JP6177564B2 - New natto bacteria with excellent low-temperature sensitivity and natto with markedly suppressed secondary fermentation - Google Patents

Description

  The present invention relates to a novel Bacillus natto that exhibits excellent low-temperature sensitivity. The present invention also relates to natto in which secondary fermentation during storage and distribution after production is significantly suppressed.

Thread-drawn natto fermented with natto bacteria (so-called natto) is a fermented food from ancient Japan, rich in soy protein and high in nutritional value. In addition to nutritional value, natto is one of the Japanese everyday foods because of its high palatability.
Furthermore, in recent years, it has been reported that natto has various health-promoting effects due to probiotic action, antibacterial action, functional ingredients, and the like, and is a food that is increasingly expected to be in demand.

  In the conventional method of producing ordinary natto, generally, spore of Bacillus natto is sprayed on cooked soybeans, and this is placed in an appropriate container. After being grown and fermented, it is produced by aging at a temperature of 10 ° C or lower. In normal natto produced in this way, 40 to 90% are spores, but the remaining 10 to 60% are present as vegetative cells, so they are distributed below 10 ° C (under refrigerated conditions). Have to sell. The reason is that when normal natto is placed at 10 ° C. or higher, the remaining vegetative cells begin to reproduce and secondary fermentation begins. That is, due to metabolism in secondary fermentation, the natto odor and ammonia odor are strengthened, the flavor is weakened, and the stringing is weakened, so that the quality of natto rapidly deteriorates.

Therefore, in conventional natto, strict temperature management is required to suppress secondary fermentation. In addition, secondary fermentation is likely to occur due to seasonal factors such as distribution and sales processes, inadequate consumer temperature management, and high summer temperatures, resulting in quality degradation.
Moreover, in the conventional natto, when sufficient aging at low temperature is not performed after fermentation, there is a problem that secondary fermentation occurs even if the low temperature is maintained during storage and distribution. Since this aging requires several days, it is a rate-limiting factor in production.

Thus, the development of a technique for preventing secondary fermentation during storage and distribution of natto has become an important issue for natto manufacturers. In addition, although an attempt was made to develop a natto strain that has suppressed the viability and fermentability in a temperature range below normal temperature (low temperature range) (see, for example, Patent Document 1), No resistant natto bacteria have been reported yet.

Japanese Unexamined Patent Publication No. 1-191655

  The present invention solves the above-mentioned problems, and develops a technology for remarkably suppressing secondary fermentation of natto in a temperature range below normal temperature (low temperature range) and remarkably preventing quality deterioration of natto during storage distribution. This is the issue.

As a result of extensive research, the present inventor has found a novel natto strain having extremely excellent low-temperature sensitivity (property in which growth and fermentation in a temperature range below room temperature are suppressed). Further, the present inventor has found that natto produced by inoculating these fermented natto strains and fermenting them in a conventional manner has significantly reduced secondary fermentation during storage and distribution. In particular, in the manufactured natto, secondary fermentation at room temperature was remarkably suppressed. Moreover, the secondary fermentation inhibitory ability was exhibited also in the natto manufactured without performing an aging process.
The flavor and stringiness of the manufactured natto were very good.

This invention is made | formed based on the said knowledge.
The invention according to [Claim 1] relates to a Bacillus natto belonging to Bacillus subtilis, characterized by having the mycological properties shown in (1) to (3) below. (1): Properties related to fermentability described in (1-1) below. (1-1): Fermented properly when inoculated into boiled soybeans or steamed soybeans and maintained the product temperature of the beans at any temperature between 37-53 ° C to produce natto with good flavor and stringing Nature that can be done. (2): Properties relating to low temperature growth inhibitory properties described in (2-1) and (2-3) below. (2-1): Inoculated on agar medium (pH 7.0) at any concentration of 3-4% (w / v) containing 10% (w / v) phyton peptone, gas phase temperature 20 ° C In the test to measure the maximum width of the colony when cultured for 48 hours at: when the maximum width of the colony cultured by inoculating K-2 strain (NITE BP-1577) is 2 to 4 mm, the natto The maximum width of colonies that have been inoculated and cultivated is 1 mm or less. (2-3): In a test for producing natto using the natto bacteria and measuring the product temperature when the natto is allowed to stand at a gas phase temperature of 15 ° C. for 48 hours; K-2 strain (NITE BP -1577), the maximum product temperature of natto produced using the Bacillus natto is 17 ° C or less when the maximum product temperature of natto produced using the natto is 17.5 to 19.5 ° C. (3) The property that the base sequence constituting 16S rDNA is the base sequence shown in SEQ ID NO: 1, or the base sequence showing 99% or more identity with the base sequence shown in SEQ ID NO: 1.
The invention according to [Claim 2] further relates to Bacillus natto according to Claim 1, which has the property relating to the low-temperature fermentation inhibition property described in (2-2) below. (2-2): After producing natto using the natto bacteria and allowing the natto to stand for 96 hours at a gas phase temperature of 15 ° C., 30 g of the natto is placed in a 2.5 L container at 20 ° C. and normal pressure. In a test where the ammonia concentration volatilized in the container is measured after standing for a minute; when the volatile ammonia concentration from 30 g of natto manufactured using K-2 strain (NITE BP-1577) is 80-120 ppm In addition, the volatile ammonia concentration from 30 g of natto produced using the natto bacteria is 60 ppm or less.
The invention according to [Claim 3] is the natto according to claim 2, wherein the concentration of volatile ammonia from 30 g of natto produced using the Bacillus natto having the property described in (2-2) is 10 ppm or less. It relates to bacteria.
The invention according to [ Claim 4] is any one of claims 1 to 3, wherein the maximum product temperature of natto produced using the natto bacteria in the properties described in (2-3) is 15.5 ° C or lower . It relates to natto bacteria described in 1.
The invention according to [ Claim 5 ] further relates to the Bacillus natto according to any one of Claims 1 to 4 , which has the properties relating to growth described in (1-2) below. (1-2): In a test in which a culture solution obtained by inoculating in a LB liquid medium and shaking culture at a liquidus temperature of 37 ° C. for 24 hours is measured with a spectrophotometer at a wavelength of 660 nm; The measured value of the culture solution obtained by inoculation is within the range of ± 20% with the measured value of the culture solution obtained by inoculating K-2 strain (NITE BP-1577).
The invention according to [ Claim 6 ] relates to Bacillus subtilis T-058 strain (NITE BP-1576).
The invention according to [ Claim 7 ] is a natto bacterium derived from the T-058 strain according to claim 6, a natto bacterium produced by mutating the T-058 strain, or a gene in the T-058 strain The present invention relates to a Bacillus natto produced by introduction and transformation, wherein the Bacillus natto has the mycological properties described in any one of (1) to (3) above.
The invention according to [ Claim 8 ] is characterized in that in producing natto, fermented by inoculating the natto bacteria according to any one of claims 1 to 7 into steamed soybeans or boiled soybeans. It is related with the manufacturing method.
The invention according to [ Claim 9 ] relates to natto produced by the method according to claim 8 .
The invention according to [ Claim 10 ] relates to natto according to claim 9 , characterized by having the following properties (A) and (B). (A): After standing at a gas phase temperature of 15 ° C. for 96 hours, 30 g of the natto is left in a 2.5 L container at 20 ° C. and normal pressure for 30 minutes, and the concentration of ammonia volatilized in the container is measured. In the test: when the volatile ammonia concentration from 30 g of natto produced using K-2 strain (NITE BP-1577) is 80-120 ppm, the volatile ammonia concentration from 30 g of natto produced using the natto Is a property of 60 ppm or less. (B): In a test for measuring the product temperature when left at a gas phase temperature of 15 ° C. for 48 hours; the maximum product temperature of natto produced using K-2 strain (NITE BP-1577) is 17.5 to 19.5 The property that the maximum product temperature of the natto is 17 ℃ or less when it is

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to suppress remarkably the secondary fermentation of natto in the temperature range (low temperature range) below normal temperature. This makes it possible to remarkably prevent quality deterioration during storage and distribution of natto. According to the present invention, it is possible to maintain the quality of a product even when the refrigerated state during storage, distribution, and sale of natto is not necessarily strictly performed.

Furthermore, in the present invention, even when the ripening process after natto fermentation is omitted and natto is produced, secondary fermentation suppression at low temperatures can be exerted, so that the production time can be greatly shortened. .
In addition, natto produced using the natto bacteria of the present invention has extremely good flavor and stringiness.

Therefore, according to the present invention, it is possible to efficiently provide natto having high quality and excellent storage stability.

In the low temperature growth test in Example 2, colonies were inoculated on 3% (w / v) agar medium (pH 7.0) containing 10% (w / v) phyton peptone and allowed to stand at 20 ° C for 48 hours. It is the photographic image figure image | photographed by the top view. In the low temperature storage test of manufactured natto in Example 3, it is the result figure which measured the product temperature of natto with time. FIG. 4 is a result diagram of calculating the maximum temperature of fermentation heat generated in a low temperature storage test of manufactured natto in Example 3. In the low temperature storage test of manufactured natto in Example 4, it is a result figure which measured the volatile ammonia density | concentration which generate | occur | produced from the natto after storage.

Hereinafter, the present invention will be described in detail with specific embodiments.
The present invention relates to a novel Bacillus natto that exhibits excellent low-temperature sensitivity. The present invention also relates to natto in which secondary fermentation during storage distribution is significantly suppressed.

[Mycological properties of new natto bacteria]
The novel Bacillus natto of the present invention is Bacillus natto that exhibits excellent cold sensitivity. Here, Bacillus natto is a bacterium classified as a variant of Bacillus subtilis (B. subtilis var. Natto, B. subtilis (natto)). Depending on the classification system, it may be classified as B. natto, a closely related species of Bacillus subtilis.
In the Bacillus natto of the present invention, the base sequence constituting 16S rRNA, which is a 16S rRNA gene, has a base sequence having identity of 99% or more, preferably 99.5% or more, more preferably 99.8% or more with the base sequence shown in SEQ ID NO: 1. Bacillus natto having the sequence in the genome. Most preferred is Bacillus natto, which has in its genome a 16S rDNA base sequence that completely matches the base sequence described in SEQ ID NO: 1.

-Morphological features The vegetative cells of the new Bacillus natto are koji molds of about 2 to 3 μm in size and have motility. Moreover, it has gram dyeability. It has the ability to form spores, and the spore shape is elliptical. The size of the spore is about 1.12 to 1.28 μm.

-Culture properties The shape of the colonies of the novel Bacillus natto on the agar plate is circular. The colony has characteristics of having wrinkles on the surface, no gloss, a color tone of opaque to milky white, and no bulge in the central part.
When the novel Bacillus natto is cultured in a liquid medium, a fungal membrane is formed on the surface of the cultured medium. Moreover, the culture solution becomes turbid.

-Carbon source assimilation The new Bacillus natto has an ability to assimilate glucose and sucrose. On the other hand, it has no assimilability for lactose and arabinose.

Physiological properties The new Bacillus natto is an aerobic bacterium, exhibits biotin requirements, and can grow on a minimal medium. It also has protease activity. It can also grow using citrate.

-Temperature characteristics The new Bacillus natto has a suitable viability in the vicinity of 37-40 ° C, which is the normal temperature range of Bacillus natto, and grows at a growth rate equivalent to that of ordinary Bacillus natto.
The growth ability of the novel Bacillus natto can be determined using the measurement result of OD660 of the culture solution as an index. Specifically, a test was performed in which a culture solution obtained by inoculating a liquid medium (for example, LB liquid medium) and shaking culture at a liquidus temperature of 37 ° C. for 24 hours was measured with a spectrophotometer at a wavelength of 660 nm. The measured value is within ± 20% of the measured value of the culture solution obtained by inoculating the K-2 strain (NITE BP-1577) in the same manner, preferably within the range of ± 15%. , Within the range of ± 10%, the growth ability can be determined using as an index.

  The novel natto bacteria have suitable natto fermentability at any temperature of 37 to 53 ° C. (preferably 40 to 50 ° C.) which is a normal natto fermentation temperature range. That is, by inoculating boiled soybeans or steamed soybeans and maintaining the product temperature of the beans for a predetermined time so that the product temperature is in the temperature range, the flavor is good and the stringing is sufficient (the amount of neva is large). Thus, it becomes possible to produce natto having a whitish color tone (a large amount of fungal membrane), a soft texture (low hardness), and good palatability.

  When the temperature is higher than 53 ° C. and lower than 55 ° C., the Bacillus natto itself can be grown, but the fermentation activity is completely stopped. In the case of 55 to 100 ° C., vegetative cells cannot grow but die, but can survive in the state of heat-resistant spores.

  When the temperature is lower than 37 ° C, the growth ability and fermentation ability are generally reduced. In particular, as a property possessed by the novel Bacillus natto, it should be noted that its ability to grow and ferment at a low temperature range (ordinary or lower) is significantly lower than that of normal bacteria.

[Details of low temperature sensitivity]
The novel Bacillus natto of the present invention is Bacillus natto having excellent low temperature sensitivity. Here, low temperature sensitivity means that fermentability at any temperature in the natto fermentation temperature zone (37-53 ° C) is equivalent to normal natto bacteria, but growth ability at low temperature temperature range (below room temperature) and It refers to the property that fermentability is extremely low. Here, the low temperature zone refers to a temperature zone below room temperature, which is a low temperature as seen from the fermentation temperature zone of natto. Specifically, it refers to 25 ° C. or lower, preferably 24 ° C. or lower, more preferably 23 ° C. or lower, further preferably 22 ° C. or lower, particularly preferably 21 ° C. or lower, and still more preferably 20 ° C. or lower. Moreover, as a minimum temperature of the said temperature range, 3 degreeC or more can be mentioned, for example.

  The degree of inhibition of low-temperature growth and fermentation inhibition of the new natto bacteria is significantly higher than that of known low-temperature-sensitive bacteria (for example, K-2 strain).

  When comparing the novel natto bacillus of the present invention with normal natto bacillus, a particularly noteworthy feature is that the normal natto bacillus actively undergoes secondary fermentation in the low temperature range (temperature range below room temperature). It can be mentioned that the growth ability and the fermentation ability at ℃ or higher, preferably 8 ℃ or higher, more preferably 10 ℃ or higher are remarkably reduced.

・ Inhibition of growth in low temperature zone The degree of growth inhibition in the low temperature zone indicated by the low temperature sensitive bacteria indicates the maximum width of colonies when the natto bacteria are inoculated on a predetermined agar medium and cultured at low temperature for a predetermined time. Can be determined.
Specifically, inoculate agar medium (pH 7.0) at any concentration of 3-4% (w / v) containing 10% (w / v) phyton peptone at a gas phase temperature of 20 ° C. In the test to measure the maximum width of colonies when cultivated for 48 hours; the maximum width of colonies cultured by inoculating K-2 strain (NITE BP-1577) is 2-4 mm, preferably 2-3 mm The maximum width of the colonies inoculated and cultured with the Bacillus natto is 1 mm or less, preferably 0.7 mm or less, more preferably 0.5 mm or less, even more preferably 0.2 mm or less, particularly preferably 0.1 mm or less, most It is preferably 0 mm (a size in which a colony cannot be visually confirmed) or can be determined as an index.

In addition, phyton peptone (Phytone Peptone) here refers to the digested material which digested soybean ground material by papain.
Here, “% (w / v)” is a value representing the content mass (g) with respect to a volume of 100 mL in%.
Moreover, the maximum width of a colony refers to the value of the maximum width of a colony milky white part.
Here, the K-2 strain is a known cold-sensitive bacterium (see Patent Document 1). In order to secure a comparative experiment with the thermosensitive bacterium of the present invention, the applicant of the present application applied for deposit of the strain to the National Institute of Technology and Evaluation (2-5-8 Kazusa Kama feet, Kisarazu City, Chiba Prefecture). This strain has been accepted as an international deposit on March 19, 2013 under the name Bacillus subtilis K-2 under the deposit number NITE BP-1577.

・ Fermentation suppression in low temperature range The degree of fermentation suppression in the low temperature range indicated by the low temperature sensitive bacteria indicates the change in product temperature (generated heat of fermentation) when natto produced using the natto bacteria is maintained at a low temperature. Can be determined.
Specifically, in a test for producing natto using the Bacillus natto and measuring the product temperature when the natto is allowed to stand at a gas phase temperature of 15 ° C. for 48 hours; K-2 strain (NITE BP-1577 The maximum product temperature of natto produced using natto is 17.5-19.5 ° C, preferably 18-19 ° C; the maximum product temperature of natto produced using the natto is 17 ° C or less (maximum fermentation heat 2 ° C or less), preferably 16.5 ° C or less (maximum fermentation heat 1.5 ° C or less), more preferably 16 ° C or less (maximum fermentation heat 1 ° C or less), more preferably 15.7 ° C or less (maximum fermentation heat 0.7 ° C or less), especially Preferably, it is 15.5 ° C. or less (maximum fermentation heat 0.5 ° C. or less), more particularly 15.4 ° C. or less (maximum fermentation heat 0.4 ° C. or less).

In addition, the degree of fermentation inhibition in the low temperature range indicated by the low temperature sensitive bacteria can be determined using as an index the amount of volatile ammonia generated when natto produced using the natto bacteria is maintained at a low temperature.
Specifically, after producing natto using the natto bacteria and allowing the natto to stand at a gas phase temperature of 15 ° C. for 96 hours, 30 g of the natto is placed in a 2.5 L container at 20 ° C. and normal pressure for 30 minutes. In a test of standing and measuring the concentration of ammonia volatilized in the container, the concentration of volatile ammonia from 30 g of natto produced using K-2 strain (NITE BP-1577) was 80 to 120 ppm, preferably 90 When the volatile ammonia concentration from 30 g of natto produced using the natto bacteria is 60 ppm or less, preferably 50 ppm or less, more preferably 40 ppm or less, further preferably 30 ppm or less, particularly preferably 20 ppm or less; Further, it is particularly preferable to determine whether it is 10 ppm or less as an index.

[Selected strains]
Examples of the method for selecting the cold-sensitive Bacillus natto of the present invention include the method described in Example 1 described later, and a method according to the following procedure can be employed.
As the selection method, it is preferable that (1) the parent strain is first subjected to mutation treatment. Here, as the mutation treatment, for example, nitrosoguanidine (NTG) treatment, ethylmethanesulfonic acid (EMS) treatment, ultraviolet treatment, etc. can be employed. Moreover, any natto bacteria can be adopted as the parent strain. For example, common commercially available bacteria such as Miyagino, Takahashi, and Naruse can be used, but various strains such as mutant strains and genetically modified strains having specific properties can also be used. In addition, the 21541 strain illustrated in the Example mentioned later is a strain which has normal temperature sensitivity, and is a strain which the applicant developed in-house.

Next, (2) the strain treated with the mutation of (1) above has a growth ability equivalent to that of normal bacteria in a temperature range where normal natto bacteria are preferably grown, and a low temperature range (below normal temperature) It is necessary to select a strain whose growth ability in the temperature zone is remarkably suppressed. For example, using a phyton peptone agar medium, it grows well on the medium at a temperature of 37 to 40 ° C., and the growth is remarkably suppressed at 10 to 25 ° C. (preferably 10 to 20 ° C.). A method of selecting the selected strain can be employed. Further, if necessary, the selection process can be repeated to select a better strain.
(3) After the number of strains is narrowed down by the selection in (2) above, it is necessary to actually produce natto and select it according to the quality of natto. If necessary, the selection of (2) above can be performed again to narrow down the cold-sensitive bacteria having good properties.
(4) About natto produced in (3), it is possible to select a better strain by evaluating the secondary fermentation inhibitory property.

  Specific examples of the cold-sensitive Bacillus natto of the present invention include the T-072 strain, the T-058 strain, and the T-101 strain. These strains are natto strains selected through a five-step selection process from 21,000 or more strains by the method described in Example 1 described later. The selected strain is a novel natto strain having properties that have extremely excellent low-temperature sensitivity and excellent quality characteristics of natto.

  Among the selected strains, the T-058 strain, in particular, was the strain with the most comprehensive properties. 2-5-8) applied for deposit. This strain has been accepted international deposit as of March 19, 2013 under the name of Bacillus subtilis T-058 under the deposit number NITE BP-1576.

  Moreover, as the low temperature sensitive natto of the present invention, natto derived from the selected bacterium, natto bacterium produced by mutating the selected bacterium, or produced by gene transfer and transformation into the selected bacterium As long as it is Bacillus natto having the above properties in terms of growth, fermentability, low temperature sensitivity, and 16SrDNA, it corresponds to the low temperature sensitive bacterium of the present invention.

[Manufacture of natto]
In this invention, it becomes possible to manufacture natto by which secondary fermentation at the time of storage distribution was suppressed notably by manufacturing natto using the said cold sensitive natto bacteria.
Here, as a method for producing natto, natto can be produced according to a conventional method except that the above-mentioned low-temperature-sensitive natto bacteria are used as natto bacteria.

-Raw material soybean In the manufacturing method of natto of this invention, any raw material which can be used for manufacture of normal natto can be used. For example, whole soybean, half soybean, cracked soybean (raw natto raw material), defatted soybean, etc. can be used. Particularly preferred are medium and large grains used in the production of high quality natto. These soybeans can be used as they are, but it is common to use those that have been dried (dried products).

In the present invention, the raw soybean is used as a steamed soybean or boiled soybean by a conventional method. Steamed soybeans are preferable in terms of preventing the component from flowing away. In addition, it is desirable to immerse the raw material soybeans in water and swell them before performing the cooking or cooking operation.
Here, as a specific preparation procedure of the steamed soybean, a method is adopted in which the soybean is immersed in water for about 6 to 24 hours, drained, and steamed at 100 to 135 ° C. for 10 to 30 minutes. be able to. Moreover, the method of pressure-steaming on the high pressure conditions of 0.12-0.22Mpa is also employable.
Moreover, as a specific preparation procedure of boiled soybeans, a method of immersing soybeans in water for about 6 to 24 hours and then simmering in 90 to 100 ° C. hot water for 20 to 50 minutes can be employed.

-Inoculation As the state of Bacillus natto when used for inoculation with Bacillus natto, it is possible to use a vegetative growth state that can be immediately proliferated and fermented, but it is usually preferable to use a spore state It is. This is because spore-shaped Bacillus natto can be stably stored and handled easily. In addition, spore natto bacteria are advantageous in that they can be prevented from contaminating beans because they do not die even when inoculated with hot boiled beans. In addition, spore-shaped Bacillus natto can be germinated quickly after inoculation with soybeans by heat shock caused by heat.

Inoculation of Bacillus natto into soybeans and the like is preferably carried out after addition (or inoculation, spraying, etc.) so that soybeans and Bacillus natto are evenly mixed in order to perform fermentation uniformly. Preferably, it is suitable to prepare a natto bacteria solution (a state in which natto bacteria are suspended in a liquid) and add and use in a liquid state.
Here, as the natto fungus solution, (i) a commercially available natto fungus spore solution or a spore-forming culture solution of various natto bacteria can be used. In addition, (ii) a culture solution obtained by cultivating Bacillus natto in a liquid medium containing a synthetic medium mainly composed of glutamic acid or glucose, soybean broth, soy milk, yeast extract and the like can also be used. In addition, (iii) natto bacteria are collected from a solid culture of natto bacteria such as soybeans (including soybean flour and defatted soybeans) and inoculated with natto bacteria (natto itself) and suspended in the solution. It can be used in a cloudy manner. In addition, the pulverized product of the solid culture can be suspended in a solution as it is and used.

The number of Bacillus natto to be inoculated is not particularly limited by the bacterial concentration according to the conventional method, but is 10 ³ to 10 ⁶ , preferably 10 ³ to 10 ⁵ , most preferably 10 ³ to 1 g of steamed soybean. 10 ⁴ is desirable.

It is preferable that the soybean inoculated with the Bacillus natto is filled in an individual container for one to several meals and then subjected to fermentation described later in the individual container. In addition, as a traditional method, it is possible to fill a boiled rice cake.
It is also possible to perform fermentation in a container of several liters volume, etc., but considering that the temperature change is difficult to be transmitted to the beans in the center when the volume value relative to the surface area increases, a larger container is used. It is not desirable to use it.

Here, any container can be used as long as it can be filled with beans. In general, a synthetic resin container using PET, PE, PP, PSP or the like generally used in natto, or a cup-shaped paper container can be used.
Moreover, the thing of the shape which can be stirred (stirring) for eating directly using the said container as a shape of a container is suitable.
Moreover, the thing of the aspect which can be sealed by a lid | cover or sealing after fermentation is suitable.

-Fermentation Fermentation of natto can be carried out by maintaining the product temperature of the beans at a normal natto fermentation temperature range of substantially 37 to 53 ° C (preferably 40 to 50 ° C).
Moreover, in this invention, it becomes possible to maintain the product temperature of the beans during fermentation in the said temperature range by maintaining room temperature in the temperature range of 30-50 degreeC (preferably 35-45 degreeC). This is because the product temperature of the beans rises due to the heat of fermentation and is maintained in the above temperature range.
Although there is no restriction | limiting in particular as predetermined time (fermentation time) which maintains the product temperature of a bean in the said fermentation temperature range, 12 to 24 hours, Preferably 16 to 20 hours can be mentioned.
Fermentation in the temperature range promotes the natto-like flavor, the soft natto-like texture, the whitish color due to the formation of the fungus, and the stringiness (neva). Is done.

  In the fermentation, “maintaining substantially in the temperature range” does not mean that the temperature range is not completely removed. For example, a slight temperature range (for example, within 2 ° C., preferably 1 If it is within a certain time (for example, within 10 minutes, preferably within 5 minutes), it means that the fermentation condition is satisfied even when the product temperature is out of the temperature range.

-Aging In the present invention, it is possible to produce natto in which secondary fermentation is remarkably suppressed even when the aging process, which is essential in the ordinary natto production method, is not performed. Therefore, in the production of natto in the present invention, it can be considered that the production is completed when the product temperature of the natto, which has been maintained in the heat treatment temperature range, is 5 ° C. or less.
In addition, when performing the aging step, the temperature is 3 ° C. or higher and lower than 10 ° C., preferably 3 ° C. or higher and lower than 8 ° C., more preferably 3 ° C. or higher and lower than 6 ° C., 6 hours to 3 days, preferably 8 hours. It is preferable to perform aging for about 2 days.

[Properties of manufactured natto]
-Secondary fermentation inhibitory properties Secondary fermentation refers to the phenomenon of secondary fermentation during storage and distribution due to the activity of vegetative cells of natto bacteria that survive in the produced natto, and is a factor that greatly degrades the quality of natto. Specifically, it refers to a reaction that causes excessive generation of ammonia or natto odor causing substances, decomposition of the stringing component and flavor component, and the like.
The natto produced in the present invention becomes a natto having a property that secondary fermentation is remarkably suppressed by the temperature characteristics of the low temperature sensitive bacteria. That is, the natto is remarkably prevented from being deteriorated during storage and distribution. In particular, secondary fermentation at room temperature is remarkably suppressed.
Here, “at the time of storage and distribution” means that when the manufacturer stores and stores the manufactured product (natto), when the manufacturer or distributor transports or delivers the product (natto), the product is sold by the seller. It means the entire time when goods such as natto are stored and displayed, when goods purchased by consumers (natto) are stored at home, and when goods such as goods are distributed.

The degree of inhibition of secondary fermentation of the manufactured natto can be determined using the product temperature (fermentation heat) when the manufactured natto is maintained at a low temperature as an index.
Specifically, in a test for measuring the product temperature when the natto is left at a gas phase temperature of 15 ° C. for 48 hours; the maximum product temperature of natto produced using K-2 strain (NITE BP-1577) Is 17.5 to 19.5 ° C, preferably 18 to 19 ° C; the maximum product temperature of the natto is 17 ° C or less (maximum fermentation heat 2 ° C or less), preferably 16.5 ° C or less (maximum fermentation heat 1.5 ° C or less) More preferably, it is 16 ° C. or less (maximum fermentation heat 1 ° C. or less), more preferably 15.7 ° C. or less (maximum fermentation heat 0.7 ° C. or less), particularly preferably 15.5 ° C. or less (maximum fermentation heat 0.5 ° C. or less), and particularly preferably Whether it is 15.4 ° C. or lower (maximum fermentation heat 0.4 ° C. or lower) can be determined as an index.

The degree of suppression of secondary fermentation of the manufactured natto can be determined using the amount of volatile ammonia generated from natto when the manufactured natto is maintained at a low temperature as an index.
Specifically, after producing natto using the natto bacteria and allowing the natto to stand at a gas phase temperature of 15 ° C. for 96 hours, 30 g of the natto is placed in a 2.5 L container at 20 ° C. and normal pressure for 30 minutes. In a test of standing and measuring the concentration of ammonia volatilized in the container, the concentration of volatile ammonia from 30 g of natto produced using K-2 strain (NITE BP-1577) was 80 to 120 ppm, preferably 90 When the volatile ammonia concentration from 30 g of natto produced using the natto bacteria is 60 ppm or less, preferably 50 ppm or less, more preferably 40 ppm or less, further preferably 30 ppm or less, particularly preferably 20 ppm or less; Further, it is particularly preferable to determine whether it is 10 ppm or less as an index.

-Quality of natto The manufactured natto has a good natto flavor, a soft texture like natto, and has sufficient stringiness (neva).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the scope of the present invention is not limited by these.

[Preparation Example 1] “Medium composition”
A medium having the following composition was prepared. Unless otherwise specified, autoclave sterilization was performed at 121 ° C. for 15 minutes. The agar concentration of the agar medium (plate) was 1.5% (w / v).

<Yeast extract medium>
Yeast extract (Mist P2G, Asahi Food and Healthcare): 20g / L
NaCl: 5g / L
pH: 7.1

<LB medium>
Tryptone: 10g / L
Yeast Extract: 5g / L
NaCl: 5g / L
pH: 7.0

<Phyton peptone medium>
Phytone Peptone (BBL Phytone Peptone, 211906, BD): 100g / L
Ion exchange water: 900g / L
pH: 7.0
(The phyton peptone medium was autoclaved at 121 ° C. for 60 minutes at an agar concentration of 3% (w / v).)

[Example 1] “Selection of low-temperature-sensitive Bacillus natto”
Bacillus natto having remarkable low-temperature sensitivity and capable of producing high-quality natto was selected. The results showing the selection process in this example are shown in Table 1.

(1) NTG mutation treatment
Bacillus natto '21541' was inoculated into 5 mL of LB liquid medium (test tube) and pre-cultured at 37 ° C and 150 rpm. 1 mL of the obtained preculture was inoculated into 100 mL of LB liquid medium (Sakaguchi flask), and cultured at 37 ° C. and 150 rpm until stationary phase. 20 mL of the culture solution was planted in 80 mL of LB liquid medium (Sakaguchi flask), and cultured under the same conditions until the logarithmic growth phase.
The obtained culture broth was collected by centrifugation and washed with 50 mM sodium phosphate buffer (pH 7). N-methyl N'-nitro-N-nitrosoguanidine (nitrosoguanidine: NTG) was added to the washed cells at a concentration of about 10 ⁷ cfu / mL to a final concentration of 160 μg / mL, and shaken for 60 minutes. Mutation treatment was performed. The survival rate at this time was 1.1%.

(2) “Primary selection: screening for cold-sensitive bacteria”
The NTG mutation-treated bacteria were smear cultured (37 ° C. overnight) on LB agar medium, and the resulting colonies (21,571 strains) were replicated on two phytonpeptone agar medium. Three replicas were made per colony.
One of the two agar media replicated was cultured overnight at 37 ° C. (high temperature conditions). On the other hand, the other agar medium was cultured at 20 ° C. (low temperature) for 48 hours.
Then, 279 strains that showed the same growth as the parent strain '21541' in 37 ° C. culture and did not grow in 20 ° C. culture or significantly poor growth were selected. In this selection, only those that matched in three replica points were selected.
Here, the selected thermosensitive bacteria were streaked again on the phyton peptone agar medium and cultured overnight at 37 ° C. A single colony was selected from the resulting colonies and replicated on LB agar medium. The replicated LB agar medium was used as a master plate in subsequent experiments.

(3) “Secondary selection: confirmation of natto productivity”
It was confirmed whether natto can be produced by adding the spore solution of 279 strains. The dried soybeans were immersed in water for 16 hours, drained, and then steamed under pressure at 1.65 kg / cm ² for 30 minutes. A natto fungus solution diluted 1,000 times to 100 g of steamed soybeans (amount that would give 5 × 10 ³ Bacillus natto spores per 1 g of steamed soybeans) was added and lightly homogenized. Then, put 50g each into a PSP natto container, cover it, and leave it in the program incubator, and leave it at 43 ° C for 8 hours, 45 ° C for 3.5 hours, 46 ° C for 5.5 hours, and 15 ° C for 1 hour. did. After standing, it was confirmed whether natto was produced by fermentation.
As a result, 83 strains having low temperature sensitivity and capable of actually producing natto were selected.

(4) “Third selection: Confirming the quality of natto”
Natto was produced using the 83 strains of the natto bacteria selected above, and the quality of the produced natto was evaluated. Natto was produced in the same manner as described in (3) above.
As a result, no matter what strain was used, the natto mycelium-forming ability, stringiness (neva), and flavor were not different from those of the parent strain '21541'. On the other hand, about the hardness of natto, the difference was seen between strains.
Therefore, 41 strains were selected by selecting as an index that the strain can produce natto that is softer than or equal to natto when using the parent strain '21541 strain'.

(5) “Fourth Selection: Screening for Secondary Fermentation Suppression”
Natto was manufactured using 41 strains of the selected 41 strains of natto, and the secondary fermentation inhibiting ability of the manufactured natto was evaluated.
After natto produced in the same way as described in (4) above (50 g of natto in a PSP tray container) is left in a temperature-controlled room set at a gas phase temperature of 4 ° C and the product temperature reaches 5 ° C , 15 trays (3 rows x 5 tiers) were stacked closely and placed in a cardboard case. Then, it was left still for 48 hours in a thermostatic chamber where the gas phase temperature was set to 15 ° C., and the product temperature of the central natto was measured over time. In the test, when the temperature difference between the gas phase temperature and the product temperature is 2 ° C. or less, it can be determined that generation of fermentation heat is suppressed, and it can be determined that secondary fermentation is suppressed. .
As a result, 14 strains (T-030, T-032, T-057, T-058, T) whose maximum product temperature during storage for 48 hours was 17 ° C or less (the temperature difference from the set temperature was 2 ° C or less). -060, T-063, T-072, T-079, T-080, T-087, T-096, T-101, T-108, T-124).

(6) “Fifth Selection: Comprehensive Evaluation”
Comprehensive evaluation of the 14 selected strains above, and 3 strains (T-072, T-058, T-101) with excellent natto productivity and remarkable low-temperature sensitivity (secondary fermentation inhibition ability) Was selected. Among these, the properties of the T-058 strain were particularly excellent.

[Example 2] “Test to check low temperature growth inhibition”
Tests were conducted to evaluate the degree of low-temperature growth inhibitory properties of the above-selected three strains, which are excellent cold-sensitive natto bacteria.

(1) Low temperature growth test
From the colonies on the agar medium in which each natto bacterium described in Table 2 was cultured, the bacteria were picked at the tip of a sterile toothpick, and a 3% (w / v) agar medium containing 10% (w / v) phyton peptone ( Replicated (inoculated) at 3 to 4 sites (pH 7.0). The plate was allowed to stand in an incubator with a gas phase temperature of 20 ° C., observed for colony formation after culturing for 48 hours, and the maximum width of the colony milky white portion was measured. The results are shown in Table 2. For some of the Bacillus natto, Fig. 1 shows a photographic image of the colony taken from the top view.

(2) Results and discussion
As a result, when cultured for 48 hours at 20 ° C. (low temperature) on an agar medium containing 10% (w / v) phytonpeptone, all the strains T-072, T-058 and T-101 selected above were cultured. A colony was not formed (see FIGS. 1A and 1B). That is, the colony size was 0 mm.
On the other hand, a comparatively small colony was formed when the K-2 strain (NITE BP-1577), which is a known cold-sensitive natto bacterium, was tested for comparison. When the maximum width of the colony of the K-2 strain (the white part where the bacterial cells grew) was measured, it was 2 to 3 mm (see FIG. 1 (B)).
Further, as a control, normal temperature-sensitive Bacillus natto (21541, Miyagino, Naruse, OUV23481) was tested, and all of them formed large colonies. When the maximum width of these colonies (milky white part where the bacterial cells grew) was measured, it was 7 to 9 mm (see FIGS. 1A and 1B).

  From these results, the selected strains (T-072 strain, T-058 strain, T-101 strain) are Bacillus natto strains whose growth at 20 ° C (low temperature) was significantly suppressed. It was also shown that it is a Bacillus natto exhibiting extremely excellent low-temperature sensitivity. That is, it was shown that natto produced using these selected strains has an excellent ability to suppress secondary fermentation.

  In addition, although the said experiment is a result at the time of using 10% (w / v) phyton peptone culture medium with agar concentration 3%, 10% (w / v) phyton peptone with agar concentration 4% (w / v) Even when the medium was employed, the same results as in Table 2 were obtained.

[Example 3] “Evaluation of secondary fermentation inhibition of manufactured natto”
Secondary fermentation inhibitory property was evaluated by producing natto using the selected excellent low-temperature-sensitive natto bacteria and measuring the product temperature when the natto was stored at a low temperature.

(1) “Measurement of product temperature over time when manufacturing natto is stored at low temperature”
The natto solution of natto described in Table 3 was inoculated to produce natto in the same manner as described in Example 1 (3). The manufactured natto (45 g of natto in a PSP tray container) is left in a thermostatic chamber set to a gas phase temperature of 4 ° C. After the product temperature reaches 5 ° C, 15 trays (3 rows x 5 rows) are placed. Closely stacked and placed in a cardboard case. Then, it left still for 48 hours in the thermostatic chamber set to 15 degreeC (low temperature) gaseous phase temperature, and the product temperature of the center natto was measured with time. The results are shown in Table 3 and Figs.
In this test, when the temperature difference between the gas phase temperature and the product temperature is within 2 ° C., it can be determined that the generation of fermentation heat is suppressed, and it can be determined that secondary fermentation is suppressed. it can.

(2) Results and discussion
As a result, in natto produced using the selected strain, the maximum fermentation heat during storage for 48 hours was 1.2 ° C. or less, indicating that secondary fermentation was significantly suppressed. In particular, when T-058 and T-072 strains were used, the maximum fermentation heat was 0.4 ° C. or less, indicating that secondary fermentation was suppressed almost completely.
On the other hand, in natto produced using K-2 strain (NITE BP-1577), a known low temperature sensitive natto bacterium, the maximum heat of fermentation was 3.5 ° C, and it was confirmed that secondary fermentation occurred. . Moreover, in natto manufactured using normal temperature-sensitive natto bacteria (21541 strain, Miyagino), the maximum heat of fermentation was 3.8 to 8.1 ° C., and it was confirmed that secondary fermentation was actively occurring.

  From these results, it was shown that in natto manufactured using the selected strains (T-072, S-058, T-101), secondary fermentation was significantly suppressed. . In particular, it was shown that natto produced using T-058 and T-072 strains can almost completely suppress secondary fermentation.

[Example 4] “Evaluation of secondary fermentation inhibition of manufactured natto”
Secondary fermentation inhibitory property was evaluated by producing natto using the selected excellent low-temperature-sensitive natto bacteria and measuring the concentration of volatile ammonia generated when the natto was stored at low temperature.

(1) "Low temperature storage of manufactured natto and measurement of volatile ammonia concentration"
The natto solution of natto described in Table 4 was inoculated to produce natto in the same manner as described in Example 1 (3). The manufactured natto (45 g of natto in a PSP tray container) is left in a thermostatic chamber set to a gas phase temperature of 4 ° C. After the product temperature reaches 5 ° C, 15 trays (3 rows x 5 rows) are placed. Closely stacked and placed in a cardboard case. Then, it was left still for 96 hours in a constant temperature room where the gas phase temperature was set to 15 ° C. (low temperature).

  30 g of natto after low-temperature storage was placed in a petri dish and placed on a disc-shaped ground glass plate having a diameter of 18 cm and a thickness of 4 mm. A 2.5-liter bell jar (bell-shaped container with two vent pipes above) was placed on the glass on which natto was placed. At that time, petrolatum was applied to the contact surface between the glass plate and the bell jar to maintain airtightness. The mixture was allowed to stand at room temperature (20 ° C.) for 30 minutes to sufficiently volatilize ammonia contained in natto. Then, it attracted | sucked through the suction cube attached to the pipe | tube of the bell jar upper part, and measured the volatile ammonia density | concentration in a bell jar with the ammonia detection pipe | tube (made by Gastec). The measurement results are shown in Table 4.

(2) Results and discussion
As a result, it was shown that natto produced using the above selected strain significantly reduces the amount of ammonia generated even after storage at 15 ° C. for 96 hours. Specifically, the volatile ammonia concentration detected under the above measurement conditions was 60 ppm or less. In particular, when T-058 and T-072 strains were used, no volatile ammonia was detected, indicating that secondary fermentation was suppressed almost completely.
On the other hand, in natto produced using K-2 strain (NITE BP-1577), a known low temperature sensitive natto bacterium, 100ppm of volatile ammonia was detected under the above measurement conditions, and secondary fermentation had occurred Was confirmed. In addition, the volatile ammonia concentration from natto produced using normal temperature-sensitive natto bacteria (21541, Miyagino, OUV23481) is 125-130ppm, confirming that secondary fermentation is actively occurring. It was.

  From these results, it was shown that in natto manufactured using the selected strains (T-072, S-058, T-101), secondary fermentation was significantly suppressed. . In particular, it was shown that natto produced using T-058 and T-072 strains can almost completely suppress secondary fermentation.

[Example 5] “Evaluation of growth in fermentation temperature range”
Tests were conducted to evaluate the viability in the fermentation temperature range for the three selected strains, which are excellent cold-sensitive natto bacteria.

(1) "37 ℃ growth test"
From the colonies on the agar medium in which each natto bacteria listed in Table 5 was cultured, the bacteria were picked at the tip of a sterile toothpick, inoculated into 5 mL of LB liquid medium (test tube), and pre-cultured at 37 ° C. and 150 rpm.
The culture suspension was measured with a spectrophotometer at a wavelength of 660 nm, adjusted to an OD660 value of 0.19 with an LB liquid medium, and then cultured at 37 ° C. and 150 rpm for 24 hours.
The OD660 value of the obtained culture suspension was measured with a spectrophotometer. The results are shown in Table 5.

(2) Results and discussion
As a result, the OD660 value of the culture suspension of K-2 strain (NITE BP-1577), which is a known cold-sensitive natto bacterium, and normal temperature-sensitive natto bacteria (21541, Miyagino, OUV23481) Was 1.95-2.10. At this time, the OD660 value of the culture suspension of the selected strains (T-072 strain, T-058 strain, T-101 strain) is 2 to 2.04, which is almost the same as that of the K-2 strain and normal bacteria. The same value was shown.

  Therefore, the selected strains (T-072, T-058, T-101) grow at the same level as the K-2 strain and normal natto bacteria (Miyagino etc.) in the fermentation temperature range. It was shown to have sex.

[Example 6] “Mycological properties of selected strains”
The bacteriological properties of the selected T-072 strain, T-058 strain, and T-101 strain were determined as follows.

(1) "Form"
Microscopic observation of vegetative cells under a microscope revealed the following form.
<Vegetable cells>
Shape: Aspergillus Size: 2-3μm
Motility: Yes Spore formation ability: Yes Gram staining: Yes

When the microscopic observation of the spores was performed under a microscope, the following form was obtained.
<Spore>
Shape: Ellipse Size: 1.12 ~ 1.28μm

(2) “Cultivation properties”
When the morphology of the colonies after culturing at 37 ° C. on the LB agar plate medium was observed, it was as follows.
<Agar plate culture>
Shape: Annular Surface: Wrinkled Raised state: No raised Color: Opaque, milky white Gloss: None Peripheral: pleated

When the cell morphology after culturing at 37 ° C. in LB liquid medium was observed, it was as follows.
<Liquid culture>
Surface growth: Bacteria film formation Turbidity: Existence

(3) “Physiological properties”
When various physiological properties were examined, the following properties were found. In the following results, “+” indicates assimilability and “−” indicates no assimilability.
<Physiological properties>
Use of citrate: +
Oxygen requirement: +
Protease activity: +
Growth in minimal medium: +
Biotin requirement: +

When the assimilation properties of various carbon sources were examined, the following properties were found. In the following results, “+” indicates assimilability and “−” indicates no assimilability.
<Utilization of carbon sources>
Glucose: +
Lactose: −
Arabinose: −
Sucrose: +

(4) Temperature characteristics
The viability and fermentability at each temperature were examined.
<Temperature characteristics>
Suitable fermentation temperature range: 37-53 ° C
Growth and fermentation ability below 20 ° C: None (remarkable low temperature sensitivity)

(5) "Determination of 16S rDNA base sequence"
After shaking culture in LB liquid medium, DNA was extracted from the collected cells and PCR was performed using a universal primer set of the 16S rDNA region. Thereafter, the PCR product was directly sequenced to determine the base sequence of 16S rDNA.
As a result, the base sequence constituting 16S rDNA from the three determined strains was a completely identical sequence (SEQ ID NO: 1). In addition, a BLAST search against the GenBank / DDBJ / EMBL database showed 100% match with the 16S rDNA region of Bacillus subtilis.

These selected strains T-072, T-058 and T-101 were judged to be new natto strains belonging to Bacillus subtilis. In particular, the T-058 strain was recognized as a comprehensive strain excellent in both low-temperature sensitivity and quality characteristics of natto.
Therefore, the applicant of the present application filed an international deposit application for the T-058 stock with the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashizu, Kisarazu City, Chiba Prefecture) on March 19, 2013 (Accession Number) : NITE BP-1576).

According to the present invention, it is possible to remarkably prevent quality deterioration during storage and distribution of natto. Therefore, according to the present invention, it is possible to provide natto having high quality and excellent storage properties, and it is expected that the technology will be usefully usable for natto manufacturers.

NITE BP-1576
NITE BP-1577

Claims (10)

A natto bacterium belonging to Bacillus subtilis, characterized by having the mycological properties shown in (1) to (3) below.
(1): Properties related to fermentability described in (1-1) below.
(1-1): Fermented properly when inoculated into boiled soybeans or steamed soybeans and maintained the product temperature of the beans at any temperature between 37-53 ° C to produce natto with good flavor and stringing Nature that can be done.
(2): Properties relating to low temperature growth inhibitory properties described in (2-1) and (2-3) below.
(2-1): Inoculated on agar medium (pH 7.0) at any concentration of 3-4% (w / v) containing 10% (w / v) phyton peptone, gas phase temperature 20 ° C In the test to measure the maximum width of the colony when cultured for 48 hours at: when the maximum width of the colony cultured by inoculating K-2 strain (NITE BP-1577) is 2 to 4 mm, the natto The maximum width of colonies that have been inoculated and cultivated is 1 mm or less.
(2-3): In a test for producing natto using the natto bacteria and measuring the product temperature when the natto is allowed to stand at a gas phase temperature of 15 ° C. for 48 hours; K-2 strain (NITE BP -1577), the maximum product temperature of natto produced using the Bacillus natto is 17 ° C or less when the maximum product temperature of natto produced using the natto is 17.5 to 19.5 ° C.
(3) The property that the base sequence constituting 16S rDNA is the base sequence shown in SEQ ID NO: 1, or the base sequence showing 99% or more identity with the base sequence shown in SEQ ID NO: 1. 2. The Bacillus natto according to claim 1, further having a property relating to low-temperature fermentation inhibition properties described in (2-2) below.
(2-2): After producing natto using the natto bacteria and allowing the natto to stand for 96 hours at a gas phase temperature of 15 ° C., 30 g of the natto is placed in a 2.5 L container at 20 ° C. and normal pressure. In a test where the ammonia concentration volatilized in the container is measured after standing for a minute; when the volatile ammonia concentration from 30 g of natto manufactured using K-2 strain (NITE BP-1577) is 80-120 ppm In addition, the volatile ammonia concentration from 30 g of natto produced using the natto bacteria is 60 ppm or less.   3. The natto bacterium according to claim 2, wherein the concentration of volatile ammonia from 30 g of natto produced using the natto bacterium in the property described in (2-2) is 10 ppm or less. The natto bacterium according to any one of claims 1 to 3 , wherein the maximum product temperature of natto produced using the natto bacterium in the properties described in (2-3) is 15.5 ° C or lower. Furthermore, the Bacillus natto according to any one of claims 1 to 4, wherein the Bacillus natto has the properties relating to the viability described in (1-2) below.
(1-2): In a test in which a culture solution obtained by inoculating in a LB liquid medium and shaking culture at a liquidus temperature of 37 ° C. for 24 hours is measured with a spectrophotometer at a wavelength of 660 nm; The measured value of the culture solution obtained by inoculation is within the range of ± 20% with the measured value of the culture solution obtained by inoculating K-2 strain (NITE BP-1577).   Bacillus subtilis strain T-058 (NITE BP-1576). Bacillus natto derived from the T-058 strain according to claim 6, Bacillus natto produced by mutating the T-058 strain, or Bacillus natto produced by gene transfer and transformation into the T-058 strain. And Bacillus natto having the bacteriological properties shown in the above (1) to (3) according to any one of claims 1 to 5 . A method for producing natto, characterized in that in producing natto, fermented by inoculating steamed soybeans or boiled soybeans with natto bacteria according to any one of claims 1 to 7 . A natto produced by the method according to claim 8 . The natto according to claim 9, which has the following properties (A) and (B).
(A): After standing at a gas phase temperature of 15 ° C. for 96 hours, 30 g of the natto is left in a 2.5 L container at 20 ° C. and normal pressure for 30 minutes, and the concentration of ammonia volatilized in the container is measured. In the test: when the volatile ammonia concentration from 30 g of natto produced using K-2 strain (NITE BP-1577) is 80-120 ppm, the volatile ammonia concentration from 30 g of natto produced using the natto Is a property of 60 ppm or less.
(B): In a test for measuring the product temperature when left at a gas phase temperature of 15 ° C. for 48 hours; the maximum product temperature of natto produced using K-2 strain (NITE BP-1577) is 17.5 to 19.5 The property that the maximum product temperature of the natto is 17 ℃ or less when it is ℃.