JPH0533973B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a soy sauce brewing method using a koji mold that has a high protease-producing ability through protoplast fusion and a high glutaminase-producing ability, and particularly relates to a soy sauce brewing method that has a high nitrogen utilization rate and a high glutamic acid content. Soy sauce has long been popular in our daily diet as a seasoning mainly based on the umami flavor of amino acids, and today it is important to provide soy sauce that contains high concentrations of nitrogen and glutamic acid, which are important flavor components. This is an important issue in the brewing world. By the way, in a soy sauce brewing method in which raw materials are enzymatically decomposed in highly concentrated saline, it is extremely difficult to free and accumulate a significant amount of nitrogen and glutamic acid in soy sauce moromi. One of the ways to solve this problem has been to adjust the concentration of the saline solution at the time of preparation, taking inhibition of enzyme action into consideration, or to add glutaminase derived from various microorganisms to the moromi, thereby adding nitrogen to the soy sauce moromi. Methods to accumulate glutamic acid and glutamic acid have been used, but both require extra steps and complicate the brewing operation. In particular, the latter method, which uses glutaminase, requires equipment costs and labor for enzyme production. It costs extra and is not necessarily economically advisable. Taking the above-mentioned current situation into consideration, the present inventors obtained a soy sauce koji mold that has the ability to freely accumulate a significant amount of nitrogen and glutamic acid in soy sauce moromi, produced soy sauce koji using this koji mold, and added it to salt. Focusing on the fact that it would be an extremely important contribution to the soy sauce brewing industry if a significant amount of nitrogen and glutamic acid could be produced and accumulated in soy sauce moromi by adding it to water, many soy sauce koji were developed to have high protease content. We searched for Aspergillus aspergillus that has production ability and high glutaminase production ability. In general, the protease production capacity of koji mold for soy sauce is 208
Unit/g koji, and glutaminase production ability is
It is known that 3.19 units/g of koji (Shoken)
Vol.7, No.4, 1981, p166-172, p169 in "Differences in brewing characteristics depending on the classification of koji mold for soy sauce"
(See “Table 4 (Differences in enzyme power of soy sauce koji)”). Conventionally, one method of obtaining such excellent koji molds is to obtain a strain with increased enzyme production ability through artificial mutation, but the obtained mutant strain does not have the ability to produce either protease or glutaminase. Even if it is higher than the parent strain, the other strain is often lower, and it has been difficult to obtain a strain that is higher than the parent strain. Therefore, as a result of various studies aimed at obtaining Aspergillus aspergillus having high protease-producing ability and high glutaminase-producing ability, the present inventors have developed a high-protease-producing strain and a high-glutaminase-producing strain belonging to the genus Aspergillus. protoplast fusion,
When the fused cells were cultured in a hypertonic regeneration medium, it was found that Aspergillus aspergillus having a high protease-producing ability and a high glutaminase-producing ability could be obtained therefrom, and based on this knowledge, the present invention was completed. That is, the present invention belongs to the genus Aspergillus.
High protease production strain of 210 units/g koji or more and 3.2
Protoplasts are fused with a strain that produces high glutaminase of 300 units/g or more of koji, and the fused cells are cultured in a hypertonic regeneration medium. This is a soy sauce brewing method characterized by separating and collecting koji molds having the above-mentioned high glutaminase production ability, inoculating and culturing the obtained koji molds into soy sauce koji raw materials to produce koji, and using this koji for preparation. . The present invention will be explained in detail below. First, the strain that belongs to the genus Aspergillus and produces a high protease of 210 units/g or more of koji used in the present invention may be any strain that belongs to the genus Aspergillus and has the above-mentioned protease production ability, such as soy sauce, miso, It can be easily obtained by searching for strains with high protease production ability from among the brewing koji molds used in the production of sake and the like. Examples include Aspergillus sojae (ATCC42249), Aspergillus sojae (ATCC42250), and Aspergillus sojae (ATCC42251), but variants or mutant strains of these can also be used.
As a strain producing glutaminase higher than that of koji, any strain belonging to the genus Aspergillus and having the above-mentioned glutaminase production ability may be used. can be obtained. For example, Aspergillus sojae 262 (FERM-P2128), which has high glutaminase production ability,
2165 (FERM-P-7280) etc., but
Furthermore, variants or mutant strains of these can also be used. Next, as the strain for protoplast fusion, any germinated strain can be used, but due to the efficiency of protoplast formation, the length of most germinated spores is 5 to 15 times the original spore outer diameter. Preferably, the cells are cultured by solid culture, preferably liquid culture, using a medium used for culturing Aspergillus oryzae according to a conventional method. The medium used for culturing may be any medium as long as it contains the nutrients required by the strain belonging to the genus Aspergillus, and a synthetic medium, a semi-synthetic medium, or a natural medium may be used. For example, as a synthetic medium, glucose, succalose, etc. are used as a carbon source, and ammonium sulfate, ammonium nitrate, etc. are used as a nitrogen source.A specific example is Czapek medium with 0.5% yeast extract and 0.2% casamino acid added to pH 6.0. For example, a culture medium adjusted to Furthermore, the composition of the natural medium includes cracked wheat, koji, etc. as a carbon source, and skim milk, defatted soybeans, etc. as a nitrogen source. In addition, appropriate inorganic salts such as phosphoric acid, potassium, magnesium, and calcium can be used as appropriate, and amino acids and vitamins necessary for the growth of bacteria can be added to the medium as necessary. When performing liquid culture as a culture method, use the appropriate medium mentioned above, such as a synthetic or semi-synthetic medium containing glucose, skim milk, etc., adjusted to a pH of around 6.5, and heat sterilized by a conventional method. Inoculate the inoculum and grow at 25 to 35°C until the germinated spores grow to 5 to 15 times the original spore outer diameter.
Cultivate aerobically for ~15 hours by standing still, shaking, or aerating. In addition, when performing solid culture, aspergillus oryzae is inoculated and mixed into a solid medium made from carbon sources such as wheat, nitrogen sources such as soybeans, and other organic or inorganic materials that have been sterilized by steaming. ,
It is preferable to culture aseptically at 25 to 38° C. for a sufficient period of time to grow the germinated spores to a length 5 to 15 times the original spore outer diameter. To obtain protoplasts from a high protease producing strain or a high glutaminase producing strain cultured in this way, 10 ⁶ to 10 ⁸ for each strain is required.
After preparing each germinated spore suspension with a concentration of germinated spores/ml (hypertonic solution) and mixing preferably equal amounts of each of the above suspensions, treatment with a cell wall lytic enzyme that has been previously sterilized, or each germinated spore suspension is performed. It is obtained by first treating a germinated spore suspension with the cell wall lytic enzyme. The cell wall lytic enzyme used here may be any enzyme as long as it has the activity of lysing the cell wall of Aspergillus oryzae, such as cellulase "Onozuka R".
-10 (manufactured by Kinki Yakult Co., Ltd.), Aspergillus cell wall lytic enzyme derived from Bacillus circulans strain IAM1165 and Streptomyces genus strain newly prepared by the present inventors, and chitinase ( (Manufactured by ICN, USA)
Examples include. These enzymes can be used alone or in combination. In particular, according to experiments conducted by the present inventors, the combined use of an enzyme derived from Bacillus circulans and chitinase, or the combined use of an enzyme derived from the genus Streptomyces and chitinase is preferable because the rate of protoplast formation can be significantly increased. When obtaining protothrust, the concentration of the cell wall lytic enzyme used is preferably a concentration sufficient to dissolve the cell wall of Aspergillus aspergillus and obtain protoplasts. 10 to 50 mg/
ml, preferably 25-35 mg/ml. In addition, when using chitinase in combination, the concentration used is 1-10
mg/ml, preferably 3 to 5 mg/ml. The time for the enzyme treatment is preferably a time sufficient to dissolve the cell wall of Aspergillus aspergillus and obtain protoplasts, and is usually preferably 30 minutes to 6 hours. Also, PH is 6.0-7.0
is preferred. Once the enzymatic treatment has finished in this way, a solution that protects the protoplasts, e.g.
Wash the protoplasts with ~1.5M sorbitol or 0.5-1.0M potassium chloride solution to obtain protoplasts in which the cell wall of Aspergillus aspergillus has been dissolved and removed. Next, the fusion treatment of the protoplasts obtained here and the regeneration of the fused protoplasts are carried out by, for example,
Anne' et al.'s method [J.Gen.Microbiol., 92 , 413
(1976)]. i.e. 0.7-1.5M sorbitol, 5-
15~ containing _100mMCaCl2 and 30~80mM glycine
In 33% polyethylene glycol 6000 (PH7.5),
The washed protoplasts of the high protease producing strain and the high glutaminase producing strain obtained above are added and mixed and treated at 20 to 30°C for 15 to 45 minutes to perform protoplast fusion. To regenerate fused protoplasts, use a hypertonic regeneration medium that can grow while protecting protoplasts (preventing damage), such as rice malt juice (or wort) containing 0.7 to 1.5M sorbitol containing 2% agar. Hypertonic regeneration agar medium (PH6.5) and 0.7~
Hypertonic minimum regeneration agar medium (PH6.5) containing 2% agar in Czapeck's solution containing 1.5M sorbitol.
Transfer the fused protoplasts obtained above to
This is carried out by culturing at 35°C for 2 to 10 days. Next, to select protoplast-fused cells from the regenerated colonies obtained in this way, the washed protoplasts of the high protease producing strain and the high glutaminase producing strain were regenerated in the same manner as above, respectively, and the obtained regenerated By understanding the characteristics of colonies such as hue, color, brightness, gloss, shape, size, etc., it is possible to select regenerated colonies with characteristics different from those. In addition, before performing the cell wall lysis treatment of Aspergillus aspergillus, each parent strain was subjected to artificial mutation treatment (irradiation with X-rays, ultraviolet rays, etc., chemical mutation treatment with nitrosoguanidine, ethyl methyl sulfate, etc.) to generate conidia of Aspergillus aspergillus. Mutations that give characteristic colors such as white, yellow, brown, and blackish brown, as well as various nutritional requirements, such as amino acid requirements such as alanine and methionine, and requirements for biotin, nicotinic acid, and para-aminobenzoic acid. Obtain a strain, use this mutant strain, perform enzyme treatment and protoplast fusion treatment in the same manner as above, and then regenerate. Since the protoplasts of the mutant strain have a characteristic color, it is possible to identify protoplast fused cells that do not have this color. can be distinguished and sorted. Furthermore, during regeneration, mutant protoplasts have difficulty or cannot grow in the absence of a specific nutrient source, so it is necessary to clearly distinguish and select protoplast fusion cells that can grow even in the absence of a specific nutrient source. I can do it. As a detection medium for regeneration, if the parent strain is not a mutant strain, a detection medium that can distinguish the colonies of each parent strain is used, and if the parent strain is a mutant strain that is auxotrophic, a minimal agar medium is generally used. It will be done. Examples of mutant strains of the high protease producing strain obtained above include Aspergillus sawjae W2-91 (FERM P-7277), which has white conidia and is endowed with a para-aminobenzoic acid requirement; Examples of mutant strains of the production strain include Aspergillus sojae M12-2-61, which has yellow conidia and is endowed with nicotinic acid auxotrophy.
(FERM P-7281), etc. Next, the protoplast fusion strain thus regenerated on a hypertonic agar medium or a hypertonic minimal agar medium is often a heterokaryon that easily reverts to the parent strain and is therefore unstable. Therefore, we applied the method of Oda et al. [Agric.Biol.Chem., 27 , 758] to the conidia of these heterokaryon strains.
(1963)], ultraviolet irradiation (distance 39 cm, time 1)
By performing a treatment for 5 minutes to 5 minutes) and testing, a stable diploid, that is, the desired koji mold, is isolated.
Can be collected. For example, Aspergillus sojae D-78 is a stable diploid strain that has a high protease-producing ability and a high glutaminase-producing ability, and was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology. Deposited as No. 7279. Next, the thus obtained strain having a high protease production ability of 300 units/g koji or more and a high glutaminase production ability of 1.0 units/g koji or more is inoculated and cultured into a normal soy sauce koji raw material to produce koji. For example, Aspergillus soya D-78 is inoculated into a mixture of watered and steamed soybeans and roasted and cracked wheat, and the mixture is cultured at 28 to 32°C for 3 to 4 days to produce soy sauce malt. In addition, all the raw materials used for soy sauce brewing can be used as soy sauce koji raw materials. Next, the above-mentioned soy sauce koji is added to a saline solution of an appropriate concentration at the usual mixing ratio for soy sauce brewing, stirred appropriately and fermented and aged for 3 to 6 months according to the usual method for soy sauce brewing, thereby producing a significant amount of nitrogen and glutamic acid. It is possible to obtain soy sauce moromi containing . Next, the aged moromi obtained as described above is subjected to conventional compression, furnace filtration, pasteurization, reduction, etc. to obtain a finished soy sauce. The present invention is obtained by culturing Aspergillus oryzae, which has a high protease production ability of 300 units/g or more of koji and a high glutaminase production of 1.0 units/g or more, which was first created by protoplast fusion of koji as described above. Since the koji used in the fermentation process is used, the present invention does not require extra steps such as adjusting the salt water concentration at the beginning of preparation in consideration of inhibition of enzyme action, and adding glutaminase derived from various microorganisms to the moromi. It is now possible to accumulate a significant amount of nitrogen and glutamic acid in soy sauce moromi by a direct method without adding any soy sauce, and because the soy sauce raw materials are decomposed very well, the total nitrogen utilization rate is about 3% higher than before. %〜
This is a much improved 10% increase, making it extremely easy to obtain soy sauce with extremely strong flavor. Experimental Example 1 High protease producing strain [Aspergillus sojae W2-91 (FERM P-7277)] and high glutaminase producing strain [Aspergillus sojae M12-2]
-61 (FERM P-7281)] was inoculated into a slant medium (rice malt juice agar medium) in a test tube, and incubated at 30°C.
After culturing for 66 hours, two types of conidia were obtained. These conidia were dispersed and mixed in a 0.01% sorbitan fatty acid ester solution "Sorgen TW-60 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.)" to a concentration of 1 to 5 x 10 ⁷ cells/ml, and 1 ml of the conidia was mixed beforehand. Add 30 ml of culture medium [obtained by adding 0.5% yeast extract and 0.2% casamino acids to Czapek medium, adjusting the pH to 6.0, and diluting this 10 times with water] in a 150 ml Erlenmeyer flask. Mix and culture with shaking at 30℃ for 12 hours until the length of the germinated spores is approximately the outer diameter of the original spores.
10 times more germinated spores were obtained. The germinated spore suspension was then centrifuged (4500 g, 20 minutes) to separate the germinated spores therefrom. Next, after washing the germinated spores with water, 1 ml of Aspergillus aspergillus cell wall lytic enzyme solution was added thereto, and cultured with gentle shaking at 27°C for 2 hours (50-60°C).
round trip/min) to obtain protoplasts. The cell wall lytic enzyme solution used here was Bacillus
A crude enzyme obtained by culturing Circulans IAM1165 in liquid by a conventional method, salting out the obtained culture filtrate by adding ammonium sulfate, and freeze-drying the obtained salting out product;
These were obtained by dissolving a commercially available chitinase agent (manufactured by ICN, USA) at a ratio of 30 mg and 5 mg in 1 ml of solution, respectively. Next, the protoplasts thus obtained were separated using a G-3 standard glass filter, washed several times with a hypertonic solution (0.8M sorbitol solution), and then the washed protoplasts were separated with 1 ml of the hypertonic solution.
Transfer to and suspend. Next, the hypertonic solution in which each protoplast was suspended was mixed and centrifuged at 20°C (700 g, 15
minutes) to obtain protoplast pellets. This contains 20% polyethylene glycol 6000 containing 0.8M sorbitol, _10mMCaCl2 and 50mM glycine.
(PH7.5) solution was mixed and a protoplast fusion reaction was performed at 25°C for 30 minutes. Next, the fused protoplast reaction solution was diluted with a hypertonic solution (0.8M sorbitol) and seeded on a rigid hypertonic minimal regeneration medium [Zapetsk agar medium (2% agar) containing 0.8M sorbitol] on a shear plate, and the dissolved soft Hypertonic minimal regeneration medium (agar
0.5%), solidified and layered, and heated to 30℃ for 4 to 7
The cells were cultured for days to regenerate protoplast fused cells. The regenerated colony is selected as a protoplast fusion strain, and this is cultured on a rice malt agar slant (at 30°C for 4 days) to obtain a heterokaryon strain with green, white, and yellow spores. The spores of the heterokaryon strain thus obtained were irradiated with ultraviolet rays for 5 minutes from a distance of 39 cm, the obtained spores were applied to a minimal agar medium (plate), and cultured at 30°C for 4 days. Aspergillus aspergillus that forms a stable colony consisting of spores is obtained. When we investigated the protease and glutaminase production ability of the koji mold obtained in this way, we found that the
The results shown in the table were obtained.

[Table] Method for measuring glutaminase activity.
Using 30g of defatted soybeans and 30g of roasted cracked wheat as raw materials, make koji in a 15cm diameter sieve in the same way as the normal soy sauce koji production method, and extract the resulting koji for 2 hours using 10 times the amount of water. However, protease activity was measured by the Anson-Hagiwara method. The activity was expressed as one unit, the activity of producing 1 μmol of tyrosine per minute per 1 g of koji. Glutaminase activity can be determined by homogenizing the koji from the extraction residue and adding the homogenized solution and L-glutamine at 30°C.
After filtration, the amount of glutamic acid in the filtrate was determined. Glutaminase activity is
The activity of producing 1 μmol of glutamic acid per minute per 1 g of koji was expressed as 1 unit. From the results in Table 1, the strains with high protease are
A typical characteristic of Aspergillus oryzae is that it has low glutaminase activity, and conversely, strains with high glutaminase activity have low protease activity, but according to the method of the present invention, by protoplast fusion, it is possible to combine the characteristics of both parents. However, it can be seen that Aspergillus aspergillus having both high protease activity and high glutaminase activity can be obtained. Example 1 A soy sauce brewing test was conducted using the koji mold of the present invention and the control koji mold (parent strain) obtained in Experimental Example 1 above. Using 300g of defatted soybeans and 300g of roasted cracked wheat as raw materials, make koji using a koji lid according to the normal soy sauce koji manufacturing method, add the resulting koji to 1200ml of 25% salt water, and manage moromi fermentation using the usual method. The moromi liquid obtained was fermented and aged for 3 months at 30°C, and the total nitrogen content (abbreviated as TN), glutamic acid content (abbreviated as Glu), Glu/TN, and nitrogen utilization rate were measured. The results are shown in Table 2.

[Table] From the results in Table 2, it can be seen that when the koji mold cells obtained in the present invention are used to make koji and produce soy sauce, the soy sauce raw materials are decomposed very well and the nitrogen utilization rate increases by about 3 to 10%. Not only is this much improved, but glutamic acid is significantly increased, so the quality of nitrogen itself is improved, and it can be seen that soy sauce with extremely strong flavor can be obtained.

Claims (1)

[Claims] 1. Protoplast fusion of a high protease producing strain of 210 units/g koji or more and a high glutaminase producing strain of 3.2 units/g koji or more belonging to the genus Aspergillus, and culturing the fused cells in a hypertonic regeneration medium;
Aspergillus aspergillus having a high protease production ability of 300 units/g koji or more and a high glutaminase production ability of 1.0 unit/g koji or more is isolated and collected from the culture, and the obtained koji mold is inoculated and cultured into soy sauce koji raw material. A soy sauce brewing method that is characterized by making koji and using this koji for preparation.