JP4428556B2 - Bacillus natto deficient in levanschlase activity and natto with high soybean oligosaccharide content - Google Patents

Description

  The present invention relates to a novel natto bacterium and a novel natto produced using the natto bacterium, and more specifically, a levanshuclase-deficient natto bacterium and a soybean oligosaccharide-rich natto produced using the natto bacterium. About.

  Soybean oligosaccharides (stachyose, raffinose) are known to proliferate intestinal bifidobacteria and have excellent health functions such as improvement of enteric fungi, suppression of intestinal spoilage products, and improvement of stool properties. (For example, refer nonpatent literature 1).

  Soybean oligosaccharide is contained in soybeans, and it is expected that the above health functions will be exerted by eating soybeans, while natto is a health food produced from soybeans, Since soybean oligosaccharide contained therein is consumed by Bacillus natto, it has been known that it contains almost no soybean oligosaccharide (see, for example, Non-Patent Document 2).

  In such a background, a method for producing natto with an increased oligosaccharide content has been developed by adding and fermenting oligosaccharides that are not consumed by Bacillus natto into the boiled natto as a raw material for natto (for example, , See Patent Document 1).

However, this method cannot sufficiently increase the content of soybean oligosaccharide (stachyose, raffinose) itself originally contained in soybean, and is sufficiently satisfactory in terms of enhancing the above-described health function of soybean oligosaccharide. It was not a thing. In this way, natto containing a high amount of soybean oligosaccharides that are originally present in soybeans but consumed by natto bacteria is produced directly by fermentation, not by strengthening soybean oligosaccharides in product natto. No successful example has been reported yet.
“Research on Health and Nutrition Foods, Volume 3, p. 89-91, 2000” "Journal of the Japan Food Industry Association, 29, pp. 105-110, 1982" JP 2003-235492 A

  An object of the present invention is to provide a method for producing natto with an increased content of soybean oligosaccharide (stachyose, raffinose) originally contained in soybean, and to achieve the original health function of soybean.

  In view of the above problems, the present inventors have studied from various directions, and in the course of research on various enzymes derived from Bacillus natto, they are also referred to as levanshuclase (levanshuclase or levansucrase. (Lase).

Thus, levanschlase is an enzyme produced by Bacillus natto, and is said to be one of the enzymes necessary for the assimilation of sucrose, the main carbon source in soybean, It is predicted that the growth of Bacillus natto will deteriorate if it is deficient.
Furthermore, since levanschlase is involved in the biosynthesis of levan, one of the stringing components of natto, the lack of the enzyme activity may cause deterioration in the quality of natto, especially stringing. Was also expected.

  Therefore, in the growth of Bacillus natto, levanschlase is an essential enzyme, and of course, natto bacillus lacking this is difficult to grow and cannot be used for the production of natto. . However, the present inventors changed the way of thinking, dared to challenge such common general knowledge, and further examined levanschlase. Then, focusing on the possibility that soybean oligosaccharides (raffinose, stachyose, etc.) contained in boiled beans are decomposed by natto-fungal levanschlase, we developed natto-fungi lacking levanschlase activity, We obtained a new idea that if natto is produced using, soybean oligosaccharides are not decomposed and new natto containing high soybean oligosaccharides can be produced.

  Therefore, based on this new idea, as a result of diligent research efforts, we succeeded in creating and obtaining natto bacteria deficient in levanschlase activity, and using it to produce natto, the soybean oligosaccharide content in natto, natto As a result of confirming the effects on fungal growth, natto quality, etc., it was confirmed that there was almost no adverse effect. Contrary to the above technical common sense, the creation of soybean oligosaccharide-containing natto was successful for the first time and the present invention was completed. It was possible.

  That is, the present invention relates to the production of new natto, which is an oligosaccharide originally contained in soybean and contains a high content of oligosaccharide consumed by Bacillus natto in the course of fermentation. For example, the following are included.

(1) A natto bacterium characterized by lacking or reducing levanschlase activity.
(2) Bacillus natto transformed with a disrupted sacB gene or a plasmid introduced with the disrupted sacB gene (for example, pSCEN1).
(3) Bacillus subtilis sacB22 (Bacillus subtilis sacB22) (FERM BP-08538).
(4) Bacillus subtilis B-1 (Bacillus subtilis B-1) (FERM BP-08584).

(5) A method for producing natto with a high soybean oligosaccharide content, comprising using the natto bacteria described in at least one of (1) to (4) above.
(6) The method according to (5), which is performed in the presence of soybean oligosaccharide and / or soybean oligosaccharide-containing material.
(7) The method according to (6) above, wherein the soybean oligosaccharide-containing material is soybean broth and / or a processed product thereof (at least one of concentrate, pasted product, dried product, and diluted product).

  (8) Natto with high soybean oligosaccharide content produced by the method according to any one of (5) to (7) above.

  According to the present invention, it is possible to provide natto containing soybean oligosaccharide originally contained in soybean at a high concentration. Further, when soybean oligosaccharide is added as necessary, soybean oligosaccharide is hardly consumed, and natto with high soybean oligosaccharide content can be provided, which is superior in health function containing soybean oligosaccharide at a higher concentration.

Hereinafter, the present invention will be described in detail.
The original natto bacillus used for breeding improvement in the present invention is not particularly limited, but natto bacillus having excellent fermentation ability usually used in natto industry, natto bacillus isolated and obtained from nature, and further improvements are repeated. It is desirable to use the excellent natto bacteria obtained from the above.

  Bacillus natto is classified into Bacillus subtilis, but it can produce the characteristics of natto such as sticky material (stringent material), and is a bacterium that forms the main body in natto fermentation, Moreover, since it has characteristics such as requiring biotin for growth, it is classified as Bacillus nutto, or Bacillus subtilis var. Natto or Bacillus subtilis (natto) may be distinguished from Bacillus subtilis. Examples of Bacillus natto include Bacillus Natto IFO3009, Bacillus subtilis IFO3335, IFO3336, IFO3936, and IFO13169, and various other Bacillus natto can be widely used.

Specific examples include the O-2 strain isolated from commercial natto and the r22 strain (see, for example, Japanese Patent Application Laid-Open No. 2000-224982) which is a mutant for improving the transformation efficiency of the strain. by Takahashi bacteria (T ₃ strains, TUA culture collection chamber) various Bacillus natto such or Miyagino bacteria (Miyagino natto Seisakusho) can be appropriately used.

  In the present invention, first of all, the Bacillus natto having a reduced consumption ability of soybean oligosaccharide is obtained by breeding the Bacillus natto that has lost the Levanschlase gene of these Bacillus natto and deactivated the enzyme activity. .

  A gene improvement method using homologous recombination can be adopted as one of the breeding methods, but the advantage of this method is that it is possible to specifically delete only the targeted gene. In microorganisms used industrially, other excellent properties can be improved by causing mutations only in the genes involved in the disadvantageous properties without breaking them.

  Furthermore, a gene inactivation method using homologous recombination ability developed by Stahl et al. In Bacillus subtilis (for example, “Journal of Bacteriology”, 158, p. 411). ˜418, 1984 ”) can also be used. This method is ultimately a method that can completely remove heterologous genes introduced into Bacillus natto for the purpose of gene disruption for breeding. It has the advantage of not becoming.

  In addition, in order to use such a gene recombination method with Bacillus natto, a transformation system for gene introduction into Bacillus natto is required, but a so-called competence that makes Bacillus natto high in gene transfer activity. Methods (see, for example, “Journal of Molecular Biology,” vol. 56, p. 209-221, 1971).

  Further, as one of the gene recombination systems of Bacillus natto, a transduction method using a phage vector has already been developed (for example, “Applied and Environmental Microbiology”). 63, p.4083-4089, 1997 "), this method can also be used in the present invention.

  In the present invention, the above-described breeding method is used to produce a target levanciclase activity-deficient natto bacillus by deleting a gene involved in the biosynthesis of natto bacillus levanshuclase. Detailed information on genes involved in levanschlase biosynthesis in fungi is not known.

  Thus, as a result of intensive studies from various directions, the present inventors have found that as possible genes, total genome information of Bacillus subtilis recently completed (for example, “Nature”, 390 volumes). , P. 249-256, 1997 "), was selected as one of the promising ones.

  It should be noted that other than gene recombination techniques as described above, that is, so-called screening methods for selecting natto bacteria already deficient in the target gene from the natural world, mutation mutation deletion of these genes by drug mutation methods, etc. Breeding is also possible by other methods such as mutation methods that have been practiced in the past.

  As the mutation treatment, a conventional method such as a mutation method using a physical treatment as well as a mutation method using the above-described drug treatment is appropriately used. Examples of the drug treatment include ethyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, N-methyl-N-nitroguanidine, ethidium bromide, nitrogen mustard, diepoxybutane, colchicine, peroxide, , Purine derivatives, nitrous acid, acridine dyes, and the like. Examples of the physical treatment include γ rays, ultraviolet rays, and temperature difference treatment.

  As a method for isolating Lebanschlase-deficient natto, the Bacillus subtilis Levanschlase-deficient strain differs from the parental strain when grown on a plate containing sucrose in a carbon source. For this reason (for example, refer to Japanese Patent Publication No. 60-550598), it can also be separated by a method of selecting different strains using the colony form as an index.

  Use of the thus-developed Bacillus natto-deficient natto for the production of natto is not particularly limited as long as a conventional method is employed.

For example, natto is generally so-called whole soybean natto manufactured using whole soybeans as a raw material, but there is also some ground natto using raw soybeans as a raw material. The manufacturing method of the whole soybean natto is that the whole soybean, which is a raw material, is immersed in cold water for more than 10 hours, and then steamed in a steaming pot using pressurized steam (1.5-2 Kg / cm ² , 128-133 ° C. ), And inoculated and mixed with natto bacteria at a high temperature (70 to 100 ° C.), filled in a predetermined container, and then brought into a fermentation chamber to be heated to a relatively high temperature (40 Generally, it is fermented for about a predetermined time (about 12 to 48 hours) at a temperature of about ~ 55 ° C, and then refrigerated at about 5 ° C (about 12 to 72 hours) for completion. Further, in the case of ground natto, it is produced in the same manner as in the case of ordinary round soybean natto, except that the previously ground soybean is soaked in water.

  In such a conventional method for producing natto, in the present invention, the natto bacteria used in the fermentation process are used in place of the levanshclase activity-deficient natto bacteria bred and improved by the above-described method.

  In this way, when comparing natto produced using natto bacteria deficient in levanshuclase activity and natto using conventional natto bacteria used in the past, it was produced with natto bacteria deficient in levanschclase activity. Natto contains soy oligosaccharides at a high concentration, and it can be confirmed that it is an excellent natto that demonstrates its original health function.

  Furthermore, in the present invention, as a method for producing natto containing soybean oligosaccharide at a higher concentration, after adding soybean oligosaccharide and / or soybean oligosaccharide-containing material to soybean, the above-mentioned Lebanshuku Fermentation can be carried out using natto bacteria deficient in the enzyme activity.

  Examples of soybean oligosaccharides to be added include soybean oligosaccharides contained in soybeans such as raffinose and stachyose, and soybean oligosaccharide products extracted and purified from soybeans, such as SOYA OLIGO (manufactured by Calpis). .

  The addition time of soybean oligosaccharide is preferably before the step of fermenting with natto bacteria, and more preferably a step of immersing soybean in water, a step of steaming soybean, or a step after cooking of soybean.

  Furthermore, as the soybean oligosaccharide-containing material, all materials containing soybean oligosaccharide are included, and for example, a soybean extract containing oligosaccharide having a low degree of purification can be used. Examples of such a low degree of purification include soybean soup obtained in the soybean cooking step and / or a processed product thereof (at least one selected from concentrate, pasted product, dried product, dried powder, and diluted product). . Natto is always produced through the steaming process of soybeans, but in the steaming process, soybean soup is generally generated, and soy soup is finally discharged outside the steamer and discarded. It is always done.

  However, in this soy broth, various active ingredients such as water-soluble saccharides in soybean, especially soybean oligosaccharides are eluted and contained. In the present invention, as a method of effectively using such soybean soup, it can be added to steamed soybean and used.

  As a method for adding soybean soup to soybeans, for example, the method disclosed in JP-A-11-225700, the method disclosed in JP-A-57-102155, JP-A-52-105245, etc. can be employed. There are no particular limitations. Although there is no special limitation also about the addition amount of soybean soup, For example, when using the double concentrated juice as soybean soup, 1-20 g with respect to 40 g of steamed soybeans, Preferably 5-15g can be added. . In the case of adding and using soybean oligosaccharide itself as well as other processed products, the amount may be determined according to the above.

  By the above method, it becomes possible to produce natto containing soybean oligosaccharide at a high concentration. Thus, according to the present invention, it is possible to obtain 400 to 500 mg or more of soybean oligosaccharide per 50 g of natto. For example, when soybean soup or other soybean oligosaccharide is added, the concentration can be further increased. It is also possible to obtain high-concentration oligosaccharide-containing natto of 800 mg or more, and in some cases equivalent to boiled beans.

  Hereinafter, the present invention will be specifically described by way of examples.

(Preparation of Bacillus natto deficient in levanschlase activity)
(1) Basic conditions As a parent strain deficient in Levanschlase activity, a Bacillus subtilis r22 strain (hereinafter also referred to as r22 strain) may be used (see, for example, JP 2000-224982 A). ) Was used. The r22 strain is a mutant strain with enhanced transformation ability obtained by subjecting the O-2 strain, which is Bacillus natto isolated from commercial natto, by a conventional method to chemical mutation treatment using nitrosoguanidine (NTG). In addition, E. coli DH5α (Escherichia coli DH5α) strain (manufactured by Takara Bio Inc.) and E. coli INV110 (Escherichia coli INV110) strain were used.

As the plasmid vector, pUC19 (manufactured by Takara Bio Inc.) and pE194 (obtained from ATCC) were used.
Unless otherwise specified, culture conditions, culture media, and other gene recombination techniques are described in “Molecular Cloning: Laboratory Manual 1”, Second Edition (1992), Cold Spring Harbor Laboratory Press, NY ” Was followed.

  Further, basic culture was performed using the NB medium shown in Table 1 below.

  Taq polymerase (manufactured by Takara Bio Inc.) was used for amplification of the DNA fragment by PCR, and the reaction conditions were in accordance with the manual attached to Taq polymerase.

The following methods were used for transformation of Bacillus natto.
That is, the chromosomal DNA of Bacillus natto was prepared using a commercially available kit, and the transformation was “Journal of Molecular Biology, 56, p.209-221, 1971”. The method was followed.

  Bacillus natto spores were prepared using the Sterlini-Madelsham replacement medium shown in Table 2 below.

(2) Vector construction FIG. 1 shows an outline of preparation of each DNA fragment used for vector construction.

  That is, primer 1 (described in SEQ ID NO: 1 (FIG. 4)) and primer 2 (described in SEQ ID NO: 2 (FIG. 5)) are used for amplification of the 5 ′ flanking region of the sacB gene of r22 strain. For the amplification of the 3 ′ flanking region, primer 3 (described in SEQ ID NO: 3 (FIG. 6)) and primer 4 (described in SEQ ID NO: 4 (FIG. 7)) were used.

  For amplification of the erythromycin resistance gene of pE194, primer 5 (described in SEQ ID NO: 5 (FIG. 8)) and primer 6 (described in SEQ ID NO: 6 (FIG. 9)) were used.

  Each primer includes the nucleotide sequence of the sacB gene of Bacillus subtilis (see, for example, “Nature, 390, pp. 249-256, 1997”), and resistance to erythromycin of pE194. The base sequence of a gene (for example, “Proceedings of National Academy of Science in USA”, vol. 77, No. 12, p. 7079-7083). , 1980 ") and designed and synthesized.

  That is, using the total DNA of the r22 strain as a template, the primer 1 (described in SEQ ID NO: 1) and primer 2 (described in SEQ ID NO: 2) are used to amplify the 5 ′ flanking region of the sacB gene. Restriction enzyme sites SacI and BamHI were introduced at both ends (DNA fragment 1) (FIG. 1). In addition, the 3 ′ flanking region of the sacB gene is amplified and restricted using primer 3 (described in SEQ ID NO: 3) and primer 4 (described in SEQ ID NO: 4) using the total DNA of the r22 strain as a template. Enzyme sites XbaI and PstI were introduced at both ends (DNA fragment 2) (FIG. 1).

  Furthermore, using pE194 as a template, primer 5 (described in SEQ ID NO: 5) and primer 6 (described in SEQ ID NO: 6) are used to amplify the erythromycin resistance gene, and restriction enzyme sites XbaI and BamHI are inserted at both ends of the gene. The introduced (DNA fragment 3) (FIG. 1).

  Subsequently, a vector was constructed using the above DNA fragment, and the outline thereof is shown in FIG. That is, DNA fragment 1 was treated with restriction enzymes Sac1 and BamHI, DNA fragment 2 was treated with XbaI and PstI, and DNA fragment 3 was treated with XbaI and BamHI. The obtained fragment was sequentially introduced between SacI and PstI on the multicloning site of pUC19 in the order of DNA fragment 1, DNA fragment 3, and DNA fragment 2 to obtain plasmid pSCEN1 (FIG. 2).

(3) Transformation The r22 strain was transformed with pSCEN1 linearized by PstI treatment. The transformant was selected by culturing at 37 ° C. for 18 hours in an LB medium supplemented with erythromycin 2 μg / ml. The transformant thus obtained was named Bacillus subtilis sacB22 (Bacillus subtilis sacB22), and internationally deposited as FERM BP-08538 at the National Institute of Advanced Industrial Science and Technology (AIST).

  The sacB gene of the resulting transformed strain Bacillus subtilis sacB22 (hereinafter also referred to as sacB22 strain) has two bases of base numbers 991 to 992 replaced with an erythromycin resistance gene. However, it was confirmed by analysis by PCR method. (Figure 3)

  In addition, it was confirmed by the following method that the levanschlase activity of the sacB22 strain was also inactivated compared to the parent strain r22.

That is, the sacB22 strain and the r22 strain were cultured in L medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 10 g / L sucrose), and the turbidity measured by OD ₆₆₀ was 1.4. And centrifuged to remove the cells and the culture supernatant was collected. The culture time required 3.75 hours for the sacB22 strain and 3.25 hours for the r22 strain.

  The collected culture supernatant was subjected to ultrafiltration with MICROCON YM-3 (manufactured by Millipore) to concentrate the protein contained in the culture solution about 5 times, and this was used as a crude enzyme solution.

  100 μl of this crude enzyme solution was added to 500 μl of 50 mM potassium phosphate buffer solution (pH 6.0) containing 8% sucrose, reacted at 37 ° C. for 40 minutes, and the amount of glucose released during the reaction was determined using glucose CII-Test Wako (Wako Pure) (Manufactured by Yakuhin). Levanschlase activity was measured using 1 U (enzyme unit) as the levanschlase activity that liberates 1 μmol of glucose per minute. Moreover, the protein amount of the crude enzyme solution was measured by protein assay (manufactured by BioRad), and the results expressed in activity (specific activity) per 1 mg of protein are shown in Table 3.

  As a result, in sacB22 strain, levanschlase activity was not detected at all, and levanschlase activity deficiency was confirmed.

(Natto prototype and quality evaluation)
According to a conventional method, spore solutions of r22 strain and sacB22 strain (FERM BP-08538) were prepared. That is, the r22 strain or sacB22 strain was inoculated with 1 platinum ear in the NB medium shown in Table 1, and after culturing at 37 ° C. overnight, the culture solution was inoculated with 1% in the spore-forming medium shown in Table 2. A spore solution was prepared by culturing at 37 ° C. for 24 hours.

  Hereinafter, each spore solution was used as an inoculum, and natto was prototyped according to a conventional method. That is, very small soybeans were immersed in tap water at 4 ° C. overnight, and then steamed in a pressure steamer at a steam pressure of 2 kpa for 8 minutes. The spore solution of the aforementioned r22 strain or sacB22 strain was diluted 100-fold with sterilized water to the cooked soybean thus prepared, seeded with 0.8 ml per 100 g of cooked soybean, and fermented at 39 ° C. for 18 hours. After fermentation, it was stored at 4 ° C. for 24 hours to prepare natto.

  When fermentation is performed using the sacB22 strain, the fermentation time is not delayed and the temperature of the natto product during the fermentation is not decreased, and the levanschlase-deficient mutation is not natto. It was found that it does not affect the growth of

Subsequently, natto obtained by trial manufacture was subjected to a sensory test.
As a result, there is no difference in quality between the parent strain (r22 strain) and the mutant strain (sacB22 strain), and the natto produced using the sacB22 strain as the inoculum has the quality required for natto. It could be confirmed.

  The analysis of leban in natto was performed by the following method. That is, 20 ml of 2.5% trichloroacetic acid is added to 10 g of natto and kept at 50 ° C. for 10 minutes. Transfer the supernatant to a 50 ml volumetric flask. Add 20 ml of 2.5% trichloroacetic acid to the remaining natto and incubate at 50 ° C. for 10 minutes. Filter with nylon mesh to remove natto.

  The filtrate is combined with the supernatant from the first extraction and then made up to 50 ml. The extracted solution is centrifuged at 12,000 rpm for 20 minutes at 30 ° C. 20 ml of supernatant is taken and adjusted to pH 7.0-7.2 with NaOH. After pH adjustment, make up to 25 ml. Take 5 ml of it, add 20 ml of ethanol and place on ice for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes, collect the precipitate, and let it air dry. The precipitate was dissolved in 20 ml of 20 mM phosphate buffer (pH 7.0) to obtain a crude extract.

To 0.6 ml of the crude extract, 0.3 ml of resorcin-thiourea reagent (resorcin 0.1 g, thiourea 0.25 g, acetic acid 100 ml) and 30% hydrochloric acid 0.21 ml were added and heated at 80 ° C. for 10 minutes. After quenching, A ₅₀₀ was measured. The concentration of levan was calculated by multiplying the calibration curve prepared using the fructose solution by 0.9 in consideration of the increase due to hydrolysis of levan.

  The results of analysis of levan in natto by the above method are shown in Table 4. The sacB22 strain produced less levan than the r22 strain, and the effect of the levanschrase gene deletion was observed. And it was found that the decrease in Leban did not affect natto stringing.

  Furthermore, soybean oligosaccharides in natto were analyzed according to the following method. That is, 1 g of natto or boiled beans was ground in a mortar and suspended in 5 ml of deionized water. 20 ml of ethanol was added to the suspension and stirred, followed by sonication (15 minutes), and further centrifuged at 3,000 rpm for 10 minutes. The supernatant after centrifugation was filtered (power size 0.45 μm) to obtain a crude extract.

  The concentration of sugar (sucrose, glucose, fructose) and soybean oligosaccharide (stachyose, raffinose) in the crude extract was measured using HPLC. HPLC was performed under the following conditions.

(HPLC conditions)
Eluent: 78% acetonitrile Column: Shodex NH2P-50 4E
Column temperature: 30 ° C
Flow rate: 1 ml / min Detector: Differential refractometer

  Table 4 shows the sugar and oligosaccharide concentrations in natto analyzed by the above method.

  As is apparent from the above results, in natto prepared using the parent strain (r22 strain), all oligosaccharides were decomposed and were not detected. On the other hand, about 70% of the soybean oligosaccharides of boiled beans remain in natto prepared using lebanshclase activity deficient natto (sacB22 strain: FERM BP-08538). Natto containing soybean oligosaccharide was realized by using deficient natto bacteria.

  In addition, sacrose degradation also occurred in the sacB22 strain, and it was found that natto has a sucrose degradation assimilation system in addition to levanschlase. Because of this system, it seems that Levanschlase deficiency did not affect the growth of Bacillus natto.

(Manufacture of soybean oligosaccharide-rich natto by the method of adding broth)
The natto was prototyped by the following method. That is, the preparation of the spore solution and the fermentation method of natto were basically the same as the method shown in Example 2.

  On the other hand, after immersing very small soybeans in tap water at 4 ° C. overnight, cooking in a pressure steamer at steam pressure of 2 kpa for 8 minutes to prepare steamed soybeans, 400 g of soybean broth collected from the pressure steamer was collected, and about 100 A double concentrated boiled juice heated and concentrated to 200 g in a hot water bath at 0 ° C. was prepared. The saccharide and oligosaccharide components in the 2-fold concentrated broth thus prepared were as shown in Table 5.

The natto was prepared below using the steamed soybean and the double concentrated soup prepared in this way.
That is, in the same manner as in Example 2, the natto was prepared by inoculating the r22 strain or sacB22 strain into the steamed soybean itself without adding the 2-fold concentrated broth.

  In addition, in the production of natto to which the double concentrated broth was added, 10 g of the double concentrated broth was added to 40 g of the steamed soybean, and then natto was prepared in the same manner as described above.

  The natto prepared by the above method was evaluated for quality by 30 skilled panelists, and the sugar composition was analyzed.

  In addition, quality evaluation is string drawing: 1 (weak)-5 (strong), thickness of fungus film: 1 (thin)-4 (thick), bean color: 2 (dark)-4.5 (light), Scents: 1 (with an off-flavor) to 3 (no off-flavor), maturity (degree of fermentation progress): 1 (unripe) to 10 (overripe), the results are shown in Table 6.

  From the results in Table 6, it was confirmed that by adding 2 times concentrated boiled juice, the fungus membrane was thick and the bean color was darkened, but the quality of natto was unquestionable did it.

  Moreover, the sugar composition in the produced natto was measured by the same method as in Example 2. The results are shown in Table 7.

  From the results in Table 7, when fermented with the r22 strain, most of the soybean oligosaccharides originally present in the steamed soybeans were consumed during fermentation and did not remain in natto, whereas sacB22 In the natto prepared using the stock, even when no soy broth was added, considerable soybean oligosaccharide remained, and it was confirmed that natto with a high content of soybean oligosaccharide could be produced. Furthermore, when soybean broth was added, it was confirmed that soybean oligosaccharides in soybean broth were added, and natto containing soybean oligosaccharides almost equivalent to steamed soybeans could be produced.

(Acquisition of mutant strains and production of soybean soybean with high oligosaccharide content)
(1) Acquisition of mutant strains Bacillus subtilis OUV23481 (hereinafter also referred to as OUV23481 strain or parent strain: internationally deposited as FERM BP-6659 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary) After shaking culture at 37 ° C. overnight in Table 1, it was centrifuged and washed with sterile physiological saline.

The washed cells were suspended in sterile physiological saline at a concentration of about 10 ⁷ cfu / ml, and N-methyl-N-nitro-N-nitrosoguanidine was added to a concentration of 20 mg / liter. The mutation treatment was performed by shaking for a minute. The survival rate at this time was about 2%.

  The mutated bacterial cells were cultured in an NB medium to express their traits, then washed with sterilized physiological saline, and then smeared on an NB medium plate containing 10% sucrose (containing 1.5% agar).

  Then, after culturing at 37 ° C. for 1 day, colonies having characteristics that do not produce levan on the surface of the colony were obtained in comparison with the parental colony.

  Levanschlase activity was measured for the obtained colonies, and a mutant strain in which the enzyme activity was completely inactivated was obtained. And this was named Bacillus subtilis B-1 (Bacillus subtilis B-1) and internationally deposited as FERM BP-08584 to the National Institute of Advanced Industrial Science and Technology patent biological depository center.

  The newly isolated mutant strain was not found to differ from the parent strain except for the lack of levanschlase activity. That is, this mutant strain was not significantly different from the parent strain in any of the form, growth state in each medium, or physiological properties, and also required biotin for growth and produced natto. No difference was observed. As for product natto, as described later, except for the point related to oligosaccharides, the natto properties such as flavor, texture, and stringiness are not significantly different from the parent strain. It was confirmed that it was none other than bacteria.

  In addition, the deficiency of levanschlase activity of Bacillus subtilis B-1 strain (hereinafter also referred to as B-1 strain) was confirmed by the following test.

That is, the turbidity of B-1 strain and parent strain OUV23481 was examined at OD ₆₆₀ using L medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 10 g / L sucrose). After culturing until 4, the cells were removed by centrifugation, and the culture supernatant was collected. The culture time was 3 hours for the B-1 strain and 3.15 hours for the parent strain OUV23481.

  The collected culture supernatant was ultrafiltered with MICROCON YM-3 (manufactured by Millipore) to concentrate the protein contained in the culture solution about 5 times, and this was used as a crude enzyme solution.

  0.1 ml of this crude enzyme solution was added to 0.5 ml of 50 mM potassium phosphate buffer (pH 6.0) containing 8% sucrose and reacted at 37 ° C. for 40 minutes, and the amount of glucose released during the reaction was determined as glucose CII− It was measured with Test Wako (manufactured by Wako Pure Chemical Industries). Levanschlase activity was measured using 1 U (enzyme unit) as the levanschlase activity that liberates 1 μmol of glucose per minute. Moreover, about the protein of the crude enzyme liquid, it measured by protein assay (made by BioRad), and the result represented with the activity per 1 mg protein (specific activity) was shown in Table 8.

  As a result, in the B-1 strain, levanschlase activity was not detected at all, and levanschlase activity deficiency was confirmed.

(2) Natto fermentation test Using the B-1 strain and the parent strain OUV23481 strain obtained above, natto was prepared as usual.
That is, the soaked soybeans were drained and steamed at 1.8 kgf / cm ² for 18 minutes. Each spore solution is inoculated so that there are 3,000 to 5,000 spores per gram of steamed soybeans, 50g each is placed in a PSP tray, the surface is covered with a thin film, a lid is applied, and then batch natto It put into the fermentation room and fermented under high humidity at room temperature 39 degreeC. After fermentation, natto was produced by aging.

  Thereafter, the contents of levan, sugar and soybean oligosaccharide (raffinose and stachyose) in natto were analyzed in the same manner as in Example 2, and the results are shown in Table 9.

  As is apparent from the above results, in natto prepared using the parent strain (OUV23481 strain), all oligosaccharides were decomposed and were not detected. On the other hand, about 70% of soybean oligosaccharides of steamed soybeans remain in natto prepared using levanshclease activity deficient natto (B-1 strain). Natto containing soybean oligosaccharide was realized by using defective natto.

It is the figure which showed the outline of DNA fragment preparation. It is a figure which shows the outline of vector construction. It is a figure which shows the outline of a sacB gene deletion process. Primer 1 is shown. Primer 2 is shown. Primer 3 is shown. Primer 4 is shown. Primer 5 is shown. Primer 6 is shown.

<110> Mitsukan Group Corporation
<120> Levansucrase Activity Lacked Bacillus subtilis and Natto Containing a Large Amount of Soybean Olygosaccharides
<130> 6768
<141> 2004-1-23
<160> 6

Claims (8)

A Bacillus natto strain that is classified into Bacillus subtilis and is capable of producing natto, characterized in that it lacks Levanschlase activity .   Bacillus subtilis sacB22 (Bacillus subtilis sacB22) (FERM BP-08538).   Bacillus subtilis B-1 (Bacillus subtilis B-1) (FERM BP-08484).   A method for producing natto with a high soybean oligosaccharide content, comprising using the Bacillus natto according to claim 1, 2 or 3.   A method for producing natto with a high content of soybean oligosaccharides, wherein the soybean oligosaccharide or soybean oligosaccharide-containing material is added and produced using the natto bacteria according to claim 1, 2 or 3.   The method according to claim 5, wherein the soybean oligosaccharide-containing material is soybean broth or a processed product thereof.   The method according to claim 6, wherein the treated product is at least one of a concentrate, pasty product, dried product, and diluted product of soybean broth. A soybean oligosaccharide-rich natto containing 400 mg or more of soybean oligosaccharide (stachyose, raffinose) per 50 g of natto , produced by the method according to any one of claims 4 to 7.

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