JPH0441995B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention is directed to Bacillus, which produces a highly active protease outside the bacterial body and produces a large amount of mucilage.
Bacillus subtilis NN-1 (hereinafter referred to as
This strain is called this strain. ). This strain secretes protease outside the bacterial body. Protease, a protein-degrading enzyme, breaks down proteins and liberates amino acids and peptides.
These substances are well known as flavor components, and glutamic acid in particular is known as an umami component and is actually used as a seasoning. In addition, this strain has the property of producing mucilage outside the bacterial body. Therefore, by using this strain, it is possible to produce highly viscous and stringy seasonings and miso, and by adding it to conventional food materials, it is possible to improve their texture. It has a wide range of uses, including being able to perform Furthermore, it can also be used industrially as a paste. [Prior Art] Conventionally, in the fermentation industry, particularly in the production of miso, Bacillus subtilis has been regarded as a harmful bacterium that contaminates and proliferates in the products, lowering the commercial value. Therefore, there is no idea of actively using Bacillus subtilis in liquid fermented foods such as seasonings and miso, and it can be said that there is no research or technological development in this direction. [Problems to be Solved by the Invention] In order to efficiently and effectively decompose raw proteins to obtain amino acids and peptides, the microorganism used must have strong protease activity. Currently, Bacillus is a strain that produces mucilage.
Bacillus natto is known. When the sticky substance is diluted with water, its viscosity and stringiness are significantly reduced. In order to culture this in liquid and obtain a solution with strong viscosity and stringiness, it is necessary to concentrate the culture solution by some method. However, this is complicated and economically leads to increased costs, so it would be advantageous to obtain a strain that does not require such a step and can produce more mucilage. Therefore, it is necessary to obtain a strain that has high protease activity and also produces sticky substances by some method. [Means for Solving the Problems] Therefore, the present inventors attempted to screen for bacterial strains having the above properties. That is,
We isolated bacterial strains that form heat-resistant spores from soil, rice straw, dried grass, etc., and selected strains with high protease activity and slime production ability from those whose colony morphology appeared to be Bacillus subtilis. As a result, a bacterial strain with both of these characteristics was isolated from rice straw. Three strains were obtained,
Protease activity ranged from 100 to 121 units/ml, and relative viscosity ranged from 26 to 60 (4-fold dilution). It is known that mucilage production is correlated with the activity of the enzyme γ-glutamyl transpeptidase (γ-GTP). The γ-GTP activity of this strain was in the range of 940 to 1508 international units/unit. Bergey's Manual of Siktematics Bacteriology
As a result of examining the properties in accordance with Volume 2 of Systematic Bacteriology, the present strain (all three strains) was identified as belonging to the taxonomic family Bacillus subtilis. There have been reports of Bacillus subtilis strains other than this strain that produce the same amount or more protease as this strain, but there is no description that this strain also produces mucilage. on the other hand,
Bacillus is currently the only bacterial strain that produces mucilage.
Natsuto is known. Therefore, Bacillus subtilis MAFF10-08103, a strain isolated from commercially available Bacillus natto, was used as a control strain in the present invention. As shown in the Examples, when the present strain and the control strain were compared in terms of protease activity and adhesive production, the present strain showed higher values in both cases. From the above results, it is concluded that this bacterial strain is a new bacterial strain. Therefore, the new bacterial strain isolated by the present inventors was named Bacillus subtilis NN-1. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its accession number is
FERM P-11319. The mycological properties of this strain are shown below. A Morphological characteristics Gram staining Positive size 0.8x2.0-3.0μm Morphology Brachypterobacterium Motile Spores Bacterial cells do not swell B Cultural characteristics Growth on substitute meat juice agar medium Colony shape The periphery is irregular. However, the colony as a whole is circular Colony color: White Colony luster: Dull glow. Growth in a dull substitute meat broth medium. Precipitation of bacterial cells. Intermediate area: No turbidity. Bacterial film is formed. Thick C Physiological properties Calatase reaction positive Does not grow under anaerobic conditions. Fouges-Proskauer test Positive Acid production Positive from glucose and mannitol, negative from arabinose and xylose Gas production Negative from glucose Hydrolysis of casein Positive Hydrolysis of gelatin Positive Hydrolysis of starch Positive Citric acid assimilation Positive Tyrosine degradation Negative Egg yolk lecithinase reaction Negative Reducing ability of salt Positive Growth in saline solution Growth occurs in 2, 5, and 7%. It will not grow at 10%. Grows at growth temperatures of 5, 10, 30, 40, and 50°C. Does not grow at 55 or 65℃. D Negative growth in the presence of 0.01% lysozyme [Example] Next, the present invention will be explained in detail using the present example. Example 1 Extra-strain protease activity of the present strain NN-1 was measured. Transfer one loopful of this bacterial strain to ordinary bouillon medium (0.5
% meat extract, 1.5% peptone, 0.5% sodium chloride, 0.5% potassium monohydrogen phosphate, PH
7.0) Inoculated into 5.0ml. After culturing at 37℃ for 26 hours, centrifuge (10000xG, 10 minutes, 5℃)
A supernatant was obtained. Protease activity was measured by the following method. The reagents used are as follows. 1 Substrate solution 1.0% acid precipitated soy protein (manufactured by Fuji Oil Co., Ltd., Fujii Piure SP-300) and
A mixed solution of 0.1N sodium chloride, the pH of which was adjusted to 7.3 with sodium hydroxide solution. 2 Reaction stop solution A mixture of 0.1M trichloroacetic acid, 0.22N sodium acetate and 0.33N acetic acid Add 0.1ml of the above supernatant to 1.0ml of the substrate solution,
The reaction was carried out at ℃ for 60 minutes. Next, add the reaction stop solution.
4.0 ml was added and left at room temperature for 60 minutes to allow undegraded protein to precipitate. The reaction solution was centrifuged (1600xG, 15 minutes, room temperature) to obtain a supernatant.
The absorbance of this supernatant at 275 nm was measured.
Note that the blank was prepared by adding the supernatant liquid after adding the reaction solution. One unit of enzyme is the amount of oxygen that liberates a trichloroacetic acid soluble substance corresponding to 1 μg of tyrosine per minute. Table 1 shows the results obtained.
It was shown to. Table 1 Strain activity (units/ml) Current strain 121 Control strain 83 As is clear from the table, this strain has a higher protease activity than the control strain. Example 2 This strain was cultured in a medium containing glutamic acid, and the viscosity of the produced mucilage was measured. That is, mucilage production medium (1.5% monosodium glutamate monohydrate, 3% sucrose, 1.5% phyton (manufactured by BBL), 0.25% KH ₂ PO ₄ , 0.17% Na ₂ PO ₄ ,
0.005% NaCl, 0.005% _MgCl2・_H2O , 100μg/
Biotin, PH7.0) was inoculated with this strain and incubated at 37℃ for 3 days.
The cells were cultured with shaking for days. After the culture was completed, the culture solution was centrifuged (8000xG, 30 minutes, 5°C) to obtain a supernatant. Three times the weight of distilled water was added to this supernatant and mixed well. Next, after degassing this liquid, the flow time was measured using an Ostwald relative viscometer (manufactured by Shibata Kagaku Kogyo Co., Ltd., viscometer No. 2). The value divided by the distilled water flow time was defined as the relative viscosity. The results are shown in Table 2. Table 2 Bacterial Strain Relative Viscosity Current strain 60.1 Control strain 16.7 As is clear from the table, the viscosity of the culture solution of this strain was higher than that of the control strain. Example 3 Oxygen γ-glutamyl transpeptidase (γ-GTP) is known to be involved in mucilage synthesis. Therefore, the enzyme activity in the culture solution of this strain was measured. That is, a loopful of this bacterial strain 1 was inoculated into a mucilage production medium having the same composition as that shown in Example 2, and cultured with shaking at 37°C for 16 hours. After the cultivation was completed, 200μ of the culture solution was inoculated into 100ml of mucilage production medium, and cultured with shaking at 37°C for 3 days. After culturing, centrifuge the culture solution (17℃, 600xG, 10 minutes,
5°C) to obtain a supernatant. γ in the obtained supernatant
- GTP activity was measured. The activity was measured using a γ-GTP measurement kit (γ-GTP manufactured by Wako Pure Chemical Industries, Ltd.).
-GTP C test (Wako) was used. One international unit of activity is the amount of enzyme that liberates p-diethylaminoaniline in 1 minute at 37°C using L-γ-glutamyl-p-diethylaminoanilide as a substrate. The results obtained are shown in Table 3.

〔Effect of the invention〕

The microorganism of the present invention has high protease activity,
Moreover, it is a microorganism with high mucilage production. Therefore, by using this strain, it is possible to ferment raw materials containing proteins in an aqueous system and produce fermented foods such as seasonings and miso that have different physical properties from conventional ones. Considering the current situation where product differentiation is desired in the food industry, this strain can be widely used as an additive in products that can impart viscosity and stringiness. In addition, this strain forms heat-resistant spores and has excellent storage stability. Therefore, it is not necessary to take any special method to distribute this strain, and it is very easy to handle. Furthermore, there are no particular difficulties in liquid culture, and it can be easily propagated. Therefore, this strain does not pose any problems when handling and is considered to be easily usable by the general public.

Claims (1)

[Claims] 1. A bacillus that has two properties: secretes protease of 100 units/ml or more when cultured in ordinary bouillon medium, and produces mucilage with a relative viscosity of 25 or more (4-fold dilution) in mucilage production medium. - Subtilis NN-1.