JP7187014B2 - Method for producing saccharifying enzyme and proteolytic enzyme using koji mold - Google Patents

Description

The present invention relates to a method for producing saccharifying enzymes and/or proteolytic enzymes using Aspergillus oryzae.

Aspergillus oryzae is a kind of useful fungi used for manufacturing food, medicine, cosmetics, etc. by brewing and fermentation. The saccharifying enzymes and proteolytic enzymes produced by Aspergillus oryzae play an important role in the reactions in the above brewing and fermentation. For this reason, there is an increasing demand for a culture technique for improving the production of the above-mentioned enzyme by koji mold.

Studies have been conducted on the effects of light irradiation on Aspergillus oryzae during cultivation of Aspergillus oryzae. For example, Non-Patent Documents 1 and 2 show that the morphology of a fungal colony changes when Aspergillus oryzae, which is a type of Aspergillus oryzae, is irradiated with light, and that the Aspergillus oryzae has a plurality of light-responsive genes. is disclosed.

HATAKEYAMA et al., Light Represses Condiation in Koji Mold Aspergillus oryzae, Biosci. Biotechnol. Biochem., 71(8), 1844-1849, 2007 R. Hatakeyama and K. Kitamoto, Jokyo (2008), Vol. 103, No. 7, 525-531

However, according to the above-mentioned Non-Patent Documents 1 and 2, although it is described that Aspergillus oryzae responds to light irradiation, it is unclear what kind of gene (especially, transcription factor) expression of Aspergillus oryzae is affected. not been to. Moreover, the change in the enzyme production of Aspergillus oryzae by light irradiation has not been investigated. That is, Non-Patent Documents 1 and 2 do not disclose a technique for improving the production of enzymes by Aspergillus oryzae by light irradiation.

Accordingly, an object of one aspect of the present invention is to realize a method for efficiently producing saccharifying enzymes and/or proteolytic enzymes using Aspergillus oryzae.

As a result of intensive studies to solve the above problems, the inventors of the present invention have found that by performing light irradiation and magnetic field application during solid culture of Aspergillus oryzae, the colony of Aspergillus oryzae grows larger and the saccharifying enzyme (α-amylase etc.) and proteolytic enzymes (acidic carboxypeptidase etc.) were found to be improved, leading to the completion of the present invention.

That is, a method according to an aspect of the present invention is a method for producing a saccharifying enzyme and/or a proteolytic enzyme, comprising a step of solid-culturing Aspergillus oryzae, wherein the solid-state culturing step includes applying a magnetic field to the Aspergillus oryzae. and a light irradiation step of irradiating the koji mold with light.

In order to solve the above problems, in the method according to one aspect of the present invention, the magnetic field application step and the light irradiation step may be performed simultaneously.

In order to solve the above problems, in the method according to one aspect of the present invention, the strength of the magnetic field may be 0.2 T or more.

In order to solve the above problems, in the method according to one aspect of the present invention, the illuminance of the light may be 50 lux or more and 2000 lux or less.

In order to solve the above problems, in the method according to one aspect of the present invention, the light may include light with a wavelength of 575 nm or more and 700 nm or less.

In order to solve the above problems, in the method according to one aspect of the present invention, the saccharifying enzyme may be α-amylase, glucoamylase, or α-glucosidase.

In order to solve the above problems, in the method according to one aspect of the present invention, the protease may be an acid carboxypeptidase.

According to one aspect of the present invention, a method for efficiently producing saccharifying enzymes and/or proteolytic enzymes using Aspergillus oryzae can be realized.

(a) is a photograph of each fungal colony when Aspergillus oryzae was solid-cultured using the method according to an example or comparative example of the present invention, and (b) is a graph showing the diameter of the fungal colony. (a) and (b) are graphs showing the relationship between the magnetic field strength and the growth enhancement rate of Aspergillus oryzae when Aspergillus oryzae is solid-cultured using a method according to an example or a comparative example of the present invention. A) is a graph when light irradiation is performed, and (b) is a graph when light irradiation is not performed. (a) is a graph showing the diameter of each fungal colony when Aspergillus oryzae was solid-cultured using a method according to an example or comparative example of the present invention, and (b) is a graph showing the diameter of each fungal colony in an example or comparative example of the present invention. Fig. 4 is a graph showing the α-amylase activity produced by each Aspergillus oryzae when the Aspergillus oryzae was solid-cultured using such a method, and (c) is a graph showing the relationship between the applied magnetic field strength and the degree of increase in the α-amylase activity. is. (a) is a graph showing the diameter of each fungal colony when Aspergillus oryzae was solid-cultured using a method according to an example or comparative example of the present invention, and (b) is a graph showing the diameter of each fungal colony in an example or comparative example of the present invention. Fig. 4 is a graph showing the acidic carboxypeptidase activity produced by each Aspergillus oryzae when the Aspergillus oryzae is solid-cultured using such a method, and (c) is a graph showing the relationship between the strength of the applied magnetic field and the degree of increase in the acid carboxypeptidase activity. is. (a) to (c) are graphs showing the diameter of each fungal colony when Aspergillus oryzae was solid-cultured using a method according to an example or a comparative example of the present invention. It is a graph when irradiated, (b) is a graph when irradiated with blue light, and (c) is a graph when irradiated with green light. Note that (d) is a graph showing the wavelength spectrum of each irradiation light.

[Embodiment 1]
An embodiment of the present invention will be described in detail below.

(Aspergillus oryzae)
Aspergillus oryzae is a type of mold used in the production of foods, medicines (such as antibiotics), and cosmetics by brewing and fermentation. Examples of the food include sake, shochu, soy sauce, miso, and vinegar. Examples of the medicines and cosmetics include enzymes, antibiotics, whitening serums, supplements, and the like.

Aspergillus oryzae is not particularly limited as long as it can produce saccharifying enzyme and/or proteolytic enzyme. , Aspergillus deflectus, Aspergillus fischerianus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus ochraceus, Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae, Aspergillus sydowii, Aspergillus tamaritus, Aspergillus versicolor, etc. Monascus microorganisms, such as Aspergillus microorganisms, Monascus purpureus, Monascus pilosus, and Monascus anka. Among these, Aspergillus oryzae, which is widely used industrially, can be preferably applied to the method of the present invention. The Aspergillus oryzae may or may not be a mutant strain such as a genetic mutation.

Aspergillus oryzae secretes digestive enzymes during the culture process to promote the above brewing and fermentation. Digestive enzymes secreted by Aspergillus are, for example, saccharifying enzymes and proteolytic enzymes. These enzymes produce various metabolites by degrading specific substrates for each enzyme.

The saccharifying enzyme is not particularly limited as long as it is a saccharifying enzyme that can be produced by Aspergillus oryzae, and examples thereof include α-amylase. α-Amylase is an enzyme that uses carbohydrates such as starch or dextrin as substrates and degrades them to produce monosaccharides such as glucose and oligosaccharides including disaccharides such as maltose. Such production of monosaccharides, oligosaccharides, etc. by Aspergillus oryzae is called saccharification, and the enzyme that performs saccharification is called saccharifying enzyme. The saccharifying enzyme is not limited to α-amylase. Examples of other saccharifying enzymes include glucoamylase and α-glucosidase.

Such saccharifying enzymes work as follows in the process of preparing rice koji in the production of sake, for example. Steamed rice, which is rich in starch, is seeded with conidia of Aspergillus oryzae to promote brewing and fermentation. At this time, the koji mold secretes the above-mentioned saccharifying enzyme outside the cell, and carbohydrates such as starch contained in the steamed rice are decomposed into monosaccharides and oligosaccharides. In this way, the steamed rice is saccharified by the saccharifying enzyme secreted by the koji mold, resulting in rice koji containing a mixture of starch, monosaccharides, and oligosaccharides.

Moreover, the protease is not particularly limited as long as it is a protease that can be produced by Aspergillus oryzae, and examples thereof include acidic carboxypeptidase. Acid carboxypeptidase is an enzyme that uses proteins and peptides as substrates and cleaves the proteins and peptides from the carboxyl terminus (C-terminus) to produce amino acids. Amino acids produced in this process are important, for example, as taste components of sake. The proteolytic enzyme is not limited to carboxypeptidases such as acidic carboxypeptidase, but includes aminopeptidase, dipeptidyl peptidase, alkaline proteinase, neutral proteinase, and the like.

The method for culturing koji mold in one embodiment of the present invention is not particularly limited as long as it is a solid culture, and various methods can be adopted depending on the purpose. These culture methods include, for example, preservation culture suitable for long-term preservation of Aspergillus oryzae, subculture suitable for subculture preservation of Aspergillus oryzae, seed koji culture suitable for obtaining a large amount of conidia of Aspergillus oryzae, It is a single fungal isolation culture suitable for isolation and examination of the properties of koji mold. In addition, brewing and fermentation using koji mold are also included in koji mold culture methods. A method according to an embodiment of the present invention will be described for a case where it is used for single-bacteria isolation culture using a solid medium, but it is not limited to this, and can be used for other koji mold culture methods.

Aspergillus is cultivated by, for example, seeding a single or arbitrary amount of conidia of Aspergillus oryzae on an agar medium in a Petri dish, and culturing at a predetermined temperature in a temperature-controlled place such as a constant temperature bath. The predetermined temperature is not particularly limited as long as it is within a temperature range where the koji mold can grow. It is preferably 20°C to 30°C, and more preferably 20°C to 30°C.

Moreover, it is preferable to appropriately adjust the humidity in the culture environment. Culturing of Aspergillus oryzae in the method according to the present embodiment includes not only agar medium but also cultivation on solid media such as wheat bran, rice, barley, and potato on which Aspergillus oryzae can grow.

The agar medium preferably contains carbohydrates such as dextrin that serve as substrates for saccharifying enzymes, and proteins such as polypeptones that serve as substrates for protease, and single or multiple amino acids. According to such a configuration, it is possible to measure the concentration of sugars and amino acids produced by decomposing the substrate during the cultivation of Aspergillus oryzae. As a result, the activities of saccharifying enzymes and proteolytic enzymes secreted by Aspergillus oryzae during cultivation can be measured.

(Magnetic field application step)
In the method according to the present embodiment, a magnetic field is applied to the Aspergillus oryzae that is being cultured (during the solid culture step) on a solid medium such as agar (magnetic field application step). For example, an electromagnet can be used as the magnetic field source. For example, a magnetic field can be applied to the Aspergillus oryzae being cultured by horizontally placing a solid culture vessel such as a petri dish between the magnetic poles of an electromagnet that is set so that the lines of magnetic force are generated horizontally with respect to the ground. Also, by changing the voltage applied to the electromagnet, the strength of the generated magnetic field can be arbitrarily adjusted. The area for generating the magnetic field in the electromagnet is preferably larger than the area of the upper surface of the agar medium. According to such a configuration, a magnetic field can be more uniformly applied to the cultured Aspergillus oryzae.

Also, the source of the magnetic field is not limited to the electromagnet, and may be, for example, a permanent magnet. According to such a configuration, a magnetic field can be easily applied to Aspergillus oryzae without requiring a power source or the like to generate the magnetic field. Moreover, even when the number of culture vessels such as Petri dishes is increased, it is easy to procure and install permanent magnets corresponding to this.

In addition, regarding the direction in which the magnetic field is applied, the lines of magnetic force in the electromagnet are generated in the horizontal direction with respect to the bottom surface of the culture vessel such as a petri dish. This is substantially the same when an electromagnet is installed on the upper surface of a culture vessel such as a petri dish. Therefore, an electromagnet may be installed on the upper surface of the culture vessel such as a petri dish. Note that the direction in which the magnetic field is applied is not limited to this. For example, an electromagnet may be arranged on the side of a culture container such as a petri dish. In this case, the lines of magnetic force are generated in a direction perpendicular to the bottom surface of the culture vessel such as a petri dish. Further, by arranging electromagnets so as to surround a culture vessel such as a petri dish, magnetic lines of force may be generated in various directions with respect to the culture vessel such as a petri dish.

The strength of the magnetic field applied to the koji mold is preferably 0.2 T (Tesla) or more. According to such magnetic field strength, saccharifying enzymes and/or proteolytic enzymes can be efficiently produced by Aspergillus oryzae in combination with the light irradiation step described later. The strength of the magnetic field is more preferably 0.4 T or more, more preferably 0.6 T or more. By increasing the intensity of the magnetic field, saccharifying enzymes and/or proteolytic enzymes can be more efficiently produced by Aspergillus oryzae.

The application of the magnetic field may be performed continuously from the start of the culture of the koji mold to the end of the culture, or may be performed only at a specific timing during the culture. For example, periodic periods of time may switch the magnetic field on and off.

(Light irradiation step)
In the method according to the present embodiment, the Aspergillus oryzae that is being cultured (during the solid culture step) on a solid medium such as an agar medium is irradiated with light (light irradiation step). For the light irradiation, for example, white light emitted from a light source such as a fluorescent lamp or an LED in the room may be used as it is. In this case, the aspergillus oryzae may be cultured at room temperature outside a constant temperature bath or the like. Also, a light source such as a fluorescent lamp may be provided in a constant temperature bath or the like. In order to efficiently irradiate the koji mold with light, the culture vessel is preferably transparent. When wheat bran, rice, barley, potato, or the like is used as a medium, it is preferable to appropriately stir the wheat bran or the like inoculated with Aspergillus oryzae in order to efficiently irradiate light.

The illuminance of the irradiated light is preferably 50 lux or more, more preferably 100 lux or more, and more preferably 200 lux or more at the upper surface of the culture vessel such as a Petri dish (that is, close to the koji mold). . Moreover, it is preferable that it is 2000 lux or less. With such illuminance, light irradiation can be performed with a standard fluorescent lamp.

The irradiated light preferably includes light with a wavelength in the red light region of 575 nm or more and 700 nm or less. In addition to applying a magnetic field, the growth of the koji mold is promoted by irradiating the koji mold with light of such a wavelength. Therefore, the yield of saccharifying enzymes and proteolytic enzymes produced by koji mold is also improved. The light to be irradiated may be white light, red light, or light of other colors as long as it contains light of such wavelengths.

In addition, when the irradiated light has a wavelength of 350 nm or more and 570 nm or less, which is in the blue light region, the growth of Aspergillus oryzae may be suppressed. However, by applying a magnetic field in addition to the irradiation with the blue light, the effect of inhibiting the growth of Aspergillus oryzae due to the irradiation with the blue light can be alleviated.

The light irradiation may be performed continuously from the start of culturing of the koji mold to the end of the culturing, or may be performed only at a specific timing during the culturing. For example, light illumination may be switched on and off according to a period of time.

In addition, the magnetic field application step and the light irradiation step may be carried out at the same timing as long as there is a time period during which the magnetic field application and the light irradiation are carried out simultaneously in the koji mold culture step, or they may be carried out independently. It may be performed according to the timing. For example, magnetic field application may be switched on and off at regular intervals, and light irradiation may be always on. Also, the timing of magnetic field application and light irradiation may be reversed.

〔Example〕
An embodiment of the invention is described below.

(Culturing conditions for Aspergillus oryzae)
First, a method for culturing Aspergillus oryzae (Aspergillus oryzae, RIB40, NBRC 100959), which is common to each of Examples and Comparative Examples, will be described. A liquid culture medium was prepared by dissolving 6 g of sodium chloride, 4 g of dextrin anhydrate, 2 g of polypeptone, 1 g of potassium dihydrogen phosphate, and 0.1 g of magnesium sulfate heptahydrate in 200 mL of distilled water. The culture solution for solid medium was prepared by adding 6 g of sodium chloride, 4 g of dextrin anhydrate, 2 g of polypeptone, 1 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, and 4 g of agar powder to 200 mL of distilled water. was prepared.

These cultures were autoclaved and sterilized at 121° C. for 15 minutes. The sterilized liquid medium was cooled sufficiently at room temperature before use. The agar medium, which is a solid medium, is obtained by cooling a sterilized solid medium culture solution to a temperature that does not solidify, distributing it in a petri dish with a diameter of 5 cm so that the thickness is about 3 mm, and solidifying at room temperature. It was used.

Next, a suspension of conidia of Aspergillus oryzae was prepared as follows. Take an appropriate amount of conidia from a koji mold strain that has been cultured in advance under sterilized conditions in a clean bench, suspend it in a sterilized liquid medium, and then irradiate it with ultrasonic waves at approximately 36 kHz for 1 minute with an ultrasonic irradiator. to disperse the conidia to obtain a conidial dispersion. Next, the dispersion was filtered with a filter to remove impurities such as mycelium. The conidial concentration was then determined by Birkerturk hemocytometer. The concentration of the dispersion was adjusted using a liquid culture medium so that the concentration of conidia in the dispersion after filtration was 6.0×10 ⁶ cells/mL, and this was used as a conidia suspension. and

Conidia were sown on the solid medium in a clean bench by the following method. An ethanol-sterilized filter paper with a diameter of 6 mm was placed in the center of a solid medium prepared in a petri dish, and 20 μL of the well-stirred conidial suspension was dropped from the conidial suspension to seed the conidia of Aspergillus oryzae. The petri dish was taken out from the clean bench after being put in a state where the sterilization condition could be maintained by putting a lid on it.

Depending on the presence or absence of magnetic field application and light irradiation, magnetic field application + light irradiation (MF + Light: Example 1), magnetic field application only (MF: Comparative Example 1), light irradiation only (Light: Comparative Example 2), and magnetic field application and light irradiation Four culture conditions were set without (Control: Comparative Example 3). Note that MF indicates Magnetic Field. Aspergillus oryzae was cultured under these culture conditions by the following method.

The petri dish seeded with the conidia was allowed to stand in a constant temperature bath maintained at 25° C. under the culture conditions corresponding to the above Examples and Comparative Examples 1 to 3, and cultured for 96 hours.

A magnetic field was applied using an electromagnet that generates a steady magnetic field, and was adjusted to a magnetic field value of 0.6 T unless otherwise specified. The lines of magnetic force in the magnetic field were generated in a horizontal direction with respect to the bottom of the petri dish. A magnetic field was applied immediately after the start of the culture, and thereafter the magnetic field was switched on and off in 12-hour cycles.

Light irradiation was performed by directly irradiating the upper surface of the petri dish with white light emitted from 9 white fluorescent lamps (36 W/unit) installed in the ceiling of the laboratory (illuminance on the upper surface of the petri dish: approximately 250 lux).

(Growth evaluation of koji mold)
After culturing for 96 hours under the culture conditions of Example 1 and Comparative Examples 1 to 3, the colonies of Aspergillus aspergillus growing concentrically on the solid medium were imaged from above and from the horizontal direction of the petri dish. Growth circle size (diameter of bacterial colony) was evaluated.

FIG. 1 shows the growth of Aspergillus oryzae when using the method according to an example or a comparative example of the present invention. FIG. It is a graph which shows the diameter of each bacterial colony. As shown in FIGS. 1(a) and 1(b), under the culture conditions of Example 1, the diameter of the bacterial colonies increased compared to the culture conditions of Comparative Examples 1-3. That is, under the culture conditions of Example 1, the growth of Aspergillus oryzae was promoted as compared with the culture conditions of Comparative Examples 1-3.

FIG. 2 is a graph showing the relationship between the magnetic field strength and the growth enhancement rate of Aspergillus oryzae when the method according to the example or comparative example of the present invention is used. FIG. 2(b) is the case without light irradiation. The growth enhancement rate in FIG. 2 indicates the increase rate of the diameter of the bacterial colony when the magnetic field is applied to the diameter of the bacterial colony when the magnetic field is not applied.

As shown in FIG. 2(a), in Example 1, the growth enhancement rate increased as the strength of the applied magnetic field increased, compared to the case where no magnetic field was applied (Comparative Example 2). On the other hand, as shown in FIG. 2B, in Comparative Example 1, the growth enhancement rate was less than 5% regardless of the strength of the applied magnetic field, compared to the case where no magnetic field was applied (Comparative Example 3). , no significant changes were observed.

(α-amylase activity evaluation)
The influence of Aspergillus oryzae on α-amylase production was evaluated by α-amylase activity measurement when the method according to the examples or comparative examples of the present invention was used. Activity measurement of α-amylase was carried out by the following method. The colony of Aspergillus oryzae together with the solid medium was crushed with a spatula and transferred to a 50 mL centrifuge tube. 10 mL of 0.01 mol/L sodium acetate aqueous solution was added to this, and ultrasonic waves of about 20 kHz were applied for 5 minutes with an ultrasonic homogenizer to further pulverize the agar and the cells. The pulverized liquid thus obtained was shaken at 30° C. for 1 hour to extract the enzyme from the agar.

Next, the pulverized liquid after enzyme extraction was centrifuged at 4° C. and 10000×g for 5 minutes, and then the supernatant was recovered. 5 mL of a 0.01 mol/L sodium acetate aqueous solution was added again to the residue precipitate, and the enzyme was extracted from the agar by shaking at 30° C. for 1 hour in the same manner as described above. After this extract was centrifuged at 4° C. and 10,000×g for 5 minutes in the same manner as above, the supernatant was collected and mixed with the previously obtained supernatant. The above extraction procedure was performed once more, and all the obtained supernatants were mixed to obtain about 20 mL of the enzyme extract.

The enzymatic activity of α-amylase was measured using an α-amylase assay kit manufactured by Kikkoman Biochemifa.

FIG. 3 is a graph showing the diameter of a koji mold colony in FIG. 3(a), and FIG. Fig. 3(c) is a graph showing the activity of the α-amylase produced (enzyme activity), and Fig. 3(c) is a graph showing the relationship between the magnetic field strength and the degree of increase in the enzyme activity of the α-amylase. The degree of increase in enzymatic activity in (c) of FIG.

As shown in (a) of FIG. 3, the diameter of the bacterial colony in Example 1 was increased compared to Comparative Examples 1-3. At this time, the diameter of the bacterial colony in Example 1 was approximately 120% of the diameter of the bacterial colony in Comparative Example 3.

In addition, as shown in FIG. 3(b), the α-amylase activity in Example 1 was greatly increased compared to Comparative Examples 1-3. At this time, the α-amylase activity in Example 1 was approximately 180% of the α-amylase activity in Comparative Example 3.

These results suggest that the increase in α-amylase activity due to the application of a magnetic field and light irradiation is caused not only by an increase in the number of fungal bodies due to the promotion of growth of Aspergillus oryzae, but also by an increase in the amount of α-amylase produced by the Aspergillus itself. rice field.

Further, as shown in FIG. 3(c), the degree of increase in α-amylase activity in Example 1 was substantially proportional to the magnetic field strength. On the other hand, compared with the α-amylase activity increase in Example 1, the α-amylase activity increase in Comparative Example 1 did not change significantly even when the magnetic field strength was changed. In other words, it was shown that both elements of appropriate magnetic field strength and light irradiation are important for increasing α-amylase activity.

(Evaluation of acid carboxypeptidase activity)
The influence of Aspergillus oryzae on acid carboxypeptidase production when using the method according to Examples or Comparative Examples of the present invention was evaluated by acid carboxypeptidase activity measurement. Acid carboxypeptidase activity was measured using an acid carboxypeptidase assay kit manufactured by Kikkoman Biochemifa Corporation by preparing an enzyme extract in the same manner as in the above α-amylase activity assay.

FIG. 4 is a graph showing the diameter of a koji mold colony in FIG. 4(a), and FIG. Fig. 4(c) is a graph showing the acid carboxypeptidase activity (enzyme activity) to be produced, and Fig. 4(c) is a graph showing the relationship between the magnetic field strength and the degree of increase in the enzyme activity of the acid carboxypeptidase. The degree of increase in enzyme activity in (c) of FIG.

As shown in FIG. 4(a), the diameter of the bacterial colony in Example 1 was increased compared to Comparative Examples 1-3. At this time, the diameter of the bacterial colony in Example 1 was approximately 120% of the diameter of the bacterial colony in Comparative Example 3.

On the other hand, as shown in FIG. 4(b), the acid carboxypeptidase activity in Example 1 was greatly increased compared to Comparative Examples 1-3. At this time, the acid carboxypeptidase activity in Example 1 was approximately 230% of the acid carboxypeptidase activity in Comparative Example 3.

From these results, the increase in acid carboxypeptidase activity due to the application of a magnetic field and light irradiation is similar to the α-amylase activity described above. It was suggested that it was due to the increase in the amount.

Moreover, as shown in FIG. 4(c), the degree of increase in acidic carboxypeptidase activity in Example 1 was substantially proportional to the magnetic field strength. On the other hand, the degree of increase in acidic carboxypeptidase activity in Comparative Example 1 showed little change compared to the degree of increase in acidic carboxypeptidase activity in Example 1. In other words, it was shown that both elements, appropriate magnetic field strength and light irradiation, are important for increasing acid carboxypeptidase activity.

(Wavelength of irradiated light)
Regarding the effect of the above-described light irradiation on Aspergillus oryzae, the wavelength dependence of light was evaluated. The wavelength of the irradiated light was in the blue light region, the green light region, or the red light region. Light of each color is obtained by combining a white light source with cellophane or plastic filters, and wavelength ranges corresponding to each color (blue light: maximum intensity wavelength 466 nm, distribution 350-570 nm; green light: maximum intensity wavelength 542 nm, distribution 470-600 nm; Red light: maximum intensity wavelength 624 nm, distribution 575 to 700 nm).

FIG. 5 is a graph showing the diameter of a fungal colony of Aspergillus aspergillus in the case of using the method according to an example or a comparative example of the present invention. ) is blue light and (c) is green light. Moreover, (d) of FIG. 5 is a graph showing the spectrum of each irradiation light.

As shown in FIG. 5(a), when red light was used for light irradiation, the diameter of the bacterial colony in Example 1 increased compared to Comparative Examples 1-3. This was the same result as when white light was used (see, for example, FIG. 1).

On the other hand, as shown in FIG. 5(b), when blue light was used for light irradiation, the diameter of the bacterial colony was slightly reduced in Comparative Example 2 compared to Comparative Example 3. On the other hand, in Example 1, the decrease in the diameter of the bacterial colony seen in Comparative Example 2 was recovered. In other words, although blue light irradiation inhibited the growth of Aspergillus oryzae, further application of a magnetic field was effective in alleviating the growth inhibition.

As shown in FIG. 5(c), when green light was used for light irradiation, no change in the diameter of the bacterial colonies was observed in Example 1 and Comparative Examples 1 to 3. That is, it was suggested that green light does not affect the growth of Aspergillus oryzae.

These results suggest that the effect of light irradiation on Aspergillus oryzae in addition to the application of a magnetic field is mainly due to light in the red light region. That is, it was suggested that the irradiation of red light, in combination with the application of a magnetic field, promotes the growth of Aspergillus oryzae, leading to an increase in the production of saccharifying enzymes and proteolytic enzymes produced by Aspergillus oryzae.

[Additional notes]
The present invention is not limited to the above-described embodiments, but can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. is also included in the technical scope of the present invention.

INDUSTRIAL APPLICABILITY The present invention can be used for brewing using Aspergillus oryzae, production of fermented products, production of medicines, and the like.

Claims (6)

A method for producing saccharifying enzymes and proteolytic enzymes, comprising a step of solid-culturing koji mold,
The solid-state culturing step includes a magnetic field applying step of applying a magnetic field to the Aspergillus oryzae and a light irradiation step of irradiating the Aspergillus oryzae with light,
The method , wherein the strength of the magnetic field is 0.2T or more and 0.6T or less . 2. The method according to claim 1, wherein said magnetic field application step and said light irradiation step are performed simultaneously. 3. The method according to claim 1 , wherein the illuminance of said light is 50 lux or more and 2000 lux or less. The method according to any one of claims 1 to 3 , wherein the light includes light with a wavelength of 575 nm or more and 700 nm or less. The method according to any one of claims 1 to 4 , wherein the saccharifying enzyme is α-amylase, glucoamylase, or α-glucosidase. The method according to any one of claims 1 to 5 , characterized in that said proteolytic enzyme is an acid carboxypeptidase.

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