JPH0265777A - Bacillus natto hos 80 - Google Patents

Abstract

NEW MATERIAL:Bacillus natto HOS 80 (FERM P-10195) belonging to Bacillus subtilis genus, having >=100 unit gamma-glutamyl transpeptidase (gamma-GTPase) activity and capable of providing aged proteins without stringiness. USE:Capable of providing fermented soybeans without stringiness having extremely low bitterness. PREPARATION:Already-known Bacillus natto is cultured, e.g., on a culture medium containing a mutantagenic agent such as acridine orange and the resultant mutants are subjected to screening to obtain strains having >=100 unit gamma-GTPase activity. Strains capable of producing fermented soybeans without stringiness are selected from the above-mentioned mutants.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to Bacillus natto, and in particular to Bacillus natto HO580, which exhibits no stringiness in foods ripened by the bacterium.

[Conventional technology and issues to be solved]

Aged natto is an excellent product that has the advantages of being not only highly digestible and nutritious due to the moderate decomposition of high-quality 1a physical protein, but also delicious, inexpensive, and long-lasting. It is a balanced food. Moreover, the value of natto has recently been recognized, as it has been discovered that natto contains a large amount of fibrinolytic enzyme, which is used to prevent and treat cerebral thrombosis.

However, since natto has a unique stringy property, when eating natto, the sticky substance may stick to the lips or tableware and cannot be easily removed, causing discomfort or disgust to people. In addition, after ripening, natto becomes difficult to handle and post-processing is not possible, and its stringy properties limit the spread of natto or limit its use as a side food, which limits its field of use.

Therefore, research was conducted on the stringiness of natto, and a strain of natto bacteria that does not have stringiness was developed. In other words, Professor Hara of Bonyo University extracted a plasmid from Bacillus natto and discovered that natto ripened by Bacillus natto without the plasmid did not become stringy.

Specifically, by extracting a plasmid from Bacillus natto, the γ-GTPase (glutamyl trans peptidase gluLamyl transp) of Bacillus natto is extracted.
eptidase) activity is approximately 0, Bacillus natto (hereinafter referred to as HO3-0 strain) has a γ-GTPase activity of approximately 0 from a Bacillus natto mutant strain obtained by mutating a known Bacillus natto. was selected and cultured, and the natto produced using the HOS-0 strain had almost no stringiness.

However, there was a problem in that the hydrophobic amino acid (tyrosine) was hardly precipitated in the natto produced by the HOS-0 strain, leaving a strong bitter taste. Therefore,
Its uses were limited, such as the need to add salt to remove the bitterness from the natto produced.

The present inventor has been conducting research on the stringiness of Bacillus natto for many years, and as a result, even though the γ-GTPase activity is more than 50% of that of the parent strain, aged natto has virtually no stringiness. Moreover, they discovered a natto bacterium with extremely low bitterness.

[Means for Solving the Problems] The gist of Bacillus natto HOS 80 according to the present invention is that Bacillus subtilis (Bucillus 5ubt.
ilis), has a 1-GTPase activity of 100 units or more, and the orbited protein has substantially no stringiness.

[Function] Here, "having substantially no stringiness" means that unprocessed aged protein, which has not been subjected to drying, freeze-drying, powder mixing, etc., has a characteristic characteristic of aged protein. It means that there is no stringiness. Also, ``aging'' means ■Protein is broken down and flavor comes out. An increase in peptides is seen. ■In the later stage, crystals of hydrophobic amino acid (tyrosine), which is an amino acid produced by decomposition, grow. ■Describes the condition of becoming soft.

Bacillus natto HO380 according to the present invention is produced from a Bacillus natto mutant strain obtained by mutating a known Bacillus natto strain.

Examples of Bacillus natto used to obtain mutant strains include Bacillus subtilis.
Biotin belonging to IIs Bacillus subtilis
Any known Bacillus natto having IJP affinity can be used as a parent strain. Specific examples include Miyagino natto bacteria, Takahashi bacteria, Asahi bacteria, Matsumura bacteria, and Naruse bacteria.

Methods of mutation include, for example, a method of contacting with a mutagen, a method of genetic manipulation, and an X-ray method.

Any known method can be employed, such as a method of irradiating with ultraviolet rays, light, etc.

According to a method of contacting a suitable mutagen as a mutation method, a known parent strain, Bacillus natto, is cultured in a nutrient medium containing a mutagen, and the resulting mutant strain produces 7-GTP.
Strains with ase activity of 100 units or more are screened. A strain capable of producing fermented foods, such as natto, using the obtained mutant strain having a γ-GTPase activity of 100 units or more, and producing natto that does not lose flavor and is substantially non-stringy. is selected.

Here, in order to precipitate tyrosine in the produced fermented food and obtain a fermented food with extremely low bitterness, it is necessary to use the mutant strain γ
- GTPase activity of 100 units or more, preferably 20 units
The parent stock is selected so that it has 0 units or more.

Furthermore, any known mutagen can be used, and examples thereof include drugs such as acridine orange, N-methyl-N'-nitro-N-nitrosoguanidine, and dimethyl sulfate.

Furthermore, the contact concentration of the mutagen varies depending on the mutagen used and is not particularly limited, but may be the same as that normally used when such operations are performed. For example, acridine orange usually has 1 to 200+mcg (microdulum)/
It is sufficient to set it to about -.

Any known nutrient medium can be used, such as meat extract, peptone, calf plasma, agar, gelatin,
Medium supplemented with salt, etc., gravy agar medium, gravy agar medium,
Meat juice gelatin medium, lidmus milk, MEM medium, etc. can be mentioned.

Cultivation may be carried out according to known methods such as static culture and shaking culture. The culture temperature and time may be the same as those for the normal culture of the parent strain Bacillus natto, and the culture temperature is usually about 30 to 45 ° C. The culture time may be about 1 to 5 days.

Specific examples of mutant strains obtained in this way include:
l1ucillus subtilis HOS 80
(It has been deposited with the Institute of Microbial Technology F+I, Agency of Industrial Science and Technology under the accession number FERM P-10195. Hereinafter referred to as rHO880 strain.)
) can be mentioned. The 80380 strain is a mutant strain obtained by mutating Miyagino natto bacteria using Miyagino natto bacteria as a parent strain.

The mycological properties of the HO380 strain are shown.

(a) Morphology Shape: Rod size; 2.3-3.5 x O, 7-0.9 μm Presence of spores; Size of spores; 0.8 x 1.6-1.8 μm Shape of spores presence or absence of ellipsoidal sporangia expansion; no spores; center Durham staining; positive (b) Growth status on ordinary agar medium (cultured at 25°C for 25 hours) Shape; annular surface; rough and wrinkled Some margins; wavy color phase; opaque, cream-colored (c) Condition of gelatin puncture culture growth; Presence or absence; + (f) Presence or absence of growth in lidmus milk; Assimilated.

decomposes casein without coagulation (g) Physiological properties catalase; deca-oxidase; + hydrolysis of starch; + hydrolysis of gelatin; deca-lysine decarboxylase; - arginine dihydrase; - ornithine decarboxylase; - formation of indole; - reduction of nitrate; + nisculin; + urease; utilization of citric acid; + phenylalanine deaminase; yolk reaction; - growth range 55°C 50°C; + 20°C/+ 7°C; 5°C from glucose Gas production: Production of acetoin; + Presence or absence of acid production from the following sugars; + Xylose; + Fructose; + Mannitol; + Maltose; + Sucrose; + Galactose; Lactose; ± β-galactosidase; + Biotin requirement ;+Growth in the presence of 5% sodium chloride;+Growth in the presence of 7% sodium chloride;+Lysozyme 0.001
Growth in the presence of χ; +Growth in the presence of 0.02 χ; -The mycological properties of the above-mentioned mutant strain are similar to that of the parent strain Miyagino Natto Bacillus, except that the cell and spore sizes are slightly different. However, strain 80380 can be clearly distinguished from the parent strain Miyagino Natto in that it can produce natto that is substantially free of stringiness.

Using the obtained Bacillus natto HO380, natto was produced according to a known method. First, after washing the soybeans, soak them in water to reduce the soybean size by about 1.5 to 3.0 times, preferably 2 times.
．． Swell the soybeans 0 to 2.5 times. Next, pressurize the soybeans to 1
10 under a pressure of 1.5 to 2.0 Kg/cm"
After boiling or directly heating for about 30 minutes, it is cooled to about 80°C or less. Thereafter, this is inoculated with i0380 and cultured for about 12 to 80 hours at a temperature of 30 to 50°C to produce natto. HO5
The ripening time at 80° C. was required to be about 1.2 times that of the parent strain.

Although the natto obtained in this way does not have the stringiness seen in the parent strain, it may have a slimy texture in some cases. Also, as natto matures,
As the protein was broken down, the umami flavor came out, the meat became softer, and an increase in peptides was observed. In the later stage, crystals of hydrophobic amino acid (tyrosine), which is an amino acid produced by decomposition, are formed. The precipitation of tyrosine is not fully explained theoretically, but Bacillus natto HO380 is 50% of the parent strain.
This is thought to be because it has the above T-GTPase activity (approximately 200 units or more). Therefore, natto with extremely low bitterness can be obtained, and the flavor is not impaired in any way.

Bacillus natto HOS 80 according to the present invention can be applied to soybeans such as natto, legumes such as glycerol, grains such as corn and wheat, seeds, potatoes, fruits, mushrooms, etc.
This method can be applied to foods containing protein such as algae.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

1 (HO380's) Miyagino natto bacteria cultured in a liquid nutrient medium (hereinafter referred to as "liquid medium") containing 1% meat extract, 1% peptone, and 0.5% salt were collected by centrifugation, and 0. ,5IIIg/IR1
1710M phosphoric acid buffer containing N-methyl-N'-nitro-N~nitrosoguanidine (NTG)
(pH 6,8) and left for 30 minutes while cooling on ice. After that, the bacteria were collected by centrifugation, washed three times with the same buffer to wash off NTG, and then mixed with 1% meat extract,
After plating on a plate medium containing 1% peptone, 0.5% salt, and 2% agar, and culturing at 40°C for 24 hours, 200 colonies that grew were picked up at random, and natto was collected for each strain. and select a strain that can produce natto with virtually no stringiness even after repeated subculturing.
Thereafter, 1-GTPase activity was measured and 7-GTPa
Bacteria with se activity of 100 units or more were selected. In this way, a stable mutant strain, H2SO4 strain, was obtained.

2 Measurement of y-GTPase activity) Measurement of 1-GTPase activity was performed using Biochimica etB.
iophysica AcLa, 73 (1963) 6
It was carried out as follows according to the method described in 79-681).

(Preparation of al sample The H2SO4 strain obtained in Example 1 was inoculated into a preculture medium consisting of the following components, and cultured at 37°C for 7 days and 14 days, respectively, with shaking at 165 r, p, and m. Shaking culture was performed. Each culture solution obtained was
The mixture was centrifuged at 00 Or, ρ, s for 5 minutes, and the supernatant was used as an enzyme solution.

Preculture medium (adjusted to pH 6,8) Peptone 1.2% Citric acid 0.2% Glycerol 2.0% NHdCI 0.7%KtHP
O,0,05% Mg5OaIHt8 0.05%FeCl1
7HzOO, 004% Biotin 0.1 μg7m (b) Enzyme activity measurement method Each component below was mixed with 50mM) Lys buffer 2 at pH 9.0.
7 dissolved in 0a1, substrate? Eight liquids were prepared.

Substrate solution t, -r-glutamyl-p-nitroanilide l hydrate (substrate); 29°C 1gMgCIz
; 41M glycylglycine; 16
6. For each of the enzyme solutions from the 7-day culture and the 14-day culture, mix 0.1 sffi of the enzyme solution and 3 ml of the obtained substrate solution, and incubate at 37°C for a predetermined period of time (0 min, 4 ml). minutes,
After the reaction (6 minutes, 8 minutes, io minutes), the absorbance at a wavelength of 410n+i was measured using an absorption photometer.

The results are shown in Figure 1.

One unit of γ-GTPase activity is equivalent to one unit of γ-GTPase activity under the above reaction conditions (3
7'C), the amount of enzyme released is 1 μmol of P-nitroanilide, and the γ-GTPase activity is the value obtained by multiplying the rate of change in absorbance over time by a constant value.

The r-GTPase activity of the HO380 strain was 2 after 7 days of culture.
91 units, and 271 units after 14 days of culture.

The results are shown in Table 1.

Under the same conditions as in Example 2, the y-GTPase activity of the parent strain Miyagino Natto Bacillus was examined.

That is, the parent strain was cultured with shaking for 7 days and 14 days under the same conditions as in Example 2, and then an enzyme solution was prepared in the same manner. The obtained enzyme solution was mixed with a substrate solution in the same manner as in Example 2, and the absorbance was measured using an absorptiometer.

The results are shown in Figure 1.

The γ-GTPase activity of the parent strain was 455 units after 7 days of culture, and 388 units after 14 days of culture. The results are shown in Table 1.

Comparison Takumi-1 Under the same conditions as in Example 2, r-GTP was
Ase activity was examined.

That is, the HO3-0 strain was cultured with shaking for 7 days and 14 days under the same conditions as in Example 2, and then an enzyme solution was prepared in the same manner. The obtained enzyme solution was mixed with a substrate solution in the same manner as in Example 2, and the absorbance was measured using an absorptiometer.

The results are shown in Figure 1.

The r-GTPase activity of the HO3-0 strain was 6 units after 7 days of culture, and 17 units after 14 days of culture. The results are shown in Table 1.

Table 1 j11-1 Among the HO380 obtained in Example 1, using a different strain from that used in Example 2, γG T P as
e activity was investigated.

(a) Sample preparation and Φ) Enzyme activity measurement method in Example 2
I did exactly the same thing. However, shaking culture was carried out only for 7 days, but not for 14 days. As a result, the relationship between absorbance and time is as shown in Figure 2, and γG T Pase
Activity was 307 units.

r-GTPa under the same conditions as Example 3 using the parent strain used in Main Example 3 to produce +(0580 strain).
se activity was examined.

As a result, the relationship between absorbance and time was as shown in FIG. 2, and the γ~G-D Pase activity was approximately 427@.

This rabbit--1 Using the parent strain used in Comparative Example 3) (O5-0 strain was produced. Using this HOS-0 strain, 1-GTPase activity was determined under exactly the same conditions as in Example 3. I looked into it.

As a result, the relationship between absorbance and time was as shown in FIG. 2, and the γ-GTPase activity was 0 units.

χ cliff spot-soil selected soybeans were washed and soaked in water until they swelled 2.0 to 2.5 times. This is heated to a pressure of 2.0 kg using pressurized steam.
/cm'' for 20 minutes, then cooled to below 80°C, and the soybeans were inoculated with the HO380 strain obtained in Example 1.The soybeans inoculated with the 80580 strain were incubated at 40°C for 24 The fermented soybeans were kept for a long time to produce aged natto.The resulting natto had a good flavor unique to natto and also had excellent storage stability.

Next, the obtained natto was examined for stringiness in the following manner.

(2) The two obtained natto were slowly pulled apart from each other, and the distance until the thread formed between the natto broke was measured.

As a result, the strings broke after only a few points of natto were pulled apart, and the strings had virtually no stringiness.

100 g of the boiled natto was dissolved in 200 aN of water, and ethyl alcohol was added thereto to a final concentration of 85%.

As a result, the solution became almost uniformly cloudy, and even when stirred with a glass rod, nothing adhered to the glass rod.

Natto was produced under the same conditions as in Example 3 using the parent strain Miyagino natto bacteria. The obtained natto was examined for stringiness in the same manner as in Example 3.

■As a result, even if the stuck natto was separated by more than 10 cm, the threads did not break.

■As a result, filaments precipitate out, and when stirred with a glass rod,
The filament adhered to the glass rod and was able to be wound up.

Program-cultured natto was produced using the Shin-Ai-1 80580 strain, the natto was stored in a refrigerator, and changes in the quality of the natto during storage were examined by visual observation and measurement of acid-soluble peptides.

(a) Preparation of sample Boiled beans were prepared by a conventional method, and the boiled beans were inoculated with the HO380 strain, then placed in a cup and subjected to programmed culture. The programmed culture was carried out at a temperature of 40°C, as shown in Figure 3.
After holding at 93% W degree for 9 hours, the temperature was gradually increased from 40°C to 52°C over 4 hours at 93% humidity, then held at 52°C and 93% humidity for 4 hours, and then, The temperature was lowered at once from 52°C to 35°C and held for 3 hours. The humidity at that time was 60%
Met.

The natto obtained through programmed culture was stored in a refrigerator, and meat U was subjected to targeted observation and acid-soluble peptide measurements over time.

(b) Method for measuring acid-soluble peptide ■ Preparation of sample solution 5 g of natto was accurately weighed out, 95 g of water was added (20-fold dilution), and homogenized thoroughly.

Add 10 g of 0.44M TCA to 10 g of this solution, mix well, extract at room temperature for 2 hours or more (40 times dilution), filter through filter paper, and then filter through a 0.45 μm filter to obtain a sample. It was made into a liquid.

(2) Measurement After adding predetermined amounts of distilled water, 0.44 M CaC01, and 2-fold diluted Folin reagent to the sample solution, the reaction was allowed to proceed at room temperature for 30 minutes, and then the absorbance was measured at 0.0.660 n-.

Measurements were performed on the first day, 4 days later, 7 days later, and 14 days later by the method described above. The measurement results are shown in Table 2 and FIG. 4. In Table 0, distilled water was used instead of the sample liquid for rank 1.

(C1 results ■Macroscopic observation Appearance-wise, the color is a little darker.

■Measurement of acid-soluble peptides Due to refrigerated storage, the amount of acid-soluble peptides increased slightly over time. From this result, it was found that the HO380 strain progresses in ripening even during refrigerated storage.

Samples were prepared under the same conditions as in Example 5 using the Ikeiban Dan HO3-0 strain, and macroscopic observations and acid-soluble peptides were measured.

■Macroscopic observation No change was observed in appearance even after 14 days.

■Measurement of acid-soluble peptides During refrigerated storage, acid-soluble peptides are measured over time.
was not increasing at all. As a result, F(O5-0
It was found that the stock rarely ripened during refrigerated storage.

Table 2 [Effects of the Invention] According to Bacillus natto HOS 80 according to the present invention, it has the excellent characteristics of the conventional Bacillus natto, which is the parent strain. Therefore, the fermented food produced by Bacillus natto HOS80 has the same flavor as conventional natto, except that it has virtually no stringiness, is highly digestible, has high nutritional value, and has a long shelf life. It has the advantage of being excellent and has extremely little bitterness, and it has become possible to provide inexpensive fermented foods. Fermented foods aged with I(O380) have virtually no stringiness, so they do not cause any discomfort to anyone, and can be used not only as a so-called side food but also for confectionery and other uses. Furthermore, the present invention has excellent effects such as being able to freely perform post-processing as a food material.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between absorbance and time for calculating the γ-GTPase activity of aged proteins. FIG. 2 shows T-GTPa in other embodiments and comparative examples of the present invention.
It is a graph showing the relationship between absorbance and time for calculating se activity. However, O is a 7-day culture of the H2SO4 strain. △ is a 7-day culture of the parent strain. M is a 14-day culture of the parent strain. Each day culture is shown. 3 [2J is a diagram for explaining programmed culture in another example of the present invention, and FIG. 4 is a graph showing the relationship between absorbance and number of days in refrigerated storage. However, O indicates HOS 80 shares and HOS -0 stock. Figure 1 Time (minutes)

Claims (1)

[Claims] (1) Bacillus subtilis
btilis) and has a γ-GTPase activity of 100%.
Bacterium natto HOS80, which is characterized in that the aged protein has substantially no stringiness.