JPH08154616A - Quality-improved natto and novel mutant strain - Google Patents

Abstract

PURPOSE: To provide NATTO (fermented soybean) improved in quality and useful in health because it has a reduced ammonia content lower than a prescribed value and contains more than a prescribed amount of γ-polyglutamic acid. CONSTITUTION: This quality-improved NATTO has less than 65mg% (W/W) ammonia content and more than 1,000mg%(W/W) γ-polyglutamic acid. This modified NATTO is produced as follows: soybeans are soaked in water in a 5-time weight/weight(W/W) of the soybean at 10-20 deg.C for 20 hours, drained, and treated with heat or an organic solvent to modify the soybean protein. Then, the modified soybean protein is adjusted in its temperature to 75-85 deg.C, inoculated with the cell bodies of nattobacillus or its spares in an amount of (0.8-1.2)×10<4> cells/g of the boiled soybeans. Then, the inoculated soybeans are cultured under conditions of 35-40 deg.C temperature and 85-95% humidity to give this NATTO. This modified nattobacillus is Bacillus subtilis MR141 (FERM- P-14692).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to quality improvement natto, more specifically, quality improvement with a small amount of ammonia and a gamma-polyglutamic acid (hereinafter referred to as PGA) content which is the same as or higher than that of the conventional one. TECHNICAL FIELD The present invention relates to natto, and a natto bacterium and a novel mutant strain used in producing natto.

[0002]

2. Description of the Related Art Cultures such as natto produced by culturing or fermenting Bacillus natto using protein-denatured beans as a medium are healthy foods, but many people dislike it. It smells like ammonia. Various methods have been proposed to improve it. That is, a new natto strain Bacillus subtilis capable of producing a natto from which ammonia is not released so much
tilis) TTCC162 (Ministry of Industrial Science and Technology Deposit No. 11052)
No. 4) (Japanese Patent Laid-Open No. 4-173069), a novel mutant strain K-2 which is growth-sensitive to low temperature
(Bacillus sp. K-2) (Deposit No. 9768), a method for suppressing secondary fermentation of natto (Japanese Patent Publication No. 5-60335), buckwheat flour, wheat germ, barley, etc. as a soybean raw material. Method of adding to
No. 154964, Japanese Patent Publication No. 60-36735)
And so on.

[0003] However, the above method is p
It is only to suppress the release of ammonium from natto by changing H, or to suppress the production of ammonia by controlling the growth of natto bacteria during storage by temperature, and the production of ammonia during natto production is essential. The amount of ammonium in natto is not so different from the conventional one because it is not controlled. As a result, in the case of long-term storage at room temperature (for example, 8 days or more), the ammonia production becomes 200 mg% (w / w) or more, and an unpleasant ammonia odor is felt. Or, because of the addition of other materials to the soybean raw material, the quality is different from the original natto. Therefore, the above-mentioned problem is not solved essentially. However, if the growth of natto bacteria is extremely suppressed during the production of natto, natto with a low ammonia content can be produced. However, since the natto thus produced has a small growth rate of Bacillus natto, the content of PGA (which is one of the indicators for the growth rate of Bacillus natto) produced due to the growth of Bacillus natto and the decomposition of soybean proteins is small. As a result, a low quality product with a strong steamed soybean odor and no natto-specific flavor and stringiness is obtained. The difficulty of producing high quality natto is there.

[0004]

SUMMARY OF THE INVENTION The object of the present invention is to solve the above-mentioned problems and has a lower ammonia content and a higher PGA content than conventional natto. An object is to provide quality-improved natto that is unchanged or more, and to provide natto bacteria and novel mutant strains that enable the production thereof.

[0005]

Means for Solving the Problems As a result of intensive studies for solving the above-mentioned problems, the present inventors have found that the ammonia production capacity is lower and the PGA production capacity is different from that of a conventionally known natto strain. The present invention has been completed based on the finding that the above problem can be solved by separating a specific natto strain having no or higher than that and using it in the production of natto.

That is, the present invention has an ammonia content of 65 mg% (w / w) or less and a PGA of 100.
It is a quality-improved natto having a content of 0 mg% (w / w) or more, and is a method for producing a quality-improved natto using a specific natto bacterium in producing such natto. In addition, a specific Bacillus subtilis (Bacillus subtilis)
lus subtilis) MR141 (Institute of Biotechnology, National Institute of Biotechnology, Industrial Technology Contract No .: FERM P-146)
92).

Hereinafter, the present invention will be described in detail. (Measurement Method) In the present invention, an ammonia and PG
The measuring method of A is as follows. Ammonia (mg%): 50 g of natto or culture and 1 part of water
After adding 00 ml and stirring thoroughly, the solid matter is removed with gauze. Next, the supernatant obtained by centrifuging this filtrate is sterilized using a membrane filter (0.45 μm) to obtain a sample solution. The liquid is measured in the same manner as in the automatic amino acid analysis method by HPLC. PGA (mg%): SD of the sample solution obtained as described above
After being placed on an S-polyacrylamide plate and subjected to electrophoresis, the plate was stained with Alcian blue, and the stained P
The chromaticity of the GA band is measured by densitometry.

The natto referred to in the present invention is defined as a culture or a fermented product obtained by denaturing a protein of beans using a known ordinary natto production method and culturing the natto bacterium in the beans. . Then, for example, it is obtained by the following method. Cultivation (fermentation) method of Bacillus natto; beans 2 to 7 times (w /
w), preferably 4-6 times (w / w) water, 5-40
C., preferably 10 to 20.degree. C., immersed for 10 to 30 hours, preferably 15 to 25 hours, and drained. This is subjected to protein denaturation treatment. Next, the modified beans have a water content of 50 to 70% (w / w), preferably 55 to 6
5% (w / w), product temperature 60 to 90 ° C., preferably 75
Adjust the temperature to ~ 85 ° C and remove the Bacillus natto cells or spores (0.5
~ 3) x 10 ⁴ pieces / g steamed soybean, preferably (0.8 ~
1.2) × 10 ⁴ seeds / g of cooked soybeans. And the product temperature is 15 to 45 ° C., preferably 35 to 40
C, humidity in the fermentation chamber 70 to 100%, preferably 85 to 9
The culture is performed at 5% for 10 to 40 hours, preferably 20 to 30 hours. If necessary, it is aged at 2 to 10 ° C. for 30 to 50 hours.

In the above method, examples of the beans include soybean, black soybean, red bean, green peas, quail bean, green bean, green beans, turquoise, peanut and the like. Examples of the modification treatment include heat treatment {steaming (for example, 1.6 to 2.0 kg / cm ² , 30 to
60 minutes), steaming after baking, steaming after frying,
Modification treatment with superheated steam and the like}, treatment with an organic solvent such as alcohol, and the like can be mentioned. The natto obtained as described above, when using a known natto bacterium, usually has an ammonia content of 70 to 2 at the time when the culture is completed.
00 mg% (w / w), PGA content is 1000-14
It is 00 mg% (w / w).

The natto strain of the present invention means that the culture obtained by culturing by the following method has an ammonium content of 65 mg%.
It is defined as a Bacillus natto having the ability to produce a culture of (w / w) or less and gamma-polyglutamic acid of 1000 mg% (w / w) or more. The content thereof is the content at the time when the culture is completed, not the one stored for a long period of time. Cultivation method: Beans are cultivated for 10 to 2 times with 5 times amount (w / w) of water.
Immerse at 0 ° C. for 20 hours and drain. This is heat-treated or treated with an organic solvent to denature the protein of pulses. Next, the temperature of the beans is adjusted to 75 to 85 ° C, and the cells or spores of Bacillus natto are (0.8 to 1.2) x
Inoculate so as to be 10 ⁴ pieces / g of steamed soybean. Then, the culture is performed at a product temperature of 35 to 40 ° C. and a humidity of the culture chamber of 85 to 95% for 25 hours.

A feature of the present invention is that the natto obtained as described above has an ammonia content of 65 mg% (w / w) or less, preferably 60 mg% (w / w) or less, and more preferably 50% (w / w). w) or less, most preferably 40% (w
/ W) and PGA of 1000 mg% (w / w) or more, preferably 1600 mg% (w / w) or more, and particularly preferably 2100 mg% (w / w) or more. And when manufacturing it, it is using the natto strain of this invention. Such a strain may be isolated from the natural world, but it is more preferable to use a commercially available natto strain as a parent strain and subject it to mutation treatment to isolate it as a novel mutant strain.

The natto has an ammonium content of 65 mg.
When it exceeds% (w / w), the amount of ammonia increases during storage to exceed 200 mg% (w / w), and the smell of ammonia becomes felt. Also, PGA is 10
When the amount is less than 00 mg% (w / w), it means that the Bacillus natto is not sufficiently growing in natto, and thus the amount of protease produced is small. Therefore, such natto has a strong steamed soybean odor, and the odor and taste peculiar to natto are greatly reduced. In addition, the stringiness of natto based on PGA is low.
As a result, the quality of natto is low. That is, the PGA content in natto is one index for estimating the growth amount of natto bacteria.

The Bacillus subtilis (Bacillus subtilis) parent strain used to obtain the novel mutant strain is Bacillus subtilis.
lus subtilis), and biotin (bio
tin) Known natto bacteria having a requirement can be used. Specific examples thereof include commercially available products such as Miyagino natto, Takahashi, Asahikawa, Matsumura and Naruse.

The above-mentioned mutation treatment method is a generally known method, for example, a method by genetic manipulation, for cells or spores,
Examples thereof include a method in which a drug having mutagenicity is brought into contact, and a method in which X-rays, ultraviolet rays, radiation, light or the like is irradiated. In the present invention, a method using a drug having mutagenicity can be preferably adopted. The drug is a known drug,
For example, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and methyl ethyl sulfate can be mentioned. When these agents are brought into contact with the cells or spores of Bacillus natto, the drug concentration is usually 10 ⁸ to 10 ⁸ cells.
10-1000 in a cell suspension of 10 ⁹ cells / ml
γ / ml. The contact is usually 0 to 50 ° C. and 1
It is 0-1400 minutes.

The mutagenized cells or spores can be obtained by using an ordinary known nutrient medium, for example, a medium containing broth, peptone, soybean flour, yeast extract, casamino acids, amino acids or a mixture thereof, or necessary nutrients. Incubate for 3 to 24 hours in a liquid medium such as an inorganic synthetic medium containing the same. After that, they are appropriately diluted and spread on agar plates of these media. Further, the cells or spores subjected to the mutation treatment may be directly smeared on these plate media. After culturing, the colonies appearing on the plate medium are picked up one by one on an agar slant medium containing broth or the like and cultured. Each of these culture strains will be cultured based on the natto strain culture method defined above. Then, the ammonia content and the PGA content of each culture are measured. Thus, a natto strain having an ability to produce a culture having an ammonia content of 65 mg% (w / w) or less and a PGA content of 1000% (w / w) or more is isolated as a novel mutant strain of the present invention. To be done. Such a separation method is called a promiscuous screening method.

In the present invention, in addition to the above-mentioned indiscriminate screening method, a method of selectively concentrating and separating the natto strain of the present invention as a novel mutant strain can be preferably used. For example, the following method can be mentioned. Mutagenized cells or spores are cultivated in the liquid medium described above, and the obtained cells are suspended in a medium containing one or more amino acids as a nitrogen source. A known concentration method for concentrating only the strain which does not produce ammonia, for example, a penicillin concentration method is performed. As the amino acid, for example, basic ones such as arginine and asparagine are preferably used.

Next, a membrane filter is placed on the surface of the nutrient plate medium, and the concentrated cell suspension is smeared on the membrane filter. When colonies are formed on the surface, the membrane filter is removed, and a pH adjusting plate medium, for example, 1 to 100 mM maleic acid-
NaOH buffer, 0.8-2.5% (w / v) agar, p
Transfer to a plate medium having a composition such as H5-7. And 0.1
Leave for 1 hour. By this operation, the pH of the membrane filter becomes 5 to 7. The membrane filter is then assayed with an assay plate medium, eg, 1-100 mM amino acid, pH 5-7, 0.8-2.5% (w / v) agar,
0.001-0.01% (w / v) bromcresol purple (BCP; bromcresol purpl)
e, pH indicator) and the like. And it incubates at 25-45 degreeC for 0.1 to 5 hours. At that time, colonies that decompose L-arginine and produce more ammonia (parent strains, strains with high ammonia-producing ability, and colonies of strains in which the properties of the parent strain do not change very much) have increased pH around them, The membrane filter around the colony turns blue. But,
The area around the colony of the mutant strain with low ammonia production capacity remains yellow without discoloration.

A large number of mutant strains having a yellow color around the colony are isolated, and a culture is produced for each of them in the same manner as in the promiscuous screening method. Then, the natto strain capable of producing the culture defined above is the natto strain of the present invention and a novel mutant strain. Needless to say, the above two screening methods are preferably used also when the natto strain of the present invention is isolated from the natural world.

Although a large number of novel mutant strains can be isolated by the above-mentioned method, as an example, MR141 and MR131 (hereinafter, these strains are simply referred to as MR141 and MR131).
Can be mentioned. MR141 and MR131 are Miyagino natto strains (Bacillus subt) as parent strains.
ilis) (hereinafter simply referred to as MR-1).

The results of comparing the mycological properties of MR141 with those of MR-1 are shown in Table 1. In addition, the experiment for investigating the mycological properties was conducted by "Method for identifying microorganisms" (published by Sanitation Technology Society, 19
1983).

Table 1 Comparison of mycological properties of MR141 and MR-1 ────────────────────────────── Property MR- 1 MR141 ────────────────────────────── 1) Vegetative cell morphology Bacillus bacilli 2) Spore formation Positive Positive 3) Gram Staining Positive Positive 4) Oxygen requirement Obligatory aerobic Oblique aerobic 5) Starch-degrading positive Positive 6) Acid production from glucose positive Positive 7) Nitrate reduction positive Positive 8) Catalase positive Positive 9) Acetoin production Positive Positive 10) Growth in medium containing 7% salt Positive Positive ───────────────────────────────

Properties other than the above, for example, biotin requirement, cell size (1 × 2 to 3 μm), spore size (0.8 × 1.6 to 1.8 μm), growth on broth agar medium Condition (cultivation at 25 ° C for 25 hours), growth condition of gelatin stab culture, growth condition in litmus milk and litmus reducibility, acid production from various sugars, optimum growth temperature range, DN
Experiments were also conducted on properties such as the GC content of A, and the properties were consistent with MR-1. Therefore, MR
141 is Bacillus subis
btilis).

However, when MR141 was cultivated by the natto strain culture method defined above, the culture had an ammonia content of 6
5 mg% (w / w) or less, 1/2 of that of MR-1
It is 1/3, and the PGA content is more than that of MR-1.
It is 1.8 to 3.5 times higher. Moreover, the protease activity of natto of MR141 is 2-3 times stronger than that of MR-1. Using the MR 141 having the above characteristics, natto having an ammonia content of 65 mg% (w / w) or less and a PGA content of 1000 mg% (w / w) or more can be produced. In addition, since the natto produced as described above has high protease productivity, the protein is well decomposed. Therefore, the flavor is excellent. This is the parent strain MR-1
Cannot be manufactured at all. This point is MR141 and MR-1
Is a clear difference. MR131 also MR1
An experiment similar to that of No. 41 was conducted to examine its properties, but it was similar to that of MR141.

When the natto is produced by culturing the natto bacterium by the above-mentioned method, the known natto bacterium strain capable of producing the one having the lowest ammonium content is 7
0 mg% (w / w) of Bacillus sp.
K-2 (Deposit No. 9768)
335) (Food and Development, Vol. 23, No. 8, 4)
7-47, 1988). Further, as a strain capable of producing natto having a high viscosity which is correlated with the PGA content, Bacillus subtilis (Baci) capable of producing 1.6 to 1.7 times higher than the parent strain.
illus subtilis) TTCC162 (Ministry of Engineering Research Deposit No. 11052) (Japanese Patent Laid-Open No. 4-173069), the ammonia content of which is 95 mg% (w / w). Thus, until now, there is no known natto or natto mutant strain capable of producing natto having an ammonia content of 65 mg% (w / w) or less and a PGA content of 1000 mg% (w / w) or more. Has not been done. So Bacillus subtilis
lus subtilis) MR141, MR131
Was identified as a new mutant strain. In particular, MR141 has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology with a deposit number of FERMP-14692.

The Bacillus subtilis natto strain and MR141 of the present invention obtained as described above have the above-mentioned characteristics. Therefore, by using them, the ammonia content is 6%.
5 mg% (w / w) or less and a PGA content of 100
It is possible to easily produce natto whose content is 0 mg% (w / w) or more. The natto strain and MR141 of the present invention are subjected to solid culture, liquid culture, etc., in addition to the production of natto of the present invention, and, for example, PGA, PGA synthase gamma-glutamyl transpeptidase (γ-glutamyl).
transpeptidase; γ-GTPase),
It can be preferably used for production of proteases and other substances that can take advantage of other characteristics of the bacterium, for example, production of protein degradation products (peptides, amino acids) and seasonings.

[0026]

The natto of the present invention is superior to the conventional natto in that
The ammonia content is low, and the PGA content is the same as or higher than that of conventional natto. In addition, even if it is stored for a long period of time, the amount of increase in the ammonia content is much lower than that of the conventional one, so that the smell of ammonia does not occur.
That is, the quality does not deteriorate. Further, since natto is a natto in which the Bacillus natto is sufficiently grown, the amount of protease produced is sufficient and soybean protein is well decomposed. The result is a high quality, improved natto with excellent flavor. Such natto is the natto strain of the present invention and the novel mutant strain M.
It becomes possible only by using R141. This is because those strains have lower ammonium production capacity than the conventionally known Bacillus natto. In addition to the production of natto, those strains may be subjected to solid culture, liquid culture, etc. to produce, for example, PGA, γ-GTPase, protease, etc., and other substances that can take advantage of the characteristics of the strain, such as protein. Degradation products (peptides, amino acids),
It can be used in the production of seasonings.

[0027]

EXAMPLES The present invention will be described below with reference to examples. Example 1 A spore suspension of (mutated) MR-1 (10 ⁶ cells / ml) was prepared, and heat-shocked at 65 ° C. for 30 minutes. This was inoculated into a 150 ml Erlenmeyer flask with pleats containing 40 ml of a preculture medium (1.0% meat extract, 0.3% yeast extract, pH 7.2) at a volume of 1/100 of the medium amount.
Then, the cells were cultured at 37 ° C. for 8 hours with shaking at 120 rpm.
NTG was added to this culture solution to a final concentration of 100 μg / ml, and the cells were further cultured for 16 hours. The cells were collected by centrifugation, washed with sterilized physiological saline, and then washed with the same saline.
It was diluted × 10 ⁴ times.

(Screening) 100 μ of the above-mentioned diluent
1% on 5% soybean flour and 1.5% agar plate medium with a membrane filter (Advantech Toyo, pore size 0.45 μm,
Smeared through a nitrocellulose membrane filter). After culturing at 37 ° C for 20 hours, the membrane filter on which colonies were formed was used as an agar plate medium for pH adjustment (10 m
M Maleic acid-NaOH buffer, 1.5% agar, pH
5.3), and allowed to stand for 30 minutes at room temperature to equilibrate the pH of the membrane filter. Next, an assay plate medium {10 mM L-arginin (L-arginin
e), 0.005% BCP, pH 5.3} and moved to 3
Incubated at 7 ° C for 2 hours. Strains that did not change color around the colony were picked up and suspended in 1 ml of physiological saline. This suspension was screened again in the same manner as above to purify the mutant strain, and the slant medium (Difco, nutrie) was purified as a novel mutant strain of the present invention.
nt agar).

(Manufacture of Natto) Small US round beans were immersed in 5 times the volume of tap water (15 ° C.) for 20 hours. This was placed in a steaming pot and steamed first for 10 minutes without pressure and then for 40 minutes at 1.8 kg / cm ² . After turning off the steam and holding it for 10 minutes,
The pressure was gradually reduced and the product was taken out. Immediately thereafter, a spore suspension of the preserved novel mutant strain (prepared by taking 1 platinum loop spore from the preserved slant medium and suspending it in 200 ml of 1% sterilized saline) was added to steamed soybeans at 80 ° C. Spores 10 ⁴
The seeds were inoculated so that the number of individual pieces / g of steamed soybean was 50 g and the mixture was filled in a styrene container (50 g volume) for natto production. Fermentation was performed for 20 hours at a product temperature of 37 ° C. and a humidity of 90% in the culture room.

Regarding natto produced with each new mutant strain,
The contents of ammonium and PGA were measured. In addition, the sensory test was performed to check the flavor, and the ammonia content was 65 mg%.
(W / w) or less and PGA content is 1000 mg%
(W / w) and above, many new mutant strains producing quality-improved natto with rich flavor were isolated. MR141 and MR131 were selected as representatives of these novel mutant strains. In particular, MR141 was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology with a deposit number of FERM P-14692.

Example 2 Using the novel mutant strains MR141 and MR131 of the present invention and its parent strain MR-1, each natto was produced by the above-mentioned method, and the quality of natto (ammonia content, PGA content, The flavor and protease activity) were investigated and compared. In the sensory test of flavor, 20 trained panelists tasted natto, asked which was preferable, and expressed it by the number of people who answered that it was preferable. The protease activity was measured by the following method. Protease activity (PU / g) measurement: Natto 50g and water 1
After adding 00 ml and thoroughly stirring, solid matter was removed with gauze. Next, the supernatant liquid obtained by centrifuging this filtrate was sterilized with a membrane filter (0.45 μm) to obtain an enzyme liquid. For this, Anson-Ogihara modified method (Y. Fukushima; Agric. Biol. C)
hem. , 49, 1643, 1985).

Table 2 ──────────────────────────────────── Strains Ammonia content PGA content Protease activity Flavor mg% (w / w) mg% (w / w) (PU / g) (Number of people) ──────────────────────────── ───────── MR141 30 2200 140 18 MR131 40 1400 130 16 MR-1 80 1000 95 2 ───────────────────────── ──────────── As can be seen from Table 2, the novel mutant strain MR14 of the present invention.
1. The natto of the present invention produced by MR131 has an ammonia content of 30 and 40 mg% (w / w), respectively, and MR-1.
From 80 mg% (w / w) of natto of
It is as low as 3 1/2. P, which is one of the indicators of natto growth
GA content is 2200 mg% (w / w) and 1400, respectively
The mg% (w / w) is 2.2 times and 1.4 times higher than that of the parent strain MR-1. Therefore, it can be seen that natto bacteria are sufficiently grown in the produced natto. The protease activity was 1.5 times higher than that of the parent strain, 1.4
Soy protein in natto was well degraded because it was twice as expensive. The results of the sensory test of flavor are much better than those of the parent strain.
From the above, it can be seen that the natto of the present invention is a quality-improved natto having an excellent flavor.

Example 3 For the natto obtained in Example 2, room temperature (27-30
A storage test was conducted at (° C) to measure the ammonia of natto.
Table 3 shows the amount of increase in ammonia.

Table 3 ────────────────────── Increasing amount of ammonia content mg% (w / w) Number of storage days MR141 MR-1 ──── ────────────────── 1 8.3 19.5 2 18.2 37.2 5 44.6 141.7 10 83.4 240.1 ──── ──────────────────

As can be seen from Table 3, even when the natto of the present invention is stored at room temperature, the amount of increase in the ammonia content is much smaller than that of MR-1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Katsumi Yuasa 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (3)

[Claims] 1. An ammonium content of 65 mg% (w /
w) or less and 10 gamma-polyglutamic acid
Quality-improved natto characterized by having a content of at least 00 mg% (w / w). 2. The culture obtained by culturing by the following method has an ammonia content of 65 mg% (w / w) or less and a gamma-polyglutamic acid content of 1000 mg%.
The method for producing a culture according to claim 1, wherein Bacillus natto having the ability to produce a culture having a (w / w) or more is used. Cultivation method: Beans are cultivated for 10 to 2 times with 5 times amount (w / w) of water.
Immerse at 0 ° C. for 20 hours and drain. This is heat-treated or treated with an organic solvent to denature the protein of pulses. Next, the temperature of the beans is adjusted to 75 to 85 ° C, and the cells or spores of Bacillus natto are (0.8 to 1.2) x
Inoculate so as to be 10 ⁴ pieces / g of steamed soybean. Then, the culture is performed at a product temperature of 35 to 40 ° C. and a humidity of the culture chamber of 85 to 95% for 25 hours. 3. A novel mutant strain Bacillus subtilis having the ability to produce the culture of claim 2.
lls subtilis) MR141 (Institute of Biotechnology, National Institute of Bioscience and Technology, Contract number: FERM P-1)
4692).