JPH01191655A - Preparation of 'natto' stable at ordinary temperature - Google Patents

Abstract

PURPOSE:To obtain the title 'NATTO' (fermented soybeans), stable at relatively high ordinary temperature and excellent in stringiness, flavor and taste without any necessity of low-temperature distribution and sales, by using a variant strain of Bacillus NATTO sensitive to low temperatures. CONSTITUTION:The aimed 'NATTO' obtained by using a variant strain K-2 of Bacillus NATTO (Bacillus sp. K-2) sensitive to low temperatures in producing the 'NATTO'. Furthermore, FERM-P 9768 is preferably used as the above- mentioned variant strain.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is characterized by the fact that at the normal natto manufacturing temperature of around 40°C, conventional commercially available Bacillus natto
It grows at almost the same speed as, but at room temperature, e.g.
Utilizing so-called cold-sensitive mutant strains that grow slowly (30°C) or do not grow rapidly (20°C) at 30°C.
This invention relates to a method for producing natto that is stable at room temperature and enables distribution and sale at room temperature.

[Conventional technology]

Conventional commercially available natto is generally made by spraying natto bacteria spores onto steamed soybeans and placing the spores in a suitable container.
After fermentation, the natto bacteria is fermented for 15 to 18 hours at a temperature of around 40℃, which is the optimum temperature for the growth of natto bacteria, to sufficiently propagate the natto bacteria, and then fermented for 1 to 2 days at a temperature of 10℃ or less. are doing. Natto made in this way is
Approximately 40 to 90% of Bacillus natto is in the form of spores, but the rest usually exists as vegetative cells. Such natto must be distributed and sold at a low temperature, usually below 10°C.

The reason for this is that when this natto is placed at a high temperature of 10°C or higher, the remaining vegetative cells begin to reproduce, and their metabolism generates ammonia, and the quality of the natto deteriorates, such as by weakening the stringiness. This is because it deteriorates rapidly.

[Problem to be solved by the invention]

As mentioned above, the rapid quality deterioration of natto at room temperature is mainly caused by the repopulation of the vegetative cells of Bacillus natto present in natto. If a natto bacterium is developed that grows at almost the same rate as conventional natto bacteria at the fermentation temperature of around 40°C, but grows slowly or does not grow at room temperature, for example, the normal outside temperature of 20 to 30°C. We focused on the possibility of using this to produce stable natto that 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 a mutant strain that grows retarded or does not grow at lower temperatures than the optimum temperature is called a cold-sensitive mutant strain. 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 creation. It has only been reported in Escherichia coli to date (J
, Bact, Vo1153, No. 1.1983 P
e8-75), 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 intensive research in accordance with the above objectives, and have found that the mutant obtained by mutating conventional commercially available Bacillus natto is a vine bacterium that achieves the desired objective. heading,
The present invention has now been completed.

The present invention uses Bacillus natto mutant strain K-2 (Bacillus sp K-2), which is sensitive to low temperatures, for 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.

I. Means for creating mutations: Conventional methods of irradiating commercially available Bacillus natto spores or vegetative cell suspensions with ultraviolet rays, X-rays, etc., or mutagenesis using N-methyl-N'-nitroso-N-nitrosoguanidine, etc. Treat with a chemical. This suspension was appropriately diluted, plated on a Petri dish of soybean broth agar medium, 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 was produced by a conventional method, and those that produced normal natto were selected and further
Store at 5°C for 5 days and check for quality deterioration.

■ Identification of Mutants As shown in the Examples below, mutant strain -2 was obtained which was able to produce high-quality natto by conventional natto production according to the above method, and which was also capable of producing natto that was stable at room temperature. . When we investigated the properties of this strain, we found that the mutant strain had -
The growth rate of No. 2 is the same as that of commercially available natto bacteria at 40°C, which is the optimum growth temperature for natto, but at 30°C, the growth rate is considerably slower than that of commercially available natto bacteria, and at 20°C, commercially available natto bacteria grows, but this 12 strains did not grow, indicating that mutant strain -2 is a so-called cold-sensitive mutant strain. Other mycological properties of commercially available natto bacteria [Rep,
Natl, Pood Res, In5t, ) No, 50
．． 18-21 (1987) and Soybean Monthly Report, December issue, 21-29 (1985)]. In other words, this mutant strain -2 is an aerobic, Durham stain-positive spore-forming rod, and has various characteristics such as bacterial size (1 x 2 to 3 μ), Cockney shape, suitable growth temperature (35 to 45°C), and various types of bacteria. Bergey's Ma
Bacillus subtilis (nua1 8th edition)
The so-called Bacillus natto (B.
acillus natto). This mutant strain K-2 (Bacillus sp K-
2) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on December 18, 1986, as Microtechnology Research Institute Deposit No. 9768.

■ Natto production and efficacy test Natto prepared using commercially available Bacillus natto and the mutant strain of the present invention -2 were stored at 25°C in a conventional manner, and changes in the amount of ammonia N in the natto and the strength and weakness of the natto were observed. The results are shown in Figures 2 and 3, respectively. Ammonia N in FIG. 2 is shown as a result measured by a distillation method. It is said that natto usually starts to smell like ammonia when the ammonia N content exceeds 200mg%, and becomes almost inedible when it exceeds 300+ng%, but commercially available natto made with natto bacteria has a strong ammonia odor within 2 days. However, it can be seen that the natto produced by the mutant strain -2 is less than 200 mg% even on the 9th day, whereas the natto becomes inedible after 3 days. The stickiness of natto in Figure 3 is determined by adding 200ml of water to 50g of natto and extracting the mucilage.
The viscosity of the obtained extract was measured using a B-type viscometer, and the results are shown below. 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 the mutant strain -2 It can be seen that good stringiness is maintained until the end of the day.

〔Effect of the invention〕

In this way, the natto obtained by the method of the present invention is stable even at high room temperatures such as 25°C, and there is no need for conventional low-temperature distribution and sales, and its quality is superior to that required for conventional low-temperature storage. The stringiness, aroma, and taste are as good as those of the original.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 percentages 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. 0.5% of this parent stock
Using a soybean broth medium (pH 6.5), in a flask,
The cells were cultured with shaking at ℃ for 2 days.

The spores were centrifuged, washed three times with sterile water, and then suspended in sterile water. This suspension was placed in a petri dish and irradiated with ultraviolet rays under a germicidal lamp (LOW) so that the survival rate was 0.1 to 0.01%. Next, an appropriate amount of the suspension was plated on a Petri dish with 0% or 5% soybean broth agar medium, and cultured at 30°C for 2 days. Mark the colonies that have formed on the agar and further incubate at 40°C.
The cells were cultured 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, select the strain that produces the best quality natto, and then use the following N-methyl-N
Mutation treatment with '-nitro N-nitrosoguanidine (NTG) was performed.

First, the bacteria were cultured for 15 to 18 hours in a flask containing 0.5% soybean broth medium (pH 6,5), then the bacterial cells were collected by centrifugation, and washed three times with sterilized 087% saline. The bacterial cells were suspended in 0.7% sterile saline, and an appropriate amount of 0.7% was added to the suspension.
A 1% NTG solution was added 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 Petri dish of 0.5% soybean broth agar medium, and cultured at 30°C 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 separated and stored in test tube slants (0.5% soybean broth agar medium).

Take 1 platinum loop of spores from a test tube slant and add 200ml
The suspension was suspended in 1% sterilized saline and sprayed onto hot steamed soybeans at about 80°C. The steamed soybeans were placed in a small styrofoam container, the soybeans were covered with a thin polyethylene film, the container was covered with a lid, and the container was fermented at 40° C. for 16 hours. then 5
After refrigerating the natto for one day at ℃, 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 mutant strain of natto bacteria that produced this natto was named -2.

[Brief explanation of the drawing]

Figure 1 shows commercially available Bacillus natto and the mutant strain of the present invention -2 cultured in a two-neat triendo medium at 40°C for 5 hours with shaking, and then the culture temperature was changed to 30°C (A) and 20°C (B). ) is a graph showing the growth of bacteria when cultured at a lower concentration. FIG. 2 is a graph showing changes in the generation of ammonia N when natto prepared using commercially available Bacillus natto and mutant strains at -2 were stored at 25°C. FIG. 3 is a graph showing changes in natto stickiness when natto prepared using commercially available Bacillus natto and mutant strains at -2 were stored at 25°C. Applicant's agent Mr. Sato

Claims (1)

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