JPH0560335B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention grows at almost the same speed as conventional commercially available Bacillus natto at around 40°C, which is the normal natto manufacturing temperature. Room temperature, e.g. 20
Concerning a method for producing natto that is stable at room temperature and enables distribution and sales at room temperature, using so-called cold-sensitive mutant strains that grow slowly (30°C) or do not grow well (20°C) at ~30°C. It is something.

[Conventional technology]

Conventional commercially available natto is generally made by spraying the spores of Bacillus natto onto steamed soybeans, placing the spores in a suitable container, and then fermenting them for 15 to 18 hours at a temperature of around 40°C, which is the optimum temperature for the growth of Bacillus natto. After allowing the natto bacteria to propagate sufficiently, it is produced by aging at a temperature of 10°C or less for 1 to 2 days. In natto produced in this way, approximately 40 to 90% of the natto bacteria are in the form of spores, while the rest usually exists as vegetative cells. Such natto must be distributed and sold at low temperatures, usually below 10°C. The reason for this is that when this natto is placed at a high temperature of 10℃ or higher, the remaining vegetative cells begin to reproduce, and their metabolism generates ammonia, and the quality of the natto deteriorates, such as the stringiness of the natto becoming weaker. This is because it deteriorates rapidly.

[Problem to be solved by the invention]

The rapid deterioration of the quality of natto at room temperature is mainly caused by the repopulation of the vegetative cells of natto bacteria present in natto, as described above.
The vegetative cells of Bacillus natto grow at almost the same rate as conventional Bacillus natto at around 40°C, which is the fermentation temperature for natto, but at room temperature, for example 20-30°C, which is the normal outside temperature.
We focused on the possibility that if a natto bacterium that grows slowly or does not grow could be developed, it could be used to produce natto that is stable and can be distributed at room temperature.

In general, mutant strains that grow slower than normal bacteria or do not grow at temperatures outside the optimal growth temperature are called temperature-sensitive mutants, and mutant strains that grow slower or do not grow at temperatures higher than the optimal temperature are called high-temperature-sensitive mutants. ,
It is known that mutants that grow retarded or do not grow at temperatures lower than the optimum temperature are called cold-sensitive mutants. High-temperature-sensitive mutant strains are thought to arise due to a decrease in the heat resistance of specific proteins necessary for growth, and examples of their creation have been reported in many bacteria including Escherichia coli and Bacillus subtilis. On the other hand, cold-sensitive mutant strains are probably caused by mutations in proteins constituting intracellular structures, which prevent the formation of these structures below a certain temperature, but there are very few examples of their production. To date, only a few cases of E. coli have been reported (J.Bact.
Vol. 153, No. 1, 1983 P66-75), there have been no reports of Bacillus subtilis, to which Bacillus natto is said to belong, as well as Bacillus natto.

Therefore, the main object of the present invention is to provide a novel method for producing natto that is stable at room temperature using a novel cold-sensitive Bacillus natto mutant strain.

[Means to solve the problem]

The present inventors have conducted extensive research in accordance with the above objectives, and have discovered that a mutant obtained by mutating conventional commercially available Bacillus natto is a bacterium that can achieve the initial objective. The present invention has now been completed.

The present invention uses Bacillus sp K-2, a mutant strain of Bacillus natto that is sensitive to low temperatures, when producing natto.
2) A method for producing natto that is stable at room temperature is provided.

The present invention will be explained in detail below.

Mutation generation means Conventional commercially available suspensions of Bacillus natto spores or vegetative cells are irradiated with ultraviolet rays, X-rays, etc., or mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine are used. Process. This suspension was appropriately diluted, plated on a soybean broth agar plate, cultured at 30°C for 2 days, the resulting colonies were marked, and further cultured at 40°C for 8 hours to create new colonies. The resulting colony is isolated as a mutant strain. Using the mutant strains obtained in this way, natto is produced using conventional methods, and those that produce normal natto are selected and stored at 25°C for an additional 5 days to examine the state of quality deterioration.

Identification of Mutants As shown in the Examples below, mutant strain K-2 was obtained which produced high-quality natto by conventional natto production according to the above method and was capable of producing natto that was stable at room temperature. Ta. When we investigated the properties of this strain, we found that
As shown in the figure, the growth rate of mutant strain K-2 is the same as that of commercially available Bacillus natto at 40°C, which is the optimum temperature for natto growth, but at 30°C the growth rate is considerably slower than that of commercially available Bacillus natto, and at 20°C Commercially available Bacillus natto grows, but this K-2 strain does not, and it was therefore found that the mutant strain K-2 is a so-called cold-sensitive mutant strain. Other mycological properties of commercially available natto bacteria [Rep. Natl. Food Res. Inst. No. 50, 18-21]
(1987) and Soybean Monthly, December issue, 21-29 (1985)
[Reference] That is, this mutant strain K-2
is an aerobic, Gram-staining positive spore-forming bacterium, which is characterized by its size (1 x 2-3μ), colony shape, suitable growth temperature (35-45℃), fermentability of various sugars,
Properties such as GC content of DNA are determined by Bergey's Manual 8.
It is the so-called Bacillus natto because it matches the properties of Bacillus subtilus, produces mucilage, and requires biotin.
natto). This mutant strain K
-2 (Bacillus sp K-2) was released on December 18, 1988.
It has been deposited with the Institute of Microbial Technology, Japan Agency of Industrial Science and Technology as Microbiology Research Institute No. 9768.

Differences from Bacillus natto mutant strain K-1 according to the earlier invention No. 9612
In the specification (name of the invention: method for producing stable natto that is difficult to cause secondary fermentation at room temperature), it was stated that the mutant strain K-1 that is difficult to cause secondary fermentation was developed. Strain K-2 and the mutant strain K-1 of the prior invention are different bacteria that differ in their properties as described below.

Mutant strain K-1 starts growing at room temperature (approximately 20 to 30 degrees Celsius), about 8 times faster than commercially available Bacillus natto.
Although there is a delay of ~20 hours, once growth begins, the growth rate itself is almost the same as that of commercially available Bacillus natto.

On the other hand, as shown by the dotted lines in Figure 1A and B, the mutant strain K-2 exhibits a tendency for the regeneration rate to be suppressed or almost flat compared to commercially available Bacillus natto at room temperature. Therefore, in terms of suppressing secondary fermentation, mutant strain K
-2 has a greater effect than mutant strain K-1.

Production and efficacy testing of natto Natto produced using commercially available Bacillus natto and mutant strain K-2 of the present invention according to the conventional method was stored at 25°C, and changes in the amount of ammonia N and stickiness of the natto were evaluated. Check the strength and weakness and apply the results to the second and third respectively.
Shown in the figure. Ammonia N in FIG. 2 is shown as a result of measurement by a distillation method. It is said that when the ammonia-N content of natto exceeds 200 mg%, the ammonia odor becomes noticeable, and when the content exceeds 300 mg%, it becomes almost inedible. It is clear that the natto produced by mutant strain K-2 is less than 200mg% even on the 9th day, whereas it smells and becomes inedible after 3 days. The stickiness of natto in Figure 3 is determined by adding 200 ml of water to 50 g of natto, extracting the mucilage, and calculating the viscosity of the resulting extract as B.
The results are shown using a type viscometer. Commercially available natto made with Bacillus natto rapidly loses its stickiness and becomes stringy when stirred after being stored for 2 days, significantly reducing its commercial value, whereas natto made with mutant strain K-2 has a yield of 10%. It can be seen that good stringiness is maintained until the end of the day.

〔Effect of the invention〕

The natto obtained by the method of the present invention is as follows:
It is stable even at high room temperatures such as ℃, so there is no need for conventional low-temperature distribution and sales, and its quality is lower than that of conventional products that require low-temperature storage.
The aroma and taste are both superior and comparable, and therefore the present invention has great industrial benefits.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples. In the present invention, all percentages are by weight.

Example A strain that produces high-quality natto was isolated from a commercially available natto starter (manufactured by Miyagino Natto Seisakusho) and used as a parent strain. This parent strain was cultured with shaking at 40°C for 2 days in a flask using 0.5% soybean broth medium (PH6.5). The spores were centrifuged, washed three times with sterile water, and then suspended in sterile water. Put this suspension in a shear dish and use a germicidal lamp (10W).
UV rays were irradiated so that the survival rate was 0.1 to 0.01%. Next, an appropriate amount of the suspension was plated on a 0.5% soybean broth agar plate and cultured at 30°C for 2 days. Colonies formed on the agar were marked and further cultured at 40°C for 8 hours. The newly generated colony was isolated as a mutant strain and transplanted into a test tube slant (0.5% soybean broth agar medium). Among the mutant strains obtained, the strain producing natto of the best quality was selected and then subjected to the following mutation treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG).

First, the bacteria were cultured for 15 to 18 hours in a flask containing 0.5% soybean broth medium (PH6.5), then the bacterial cells were collected by centrifugation, washed three times with sterilized 0.7% saline, and then was suspended in 0.7% sterile saline, an appropriate amount of 0.1% NTG solution was added thereto, and the mixture was shaken at 40°C for 30 minutes. The cells were then collected by centrifugation, washed three times with 0.7% sterile saline, and then suspended in 0.7% sterile saline.

Next, an appropriate amount of this suspension was plated on a 0.5% soybean broth agar medium and incubated at 30°C.
The cells were cultured for 2 days. Colonies generated on the agar were marked and cultured for an additional 8 hours at 40°C, and newly generated colonies were isolated and stored in test tube slants (0.5% soybean broth agar medium).

Take one platinum loop of spores from the examiner's slant,
It was suspended in 200 ml of 1% sterilized saline and sprayed onto steamed soybeans heated to about 80°C. Place the steamed soybeans in a small styrofoam container, cover the soybeans with a thin polyethylene film, cover with a lid, and heat at 40℃ for 16 hours.
Fermented for an hour. After refrigerating the natto for one day in a refrigerator at 5°C, the quality of the natto was examined and bacteria that produced high-quality natto were selected. After storing selected high-quality natto at 25°C for 7 days, changes in quality were examined. After 7 days, one natto that maintained good quality was found, and the natto bacteria that produced this natto was named mutant strain K-2.

[Brief explanation of drawings]

FIG. 1 is a graph showing the growth of commercially available Bacillus natto and the mutant strain K-2 of the present invention, and the solid line shows the growth of the bacteria when both strains were cultured with shaking at 40°C in a nutrient medium. , the dotted line is initially 40℃
This is a graph showing the growth of bacteria when the culture temperature was lowered to 30°C A and 20°C B from the points indicated by the arrows after 5 hours of shaking culture. Figure 2 is a graph showing changes in ammonia N generation when natto produced using commercially available Bacillus natto and mutant strain K-2 were stored at 25°C. Figure 3 shows natto produced using commercially available Bacillus natto and mutant strain K-2 at 25°C.
This is a graph showing changes in the stickiness of natto when stored in .

Claims (1)

[Scope of Claims] 1. A method for producing natto that is stable at room temperature, which comprises using a mutant strain of Bacillus natto K-2 (Bacillus sp K-2) that is sensitive to low temperatures. 2. Low-temperature sensitive Bacillus natto mutant strain K-2 (Bacillus sp K-2) that can produce natto that is stable at room temperature.