), magnesium sulfate heptahydrate 0.05% (w / vol), iron sulfate heptahydrate 0.02% (w / vol) and water The mixture was placed in a 500 ml Erlenmeyer flask with a baffle and autoclaved at 121 ° C. for 15 minutes to prepare a control group. 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 / ml, and cultured with shaking at 30 ° C. for 72 hours at 100 rpm.

2. Measurement method: After completion of the culture, glucoamylase activity (GA) and α-amylase activity (AA) in each culture supernatant were measured. The measurement of glucoamylase activity (GA) was performed using a saccharification power fraction determination kit (manufactured by Kikkoman), and the α-amylase activity was performed using an α-amylase measurement kit (manufactured by Kikkoman).

3. Results; The results of measuring the enzyme activity values in the culture supernatant are shown in FIG.
Glucoamylase was hardly produced in test group 1 that did not contain sodium nitrate, test group 3 that did not contain potassium dihydrogen phosphate, and test group 6 that did not contain both magnesium sulfate and iron sulfate (FIG. 2 (a)). . On the other hand, α-amylase was hardly produced in the test group 1, and it was confirmed that the production was suppressed in the test groups 3, 5, and 6 (FIG. 2 (b)).
Thus, it became clear that the enzyme productivity in the liquid koji of yellow koji was greatly influenced by the salts contained in the medium. That is, in order to simultaneously produce both enzymes at a high yield, it is essential to use cereals with a surface covered with husk, such as 98% polished wheat, sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate or iron sulfate. It was done.

Example 3 (Effect of sulfate on enzyme production in liquid koji production of yellow koji mold)
In Example 2, both the magnesium sulfate and the iron sulfate in the test group 6 were not observed in the magnesium sulfate-deficient medium in the test group 4 and the iron sulfate-deficient medium in the test group 5 although no tendency to decrease the enzyme productivity was observed. It was confirmed that there was a marked decrease in the medium lacking. Therefore, a test was conducted to confirm the effect of sulfate for the purpose of searching for essential factors of enzyme productivity in liquid koji mold.

1. Liquid soot production method: Liquid soot was produced by the following method, and the enzyme activity was measured.
That is, water, 98% refined barley (Australian schooner) 2.0% (w / vol), potassium nitrate 0.2% (w / vol), potassium dihydrogen phosphate 0.3% (w / vol), sulfuric acid After preparing 100 ml of liquid culture medium of 3 test sections as shown in Table 2 containing 0.05% (w / vol) of magnesium heptahydrate or 0.041% (w / vol) of magnesium chloride hexahydrate, The flask was put into a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. The amount of magnesium chloride hexahydrate added was calculated from 4.06 mM, which is a molar concentration corresponding to 0.05% magnesium sulfate heptahydrate, and blended so that the magnesium concentration in each medium was equal. did.
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 100 rpm.

2. Measurement method: After completion of the culture, glucoamylase activity (GA) and α-amylase activity (AA) in each culture supernatant were measured. The glucoamylase activity (GA) was measured using a saccharification power fractionation kit (manufactured by Kikkoman), and the α-amylase activity was measured using an α-amylase measurement kit (manufactured by Kikkoman).

3. Results; The enzyme activity measurement results are shown in FIG. The glucoamylase activity was significantly increased only in test group 1 in which the medium contained magnesium sulfate. On the other hand, the activity increase was not seen in the test group 2 which is a magnesium chloride addition group (FIG. 3 (a)). α-Amylase produced the highest amount in the test group 1 containing magnesium sulfate, although the enzyme production behavior was slightly different (FIG. 3B).
From the above results, it was suggested that the main body of the addition effect of magnesium sulfate is the sulfate radical.

Example 4 (Influence of the amount of salt added)
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 liquid koji mold of yellow koji. Therefore, it was investigated whether enzyme productivity could be enhanced by increasing the amount of inorganic salts added.

1. Liquid soot production method: Liquid soot was produced by the following method, and the enzyme activity was measured.
That is, 98% refined barley (Australian schooner), sodium nitrate, potassium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, iron sulfate heptahydrate and water were blended in the composition shown in Table 3. 100 ml of the liquid medium was put into a 500 ml Erlenmeyer flask with a 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 100 rpm.

2. Measurement method: After completion of the culture, glucoamylase activity (GA) and α-amylase activity (AA) in each culture supernatant were measured. The glucoamylase activity (GA) was measured using a saccharification power fractionation kit (manufactured by Kikkoman), and the α-amylase activity was measured using an α-amylase measurement kit (manufactured by Kikkoman).

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 is doubled compared to the control group, and test group 2 is enhanced four times. The glucoamylase activity at that time was the highest in the test group 2 where the inorganic salt concentration was enhanced 4 times, and it was shown that the added amount of inorganic salts contributed to the improvement of enzyme productivity (FIG. 4 (a)). α-Amylase also showed the highest activity in test group 1 where the inorganic salts were enhanced twice as much as the control group, and it was confirmed that the added amount of inorganic salts affected the enzyme productivity (FIG. 4 (b)). ).

Example 5 (Influence of culture temperature)
A test was 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.

1. Method for producing liquid koji: Under the same medium conditions as in test group 2 of Example 4, a koji mold was produced at different culture temperatures, and the enzyme activity was measured.
That is, 98% refined barley (Australian schooner) 2.0% (w / vol), sodium nitrate 1.2% (w / vol), potassium chloride 0.8% (w / vol), potassium dihydrogen phosphate 500 ml of 100 ml of liquid medium containing 0.4% (w / vol), magnesium sulfate heptahydrate 0.2% (w / vol), iron sulfate heptahydrate 0.08% (w / vol) and water The flask was placed in an Erlenmeyer flask with a baffle and sterilized by autoclave at 121 ° C. for 15 minutes. Subsequently, the medium was inoculated with Aspergillus oryzae RIB40 at 1 × 10 ⁶ cells / ml.
The culture conditions were as follows: control group 72 hours at constant 30 ° C, test group 1 at constant 37 ° C for 72 hours, test group 2 at 30 ° C from the start of culture to 24 hours, culture 24th to 72 hours. Until then, the cells were cultured at 37 ° C. The stirring conditions were 100 rpm shaking culture in all test sections.

2. Measurement method: After completion of the culture, glucoamylase activity (GA) and α-amylase activity (AA) in each culture supernatant were measured. The glucoamylase activity (GA) was measured using a saccharification power fractionation kit (manufactured by Kikkoman), and the α-amylase activity was measured using an α-amylase measurement kit (manufactured by Kikkoman).

3. Results; FIG. 5 shows the results of measurement of enzyme activity in each test group.
In the test group 1 in which the culture temperature was kept constant at 37 ° C., a tendency that the enzyme productivity slightly decreased was confirmed, but in the test group 2 in which the culture temperature was shifted from 30 ° C. to 37 ° C., glucoamylase (FIG. 5A) ) And α-amylase (FIG. 5B) were enhanced.
In test group 2, it is presumed that cell growth was carried out at a culture temperature of 30 ° C. and enzyme production was carried out at a culture temperature of 37 ° C., and the culture temperature in accordance with the growth phase of such yellow koji molds. It was suggested that control is effective in enhancing enzyme activity.
In addition, the enzyme activity of test group 2 is 106.5 U / ml of glucoamylase and 563.5 U / ml of α-amylase, which is considered to be an enzyme amount sufficient for producing fermented foods and drinks such as rice shochu and sake. .

Example 6 (Manufacture of dried liquid rice cake)
(I) Manufacture of dried liquid soaked product 200 ml of liquid soot produced by the method of test section 2 described in Example 5 was pre-frozen at −30 ° C. for 2 hours and then dried at 25 ° C. and a vacuum of 0.5 Torr for 24 hours. As a result, 2.5 g of a liquid koji dried product (liquid koji vacuum freeze-dried product) was obtained.

(II) Measurement of enzyme activity About the liquid koji which does not perform the freeze-drying process manufactured by the method of the test section 2 of Example 5, and the liquid koji dried product obtained by said (I), glucoamylase (GA) And α-amylase (AA) activity was measured.

The glucoamylase activity was measured using a saccharification power fractionation kit (manufactured by Kikkoman), and the α-amylase activity was measured using an α-amylase measurement kit (manufactured by Kikkoman).
The enzyme activity of the dried liquid koji product was measured using a solution obtained by dissolving 250 mg of dried koji product in 20 ml of 10 mM acetate buffer (pH 5).

Table 4 below shows the enzyme activity measurement results of the liquid koji that was not subjected to freeze-drying and produced by the method of Test Section 2 described in Example 5 and the dried liquid koji product obtained in (I) above.
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 drinks.

Example 7 (Method for producing shochu)
The charging composition is as shown in Table 5. The rice was made from 90% polished rice (Koshihikari from Ibaraki Prefecture), washed for 15 minutes, drained for 10 minutes, steamed for 30 minutes, and the liquid koji produced by the method of Test Zone 2 described in Example 5 Lactic acid and water were added. This was inoculated with 50 μl of shochu yeast (Kagoshima yeast) that had been statically cultured in YPD medium at 30 ° C. for 48 hours. Fermentation conditions were fixed at 25 ° C., and fermentation was performed for 18 days.

Fermentation proceeded smoothly, and the alcohol content of the obtained final moromi (shochu moromi) was 17.6%.
As a result of sensory evaluation of shochu raw sake produced by distilling this shochu moromi by vacuum distillation with six specialist panels, there is a comment that “clean and light”, and that a rice shochu with a refreshing flavor can be obtained. all right.
From the above results, it was shown that shochu can be produced using liquid soot.

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), dipping for 15 minutes, draining for 10 minutes, cooking for 30 minutes, 1630 ml of water, a liquid produced by the method of Test Zone 2 described in Example 5 500 ml of sputum and 1.6 ml of 90% lactic acid were mixed. To this was added 100 μl of sake yeast (Association No. 7) shake-cultured in YPD medium for 24 hours at 30 ° C., and fermentation was performed at 25 ° C. Three days later, 750 g of 90% polished rice (Koshihikari from Ibaraki Prefecture) cooked into 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 as shown in Table 6 below.

The final mash obtained above was filtered to obtain sake. As a result of sensory evaluation with 10 panelists, this sake had a rich taste and an evaluation of a fruity fragrance.
From the above, it has been clarified that sake can be produced using liquid koji by the method of the present invention.

Example 9 (Production of soy sauce)
Preparation: The preparation composition was as shown in Table 7 below. The whole soybeans were washed, immersed in water overnight, drained for 60 minutes, and steamed for 4 hours, and then crushed by a food processor. Wheat (Agriculture No. 61) was roasted and then ground. Sodium chloride 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 whole soybeans and wheat treated as described above were added thereto. The culture solution of Z. rouxii used was a shaking culture in YPD medium at 30 ° C. for 24 hours.

Fermentation conditions: After 30 days of fermentation at 15 ° C, the temperature was raised to 2 ° C / day to 30 ° C. After reaching the temperature, fermentation was further performed at 30 ° C. for 3 months. Up to this point, the mixture was properly stirred. Thereafter, the mixture was aged at 25 ° C. for 2 months without stirring to obtain a final mash.
The analysis values of the final moromi obtained are as shown in Table 8 below.

＊食塩以外の可溶性固形分

* Soluble solids other than salt

Filtration: The final mash obtained was squeezed with a nylon filter cloth.
Post-treatment: Fired at 80 ° C. for 30 minutes and dragged to obtain the final product.
The sensory evaluation of the soy sauce obtained as described above was conducted by 6 panelists specializing in alcoholic beverages.
From the above, it has been found that according to the method of the present invention, it is possible to produce soy sauce using a liquid koji.

Example 10 (Production of miso)
Preparation and aging;
The charging composition was as shown in Table 9. The whole soybeans were washed, immersed in water overnight, drained for 60 minutes, boiled for 4 hours, and crushed (crushed) by a food processor. For yeast, Zygosaccharomyces rouxii (NBRC0510) was shaken and cultured in 10 ml of YPD medium at 30 ° C. for 24 hours, and then collected by centrifugation, and the resulting cells were washed twice with sterile water.
To the liquid koji produced by the method of Test Zone 2 described in Example 5, salt, the yeast, and the whole soybeans treated as described above were added. Fermentation conditions were fixed at 25 ° C., and fermentation and aging were performed for 6 months to obtain miso.

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

Moreover, when the sensory evaluation of the obtained barley miso was performed by six specialist panels, it was judged 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.

Example 11 (Production of mirin)
Preparation; Preparation preparation was as shown in Table 11. The glutinous rice (chicken rice, 90% whitened) was washed, immersed in water for 60 minutes, drained for 30 minutes, steamed for 1 hour, and allowed to cool. Steamed glutinous rice, 45% raw alcohol, and liquid koji produced by the method of Test Zone 2 described in Example 5 were mixed.

Saccharification and aging conditions: The mixture was left at 25 ° C. for 2 months.
Filtration: Squeezed with a nylon filter cloth.
Post-processing; dragging and final product.
Table 12 shows the result of the component analysis of the moromi after completion of the aging step.

The sensory evaluation of the liquid koji mirin prepared as described above was carried out by six panelists specializing in alcoholic beverages, and it was judged that the sweetness and umami taste were good and the quality could be used sufficiently as mirin.
From the above, it has been clarified that the method of the present invention can produce mirin using liquid soot.

Example 12 (Production of grain vinegar)
(I) Alcohol fermentation using liquid koji The mixing composition was as shown in Table 13. As the round barley (domestic 2 barley barley, 70% koji ratio), what was dipped for 60 minutes, drained for 30 minutes and boiled for 60 minutes after washing was used. As the yeast, shochu yeast (Kagoshima yeast) was used, and 50 μl of an inoculated yeast cultured at 30 ° C. for 48 hours in a YPD medium was inoculated.
To the liquid koji produced by the method of Test Section 2 described in Example 5, the yeast, the barley treated as described above, 90% lactic acid, and water were added. Fermentation conditions were fixed at 25 ° C., and alcohol fermentation was performed for 10 days to obtain a liquid koji alcoholic fermentation broth.

(II) Production of cereal vinegar using liquid koji alcoholic fermentation liquid (1) Acetic acid bacteria used; Acetobacter aceti subsp. Aceti (NBRC3284) was used. Acetic acid bacteria were cultured in ordinary bouillon + 1% glucose medium.
(2) Preparation: The liquid koji fermented liquid obtained in (I) above was centrifuged, 1 ml of the acetic acid bacteria culture liquid obtained in (1) above was added to the supernatant, and the mixture was cultured at 30 ° C. for 3 months. .
(3) Lowering; After culturing, the fungus on the surface was removed and centrifuged to obtain the final product.
The acidity of the grain vinegar obtained as described above was 6.1%, and the pH was 3.1.
When the sensory evaluation of this grain vinegar was conducted by six specialist panels, it was judged that the quality could be used as grain vinegar.

Example 13 (Production of amazake)
Using the liquid koji produced by the method of test section 2 described in Example 5, amazake was produced as follows.
The charging recipe was as shown in Table 14. The rice was 90% polished rice (Koshihikari from Ibaraki Prefecture), washed, immersed in water for 60 minutes, drained for 30 minutes, cooked for 1 hour, and allowed to cool. Saccharification was performed at 55 ° C. for 16 hours using the raw materials having the following composition to produce amazake.

The resulting liquor charged with liquid koji had a Brix of 18.5.
Moreover, when the sensory evaluation of the obtained liquor prepared with the liquid koji was conducted by 6 panelists specializing in alcoholic beverages, it was judged that the quality was sufficient for use as amazake.

Example 14 (Method for producing ethanol)
The charging composition is as shown in Table 15. The rice was prepared by the method of Test Zone 2 described in Example 5 using 90% polished rice (Koshihikari from Ibaraki Prefecture), washed with rice for 15 minutes, drained for 10 minutes, and steamed for 30 minutes. Liquid koji, lactic acid and water were added. This was inoculated with 50 μl of shochu yeast (Kagoshima yeast) that had been statically cultured in YPD medium at 30 ° C. for 48 hours. Fermentation conditions were fixed at 25 ° C. and fermentation was performed for 16 days.

The fermentation proceeded smoothly, and the alcohol content of the final mash obtained was 17.8%.
The mash after the fermentation obtained above was continuously distilled with a precision distillation machine (manufactured by Shibata Kagaku Co., Ltd., HP-1000T special model) 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 clear that industrial alcohol (ethanol) can be produced using liquid koji mold of yellow koji mold.

In Example 1, it is a graph which shows the enzyme activity at the time of cultivating jaundice in the liquid culture medium containing various carbon sources. (A) is a measurement result of glucoamylase, (b) is a measurement result of α-amylase. In Example 2, it is a graph which shows the enzyme activity at the time of cultivating jaundice in the liquid culture medium except various salts. (A) is a measurement result of glucoamylase, (b) is a measurement result of α-amylase. In Example 3, it is a graph which shows the enzyme activity at the time of cultivating jaundice in the liquid culture medium except various salts. (A) is a measurement result of glucoamylase, (b) is a measurement result of α-amylase. In Example 4, it is a graph which shows the enzyme activity at the time of cultivating jaundice in the liquid culture medium which changed the addition amount of inorganic salt. (A) is a measurement result of glucoamylase, (b) is a measurement result of α-amylase. In Example 5, it is a graph which shows the enzyme activity at the time of changing the culture | cultivation temperature and carrying out liquid culture | cultivation of yellow gonococcus. (A) is a measurement result of glucoamylase, (b) is a measurement result of α-amylase.

Claims (9)

A method for producing liquid koji, wherein the entire surface is covered with cereal (excluding pulverized material) , one or two types selected from sodium nitrate and potassium nitrate, nitrate, potassium dihydrogen phosphate And one or two phosphates selected from ammonium phosphate, and one or three sulfates selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate A method for producing liquid koji characterized by culturing yellow koji molds in a liquid medium. The method for producing a liquid koji according to claim 1, wherein the liquid medium contains one or two kinds of nitrates selected from sodium nitrate and potassium nitrate at a concentration of 0.1 to 2.0% (w / v). The liquid medium contains one or two kinds of phosphates selected from potassium dihydrogen phosphate and ammonium phosphate at a concentration of 0.05 to 1.0% (w / v). Method for producing liquid koji. The liquid medium contains one to three kinds of sulfates selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate at a concentration of 0.01 to 0.5% (w / v). The method for producing a liquid bag according to claim 1.   2. The liquid koji according to claim 1, wherein the culture temperature when culturing 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. Manufacturing method.   The method for producing a liquid koji according to claim 1, wherein at least glucoamylase and α-amylase are simultaneously produced and accumulated in the liquid koji. 2. The method for producing a liquid rice bran according to claim 1, wherein the cereals whose entire surface is covered with husk are brown rice, rice with all or part of rice husks and wheat with an unpolished ratio of 92% or more. The method for producing a liquid koji according to claim 1, wherein the yellow koji mold is Aspergillus oryzae. 1 type selected from cereals whose surface is entirely covered with husks (excluding pulverized products), 1 or 2 types of nitrates selected from sodium nitrate and potassium nitrate, potassium dihydrogen phosphate and ammonium phosphate Or culturing jaundice in a liquid medium containing two kinds of phosphates and one or three kinds of sulfates selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate A method for producing glucoamylase and / or α-amylase characterized by the above.

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