JPH0116465B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing soy sauce with a high glutamic acid content, and more particularly, to a method for producing soy sauce with a high glutamic acid content by using glutaminase-producing bacteria belonging to the genus Brera in combination with soy sauce koji. [Prior art] Generally, in the production of soy sauce, proteins in raw materials are enzymatically decomposed during the maturing process of moromi by the action of proteolytic enzymes derived from soy sauce koji, and are converted into peptides to produce amino acids, mainly glutamic acid. leading to. The production of glutamic acid by the enzymatic decomposition is due to the fact that the glutamine produced by the decomposition is converted into glutamic acid by the action of glutaminase. Therefore, if glutaminase is not present in the system during the soy sauce manufacturing process or its activity is low, soy sauce with the desired glutamic acid content cannot be obtained. It is known that in systems where glutaminase does not exist or its activity is low, the glutamine produced by the enzymatic decomposition described above is converted non-enzymatically to pyroglutamic acid; In the soy sauce that is prepared, the amount of glutamic acid is approximately
It has been reported that up to 25% of pyroglutamic acid is detected (Enzyme Engineering Journal, Vol. 47, No.
693-700, 1969). Incidentally, pyroglutamic acid has a phenolic odor and a bitter taste, and its presence is unfavorable in terms of the quality of soy sauce. Therefore, the production of pyroglutamic acid due to the lack of glutaminase activity as described above is due to the fact that the glutaminase produced by the koji mold in soy sauce koji made by adding only seed koji using the usual method is (1) extremely unstable; The pH of moromi is 5.0 to 5.5 because it quickly loses its activity in hot moromi, 2 the activity is significantly inhibited by the salt in moromi, and 3 the optimal pH for its action is 7.0 to 8.0. This is thought to be due to insufficient activity due to reasons such as weakening of the enzymatic reaction. In view of the above-mentioned circumstances, there have conventionally been methods for reducing the salt content in moromi for the purpose of increasing the activity of glutaminase produced by Aspergillus oryzae, and methods for adjusting the pH of moromi to approximate the optimal pH of the glutaminase. Although these methods have been proposed, they are not practical because they have drawbacks such as bacterial contamination during the ripening process of moromi and deterioration of flavor due to ripeness. Furthermore, since filamentous fungi, yeasts, and bacteria have been searched for as microorganisms capable of producing glutaminase in addition to Aspergillus oryzae, attempts have been made to use glutaminase produced by such glutaminase-producing bacteria in combination with Aspergillus oryzae. (b) A method of adding peptide glutaminase produced by a strain belonging to the genus Bacillus to moromi (Japanese Patent Publication No. 48-35459), (b) A method of decomposing protein raw materials using a combination of protease and glutaminase without making koji ( Special Public Interest Publication 1977-34898),
(c) During the process of decomposing a protein-containing raw material with a proteolytic enzyme, or in the decomposition product obtained by the decomposition, inoculating and culturing salt-tolerant lactic acid bacteria in the presence of 0 to 12% common salt, or culturing the lactic acid bacteria. A method of bringing the bacterial cells into contact with each other (Japanese Patent Publication No. 50-34640), a method of adding bacterial cells belonging to the genus (d)Rhodotorula to koji for making soy sauce malt and moromi (Japanese Patent Publication No. 57-58870), etc. have been proposed. However, although the above method (a) is intended for the production of soy sauce, the culture solution of bacteria belonging to the genus Bacillus is prepared separately from the soy sauce koji and added to the moromi after the soy sauce koji is prepared. It is necessary to provide a new device for preparing the , which has the disadvantage of increasing costs. The methods (b) and (c) involve enzymatically hydrolyzing a protein-containing raw material to obtain an amino acid mixture with a high glutamic acid content, and are essentially different from the method for producing soy sauce. [Problems to be solved by the invention] From the above-mentioned circumstances, in order to obtain soy sauce with a high glutamic acid content, the activity of glutaminase acting in soy sauce moromi is inhibited by the salt concentration (15 to 17%) in soy sauce moromi. However, it is necessary that there is no need to newly provide a process and equipment for producing glutaminase, and that the aroma of moromi after ripening is not adversely affected. [Means for Solving the Problems] Therefore, the present inventor conducted intensive research on this issue and found that a strain belonging to the genus Rubula has the ability to produce glutaminase, and that the glutaminase produced by this strain satisfies the above conditions. The present invention has been completed by finding that the following requirements are satisfied. That is, the present invention provides a method for producing soy sauce with a high glutamic acid content, which is characterized in that soy sauce koji is prepared in the coexistence of glutaminase-producing bacteria belonging to the genus Rubula. The present invention will be explained in detail below. The glutaminase-producing bacteria belonging to the genus Bullera used in the present invention include Bullera alba (Bullera alba).
alba), Bullera grandispora (Bullera
grandispora), Bullera tuge (Bullera
tsugae), etc. These bacterial species are known and their strains can be obtained from public microbial repositories such as the Institute of Fermentation (IFO) and the Faculty of Agriculture, Kyoto University (AKU). As mentioned above, the glutaminase-producing bacteria used in the present invention belong to the known species of the genus Brera, and their mycological properties are described in J.
It is described in detail in LODDER's "THE YEASTS" (1967). For example, the main mycological properties of Brera alba used in the Examples of the present invention are as follows. (1) Characteristics Propagates by multipolar budding. Cells are oval to spherical. Neither pseudohyphae nor hyphae are formed. Ejected spores are symmetrical, spherical, oval, and ejected from the petite.
The bacterial body is pale yellow to cream color. In liquid media, it grows on the surface. Assimilates sugars oxidatively and does not assimilate nitrates. (2) Physiological properties Assimilation of potassium nitrate None Ethanol as sole carbon source No growth Degradability of arbutin Negative Fermentability Negative Assimilation of sugar Glucose + Galactose + Satucarose + Maltose + Lactose + The above bacteria used in the present invention is Aspergillus oryzae Unlike bacteria belonging to the genus Bacillus, it does not suppress the growth of Aspergillus aspergillus even when coexisting with soy sauce raw materials processed in a conventional manner, and has the property of producing glutaminase. has the property that its activity is not easily inhibited by salt.
Therefore, in the present invention, the glutaminase-producing bacteria belonging to the genus Brera can be added to the raw material for koji production together with seed koji during the production of soy sauce koji, and the glutaminase-producing bacteria can proliferate and coexist with the koji mold. It can be prepared as soy sauce koji. In this case, it is particularly preferable to add the above-mentioned glutaminase-producing bacteria together with the seed koji when adding the koji for making koji. As described above, according to the present invention, the glutaminase-producing bacteria belonging to the genus Brera can be used in the form of soy sauce koji coexisting with koji mold, so it is necessary to newly provide a device for separately cultivating the glutaminase-producing bacteria. do not have. However, from the viewpoint of utilizing glutaminase produced by a glutaminase-producing bacterium belonging to the genus Brera, it is of course possible to prepare a culture solution of the bacterium separately and prepare it together with soy sauce koji. That is, the present invention prepares a glutaminase-producing bacterium belonging to the genus Brella in coexistence with soy sauce koji, thereby utilizing the combined enzymatic action of the proteolytic enzyme of the koji mold and the glutaminase produced by the glutaminase-producing bacterium. It significantly increases the glutamic acid content of. Next, the results of testing the glutaminase activity produced by the glutaminase-producing bacteria belonging to the genus Brera used in the present invention are shown. Test method: Brela alba IFO as a strain belonging to the genus Brela
1192, the strain was inoculated into a normal liquid medium for yeast, cultured with shaking at 30℃ for 72 hours, and this culture solution was inoculated with seed koji in a flask koji using equal amounts of defatted soybeans and wheat. The above-mentioned bacterial strain was added to 1 g of raw material so that the number of bacteria was 1×10 ⁷ cells, and koji was made for 3 days.
Next, double the amount of 15% saline was added to the de-koji and digested at 40°C for 5 days, and the resulting liquid was analyzed for glutamic acid using a conventional method. The results are shown in Table 1.

〔Effect of the invention〕

As described above, according to the present invention, by using glutaminase-producing bacteria belonging to the genus Brella in coexistence with soy sauce koji, it is possible to obtain soy sauce with a significant glutamic acid content without installing a separate device in a conventional soy sauce production device. becomes possible. [Example] Next, the present invention will be explained in more detail with reference to Examples. Example 1 10ml of soy sauce juice, 1g of glucose, and 90ml of water
After adjusting the pH to 5.5 and sterilizing, the mixture was inoculated with Bullera alba IFO 1192 and cultured with shaking at 25°C for 72 hours to obtain a culture solution. Separately, 120% water was removed from 500g of defatted soybeans, and 1.2Kg/
Steamed at cm ² for 20 minutes, mixed with 500 g of roasted and crushed raw wheat, and at the same time, the above culture solution was mixed with koji starter for soy sauce brewing at 1x per gram of raw material.
The bacteria were spread to a number of 1×10 ⁸ to 10 ⁵ and made into koji for 3 days using a conventional method to obtain koji. Further to the koji
Salt water was added to make it 11.7 ml of water, and the mixture was aged at 30°C for 3 months to give a raw soy sauce. Table 2 below shows the results of analyzing the components of the de-koji and raw soy sauce obtained as described above. The protease activity of the brewed koji was determined using the ``Enzyme Research Method'' (1956) written by Bunji Hagiwara, and the analysis of raw soy sauce was conducted using the ``Standard Soy Sauce Analysis Method'' published by the Japan Soy Sauce Technical Association (1966). The potency is dried koji.
One unit was defined as the amount of enzyme that produced a TCA-soluble substance equivalent to 1 μmole of tyrosine per minute using a 10% solution. The titer of glutaminase was defined as the amount of enzyme that produced 1 μmole of glutamic acid per minute using a 10% dry matter solution of homogenized koji as 1 unit.
(Nichishoken Vol. 7, p. 74, 1981). The number of yeast in the released koji was determined from the number of colonies after culturing at 25° C. for 4 days using the plate culture method using YM medium.

[Table] * Glutamic acid = total nitrogen As shown in Table 2, it has almost no effect on the protease activity used to judge the quality of koji during de-koji production, and glutaminase activity varies depending on the amount of Brera bacteria added. As can be seen from the number of yeast, strains of the genus Brella increased up to about 200 times during koji production using the normal koji production method without affecting the koji mold in any way. Furthermore, analysis of raw soy sauce showed that glutamic acid increased by up to 31% without any effect on the elution of other components of soy sauce. Example 2 90 ml of water was mixed with 10 ml of soy sauce raw juice and 1 g of glucose, and after sterilization, this mixture was inoculated with Bullera alba (IFO 1192) and incubated at 25°C for 72 hours.
A culture solution was obtained by shaking culture for hours. Separately, 120% water was removed from 500g of defatted soybeans, and the water was steamed at 1.2Kg/cm ² for 20 minutes.
500g of raw wheat roasted and crushed is mixed with this, and at the same time, the above culture solution is sprinkled with seed koji for soy sauce brewing so that the number of particles is 1 x 10 ⁶ and 1 x 10 ⁷ per 1 g of raw material. Then, it was made into koji for 3 days as usual. 1,300 g of this de-koji and 130 g of wheat gluten were mixed, mixed with 11.7 hours of water, aged at 30°C for 3 months, and subjected to moromi to give raw soy sauce. The protease activity of the koji at this time was 3.5 U/g koji with no addition, 3.4 U/g koji with the addition of 1×10 ⁶ proteases, and 3.3 U/g koji with the addition of 1×10 ⁷ proteases. The koji was good. Table 3 shows the results of analyzing the raw soy sauce obtained as described above.

[Table] As shown in Table 3, raw soy sauce with a high glutamic acid content can be obtained even with moromi, which is a mixture of wheat gluten and soy sauce malt that is not made into koji, and even with different strains of Brera genus. No effect was observed on flavor and flavor.

[Brief explanation of drawings]

Figure 1 shows the relationship between the salt concentration (%) in the reaction solution and the specific activity of glutaminase at pH 7.5.
Figure 2 shows the relationship between the pH of the reaction solution and the specific activity of glutaminase, and Figure 3 shows the amount of the test bacterial strain added (individuals/
The relationship between the specific activity of glutaminase in raw material g) and koji and the glutamic acid/total nitrogen in the digestive fluid is shown.

Claims (1)

[Scope of Claims] 1. A method for producing soy sauce with a high glutamic acid content, which comprises preparing soy sauce koji in coexistence with glutaminase-producing bacteria belonging to the genus Brera. 2. The production method according to claim 1, wherein glutaminase-producing bacteria belonging to the genus Brera are allowed to coexist with soy sauce koji in a mixed state. 3. The production method according to claim 2, wherein glutaminase-producing bacteria belonging to the genus Brera are mixed with soy sauce koji by adding them together with seed koji during the production of soy sauce koji.