JP2009095279A - Koji mold, method for breeding the same, and method for producing soy sauce - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To breed a Koji mold having the protease productivity of ≥1,100 U/g Koji and the glutaminase productivity of ≥2.0 U/g Koji, and to provide soy sauce having a high nitrogen utilization ratio, total nitrogen and glutamic acid content using the Koji mold. <P>SOLUTION: The Koji mold is obtained by irradiating a strain belonging to the genus Aspergillus with a heavy ion beam and separating the Koji mold having protease productivity of ≥1,100 U/g Koji and glutaminase productivity of ≥2.0 U/g Koji from the irradiated strain or a cultured product thereof. The resultant Koji mold is inoculated into raw materials for soy sauce Koji and cultured to prepare the Koji, and the resultant Koji is used to carry out preparation for brewing. As a result, the soy sauce is obtained. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a koji mold having a protease producing ability of 1,100 U / g 麹 or more and a glutaminase producing ability of 2.0 U / g 麹 or more, and a breeding method thereof. The present invention also relates to a method for obtaining soy sauce having a high total nitrogen and glutamic acid content using the koji mold.

  Since soy sauce is a seasoning containing amino acids and peptides as flavoring ingredients, in the brewing of soy sauce, the utilization rate of nitrogen is improved, the amount of amino acids and peptides that are important as taste ingredients is increased, and glutamic acid is used. The production of soy sauce with a strong taste by increasing the production amount of soy sauce is of great significance, and therefore, the emergence of koji mold capable of producing powerful proteases and glutaminases has been strongly desired for the soy sauce industry.

  However, in the method for producing soy sauce in which the raw material is enzymatically decomposed in high-concentration saline, it is extremely difficult to release and accumulate significant amounts of nitrogen, peptides and glutamic acid in the soy sauce moromi. One solution to this problem is to adjust the initial saline concentration taking into account the inhibition of enzyme action, or to add glutaminases of various microorganisms to the moromi so that nitrogen is added to the soy sauce moromi. However, both methods require extra steps and become complicated, and the method of adding the latter glutaminase is particularly expensive. And it costs extra labor and is not always economical.

  In view of the above situation, the present inventors have obtained a soy sauce koji mold having the ability to release and accumulate significant amounts of nitrogen and glutamic acid in soy sauce moromi. Paying attention to the fact that it can make a significant contribution to the soy sauce industry if it can be added to water and produce and accumulate significant amounts of nitrogen and glutamic acid in soy sauce moromi, it has a high protease-producing ability for many koji molds, and glutaminase The breeding method of Aspergillus oryzae with high productivity was examined.

Generally, koji molds used in the production of soy sauce have a protease production ability of 500 to 650 U (sometimes referred to as units or units) / g koji, and a glutaminase production ability of 0.25 to 1.5 U / g Strains having a glutaminase-producing ability as low as cocoons and a high glutaminase producing ability of 3.0 to 8.0 U / g 麹 tended to have a protease producing ability as low as 100 to 400 U / g （(see FIG. 1). .
For this reason, in the production of soy sauce so far, if a strain with a high protease-producing ability is used, the soluble utilization rate of soluble nitrogen in the soy sauce raw material is improved, but the production of glutamic acid in moromi soup is low due to low glutaminase-producing ability. , There is a tendency that soy sauce rich in umami tends to be difficult to obtain, and if a strain with high glutaminase producing ability is used, the protease utilization ability is low, so that the soluble utilization of soluble nitrogen in soy sauce cannot be improved. .

  Until now, in order to obtain an excellent koji mold having high protease-producing ability and glutaminase-producing ability, attempts have been made to artificially treat koji mold (Patent Document 1). Protease production ability is improved about 3 times or more compared to the parent strain (former strain), but glutaminase production ability is known to decrease to half or less, and protease production ability is almost reduced without reducing glutaminase production ability. It was very difficult to breed mutant strains that markedly improved.

Conventionally, as a method for solving this drawback, protoplast fusion of a high protease production strain and a high glutaminase production strain belonging to Aspergillus soya is performed, and the fusion diploid strain is haploidized to obtain the mutant strain in question. There is a known method (see, for example, Patent Document 2).
However, in this method, it is described that a koji mold having a protease producing ability higher than 650 U / g koji and a glutaminase producing ability higher than 3.5 U / g koji can be bred, but the obtained koji mold is a protease. Aspergillus soya Ben-1 (FERM P-7522), which has the highest production ability of 673 U / g 麹, is known for the breeding of Aspergillus oryzae having a higher protease production ability. Absent.
Japanese Examined Patent Publication No. 3-37,913 Japanese Examined Patent Publication No. 3-73,271

  An object of the present invention is to breed a koji mold having a protease producing ability higher than 1,100 U / g ／ and having a glutaminase producing ability of 2.0 U / g ／ or more. Another object of the present invention is to obtain soy sauce having a high total nitrogen and glutamic acid content using the koji mold.

  As a result of intensive studies in order to solve the above-mentioned problems, the present inventors have found that conidia of a strain belonging to the genus Aspergillus and having a high glutaminase-producing ability (for example, Aspergillus soja ATCC42251) are ion sources such as C, Ne, and He. When the heavy ion beam was irradiated, it was discovered that among the resulting conidia, there are strains that have a high glutaminase-producing ability and a markedly higher protease-producing ability than the parent strain. Was completed.

The present invention is the following koji mold and its breeding method.
(1) A strain belonging to the genus Aspergillus and having a protease producing ability of 1,100 U / g 麹 or more and a glutaminase producing ability of 2.0 U / g 麹 or more.
(2) Aspergillus according to (1), wherein the strain belonging to Aspergillus is a strain belonging to Aspergillus soja.
(3) Aspergillus according to (2), wherein the strain belonging to Aspergillus soja is Aspergillus soya I67-1 (NITE AP-430).
(4) A strain belonging to the genus Aspergillus is irradiated with a heavy ion beam, and has a protease producing ability of 1,100 U / g 麹 or more from the irradiated body or its culture, and a glutaminase of 2.0 U / g 麹 or more. A method for breeding koji molds, which comprises separating koji molds having production ability.
(5) A strain belonging to the genus Aspergillus is irradiated with a heavy ion beam, and has a protease producing ability of 1,100 U / g 麹 or more from the irradiated body or its culture, and a glutaminase of 2.0 U / g 麹 or more. A method for producing soy sauce, comprising separating koji molds having production ability, inoculating and culturing the obtained koji molds in a soy sauce koji raw material to prepare koji, and charging using the koji.

  According to the present invention, a koji mold having a protease producing ability of 1,100 U / g 麹 or more and a glutaminase producing ability of 2.0 U / g 麹 or more can be obtained. According to this koji mold, soy sauce having a high nitrogen utilization rate and a high total nitrogen and glutamic acid content can be obtained.

Hereinafter, the present invention will be described in detail.
Examples of the strain belonging to the genus Aspergillus include any strain belonging to Aspergillus oryzae, Aspergillus soya, Aspergillus niger, Aspergillus awamori, etc. Jaundice is preferred.
Such a strain can be obtained by isolating a strain having a glutaminase producing ability of 2.5 U / g 麹 or more from koji molds used for brewing conventional soy sauce, miso or sake. In addition, Aspergillus soja 262 (FERM P-2188), Aspergillus soya 2165 (FERM P-7280), Aspergillus oryzae (IAM2638), Aspergillus soja ATCC 42251, etc. can be used.

Next, in order to irradiate with a heavy ion beam, the spore suspension of the above strain is applied to a normal agar plate culture medium, for example, a malt medium (malt extract 9%, yeast extract 0.5%, agar 2% pH 6) or the like. 0.2 to 20 × 10 ⁶ are applied, and irradiation is performed at an intensity of 25 to 2,500 Gy using ¹² C ⁵⁺ as an ion source.
For the irradiation of the heavy ion beam, for example, TIARA of Takasaki Quantum Application Laboratory can be used.

  After irradiation, the cells are cultured in the same medium at 25 to 35 ° C. for 2 to 6 days to allow conidia to grow, and these conidia are collected and screened for high protease mutants by casein plate.

  In this manner, koji molds having a protease producing ability of 1,100 U / g 麹 or more and a glutaminase producing ability of 2.0 U / g 麹 or more are separated from the culture.

  In this way, according to the present invention, the target Aspergillus oryzae Aspergillus soya I67-1 (NITE AP-430) can be easily obtained.

Next, the koji mold thus obtained is inoculated and cultured in ordinary soy sauce koji materials to make koji.
For example, aspergillus soya I67-1 (NITE AP-430) is inoculated and mixed with a mixture of soyed and steamed soybeans and fried rice cracked wheat, and cultured at 25-35 ° C. for 3-4 days. to make. In addition, as a soy sauce koji raw material, all the raw materials used to manufacture soy sauce can be used.

  Next, the above-mentioned soy sauce cake is added to a salt solution of an appropriate concentration at a normal charge ratio in soy sauce brewing, and is appropriately stirred and fermented and aged for 3 to 6 months according to a conventional method of soy sauce brewing. Water-soluble nitrogen and glutamic acid Soy sauce moromi containing a significant amount of.

  Next, the ripening moromi obtained as described above is subjected to normal pressing, filtration, burning, orientation pulling and the like to obtain product soy sauce.

In addition, the measuring method of protease activity and glutaminase activity is based on the following method.
(Measurement method of protease activity)
Ten times the amount of distilled water was added to the bowl, mixed well, and allowed to stand at room temperature for 2 hours. This is NO. The mixture was filtered with No. 2 filter paper (manufactured by Advantech), and the obtained extract was used as an enzyme sample and measured according to the method described in “Soy Sauce Test Method” (Japan Soy Sauce Research Institute, 1985).
The protease activity shown in Table 1 and FIG. 1 shows 1 U (unit or unit) as an activity to produce 1 μmol of tyrosine per gram per gram.

(Method for measuring glutaminase activity)
An enzyme sample was prepared by adding 50 times the amount of distilled water to the koji and crushing with polytron for 1 minute. 5 ml of 0.1 M L-glutamine solution (1 M Tris, 0.1 M hydroxyamine hydrochloride pH 8) was added to 7 ml of this enzyme sample, mixed well, and reacted at 30 ° C. for 1 hour. The reaction was stopped by adding 2 ml of stop solution (50 g ferric chloride / 1000 ml 8.4 N hydrochloric acid). This solution is NO. The mixture was filtered with 5C filter paper (manufactured by Advantech). The absorbance of this filtrate was measured at 420 nm. After subtracting the absorbance of the enzyme blank and water blank from the absorbance of the test, the activity was converted to U / g と し て by multiplying the coefficient by 6.03. The glutaminase activity in Table 1 and FIG. 1 shows 1 U (unit or unit) as an activity for producing 1 μmol of glutamic acid per minute per gram of cocoon.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these at all.

(Breeding Neisseria gonorrhoeae to heavy ion beam irradiation)
Aspergillus soja ATCC 42251 was used as the parent strain.
This strain was a koji mold having a protease producing ability of 551 units / g 麹 and a glutaminase producing ability of 2.62 units / g 麹.
About 2 × 10 ⁶ conidia of this strain are applied to Malt's medium (Maltz extract 9%, yeast extract 0.5%, agar 2% pH 6), and irradiated with an intensity of 250 Gy using ¹² C ⁵⁺ as an ion source. It was.
Heavy ion beam irradiation was performed using TIARA of the Japan Atomic Energy Agency Takasaki Quantum Application Laboratory.
After irradiation, the cells were cultured at 30 ° C. for 4 days to allow conidia to grow. The conidia were collected and screened for high protease mutants by casein plate.

(Screening with casein medium)
The collected conidia are diluted with sterilized water so as to be about 400 cells / ml, and 50 μl of this diluted solution is diluted with casein medium (milk casein 0.4%, casamino acid 0.05%, potassium monophosphate 0.36). %, Disodium phosphate 1.43%, magnesium sulfate 0.5%, ferric sulfate 0.002%, agar 2% pH 6.5) and cultured at 30 ° C. for 4 days.
After the culture, a strain having a large clear zone formed around the colony was selected, and Aspergillus soja I67-1 (NITE AP-430) was bred.
This koji mold was a koji mold having a protease producing ability of 1,929 U / g 麹 and a glutaminase producing ability of 2.38 U / g 麹.

(Production example of soy sauce cake)
Mixing steamed defatted soybeans and fried rice cracker wheat at a ratio of 50:50, add 0.1% (w / w) of koji seeds here, and knead them for 3 days, and analyze the soy sauce koji obtained The results are shown in Table 1. Ben-1 and PFA-118 are strains described in Japanese Patent Publication No. 3-73,271, and data are also reprinted from the same patent.

  From the results of Table 1, it can be seen that according to the present invention, a koji mold having a protease producing ability of 1,100 U / g 麹 or more and a glutaminase producing ability of 2.0 U / g 麹 or more can be obtained.

(Digestibility test of soy sauce cake)
The digestibility of the soy sauce cake obtained in Example 2 was measured by a conventional method. The results are shown in Table 2.

  From the results in Table 2, it can be seen that Aspergillus soja I67-1 bacilli show higher digestibility than other strains. From this, it can be seen that according to the present invention, the soy sauce raw material has a high nitrogen utilization rate, and soy sauce with a high nitrogen concentration can be produced.

(Example of soy sauce production)
Using the koji mold Aspergillus soya I67-1 (present invention) obtained in Example 1 above, a soy sauce production test was conducted as follows. For comparison, a soy sauce production test was conducted in the same manner except that the parent strain Aspergillus soja ATCC 42251 was used instead of the inventive strain.
Steamed defatted soybeans and fried rice cracker wheat are mixed at a ratio of 50:50, 0.1% (w / w) of koji is added to the koji, and the koji is smelted for 3 days. Therefore, soy sauce cake and salt water were mixed and charged, fermented and matured at 30 ° C. for 5 months, and pressed to obtain soy sauce. About the obtained soy sauce, total nitrogen% (w / v) (abbreviated as TN), glutamic acid% (w / v) (abbreviated as Glu), Glu / TN and nitrogen utilization rate (%) were measured.
The results are shown in Table 3.

  From the results shown in Table 3, when the koji mold obtained in the present invention (strain of the present invention) was used to produce soy sauce, the soy sauce raw material was decomposed well and the nitrogen utilization increased by about 3%. It can also be seen that the total nitrogen concentration is high, the glutamic acid content is high, the quality of the nitrogen itself is improved, and soy sauce with a very strong taste is obtained.

(Correlation diagram between protease production ability and glutaminase production ability of Aspergillus oryzae)
The protease-producing ability and glutaminase-producing ability of Aspergillus sojae ATCC 42251 (parent strain) and Aspergillus sojae 167-1 (NITE AP-430) of the present invention bred in Example 1 were measured to produce FIG.
For comparison, the relationship between the protease-producing ability and glutaminase-producing ability of Aspergillus sojae belonging to conventional Aspergillus sojae (conventional strain) was examined. The results are shown in FIG.

  From the results shown in FIG. 1, conventionally known koji molds have a high protease-producing ability of 500 to 650 U / g 麹 and glutaminase producing ability is as low as 0.25 to 1.5 U / g１．５, and 3.0 It can be seen that a strain having a high glutaminase producing ability of ˜8.0 U / g 麹 tends to have a protease producing ability as low as 100 to 400 U / g 麹. In addition, when a high protease-producing strain and a high glutaminase-producing strain belonging to Aspergillus soja are protoplast-fused and the fusion diploid strain is haploidized, a protease-producing ability higher than 650 units / g 麹 and 3 It can be seen that gonococci having a glutaminase-producing ability higher than 5 units / g 麹 can be bred. However, it is found that the resulting koji mold is Aspergillus soja Ben-1 (FERM P-7522) having a production ability of 673 units / g 麹 at the highest in terms of protease production ability. On the other hand, according to the present invention, it is understood that a koji mold having a protease producing ability higher than 1,100 U / g 麹 and having a glutaminase producing ability of 2.0 U / g 麹 or more is obtained.

FIG. 1 shows the correlation between protease-producing ability and glutaminase-producing ability of each koji mold.

Claims (5)

  A strain belonging to the genus Aspergillus, having a protease producing ability of 1,100 U / g 麹 or more and having a glutaminase producing ability of 2.0 U / g 麹 or more.   The gonococcus according to claim 1, wherein the strain belonging to the genus Aspergillus is a strain belonging to Aspergillus soja.   The gonococcus according to claim 2, wherein the strain belonging to Aspergillus sojae is Aspergillus sojae 167-1 (NITE AP-430).   A strain belonging to the genus Aspergillus is irradiated with a heavy ion beam, and has a protease producing ability of 1,100 U / g ／ or more and a glutaminase producing ability of 2.0 U / g 麹 or more from the irradiated body or its culture. A method for breeding koji molds, which comprises separating koji molds having the koji molds.   A strain belonging to the genus Aspergillus is irradiated with a heavy ion beam, and has a protease producing ability of 1,100 U / g ／ or more and a glutaminase producing ability of 2.0 U / g 麹 or more from the irradiated body or its culture. A method for producing soy sauce, wherein the koji molds are separated and bred, the obtained koji molds are inoculated and cultured in a soy sauce koji raw material to make koji, and the koji is used for preparation.

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