JPH06261744A - Bacillus natto strain and natto produced using the same - Google Patents

Abstract

PURPOSE:To provide specific Bacillus natto strains having high natto kinase productivity, excellent in flavor, properties and quality, suitable for e.g. producing natto (fermented soybean). CONSTITUTION:The Bacillus natto strains, a T0-9 strain which belongs to Bacillus subtilis natto (FERM P-13164) and analogous strains thereof. The strains of the present invention have the following mycological characteristics: (1) capable of producing natto kinase (an enzyme having high fibrin-dissolving ability) about six times higher than conventional analogous strains; (2) a colony shape therefrom on an agar flat plate medium is of circularly rising type, in contrast to of flat plate type in the case of conventional analogous strains; (3) Gram-positive sporulated fungi; and (4) for various enzymatic activities, at least the activity on either D-raffinose, N-acetyl-glucosamine, amidon, or D,L-xylose differs from that of conventional Bacillus natto strains.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a natto strain and a natto produced by using the natto strain. More specifically, the present invention relates to a novel natto strain having a high nattokinase-producing ability and a natto-kinase-rich natto produced using this natto strain.

[0002]

2. Description of the Related Art Natto, which is one of the traditional foods, has been known to have a high nutritional value. Recently, one of the enzymes (nattokinase) contained in natto has a thrombolytic action. It has been reported that it has (Agricultural Congress, 1987, p. 549, 1988), and its functionality has attracted particular attention.

This nattokinase is an enzyme produced by Bacillus Subtilis natto used in the production of natto. It exhibits high fibrinolytic ability and strong persistence, and is a leading cause of death in Japan and other developed countries. It is said to be effective in preventing organ diseases (thrombosis, stroke, myocardial infarction, etc.). In addition, a therapeutic effect on senile dementia and deafness, which is considered to be caused by blood clots in the capillaries of the brain, is expected. Moreover, this nattokinase has a mild thrombolytic action as compared with urokinase used as a therapeutic component for the above cardiovascular diseases, but has a long half-life in blood and the effect after ingestion lasts for a long time. It also has the excellent characteristics that This nattokinase contains about 1,600 IU (international unit) per gram of wet weight in the case of natto produced using a conventional commercially available natto strain (eg, Miyagino bacterium, Naruse bacterium, Takahashi bacterium, etc.). . Therefore, in terms of the above-mentioned clinical dose of urokinase, in the case of the conventional product, in order to obtain a sufficient thrombolytic effect, it is necessary to ingest about 100 g of natto per day for an adult. However, the amount of natto used for a normal diet is about 40 to 50 g, and it is practically impossible to continue to consume a large amount of 100 g every day.

For this reason, natto containing a large amount of nattokinase has been manufactured and sold by using a natto strain having a high nattokinase-producing ability (for example, "Nattokinase Natto (trademark)" manufactured by Yamada Foods Co., Ltd.). Made; "My Natto (trademark)", Asahi Kogyo Co., Ltd.),
In the case of these conventional high-enzyme-active strains, strict temperature control and low-temperature long-time fermentation are required, so that the production process is complicated and the production cost is inevitably increased.

The present invention has been made in view of the above circumstances, and produces nattokinase in a much larger amount than that of a normal natto bacterium, and produces natto without using the above-described special process. The purpose is to provide a new natto strain that can be used. Another object of the present invention is to provide a new natto containing a large amount of nattokinase.

[0006]

The inventors of the present invention have
Repeatedly researched to solve the above-mentioned problems, by collecting Natto strains from various rice straws widely obtained from nature and extensively screening their enzyme activity, biochemical properties and morphology, conventionally known We succeeded in isolating a new strain of natto that did not exist.

Further, the inventors of the present invention have found that there is a strong positive correlation between the content of nattokinase in natto and the taste, flavor, and characteristic properties (color tone, hardness, viscosity, etc.) of natto. Found and completed this invention. That is, this invention is a T belonging to Bacillus subtilis Natto.
The present invention provides an o-9 strain (Mikken Kenkyusho No. 13164), a natto strain that is a similar strain thereof, and natto characterized by being produced using this natto strain.

Hereinafter, the selection and isolation process of the natto strain of the present invention will be described. (1) Collection / isolation of Bacillus subtilis natto The inventors of the present invention collected Bacillus subtilis natto by the following method using rice straw obtained from Nishinasuno-cho, Tochigi Prefecture and Tsukuba-shi, Ibaraki Prefecture. First, using each of the rice straws described above, a so-called "warazuto" was prepared, heat-treated with hot water at 95 to 100 ° C, and 100 g of each soybean was steamed at 126 ° C for 30 minutes. It was filled and fermented in an incubator at 39 ° C. for 18 hours to produce natto.

Then, the natto thus produced was suspended in physiological saline and the suspension was applied to an agar plate medium having the following composition and cultured at 37 ° C. for 18 hours to form a natto colony. Composition of agar plate medium Meat extract 5 g Peptone 10 g Glucose 1 g NaCl 5 g Agar 15 g Distilled water 1000 ml pH 7.0 Various forms were selected from the colonies thus formed, and 200 strains were isolated and The broth liquid medium having the composition was inoculated into each and cultivated with shaking at 39 ° C for 16 hours.

Composition of broth liquid medium Meat extract 3 g Peptone 10 g NaCl 5 g Distilled water 1000 ml pH 7.0 Then, the number of viable bacteria in each culture solution was measured, and 1 × 10 ⁵ cells / g were added to the above-mentioned cooked soybeans. Inoculate with fungus, 39 ℃
Fermented in an incubator for 18 hours to produce natto.

Further, these natto were suspended in physiological saline, and the suspension was applied on the same agar plate medium as above to form monocolonies, and the strains were purified to make several strains of natto. The strain was isolated. (2) Measurement of nattokinase activity Among the Bacillus natto strains isolated in (1) above, 6 strains derived from rice straw produced in Nishinasuno-machi, Tochigi Prefecture (To-3 to 5, 7 to
9) and three strains (Ib-1, 4, 7) derived from rice straw produced in Tsukuba, Ibaraki Prefecture, were used to produce natto in the same manner as in Example 1.

Then, nattokinase was extracted from these natto and the activity thereof was measured by the following fibrin plate method (see page 82 of "Natto test method" edited by the Ministry of Agriculture, Forestry and Fisheries Research Institute). Fibrin plate method Immerse 5 g of natto in 100 ml of 0.01 M phosphate buffer (pH 7.5) to completely remove the fungal moss and viscous substances on the surface layer, while stirring regularly to prevent enzyme inactivation. Four
It was stored in a cold place at ℃ for 1.5 hours.

Then, the natto additive solution is filtered with a filter paper,
1 ml of the above-mentioned buffer solution was added to 1 ml of the filtrate to dilute it, and 10 μl of this diluted solution was poured into a fibrin plate (manufactured by Kitaken Co., Ltd.) and reacted in a humid box at 37 ° C. for 4 hours. The nattokinase activity (casein unit: Cu / g) was determined from the standard curve.

As a control, the nattokinase activity was also measured by the above-mentioned fibrin plate method for natto produced in the same manner using commercially available natto bacteria (M strain and I strain) and commercially available natto (O). The measurement results are as shown in Table 1. Of the 12 types of natto tested, To-
Natto produced using strains 7-9 and Ib-7 showed significantly higher enzyme activity.

[0015]

[Table 1]

(3) Sensory test Twelve kinds of natto whose enzyme activity was measured in the above (2) were evaluated for flavor (taste, fragrance, etc. unique to natto) and property characteristics (color tone, hardness, viscosity, etc.). As a result, it was confirmed that natto (Sample Nos. 4 to 6 and 9) which showed high nattokinase activity in the above (2) had extremely good flavor and properties (particularly strong viscous force). (4) Presence or absence of enzyme activity for various carbon sources Excellent results were shown in (2) and (3) above.
Of the 7 species of Natto strains, To-7 and 9 strains have API
The presence or absence of enzyme activity against various carbon sources shown in Table 2 was tested using 50 CHB. As a control, a commercially available B. natto (M strain) was also tested.

The results are shown in Table 2, and the isolates To-7 and 9 of the present invention were D-Raffin marked with * in the table.
ose, N-Acetyl glucosamine, Amidon, and D, L
-Xylose was confirmed to have an enzyme activity different from that of the conventional Natto strain.

[0018]

[Table 2]

Of the Bacillus natto strains of the present invention isolated through the various screening steps as described above, To-9 strain was deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology on September 17, 1992, and subjected to microfabrication. The accession number of Kenkyusho No. 13164 was obtained. The To-9 strain and the natto strain of the present invention isolated in (1) to (3) above have the following mycological properties.

An enzyme (nattokinase) having a high fibrinolytic ability is produced about 6 times as much as a conventional strain. A colony is formed on an agar plate medium having the above composition and grows well. The colony shape on the agar plate medium is a circular ridge type, unlike the flat disc type of conventional strains.

It is a Gram-positive spore-forming bacterium. At least D-Raffinose for various enzyme activities
, N-Acetyl glucosamine, Amidon, and D, L-X
The activity against any of ylose is different from that of the conventional Natto strain. Further, the natto of the present invention produced by using such a novel natto strain contains about 6 times as much nattokinase as the conventional product, and has excellent quality in the unique flavor and properties of natto. .

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following examples.

[0023]

【Example】

Example 1 60 kg of Chinese small soybeans were immersed in water at 18 ° C. for 16 hours, put in a steamer, and steamed under pressure at a steam pressure of 1.5 kg / cm ² for 40 minutes. Then, this steamed soybean was inoculated with 3 × 10 ⁶ viable bacteria of the Bacillus natto To-9 strain per 1 g of soybean,
It is filled in a commercially available natto container and the product temperature is up to 50 ° C in the fermentation chamber.
It was fermented and aged for 18 hours at the following temperature, and then further aged for 24 hours at a low temperature in a refrigerator at 5 ° C. to produce natto.

The nattokinase activity of the thus-produced natto was measured by the aforementioned fibrin plate method. As a control, the activity of nattokinase was also determined for natto (control 1) and commercial natto (control 2) produced by the same procedure as above using a commercially available M. natto strain.

The results are shown in Table 3, and the natto produced in Example 1 had a much higher nattokinase activity than the controls 1 and 2. From the results of the sensory test, the quality of each product was almost the same, but the natto of Example 1 was remarkably excellent in viscosity.

[0026]

[Table 3]

Example 2 30 kg of Chinese small soybeans soaked in water at 18 ° C. for 16 hours
And 25 kg of thawed frozen sweet corn were put in a steamer and mixed, and after steaming under the same conditions as in Example 1, the Bacillus natto To-7 strain of the present invention was inoculated in the same manner as in Example 1 and filled in a container. Then, it was fermented and matured and refrigerated overnight to produce natto with corn.

The nattokinase activity of this natto containing corn was measured in the same manner as in Example 1 to find that it was 1,8.
An activity value of 50 cu / g (5.3 times that of the conventional product) was obtained. Moreover, the result of the sensory test was also good, and particularly excellent evaluation was obtained regarding the viscosity. Example 3 90 kg of dehulled soybean (ground soybean) soaked in water at 18 ° C. for 2.5 hours was put into a steaming pot and heated and steamed at a steam pressure of 1.0 kg / cm ² for 10 minutes, and the To-8 strain was added thereto. Was inoculated under the same conditions as in Example 1 and filled in a container. Next, the product was fermented and matured for 17 hours at a temperature at which the product temperature reached a maximum of 48 ° C., and refrigerated overnight to produce ground natto.

The nattokinase activity of this ground natto was measured in the same manner as in Example 1.
An activity value of 0 cu / g (5 times that of the conventional product) was obtained. Moreover, the result of the sensory test was also good. Furthermore, what is noteworthy about the ground natto produced in Example 3 is its excellent stability. That is, in the case of the conventional fluffy natto, ammonia is generated by the autodigestive action of the natto bacterium over time, and the precipitation of tyrosine is observed particularly after 5 to 6 days after the production. In the fluffy natto produced using the same, no precipitation of tyrosine was observed even after 8 days from the production, and no offensive odor due to ammonia was generated.

[0030]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a novel natto strain having a high nattokinase-producing ability. This makes it possible to produce natto having a significantly high nattokinase content and excellent quality in the same fermentation process as in the case of using a conventional bacterium.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Rumiko Tomoyori 1-1-16 Nagatsuda, Midori-ku, Yokohama-shi, Kanagawa 1-102

Claims (2)

[Claims] 1. To belonging to Bacillus Subtilis Natto
-9 strains (Microtech Lab. No. 13164) and its similar strains, Natto strain. 2. Natto produced by using the natto strain according to claim 1.