JP2018134029A - Enzyme-containing liquid culture method and method of producing enzyme-containing powder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme-containing liquid culture which does not require pre-culturing for growth of bacteria, and an enzyme-containing liquid culture method capable of producing an enzyme without using indigestible carbohydrates in a liquid medium.SOLUTION: The enzyme-containing liquid culture method comprises the steps of: adding a first cereal koji into a conditioned liquid medium; performing a first aeration culture using the conditioned liquid medium to which the first cereal koji was added; adding a second cereal koji to the conditioned liquid medium to which the first aeration culture was performed; and performing a second aeration culture using the conditioned liquid medium to which the second cereal koji was added. Preferably, the first cereal koji is selected from at least one of brown rice koji, soybean koji, rice bran koji, millet and rice koji, and the second cereal koji is soybean koji.SELECTED DRAWING: None

Description

  The present invention relates to an enzyme-containing liquid culture method and an enzyme-containing powder production method.

  The culture method for secretory production of various enzymes in koji molds includes a solid culture method in which koji molds are inoculated and cultured on raw materials after steaming and the like, and a liquid medium is prepared by adding raw materials and other nutrient sources to water. There is a liquid culture method of inoculating and culturing a liquid medium that has been adjusted and then pre-cultured koji molds. The solid culture method has been established as a culture method capable of producing many kinds of enzymes in a large scale on a large scale. However, there is a problem that the manufacturing equipment is enlarged and it is difficult to stably operate and control temperature and humidity. On the other hand, it is said that the liquid culture method is easy for culture control and quality control.

  However, in the conventional liquid culture method, in order to secrete and produce a sufficient amount of enzyme, it is necessary to perform pre-culture prior to the main culture for the purpose of increasing the number of koji molds in advance. That is a problem.

  Furthermore, as a problem of the conventional liquid culture method, a decrease in productivity of amylase in the medium is mentioned. Neisseria gonorrhoeae produces many enzymes, including amylase, when it grows. In particular, koji mold for sake produces a large amount of amylase, particularly α-amylase. However, starch in the medium is decomposed and saccharified by amylase produced by Aspergillus oryzae, and amylase in the medium is consumed accordingly. In order to solve this problem, a method of replacing starch with an indigestible substance such as indigestible starch, brown rice, rice bran, rice bran, or amaranth has been reported.

  For example, Patent Document 1 discloses a method for culturing koji mold using a liquid medium containing a hardly digestible dextrin, a polydextrose, a water-soluble cellulose, pullulan and the like, which are non-degradable carbohydrates of koji mold.

  Further, Patent Document 2 discloses that glucoamylase activity is obtained by culturing gonococci using a liquid medium containing indigestible dextrin, polydextrose, water-soluble cellulose, pullulan and the like which are refractory saccharides of the gonococcus. Is disclosed to provide a high koji mold culture.

  Thus, attempts have been made to increase the productivity of enzymes by using indigestible dextrin or the like as a hardly degradable saccharide in a liquid culture medium.

JP 2005-295871 A Japanese Patent No. 4087624

  An object of the present invention is to provide an enzyme-containing liquid culture method that does not require preculture for the purpose of bacterial growth. A further object of the present invention is to provide an enzyme-containing liquid culture method capable of producing an enzyme without using an indigestible carbohydrate in a liquid medium.

  The present inventor has found that the above-mentioned object can be achieved by adding cereal meal instead of the hardly digestible sugar as a carbon content to the conditioned liquid medium. Furthermore, the present inventors have found that the enzyme obtained by the enzyme-containing liquid culture method of the present invention has significantly higher enzyme activity than the enzyme obtained by the conventional method.

For example, the present invention is as follows.
1. The following steps:
(I) adding the first cereal meal to the conditioned liquid medium;
(Ii) performing a first aeration culture using the conditioned liquid medium supplemented with the first cereal meal,
(Iii) adding a second cereal meal to the conditioned liquid medium subjected to the first aeration culture; and (iv) using the conditioned liquid medium to which the second cereal meal is added Performing aeration culture,
An enzyme-containing liquid culture method comprising:

  2. The enzyme-containing product according to 1, wherein the first cereal meal is selected from at least one of brown rice meal, soybean meal, rice meal, meal, and meal, and the second cereal meal is soybean meal Liquid culture method.

  3. The first cereal meal is added in an amount of 2% to 15% of the volume of the conditioned liquid medium, and the second cereal meal is in an amount of 1% to 10% of the volume of the conditioned liquid medium. 3. The enzyme-containing liquid culture method according to 1 or 2, which is added.

  4). The enzyme-containing liquid culture method according to any one of 1 to 3, wherein the first and second cereal meals become carbon in the conditioned liquid medium.

  5. Any of 1-4 wherein the first and second cereal meals are produced by Aspergillus oryzae, Aspergillus kawachi, Alpergillus niger, Aspergillus awamori, and Aspergillus soya The enzyme-containing liquid culture method according to claim 1.

6). The first aeration culture is performed at a temperature of 20 ° C. to 40 ° C. for 24 hours to 60 hours, and the second aeration culture is performed at a temperature of 10 ° C. to 40 ° C. for 12 hours to 30 hours. The enzyme-containing liquid culture method according to any one of 1 to 5.
7). The enzyme-containing liquid culture method according to any one of 1 to 6, wherein the enzyme is at least one of α-amylase, glucoamylase, α-glucosidase, and protease.

8). The following steps:
(I) obtaining a filter cloth filtrate by filtering the culture solution containing the enzyme produced by the enzyme-containing liquid culture method according to any one of 1 to 7,
(Ii) obtaining a microfiltrate by microfiltration of the filter cloth filtrate,
(Iii) obtaining a concentrate by ultrafiltration of the microfiltrate,
(Iv) adding ethyl alcohol to the concentrate and stirring;
(V) obtaining a precipitate by cooling the mixture of the stirred ethyl alcohol and the concentrated liquid;
(Vi) isolating the precipitate from the cooled mixture;
(Vii) obtaining a solid content by centrifuging the isolated precipitate; and (Viii) obtaining a powder by drying the solid content,
A method for producing an enzyme-containing powder comprising

9. The following steps:
(I) obtaining a soybean-derived powder additive liquid by adding soybean-derived powder to the microfiltrate according to 8 and stirring;
(Ii) obtaining a precipitate by cooling the soybean-derived powder additive solution;
(Iii) removing the supernatant from the precipitate;
(Iv) obtaining a solid by centrifuging the precipitate from which the supernatant has been removed, and (V) obtaining a powder by drying the solid,
A method for producing an enzyme-containing powder comprising

  10. 10. The method for producing an enzyme-containing powder according to 9, wherein the soybean-derived powder is selected from at least one of okara powder, soy milk powder, and kinako powder.

  11. The method for producing an enzyme-containing powder according to 9 or 10, wherein the soybean-derived powder is added in an amount of 2% to 20% of the volume of the microfiltrate.

  12 The method for producing an enzyme-containing powder according to any one of 8 to 11, wherein the enzyme-containing powder contains at least one of α-amylase, glucoamylase, α-glucosidase, and protease.

  The present invention enables an enzyme-containing liquid culture method that does not require pre-culture. Furthermore, the present invention enables an enzyme-containing liquid culture method that produces enzymes without using indigestible carbohydrates in a liquid medium.

Hereinafter, an embodiment which is an application example of the present invention will be described.
(Cereal meal)
The cereal meal concerning this embodiment is obtained by the following steps (i) to (V).
(I) Dividing cereals into several pieces (for example, 2 to 4 divisions) or crushing.
(Ii) Soaking the cereals in room temperature water for about 12 hours to about 48 hours (about 48 hours in winter), and then draining for about 1 hour.
(Iii) Softening the cereal after draining by steaming, and then allowing to cool to about 20 ° C to about 40 ° C.
(IV) Sprinkling aspergillus oryzae selected from at least one of Aspergillus oryzae, Aspergillus kawachi, Alpergillus niger, Aspergillus awamori, and Alpergillus soja on the cereals that have been allowed to cool.
(V) The cereals sprayed with the gonococcus are allowed to stand in a straw chamber whose temperature is controlled to 40 ° C. or less for about 48 hours to about 72 hours, and then brewed.
In addition, the cereal meal concerning this embodiment may be manufactured by methods other than the steps (i) to (V), or may be commercially available.

  Since the cereal meal according to the present embodiment is already in a state where the koji mold has been grown in the cereal, in the enzyme-containing liquid culture method described later, by adding the cereal meal according to the present embodiment to the conditioned liquid medium, Enzymes can be produced without culturing.

  The cereal meal according to the present embodiment is obtained by multiplying cereals with koji mold effective for food fermentation and the like, but is not limited thereto, and includes, but is not limited to, brown rice cake, soybean meal, rice cake, rice cake, and rice cake. It is done.

  The koji mold according to the present embodiment produces and releases various enzymes that degrade starch, protein, and the like from the tip of the mycelium. The produced and released enzyme decomposes the carbon content in the medium to produce glucose and amino acids. Neisseria gonorrhoeae grows using the glucose and amino acids produced here as nutrient sources. The koji mold according to this embodiment is a group of incomplete bacteria classified into the genus Aspergillus, including, but not limited to, Aspergillus oryzae, Aspergillus kawachi, Alpergillus niger, Aspergillus awamori, Alpergillus soya, etc. Can be mentioned. Particularly preferred Aspergillus is Aspergillus oryzae, Aspergillus kawachi, or Aspergillus niger.

(Enzyme-containing liquid culture method)
The present embodiment is an enzyme-containing liquid culture method including the following steps.
(I) Adding the first cereal meal to the conditioned liquid medium.
(Ii) Performing a first aeration culture using the conditioned liquid medium to which the first cereal meal is added.
(Iii) adding a second cereal meal to the conditioned liquid medium subjected to the first aeration culture.
(Iv) Performing a second aeration culture using the conditioned liquid medium to which the second cereal meal is added.

The conditioned liquid medium according to the present embodiment includes, for example, polypeptone (registered trademark), yeast extract powder, cyclose, sodium nitrate, potassium nitrate, magnesium sulfate, iron sulfate heptahydrate, and components necessary for the growth of Aspergillus oryzae. Is a medium adjusted to have a pH of 3 to 6 (preferably a pH of 4 to 5) by adding adjusted water or the like to the composition of the concentrated medium.

  Polypeptone (registered trademark) is a culture substrate made from soybean products sold by Nippon Pharmaceutical Co., Ltd., and is added to the medium to supply a degradation product of vegetable protein. Not limited to this, peptone derived from other plants or animals can also be used.

  Enzyme extract powder is a yeast autolysate and is added to the medium to supply amino acids and the like. Not limited to this, extracts derived from other plants or animals can also be used.

  Cyclos is added to the medium to supply carbohydrates.

  Sodium nitrate, potassium nitrate, magnesium sulfate, and iron sulfate heptahydrate are added to the medium to supply minerals and the like. Not only these but mineral sources, such as other inorganic salts, can also be used.

  For example, a conditioned liquid medium for 20 L is obtained by adjusting the concentrated medium composition shown in Table 1 to pH 4.4 with the adjusting liquid shown in Table 2. The amount and ratio of each component can be appropriately changed as long as the culture is not inhibited.

  The conditioned liquid medium according to this embodiment may have a composition other than those described above. For example, a Czapek-Dox (CD) medium generally used for culturing koji molds may be used.

  The conditioned liquid medium according to this embodiment is preliminarily sterilized by heating at 80 ° C. to 95 ° C. for 10 minutes to 40 minutes, preferably 15 minutes to 35 minutes, more preferably 20 minutes to 30 minutes, and then 20 ° C. to 50 ° C. , Preferably 25 ° C to 45 ° C, more preferably cooled to 30 ° C to 40 ° C.

  The first cereal meal according to this embodiment can be added to the conditioned liquid medium in an amount of 2% to 15%, preferably 3% to 12%, more preferably 5% to 10% of the volume of the conditioned liquid medium. . The second cereal meal according to this embodiment can be added to the conditioned liquid medium in an amount of 1% to 10%, preferably 2% to 8%, more preferably 3% to 5% of the volume of the conditioned liquid medium. .

  The aeration culture according to this embodiment is a culture with stirring, and the purpose of the aeration is to give air to koji molds. The time for performing the first aeration culture is set between 24 hours and 60 hours, preferably between 24 hours and 54 hours, more preferably between 24 hours and 48 hours, depending on the propagation condition of the koji mold, and the second aeration is performed. The time for culturing can be set between 12 hours and 30 hours, preferably between 12 hours and 27 hours, more preferably between 12 hours and 24 hours, depending on the propagation condition of the koji mold. The temperature of the first aeration culture is set to 20 ° C to 40 ° C, preferably 25 ° C to 40 ° C, more preferably 30 ° C to 40 ° C, and the temperature of the second aeration culture is 10 ° C to 40 ° C. C, preferably 15C to 35C, more preferably 20C to 30C.

  The first and second cereal cocoons according to the present embodiment may be made by a koji mold classified into the genus Aspergillus. The koji mold according to the present embodiment is not limited thereto, but may be a koji mold selected from at least one of Aspergillus oryzae, Aspergillus kawachi, Aspergillus niger, Aspergillus awamori, and Aspergillus soya. Particularly preferred Aspergillus is Aspergillus oryzae, Aspergillus kawachi, or Aspergillus niger.

  The first and second cereal meals according to the present embodiment are not limited to these, but can be selected from at least one of brown rice cake, soybean meal, rice cake, rice cake, and rice cake, respectively. Preferably, the first cereal meal according to the present embodiment is selected from at least one of brown rice meal, soybean meal, rice meal, meal, and rice cake, and the second cereal meal is soybean meal.

  Since the 1st and 2nd cereal meal concerning this embodiment itself becomes carbon content in a conditioned liquid culture medium, it can produce an enzyme, without using indigestible saccharides as carbon content. Moreover, the culture solution obtained by this embodiment contains many kinds of enzymes including amylase and protease, especially α-amylase, glucoamylase, α-glucosidase and protease.

(Method 1 for producing enzyme-containing powder)
The enzyme-containing powder according to this embodiment is obtained by the following steps.
(I) A filter cloth filtrate is obtained by filtering the culture medium containing the enzyme produced by the enzyme-containing liquid culture method.
(Ii) A microfiltrate is obtained by microfiltration of the filter cloth filtrate.
(Iii) Obtaining a concentrate by ultrafiltration of the microfiltrate.
(Iv) Add ethyl alcohol to the concentrated solution and stir.
(V) A precipitate is obtained by cooling the stirred mixture of the ethyl alcohol and the concentrated liquid.
(Vi) isolating the precipitate from the cooled mixture.
(Vii) Obtaining solid content by centrifuging the isolated precipitate.
(Viii) Obtaining a powder by drying the solid content.

  The culture solution according to the present embodiment is produced by the enzyme liquid culture method described above, and a filter cloth of 100 mesh to 400 mesh, preferably 150 mesh to 350 mesh, more preferably 200 mesh to 300 mesh is used. The filter cloth filtrate is obtained by filtration. The filter cloth filtrate is then placed in a precipitation tank whose internal temperature is set to 4 ° C to 14 ° C, preferably 6 ° C to 12 ° C, more preferably 8 ° C to 10 ° C.

  The precipitation tank containing the filter cloth filtrate should be allowed to stand in the refrigerator for 12 hours to 60 hours, preferably 18 hours to 54 hours, more preferably 24 hours to 48 hours without using a reducing agent. Is lowered. The filter cloth filtrate that has been starched in the refrigerator is microfiltered to obtain a microfiltrate. Further, the microfiltrate is ultrafiltered to obtain a concentrated solution. In ultrafiltration, the amount of the concentrate is 1/5 to 25 times the volume of the microfiltrate, preferably 1/8 to 20 times, more preferably 1/10 to 15 times the volume. In the ultrafiltration, the liquid temperature is set to 15 ° C. or lower, preferably 12 ° C. or lower, more preferably 10 ° C. or lower. The concentrate concentrated by ultrafiltration is placed in a sterilization tank with ethyl alcohol and gently stirred.

  Here, ethyl alcohol is put in the sterilization tank so that the concentration of ethyl alcohol in the sterilization tank is at least 70% or more (for example, 75%). Thereafter, the mixture of ethyl alcohol and concentrated liquid in the sterilization tank is allowed to stand in the refrigerator for 12 to 60 hours, preferably 18 to 54 hours, more preferably 24 to 48 hours. At this time, in the sterilization tank, the enzyme, protein, or other substance in the mixture of ethyl alcohol and the concentrated liquid crystallizes and precipitates. The precipitate is then separated from the mixture of ethyl alcohol and concentrate.

  The separated precipitate is 2000 to 4500 rotations, preferably 2500 to 4000 rotations, more preferably 3000 to 3500 rotations, and 3 to 20 minutes, preferably 5 to 15 minutes, more preferably 7 minutes. By centrifuging for 10 minutes, it is separated into a layer containing the enzyme and a supernatant layer containing ethyl alcohol. The supernatant layer containing ethyl alcohol is removed from the layer containing the enzyme to obtain a solid content. The solid content obtained here is dried at room temperature in a dryer and further pulverized to obtain enzyme-containing powder.

(Method 2 for producing enzyme-containing powder)
The enzyme-containing powder according to this embodiment is obtained by the following steps.
(I) A soybean-derived powder additive liquid is obtained by adding soybean-derived powder to the microfiltrate described in Production Method 1 for Enzyme-Containing Powder and stirring.
(Ii) A precipitate is obtained by cooling the soybean-derived powder additive solution.
(Iii) removing the supernatant from the precipitate.
(Iv) Obtaining a solid content by centrifuging the precipitate from which the supernatant has been removed.
(V) Obtaining a powder by drying the solid content.

  The soybean-derived powder according to this embodiment is obtained by drying okara produced as a by-product when making soybeans, soy milk, or tofu, and making it into a finer powder. The soybean-derived powder according to this embodiment is not limited to these, but may be okara powder, soy milk powder, and kinako powder.

  In the soybean-derived powder additive liquid according to this embodiment, the soybean-derived powder is 2% to 20%, preferably 3% to 15%, more preferably 5% to 10% of the volume of the microfiltrate. It is obtained by adding to The obtained soybean-derived powder additive solution is allowed to stand in a refrigerator for 3 hours to 40 hours, preferably 6 hours to 30 hours, and more preferably 12 hours to 24 hours to obtain a precipitate. Enzymes and the like contained in the microfiltrate can be adsorbed to the soybean-derived powder during this time. By removing the supernatant from the resulting precipitate, a wet precipitate is obtained. The precipitate from which the supernatant liquid has been removed is 2000 to 4500, preferably 2500 to 4000, more preferably 3000 to 3500, 1 minute to 10 minutes, preferably 2 to 8 minutes, and more preferably. Is centrifuged for 3-7 minutes and dehydrated. Thereafter, the precipitate in a semi-dry state is dried at room temperature in a dryer and further pulverized to obtain an enzyme-containing powder.

  The enzyme-containing powder obtained by the production methods 1 and 2 of the present embodiment includes many types of enzymes including gonococcal products from low to high molecules. In particular, there are many α-amylases, glucoamylases, α-glucosidases and proteases.

Next, although an Example is shown, this invention is not limited to these Examples.
(Manufacturing method of brown rice bran)
5 kg of brown rice was divided into 2 to 4 parts. The divided brown rice was immersed in room temperature water for 48 hours, then drained for about 1 hour, and then steamed until the brown rice became soft. The brown rice after post-steaming was allowed to cool to about 30 ° C., and then Aspergillus oryzae was sprayed on the brown rice. Thereafter, the brown rice sprinkled with gonococcus was placed in the cocoon room and monitored every 6 to 8 hours so that the temperature in the cocoon room did not exceed 40 ° C. After 48 hours, 5 kg of brown rice bran was put out.

(Method for producing soybean meal)
2 kg of soybean was divided into 2 to 4 parts. The divided soybeans were immersed in room temperature water for 24 hours, then drained for about 1 hour, and then steamed so that the soybeans could be crushed with fingers. The soybean after the post-steaming was allowed to cool to about 20 ° C., and then Aspergillus oryzae was sprayed on the soybean. After that, soybeans sprayed with koji molds were put in a soybean koji box with the koji lid placed on top. Monitoring was performed so that the temperature in the soybean meal box was maintained at about 25 ° C to about 27 ° C. After 72 hours, 2 kg of soybean meal was produced.

(Enzyme-containing liquid culture method)
The concentrated medium composition shown in Table 3 was adjusted to pH 4.4 with the adjusting solution shown in Table 4 to obtain a conditioned liquid medium.

  2000 mL of conditioned liquid medium was sterilized by heating at 95 ° C. for 30 minutes in advance, and then cooled to 35 ° C. To this cooled conditioned liquid medium, 200 g of brown rice bran was added as the first cereal bran. The first aeration culture was performed at 35 ° C. to 37 ° C. for 48 hours while stirring the conditioned liquid medium to which the brown rice bran was added. After confirming that the brown rice koji mold was sufficiently propagated, 100 g of soybean koji was added as a second cereal koji to the prepared liquid medium subjected to the first aeration culture. A second aeration culture was performed at 20 ° C. to 30 ° C. for another 24 hours while stirring the conditioned liquid medium to which soybean meal was added.

(Method 1 for producing enzyme-containing powder)
A 10 L culture solution containing the enzyme produced in the enzyme-containing liquid culture method was filtered using a 300 mesh filter cloth to obtain a 7 L filter cloth filtrate. This filter cloth filtrate was put into a precipitation tank and then allowed to stand for 24 hours in a refrigerator to lower the starch. Thereafter, the filter cloth filtrate is filtered using a filter having a pore diameter of 1 μm, pre-filtered using a 0.45 μm cartridge filter, and then subjected to microfiltration using a 0.45 μm membrane cartridge filter. A liquid was obtained.

  A portion (1 L) of the microfiltrate was collected for use in Method 2 for producing enzyme-containing powder described below. After fractionation, the remaining 5 L of microfiltrate was subjected to ultrafiltration (molecular weight: 6,000 to 100,000) to obtain 500 mL of a concentrated solution. During ultrafiltration, the liquid temperature was not allowed to exceed 15 ° C. The concentrated solution after ultrafiltration and cooled 88% ethyl alcohol were placed in a sterilization tank and gently stirred. At this time, the concentration of ethyl alcohol in the sterilization tank was kept from 70% or less. Thereafter, the mixture of ethyl alcohol and concentrated liquid in the sterilization tank was allowed to stand in the refrigerator for 24 hours.

  At this time, in the sterilization tank, enzymes, proteins, or other substances in the mixture of ethyl alcohol and concentrate were crystallized and precipitated. This precipitate was separated from the mixture of ethyl alcohol and concentrate. Furthermore, this precipitate was separated into a layer containing enzymes and a supernatant layer containing ethyl alcohol by centrifuging at 3500 rpm for 10 minutes. And the solid content was obtained by removing a supernatant layer from the layer containing an enzyme etc. The solid content obtained here was dried at room temperature in a dryer and further pulverized to obtain 5 g of enzyme-containing powder.

(Method 2 for producing enzyme-containing powder)
Okara powder was obtained by adding 100 g of okara powder to 1 L of the microfiltrate collected in Production method 1 of the enzyme-containing powder and stirring it. The obtained okara powder additive solution was allowed to stand in a refrigerator for 12 hours to obtain a precipitate. Thereafter, the supernatant of the precipitate was removed, and the precipitate in a wet state was dehydrated by centrifuging at 3500 rpm for 5 minutes. Thereafter, the precipitate after centrifugation in a semi-dry state was dried at room temperature in a drier and further pulverized to obtain 95 g of enzyme-containing powder.

  The enzyme-containing powder obtained by the method 1 for producing the enzyme-containing powder and the method 2 for producing the enzyme-containing powder includes many types of enzymes, particularly α-, including koji mold products from low to high molecules. A lot of amylase, glucoamylase, α-glucosidase and protease were contained.

(Measurement of enzyme activity)
The glucoamylase activity, α-glucosidase activity, α-amylase activity, and acidic / neutral / alkaline protease activity of the microfiltrate obtained in this example were measured. For comparative verification, each activity of a microfiltrate obtained from brown rice bran, a microfiltrate obtained from soybean koji, and a microfiltrate obtained by a known liquid culture method was also measured.

Preparation of microfiltrate for comparative verification
0.5% saline and brown rice bran were mixed at a volume ratio of about 5: 1. This mixed solution of saline and brown rice bran was stirred in a refrigerator periodically and allowed to stand for about 4 hours. Thereafter, the mixed solution of the saline solution and the brown rice bran is filtered using a 300 mesh filter cloth and a filter having a pore size of 1 μm, and then finely filtered using a 0.45 μm membrane filter. A filtrate was obtained.

Soybean cake:
0.5% saline and soybean meal were mixed in a volume ratio of about 5: 1. This mixed solution of saline and soybean cake was stirred regularly in the refrigerator and allowed to stand for about 4 hours. Thereafter, the mixed solution of the saline solution and soybean meal is filtered using a 300 mesh filter cloth and a filter having a pore size of 1 μm, and then fine filtered using a 0.45 μm membrane filter. A filtrate was obtained.

Known liquid culture methods:
Aspergillus oryzae was precultured, and after the aspergillus was sufficiently propagated, the preculture was mixed with the main culture. Aeration culture was performed while stirring the mixed culture for 48 hours. After completion of the aeration culture, the culture solution was filtered using a 300-mesh filter cloth and a filter having a pore diameter of 1 μm, and then subjected to microfiltration using a 0.45 μm membrane filter to obtain a microfiltrate for comparative verification.

Measurement result Saccharification power:
The saccharification power (glucoamylase activity and α-glucosidase activity) was measured with a Kikkoman saccharification power measurement kit. The microfiltrate obtained by a known liquid culture method is 16 U / g to 19 U / g, the microfiltrate obtained from brown rice bran is 15 U / g to 17 U / g, and the precision obtained by this example. The filtrate was a result of 25 U / g-27 U / g.

α-Amylase activity:
Α-Amylase activity was measured by a standard analysis method of the Incorporated Administrative Agency of Liquors Research Institute. The microfiltrate obtained by a known liquid culture method is 70 U / g to 120 U / g, the microfiltrate obtained from brown rice bran is 65 U / g to 120 U / g, and the precision obtained by this example. The filtrate was a result of 180 U / g-200 U / g.

Acidic / neutral / alkaline protease activity:
Protease was measured by the standard analysis method of the National Research Institute for Liquors. The microfiltrate obtained from soybean meal is acidic: 1 U / g to 3 U / g, neutral: 8 U / g to 10 U / g, alkaline: 7 U / g to 9 U / g, and obtained by this example. The obtained microfiltrate had the results of acidity: 7 U / g to 10 U / g, neutrality: 8 U / g to 12 U / g, and alkalinity: 4 U / g to 8 U / g.

  From the above results, it can be seen that the microfiltrate obtained in this example has higher enzyme activity than any microfiltrate used in the comparative verification, and in particular has significantly higher α-amylase activity. It was.

Examples of the cereal meal according to this embodiment include brown rice cake, soybean meal, rice cake, rice cake, and rice cake.

Claims (12)

The following steps:
(I) adding the first cereal meal to the conditioned liquid medium;
(Ii) performing a first aeration culture using the conditioned liquid medium supplemented with the first cereal meal,
(Iii) adding a second cereal meal to the conditioned liquid medium subjected to the first aeration culture; and (iv) using the conditioned liquid medium to which the second cereal meal is added Performing aeration culture,
An enzyme-containing liquid culture method comprising:   The first cereal meal is selected from at least one of brown rice meal, soybean meal, rice meal, meal, and meal, and the second cereal meal is soybean meal. Enzyme-containing liquid culture method.   The first cereal meal is added in an amount of 2% to 15% of the volume of the conditioned liquid medium, and the second cereal meal is in an amount of 1% to 10% of the volume of the conditioned liquid medium. The enzyme-containing liquid culture method according to claim 1 or 2, which is added.   The enzyme-containing liquid culture method according to any one of claims 1 to 3, wherein the first and second cereal meals become carbon in the conditioned liquid medium.   The first and second cereal meals are produced by a koji mold selected from at least one of Aspergillus oryzae, Aspergillus kawachi, Alpergillus niger, Aspergillus awamori, and Alpergillus soya. The enzyme-containing liquid culture method according to any one of the above.   The first aeration culture is performed at a temperature of 20 ° C. to 40 ° C. for 24 hours to 60 hours, and the second aeration culture is performed at a temperature of 10 ° C. to 40 ° C. for 12 hours to 30 hours. The enzyme-containing liquid culture method according to any one of claims 1 to 5.   The enzyme-containing liquid culture method according to any one of claims 1 to 6, wherein the enzyme is at least one of α-amylase, glucoamylase, α-glucosidase, and protease. The following steps:
(I) obtaining a filter cloth filtrate by filtering the culture solution containing the enzyme produced by the enzyme-containing liquid culture method according to any one of claims 1 to 7,
(Ii) obtaining a microfiltrate by microfiltration of the filter cloth filtrate,
(Iii) obtaining a concentrate by ultrafiltration of the microfiltrate,
(Iv) adding ethyl alcohol to the concentrate and stirring;
(V) obtaining a precipitate by cooling a mixture of the stirred ethyl alcohol and the concentrated liquid;
(Vi) isolating the precipitate from the cooled mixture;
(Vii) obtaining a solid content by centrifuging the isolated precipitate; and (Viii) obtaining a powder by drying the solid content,
A method for producing an enzyme-containing powder comprising The following steps:
(I) obtaining a soybean-derived powder additive solution by adding soybean-derived powder to the microfiltrate according to claim 8 and stirring the mixture;
(Ii) obtaining a precipitate by cooling the soybean-derived powder additive solution;
(Iii) removing the supernatant from the precipitate;
(Iv) obtaining a solid by centrifuging the precipitate from which the supernatant has been removed, and (V) obtaining a powder by drying the solid,
A method for producing an enzyme-containing powder comprising   The method for producing an enzyme-containing powder according to claim 9, wherein the soybean-derived powder is selected from at least one of okara powder, soy milk powder, and kinako powder.   The method for producing enzyme-containing powder according to claim 9 or 10, wherein the soybean-derived powder is added in an amount of 2% to 20% of the volume of the microfiltrate.   The method for producing an enzyme-containing powder according to any one of claims 8 to 11, wherein the enzyme-containing powder contains at least one of α-amylase, glucoamylase, α-glucosidase, and protease.