JP2009213423A - Bacillus natto with suppressed ammonia production, and natto (fermented soybean) produced using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide Baillus natto with suppressed ammonia production, suppressing the ammonia production itself of natto, and producing natto highly suppressed in ammonia odor emission, needless to say, in undergoing normal fermentation, even in undergoing overfermentation, refermentation or under long-term storage, and to provide natto obtained using the Bacillus natto. <P>SOLUTION: A TTCC999 strain (FERM P-21498) or TTCC1000 strain (FERM P-21499) belonging to Bacillus subtilis is newly created. The objective natto is obtained by fermenting soybeans using the Bacillus natto with suppressed the ammonia production. Thus, ammonia odor emission from the above Bacillus natto can be suppressed to extremely low levels compared to natto produced from parent strain, i.e. conventional natto. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a natto bacterium in which the ammonia odor generated from natto is suppressed from the conventional one, and to natto in which ammonia production is suppressed using the bacterium. More specifically, the present invention relates to a natto bacterium that reduces ammonia produced by using arginine as a starting material by not assimilating arginine, and natto with reduced odor of ammonia using the bacterium.

  Natto, which has been eaten since ancient times in Japan, is widely eaten as a traditional nutritious health food that is rich in plant protein, dietary fiber, calcium, iron, vitamins and the like. On the other hand, it is also recognized as a fermented food with a strong smell.

  In addition, when the natto product is left at room temperature in the distribution process, fermentation starts again, and the product may deteriorate significantly. In the natto industry, this phenomenon is generally called re-fermentation or secondary fermentation. This re-fermentation also gives a strong ammonia odor and is one of the causes of discomfort.

  This ammonia odor is often the reason why consumers don't like natto. Therefore, development of natto with reduced ammonia odor is expected.

For this reason, several attempts have been made to reduce the ammonia odor. For example, Patent Document 1 describes a natto that has less ammonia odor and stuffy odor and has good stringiness. The natto of patent document 1 adds a fructose polymer to the soybean at the time of natto manufacture, or the natto after manufacture.
JP 2005-333856 A

  Furthermore, Patent Document 2 bred and developed a natto bacterium that can sufficiently ferment natto and produce natto with good quality, and can produce natto with low ammonia odor and low ammonia odor. A natto bacterium that produces natto of good quality with a reduced ammonia odor by producing natto using the natto bacterium, and natto using the natto are described.

The Bacillus natto of patent document 2 isolates and identifies the glutamine synthetase gene of Bacillus natto, obtains the Bacillus natto having enhanced activity of the glutamine synthetase by enhancing the expression of the gene, and preferably 0.01 unit / It is Bacillus natto in which the enzyme activity is increased to more than mg protein. Specifically, FERM BP-10446 is mentioned.
JP 2007-143467 A

  Furthermore, Patent Document 3 describes a quality-improved natto having a low ammonia content and a gamma-polyglutamic acid content that is the same as or higher than that of conventional natto, its production method, and a natto strain used for its production. ing.

The natto strain of Patent Document 3 can be obtained by screening from nature (paragraph 0011) or by mutation treatment and selecting natto bacteria having a reduced ammonia content (paragraphs 0011 and 0013 to 0018). Specifically, the natto strain having the accession number FERM P-14392 (Example 1) obtained from the Miyagino natto strain is mentioned.
JP-A-8-154616

  However, the natto of Patent Document 1 is the one that ammonia odor is selectively adsorbed on the fructose polymer by adding an additive (fructose polymer) to reduce the ammonia odor (paragraph 0034). It cannot be said that the generation of odor is suppressed. Therefore, the manufacturing process and cost of natto are increased.

  In addition, natto of Patent Document 2 activates glutamine synthase and lowers the ammonia content in natto by consuming the ammonia once produced (paragraph 0010). Focusing on this stage, it does not suppress the generation of ammonia odor. In addition, since it is a Bacillus natto strain into which a recombinant gene has been introduced, it cannot be used in the production of natto and sold.

  Furthermore, the amount of ammonia in Patent Document 3 is the ammonia concentration (paragraph 0007) in the supernatant obtained by suspending natto and culture in water, removing solids, and centrifuging (paragraph 0007), and released outside the cells. Yes, the amount of ammonia. In addition, the decrease in ammonia odor is the content at the end of the culture and is not the value when stored for a long time (paragraph 0010). Furthermore, when the ammonia content exceeds 65 mg% (w / w), ammonia is increased during storage to exceed 200 mg% (w / w), and an ammonia odor can be felt (paragraph 0012). Therefore, it can be said that the generation of ammonia cannot be suppressed during over-fermentation and re-fermentation.

  Therefore, the present invention suppresses the generation of ammonia in natto itself, and produces natto in which the generation of ammonia odor is extremely suppressed not only during normal fermentation but also during over-fermentation and re-fermentation. An object is to provide fungi and natto using the same.

  In order to solve the above problems, the inventors focused on ammonia produced using arginine as a starting material, and as a result of intensive research, produced natto using natto bacteria that cannot be arginine-assimilated. As a result, the present inventors have found that the ammonia odor generated from the water can be drastically reduced, and further research has been completed.

  That is, the present invention is a natto bacterium with suppressed ammonia production characterized by reduced or deficient arginine assimilation ability, characterized in that it cannot grow using arginine as the sole carbon source and nitrogen source. The natto bacterium in which the ammonia production is suppressed, and the arginine concentration in the growth medium is 0.7% or more, and the natto bacterium in which the ammonia production is suppressed as described above.

  Furthermore, the Bacillus subtilis is TTCC999 strain (Accession No. FERMP-21498) belonging to Bacillus subtilis, wherein the Bacillus subtilis is Bacillus natto with suppressed ammonia production. It was set as the Bacillus natto which suppressed ammonia production characterized by being TTCC1000 strain (accession number FERMP-21499) which belongs to Subtilis (Bacillus subtilis).

  More specifically, soybeans are fermented (overfermented) at 38 ° C. to 42 ° C. for 24 hours, and 50 g of natto is placed in a sealed container (2.7 L) and left in a sealed container for 30 minutes at 25 ° C. Any one of the above-mentioned natto bacteria with suppressed ammonia production, wherein the ammonia concentration is 50 ppm or less, and the soybean is fermented (normally fermented) at 38 ° C. to 42 ° C. for 18 hours, followed by 5 3 days at 10 ° C, 12 hours at 10 ° C, 24 hours at 25 ° C (re-fermented) 50 g of natto was placed in a closed container (2.7 L) and left at 25 ° C for 30 minutes. The ammonia concentration is 50 ppm or less, wherein the ammonia production is suppressed in any one of the above-mentioned natto bacteria.

  In addition, a natto composition characterized by fermenting beans using natto bacteria with suppressed ammonia production as described above.

  Since this invention is the above structure, the following effects are acquired. In other words, not only normal fermentation, but also over fermenting natto, re-fermenting, and long-term storage, the generation of ammonia odor of 40 ppm or less, lower than 50 ppm, which is said to be clearly felt by humans, and the conventional stringing and umami. It is possible to provide natto bacteria that produce natto that does not change, and natto produced using the same. Furthermore, since the generation of ammonia odor is extremely suppressed, restrictions on fermentation control, distribution temperature, etc. are relaxed, making it possible to easily manufacture and sell natto.

  In addition, the parent strain (natto bacteria separated from commercially available natto is UV-treated to obtain the natto bacteria of the present invention, so that natto can be manufactured and sold using the natto bacteria of the present invention, and safe ammonia Natto with reduced odor can be provided.

  Bacillus natto that suppresses ammonia production in natto and produces natto with extremely low ammonia odor during normal fermentation as well as over-fermentation, re-fermentation, or long-term storage And, for the purpose of providing natto using the same, a natto bacterium with suppressed ammonia production, characterized by being a TTCC999 strain (accession number FERMP-21498) belonging to Bacillus subtilis, or Bacillus subtilis -It realized by setting it as the structure of the Bacillus natto which suppressed ammonia production characterized by being the TTCC1000 strain (accession number FERMP-21499) which belongs to Subtilis (Bacillus subtilis).

  Specifically, the concentration of ammonia in the sealed container when soybean is fermented (overfermented) at 38 ° C. to 42 ° C. for 24 hours, 50 g of natto is placed in a sealed container (2.7 L) and left at 25 ° C. for 30 minutes. The natto bacteria with suppressed ammonia production as described in any one of the above, wherein the soybean is fermented at 38 ° C. to 42 ° C. for 18 hours (normal fermentation), followed by 5 ° C. 3 days at 10 ° C. for 12 hours and at 25 ° C. for 24 hours (re-fermented), 50 g of natto was placed in a sealed container (2.7 L), and the ammonia concentration in the sealed container when left at 25 ° C. for 30 minutes The Bacillus natto having suppressed ammonia production as described in any one of the above, wherein the content is 50 ppm or less.

  Furthermore, it realized by setting it as the structure of natto characterized by fermenting beans using the natto bacteria in which ammonia production in any one of the above was suppressed. As a result, the ammonia odor could be suppressed to be extremely low compared to the parent strain.

  Here, in Natto, it is considered that there is a route shown in FIG. 1 as a part of a mechanism for generating ammonia starting from arginine.

  Therefore, the inventors tried to obtain a natto strain with reduced or deficient arginine utilization ability in order to suppress the generation of ammonia odor from natto.

  Arginine assimilation ability is reduced or lost means that arginine cannot be grown using arginine as the only nitrogen source. It can be obtained by screening from nature, gene recombination techniques, and the like.

  Mutant means natto bacteria that exist in nature and natto bacteria obtained by mutating commercially available natto bacteria. Mutation treatment is not particularly limited, and conventional techniques are used. it can. For example, a method of contacting with a mutation agent such as nitrosoguanidine, such as ultraviolet ray and gamma ray irradiation, can be mentioned.

  Thus, in the present invention, any arginine non-assimilating natto strain or the like can be used regardless of whether it is obtained, bred or produced.

  Hereinafter, the natto bacteria in which ammonia production is suppressed and natto produced using the same will be described in detail. In addition, this invention is not limited at all by the following examples. First, the production of Bacillus natto with reduced or deficient arginine utilization is described.

[Mutation processing]
Bacillus natto isolated from commercially available natto (Takano Foods Co., Ltd., trade name: Mini 3) was used as the parent strain.

The parent strain is cultured in a broth medium at 37 ° C. for 4 hours, collected, made into a cell suspension of about 1.0 × 10 ^{6 to 7} cells / ml, placed under an ultraviolet lamp (distance 30 cm), and exposed to ultraviolet light. Irradiated for 3.5 minutes. The survival rate at that time was about 0.1%.

[Primary screening]
The above-mentioned cell suspension subjected to the mutation treatment was put into an arginine medium (a medium obtained by replacing agar with water from the selection medium shown in FIG. 2) and cultured (amplify) at 37 ° C. for 30 minutes. Thereafter, ampicillin was added to a final concentration of 100 μg / ml and cultured overnight at 37 ° C. (ampicillin concentration method).

  Ampicillin is an antibiotic that acts during the growth of bacteria, and only those that can assimilate arginine are first grown, and those that can assimilate arginine by ampicillin are preferentially killed. In addition, bacteria that cannot assimilate arginine (target bacteria) cannot grow during this process, and since they do not grow, ampicillin does not act, and it is easy to survive to the end. Went.

In addition, Bact shown in FIG.
Yeast Nitrogen Base W / O Amino Asids and
Ammonium Sulfate (Difco, product number 233520) is a medium used for classification based on the nitrogen source and carbon source requirements of yeast, and contains essential nutrients and vitamins necessary for yeast culture. However, amino acids, ammonium sulfate, and carbon sources are excluded.

[Secondary screening]
Subsequently, the cells cultured overnight were collected, washed with physiological saline, and ampicillin was removed. Then, the cultured cells were suspended in a small amount of physiological saline, applied to the broth medium shown in FIG. 3, and cultured at 37 ° C. for 16 hours to form colonies. At this time, a total of 25538 colonies were formed.

  Subsequently, the formed coro was inoculated into the arginine medium of FIG. 2 and the broth medium of FIG. 3, respectively, and a strain that grew only on the broth medium was selected. At this time, 84 strains did not grow on the arginine medium. The target strain of the present invention is included in the 84 strains.

[Third screening]
About 84 strains obtained in this manner, natto was prepared by ordinary fermentation (38-42 ° C., 18 hours) and over-fermentation (38 ° C.-42 ° C., 24 hours) according to the conventional method shown below. Thereafter, the ammonia odor was measured by the following method. Further, natto for normal fermentation was subsequently allowed to stand (re-fermentation) at 5 ° C. for 3 days, 10 ° C. for 12 hours, and 25 ° C. for 24 hours, and similarly the ammonia odor was measured. As a result, it was confirmed that 10 strains suppressed ammonia odor compared to natto prepared with the parent strain. Among them, two strains of natto strain in which ammonia odor was greatly suppressed were obtained.

  They are TTCC999 strain (accession number FERMP-21498) and TTCC1000 strain (accession number FERMP-21499) belonging to Bacillus subtilis, respectively. These strains have been applied for deposit at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary and have been entrusted as of February 1, 2008.

Hereinafter, a method for producing natto will be described. The raw material soybean soaked in water was steamed at high pressure, and each soybean was inoculated with fermented boiled beans. The soaked soybeans soaked at 15 ° C. to 20 ° C. for 18 to 14 hours were placed in a pressure kettle and cooked in about 20 minutes. The inoculation was carried out using natto fungal spores so that 10 ³ to 10 ⁴ cells / g of steamed soybeans were obtained. After filling this into a natto PSP container, the surface was covered with a perforated polyethylene film, covered with a lid, and fermented at a temperature of 38 ° C. to 42 ° C. for the predetermined time.

  The ammonia concentration in natto was measured by the following method. The product temperature was set to room temperature (25 ° C.), and 50 g of well-stirred natto was placed in a petri dish having a diameter of 9 cm, placed in the following container, and allowed to stand for a certain time.

  Specifically, natto is put in a container consisting of a plastic tapper (with a capacity of 2.7 liters, a length of about 21 cm, a width of about 16 cm, and a height of about 9 cm) with tubes (suction ports and suction ports) installed on the lid. The mixture was sealed and allowed to stand at room temperature (25 ° C.) for 30 minutes.

  Then, lightly shake the container to make the ammonia in the container uniform, and set the gas sampling device (manufactured by Gastec Co., Ltd.) on the suction port tube. 3L, 3La, 3M), the handle of the gas sampler was pulled, and the ammonia concentration was measured according to the instruction manual. The tube installed on the lid was opened only during measurement, and was closed with a pinch cock at other times.

[Ammonia measurement result of natto made by mutant strain]
The ammonia odor measurement result of the natto obtained by the above natto bacteria is shown in FIG. The ammonia odor of natto obtained from the TTCC999 and TTCC1000 strains of the present invention obtained this time is 10% after normal fermentation, about 7% after over-fermentation, and about 25% after re-fermentation (40 ). From the results of FIG. 4, it can be seen that the Bacillus natto obtained in the present invention is Bacillus natto with significantly suppressed ammonia production compared to conventional natto. In addition, the natto obtained by these 2 strains was the same as the natto obtained from the parent strain except for the ammonia odor.

  From the above, using the natto strain of the present invention, it is possible to produce natto in which production of ammonia odor starting from arginine is suppressed in natto.

In addition, the natto produced using the natto bacteria with suppressed ammonia production of the present invention, as the natto bacteria, the method and conditions for producing natto other than using the natto bacteria with suppressed ammonia production according to the present invention, It does not restrict | limit in particular, A suitable method and conditions can be used arbitrarily. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce and provide natto with reduced ammonia production by suppressing the decomposition of arginine in the fermentation process of natto.

  That is, the present invention immerses the washed raw soybeans in water, then steams them, inoculates the natto bacteria with suppressed ammonia production according to the present invention, fills them in a predetermined container, and at a predetermined temperature. A normal method for producing natto for fermentation may be used.

It is a figure which shows a part of ammonia generation path | route. It is a composition of an arginine plate medium. It is a composition of a gravy plate culture medium. It is an ammonia concentration measurement result.

Claims (8)

  A natto bacterium with suppressed ammonia production, characterized in that the ability to assimilate arginine is reduced or lost. Bacillus natto with suppressed ammonia production, characterized by being unable to grow using arginine as the only carbon and nitrogen source. The natto bacteria with suppressed ammonia production according to claim 2, wherein the growth medium has an arginine concentration of 0.7% or more.   The natto with reduced ammonia production according to any one of claims 1 to 3, wherein the natto is TTCC999 strain (Accession No. FERMP-21498) belonging to Bacillus subtilis. Fungus.   The natto with suppressed ammonia production according to any one of claims 1 to 3, wherein the natto is TTCC1000 strain (accession number FERMP-21499) belonging to Bacillus subtilis. Fungus.   Soybeans are fermented (overfermented) at 38 ° C. to 42 ° C. for 24 hours, and 50 g of natto is put in a sealed container (2.7 L) and left at 25 ° C. for 30 minutes, the ammonia concentration in the sealed container is 50 ppm or less. The Bacillus natto in which ammonia production is suppressed according to any one of claims 1 to 5.   Soybeans were fermented at 38 ° C. to 42 ° C. for 18 hours (ordinary fermentation), and then left at 50 ° C. for 3 days, 10 ° C. for 12 hours, and 25 ° C. for 24 hours (re-fermented). 2.7L), the ammonia concentration in the sealed container when left at 25 ° C. for 30 minutes is 50 ppm or less, and ammonia production according to any one of claims 1 to 6 is suppressed. Natto bacteria.   A natto obtained by fermenting beans using the Bacillus natto with suppressed ammonia production according to any one of claims 1 to 7.

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