A novel koji mold with high saccharification power, low tyrosinase activity, and suitable for seed koji production

Abstract

To provide a novel Aspergillus oryzae having high saccharifying power and low tyrosinase activity that enable the production of high-quality Amazake and having the high productivity of seed Koji.SOLUTION: The present invention relates to an Aspergillus oryzae NGA3 strain (NITE P-03363). According to the present invention, an Aspergillus oryzae can be provided which has sufficient activity necessary for saccharification as compared to AOK3006 strain which is a parent strain of the Aspergillus oryzae, has much less browning when producing Koji, and has the high productivity of seed Koji so as to enable the stable supply of seed Koji as compared to CK33-3 strain which is a similar Aspergillus oryzae strain of the present applicant.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a novel Aspergillus oryzae. More specifically, the present invention relates to a novel Aspergillus oryzae that has excellent aroma of the resulting koji rice, less browning when producing koji, high productivity of seed koji, and is particularly suitable for producing amazake.

Background Art

[0002] Riding on the wave of the recent fermented food boom, various types of amazake have been developed and put on the market throughout the country. In the sensory evaluation of amazake, not only the aroma and taste but also the whiteness of the appearance are emphasized, and these sensory characteristics are largely due to Aspergillus oryzae. Among Aspergillus oryzae, it is known that those with high saccharifying power (glucoamylase activity + α-glucosidase activity, the same hereinafter) can produce amazake with a strong sweetness, and further, those with lower tyrosinase activity can produce amazake with a whiter color tone. Therefore, in order to produce high-quality amazake, the development of Aspergillus oryzae with high saccharifying power and low tyrosinase activity is required. Such Aspergillus oryzae has already been obtained and put into practical use by the applicant through obtaining a large number of artificial mutants from existing Aspergillus oryzae using transposon transfer and screening these artificial mutants using tyrosinase activity etc. as indicators (Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When Aspergillus oryzae is used in the production of fermented foods such as sake, miso, and soy sauce including amazake, in most cases, seed koji mainly composed of conidia of Aspergillus oryzae, which is the basis of koji, is used. However, since the Aspergillus oryzae strain (CK33-3 strain of Patent Document 1) with high saccharification ability and low tyrosinase activity, which has been put into practical use by the applicant, has weak conidia-forming ability, the seed koji productivity is low, and the supply of seed koji to fermented food manufacturers is also unstable.

[0005] Therefore, an object of the present invention is to provide a novel Aspergillus oryzae strain having high saccharification ability, low tyrosinase activity, and high seed koji productivity, which enables the production of high-quality amazake.

Means for Solving the Problems

[0006] As a result of intensive studies to solve the above problems, the present inventor used the AOK3006 strain (an unpublished strain owned by the applicant of the present application) with high saccharification ability as the parent strain, examined the stress conditions during transposon mutagenesis, and further examined the medium and screening method for culturing the obtained mutant strains. As a result, the present inventor succeeded in selecting a mutant strain with low tyrosinase activity and improved seed koji productivity while maintaining high saccharification ability, and thus reached the present invention. That is, the present invention is as follows. 1. Aspergillus oryzae NGA3 strain (NITE P-03363). 2. Seed koji comprising conidia of the Aspergillus oryzae of claim 1.

Effects of the Invention

[0007] According to the present invention, compared with the AOK3006 strain, which is the parent strain of Aspergillus oryzae, it has sufficient activity required for saccharification, and the browning property when producing koji is much less. Compared with the CK33-3 strain, which is a similar Aspergillus oryzae strain of the applicant of the present application, it is possible to provide an Aspergillus oryzae strain with high seed koji productivity and stable supply of seed koji.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0009] Hereinafter, the present invention will be described in detail. 1. Regarding the koji mold of the present invention The koji mold Aspergillus oryzae NGA3 strain of the present invention (hereinafter, the koji mold NGA3 strain, or simply the NGA3 strain) is a koji strain selected in line with the object of the present invention from among those obtained by mutating the koji mold AOK3006 strain, an unpublished strain held by the applicant. Similar to the parent strain of koji mold AOK3006, it has a high saccharifying power that is considered to greatly contribute to the flavor of amazake, and a high α-amylase activity that dissolves starch, and has sufficient activity required for saccharification. Moreover, different from the parent strain, it has a lower tyrosinase activity that causes browning compared to the parent strain, and has a lower browning property when producing koji. The saccharifying power is 400 - 800 U / g dry koji when producing koji by culturing for about 20 - 24 hours at an inlet temperature of 30 - 35°C using raw rice that has been milled to about 90% of its original weight, then performing maintenance, and further culturing at 30°C for about 20 - 24 hours. The tyrosinase relative activity when produced in the same way is between 0.2 - 0.6 as a relative value to the parent strain AOK3006 strain and between 0.4 - 1.2 as a relative value to the CK-33 strain. The koji strain CK33-3, which is the same as the one previously reported by the applicant (see Patent Document 1), surprisingly, compared with this koji strain, the productivity of the starter koji of the koji strain of the present invention is more than twice, more specifically, about 2 to 3 times. For example, when using the same 120 kg of brown rice, only 2.0 kg of starter koji can be produced with the CK33-3 strain, while 4.4 kg can be produced using the koji mold of the present invention, and the effect is very significant. Furthermore, compared with the CK33-3 strain, it has also been found that when making amazake using the koji strain of the present invention, the koji rice grains are less likely to remain.

[0010] 2. Acquisition of the koji strain of the present invention A koji mold mutant is created, and the target koji strain is screened from it to obtain the koji strain of the present invention.

[0011] (1) Creation of a koji mold mutant using the transfer of an endogenous transposon The koji mold strain AOK3006 is an unpublished strain held by the applicant. Compared with ordinary koji molds, it has high saccharifying power and α-amylase activity that greatly contributes to the flavor of amazake, which can dissolve starch. However, this strain has the drawback of browning. Using this AOK3006 strain as the parent strain, while maintaining sufficient activity required for saccharification, the aim is to obtain a mutant strain that is less likely to brown during koji production and has high productivity of starter koji. As methods for obtaining artificial mutants of Aspergillus oryzae, various methods such as irradiation with radiation or ultraviolet rays and treatment with chemical agents have been attempted, but a method of obtaining mutant strains by utilizing the transfer of endogenous transposons is known. The transposon gene consists of units of 1 to 2 kbp, has inverted repeat sequences TIR of 20 to 30 bp at both ends and direct repeat sequences TSD of about 2 to 10 bp outside thereof, and the sequence encoding Transposase is located in the internal region of TIR. At the time of transfer, it is presumed that Transoposase recognizes TIR, excises the transposon from the genome, and inserts it at another position. The transposon gene Crawler of Aspergillus oryzae has already been reported, and several practical research examples have also been made (Reference 1: K. Ogasawara, Journal of the Brewing Society of Japan, Vol. 105(6), p334 - 342(2010). Reference 2: K. Ogasawara et al., Report of Akita Prefectural Food Research Center, No. 15, p19 - 28(2013), http: / / www.arif.pref.akita.jp / download / houkoku / houkoku15.pdf). Strain AOK3006 has multiple copies of the active DNA transposon Crawler, and Crawler transfers by strong stress treatment to conidia. Therefore, mutants of strain AOK3006 are induced by the transposon Crawler of Aspergillus oryzae to create a mutant bank. The transposon mutation of strain AOK3006 of the present invention is carried out, for example, by stress treatment with heat or chemical agents or a combination thereof. Examples of chemical agents include copper ions, hydrogen peroxide, and acid treatment at about pH 3.0, and copper ions are preferred. Treatment with chemical agents is carried out, for example, by adding a chemical agent to a conidial suspension adjusted so that the concentration of conidia of the parental strain is about 10 5 ~10 7 cells / ml, and shaking at 27 - 35°C for 4 - 8 hours. The concentration of the chemical agent is usually about 15 - 40 mM for copper ions, more preferably 30 - 40 mM, 0.05 - 0.2% for hydrogen peroxide, and 0.2 M for sodium acetate buffer (pH 3.0). Heat treatment is usually carried out by shaking at 50 - 52°C for 6 - 8 hours. In Patent Document 1, mutations were also induced by Aspergillus oryzae transposon Crawler to obtain mutant strains. However, it does not mean that the same mutant strains can be obtained under the same stress conditions. Furthermore, it is expected that more different mutant strains can be obtained by adjusting the stress conditions. In the invention of the present application, the stress conditions were studied, and by increasing the copper ion concentration compared to Patent Document 1, the creation of the target Aspergillus oryzae mutant strain is expected.

[0012] (2) Selective culture The stress-treated conidial suspension is applied to a minimal agar medium (CD-KClO containing potassium perchlorate) 3、 Czapek-Dox medium containing KClO3), and cultured at 30 °C for 7 to 14 days. The grown single colonies are randomly collected and inoculated onto fresh CD-KClO3, and after growth, they are stored as transposon mutation candidate strains (Aspergillus oryzae mutant bank). The medium used this time is one type of selective medium for nitrate reductase (niaD) -deficient strains. Resistant strains that grow on the selective medium for nitrate reductase -deficient strains are likely to have some mutation introduced into normal nitrogen metabolism, but at the same time, there is also a high possibility of obtaining strains with reduced tyrosinase activity (Reference 3: Japanese Patent Laid-Open No. 2003-339370). In the invention of the present application, the mutant strains are cultured on a selective medium containing potassium perchlorate, which is a selective medium for nitrate reductase -deficient strains, to selectively culture Aspergillus oryzae strains that are more in line with the purpose. Among the AOK3006 strain transposon mutant strains, collect strains with good growth on the selective medium or hyphae showing conidiospore formation. By collecting strains with good growth, highly productive Aspergillus oryzae strains can be obtained.

[0013] (3) Screening of Aspergillus oryzae mutant strains First, primary screening is performed using tyrosinase activity as an index from the created AOK3006 mutant bank. It is known that tyrosinase activity is correlated with browning in Aspergillus oryzae, and the lower the activity, the less browning of koji and amazake. (I) Production of a small amount of koji Many of the enzyme activities associated with the practical use of Aspergillus oryzae are specific to rice koji culture. In particular, the tyrosinase targeted this time is an example, and evaluation using rice koji is essential. However, the conventional koji production using petri dishes and koji lids is not suitable for screening due to space and equipment limitations. Therefore, the inventor of the present invention examined a method for producing a small amount of koji using disposable 50 ml conical tubes. That is, a certain amount of conidiospores of Aspergillus oryzae grown on a potato dextrose agar (PDA) plate was collected with a sterilized microspatula, suspended in a 0.01% (W / V) Tween 80 solution, and a spore suspension was prepared. 3 g of gelatinized rice was placed in a conical tube, the spore suspension was added so that the moisture content was around 35%, and it was stirred well so that the moisture was uniform. The cap of the conical tube was lightly tightened, cultured at 30 °C for 20 to 24 hours, then covered with a cellulose filter instead of the cap, and further cultured for about 24 hours. The tyrosinase activity of the koji obtained in this way was higher than that of koji production at the site, but it was sufficient for screening low-tyrosinase activity mutant strains. As a result, it was possible to establish a rate-limiting method for screening the target transposon mutant bank. In addition, compared with Patent Document 1, by using gelatinized rice instead of steaming rice every time, the quality is stabilized, the accuracy of the results is improved, and those with lower tyrosinase activity can be selected. (II) Measurement of tyrosinase activity Measure by the following two methods and narrow down the candidate strains. First, add DOPA (Dihydroxyphenylalanine), a substrate of tyrosinase, to rice koji, and visually evaluate the degree of browning after the enzyme reaction to narrow down the candidate strains. Next, after crushing the rice koji of the shortlisted candidate strains with beads, centrifuge to collect the supernatant, add DOPA (Dihydroxyphenylalanine), measure the absorbance after the enzyme reaction to measure the tyrosinase activity, and further narrow down the candidate strains. In this way, the Aspergillus oryzae NGA3 strain of the present invention is obtained.

[0014] 3. Inoculum of the koji mold strain of the present invention An inoculum is used by spraying it on steamed raw materials when producing koji, and mainly consists of conidia of koji mold. In the inoculum of the present invention, in addition to these conidia, for example, bulking agents such as α-starch may be mixed, or a part other than the conidia of koji mold may be mixed in. The inoculum is produced by culturing koji mold using rice as a raw material, allowing conidia to sufficiently grow, and then drying.

[0015] 4. Koji obtained by using the koji mold and inoculum of the present invention The koji of the present invention is produced using either or both of the koji mold and inoculum of the present invention. Koji may be produced by mixing koji with steamed rice, or by inoculating steamed rice with koji mold, or by spraying the inoculum of the koji mold strain of the present invention on steamed rice to produce koji. However, generally, an inoculum is often used when producing koji.

[0016] 5. Processed foods using the koji mold of the present invention Processed foods containing koji obtained by using the koji mold or inoculum of the present invention as a raw material include, for example, amazake, sake, miso, soy sauce, koji pickles, amazake blended beverages, etc., and furthermore, processed fishery products such as rice sushi and fish soy sauce that utilize koji are also included. It includes not only processed foods whose manufacturing process includes a fermentation process, but also those that use koji for seasoning or flavoring. To produce amazake using the koji mold of the present invention, it can be done in exactly the same way as with conventional koji mold. The koji mold can of course be used alone as an inoculum for amazake, and it can also be used in combination with other koji molds according to the purpose. In the amazake thus obtained, there is little browning, the color tone of the amazake itself is good, it has sufficient sweetness, and it is finished into a high-quality product. Also, when producing processed foods other than amazake using the koji mold of the present invention, it can be done using the conventional well-known manufacturing method as it is.

Examples

[0017] Example 1 Preparation of a bank of koji mold mutant strains (1) Preparation of conidia of the parental strain (AOK3006) The conidia of strain AOK3006 were spread on a potato dextrose agar medium (PDA) and cultured at 30 °C for 7 to 10 days. The Aspergillus mycelium on the PDA where the conidia had grown was suspended in a 0.1% Tween 80 solution, and then the Aspergillus hyphae, agar medium, etc. were filtered through Miracloth, and the filtrate, which is the conidia suspension, was collected. Sterile water was added to the collected conidia suspension, and after centrifugation, the supernatant was removed. After washing several times with sterile water, it was resuspended in sterile water so that the concentration of the conidia was about 10 6 cells / ml, and a conidia suspension was prepared.

[0018] (2) Stress treatment (transposon transfer) The stress treatment was performed by adding copper ions. For the copper ion treatment, copper sulfate (CuSO4) was added to the conidia suspension to a concentration of 18 mM or 33 mM, and it was cultured with shaking at 30 °C. After completion, it was washed with a 0.01% Tween 80 solution and centrifuged. Furthermore, the collected conidia were washed several times with sterile water and resuspended in sterile water to obtain a conidia suspension treated with copper ion stress. In the above treatment, the survival rate was around 20% in the 18 mM treatment group and 0.1 - 3% in the 33 mM treatment group. Generally, the lower the survival rate due to the mutation treatment, the higher the possibility that gene mutations have occurred. It is considered that transposon transfer occurs more frequently in the 33 mM treatment group. Therefore, there is a high possibility that conidia with transposon transfer exist in the conidia suspension treated with 33 mM copper ion stress, which has a lower survival rate.

[0019] (3) Selection culture The stress-treated conidial suspension at 33 mM was spread on a minimal agar medium containing potassium perchlorate (CD-KClO₃; Czapek-Dox medium containing 6.13% potassium perchlorate (CD medium)) (0.19% sodium glutamate, 1.0% glucose, 0.1% dipotassium hydrogen phosphate, 0.1%, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.0001% iron(II) sulfate heptahydrate, 0.0088% zinc(II) sulfate heptahydrate, 0.00004% copper(II) sulfate pentahydrate, 0.00002% manganese(II) sulfate pentahydrate, 0.00001% sodium tetraborate decahydrate, 0.00001% ammonium molybdate(VI) tetrahydrate) and cultured at 30 °C for 7 to 14 days. The grown single colonies were randomly collected and inoculated onto fresh CD-KClO₃, and after growth, they were stored as transposon mutant candidate strains. (Aspergillus mutant bank)

[0020] Example 2 Screening for low tyrosinase activity strains from an Aspergillus mutant bank (1) Preparation of koji from Aspergillus contained in the Aspergillus mutant bank The conidial suspension of the Aspergillus mutant bank was inoculated onto potato dextrose agar (PDA) prepared in a 24-well plate and cultured at 30 °C for 5 to 7 days. After the grown Aspergillus conidia were collected in a certain amount with a sterilized microspatula, they were suspended in a 0.01% (W / V) Tween 80 solution to prepare a spore suspension. 3 g of gelatinized rice was put into a conical tube, the spore suspension was added so that the moisture content was around 35%, and it was stirred well to make the moisture uniform. After stirring, the cap of the conical tube was lightly tightened and cultured at 30 °C for 20 to 24 hours, then covered with a cellulose filter instead of the cap and cultured for about another 24 hours to prepare koji. (2) Screening for low tyrosinase activity strains The tyrosinase activity of the obtained koji was evaluated, and screening for candidate strains with low tyrosinase activity was carried out. First, enzyme activity evaluation was performed by adding DOPA (Dihydroxyphenylalanine, a substrate for tyrosinase) containing 10% ethanol to koji made from rice, and visually evaluating the degree of browning at 40°C ([Figure 1] above). Strains with less browning were shortlisted as candidate strains. Next, a crushed extract of the koji of the shortlisted candidate strains (20 mM sodium acetate buffer containing 0.5% NaCl, pH 5.0, crushed with beads) was prepared, an enzyme reaction solution containing DOPA was added, and the oxidation activity was measured. The results were shown as relative activity to the parent strain ([Figure 1] below). Strains with low activity were selected to further narrow down the candidate strains. As a control for both measurements, the Aspergillus oryzae CK33-3 strain with low tyrosinase activity (Patent Document 1) was used. Also, NGA1, 8, and 10 are other candidate mutant strains. From [Figure 1], one strain (NGA3 strain) was found that had lower tyrosinase relative activity and a lower degree of browning visually than the parent strain AOK3006, and showed an enzyme activity comparable to that of the CK33-3 strain, which is a low-tyrosinase-activity strain. In this way, it was possible to select strains with low tyrosinase activity.

[0021] Example 3: Test for producing mother koji of excellent selected strains Using the NGA3 strain, which had a low tyrosinase relative activity in Example 2, a test for producing mother koji was conducted. The production of mother koji was carried out as follows. The brown rice was polished to a head rice yield of 97 - 98%, washed, and soaked in a sufficient amount of water. After draining the water, it was steamed in an autoclave and then cooled. The flask-cultured koji spore-forming bacteria were sprayed onto the cooled steamed rice, and it was stirred well to make the koji spores uniform. After standing overnight at 30 - 35°C, it was put into a sterilized wooden koji trough. In the first half of koji making, temperature control (around 35°C) was carried out for the purpose of koji mold growth, and in the second half of koji making, temperature and humidity control (around 30°C for temperature and saturated humidity) was carried out to promote conidiation of the koji mold, and koji making was carried out for 5 - 6 days. After the koji after koji making was dried until the moisture content was 10% or less, it was sieved to remove hyphae and grains, and only conidia were collected. The weight of the obtained conidia was divided by the weight of the brown rice used to obtain the productivity of the koji spores. That is, "Productivity of koji spores = Weight of conidia / Weight of brown rice used". Finally, α-starch (rice starch or potato starch) was added to the conidia as a bulking agent to prepare koji spores. The productivity of koji spores of each koji mold strain is shown in [Table 1]. It was revealed that the productivity of koji spores of the NGA3 strain was more than twice as high as that of the CK33-3 strain, which is a koji mold strain with high saccharification ability and low tyrosinase activity obtained so far.

Table 1

[0022] Example 4 Practical Koji Manufacturing Test of Excellent Selected Strains The koji spores obtained in the koji spore manufacturing test were inoculated into 1.8 kg of steamed rice prepared from 90% polished rice, and a koji manufacturing test was carried out according to the standard method using a koji lid. There were no particular problems in the koji making operation process, and the work could be advanced in synchronization with the control strain CK33-3. The relative tyrosinase activity of the manufactured koji was measured in the same manner as in Example 2. However, the relative tyrosinase activity of the rice koji using the NGA3 strain was as low as that of CK33-3, which is a low tyrosinase activity strain ([Figure 2]), and the appearance of the koji after production was also white. Furthermore, the saccharifying power (glucoamylase activity + α-glucosidase activity) and α-amylase activity were evaluated as follows. That is, 10 g of koji was extracted with 50 ml of 20 mM sodium acetate buffer (pH 5.0, 0.5% NaCl) at room temperature for 3 hours according to the prescribed analysis method of the National Tax Agency to prepare an enzyme extract for activity measurement. Then, the saccharifying power and the activity of α-amylase were measured using a brewing analysis kit manufactured by Kikkoman Biochemifa Co., Ltd. (saccharifying power measurement kit: product code 60211, α-amylase measurement kit: product code 60213) ([Figure 3]). From [Figure 3], compared with CK33 which is the control strain, the enzyme activity was slightly decreased, but all showed high activity values, and the ability to convert starch into glucose, which is the element of sweetness, was sufficient. Furthermore, a sensory evaluation was conducted using 8 commercially available kojis (A to H) using the CK33-3 strain as a control ([Table 2]). The sensory evaluation panel consisted of 8 people, and the evaluation items were 5 items: color, haziness, aroma, taste, and texture. The evaluation was conducted using a 5-point scale (1: very good, 2: good, 3: normal, 4: difficult, 5: very difficult), and the average value was calculated. The lower the score, the higher the evaluation of the koji. As shown in [Table 2], the score of the rice koji using the NGA3 strain was 2.4, with an evaluation of "normal to good", and the ranking was also the second. In terms of quality, it was not inferior compared to the commercially available kojis using other CK33-3 strains.

Table 2

[0023] Example 5 Sweet sake production test and sensory test using excellent selected strains Using the rice koji prepared in Example 3, a sweet sake production test was conducted. The sweet sake was prepared by adding 200 g of water to 100 g of rice koji and incubating at 58 °C for 4 hours. The Brix sugar degree and glucose concentration of the sweet sake were equivalent to those of CK33-3 which is the control strain, and sufficient saccharification was carried out ([Table 3]).

Table 3

Table 4

Industrial Applicability

[0024] According to the present invention, it is possible to provide an Aspergillus oryzae having sufficient activity necessary for saccharification, less browning property, and high productivity of seed koji. Therefore, it is particularly useful for the production of seed koji for sweet sake and the production of sweet sake.

Deposit Number

[0025] Aspergillus oryzae NGA3 NITE P-03363

Claims

1. Aspergillus oryzae NGA3 strain (NITE P-03363).

2. A seed koji comprising conidia of the koji fungus of claim 1.

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