JP4083194B2 - Method for producing liquid cake - Google Patents

Description

  The present invention relates to a method for producing a liquid koji, and more particularly to a method for producing a liquid koji having enhanced enzyme activity.

  The koji used in the production of alcoholic beverages, etc., is prepared by inoculating and cultivating spores of filamentous fungi on the raw material after processing such as cooking, and preparing a liquid medium by adding the raw material and other nutrients to water, There is a liquid koji that inoculates and cultivates gonococcal spores or precultured hyphae.

  In the production of conventional fermented foods and beverages such as alcoholic beverages, such as sake, shochu, soy sauce, miso, mirin, etc., so-called solid koji made by a solid culture method is widely used. Aspergillus kawachii, Aspergillus awamori, Aspergillus niger (Aspergillus niger), Aspergillus sperm, Aspergillus sperm Is sprayed onto a solid raw material such as steamed cereals, and the koji mold is grown on the surface thereof.

  For example, in the production of shochu, solid soot such as Aspergillus kawachii and Aspergillus awamori is widely used. However, since the solid culture method is a culture system in which raw materials and koji molds are dispersed non-uniformly, it is difficult to make uniform factors such as temperature, water content, and various nutrient components, and the culture control is very complicated. . In addition, iron is often produced in an open state, and in this case, attention is required in terms of quality control such as contamination by various bacteria. Therefore, it can be said that the method is not suitable for large-scale manufacturing.

  On the other hand, the liquid culture method is easy to control culture and quality control, and is a culture form suitable for efficient production. However, there are few examples in which a culture obtained by liquid culture of koji mold is actually used as shochu because, for example, the enzyme activity necessary for shochu brewing cannot be sufficiently obtained. Here, the culture obtained by the liquid culture method refers to the culture itself obtained by the liquid culture method (hereinafter sometimes referred to as “liquid koji”), a culture solution, microbial cells, a concentrate thereof, or their It may be a dry product.

  As a major reason why the culture obtained by the liquid culture method is not used for the production of fermented foods and beverages such as shochu, in addition to the above reasons, the enzyme production behavior of amylase, cellulase, etc. of Aspergillus is significantly different in liquid culture Moreover, it is known that productivity generally decreases (see Non-Patent Documents 1 and 2).

  Usually, in the production of alcoholic beverages including shochu, alcohol is produced by parallel double fermentation. Therefore, the saccharide-degrading-related enzymes of koji mold that affect glucose supply to koji molds, in particular glucoamylase and acid-resistant α-amylase, are key enzymes in alcohol fermentation. However, it is known that in the culture obtained by the liquid culture method, the activity of glucoamylase is remarkably low, and the production behavior is also greatly different from that of solid culture (see Non-Patent Documents 3 to 6).

  As a method for improving the glucoamylase activity of Aspergillus oryzae, a method of cultivating Aspergillus oryzae while stressing the growth of mycelia (see Patent Document 1) or a method of adding roasted cereals to an Aspergillus culture medium (see Patent Document 2) It has been reported. The method disclosed in Patent Document 1 is a method in which a novel gene glaB encoding glucoamylase is expressed by culturing in a entrapping immobilization agent on a porous membrane or having voids, thereby enhancing the enzyme activity. Special culture equipment is required and is not practical. In addition, the method disclosed in Patent Document 2 is a method for culturing koji molds in a liquid medium using cereals roasted as at least a part of the raw material, and adds a new manufacturing process of roasting cereals. .

  Therefore, the present inventors have proposed an invention relating to a method for culturing koji molds using a liquid medium containing a saccharide that is hardly degradable for koji molds (see Patent Document 3). According to this invention, in a liquid culture of Aspergillus, it is possible to easily and inexpensively obtain an Aspergillus culture that has a high activity of a saccharide degradation-related enzyme such as glucoamylase that can be used in the production of fermented foods and drinks such as alcoholic beverages. Can do.

  On the other hand, regarding acid-resistant α-amylase, molecular biological analysis has recently started in detail (see Non-Patent Document 7). According to it, white mold has two types of amylase genes with different properties, non-acid-resistant α-amylase and acid-resistant α-amylase, but their expression patterns are greatly different. Although acid-resistant α-amylase is sufficiently produced, it is reported that acid-resistant α-amylase, which is a key enzyme for shochu brewing, is hardly produced.

  In shochu production, brewing is performed under a low pH environment in order to prevent the shochu mash from becoming rotted. However, non-acid resistant α-amylase is rapidly inactivated under low pH conditions, and therefore hardly contributes to the degradation of carbohydrates in shochu brewing. For this reason, it is indispensable for producing shochu to produce a large amount of acid-resistant α-amylase, which is thought to contribute to the degradation of carbohydrates in shochu brewing, by liquid culture of koji mold.

In the past, although there have been reports that have examined in detail the production behavior of acid-resistant α-amylase in liquid culture of Aspergillus oryzae, the method uses a synthetic medium containing peptone or citrate buffer, and the culture time is It is difficult to say that it is a method for producing liquid koji that can be applied to actual shochu brewing, such as taking more than 100 hours (see Non-Patent Documents 8 to 10).
Thus, it is generally considered that acid-resistant α-amylase is basically an enzyme that is not produced in liquid culture, and a liquid koji with high activity of acid-resistant α-amylase has not been developed so far. .

Japanese Patent Laid-Open No. 11-225746 JP 2001-321154 A JP 2003-265165 A Iwashita K. et a1: Biosci. Biotechno1. Bioche., 62, 1938-1946 (1998) Yuichi Yamane et al .: Japan Brewing Association Journal, 99, 84-92 (2004) Hata Y. et. Al .: J. Ferment. Bioeng., 84, 532-537 (1997) Hata Y. et. A1 .: Gene., 207, 127-134 (1998) Ishida H. et. Al .: J. Ferment. Bioeng., 86, 301-307 (1998) Ishida H. et. A1: Curr. Genet., 37, 373-379 (2000) Nagamine K. et.a1 .: Biosci. Biotechno1. Biochem., 67, 2194-2202 (2003) Sudo S. et al: J. Ferment. Bioeng., 76, 105-110 (1993) Sudo S. et al: J. Ferment. Bioeng., 77, 483-489 (1994) Shigetoshi Sudo et al .: Journal of the Japan Brewing Association, 89, 768-774 (1994)

The inventors of the present invention can produce shochu such as glucoamylase and acid-resistant α-amylase by culturing koji molds in a liquid medium containing, as a raw material, cereals whose surfaces are all or partly covered with husks. It has been found that a liquid soot sufficiently containing the necessary enzyme activity can be produced, and patent applications have already been filed (see Japanese Patent Application Nos. 2004-350661 and 2004-352320).
However, enzyme production behaviors other than glucoamylase and acid-resistant α-amylase by these methods have not been known so far.

  The object of the present invention is to develop a method for enhancing amylolytic enzymes such as glucoamylase and acid-resistant α-amylase and other enzyme activities in liquid koji, and in particular, optimizing the composition of the liquid medium. By doing so, it is providing the method of manufacturing a liquid rice cake with high enzyme activity.

  As a result of intensive studies on the combined effect of the above-mentioned culture raw materials and various nutrient sources, the present inventors aim to further increase enzyme production in liquid koji, and as a result, the liquid medium contains a specific nitrogen source. Furthermore, it has been found that the coexistence of sulfate and phosphate improves the productivity of amylolytic enzyme glucoamylase, cellulase cellulose degrading enzyme, and acid carboxypeptidase proteolytic enzyme. The present invention has been completed.

That is, the present invention according to claim 1 is one or more kinds selected from cereals whose surfaces are entirely covered with cereals , and potassium nitrate, sodium nitrate, yeast cells, processed yeast cells, cereal husks, and cereals. A method for producing a liquid koji with enhanced enzyme activity, comprising culturing white koji mold or black koji mold in a liquid medium containing a nitrogen source.
The present invention according to claim 2 is the method for producing a liquid koji having enhanced enzyme activity according to claim 1, wherein the enzymes are at least glucoamylase and acid-resistant α-amylase.
In the present invention according to claim 3, the liquid medium is composed of cereals whose entire surface is covered with husk, one or two kinds selected from potassium nitrate and sodium nitrate, potassium dihydrogen phosphate and ammonium phosphate. 2. The enzyme activity according to claim 1, comprising one or two selected, and one or three selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate, and ammonium sulfate. This is a method for producing an enhanced liquid koji .
The present invention according to claim 4, liquid medium, according to one or two elements selected from potassium nitrate and sodium nitrate to claim 3 containing at a concentration of 0.1 ~2.0% (w / vol) This is a method for producing a liquid koji with enhanced enzyme activity.
In the present invention according to claim 5 , the liquid medium contains one or two selected from potassium dihydrogen phosphate and ammonium phosphate at a concentration of 0.05 to 1.0% (w / vol). Item 4. A method for producing a liquid koji having enhanced enzyme activity according to Item 3 .
In the present invention according to claim 6 , the liquid medium is 0.01 to 0.5% (w / vol) of one to three selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate. It is a manufacturing method of the liquid rice cake with which the enzyme activity was enhanced of Claim 3 contained in the density | concentration.

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. 4. A method for producing a liquid koji having an enhanced enzyme activity.
The present invention according to claim 8 is the method for producing a liquid koji with enhanced enzyme activity according to claim 1, wherein the white mold is Aspergillus kawachi.
The present invention according to claim 9 is the production of a liquid koji having enhanced enzyme activity according to claim 1, wherein the black koji mold is one or two selected from Aspergillus awamori and Aspergillus niger. Is the method.
The present invention according to claim 10 is the method for producing a liquid koji having enhanced enzyme activity according to claim 1, wherein the cereal is barley.

The present invention according to claim 11 is a cereal whose entire surface is covered with husk, and at least one nitrogen selected from potassium nitrate, sodium nitrate, yeast cells, processed yeast cells, cereal husks and cereal straws. A method for producing one or more enzymes selected from glucoamylase and acid-resistant α-amylase, wherein the enzyme is produced by culturing white koji mold or black koji mold in a liquid medium containing a source.
The present invention according to claim 12 is a cereal whose entire surface is covered with husk, one or two selected from potassium nitrate and sodium nitrate, and one selected from potassium dihydrogen phosphate and ammonium phosphate Or, an enzyme is produced by culturing white or black koji molds in a liquid medium containing two kinds and one to three kinds selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate. Is a method for producing one or more enzymes selected from glucoamylase, acid-resistant α-amylase, cellulase and acid carboxypeptidase .
The present invention according to claim 13, a liquid medium, according to claim 12 containing a concentration of one or two elements selected from potassium nitrate and sodium nitrate 0.1 ~2.0% (w / vol) This is an enzyme production method.
In the present invention according to claim 14 , the liquid medium contains one or two selected from potassium dihydrogen phosphate and ammonium phosphate at a concentration of 0.05 to 1.0% (w / vol). Item 13. A method for producing the enzyme according to Item 12 .
In the present invention according to claim 15 , in the liquid medium, 0.01 to 0.5% (w / vol) of one to three kinds selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate It is a manufacturing method of the enzyme of Claim 12 contained in the density | concentration.
The present invention according to claim 16, cereals entire surface is covered with grain skins, brown rice, claim 11 chaff is the polishing ratio of 92% or more of wheat from the United States and untreated polished marked with all or part Or the method for producing an enzyme according to 12 .
The present invention according to claim 17 is the method for producing an enzyme according to claim 11 or 12 , wherein the white mold is Aspergillus kawachi.
The present invention according to claim 18 is the method for producing an enzyme according to claim 11 or 12 , wherein the black koji mold is one or two selected from Aspergillus awamori and Aspergillus niger.
The present invention according to claim 19 is the method for producing an enzyme according to claim 11 or 12, wherein the cereal is barley.

  According to the present invention, a specific organic substance and / or inorganic substance is added as a nitrogen source to a liquid medium using at least a part of the surface covered with cereal as a culture raw material, and further sulfate and phosphate are added. And culturing the koji mold in the liquid medium not only can significantly improve the productivity of starch-degrading enzyme in the liquid koji, but also a liquid koji with high production of cellulolytic enzyme and proteolytic enzyme. Can be manufactured. In addition to the above enzymes, the productivity of all enzymes produced by Aspergillus is considered to increase.

  When fermented foods and drinks such as shochu are produced using the liquid koji produced according to the present invention, cellulolytic enzyme activity is high, so that favorable fermentation is performed due to a decrease in moromi viscosity, and an increase in alcohol yield can be expected. Moreover, the amount of amino acid production increases by high proteolytic enzyme activity, and the fermented food / beverage products which have a gorgeous fragrance can be manufactured.

Furthermore, since liquid culture allows stricter culture control than solid culture, a liquid koji with stable quality can be produced at low cost.
Moreover, since the cereals used in the present invention are not whitened or have been at least polished to the extent that the husk is left on the surface of the grain, an improvement in raw material utilization rate and yield can be expected.

  Hereinafter, the present invention will be specifically described.

The method for producing liquid koji in the present invention includes a step of culturing koji molds in a liquid medium prepared by adding raw materials such as cereals and nitrogen sources to produce liquid koji having enhanced enzyme activity. .
That is, in the present invention, the koji mold is cultured using a liquid medium containing cereals whose whole or part of the surface is covered with husks as a culture raw material, so it takes time to saccharify starch in the cereals. The sugar release rate to the culture system is suppressed, and the enzyme activity of the liquid koji is enhanced. Furthermore, since a liquid medium containing a specific nutrient source is used, various enzymes are highly produced by Aspergillus.

  Here, examples of the enzyme produced by Aspergillus include starch-degrading enzymes such as glucoamylase and α-amylase, cellulose-degrading enzymes such as cellulase and β-glucosidase, and proteolytic enzymes such as acid carboxypeptidase and acid protease. However, it is not necessarily limited to these.

In the present invention, examples of cereals used as a culture raw material 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 cereal is barley, the unpolished milling rate is 100%, or the unpolished milling rate is 100%, and the unmilled milling rate (100%) is used to obtain the barley grain ratio (general Is a ratio obtained by subtracting 7-8%), that is, a fineness ratio of about 92-93% or more.

  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 includes unpolished barley to those that have been refined to such an extent that the husk remains on the surface of the grain, that is, those having a milling ratio of 90% or more. Moreover, a grain skin means the outer side part which has covered the surface of the grain of cereals.

  The above culture raw materials are used alone or in combination of two or more for preparing the following liquid medium. That is, the cultivated raw material grain is mixed with water together with a nitrogen source described later to prepare a liquid medium. The blending ratio of cereals is adjusted so that enzymes such as amylolytic enzymes, cellulose degrading enzymes, and proteolytic enzymes are selectively generated and accumulated in the koji mold culture.

For example, when barley is used as a culture raw material, it is prepared in a liquid medium in which 1 to 20% (w / vo1) of brown barley is added to water. Moreover, when unpolished barley is used as brown wheat, it is more preferably prepared in a liquid medium with 8 to 10% (w / vol) added. More preferably, it is prepared in a liquid medium supplemented with 1 to 4% (w / vo1).
Moreover, when brown rice except rice husk is used as a culture raw material, brown rice is 1 to 20% (w / vo1), preferably 5 to 13% (w / vo1), more preferably 8 to 10% with respect to water. Prepared in a liquid medium supplemented with% (w / vo1).
Similarly, when other cereals are used, they are prepared in a liquid medium supplemented with 1 to 20% (w / vo1) of water.

As described above, the optimum blending amount varies depending on the degree of milling of the raw material used, the koji 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 Neisseria gonorrhoeae becomes insufficient, and the oxygen concentration in the culture decreases. This is not preferable because the culture is difficult to proceed. On the other hand, if the amount of the raw material used is less than the lower limit, the target enzyme will not be produced at a high rate.

  The starch contained in the culture raw material may be gelatinized before the culture. 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 heating is performed at a temperature higher than the gelatinization temperature of starch by high-temperature high-pressure sterilization or the like, gelatinization of starch is simultaneously performed by this treatment.

In the liquid medium, an organic substance, an inorganic substance, or the like is added and contained as a nitrogen source in addition to the above-described culture raw material. These nitrogen sources are not particularly limited as long as the koji mold grows and the target enzyme is sufficiently produced. Examples of the organic substance include yeast cells or processed products thereof (for example, yeast cell decomposition products, yeast extract and the like), cereal husks, cereals and the like, and examples of inorganic substances include nitrates.
As the nitrate, potassium nitrate, sodium nitrate or the like can be used, and potassium nitrate is particularly preferable.

These nitrogen sources may be used alone or in combination of two or more organic substances and / or inorganic substances.
The amount of nitrogen source added is not particularly limited as long as it promotes the growth of Aspergillus, but it is 0.1 to 2% (w / vol), preferably 0.5 to 1.0% (w / Vol). Moreover, the addition amount of nitrate as an inorganic substance is 0.05 to 2.0% (w / vol), preferably 0.1 to 2.0% (w / vol), most preferably 0.2 to 1.5. % (W / vol).
When a nitrogen source is added exceeding the upper limit, growth of koji mold is inhibited, which is not preferable. Moreover, when the addition amount is less than the lower limit, enzyme production is not promoted, which is also not preferable.

  Yeasts used as a kind of nitrogen source in the present invention include beer yeasts, wine yeasts, whiskey yeasts, shochu yeasts, sake yeasts, baker's yeasts used in brewing processes and food production, as well as Saccharomyces genus, Candida ( Candida, Torulopsis, Hanseniaspora, Hansenula, Debaryomyces, Saccharomycopsis, Saccharomycos, Saccharomycos Examples include yeast cells of the genus Pichia and Pachysolen.

  These yeasts can be used as a nitrogen source, but can also be used as a yeast cell decomposition product or yeast extract. Yeast cell degradation products or yeast extracts are produced by self-digestion of yeast cells (method of solubilizing cells using the proteolytic enzyme inherent in yeast cells), enzyme decomposition methods (from microorganisms and plants) (Methods of solubilization by adding enzyme preparations, etc.), hot water extract method (method of soaking yeast cells in hot water for a certain period of time), acid or alkali decomposition method (various acids or alkalis) Additive solubilization method), physical crushing method (sonication, high-pressure homogenization method, crushing method by mixing and stirring solid materials such as glass beads), freeze-thawing method (freezing / thawing) For example, by crushing once or more).

Moreover, cereal meals, such as rice bran, can also be used as a nitrogen source, and this is a by-product produced when grain is refined. Cereal seeds are made up of the epidermis, embryo, and endosperm, and the rice hulls that protect them. Of these, the combination of the embryo and the epidermis is the cocoon.
Furthermore, in the present invention, the cereal husk, that is, the skin of the cereal, can be used as a nitrogen source, and usually the same kind of cereal husk as the cultivated raw material cereal is used. These cereal meals and cereal husks can be used in combination with other nitrogen sources.

The liquid medium used in the present invention can contain sulfate and phosphate in addition to the aforementioned culture raw material and nitrogen source. By using these inorganic salts in combination, it is possible to enhance enzyme activities such as amylolytic enzymes, cellulolytic enzymes, and proteolytic enzymes.
For example, as the sulfate, magnesium sulfate heptahydrate, iron sulfate heptahydrate, ammonium sulfate and the like can be used, and magnesium sulfate heptahydrate is particularly preferable. As the phosphate, potassium dihydrogen phosphate, ammonium phosphate or the like can be used, and potassium dihydrogen phosphate is particularly preferable.
These inorganic salts can be used alone or in combination of two or more.

The concentration of the inorganic salts in the liquid medium is adjusted to such a level that enzymes such as amylolytic enzymes, cellulolytic enzymes, and proteolytic enzymes are selectively generated and accumulated in the koji mold culture. For example, in the case of sulfate, 0.01 to 0.5%, preferably 0.02 to 0.1%, and in the case of phosphate, 0.05 to 1.0%, preferably 0.1 to 0.00. 5% (all w / vol).
The above inorganic salts may be used alone or in combination of two or more.

To the liquid medium, organic substances other than the aforementioned nitrogen source and 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 koji molds, but organic substances such as wheat koji, corn steep liquor, soybean koji, defatted soybeans, etc., and inorganic salts such as ammonium salt and potassium Water-soluble compounds such as salts, calcium salts and magnesium salts can be mentioned, and two or more kinds of organic substances and / or inorganic salts may be used simultaneously.
These addition amounts are not particularly limited as long as they promote the growth of Neisseria gonorrhoeae, but are about 0.1 to 5% (w / vol) as organic substances and 0.1 to 1% (w / vol.) As inorganic salts. It is preferable to add about vol).
When these nutrient sources are added in excess of the upper limit value, the growth of koji mold is inhibited, which is not preferable. Moreover, when the addition amount is less than the lower limit, enzyme production is not promoted, which is also not preferable.

  The liquid medium of Aspergillus oryzae obtained by mixing the above culture raw material and nitrogen source with water 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.

After the sterilized liquid medium is cooled to the culture temperature, white and / or black koji molds are inoculated into the liquid medium.
As the koji mold used in the present invention, glucoamylase, acid-resistant α-amylase, starch-degrading enzyme such as α-amylase, cellulolytic enzyme such as cellulase and β-glucosidase, proteolytic enzyme such as acid carboxypeptidase, acid protease, etc. Neisseria gonorrhoeae having productivity is preferred. Specifically, Aspergillus kawachii and the like as white mold, and Aspergillus awamori and Aspergillus niger and the like as black gonorrhoeae.
Moreover, the form of the koji mold inoculated into the medium is arbitrary, and spores or hyphae can be used.

These 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. There is no problem with using any form of spores or hyphae obtained by preculture, but it is preferable to use hyphae because the time required for the logarithmic growth phase is shortened.
There is no particular limitation on the amount of koji mold inoculated into the liquid medium, but about 1 × 10 ⁴ to 1 × 10 ⁶ spores per 1 ml of the liquid medium, and 0.1 to 10 preculture solution for mycelia. It is preferable to inoculate about 1%.

The culture temperature of the 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 is delayed, and contamination with various bacteria is likely to occur. The culture time is suitably 24 to 72 hours.
Any culture apparatus may be used as long as it can perform liquid culture. However, since Neisseria gonorrhoeae needs to perform aerobic culture, it needs to be performed under aerobic conditions in which oxygen and air can be supplied into the medium. Moreover, it is preferable to stir so that the raw material, oxygen, and koji mold in the medium are uniformly distributed in the apparatus during the culture. The stirring conditions and the aeration amount may be any conditions as long as the culture environment can be maintained aerobically, and may be appropriately selected depending on the culture apparatus, the viscosity of the medium, and the like.

By culturing by the above culture method, enzymes such as starch degrading enzyme, cellulose degrading enzyme, and proteolytic enzyme are produced at high yield, and liquid koji having enzyme activity that can be used for the production of shochu and the like 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.

As described above, according to the culture method described above, enzymes such as starch degrading enzymes, cellulose degrading enzymes, and proteolytic enzymes can be produced at a high rate.
Therefore, the method for producing the enzyme according to claim 14 is the same as the method for producing the liquid koji described above.

The liquid koji obtained by the production method of the present invention can be suitably used for the production of fermented foods and drinks such as shochu. 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 In the preparation stage, when producing mirin, in the preparation stage, when producing amazake, in the preparation stage, liquid rice cake can be used instead of solid rice cake.
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 food / beverage products, such as shochu, 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 performed in a liquid phase, for example, when producing shochu, corn, wheat, rice, potatoes, sugar cane, etc. are used as raw materials, and the raw materials are heat-resistant at a high temperature of about 80 ° C. A method for producing a mash that has been liquefied by dissolving it using a sexual enzyme agent, and then the above-mentioned liquid koji and moromi fermented with alcohol by adding yeast, by distillation using an atmospheric distillation method or a vacuum distillation method, etc. Is mentioned.

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 to prepare a preparation by a conventional method.
In addition, by using a promoter region of a gene such as starch-degrading enzyme of Aspergillus oryzae, it is possible to produce a desired heterologous protein at high production in the Aspergillus culture.

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

Example 1 (Addition of inorganic nitrogen in the production of liquid soot)
The effect of adding potassium nitrate as an inorganic nitrogen to the liquid medium was examined by the following method.
In other words, the raw brown wheat used as a raw material becomes 2% (w / vol) in water added with no addition (control), 0.2% (w / vol) or 0.4% (w / vol) potassium nitrate. Three types of liquid media added to were prepared.
100 ml of each liquid medium is placed in a 500 ml baffled Erlenmeyer flask, sterilized by autoclave and then pre-cultured in liquid medium (Aspergillus)
Kawachii IFO4308) was inoculated to 1% (v / vol) with respect to the liquid medium. The brown barley used was an Australian Stirling 95% polished white (the following examples are also basically the same).

Thereafter, the cells were cultured for 48 hours at a temperature of 37 ° C. and a shaking speed of 100 rpm. After completion of the culture, the activities of glucoamylase and acid-resistant α-amylase were measured for each of the obtained cultures. Table 1 and FIG. 1 show the activities of glucoamylase and acid-resistant α-amylase in the cultures obtained by culturing koji molds in liquid media according to the amount of potassium nitrate used.
In addition, the saccharification power fractionation determination kit (made by Kikkoman) was used for the measurement of the enzyme activity of glucoamylase. In addition, the enzyme activity of acid-resistant α-amylase was measured by slightly improving the method described in Non-Patent Document 7, inactivating the non-acid-resistant α-amylase by acid treatment of the culture, Acid resistance α-amylase activity was measured using an amylase measurement kit (manufactured by Kikkoman). More specifically, 9 ml of 100 mM acetate buffer (pH 3) was added to 1 ml of the culture solution, and acid treatment was performed at 37 ° C. for 1 hour, followed by measurement using an α-amylase measurement kit (manufactured by Kikkoman).

  As shown in Table 1 and FIG. 1, the cultures obtained by adding potassium nitrate, which is an inorganic nitrogenous substance, to the liquid medium and cultivating both the 0.2% added group and the 0.4% added group compared to the target group without addition. It can be seen that the activities of both amylase and acid-resistant α-amylase are greatly increased, and that the balance between glucoamylase and acid-resistant α-amylase is good.

Example 2 (Addition of a plurality of inorganic substances in the production of liquid soot)
Next, the effect of adding a plurality of inorganic substances was examined as follows.
That is, potassium nitrate or sodium nitrate as an inorganic nitrogen substance and potassium dihydrogen phosphate as an inorganic salt were added to water in the formulation shown in Table 2. The amount of sodium nitrate added was calculated from 20 mM, which is a molar concentration corresponding to 2.0% potassium nitrate, and 1.7% was blended so that the nitrate ion concentrations were equal. As a control, inorganic nitrogen and water containing no inorganic salt were used.
Four types of liquid culture media were prepared by adding the raw wheat as a culture raw material to 2% (w / vol) to the raw material water prepared as described above, and liquid culture of white koji molds under the same conditions as in Example 1 Was done. Thereafter, glucoamylase activity and acid resistant α-amylase activity were measured in the same manner as in Example 1. The results are shown in Table 2 and FIG.

  As shown in Table 2 and FIG. 2, the activity of both enzymes, glucoamylase and acid-resistant α-amylase, increased when inorganic nitrogen and inorganic salt were added as compared to the control group without addition.

Example 3 (Addition of yeast cells or yeast autolysate in the production of liquid koji)
A liquid koji was produced using a liquid medium supplemented with yeast cells or yeast autolysate (yeast extract).

(1) Preparation of yeast cell to be added or yeast autolysate The brewer's yeast recovered from the beer brewing process is treated under the following conditions to beer yeast cells and yeast autolysate (1 ), (2).

Yeast cell: beer yeast cell autolysate obtained by dehydrating beer yeast to a water content of about 70% by centrifugation at 5,000 × g for 15 minutes (1): equivalent amount of water Yeast autolysate obtained by treatment at 52 ° C. for 18 hours after suspension in yeast (2): beer yeast cells suspended in an equal amount of 1% lactic acid and then treated at 52 ° C. for 18 hours Yeast autolysate obtained

(2) Preparation of liquid koji using liquid medium supplemented with yeast The prepared yeast cells and yeast autolysates (1) to (2) were 0.20%, 0.50%, 1% (v), respectively. / Vol) was added to water to prepare raw water. A liquid medium was prepared by adding cultivated raw barley to these raw waters to 2% (w / vol), and then liquid culture of white koji molds was performed under the same conditions as in Example 1. Thereafter, glucoamylase activity and acid-resistant α-amylase activity were measured in the same manner as in Example 1.

  On the other hand, as a control group, liquid culture (No. 1) prepared by adding only 2% (w / vol) of brown barley to water was inoculated with white birch and liquid culture was performed as in Example 1. The glucoamylase activity and acid-resistant α-amylase activity of the obtained liquid koji were measured in the same manner. The results are shown in Table 3 and FIG.

＊：単位はｖ／ｖｏｌ

*: Unit is v / vol

(3) Results As shown in Table 3 and FIG. 3, both the test group to which the yeast cells themselves were added and the test group to which the yeast autolysate was added were more glucosylated than the non-added control group (No. 1). Both amylase activity and acid-resistant α-amylase activity are increased. In particular, the No. 7 test plot showed good results. Moreover, in any test section, glucoamylase activity and acid-resistant α-amylase activity are increased in proportion to the amount of yeast cells or yeast autolysate added.

Example 4 (Addition of a combination of inorganic nitrogen and / or inorganic salt and yeast)
Potassium nitrate, potassium dihydrogen phosphate and yeast cells were combined as shown in Table 4 and added to water to prepare raw water. The yeast cells used were yeast cells themselves (yeast cells of Example 3) obtained by dehydrating beer yeast recovered from the beer brewing process to a water content of about 70% by centrifugation. As a control (No. 1), additive-free raw water was used.
Using the liquid medium prepared by adding 2% (w / vol) of barley to the raw water prepared in the combination of Table 4, inoculating white birch fungi in the same manner as in Example 1, and performing liquid culture, The glucoamylase activity and acid-resistant α-amylase activity were measured. The results are shown in Table 4 and FIG.

  From Table 4 and FIG. 4, it can be seen that in all the test groups, both the glucoamylase activity and the acid-resistant α-amylase activity are much higher than the control group without addition (No. 1). In particular, the test groups No. 15 to 18 had very high activity for both enzymes. From this, it is considered that the nutrient balance in the liquid medium was improved by the combined use of inorganic nitrogen and / or inorganic salt and yeast cells, and enzyme production by filamentous fungi was actively performed.

Example 5 (combination of barley meal, yeast cells, inorganic substances)
Barley koji, yeast cells, potassium nitrate, and potassium dihydrogen phosphate were combined as shown in Table 5 and added to water to obtain raw material water for the liquid medium. The barley koji used was recovered from the koji process of 70% refined barley (Australian Stirling) and contains barley husk and koji. Moreover, the used yeast cell is the yeast cell itself (yeast cell of Example 3) which dehydrated the beer yeast collect | recovered from the beer brewing process to about 70% of water content by centrifugation. On the other hand, additive-free raw water was used as a control.
Using the liquid medium prepared by adding 2% (w / vol) of barley to the raw water prepared in the combination of Table 5, inoculated with white birch fungi in the same manner as in Example 1, Glucoamylase activity and acid resistant α-amylase activity were measured in the same manner. The results are shown in Table 5 and FIG.

○：硝酸カリウム０．２％（ｗ／ｖｏｌ）とリン酸２水素カリウム０．３％（ｗ／ｖｏｌ）を添加した試験区

○: Test group to which potassium nitrate 0.2% (w / vol) and potassium dihydrogen phosphate 0.3% (w / vol) were added

From Table 5 and FIG. 5, it can be seen that, in all the test groups, both the glucoamylase activity and the acid-resistant α-amylase activity are much higher than the control group without addition (No. 1). In particular, in test group No. 4, both enzymes were very active and well balanced. From this, it is considered that the nutritional balance in the liquid medium was improved by the combined use of barley koji and yeast cells, and enzyme production by filamentous fungi was actively performed.
In addition, combined use (No. 8, No. 9) of barley koji or yeast cells with inorganic nitrogen compounds and inorganic salts also showed good results.

Example 6 (Addition of a combination of barley husk and yeast cells)
Potassium nitrate, potassium dihydrogen phosphate, barley husk and yeast cells were combined as shown in Table 6 and added to the raw water to obtain raw water for the liquid medium. The barley husk used was obtained by passing the barley husk obtained from the milling process of 70% white barley through a 2 mm mesh sieve and collecting only the barley husk. Moreover, the used press yeast is the yeast cell itself (yeast cell of Example 3) which dehydrated the beer yeast collect | recovered from the beer brewing process to about 70% of water content by centrifugation. On the other hand, as a control, additive-free raw water (No. 1) was used.
Using the liquid medium prepared by adding 2% (w / vol) of barley to the raw water prepared in the combination of Table 6, inoculated with white birch fungi in the same manner as in Example 1 and obtained by liquid culture The glucoamylase activity and acid-resistant α-amylase activity of the liquid koji were measured. The results are shown in Table 6 and FIG.

  From Table 6 and FIG. 6, it can be seen that both the glucoamylase activity and the acid-resistant α-amylase activity are much higher in all the test groups than in the control group without addition (No. 1). In particular, in test plots Nos. 6-9, both enzymes were very active and well balanced. From this, it is considered that the nutritional balance in the liquid medium was improved by the combined use of barley husk and yeast cells, and enzyme production by filamentous fungi was actively performed.

Example 7 (Sulfate addition effect in liquid koji production)
Liquid rice cakes were produced by the following method, and their enzyme activities were measured.
1. Pre-culture method
8 g of 65% white wheat (Australian Stirling) and 100 ml of water were put into a 500 ml Erlenmeyer flask with a baffle, and autoclaved at 121 ° C. for 15 minutes. After standing to cool, this preculture medium was inoculated with 1 × 10 ⁶ white aspergillus (Aspergillus kawachii NBRC4308) / ml and shake-cultured at 37 ° C. for 24 hours at 100 rpm to prepare a preculture solution.

2. Main culture method
98% refined wheat (brown wheat, Australian Stirling) 2.0% (w / vol), potassium nitrate 0.2% (w / vol), potassium dihydrogen phosphate 0.3% (w / vol), magnesium sulfate 100 ml of a liquid medium of 5 test sections containing 0.1% (w / vol) of heptahydrate and 0.082 (w / vol) of magnesium chloride hexahydrate in the composition ratio shown in Table 7 was prepared. These liquid media were put in a 500 ml baffled conical flask and autoclaved at 121 ° C. for 15 minutes.
After standing to cool, 1 ml of the preculture was inoculated into this main culture medium, and cultured with shaking at 37 ° C. for 48 hours at 100 rpm. The amount of magnesium chloride hexahydrate added was calculated from 8.12 mM, which is a molar concentration corresponding to 0.1% of magnesium sulfate heptahydrate, so that the magnesium concentration in the medium of each test group was equal. Blended into

3. Enzyme activity measurement method After completion of the culture, glucoamylase activity (GA) and acid-resistant α-amylase activity (ASAA), which are amylolytic enzymes, were measured.
The glucoamylase activity (GA) was measured using a saccharification power fractionation kit (manufactured by Kikkoman).
Measurement of acid-resistant α-amylase activity (ASAA) is <Sudo S. et al: J. Ferment. Bioeng., 76, 105-110 (1993), Sudo S. et al: J. Ferment. Bioeng., 77, 483 -489 (1994), Shigetoshi Sudo et al .: Japan Brewing Association Journal., 89, 768-774 (1994)> slightly improved, non-acid-resistant α-amylase activity by acid treatment of the culture After inactivation, an α-amylase measurement kit (manufactured by Kikkoman) was used. More specifically, 9 ml of 100 mM acetate buffer (pH 3) was added to 1 ml of the culture solution, and after acid treatment at 37 ° C. for 1 hour, measurement was performed using an α-amylase measurement kit (manufactured by Kikkoman).

Cellulase activity (CEL), which is a cellulolytic enzyme, and acid carboxypeptidase activity (ACP), which is one of proteolytic enzymes, were also measured at the same time.
Cellulase activity (CEL) was determined by quantifying the amount of reducing sugar produced by hydrolysis using carboxymethylcellulose (CMC) as a substrate by the dinitrosalicylic acid (DNS) method. More specifically, 1 ml of 1% CMC substrate solution (dissolved by Sigma low viscosity (trade name) in 100 mM acetate buffer (pH 5)) was added to 1 ml of the culture solution, and the enzyme was accurately incubated at 40 ° C. for 10 minutes. After the reaction, add 4 ml of DNS reagent (including 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% sodium potassium tartrate tetrahydrate, 0.3% lactose monohydrate) and mix well. Stopped. In order to quantify the amount of reducing sugar contained in the reaction stop solution, the reaction stop solution was accurately heated in a boiling water bath for 15 minutes. Subsequently, after cooling to room temperature, the amount of reducing sugar corresponding to glucose was quantified by measuring the absorbance at 540 nm. One unit of cellulase activity (CEL) was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 μmol of glucose per minute.
Acid carboxypeptidase activity (ACP) was measured using an acid carboxypeptidase measurement kit (manufactured by Kikkoman).
The measurement results are shown in FIG.

4). Result As shown to FIG. 7 (A), the glucoamylase activity rose notably in the test group 2 which is a magnesium sulfate addition group. Moreover, as shown in FIG.7 (C) and FIG.7 (D), in the test group 2 which is a magnesium sulfate addition group, cellulase and acidic carboxypeptidase activity also rose. On the other hand, activity did not increase in test group 3 to which magnesium chloride, which is the same magnesium salt, was added, suggesting that the main body of these enzyme productivity increasing effects is sulfate groups.
In addition, in the test sections 4 and 5 in which magnesium sulfate was added but potassium nitrate or potassium dihydrogen phosphate was deficient, the effect of increasing enzyme productivity was not confirmed. It was found that enzyme productivity was significantly improved when both salts were included.

In this way, by culturing koji mold using cereals (brown wheat) whose surface is covered with husks and a liquid medium containing nitrate, phosphate and sulfate, glucoamylase and acid-resistant α-amylase, etc. In addition to the enzyme group necessary for the production of shochu, etc., a liquid koji can be produced in which cellulase, which is a cellulose-degrading enzyme, and acidic carboxypeptidase, which is a proteolytic enzyme, are produced at the same time.
High production of cellulose-degrading enzyme can be expected to reduce mash viscosity and increase alcohol yield in shochu production, and high production of proteolytic enzyme will increase the amino acid component of shochu mash. It also becomes possible to produce shochu with a gorgeous scent.
In addition, by the method of the present invention, it is highly likely that enzyme groups produced by koji molds such as starch-degrading enzyme groups, cellulose-degrading enzyme groups, and proteolytic enzyme groups other than the enzymes measured this time are generally produced at high yield I think that the.

According to the present invention, not only can the productivity of amylolytic enzymes in liquid koji be remarkably improved, but also a liquid koji with high production of cellulolytic enzymes and proteolytic enzymes can be produced. In addition, since liquid culture allows stricter culture control than solid culture, a liquid koji with stable quality can be produced efficiently and inexpensively.
By using the liquid koji produced according to the present invention for the production of fermented foods and beverages such as shochu, the alcohol yield and the amount of amino acids produced are increased, and a gorgeous flavored fermented food and beverages can be efficiently produced.
Moreover, since the cereals used in the present invention are not whitened or have been at least polished to the extent that the husk is left on the surface of the grain, an improvement in raw material utilization rate and yield can be expected.

The activity of glucoamylase and acid-resistant α-amylase of a culture obtained by culturing in a liquid medium using potassium nitrate as a nitrogen source is shown. The black bars indicate glucoamylase activity (U / ml), and the white bars indicate acid-resistant α-amylase activity (U / ml). The activity of glucoamylase and acid-resistant α-amylase of the culture obtained by culturing in a liquid medium using inorganic nitrogen and inorganic salt is shown. The black bars indicate glucoamylase activity (U / ml), and the white bars indicate acid-resistant α-amylase activity (U / ml). The activity of glucoamylase and acid-resistant α-amylase of a culture obtained by culturing in a liquid medium using yeast cells or yeast autolysate as a nitrogen source is shown. The black bars indicate glucoamylase activity (U / ml), and the white bars indicate acid-resistant α-amylase activity (U / ml). The activity of glucoamylase and acid-resistant α-amylase of a culture obtained by culturing in a liquid medium using a combination of inorganic nitrogen, inorganic salt and yeast cells is shown. Moreover, a black bar shows glucoamylase activity (U / ml) and a white bar shows acid-resistant α-amylase activity (U / ml). The activity of glucoamylase and acid-resistant α-amylase of a culture obtained by culturing in a liquid medium using a combination of barley meal, yeast cells and inorganic nitrogen as a nitrogen source is shown. The black bars indicate glucoamylase activity (U / ml), and the white bars indicate acid-resistant α-amylase activity (U / ml). The activity of glucoamylase and acid-resistant α-amylase of a culture obtained by culturing in a liquid medium using a combination of barley husk and yeast cells as a nitrogen source is shown. The black bars indicate glucoamylase activity (U / ml), and the white bars indicate acid-resistant α-amylase activity (U / ml). It is a graph which shows the various enzyme activities in the koji mold culture obtained by culture | cultivating with the liquid culture medium which uses a sulfate, nitrate, and a phosphate. (A) is glucoamylase (GA), (B) is acid-resistant α-amylase (ASAA), (C) is cellulase (CEL), (D) is acid carboxypeptidase (ACP) activity (U / ml). Each is shown.

Claims (19)

Bacillus subtilis in a liquid medium containing one or more nitrogen sources selected from cereals whose entire surface is covered with cereals and potassium nitrate, sodium nitrate, yeast cells, processed yeast cells, cereal husks and cereals Alternatively, a method for producing a liquid koji having enhanced enzyme activity, comprising culturing black koji mold.   The method for producing a liquid koji with enhanced enzyme activity according to claim 1, wherein the enzymes are at least glucoamylase and acid-resistant α-amylase. The liquid medium is a cereal whose entire surface is covered with husk, one or two selected from potassium nitrate and sodium nitrate, one or two selected from potassium dihydrogen phosphate and ammonium phosphate, The method for producing a liquid koji with enhanced enzyme activity according to claim 1, comprising one to three kinds selected from magnesium sulfate heptahydrate, iron sulfate heptahydrate and ammonium sulfate. . The liquid medium having enhanced enzyme activity according to claim 3 , wherein the liquid medium contains one or two kinds selected from potassium nitrate and sodium nitrate at a concentration of 0.1 to 2.0% (w / vol). Production method. The enhancement of enzyme activity according to claim 3 , wherein the liquid medium contains one or two kinds selected from potassium dihydrogen phosphate and ammonium phosphate at a concentration of 0.05 to 1.0% (w / vol). Method for producing a liquid bowl. Liquid medium, the magnesium sulfate heptahydrate to claim 3 in a concentration of from one not selected from iron sulfate heptahydrate and ammonium sulfate Three 0.01~0.5% (w / vol) A method for producing a liquid koji with enhanced enzyme activity.   2. The enzyme activity according to claim 1, wherein the cereals having the entire surface covered with husk are brown rice, rice with all or part of rice husks, and wheat with an unpolished to polished ratio of 92% or more. A method for producing a liquid bag.   2. The method for producing a liquid koji with enhanced enzyme activity according to claim 1, wherein the white mold is Aspergillus kawachi.   The method for producing a liquid koji with enhanced enzyme activity according to claim 1, wherein the black koji mold is one or two selected from Aspergillus awamori and Aspergillus niger. The method for producing a liquid koji with enhanced enzyme activity according to claim 1, wherein the cereal is barley. In a liquid medium containing one or more nitrogen sources selected from cereals whose entire surface is covered with cereals and potassium nitrate, sodium nitrate, yeast cells, processed yeast cells, cereals cereals and cereal meals, Alternatively, a method for producing at least one enzyme selected from glucoamylase and acid-resistant α-amylase, wherein the enzyme is produced by culturing black koji mold. Cereals whose entire surface is covered with husk, one or two selected from potassium nitrate and sodium nitrate, one or two selected from potassium dihydrogen phosphate and ammonium phosphate, and magnesium sulfate 7 water A glucoamylase, acid-resistant, characterized in that an enzyme is produced by culturing white or black koji molds in a liquid medium containing one or three kinds selected from Japanese, iron sulfate heptahydrate and ammonium sulfate A method for producing at least one enzyme selected from sex α-amylase, cellulase and acid carboxypeptidase. The method for producing an enzyme according to claim 12 , wherein the liquid medium contains one or two selected from potassium nitrate and sodium nitrate at a concentration of 0.1 to 2.0% (w / vol). The method for producing an enzyme according to claim 12 , wherein the liquid medium contains one or two selected from potassium dihydrogen phosphate and ammonium phosphate at a concentration of 0.05 to 1.0% (w / vol). . Liquid medium, the magnesium sulfate heptahydrate to claim 12 containing in a concentration of from one not selected from iron sulfate heptahydrate and ammonium sulfate Three 0.01~0.5% (w / vol) A method for producing the described enzyme. The method for producing an enzyme according to claim 11 or 12 , 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 to unpolished ratio of 92% or more. . The method for producing an enzyme according to claim 11 or 12 , wherein the white mold is Aspergillus kawachi. The method for producing an enzyme according to claim 11 or 12 , wherein the black koji mold is one or two selected from Aspergillus awamori and Aspergillus niger. The method for producing an enzyme according to claim 11 or 12, wherein the cereal is barley.

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