JP4906648B2 - Method for producing filamentous fungus culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling the productivity of an enzyme, in particular, a starch digesting enzyme, a vegetable fiber digesting enzyme or a protease of a fungal culture by controlling the speed of releasing a nutrient in culture materials into the culture system for the production of the fungal culture by culturing a fungus in a liquid medium containing cereals. <P>SOLUTION: Disclosed is a method for producing a fungal culture, wherein the productivity of an enzyme in the fungal culture is controlled by culturing the fungus while controlling the speed of releasing the nutrient of the cereals into the culture system with controlling the milling rate of the cereals using a liquid medium containing cereals which whole surface is covered with husk and inorganic salts. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a filamentous fungus culture using a liquid medium, and in particular, by producing a filamentous fungus while culturing the filamentous fungus while controlling the release rate of nutrients in the culture raw material to the culture system. The present invention relates to a method for producing a filamentous fungus culture that adjusts its properties.

  For the production of fermented foods and beverages such as shochu, koji molds, which are a type of filamentous fungi, are used. The culture form is based on a solid culture method in which koji molds are grown on the cereal surface and is called a solid koji method. Such a method using a solid koji is a traditional production method, but can be said to be a method unsuitable for large-scale production because of a special culture form called solid culture.

  On the other hand, liquid koji, which is a koji mold culture obtained by culturing koji mold, is easy to control and can be said to be a culture method suitable for efficient production. It is well known that the enzymes necessary for the production cannot be sufficiently obtained (see Non-Patent Documents 1-4), and so far there have been few examples used in actual production.

  In enzyme production by liquid culture of filamentous fungi including koji molds, it is known that enzyme productivity is improved by controlling the concentration of nutrients such as glucose in the culture system low. Conventionally, the concentration of nutrients such as sugar has been reduced by a fed-batch culture method in which nutrients such as sugar are added little by little from outside the culture system, but the development of a simpler method has been desired (non- (See Patent Documents 5-6).

  We have developed a method for producing koji mold cultures that contain sufficient enzymes such as glucoamylase and acid-resistant α-amylase by culturing koji molds in a liquid medium using raw materials with husks and husks. Patent applications have already been filed (Japanese Patent Application No. 2004-350661, Japanese Patent Application No. 2004-352320, Japanese Patent Application No. 2004-352324, Japanese Patent Application No. 2004-378453, Japanese Patent Application No. 2005-290651). No. specification, see Japanese Patent Application No. 2005-290648). However, the enzyme high production mechanism of this production method, the method for adjusting the enzyme productivity of the koji mold culture based thereon, and the method for adjusting the enzyme productivity in filamentous fungi other than koji molds are not yet known.

  On the other hand, a method of liquefying uncooked starch using an enzyme solution obtained by culturing a new Cortycium sp. Strain having a particularly high uncooked starch saccharification ability in a liquid medium containing an uncooked raw material and minerals (Patent Document 1) And a method for producing sake by making the enzyme solution act on an uncooked raw material (see Patent Document 2). However, the basidiomycete Cortecium spp. Is significantly different from the koji mold widely used for the production of fermented foods and drinks. Moreover, Patent Documents 1 and 2 describe that Aspergillus awamori and Rhizopus do not have sufficient saccharification. Furthermore, it is unclear whether the enzyme solution has sufficient acid-resistant α-amylase activity.

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) Bhargava S. et al: Biotechnol Bioeng., 82 (1), 111-7 (2003) Pedersen H. et al: Appl Microbiol Biotechnol., 53 (3): 272-7 (2000) Japanese Patent Publication No. 5-68237 Japanese Patent Publication No. 6-53059

  An object of the present invention is a liquid medium using at least one kind selected from cereals, beans, potatoes, amaranthus, and quinoa as a culture raw material, and the release rate of nutrients in the cereal as a culture raw material to the culture system. It is intended to provide a method for adjusting the productivity of filamentous fungal culture enzymes, particularly starch-degrading enzymes, plant fiber-degrading enzymes, and proteolytic enzymes, by culturing filamentous fungi while controlling them.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have adjusted the whitening rate when using a raw material with a husk as a raw material for a liquid medium, thereby adjusting glucose, which is one of nutrients. It has been found that the release rate of can be controlled into the medium. Moreover, as a result of culturing filamentous fungi using raw materials having different polishing ratios, it was found that enzyme productivity in the filamentous fungus culture can be controlled by polishing ratio.
In addition, the present inventors have cultivated koji molds in a liquid medium containing a starch raw material from which the husk or hull has been removed and not pregelatinized, so that glucoamylase necessary for the production of fermented food and drink and It has been found that a liquid soot containing a large amount of acid-resistant α-amylase can be produced. This is because even raw materials that are not encased in husks or husks are not easily degraded unless they are pregelatinized, and the release of sugars and amino acids into the culture system is suppressed, resulting in the necessary enzyme activity. It is expected to be.
The present inventors have completed the present invention based on these findings.

That is, the present invention according to claim 1 uses a liquid medium containing barley and an inorganic salt of a whitening ratio or higher that has been polished to the extent that the husk is left on the surface of the grain, and the nutrients in the barley It is a method for producing a filamentous fungus culture, characterized in that the enzyme productivity of the filamentous fungus culture is adjusted by culturing the filamentous fungus while controlling the release rate to the culture system.
The present invention according to claim 2, nutrient is an amino acid derived from a protein of sugar and / or barley derived starch in barley, a method for producing filamentous fungus culture product according to claim 1.
The present invention according to claim 3 is the method for producing a filamentous fungus culture according to claim 1, wherein the enzyme is at least one selected from starch degrading enzymes, plant fiber degrading enzymes, and proteolytic enzymes.
The present invention according to claim 4 is the method for producing a filamentous fungus culture according to claim 1, wherein the filamentous fungus is at least one selected from Neisseria gonorrhoeae, Trichoderma spp. And white rot fungi.

This invention which concerns on Claim 5 is a manufacturing method of the enzyme agent using the filamentous fungus culture obtained by the method of any one of Claims 1-4.
This invention which concerns on Claim 6 is a manufacturing method of fermented food / beverage products using the filamentous fungus culture obtained by the method of any one of Claims 1-4.
The present invention according to claim 7 is a method for producing an enzyme using the filamentous fungus culture obtained by the method according to any one of claims 1 to 4.

  According to the present invention, by controlling the release rate of nutrients in the culture raw material to the culture system, the nutrient concentrations of sugars and amino acids in the culture system are kept low, and the enzyme productivity of the filamentous fungus culture is adjusted. can do.

  According to the present invention, the release rate of glucose into the medium in the culture system could be controlled by adjusting the milling rate. As a result of producing a koji mold liquid culture using raw materials with different polishing ratios, it was found that the enzyme productivity of starch-degrading enzymes and plant fiber-degrading enzymes can be controlled by the polishing ratio. In addition, it has been found that the productivity of other enzymes produced by filamentous fungi other than koji molds can be controlled by the same method.

According to the present invention, a filamentous fungus culture containing a balance of glucoamylase and acid-resistant α-amylase necessary for the production of fermented foods and beverages such as shochu is produced using a raw material to which no husk or hull is attached. be able to.
Therefore, for example, when manufacturing wheat shochu and the like, the same raw material can be used in the liquid koji manufacturing process and the fermentation process, and the manufacturing cost can be reduced. In addition, barley hulls and straw may have an adverse effect on the quality of sake. Furthermore, since the raw material is used without being heated, it leads to energy saving.
Moreover, it becomes easy to make a variety of fermented foods and drinks by using a filamentous fungus culture produced by combining filamentous fungus cultures using various raw materials and filamentous strains.

By the method of the present invention, it is expected that the release rate of many nutrients contained in cereals such as various sugars and amino acids in addition to glucose is similarly controlled. Enzyme production that is subject to catabolite suppression by concentration is expected to be widely regulated.
In addition, it is highly applicable to heterogeneous protein production using promoter regions such as amylolytic enzyme genes.

  Further, according to the present invention, even in batch culture that is simpler than fed-batch culture, the concentration of nutrients such as sugar in the medium can be adjusted by adjusting the release rate of nutrients from the raw material already added in the culture system. Therefore, it is possible to achieve the same culture effect as fed-batch culture. Therefore, the culture method of the present invention is considered to be a new culture mode that has not been reported so far.

  Furthermore, since liquid culture enables stricter culture control than solid culture, according to the present invention, a filamentous fungus culture with stable quality can be produced at low cost.

Hereinafter, the present invention will be specifically described.
The present invention relates to a method for producing a filamentous fungus culture, and using a liquid medium containing cereals whose entire surface is covered with husks, while controlling the release rate of nutrients in the culture raw material to the culture system, the filamentous fungi Is characterized in that the enzyme productivity of the filamentous fungus culture is adjusted.

In the present invention, by controlling the release rate of nutrients contained in the culture raw material to the culture system, the concentration of nutrients such as sugars and amino acids in the culture system is kept low, and the enzyme activity in the filamentous fungus culture is enhanced. Is done.
As a means for controlling the release rate of nutrients in the culture raw material to the culture system, a method by adjusting the polishing ratio of cereals, a method using beans, mosses, amaranthus, or quinoa to which the husk is attached as a culture raw material, A method of using a culture raw material from which the outer skin has been removed but not pregelatinized, a method of adjusting the concentration of inorganic salt when heat-treating the liquid medium, and an edible film on the cereal surface is artificially formed, Examples include, but are not limited to, methods for protecting cereal starches and cereal proteins. It is also possible to control the nutrient release rate by suppressing physical disruption of the culture raw material in the culture solution. For example, by using a culture device with weak stirring shear force, the nutrient release rate can be controlled. Can be suppressed.

  Here, examples of the enzyme produced by the filamentous fungus include an enzyme group whose productivity is catabolite-suppressed depending on the concentration of a sugar such as glucose or a proteolysate such as an amino acid in the culture system. Specific examples include starch-degrading enzymes such as glucoamylase, acid-resistant α-amylase and α-amylase, plant fiber-degrading enzymes such as cellulase, xylanase and β-glucosidase, and proteases such as protease, peptidase and glutaminase. However, it is not necessarily limited to these.

In the present invention, the raw material for cultivation includes barley, rice, wheat, buckwheat, millet, millet, millet, cucumber, corn and other grains, soybeans, red beans and other beans, sweet potatoes and other cereals, amaranth, quinoa and other cereals Can be used.
Here, amaranthus is a general term for Amaranthaceae, 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 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.
These culture raw materials can be used alone or in combination of two or more. Moreover, the shape of these raw materials is not specifically limited.

The culture raw material is mixed with water to prepare a liquid medium.
The mixing ratio of the culture raw material is adjusted so that the target enzyme to be accumulated in the filamentous fungus culture is selectively generated and accumulated.
For example, in order to produce glucoamylase and acid-resistant α-amylase in a well-balanced and high production, when barley is used as a raw material, a liquid medium containing 1 to 20% (w / vol) of brown barley to water is used. Prepared. In addition, when unpolished barley is used as brown wheat, it is more preferably prepared in a liquid medium supplemented with 8 to 10% (w / vol). More preferably, it is prepared in a liquid medium supplemented with 1 to 4% (w / vol).
When the amount of brown barley used exceeds 20% (w / vol), the viscosity of the culture solution becomes high, and the supply of oxygen and air necessary for aerobic culture of filamentous fungi becomes insufficient, so oxygen in the culture This is not preferable because the concentration decreases and the culture becomes difficult to proceed.

  Next, when rice is used as a culture raw material, rice is 1 to 20% (w / vol), preferably 5 to 13% (w / vol), more preferably 8 to 10% (w / Vol) Prepared in added liquid medium.

  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 in which moss is added to 1 to 10% (w / vol) with respect to water.

  For example, when amaranth is used as a culture raw material, it is 1.5 to 15% (w / vol), preferably 2 to 10% (w / vol), more preferably 2 to 8% (w / Vol) Prepared in added liquid medium. On the other hand, in the case of quinoa, a liquid added with 1.5 to 7% (w / vol), preferably 2 to 6% (w / vol), more preferably 2 to 4% (w / vol) with respect to water. Prepared in medium.

  As described above, since the optimum blending amount varies depending on the target enzyme, the degree of whitening of the raw material used, the filamentous strain used, the type of raw material, and the like, it may be arbitrarily selected.

In addition to the aforementioned raw materials, it is preferable to add organic substances, inorganic salts, and the like as nutrient sources to the liquid medium.
For example, when using Aspergillus kawachii, etc. as a filamentous fungus, and Aspergillus awamori, Aspergillus niger, Aspergillus niger, etc., use nitrate and phosphate together. More preferably, a sulfate is used together with these. Here, sodium nitrate, potassium nitrate, or the like can be used as the nitrate, and potassium 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 also be used in combination of multiple types.

  The concentration of the inorganic salt in the liquid medium in the case of using the white koji mold or the black koji mold is adjusted so that glucoamylase and acid-resistant α-amylase are selectively generated and accumulated in the koji mold culture. The 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% (all are w / vol).

  In addition, when using yellow koji mold such as Aspergillus oryzae or Aspergillus sojae as a filamentous fungus, it is preferable to use nitrate, phosphate and sulfate in combination in a liquid medium. Here, sodium nitrate, potassium nitrate or the like 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 also be used in combination of multiple types.

  The concentration of the inorganic salt in the liquid medium in the case of using the yellow koji mold is adjusted to such a level that glucoamylase and acid-resistant α-amylase are selectively generated and accumulated in the koji mold culture. 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% (all are w / vol).

In the liquid medium in the present invention, organic substances other than the above-mentioned inorganic salts, inorganic salts, and the like can be added as a nutrient source as appropriate. 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 filamentous fungi, but is about 0.1 to 5% (w / vol) as an organic substance, 0.1 to 1% (w / vol) is preferably added.
When these nutrient sources are added in excess of the upper limit value, the growth of filamentous fungi is inhibited, which is not preferable. Moreover, when the addition amount is less than the lower limit, the enzyme is not sufficiently produced, which is also not preferable.
In addition to these nutrient sources, additives such as antibiotics and preservatives can be used in the liquid medium as necessary.

In the present invention, when using a heat-treated liquid medium, the release rate of nutrients in the culture raw material to the culture system can also be adjusted by appropriately adjusting the inorganic salt concentration of the liquid medium during the heat treatment. Can be controlled.
That is, it is possible to suppress the release of nutrients in the culture raw material into the culture system by increasing the inorganic salt concentration of the liquid medium during the heat treatment. This is considered to be because the physical collapse of the raw material is suppressed by heating the culture raw material in the presence of an inorganic salt.

Here, there is no restriction | limiting in particular as an inorganic salt, Although what is normally used for the liquid culture medium for filamentous fungi as mentioned above can be used, Nitrate, a phosphate, and a sulfate are more preferable.
Moreover, as an inorganic salt density | concentration in the liquid culture medium at the time of heat processing, it can be set as about 1 to 10 times the suitable density | concentration in an already described liquid culture medium.
If the inorganic salt concentration in the liquid medium during the heat treatment exceeds the concentration range suitable for the cultivation of filamentous fungi, dilute it after the heat treatment and adjust the inorganic salt concentration as appropriate, and then subject to culture. Can do.

  As said heat processing conditions, it is 80-130 degreeC, Preferably it is 100-121 degreeC, 5-120 minutes, Preferably it can be set as 10-30 minutes. In particular, it is preferable that the temperature is 110 to 121 ° C. for 5 to 20 minutes, which is a general condition for heat sterilization of a liquid medium using an autoclave or the like, because the medium can be sterilized at the same time.

  Next, the filamentous fungus is inoculated into the liquid medium. As the filamentous fungus used in the present invention, a filamentous fungus that produces an enzyme that is catabolite-suppressed by the concentration of nutrients such as sugars and amino acids in the culture system can be widely used. Aspergillus, Trichoderma, white rot fungus Ilpex lacteus can be exemplified. Specific examples of the genus Aspergillus include, for example, Aspergillus kawachii and the like Aspergillus, Aspergillus oryzae, Aspergillus sojae and Aspergillus sojae Examples include Aspergillus awamori, Aspergillus niger, Aspergillus such as Aspergillus niger, Aspergillus aculeatus, and the like. Specific examples of the genus Trichoderma include cellulase-producing bacteria such as Trichoderma viride and Trichoderma reesei.

These filamentous fungi can be used either by culturing with one kind of strain or mixed culturing with two or more kinds of the same or different kinds of strains. There is no problem whether these are used in the form of spores or hyphae obtained by preculture, but it is preferable to use hyphae because the time required for the logarithmic growth phase is shortened. The inoculation amount of the filamentous fungus into the liquid medium is not particularly limited, 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 the mycelia. It is preferable to inoculate about 10%.

  The culture temperature of the filamentous fungus 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 filamentous fungi is slowed down, and contamination with various germs is likely to occur. The culture time is preferably 24 to 120 hours. Any culture apparatus may be used as long as it can perform liquid culture. However, since filamentous fungi 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 in a culture medium, oxygen, and a filamentous fungus may distribute uniformly in an apparatus during culture | cultivation. 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.

In the method for producing a filamentous fungus culture described above, the present invention uses cereals that are at least partially or entirely covered with husks as a culture raw material, and adjusts the milling rate of the cereals to adjust the milling ratio in the cereals. It controls the release rate of nutrients into the culture system.
In the present invention, as the shape of the cereal, it is necessary that all or part of the surface is covered with at least the husk, and the unmilled product, or at least the husk is left on the surface of the grain. Those that are higher than the degree of whitening that has been polished to the extent can be used, and brown rice, brown wheat, etc. can also be used. For example, when the cereal is barley, the unpolished milling rate is 100%, or the unpolished milling rate is 100%, and the unpolished milling rate (100%) is used to obtain the barley grain ratio (general Is a ratio obtained by subtracting 7 to 8%), that is, a fineness ratio of about 92 to 93% or more.

  In the present invention, the rate of release of nutrients to the culture system is controlled by adjusting the grain polishing rate, thereby enhancing the enzyme activity in the filamentous fungus culture. Therefore, an optimum milling ratio is selected according to the enzyme to be produced, the type of raw material grain, and the like. For example, when producing glucoamylase or acid-resistant α-amylase, and using barley as a raw material, the milling ratio is 90 to 100%, preferably 98%. can do.

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

  Starch contained in cereals 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.

  The liquid medium of the present invention is prepared by mixing water, the above-described culture raw materials, and other medium components. The liquid medium may be sterilized as necessary, and the processing 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.

  In another aspect of the present invention, in the above method for producing a filamentous fungus culture, the nutrients in the culture raw material can be obtained by using a raw material from which the husk or hull has been removed and which has not been pregelatinized. Is to control the release rate into the culture system.

In the present invention, the culture raw material needs to have the husk or outer skin removed and not pregelatinized.
For example, when the culture raw material is barley, a ratio obtained by subtracting the grain ratio (generally 7 to 8%) from the unmilled whitening ratio (100%), that is, a polishing ratio of about 92 to 93% or less. Can be used, and can also be used barley (milling ratio 65%).
The culture raw material is not subjected to a treatment such as heating to gelatinize the starch, but may be subjected to a treatment such as threshing, milling, peeling, washing, shredding, shredding, freezing and the like as necessary.

In the present invention, the mixing ratio of the culture raw material in the liquid medium is such that glucoamylase and acid-resistant α-amylase are selectively generated and accumulated in the filamentous fungus culture. Specifically, 1 to 10% (w / vo1), preferably 2 to 6% (w / vo1) of the culture raw material may be added to the liquid medium. The optimum blending ratio differs depending on the condition, and may be appropriately selected in consideration of these.
When the amount of culture raw material used exceeds the upper limit, when using aerobic filamentous fungi, the viscosity of the culture solution becomes high, and supply of oxygen and air necessary for aerobic culture of filamentous fungi becomes insufficient, and the culture product This is not preferable because the oxygen concentration in the medium is lowered and the culture becomes difficult to proceed. On the other hand, if the usage-amount of this raw material is less than a lower limit, glucoamylase and acid-resistant alpha-amylase will not be produced highly.

The liquid medium of the present invention is prepared by mixing water, the above-described culture raw materials, and other medium components. At this time, if necessary, after the medium components other than the culture raw material are mixed with water and sterilized in advance, a starch raw material that has not been pregelatinized can be further added. Alternatively, a method in which a part of the culture raw material and other medium components are mixed with water and sterilized in advance, and then the remaining culture raw material that has not been pregelatinized can be further added.
The sterilization 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.

  By culturing filamentous fungi by the above-described culture method, a filamentous fungus culture in which the target enzyme is efficiently produced and accumulated can be obtained. In the present invention, the filamentous fungus culture includes, in addition to the cultured itself, a culture solution obtained by centrifuging the culture, a concentrate, a purified product, a dried product, and the like. .

As described above, according to the culture method described above, a group of enzymes whose productivity is catabolite-suppressed can be highly produced by the concentration of sugars such as glucose and proteolysates such as amino acids in the culture system.
Therefore, the method for producing the enzyme according to claim 7 is the same as the method for producing the filamentous fungus culture described above.

The filamentous fungus culture obtained by the present invention can be used not only for the production of fermented foods and beverages but also for the production of sugars, amino acids and derivatives thereof, enzyme agents, pharmaceutical digestive agents and the like. 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 brewed vinegar, in the preparation stage, when producing mirin, in the preparation stage, when producing amazake, in the preparation stage, the koji mold culture is Can be used instead. In addition, the fermentation raw material (hanging raw material) used for manufacture of these fermented food / beverage products may or may not be pregelatinized.
A part of the obtained filamentous fungus culture can also be used as a starter in the production of the next filamentous fungus culture. Thus, by continuously producing the filamentous fungus culture, stable production can be achieved, and at the same time, the production efficiency can be improved.

  As a method for producing an enzyme agent from a filamentous fungus culture in the present invention, the culture can be used as it is, or its filtrate, centrifugation supernatant or the like can be used as a liquid enzyme agent, or dried or fixed on a carrier by a conventional method. By doing so, it can also be set as a powdered and granular enzyme agent. At this time, an appropriate excipient or the like may be added as necessary.

  Moreover, when manufacturing fermented foods and drinks, such as liquor, using said filamentous fungus culture, all the processes can be performed in a liquid phase. For example, when producing shochu, corn, wheat, rice, potato, sugar cane, etc. are used as raw materials, and the raw materials are dissolved and liquefied using a heat-resistant enzyme agent at a high temperature of about 80 ° C. The mash that has been subjected to alcoholic fermentation by adding the koji mold culture and yeast can be produced by distillation using an atmospheric distillation method or a vacuum distillation method.

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

<Experimental example 1> Measurement of glucose release rate in barley with different milling ratio
Barley (Australian Stirling varieties) with different milling ratios of 65% to 98% were reacted with an enzyme derived from a koji mold culture, and the glucose release rate from barley was measured.

  Specifically, weigh 2g each of 65% white wheat, 83% white wheat, 92% white wheat, 95% white wheat, 98% white wheat and 98% white wheat, and add 50ml water to a 200ml Erlenmeyer flask. I put it in. This was sterilized with an autoclave (121 ° C., 15 minutes) to prepare a “barley substrate solution”.

Subsequently, the koji mold culture produced using barley (Australian Stirling) as a culture raw material was subjected to solid-liquid separation by filter paper filtration to obtain a “koji mold supernatant”.
The specific method for producing the koji mold culture used in this test was as follows.

1. Pre-culture method
8 g of 65% white wheat and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask, 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 barley becomes 2.0% (w / vol) in water to which potassium nitrate 0.2% (w / vol) and potassium dihydrogen phosphate 0.3% (w / vol) are added. A liquid medium added as described above was prepared. 3000 ml of the prepared liquid medium was put on a jar fermenter (manufactured by Maruhishi Bioengineering Co., Ltd.) with a capacity of 5000 ml, sterilized by autoclave (121 ° C., 15 minutes) and then pre-cultured in liquid medium by the above method (Aspergillus kawachii) NBRC4308) 30 ml was inoculated. Thereafter, the cells were cultured for 42 hours at a temperature of 37 ° C., a stirring speed of 300 rpm and an aeration rate of 0.5 vvm, and filtered through a filter paper (Toyo Filter Paper No. 2) to obtain a “Koji mold culture supernatant”.

3. Measurement method 50 ml of the barley substrate solution and 50 ml of the koji mold culture supernatant thus prepared were each incubated at 37 ° C. for 5 minutes, and then the reaction was started by mixing them. The glucose concentration in the reaction solution sampled 1 hour, 2 hours, 3 hours, and 4 hours after the start of the reaction was measured using Glucose C-II Test Wako (manufactured by Wako Pure Chemical Industries).

4). Results The time course of the glucose concentration in the reaction solution is as shown in FIG. 1, and it was confirmed that the amount of released glucose was different depending on the polishing ratio of barley used in the barley substrate solution. The bark percentage of barley is usually about 10%, and the 65% white and 83% white wheat used in this test have no skin on the surface. On the other hand, 92% refined wheat, 95% refined wheat and 98% refined wheat are in a state where the skin remains on the surface.
As shown in FIG. 1, in the test plots using 65% and 83% refined wheat and 98% refined wheat that had no husk on the surface, all showed high glucose concentrations, whereas 92% and 95% In the test plots using 100% and 98% polished wheat, it can be seen that the lower the polished ratio, the lower the glucose concentration. From this, it was shown that the amount of glucose released is adjusted by the presence of the husk.

FIG. 2 shows the calculated glucose release rate in the first hour of the reaction in this test. From this figure, it was confirmed that the lower the milling rate, the lower the glucose release rate is controlled.
On the other hand, in the test section using 98% polished white barley, the glucose release rate increased to the same level as that of 65% polished wheat, and the husks physically covered the barley starch. It was suggested that this was the main factor adjusting the speed.

<Example 1> Manufacture of birch fungus culture using barley with different milling ratio Using various milled barley (Australian Stirling), birch fungus culture was produced by the following method and their enzyme activity was measured. did.

1. Pre-culture method
8 g of 65% white wheat and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask, 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
2% of 65% white wheat, 83% white wheat, 92% white wheat, 95% white wheat, 98% white wheat, 0.2g potassium nitrate, 0.3g potassium dihydrogen phosphate and 100ml water with 500ml baffle The flask was placed in an Erlenmeyer 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.

3. Measurement method After completion of the culture, glucoamylase activity (GA), α-amylase activity (AA) and acid-resistant α-amylase activity (ASAA), which are amylolytic enzymes, were measured.
The glucoamylase activity (GA) was measured using a saccharification power fractionation quantification kit (manufactured by Kikkoman), and the α-amylase activity (AA) was measured using an α-amylase measurement kit (manufactured by Kikkoman). In addition, the 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 .: Journal of the Japan Brewing Association, 89, 768-774 (1994)>, a slightly improved method for acid-free α-amylase by acid treatment of the culture After the activity was deactivated, 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) and β-glucosidase activity (BGL), which are cellulolytic enzymes, were also measured simultaneously. 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 (Sigma low viscosity dissolved in 100 mM acetate buffer (pH 5)) is added to 1 ml of the culture solution, and the enzyme reaction is performed at 40 ° C. for exactly 10 minutes. 4 ml of DNS reagent (including dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3%) may be added. Mix and stop the reaction. 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.

β-glucosidase activity was measured by the following method. 1 mM p-nitrophenyl-β-D-glucopyranoside (PNPG) was used as a substrate, and the enzyme reaction was performed at 37 ° C. for exactly 10 minutes in 50 mM acetate buffer (pH 5). The amount of p-nitrophenol was quantified and the enzyme activity was calculated. The reaction was stopped by adding 200 mM sodium carbonate solution twice the amount of the reaction solution. One unit of activity was defined as the activity of releasing 1 μmol of glucose per minute.
The measurement results are shown in FIG. 3 and FIG.

4). Results As shown in FIG. 3, the productivity of starch-degrading enzymes increased as the polishing rate decreased. In addition, even when 98% polished wheat having a low milling rate was used, enzyme productivity was significantly reduced when pulverized. Furthermore, as shown in FIG. 4, it was confirmed that the productivity of the cellulolytic enzyme tends to increase as the milling rate decreases. Thus, the tendency of the enzyme productivity of the white koji mold culture to be inversely correlated with the glucose release rate shown in Experimental Example 1 is confirmed, and the enzyme productivity of the white koji mold culture can be controlled by changing the barley milling ratio. It has been shown.

<Example 2> Manufacture of black koji fungus culture using barley with different milling ratios Using various milled barley (Australian Stirling), black koji fungus cultures were produced by the following method and their enzyme activities were measured. did.

1. Pre-culture method
8 g of 65% white wheat and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask, and autoclaved at 121 ° C. for 15 minutes. After standing to cool, the preculture medium was inoculated with Aspergillus awamori NBRC4388 at 1 × 10 ⁶ cells / ml and cultured with shaking at 37 ° C. for 24 hours at 100 rpm to obtain a preculture solution.

2. Main culture method
2% of 65% white wheat, 83% white wheat, 92% white wheat, 95% white wheat, 98% white wheat, 0.2g potassium nitrate, 0.3g potassium dihydrogen phosphate and 100ml water with 500ml baffle The flask was placed in an Erlenmeyer 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.

3. Measurement method After completion of the culture, glucoamylase activity (GA), α-amylase activity (AA) and acid-resistant α-amylase activity (ASAA), which are amylolytic enzymes, were measured.
The glucoamylase activity (GA) was measured using a saccharification power fractionation quantification kit (manufactured by Kikkoman), and the α-amylase activity (AA) was measured using an α-amylase measurement kit (manufactured by Kikkoman). In addition, the 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 .: Journal of the Japan Brewing Association, 89, 768-774 (1994)>, a slightly improved method for acid-free α-amylase by acid treatment of the culture After the activity was deactivated, 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).
The measurement results are shown in FIGS.

4). Result As shown in FIGS. 5-7, the productivity of the amylolytic enzyme became higher as the milling ratio decreased. In addition, even when 98% polished wheat having a low milling rate was used, enzyme productivity was significantly reduced when pulverized. Thus, the enzyme productivity of the black koji mold culture was confirmed to have an inverse correlation with the glucose release rate shown in Experimental Example 1, and the enzyme productivity of the black koji mold culture can be controlled by changing the barley milling ratio. It has been shown.

<Example 3> Manufacture of koji mold culture using barley with different milling ratios Using various milled barley (Australian Stirling), koji mold culture was produced by the following method, and their enzyme activities were measured. did.

1. Culture method
65% white wheat, 83% white wheat, 92% white wheat, 95% white wheat, 98% white wheat 2g any 2g, sodium nitrate 1.2% (w / vol), potassium chloride 0.8% (w / Vol), potassium dihydrogen phosphate 0.4% (w / vol), magnesium sulfate heptahydrate 0.2% (w / vol), iron sulfate heptahydrate 0.08% (w / vol) Then, 100 ml of a medium containing water and water was put into a 500 ml baffled Erlenmeyer flask, and autoclaved at 121 ° C. for 15 minutes. After standing to cool, Aspergillus oryzae RIB40 was inoculated into this medium 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), which are amylolytic enzymes, were measured.
The glucoamylase activity (GA) was measured using a saccharification power fractionation quantification kit (manufactured by Kikkoman), and the α-amylase activity (AA) was measured using an α-amylase measurement kit (manufactured by Kikkoman).
The measurement results are shown in FIGS.

3. Results As shown in FIGS. 8 and 9, the productivity of amylolytic enzymes increased as the milling rate decreased. In particular, the activity of glucoamylase was significantly increased in 98% polished wheat. In addition, even when 98% polished wheat having a low milling rate was used, enzyme productivity was significantly reduced when pulverized. Thus, the enzyme productivity of the koji mold culture is greatly influenced by the glucose release rate shown in Experimental Example 1, and the enzyme productivity of the koji mold culture can be controlled by changing the barley milling ratio. Indicated.

<Example 4> Manufacture of a filamentous fungus (Trichoderma viride) culture using barley having different milling ratios Using various milled barley (Australian Stirling), filamentous fungi having the ability to produce cellulolytic enzymes by the following method (Trichoderma viride) cultures were produced and their enzyme activity was measured.

1. Pre-culture method Glucose 2%, yeast extract 0.5%, potassium nitrate 0.1%, potassium hydrogen phosphate 0.1%, ammonium sulfate 0.07%, magnesium sulfate heptahydrate 0.03%, calcium chloride 0 100 ml of a medium containing 0.02% (both w / vol) and water was put into a 500 ml baffled Erlenmeyer flask, and autoclaved at 121 ° C. for 15 minutes. After cooling, the preculture medium was inoculated with Trichoderma viride NBRC31137 at 1 × 10 ⁶ cells / ml and shake-cultured at 30 ° C. for 24 hours at 100 rpm to obtain a preculture solution. .

2. Main culture method: Refined barley 2%, Tryptone 0.08%, Ammonium sulfate 0.25%, Ammonium phosphate 0.1%, Calcium chloride 0.03%, Magnesium sulfate heptahydrate 0.03%, Potassium nitrate 0.12 100 ml of a medium containing% (both w / vol) and water was placed in a 500 ml Erlenmeyer flask equipped with a baffle and autoclaved at 121 ° C. for 15 minutes.
In addition, as said refined barley, what grind | pulverized 65% refined wheat, 83% refined wheat, 92% refined wheat, 95% refined wheat, 98% refined wheat, or 98% refined wheat is used.
After standing to cool, 10 ml of the preculture solution was inoculated into this main culture medium, and cultured with shaking at 30 ° C. for 90 hours at 100 rpm.

3. Measurement method After completion of the culture, cellulase activity (CEL), which is a cellulolytic enzyme, was measured. 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 (Sigma low viscosity dissolved in 100 mM acetate buffer (pH 5)) is added to 1 ml of the culture solution, and the enzyme reaction is performed at 40 ° C. for exactly 10 minutes. 4 ml of DNS reagent (including dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3%) may be added. Mix and stop the reaction. 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.

4). Results The measurement results are shown in FIG. Also in Trichoderma viride, which is a filamentous fungus other than Neisseria gonorrhoeae, it was confirmed that a difference in cellulase productivity was caused by the polishing rate. The highest enzyme productivity was obtained when 98% polished barley was used, but the enzyme activity was significantly reduced in the pulverized product, suggesting that the effect of inhibiting the release of nutrients by barley husk contributes to high cellulase production. It was.

<Example 5> Manufacture of filamentous fungus (Trichoderma reesei) culture using barley with different milling ratio Using various milled wheat (Australian Stirling), filamentous fungus having cellulose-degrading enzyme-producing ability as follows. (Trichoderma reesei) cultures were prepared and their enzyme activity was measured.

1. Culture method (1) Pre-culture method Glucose 2%, yeast extract 0.5%, potassium nitrate 0.1%, potassium hydrogen phosphate 0.1%, ammonium sulfate 0.07%, magnesium sulfate heptahydrate 0.03 %, Calcium chloride 0.02% (both w / vol) and water (100 ml) were placed in a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. After standing to cool, Trichoderma ressei NBRC31326 was inoculated to this preculture medium at 1 × 10 ⁶ cells / ml, and cultured with shaking at 30 ° C. for 72 hours at 100 rpm to obtain a preculture solution. .

(2) Main culture method Refined barley 2%, tryptone 0.08%, ammonium sulfate 0.25%, ammonium phosphate 0.1%, calcium chloride 0.03%, magnesium sulfate heptahydrate 0.03%, potassium nitrate 100 ml of a medium containing 0.12% (both w / vol) and water was put into a 500 ml baffled Erlenmeyer flask, and autoclaved at 121 ° C. for 15 minutes.
In addition, what grind | pulverized 65% refined barley, 83% refined wheat, 95% refined wheat, 98% refined wheat, or 98% refined wheat as said refined barley was used.
After allowing to cool, 10 ml of the preculture was inoculated into the main culture medium, and cultured with shaking at 30 ° C. for 96 hours at 100 rpm.

2. Enzyme activity measurement method After completion of the culture, the supernatant was collected from the culture broth by centrifugation, and the activity of the plant fiber-degrading enzyme in the supernatant was measured.

(1) Cellulase activity measurement method 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 (Sigma low viscosity dissolved in 100 mM acetate buffer (pH 5)) is added to 1 ml of the culture solution, and the enzyme reaction is performed at 40 ° C. for exactly 10 minutes. 4 ml of DNS reagent (including dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3%) may be added. Mix and stop the reaction. 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.

(2) Xylanase Activity Measurement Method Next, xylanase activity (XYL) was quantified by increasing the absorbance at 540 nm by reacting reducing sugars produced by enzymatic hydrolysis using oat spelts-derived xylan as a substrate. More specifically, 1% xylan substrate solution [Sigma Xylan, from oat spelts dissolved in 200 mM acetic acid buffer (pH 4.5)] 0.1 ml of culture solution was added to 1.9 ml, and exactly 10 at 40 ° C. After performing the enzyme reaction for 1 minute, it contains DNS reagent (dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3% 4 ml was added and mixed well to stop the reaction. 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 xylose was quantified by measuring the absorbance at 540 nm. One unit of xylanase activity was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 μmol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes.

(3) β-glucosidase activity measurement method β-glucosidase activity (BGL) was measured by the following method. 1 mM p-nitrophenyl-β-D-glucopyranoside (PNPG) was used as a substrate, and the enzyme reaction was performed at 37 ° C. for exactly 10 minutes in 50 mM acetate buffer (pH 5). The amount of p-nitrophenol was quantified and the enzyme activity was calculated. The reaction was stopped by adding 200 mM sodium carbonate solution twice the amount of the reaction solution. One unit of activity was defined as the activity of releasing 1 μmol of glucose per minute.

3. Results The measurement results are shown in FIGS. Also in Trichoderma reesei, which is a filamentous fungus other than Aspergillus, it was confirmed that there was a difference in plant fiber-degrading enzyme productivity due to milling ratio. High enzyme productivity was obtained when 95% or 98% polished wheat was used, but the enzyme activity was significantly reduced in 98% polished wheat and crushed products. It was suggested that it contributes to high production.

<Example 6> Production of filamentous fungus (Aspergillus accumulator) culture using barley with different milling ratio Using various milled wheat (Australian Stirling), filamentous fungi having the ability to produce cellulolytic enzyme by the following method (Aspergillus accumulator) cultures were produced and their enzyme activity was measured.

1. Culture method (1) Pre-culture method Glucose 2%, yeast extract 0.5%, potassium nitrate 0.1%, potassium hydrogen phosphate 0.1%, ammonium sulfate 0.07%, magnesium sulfate heptahydrate 0.03 %, Calcium chloride 0.02% (both w / vol) and water (100 ml) were placed in a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. After allowing to cool, Aspergillus aculeatus NBRC3530 was inoculated into this preculture medium at 1 × 10 ⁶ cells / ml and shake-cultured at 30 ° C. for 72 hours at 100 rpm to obtain a preculture solution. .

(2) Main culture method Refined barley 2%, tryptone 0.08%, ammonium sulfate 0.25%, ammonium phosphate 0.1%, calcium chloride 0.03%, magnesium sulfate heptahydrate 0.03%, potassium nitrate 100 ml of a medium containing 0.12% (both w / vol) and water was put into a 500 ml baffled Erlenmeyer flask, and autoclaved at 121 ° C. for 15 minutes.
In addition, what grind | pulverized 65% refined barley, 83% refined wheat, 95% refined wheat, 98% refined wheat, or 98% refined wheat as said refined barley was used.
After allowing to cool, 10 ml of the preculture was inoculated into the main culture medium, and cultured with shaking at 30 ° C. for 96 hours at 100 rpm.

2. Enzyme activity measurement method After completion of the culture, the supernatant was collected from the culture broth by centrifugation, and the activity of the plant fiber-degrading enzyme in the supernatant was measured.

(1) Cellulase activity measurement method 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 (Sigma low viscosity dissolved in 100 mM acetate buffer (pH 5)) is added to 1 ml of the culture solution, and the enzyme reaction is performed at 40 ° C. for exactly 10 minutes. 4 ml of DNS reagent (including dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3%) may be added. Mix and stop the reaction. 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.

(2) β-glucosidase activity measurement method β-glucosidase activity (BGL) was measured by the following method. 1 mM p-nitrophenyl-β-D-glucopyranoside (PNPG) was used as a substrate, and the enzyme reaction was performed at 37 ° C. for exactly 10 minutes in 50 mM acetate buffer (pH 5). The amount of p-nitrophenol was quantified and the enzyme activity was calculated. The reaction was stopped by adding 200 mM sodium carbonate solution twice the amount of the reaction solution. One unit of activity was defined as the activity of releasing 1 μmol of glucose per minute.

3. Results The measurement results are shown in FIGS. It was also confirmed that plant fiber-degrading enzyme productivity was different depending on the polishing rate even in Aspergillus accumulator, which is a filamentous fungus other than Neisseria gonorrhoeae. When 98% polished barley was used, high enzyme productivity was obtained. On the other hand, the CEL activity and BGL activity decreased in the pulverized product, suggesting that the effect of inhibiting the release of nutrients by barley husk contributes to the high production of these enzymes.

<Example 7> Manufacture of white rot fungus culture using barley with different milling ratios Using various milled barley (Australian Stirling), a white rot fungus culture having cellulolytic enzyme producing ability was prepared as follows. Produced and their enzyme activity was measured.

1. Culture method (1) Pre-culture method Glucose 2%, yeast extract 0.5%, potassium nitrate 0.1%, potassium hydrogen phosphate 0.1%, ammonium sulfate 0.07%, magnesium sulfate heptahydrate 0.03 %, Calcium chloride 0.02% (both w / vol) and water (100 ml) were placed in a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. After standing to cool, 30 5 mm square mycelial mats of Irpex lacteus NBRC5367 were inoculated into the preculture medium, and cultured with shaking at 28 ° C. for 96 hours at 120 rpm to obtain a preculture solution.

(2) Main culture method White barley 2%, polypeptone 0.1%, ammonium sulfate 0.14%, potassium dihydrogen phosphate 0.2%, urea 0.03%, magnesium sulfate heptahydrate 0.03%, A medium containing 100% calcium chloride, 0.1% Tween 80 (w / vol) and water was placed in a 500 ml baffled Erlenmeyer flask, and autoclaved at 121 ° C. for 15 minutes.
In addition, what grind | pulverized 65% polished wheat, 83% polished wheat, 98% polished wheat, or 98% polished wheat was used as said polished wheat.
After allowing to cool, 10 ml of the preculture was inoculated into this main culture medium, and cultured with shaking at 120 rpm at 28 ° C. for 96 hours.

2. Enzyme activity measurement method After completion of the culture, the supernatant was collected from the culture broth by centrifugation, and the activity of the plant fiber-degrading enzyme in the supernatant was measured.

(1) Cellulase activity measurement method 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 a culture solution is added to 1 ml of a 1% CMC substrate solution (low viscosity made by Sigma dissolved in 100 mM acetate buffer (pH 5)), and the enzyme reaction is carried out at 40 ° C. for exactly 10 minutes. 4 ml of DNS reagent (including dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3%) may be added. Mix and stop the reaction. 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.

(2) Xylanase Activity Measurement Method Next, xylanase activity (XYL) was quantified by increasing the absorbance at 540 nm by reacting reducing sugars produced by enzymatic hydrolysis using oat spelts-derived xylan as a substrate. More specifically, 1% xylan substrate solution [Sigma Xylan, from oat spelts dissolved in 200 mM acetic acid buffer (pH 4.5)] 0.1 ml of culture solution was added to 1.9 ml, and exactly 10 at 40 ° C. After performing the enzyme reaction for 1 minute, it contains DNS reagent (dinitrosalicylic acid 0.75%, sodium hydroxide 1.2%, sodium potassium tartrate tetrahydrate 22.5%, lactose monohydrate 0.3% 4 ml was added and mixed well to stop the reaction. 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 xylose was quantified by measuring the absorbance at 540 nm. One unit of xylanase activity was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 μmol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes.

3. Results The measurement results are shown in FIGS. Even in white rot fungi, it was confirmed that there was a difference in plant fiber-degrading enzyme productivity depending on the whitening ratio. The highest enzyme productivity was obtained when 98% refined barley was used, but the enzyme activity was significantly reduced in the pulverized product, and the effect of inhibiting the release of nutrients by barley husk contributed to the high production of plant fiber-degrading enzymes. Was suggested.

<Example 8> Production of white birch culture using non-α-modified barley (1) Pre-culture method; 8 g of round barley (Australian Stirling species) and 100 ml of water were placed in a 500 ml baffled Erlenmeyer flask, 121 ° C, Autoclaved for 15 minutes. This preculture medium was inoculated with 1 × 10 ⁶ cells / ml of Aspergillus kawachii NBRC 4308 and cultured at 37 ° C. for 24 hours with shaking at 100 rpm to obtain a preculture solution.

(2) Main culture method: 0.2 g of KNO ₃ , 0.3 g of KH ₂ PO ₄ and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask and sterilized by autoclave at 121 ° C. for 15 minutes. After cooling, chloramphenicol (Wako Pure Chemical Industries, Ltd.) was added to 50 μg / ml, and 2 g of unheated barley was added. The main culture medium was inoculated with 1 ml of the preculture and cultured at 37 ° C. for 72 hours with shaking at 100 rpm to obtain a koji mold culture.
As a control, a koji mold culture was produced using a gelatinized culture raw material. That is, 2 g of barley, 0.2 g of KNO ₃ , 0.3 g of KH ₂ PO ₄ and 100 ml of water were placed in a 500 ml Erlenmeyer flask with a baffle, and autoclaved at 121 ° C. for 15 minutes. After cooling, chloramphenicol was added to 50 μg / ml. The main culture medium was inoculated with 1 ml of the preculture solution and cultured at 37 ° C. for 72 hours with shaking at 100 rpm to obtain a koji mold culture.

(3) Measuring method About the koji mold culture obtained in each test section, the activity of glucoamylase, acid-resistant α-amylase and α-amylase was measured. That is, the glucoamylase activity was measured using a saccharification power fractionation kit (manufactured by Kikkoman). The acid-resistant α-amylase activity was measured by <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 Society Journal, 89, 768-774 (1994)> slightly improved, acid-treated culture to inactivate non-acid-resistant α-amylase activity. Thereafter, 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, an α-amylase measurement kit (manufactured by Kikkoman) was used. The α-amylase activity was measured using an α-amylase measurement kit (manufactured by Kikkoman). The results are shown in Table 1 below.

(4) Results In the raw round barley fungus culture of the present invention, both glucoamylase and acid-resistant α-amylase were produced in good balance. On the other hand, α-amylase had a slightly low production amount.
In contrast, acid-resistant α-amylase and α-amylase were produced in a relatively large amount using a pre-gelatinized raw material, but due to the presence of KNO ₃ and KH ₂ PO ₄ , the nutritional conditions were in a suitable range. It is thought that it is maintained.
Therefore, it became clear that the koji mold culture which can be used for manufacture of fermented food / beverage products can be manufactured by this invention.

<Example 9> Manufacture of barley shochu by a white birch culture using non-α-modified barley (1) Pre-culture method; 8 g of barley (Australian Stirling seed) and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask, Autoclaved at 121 ° C for 15 minutes. The preculture medium was obtained by inoculating Bacillus subtilis (Aspergillus kawachii NBRC 4308) at 1 × 10 ⁶ cells / ml in this preculture medium and shaking culture at 37 ° C. for 24 hours at 100 rpm.
(2) Main culture method: 0.5 g of barley, 0.2 g of KNO ₃ , 0.3 g of KH ₂ PO ₄ and 100 ml of water were placed in a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. 1 ml each of the preculture was inoculated into this main culture medium and cultured with shaking at 37 ° C. for 24 hours at 100 rpm. Thereafter, 1.5 g of unheated barley was added, and further shake-culture was performed at 37 ° C. for 48 hours at 100 rpm to obtain a koji mold culture.
(3) Yeast; Kagoshima yeast was shake-cultured overnight at 100 rpm in 1 ml of YPD medium, collected after centrifugation, and washed twice with sterile water.
(4) Preparation: The preparation composition is shown in Table 2 below. As the wheat, the barley was washed, immersed in water for 60 minutes, drained for 30 minutes, and steamed for 40 minutes. The yeast used the whole thing mentioned above.

(5) Fermentation conditions: Fermentation was carried out at 25 ° C. for 20 days. Secondary charging was performed 3 days after the primary charging.
(6) Distillation: Distillation was carried out under reduced pressure at -650 mmHg.

(7) Results Fermentation progressed smoothly. The alcohol content of the mash after the fermentation was 17.5%.
When the sensory evaluation of the sample after distillation was conducted by 6 panelists specializing in alcoholic beverages, it was clean and the evaluation was high.
From this result, it was clarified that the method of the present invention can produce a barley shochu having no problem.

<Example 10> Manufacture of a koji mold culture using non-α-modified barley (1) Pre-culture method; 8 g of round barley (Australian Stirling species) and 100 ml of water were placed in a 500 ml baffled Erlenmeyer flask, 121 ° C, Autoclaved for 15 minutes. The pre-culture medium was inoculated with Aspergillus oryzae NRIB40 at 1 × 10 ⁶ cells / ml, and cultured with shaking at 37 ° C. for 24 hours at 100 rpm to obtain a pre-culture solution.

(2) Main culture method: 0.8 g of KNO ₃ , 1.2 g of KH ₂ PO ₄ , 0.2 g of MgSO ₄ and 100 ml of water were placed in a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. After cooling, chloramphenicol (Wako Pure Chemical Industries, Ltd.) was added to 50 μg / ml, and 2 g of barley was added. 1 ml of the preculture solution was inoculated into this main culture medium, and cultured at 37 ° C. for 72 hours with shaking at 100 rpm to produce a koji mold culture.
As a positive control, a koji mold culture was produced by the following method. That is, 2 g of 95% barley (Australian Stirling seed), 0.8 g of KNO ₃ , 1.2 g of KH ₂ PO ₄ , 0.2 g of MgSO ₄ and 100 ml of water were placed in a 500 ml baffled Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. . After cooling, chloramphenicol was added to 50 μg / ml. 1 ml of the preculture solution was inoculated into this main culture medium, and cultured at 37 ° C. for 72 hours with shaking at 100 rpm to produce a koji mold culture.

(3) Result About the koji mold culture obtained in each test section, it carried out similarly to Example 8, and measured the activity of glucoamylase and alpha-amylase. The results are shown in Table 3 below.
In the raw round barley koji mold culture, glucoamylase was produced satisfactorily and α-amylase activity was slightly low. Both enzymes are inferior in activity to the positive control, but are produced in a well-balanced manner, and it has been clarified that a koji mold culture that can be used for producing fermented foods and drinks can be produced.

<Experimental example 2> Measurement of glucose release rate in barley substrate solutions with different inorganic salt concentrations during sterilization Barley substrate solutions obtained by autoclaving barley-containing inorganic salt aqueous solutions with different salt concentrations were obtained from enzymes derived from koji molds. After reacting, the rate of glucose release from barley was measured.

1. Barley Substrate Solution Preparation Method First, 2 g of 98% polished barley (Australian Stirling) was weighed and placed in a 500 ml Erlenmeyer flask together with 50 ml of water. This was sterilized by autoclaving (121 ° C., 15 minutes) to prepare a barley substrate solution, which was designated as “No. 1 control group”.
In the “No. 2 salt use group”, autoclave sterilization was performed using 50 ml of an inorganic salt aqueous solution containing 0.1 g of potassium nitrate and 0.15 g of potassium dihydrogen phosphate instead of water in the “No. 1 control group”. That is, the salt concentration during autoclave sterilization is 0.2% potassium nitrate and 0.3% potassium dihydrogen phosphate.
In “No.3 high-concentration salt use zone”, after “autoclave sterilization using 10 ml of an inorganic salt solution containing 0.1 g of potassium nitrate and 0.15 g of potassium dihydrogen phosphate instead of water in“ No.1 control zone ” 40 ml of sterilized water was added. In other words, the salt concentration at the time of sterilization is 1.0% potassium nitrate which is 5 times the amount of No.2 and 1.5% potassium dihydrogen phosphate, but the salt concentration after sterilization and addition is 0.2% potassium nitrate which is the same as No.2 and phosphorus. It was prepared to be 0.3% potassium dihydrogen acid.

2. Method for preparing gonococcal culture supernatant Subsequently, the gonococcal culture prepared using barley (Australian Stirling) as a culture raw material was subjected to solid-liquid separation by filter paper filtration to obtain a “gonococcal culture supernatant”.
The specific method for producing the koji mold culture used in this test was as follows.

(1) Pre-culture method
8 g of 65% white wheat and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask, 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 barley is 2.0% (w / vol) in water to which potassium nitrate 0.2% (w / vol) and potassium dihydrogen phosphate 0.3% (w / vol) are added. The liquid culture medium added so that it might become was prepared. 3000 ml of the prepared liquid medium was put on a jar fermenter (manufactured by Maruhishi Bioengineering Co., Ltd.) with a capacity of 5000 ml, sterilized by autoclave (121 ° C., 15 minutes) and then pre-cultured in liquid medium by the above method (Aspergillus kawachii) NBRC4308) 30 ml was inoculated. Thereafter, the cells were cultured for 42 hours at a temperature of 37 ° C., a stirring speed of 300 rpm and an aeration rate of 0.5 vvm, and filtered through a filter paper (Toyo Filter Paper No. 2) to obtain a “Koji mold culture supernatant”.

3. Method for Measuring Glucose Release Rate 50 ml of barley substrate solution and 50 ml of Aspergillus culture supernatant were each incubated at 37 ° C. for 5 minutes, and then the reaction was started by mixing them. The glucose concentration in the reaction solution sampled 3 hours after the start of the reaction was measured using Glucose C-II Test Wako (manufactured by Wako Pure Chemical Industries), and the glucose release rate was calculated.

4). Results The glucose release rate calculated from the glucose concentration measurement result in the reaction solution is as shown in FIG. 18, and it was confirmed that the glucose release rate differs depending on the salt concentration at the time of preparing the barley substrate solution. In Experimental Example 1 above, it has already been shown that the rate of glucose release can be adjusted by the milling rate of barley, but this test shows that the glucose release rate is low due to the presence of salts during heat treatment such as autoclave sterilization. It was confirmed that It was inferred that this was due to the fact that salts were present during the heat treatment of barley, thereby suppressing the physical breakdown of barley.

<Example 11> Production of white birch culture using barley with different inorganic salt concentrations during sterilization Using various kinds of white wheat (Australian Stirling), white birch cultures were produced by the following method, Enzyme activity was measured.

1. Pre-culture method
8 g of 65% white wheat and 100 ml of water were put into a 500 ml baffled Erlenmeyer flask, 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
Weigh out 2g of 98% refined wheat (Australian Stirling) and place it in a 500ml Erlenmeyer flask with 100ml of water and sterilize it in an autoclave (121 ° C, 15 minutes) to prepare the main culture medium. .1 Control zone ".
In the “No. 2 salt use group”, autoclave sterilization was performed using 100 ml of an inorganic salt aqueous solution containing 0.2 g of potassium nitrate and 0.3 g of potassium dihydrogen phosphate instead of water in the “No. 1 control group”.
In “No.3 high-concentration salt use zone”, after “autoclave sterilization using 20 ml of inorganic salt aqueous solution containing 0.2 g of potassium nitrate and 0.3 g of potassium dihydrogen phosphate instead of water in“ No.1 control zone ” 80 ml of sterilized water was added. In other words, the salt concentration at the time of sterilization is 1.0% potassium nitrate, which is 5 times the amount of No. 2, and 1.5% potassium dihydrogen phosphate, but the salt concentration after sterilization and addition is 0.2% of potassium nitrate, the same as No. 2. It was prepared to be 0.3% potassium dihydrogen phosphate.
The main culture medium prepared as described above was allowed to cool, then 1 ml of the preculture was inoculated and cultured at 37 ° C. for 48 hours with shaking at 100 rpm.

3. 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).
In addition, the 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 .: Journal of the Japan Brewing Association, 89, 768-774 (1994)>, a slightly improved method for acid-free α-amylase by acid treatment of the culture After the activity was deactivated, 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).

4). Results The measurement results of glucoamylase activity are shown in FIG. 19, and the measurement results of acid resistance α-amylase activity are shown in FIG.
As shown in FIGS. 19 and 20, enzyme productivity was improved in No. 3 having a high salt concentration during sterilization compared to No. 2 having a similar salt concentration during culture. In Experimental Example 2, it was confirmed that the higher the inorganic salt concentration of the barley substrate solution during the heat treatment, the lower the sugar release rate from the barley raw material. Thus, this suggests that the rate of sugar release affects enzyme productivity in addition to the effect of salts on the growth of Aspergillus. In No. 1 containing no salt, the growth of koji molds was suppressed, and it was considered that enzyme production was remarkably suppressed due to the high sugar release rate.
Until now, it was thought that the inorganic salts added at the time of medium preparation were involved only in the growth of koji molds during culture. However, the present example suggested that enzyme production in the liquid culture of Aspergillus oryzae may be promoted also by the sugar release inhibitory effect from the barley raw material.

In the method of the present invention, by controlling the nutrient concentration in the culture system to be low, it is equivalent to fed-batch culture in a simple batch culture without performing fed-batch culture in which nutrients are added little by little from outside the culture system. Culture mode is possible.
In addition, according to the present invention, not only the filamentous fungus culture used for the production of fermented foods and drinks, but also the filamentous fungus culture used in a wide range of industries, and the enzymes and enzyme agents produced thereby can be produced stably and inexpensively. A method is provided.
Furthermore, the present invention is expected to be applied to heterologous protein production using a promoter region such as amylolytic enzyme gene.
Therefore, the present invention can be used in a wide range of industries such as food manufacturing industry, fermentation industry, and pharmaceutical manufacturing industry.

It is a figure which shows the time-dependent change of the glucose concentration in a reaction liquid when the barley substrate solution of each milling ratio and the koji mold culture supernatant liquid are made to react. It is a figure which shows the glucose release rate in the first 1 hour when the barley substrate solution of each polishing ratio and the koji mold culture supernatant liquid are reacted. It is a figure which shows the enzyme activity in the birch culture using barley of each refined ratio as a culture raw material. (A) shows the enzyme activity of glucoamylase (white bar) and α-amylase (black bar), and (B) shows the enzyme activity of acid-resistant α-amylase. It is a figure which shows the enzyme activity in the birch culture using barley of each refined ratio as a culture raw material. (A) shows cellulase activity, and (B) shows the enzyme activity of β-glucosidase. It is a figure which shows the glucoamylase activity (GA) in the black koji mold | fungi culture which used the barley of each milling ratio as a culture raw material. It is a figure which shows (alpha) -amylase activity (AA) in the black koji mold | fungi culture which used the barley of each refined ratio as a culture raw material. It is a figure which shows the acid-resistant alpha-amylase activity (ASAA) in the black koji mold | fungi culture using barley of each refined ratio as a culture raw material. It is a figure which shows the glucoamylase activity (GA) in the koji mold culture which used the barley of each milling ratio as a culture raw material. It is a figure which shows (alpha)-amylase activity (AA) in the yellow gonococcus culture using the barley of each milling ratio as a culture raw material. It is a figure which shows the cellulase activity (CEL) in the Trichoderma viride culture using the barley of each milling ratio as a culture raw material. It is a figure which shows the cellulase activity (CEL) in the Trichoderma reesei culture which used the barley of each milling ratio as a culture raw material. It is a figure which shows the xylanase activity (XYL) in the Trichoderma reesei culture which used the barley of each milling ratio as a culture raw material. It is a figure which shows (beta) -glucosidase activity (BGL) in the Trichoderma reesei culture which used the barley of each milling ratio as a culture raw material. It is a figure which shows the cellulase activity (CEL) in the Aspergillus accumulator culture | cultivation which used the barley of each milling ratio as a culture raw material. It is a figure which shows (beta) -glucosidase activity (BGL) in the Aspergillus accumulator culture | cultivation which used the barley of each milling ratio as a culture raw material. It is a figure which shows the cellulase activity (CEL) in the white rot fungus culture using the barley of each milling ratio as a culture raw material. It is a figure which shows the xylanase activity (XYL) in the white rot fungus culture | cultivation which used the barley of each milling ratio as a culture raw material. It is a figure which shows the glucose concentration in a reaction liquid when the barley substrate solution in which the salt density | concentration at the time of sterilization and the koji mold culture supernatant liquid are made to react. It is a figure which shows the glucoamylase activity (GA) in the white koji mold culture using the liquid culture medium from which the salt concentration at the time of sterilization differs. It is a figure which shows the acid-resistant alpha-amylase activity (ASAA) in the white gonococcus culture using the liquid culture medium from which the salt concentration at the time of sterilization differs.

Claims (7)

While controlling the release rate of nutrients in the barley to the culture system, using a liquid medium containing barley and mineral salt of the whitening ratio or higher that has been refined to the extent that the husk is left on the surface of the grain A method for producing a filamentous fungus culture, comprising adjusting the enzyme productivity of the filamentous fungus culture by culturing the filamentous fungus. Nutrient is an amino acid derived from a protein of sugar and / or barley derived starch in barley, the manufacturing method of the filamentous fungus culture product according to claim 1.   The method for producing a filamentous fungus culture according to claim 1, wherein the enzyme is at least one selected from starch degrading enzymes, plant fiber degrading enzymes, and proteolytic enzymes.   The method for producing a filamentous fungus culture according to claim 1, wherein the filamentous fungus is at least one selected from Neisseria gonorrhoeae, Trichoderma spp. And white rot fungi.   The manufacturing method of the enzyme agent using the filamentous fungus culture obtained by the method of any one of Claims 1-4.   The manufacturing method of fermented food / beverage products using the filamentous fungus culture obtained by the method of any one of Claims 1-4.   The manufacturing method of the enzyme using the filamentous fungus culture obtained by the method of any one of Claims 1-4.

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