JPH10262655A - New bacillus natto and its acquisition and poly-gamma-glutamic acid produced with new bacillus natto and seasoning utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new Bacillus natto producing a system comprising poly γ-glutamic acid containing L-glutamic acid in a large amount by belonging to Bacillus subtilis and lacking a glutamate recemase. SOLUTION: This Bacillus natto belongs to Bacillus subtilis, lacks a glutamate racemase, and produces poly-γ-glutamic acid wherein >=70% of the consisting units is L-glutamic acid. The new Bacillus natto is deposited as FERM P-16147, and is obtained by subjecting a wild strain belonging to Bacillus subtilis to a mutation treatment, selecting bacteria capable of growing in a perfect culture medium but not growing in a minimal culture medium from the mutated bacteria, and further selecting a bacterium capable of growing in a minimal culture medium containing glutamic acid from the selected bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel Bacillus subtilis belonging to Bacillus subtilis, a method for obtaining the same and a use thereof.

[0002]

The natto is not only good in digestibility and high in nutritive value, but also delicious and inexpensive, in addition to having good digestibility by decomposing the high-quality vegetable protein contained in soybean. It is an excellent balanced food with advantages such as being excellent.

[0003] Natto is characterized by its stickiness, that is, stringiness, and the amount of natto yarn and the strength of the stickiness increase as the ripening proceeds. This thread is obtained by Bacillus natto digesting the protein contained in natto, contains poly-γ-glutamic acid composed of D-glutamic acid and L-glutamic acid, and is also a component of natto umami.

The molecular structure of poly-γ-glutamic acid produced by Bacillus natto was examined, and D-glutamic acid and L-glutamic acid were examined.
-There is also a research example that clarifies the ratio of glutamic acid (Osaka City University, Rieko Ito et al., March 1996).

[0005] From these studies and the study of the present inventors, poly-γ-glutamic acid produced by Bacillus natto of normal natto currently on the market has a D-glutamic acid ratio of about 50 to 80 in the constituent units. %, And it is clear that this ratio increases as natto ripens. However, what actually contributes to the taste of natto is poly-
It is only L-glutamic acid in γ-glutamic acid, and D-glutamic acid, which increases with age, is tasteless.

[0006] It is an object of the present invention to obtain a natto that draws a thread having an even greater flavor than conventional natto.

Another object of the present invention is to obtain poly-γ-glutamic acid containing a large amount of L-glutamic acid, and to obtain a seasoning using the obtained poly-glutamic acid.

[0008] For this purpose, it is a further object of the present invention to obtain a Bacillus natto that produces a thread comprising poly-γ-glutamic acid, which contains a large amount of L-glutamic acid.

[0009]

Therefore, the present inventors have found that glutamic acid or its precursors originally contained in soybeans are all L-forms.
We focused on the mechanism by which glutamic acid is converted to D-glutamic acid. That is, assuming that Bacillus natto that does not perform such conversion in the cells produces poly-γ-glutamic acid consisting only of L-glutamic acid, as a result of intensive studies on such Bacillus natto, This led to the invention.

The gist of the novel natto bacterium according to the present invention resides in that it belongs to Bacillus subtilis and lacks glutamase-tracemase.

The gist of the novel natto fungus according to the present invention belongs to Bacillus subtilis and has a structural unit of about 7
0% or more is to produce poly-γ-glutamic acid which is L-glutamic acid.

[0012] A novel natto bacterium according to the present invention, wherein any of the natto bacterium described above preferably belongs to Bacillus subtilis and exhibits glutamic acid requirement.

The gist of the poly-γ-glutamic acid according to the present invention is that it is produced by a novel Bacillus natto, and that about 70% or more of its constituent units are L-glutamic acid.

The gist of the seasoning according to the present invention is that a poly-containing seasoning containing about 70% or more of L-glutamic acid is used.
γ-glutamic acid.

The gist of the novel method for obtaining Bacillus natto according to the present invention described in any of the above is that Bacillus su
a step of mutating a wild strain belonging to btilis; a step of selecting, from the mutated strains, a bacterium that grows on a complete medium and does not grow on a minimal medium; and a medium obtained by adding glutamic acid to the minimal medium from the selected bacteria. And further selecting a bacterium that grows in the above.

The term "natto bacterium" in the present invention refers to Bacillus sp.
It refers to a bacterium belonging to subtilis and exhibiting a requirement for biotin, and can be used for production of natto, which is generally commercially available.

The term "deficiency of glutamase-tracemase" in the present invention refers to a complete lack or a partial lack of an enzyme required for the conversion of D-glutamic acid to L-glutamic acid or vice versa. . Specifically, the lack of a part of the enzyme specifically means that the amount of the enzyme is about 通常 of the amount of the enzyme that a normal wild strain has
Hereafter, preferably, it has only 1/5 or less enzyme level.

The term "glutamic acid-requiring bacterium" of the present invention
The term “bacteria” refers to a bacterium that needs to contain glutamic acid in a medium for the growth of the bacterium. Such a bacterium does not grow at all or cannot grow sufficiently in a medium containing no glutamic acid.

As used herein, the term "wild strain" refers to a strain having a phenotypic trait normally found in nature.

The term "seasoning" in the present invention includes, for example, umami seasoning, tressing, mayonnaise, various soups, and sauces.

[0021] The term "thread" in the present invention refers to a viscous substance produced when inoculated with Bacillus natto on an appropriately treated soybean or medium, and in the present invention, terms interchangeable with thread and viscous substance are used. It is.

The term "complete medium" in the present invention refers to a medium in which both prototrophic bacteria and auxotrophs can grow, and contains all of trace components such as carbon sources, nitrogen sources, and minerals.
Usually contains organic matter such as yeast extract or peptone. The components and ratios are not particularly limited as long as they are within the range of common knowledge of those skilled in the art.

As used herein, the term "minimal medium" refers to a synthetic medium that contains the minimum number of nutrients required for the growth of prototrophy.
For example, it contains a carbon source such as a saccharide, nitrogen, and an inorganic compound at a certain ratio. Since biotin is essential for culturing Bacillus natto, the minimum medium always contains biotin.

In the present invention, the term "structural unit" refers to poly-γ
-Refers to a monomer that constitutes glutamic acid.

Further, in the present invention, "about 70% or more of the constitutional unit" means substantially a monomer in poly-γ-glutamic acid.
About 70% or more of
This takes into account errors and the like that occur when measuring the proportion of monomers in the polymer.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION The Bacillus natto according to the present invention is selected from mutants obtained by mutating a known Bacillus natto.

As the Bacillus natto used to obtain the mutant strain, any known Bacillus natto belonging to Bacillus subtilis having biotin requirement can be used as a parent strain. Specific examples thereof include Miyagino natto bacteria, Takahashi bacteria, Asahikawa bacteria, Matsumura bacteria, and Naruse bacteria.

For cultivation of such Bacillus natto, any complete medium known to those skilled in the art can be used. For example, a medium supplemented with meat extract, peptone, calf plasma, agar, gelatin, salt, or the like, a broth medium, a broth agar medium, a broth gelatin medium, a litmus milk, a MEM medium, an east extract medium, and the like are used.

The culture may be performed according to a known method such as stationary culture or shaking culture. The culture temperature and time
The culture may be the same as the usual culture, and the culture time at 30 ° C. to 45 ° C. may be about 1 day to 5 days.

Examples of the mutation method include any known methods such as a method of contacting a chemically synthesized substance as a mutagen, a method of genetic manipulation, and a method of irradiating with X-rays, ultraviolet rays, or light. Can be adopted.

As a method of mutation, a method of bringing a mutagen into contact with a bacterium is, for example, suspending a known natto bacterium as a parent strain in a buffer containing a mutator,
Alternatively, this means culturing in a medium containing a mutagen.

From the bacteria subjected to the mutation treatment, bacteria having no glutamase-tracemase activity can be selected. Selection is performed, for example, by selecting a bacterium that does not grow on the minimal medium among the bacteria grown on the complete medium by a method known to those skilled in the art, and then taking the bacterium that grows on a medium obtained by adding glutamic acid to the minimal medium. Can be done by

As a specific example of the mutant strain thus obtained, Bacillus subtilis M-28 (deposited with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-16147. -28)
Can be mentioned. M-28 is a mutant strain obtained by mutating this Miyagi natto using Bacillus natto as a parent strain.

The bacteriological properties of the M-28 strain are shown. (A) Form Shape; rod size; 2.3 to 3.5 × 0.7 to 0.9 μm presence of spores; spore size; 0.8 × 1.6 to 1.8 μm spore shape Oval spore swelling presence / absence; spore-free site; central Gram staining; positive (b) Growth on normal agar medium (cultured at 25 ° C for 25 hours) Shape; annular surface; rough and wrinkled (C) gelatin puncture culture Growth state; + liquefaction; layered (d) growth on anaerobic agar medium;-(e) growth on sabrose-sucrose medium Presence or absence; + (f) presence or absence of growth in litmus milk;

Decomposes casein without coagulation. (G) physiological properties catalase; + oxidase; + starch hydrolysis; + gelatin hydrolysis; + lysine decarboxylase;-arginine dihydrase;-ornithine decarboxylase; Formation;-reduction of nitrate; + esculin; + urease;-utilization of citric acid; + phenylalanine deaminase;-egg yolk reaction; -Production of gas from glucose;-Production of acetoin; + β-galactosidase; + Biotin requirement; + Glutamate requirement; + Glutamase-Tracemase activity;

The bacteriological properties of M-28 are almost the same as those of the parent strain, Miyagino natto, but show the requirement for glutamate, do not have glutamase-tracemase activity,
It can be clearly distinguished from the parent strain in that it produces poly-γ-glutamic acid in which about 70% or more of the structural units are L-glutamic acid.

Furthermore, the present invention includes mutants other than M-28. Their common property is that they have no glutamate-tracemase at all, or even if they do, they are at most 1/2 or less, preferably at most 1/5, the amount of normal cells. The mutated Bacillus natto of the present invention has a poly-γ-type in which about 70% or more of the constituent units are L-glutamic acid.
Produces glutamic acid.

Furthermore, both the bacterium that requires glutamic acid, such as M-28, and the bacterium that no longer requires glutamic acid due to reversion are included in the novel natto bacteria of the present invention. Glutamate can be made by an alternative route, even in the absence of glutamase-tracemase. Therefore, among the mutant Bacillus natto selected according to the method of the present invention with the glutamate auxotrophy as a marker, only the glutamate auxotrophic phenotype is restored while the phenotype lacking glutamate-tracemase activity remains. It may disappear due to mutation. Such bacteria are advantageously used in the present invention.

The mutant natto thus obtained is preferably used for producing natto. The production of natto using the mutated Bacillus natto is carried out by a method known to a person skilled in the art.

For example, soybeans are immersed in water and heated. The heated soybeans are cooled appropriately, inoculated with mutant Bacillus natto, and allowed to stand for an appropriate time, for example, overnight. The heating of soybeans can be achieved, for example, by steaming with pressurized steam for 10 to 30 minutes or by direct heating. After inoculation, the cells can be cultured at a temperature of, for example, 30 ° C to 50 ° C for about 12 to 80 hours.

The mucilage contained in the natto thus obtained, that is, the thread, is poly-γ-glutamic acid, and about 70% or more, preferably about 80% or more of its constituent units is L-glutamic acid.

In order to confirm the constituent units of the components of the yarn contained in natto, it is necessary to first separate the yarn. For separation of the yarn, for example, washing with water, purification by ethanol, separation by centrifugation, or a combination thereof is suitably used.

Specifically, for example, the yarn is washed out of natto with water, and an appropriate amount of ethanol is added, for example, 70% of the total amount.
%, And the yarn is extracted by winding it with a glass rod or the like or by centrifugation.

The obtained natto yarn is neutralized by hydrolysis known to those skilled in the art, for example, by acid treatment.
Using the hydrolyzed solution as a sample, the amounts of glutamic acid and L-glutamic acid are measured.

For measurement of the amount of glutamic acid, for example, ninhydrin colorimetry, paper chromatography, and HPLC are preferably used.

When HPLC is used for the measurement, the column is
Any column suitable for separating proteins or amino acids can be used. For example, Shodex Asahi
For example, pak ES502C (manufactured by Showa Denko KK) is used, and water or hot water can be preferably used for extraction. For detection, any column for detection known to those skilled in the art can be used.

For measuring the amount of L-glutamic acid, an enzymatic method is preferably used, but not limited thereto. As an enzymatic method, for example, L-glutamic acid is added to a sample containing L-glutamic acid.
An enzyme using only glutamic acid as a substrate is allowed to act, and the absorbance of the obtained reaction solution is measured. The amount of L-glutamic acid is calculated from the absorbance.

The amount of L-glutamic acid can be easily measured by an enzymatic method using a commercially available kit. For example, this kit is an F-type manufactured by Behringer Mannheim.
It is a kit.

It can be seen that the mutant strain produces a thread containing L-glutamic acid at a higher ratio than the wild strain, that is, poly-γ-glutamic acid.

To determine the nature of the mutant Bacillus natto, glutamase-transasease activity is measured.

The method for measuring glutamase trasemase can be, for example, a method for measuring the rate of change from D-glutamic acid to L-glutamic acid.

Specifically, for example, a mutant Bacillus natto or a wild strain is appropriately taken, suspended in water and crushed to obtain a cell extract. This extract is diluted appropriately and a part of it is
-Put in a solution containing glutamic acid, react at an appropriate temperature for several hours, and then measure the amount of L-glutamic acid. The amount of L-glutamic acid can be measured, for example, with an F-kit.

The mutated Bacillus natto can be cultured in any ordinary medium such as an ordinary complete medium or a minimal medium containing glutamic acid. Even when cultured in such a medium, poly-γ-rich L-glutamic acid is contained.
Produces glutamic acid.

Preferably, the mutant Bacillus natto is cultured in a medium rich in L-glutamic acid to produce poly-γ-glutamic acid rich in L-glutamic acid.

A method for obtaining the mutant Bacillus natto of the present invention, obtaining poly-γ-glutamic acid from a thread produced by the Bacillus natto,
In the following examples, and methods for using the same, the method of mutation treatment, the medium to be used, etc., within the scope not departing from the gist, various improvements, corrections, modifications based on the knowledge of those skilled in the art Can be implemented.

[0056]

Next, examples and comparative examples of obtaining the Bacillus natto according to the present invention, the properties of the slime produced with the Bacillus natto, and utilization of the slime are described. In addition,% represents weight% unless otherwise specified.

Example 1 A mutant strain requiring glutamic acid was obtained by subjecting a wild natto fungus to a treatment for inducing mutation.

First, yeast extract 0.5%, peptone 1
%, Glucose 1%, 0.1M phosphate buffer-
Natto cultivated in a liquid nutrient medium (hereinafter referred to as "liquid medium") adjusted to pH 7.0 with 10,000
The cells were collected by centrifugation at 10 minutes at rpm.

This bacterium was suspended in 1/10 M phosphate buffer (pH 6.8) containing 0.5 mg / ml of N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and cooled on ice. For 30 minutes. Next, the bacteria were collected by centrifugation, washed three times with 1/10 M phosphate buffer, and
TG was washed off. Then, at 35 ° C in the above liquid medium, 2
After culturing for an hour, the cells were collected again by centrifugation, washed with a buffer, and stored at 5 ° C.

The bacterium obtained by the mutation treatment was transferred to a complete plate medium containing 0.5% yeast extract, 1% peptone, 1% glucose and 1.5% agar and prepared with a pH 7.1 buffer. After growing overnight at 35 ° C., the grown colonies were replicated on a minimal medium. The minimum medium is 0.5% glucose, 0.2% (NH ₄ ) ₂ SO ₄ , K ₂ H
PO ₄ 0.5%, sodium citrate 0.1%, MgS
O ₄ 0.02% biotin 0.001%, and agar 2
% Was adjusted to pH 7.0. By comparing the bacteria on the replicated medium with the bacteria on the complete medium, strain 32004 that did not grow on the minimal medium was selected from the complete medium. The selected bacteria were inoculated on a minimal medium supplemented with L-glutamic acid and allowed to stand at 35 ° C. overnight. As a result, 87 strains of bacteria growing on the culture medium were obtained as mutant natto bacteria showing glutamic acid requirement.

Example 2 Using the Bacillus natto obtained in Example 1, natto was produced, and its viscous material, thread, was analyzed.

Wash soybeans and soak in water overnight
Heated at ℃ ° C for 15 minutes. The heated soybeans were cooled appropriately, the natto bacteria obtained in Example 1 were inoculated, and left at 40 ° C. overnight. At this time, 200 g of whole soybeans
Each was used as a raw material. The thread was washed out from the natto with 400 g of water. Further, ethanol was added so as to make up 70% of the whole, and the yarn was extracted by winding it with a glass rod or by centrifugation.

0.2 g of the natto yarn thus obtained
After 2 ml of 1N HCl was added thereto to hydrolyze the yarn, 1N NaOH was neutralized with 2 ml. 1 ml of water was added to the hydrolyzed solution to make 5 ml, which was used as a sample for measuring the amounts of glutamic acid and L-glutamic acid.

(1) Measurement of the amount of total glutamic acid After diluting the sample with water five-fold, the diluted sample was subjected to HPLC in increments of 10 μl to determine the amount of total glutamic acid in the sample. The column was Shodex SUGAR SC110
1 and extraction was performed with water. The flow velocity at this time is
The column temperature was 80 ° C. at 1.0 ml / min. Shodex RI SE-61 was used for detection. The result is shown in FIG.

(2) Measurement of the amount of L-glutamic acid After diluting the sample 5 times with water, 200 μl of the sample was diluted.
Then, 600 μl of solution 1 (potassium phosphate / triethanolamine buffer, pH 8.6) of F kit (manufactured by Boehringer Mannheim Co., Ltd.) and solution 2 (diaphora)
, Β-NAD and stabilizer), 200 μl, solution 3
(Indonitroterazolium chloride) and 200 μl of water were added. This mixture is added at 25 ° C. for 2 hours.
Allowed to react for minutes. The absorbance was measured at a wavelength of 492 nm. And its value as E _1.

Thereafter, 30 μl of solution 4 (glutamate dehydrogenase) was added to this solution, and reacted at 25 ° C. for 15 minutes. The absorbance was measured again at a wavelength of 492 nm, and the value was defined as E ₂ .

Similarly, E ₁ was used starting from 200 μl of water.
And E ₂ and the measured, which was the value of the blank.

E of Sample and Blank₁And E_TwoBased on
Then, the amount of L-glutamic acid was measured. That is, △
E = (Sample E_Two-Sample E₁)-(Blank E_Two−Bran
Qu E ₁) And the amount of L-glutamic acid is 0.112 × ΔE
(Mg / ml).

The amount of thread produced when a representative bacterium among the mutant natto bacteria thus obtained was planted in 200 g of soybean, the total glutamic acid content of poly-γ-glutamic acid, L
-Glutamic acid amount and L- based on total glutamic acid
The proportions of glutamic acid are shown in Tables 1, 2 and 3.
The weight of the natto yarn is the dry weight of the yarn.

[0070]

[Table 1]

[0071]

[Table 2]

[0072]

[Table 3]

As a result, it was found that all of the obtained mutants produced a thread containing L-glutamic acid at a higher ratio than the wild-type strain, that is, poly-γ-glutamic acid.

Example 3 The mutant natto strain obtained in Example 1 was measured for glutamase-transasease activity.

A wild strain and each glutamic acid-requiring strain were picked up with one platinum loop, suspended in 1 ml of water and disrupted to obtain a cell extract. This extract was diluted 10-fold, and 1 ml of it was diluted with 4 ml containing 1 mg / ml D-glutamic acid.
After reacting at 30 ° C. for 2 hours, the amount of L-glutamic acid was measured. The amount of L-glutamic acid was measured in the same manner as in Example 2. As a result, all the mutant Bacillus natto had no glutamate-tracemase activity, or １ ／ or less, preferably 1/5 or less of the wild strain. Table 4 shows the results.

[0076]

[Table 4]

Example 4 Poly-γ which is a component of the natto yarn obtained in Example 2
-The molecular weight of glutamic acid was measured.

First, natto obtained from 200 g of soybeans was washed with about 500 g of water to obtain a washing thread. Ethanol was added to this yarn so as to be 70% of the total amount, and a viscous substance was removed by centrifugation. This operation using ethanol is repeated several times,
A purified yarn was obtained. To measure this molecular weight, Sho
H using dex PROTEIN KW-802.5
Gel filtration by PLC was performed. Gaitamate dehydrogenase (molecular weight 290,00) as a molecular weight marker
0), lactate dehydrogenase (molecular weight 142,0
00), enolase (molecular weight: 67,000), adenylate tokinase (molecular weight: 32,000), cytochrome c
(Molecular weight: 12,4000), and the molecular weight was calculated from each retention time.

As a result, the molecular weight of the poly-γ-glutamic acid contained in the natto which was strongly threaded and the natto which was normally threaded was in the high molecular range near 300,000. On the other hand, the molecular weight of poly-γ-glutamic acid contained in the natto which had poor thread pulling due to weak thread pulling was 10,000 or less.

Example 5 One of the mutated Bacillus natto obtained in Example 1 was liquid-cultured in a special medium to produce poly-γ-glutamic acid containing a large amount of L-glutamic acid.

A wild strain of Miyagino natto and one of the natto obtained in Example 1 were prepared by adding 5% glucose, 3% polypeptone, 0.5% yeast extract and 1% glutamic acid,
Culture in a liquid medium (medium 1) adjusted to pH 7.0 and a liquid medium (medium 2) adjusted to pH 7.0 containing maltose 5%, polypeptone 3%, yeast extract 0.5%, glutamic acid 1%. did. 100 ml of each medium was used, and the culture was performed at 400 ° C. for 12 hours. Ethanol was added to the liquid after the cultivation so that the concentration became 70%, and the thread contained in the culture liquid was wound up with a glass rod.

To analyze the structure of poly-γ-glutamic acid which is a component of the yarn, the obtained yarn was treated with 1N NaOH.
Then, the amount of total glutamic acid and the amount of L-glutamic acid were measured in the same manner as in Example 2, and the ratio of L-glutamic acid was examined.

As a result, the weight of the yarn obtained from the culture medium 1 was 0.62 g for the wild strain and 1.03 g for the mutant strain. The ratio of L-glutamic acid in the thread was 52.6 in the wild strain.
% And 90.5% of mutants. The weight of the yarn obtained from the culture medium 2 was 0.71 g for the wild strain and 1.12 g for the mutant strain.
Met. The ratio of L-glutamic acid in the thread was 5 in the wild strain.
1.5% and 90.6% mutants.

Therefore, it was found that the mutant strain of the present invention produced a large amount of thread from a medium containing a large amount of L-glutamic acid, and that the thread contained mostly L-glutamic acid.

Example 6 A seasoning with a strong umami was produced using natto yarn produced by a mutated Bacillus natto.

Wash the whole soybeans and soak in water overnight.
Heated at ℃ ° C for 15 minutes. 200 g of heated soybeans were cooled appropriately and inoculated with the Bacillus natto M-28 obtained in Example 1,
It was left at 40 ° C. overnight. Next, from this natto water 40
The thread was washed out with 0 g. Furthermore, ethanol was added to the total amount of 70%.
%, And the yarn was wound up with a glass rod and extracted.

The natto yarn obtained by the above method was used as a dressing material. That is, vinegar, soy sauce, salad oil, and pepper were mixed, and natto thread was added so as to be 1% of the whole instead of the chemical seasoning and the amino acid additive. For comparison, a dressing was made by inserting a natto thread made using a wild strain obtained in the same manner. In the dressing thus obtained, L-
The amount of glutamic acid was examined by the method of Example 2. The results are shown in the table.

[0088]

[Table 5]

Further, when the dressing was subjected to a sensory test, it was found that the dressing had a stronger taste than the conventional dressing.

[0090]

According to the present invention, glutamase trasema-
A novel natto-deficient natto bacterium can be obtained.

According to the present invention, Bacillus natto producing poly-γ-glutamic acid having a large proportion of L-glutamic acid as a constituent unit can be obtained.

According to the present invention, any of the above Bacillus natto, and a novel Bacillus natto requiring glutamic acid can be obtained.

Further, according to the present invention, natto having an increased umami taste can be obtained by using such a mutant natto.

Further, according to the present invention, poly-γ-glutamic acid containing a large amount of L-glutamic acid can be obtained.

Further, according to the present invention, poly-γ-glutamic acid containing a large amount of L-glutamic acid as a constitutional unit can be obtained, and a seasoning having a strong umami can be produced by using it.

[0096]

[Brief description of the drawings]

FIG. 1 is a graph showing the amount of glutamic acid produced by the mutant Bacillus natto of the present invention.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 1/20 C12R 1: 125) (C12P 13/00 C12R 1: 125)

Claims (7)

[Claims] 1. Bacillus subtiris (Bacillus subti)
nat), which is deficient in glutamase-tracemase. 2. Bacillus subtilis (Bacillus subti)
lis), wherein about 70% or more of the structural units produce poly-γ-glutamic acid, which is L-glutamic acid. 3. Bacillus subtiris (Bacillus subti)
lis), and exhibit glutamic acid auxotrophy. 4. A Bacillus subtilis FERM deficient in glutamase trasemase.
P-16147. 5. The method for obtaining a novel Bacillus natto according to any one of claims 1 to 4, wherein the wild-type strain belonging to Bacillus subtilis is subjected to mutation treatment; A step of selecting a bacterium that grows on the medium and does not grow on the minimal medium; and a step of further selecting, from the selected bacteria, a bacterium that grows on a medium obtained by adding glutamic acid to the minimal medium. 6. Poly-γ-glutamic acid produced by a novel Bacillus natto, wherein about 70% or more of the constituent units are L-glutamic acid. 7. A seasoning comprising the poly-γ-glutamic acid according to claim 6.