JP5617102B2 - Natto strain, natto and method for producing the same - Google Patents

Description

  The present invention relates to a natto strain belonging to Bacillus subtilis and uses thereof. In particular, a natto strain capable of producing natto with a soft texture and excellent umami and aroma using a colored soybean such as black, brown, red, or green, referred to herein as “pigmented soybean” , Natto and its manufacturing method.

  Natto is a traditional Japanese fermented food that is nutritious and familiar on the table. It is produced by inoculating fermented natto bacteria on steamed soybeans. The raw soybean is generally yellow soybean, and there are only three types of natto bacteria used nationwide, excluding those developed by major manufacturers. Of these three types, the one with the highest utilization rate nationwide is Miyagino fungus sold by the Miyagino Natto Factory. However, there are pigmented soybeans such as black, brown, and red in addition to yellow, but the texture of the soybeans is stiff regardless of any of these three types of natto bacteria. Further, there are problems such as poor umami and a soybean odor remaining when fermentation is insufficient. Some yellow soybeans still have problems with existing Bacillus natto, such as those with hard skin and those lacking in taste, depending on the country of origin and the climate and variety of the year. As a method for producing black soybean natto, for example, a technique described in Patent Document 1 is disclosed. However, it is unavoidable to introduce equipment for freezing steamed soybeans, which incurs capital investment costs and increases the time and effort due to the addition of new processes in natto production. Moreover, in order to solve the problem that the skin is hard and the food texture is bad, for example, Patent Documents 2 and 3 partially decompose soybean epidermis components using bacteria having high ability to decompose cellulose and hemicellulose. A method is disclosed. However, these methods alone are not sufficient for pigmented soybeans. In order to sufficiently ferment pigmented soybeans and to produce natto excellent in taste and aroma, natto bacteria having higher protease activity than conventional natto bacteria are required.

JP 2009-201466 A JP 2009-39080 A Japanese Patent Laid-Open No. 9-09903

  Pigmented soybeans, especially black soybeans, have thick epidermis that contains components that are difficult to decompose, so conventional natto strains are poorly fermented, and the finished natto is the epidermis that appears to be due to insufficient fermentation. The crunchy texture remains and is hardened. In addition, it has a problem that it has poor taste and a soybean odor. Therefore, there is a need for a technique for producing natto that feels soft enough so that the epidermis does not remain in the mouth, feels good enough, has no soy odor, and has a good fermentation odor. The present invention solves the problems of the prior art and provides a novel natto strain applicable to pigmented soybean, which is difficult to ferment with the conventional natto strain, and is produced using this new natto strain. It aims at providing natto and its manufacturing method.

  In order to solve the above-mentioned problems, the first means of the present invention is characterized in that the Bacillus subtilis (Bacillus subtilis) Bacillus natto mutant IBARARAKI C-1 strain (FERMP-22078).

  The second means is capable of producing natto using soybeans selected from the group consisting of black soybeans, brown soybeans, red soybeans and green soybeans as a raw material. It is characterized by being.

  The third means is a mixture of the natto strain described in the first means and the natto strain IBARRAKI P-1 strain (FERM P-22079) belonging to Bacillus subtilis at a mass ratio of 8: 2. It is characterized by being a natto strain.

  The fourth means is a method for producing natto characterized by using a natto mutant described in the first or second means or a mixed natto strain described in the third means.

  The fifth means is natto obtained by the production method described in the fourth means.

  According to the present invention, a Bacillus subtilis IBARAKI C-1; IBARAKI C-1 bacterium belonging to a novel Bacillus subtilis is provided. IBARAKI C-1 bacteria can be used for the production of natto. Natto can be produced by inoculating and fermenting IBARAKI C-1 bacteria in boiled beans. Since IBARARA C-1 bacteria have both high cellulase activity and protease activity, the decomposition of soybean epidermis by cellulase promotes the ability of proteases to work on seeds, so that not only the epidermis but also the seeds are soft. Furthermore, it is possible to make natto with strong taste by producing abundant peptides and amino acids which are protein degradation products. In addition, when the fermentation proceeds sufficiently, the soybean odor felt when the fermentation is insufficient is suppressed, and natto with a good fermentation odor can be produced. Because of these effects, the epidermis often used for natto production can be used for the production of natto for yellow soybeans, but it is also soft against the pigmented soybeans that were difficult to ferment with conventional natto bacteria. It becomes possible to produce natto that feels finished, feels good, and feels a good fermented odor that is not a soy odor. Furthermore, by using a natto strain obtained by mixing the natto strain and Bacillus subtilis IBARAKI P-1 strain, it is possible to produce natto having higher stringiness than the case of IBARAKI C-1 bacterium alone. .

  The time-dependent change of the cellulase activity of IBARAKI C-1 bacteria and Miyagino is shown.   The time-dependent change of the protease activity of IBARAKI C-1 bacteria and Miyagino is shown.

  Below, isolation | separation, culture | cultivation, and a use of the Bacillus natto mutant (IBARAKI C-1 microbe) of this invention are demonstrated in detail.

  The IBARAKI C-1 bacterium, which is a novel Bacillus natto of the present invention, was obtained as follows. One rice straw after harvesting was put into a sterilized 50 ml centrifuge tube, and sterilized water was poured into it and suspended. In order to selectively obtain only heat-resistant spores, this was treated at 95 ° C. for about 5 minutes. 1 ml of this suspension was poured into 10 ml of TSB (tryptic soy broth) and cultured overnight at 37 ° C. with shaking. The culture was diluted 10,000 times and 100 μl was applied to a TSA (tryptic soy agar) plate medium. After culturing at 37 ° C. for 24 hours, the separated bacteria were applied to a TSA plate medium and purified.

  A natto was prototyped for all the isolated bacterial strains, and a bacterial strain having suitability for natto processing was selected from the viewpoints of fungus covering, bean hardness, stringing, and thread knotting. The selected bacterial strains were analyzed for 16S rRNA gene, as well as phylogenetic analysis tests such as AFLP and biotin requirement test, and the results confirmed that they were Bacillus subtilis belonging to Bacillus subtilis. The difference between Bacillus natto and Bacillus subtilis other than Bacillus natto is that Bacillus natto has biotin requirements and has the ability to produce mucilage, whereas Bacillus subtilis other than Bacillus natto has these characteristics. Not to show.

  Next, an operation for inducing mutation was performed on these bacterial strains. It has been reported that a strain in which Bacillus subtilis is cultured in a rifampicin-containing medium and has grown, that is, a strain that has acquired resistance to rifampicin, is mutated into RNA polymerase and several enzyme activities are increased. This method was applied to Bacillus natto, a kind of Bacillus subtilis. Specifically, it is as follows.

And 6g sodium glutamate monohydrate, and 12g sucrose, and 6g of phytone peptone, and a 1g of _KH 2 PO _4, and 0.68g of Na 2 HPO _4, and NaCl 0.2g, MgCl ₂ · 7H _After 0.2 g of ₂ O and 6 g of agar were dissolved in 400 ml of reverse osmosis water (GSP medium) and sterilized, a medium to which rifampicin was added at a rate of 2 μg / ml was prepared. Among the strains subjected to genetic analysis, one strain that was particularly suitable for natto processing was cultured overnight in LB medium with shaking. After culturing, it was diluted 2-fold, applied to 100 μl of the prepared GSP medium containing rifampicin, and cultured at 37 ° C. overnight. A strain capable of producing γ-polyglutamic acid (γ-PGA), which is a viscous substance corresponding to the stickiness of natto, was isolated and purified on an LB agar medium. Thereby, Bacillus subtilis IBARAKI C-1 (IBARAKI C-1 bacterium) of the present invention was obtained. This Bacillus natto mutant is Bacillus subtilis IBARAKI C-1 deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the deposit number of FERM P-22078. In addition, about the parental strain before the mutation of the natto bacillus mutant of the present invention, the IS4Bsu1 and IS256Bsu1 that are always possessed by commercially available natto starter strains such as Miyagino are not retained, and the AFLP test Confirmed different.

  Cellulase and protease activities were measured and compared for the IBARAKI C-1 bacterium of the present invention and Miyagino bacterium, a commercially available natto starter strain. First, IBARAKI C-1 and Miyagino were cultured overnight at 37 ° C. in LB medium. 1 ml of the culture solution was poured into a 300 ml baffled flask poured with 100 ml of LB medium. This culture solution was collected at certain intervals during the culture, centrifuged at 15,000 rpm for 10 minutes to obtain a supernatant, and the activity was measured using this culture supernatant as an enzyme solution.

  The method for measuring cellulase activity is as follows. A 1% Azo-CMC solution (PH6) was used as a substrate. The reaction vessel used was a 1.5 ml Eppendorf tube. 100 μl of the enzyme solution (culture supernatant) was added to 100 μl of the substrate solution, mixed quickly, and the sample tube was incubated at 50 ° C. for 30 minutes. After incubation, 1000 μl of 99.5% ethanol was added to stop the reaction. This was allowed to stand at room temperature for 10 minutes and then centrifuged at 14,000 rpm for 10 minutes to obtain a supernatant. The degree of Azo-CMC degradation was measured by measuring the absorbance at 590 nm, and the cellulase activity was evaluated. In addition, after mixing a substrate solution and an enzyme solution, what added ethanol immediately was set as the background.

  The results are shown in FIG. According to Table 1, it was confirmed that the IBARAKI C-1 bacterium had an enzyme activity rising, that is, the absorbance (590 nm) exceeded 0.05 earlier than the Miyagi bacterium by about 3 hours. In addition, it is confirmed from FIG. 1 that there is a time zone in which the IBARAKI C-1 bacterium has a maximum value of 15% or more higher in several hours around 9 hours of culture when the enzyme activity of the IBARAKI C-1 bacterium has reached its peak. It was done. From this, since more epidermal components can be decomposed until the completion of fermentation, the skin can be finished softly. Moreover, it can be expected that the decomposition of the epidermis component facilitates the action of enzymes such as protease described later and facilitates the progress of fermentation.

  The method for measuring protease activity is as follows. 0.5% azocasein (100 mM Tris-HCl (PH8)) was used as a substrate. The reaction vessel used was a 1.5 ml Eppendorf tube. 100 μl of the enzyme solution (culture supernatant) was added to 500 μl of the substrate solution, mixed quickly, and the sample tube was incubated at 37 ° C. for 30 minutes. After incubation, 400 μl of 15% TCA was added to stop the reaction. After centrifugation at 10,000 rpm for 10 minutes, 900 μl of the supernatant was dispensed into a new Eppendorf tube, and 45 μl of 10M NaOH was added thereto. The degree of degradation of azocasein was carried out by measuring the absorbance at 440 nm, and the protease activity was evaluated. In addition, after mixing a substrate solution and an enzyme solution, what added 15% TCA immediately was set as the background.

  The result is shown in FIG. IBARAKI C-1 bacteria not only shows a value 1.5 times or more immediately after the rise of protease activity, but is always 2 in the time zone up to about 18 hours after the start of fermentation important for fermentation. It was confirmed that the activity was doubled. From this result, as more protein is decomposed, the beans themselves are finished softer, and more peptides and amino acids are produced, resulting in a more delicious natto.

Using soybean natto (IBARAKI C-1) according to the present invention and Miyagino fungus, which is a commercially available natto starter strain, black soybean natto was produced as a prototype. First, after soaking black soybeans in 3 times the amount of water (20 ° C.) for 16 hours, the water was thoroughly drained and steamed for 30 minutes at 0.18 MPa. Next, the Bacillus natto spore suspension was sprayed and stirred well so that the number of Bacillus natto bacteria was 10 ³ per 1 g of steamed soybeans. A predetermined amount of this was filled in a polystyrene foam container, covered with a polystyrene film having a small hole, and capped. Next, after 18 hours of fermentation treatment at 39 ° C. and 90% humidity, heat was taken at 20 ° C. and 50%, and aging was carried out by refrigeration at 5 ° C. for 1 day or longer to obtain the desired natto.

  The natto thus obtained was subjected to hardness evaluation and sensory test. In addition, the said hardness evaluation and sensory test were done by the method shown below.

(1) Natto Hardness Evaluation Test The natto hardness evaluation test was carried out by the method described in “Natto Test Method” (edited by the Natto Test Study Group, published by Kotsusha, March 8, 1990). In other words, natto is placed on an upper clock balance, and the test adapter (2 cm in length, 2 cm in width, 2 mm in thickness, thins from the tip of 3 mm, and the contact surface with natto is 1 mm in thickness) in the uniaxial direction of soybean Cutting was performed, and the weight (g) at the time of cutting was defined as the cutting strength. In addition, this cutting strength test was done about 50 natto grains and the average value is shown in Table 2. From this, it was confirmed that the use of IBARAKI C-1 bacteria had a lower cutting strength, and that natto was softly finished due to the effects of cellulase and protease.

(2) Sensory test Two types of natto were compared and evaluated by 26 people of various ages and genders. The results are shown in Table 3. Ibaraki C-1 Bacteria is better than Miyagi Bacteria in all the items examined, and Ibaraki C-1 Bacteria of the present invention is used to make the epidermis soft without leaving it in the mouth and feel sufficient umami It has become possible to make natto that feels a good fermented odor, not a soy odor.

Bacillus subtilis Ibaraki P. one strain deposited at the National Institute of Advanced Industrial Science and Technology (AIST) under the accession number of FERRM P-22079, the parent strain before mutation of IBARAKI C-1 and IBARRAKI C-1 (Bacillus subtilis IBARAKI P-1; IBARAKI P-1 bacterium) was used to produce natto as shown below. That is, first, after soaking yellow soybeans in 3 times the amount of water (20 ° C.) for 16 hours, the water was thoroughly drained and steamed for 30 minutes under the condition of 0.18 MPa. Next, natto fungal spore suspension is mixed with 80% IBARAKI C-1 bacterium at 20% IBARAKI P-1 bacterium so that the number of Bacillus natto bacteria is 10 ³ per 1 g of steamed soybeans. Was sprayed and stirred well. A predetermined amount of this was filled in a polystyrene foam container, covered with a polystyrene film having a small hole, and capped. Next, after fermenting for 10 hours at 39 ° C. and 90% humidity, heat was taken at 20 ° C. and 50%, and aging was carried out by refrigeration at 5 ° C. for 1 day or longer to obtain the desired natto. As a result, fermentation usually takes about 18 to 20 hours, but by using mixed natto bacteria, it was possible to produce natto in a shorter time than usual. In addition, the mixed bacteria can produce natto with stringing stronger than IBARAKI C-1 bacteria alone.

  FERM P 22078

  FERM P 22079

Claims (5)

  A Bacillus subtilis natto bacillus Ibaraki C-1 strain (FERM P-22078) belonging to Bacillus subtilis. The Bacillus natto mutant IBARARA C-1 strain (FERM ) according to claim 1, wherein natto can be produced using soybeans selected from the group consisting of black soybeans, brown soybeans, red soybeans and green soybeans. P-22078) . The Bacillus natto mutant IBARARAKI C-1 strain (FERM P-22078) according to claim 1 and the Bacillus subtilis natto strain IBARARAKI P-1 strain (FERM P-22079) of 8: 2 Natto bacillus mixed by mass ratio . The Bacillus natto mutant IBARARA C-1 strain (FERM P-22078) according to claim 1 or 2, or the Bacillus natto obtained by mixing according to claim 3. A method for producing natto, characterized in that   Natto obtained by the production method according to claim 4.

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