Bacillus subtilis, and food and fermented product containing the same

Abstract

To provide a Bacillus subtilis that can survive and proliferate in an acidic environment.MEANS FOR SOLVING THE PROBLEM: In order to solve the above problem, a Bacillus subtilis is provided herein that is capable of proliferating at pH 4.5 and surviving at pH 2.8. Specifically, the Bacillus subtilis provided herein is identified as strain TN19-3 (Accession No.: NITE AP-04010) or strain TN32-3 (Accession No.: NITE AP-04011).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to Bacillus subtilis and foods containing the same.

Background Art

[0002] Due to the increasing health consciousness in recent years, the concept of probiotics has become widely known. Probiotics are defined in the FAO (Food and Agriculture Organization of the United Nations) / WHO (World Health Organization) guidelines as "live microorganisms that, when ingested in appropriate amounts, confer beneficial effects on the health of the host." The beneficial effects are expected to include improvement of the intestinal environment, prevention of intestinal infections, and regulation of immune function.

[0003] In Japan, soy fermented foods have been consumed since ancient times, and natto is a typical example. Natto is produced by fermenting soybeans using Bacillus subtilis natto contained in Bacillus subtilis. A characteristic of natto is its stickiness, which is also called "stringiness." Patent Document 1 describes that stringiness is a major element of the texture of natto, that the strength of the stringiness mainly depends on PGA (polyglutamic acid) produced by Bacillus subtilis natto, that PGA has health-promoting effects such as an effect of promoting calcium absorption and an effect of suppressing the rise in postprandial blood glucose levels, and that a novel natto bacterium with high PGA productivity was obtained by inducing mutations in the Namegata-2-2 strain by X-ray irradiation.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Among microorganisms, there are those with limited environmental conditions for growth and survival, and many are particularly sensitive to pH. On the other hand, from the perspective of probiotics, it is preferable to deliver live bacteria to the intestine. The present invention aims to provide Bacillus subtilis that can survive and grow in an acidic environment, as well as foods and fermented products containing the same.

Means for Solving the Problems

[0006] To achieve the above object, the present invention provides Bacillus subtilis that can grow at pH 4.5 and can survive at pH 2.8.

[0007] The present invention also provides Bacillus subtilis that is strain TN19-3 (accession number: NITE AP-04010) or strain TN32-3 (accession number: NITE AP-04011).

[0008] The present invention also provides foods and fermented products using these Bacillus subtilis.

Effects of the Invention

[0009] The above-mentioned Bacillus subtilis can survive and grow in an acidic environment.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0011] Hereinafter, modes for carrying out the present invention will be described based on the drawings. The following description of the preferred embodiments is merely illustrative in nature and is not intended to limit the present invention, its application method, or its uses.

[0012] Bacillus subtilis Bacillus subtilis is a non-toxic Gram-positive bacterium and a spore-forming bacterium.

[0013] The present invention provides a Bacillus subtilis that satisfies (a) being able to grow at pH 4.5 and (b) being able to survive at pH 2.8. By being able to survive or grow in an acidic environment like this, vegetative cells can pass through the stomach while alive.

[0014] "Being able to grow at pH 4.5" means that when a colony with a loop size (diameter of about 2.5 mm) of 1 μL loop is inoculated into 5 mL of TSB liquid medium at pH 4.5 and statically cultured at 40 °C for 48 hours, it means satisfying at least one of the following two conditions: (1) the McFarland turbidity becomes 2 or more, or (2) a visible film is formed, and it is preferable to satisfy both. The McFarland turbidity can be determined by visual comparison with a standard solution.

[0015] "Being able to survive at pH 2.8" means that when statically cultured at 40 °C in TSB liquid medium at pH 2.8, the survival rate after 6 hours is 50% or more, and it is preferable that this survival rate is 60% or more. Furthermore, it is preferable that the survival rate after 24 hours is 50% or more, or 60% or more, and it is preferable that the survival rate after 48 hours is 50% or more, or 60% or more.

[0016] Here, the survival rate is measured as follows. The bacterial cells are suspended in a normal TSB liquid medium (about pH 7.3 ± 0.2) and adjusted to 10 6 CFU / mL. CFU means that a McFarland turbidity of 1 is 3×10 8It may be adjusted assuming it is CFU / mL. The McFarland turbidity can be determined by visual comparison with a standard solution. 100 μL of this suspension is mixed with 4.9 mL of TSB liquid medium adjusted to pH 2.8, and then statically cultured at 40°C. From the start of the culture, after 6, 24, and 48 hours, after stirring, the culture solution is sampled, serially diluted, and 100 μL of the diluted culture solution and the undiluted culture solution are individually spread on TSB agar medium and cultured at 40°C for 24 hours. The number of colonies obtained is counted, and the colony-forming units per unit volume (CFU / mL) of the culture solution are calculated based on the dilution ratio. Calculate the percentage of the value (CFU / mL) thus calculated with respect to the colony-forming units per unit volume at the initial stage of culture at low pH, 2×10 4 (CFU / mL). Assuming the number of samples per strain is 3, the average value of the obtained values is taken as the survival rate (%).

[0017] Bacillus subtilis can be identified by 16S rRNA analysis. Specifically, for the sequence of the V3-V4 region of 16S rRNA, a BLAST (Basic Local Alignment Search Tool) homology search is performed, and it is sufficient if the homology rate with Bacillus subtilis is 98% or more. In addition, as Bacillus subtilis, when the data obtained by decoding the draft genome sequence by short reads using DNBSEQ is analyzed by SpeciesFinder at the Center for Genomic Epidemiology, it is preferably determined to be Bacillus subtilis.

[0018] In addition, the Bacillus subtilis of the present invention preferably corresponds to Sequence Type (ST) ST4 based on seven genes (glpF, ilvD, pta, purH, pycA, rpoD, tpiA) contained in housekeeping genes in molecular epidemiological classification analysis (MLST: Multi-Locus Sequence Typing). That is, it is preferable that the above genes correspond to the following numbers: glpF 3, ilvD 2, pta 2, purH 6, pycA 4, rpoD 3, tpiA 4.

[0019] The present invention provides, as Bacillus subtilis, the TN19-3 strain (accession number: NITE AP-04010) and the TN32-3 strain (accession number: NITE AP-04011). These strains can meet the conditions of the above-mentioned Bacillus subtilis.

[0020] The TN19-3 strain (accession number: NITE AP-04010) and the TN32-3 strain (accession number: NITE AP-04011) were first isolated by the inventor from Acer palmatum Thunb.

[0021] The TN19-3 strain (accession number: NITE AP-04010) and the TN32-3 strain (accession number: NITE AP-04011) are deposited as follows.

[0022] Depositary institution name: National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center Contact address: Room 122, 2-5-8 Kazusa Kamashidzu, Kisarazu City, Chiba Prefecture 292-0818 Accession number / identification display: NITE AP-04010 / TN19-3, NITE AP-04011 / TN32-3 Receiving date: November 9, 2023 The TN19-3 strain (accession number: NITE AP-04010) and the TN32-3 strain (accession number: NITE AP-04011) can be cultured in TSB medium, but the medium is not limited thereto.

[0023] 〔Food〕 The present invention also provides a food containing the above-mentioned Bacillus subtilis. The food may be either solid or liquid. The food preferably contains legumes, and particularly preferably contains soybeans.

[0024] The food is preferably a fermented food. A fermented food is a food containing Bacillus subtilis and a fermentation product which is a substance obtained by fermenting a raw material with Bacillus subtilis. As the raw material, beans, particularly soybeans, are preferred, but not limited thereto. As described above, since the Bacillus subtilis of the present invention can survive and grow in an acidic environment, there is a possibility that it can reach the intestine in a living state. Further, the food preferably contains PGA.

[0025] 〔Fermentation product〕 A fermentation product is a substance obtained by fermenting a raw material with Bacillus subtilis. The fermentation product of Bacillus subtilis can be applied to other than foods. For example, Japanese Patent Application Laid-Open No. 2023-116694 discloses using Bacillus subtilis as an agricultural bioactive substance. An agricultural bioactive substance includes a biostimulant of a fungicide, an insecticide, a nematicide, a herbicide, a herbicide-phytotoxicity reducing agent, a plant growth regulator and / or a plant health promoter, or a combination thereof.

Examples

[0026] Hereinafter, unless otherwise specified, the medium was obtained by general composition and operation, and its pH was a general value (about pH 7.0 ± 0.2 for LB medium and about 7.3 ± 0.2 for TSB).

[0027] (Isolation of Bacillus subtilis) Leaves were collected from Momijibafu within the campus of Hiroshima University (3-2, Kagamiyama 1-chome, Higashi-Hiroshima City, Hiroshima Prefecture) and boiled for 30 minutes. After boiling, the leaves were taken out, and 4 to 8 leaves were used to wrap the whole of about 10 commercially available domestic boiled soybeans so as to cover them. The outside of the leaves wrapping the soybeans was covered with food wrap film except for both ends where the soybeans were not located, and incubated at 40 °C for 72 hours. In this way, by covering with wrap, the leaves were brought into close contact with the soybeans to maintain a moist environment, and ventilation was ensured by exposing a part from the wrap. After incubation, the soybeans were aseptically taken out and seeded one by one on an LB agar medium. After culturing at 40 °C for 24 hours, the growth of the bacteria was confirmed and subcultured on a TSB agar medium. 33 single colonies were picked from the TSB agar medium, cultured in a TSB liquid medium at 40 °C for 24 hours, and then cryopreserved for subsequent tests.

[0028] The 75 isolated strains were designated as BN9-1 to 4, BN10-1 to 4, BN11-1 to 4, BN12-1 to 3, BN13-1 to 3, BN14-1 to 3, BN15-1 to 3, BN16-1 to 3, BN17-1 to 3, BN18-1 to 3, BN19-1 to 2, TN19-3, BN20-1 to 3, BN21-1 to 3, BN22-1 to 3, BN23-1 to 3, BN24-1 to 3, BN25-1 to 3, BN26-1 to 3, BN27-1 to 3, BN28-1 to 3, BN29-1 to 3, BN30-1 to 3, BN31-1 to 3, BN32-1 to 2, and TN32-3, respectively.

[0029] (Low pH growth test) The isolated strains and the Miyagino strain as a control (shown as M-1 in Fig. 1) were precultured at 40 °C on TSB agar medium. One colony with a loop size of 1 μL loop (diameter approximately 2.5 mm) was inoculated into 5 mL of TSB liquid medium adjusted to pH 5.7, 5.1, 4.7, and 4.5, and statically cultured at 40 °C for 48 hours. After culturing, growth (turbidity and film) was confirmed. If at least one of the conditions that the MacFarland turbidity is 2 or more or a film is observed is satisfied, it was determined that growth was possible. The MacFarland turbidity was determined by visual comparison with a standard solution. The results are shown in Fig. 1.

[0030] As shown in Fig. 1, the Miyagino strain grew at pH 5.7 but could not grow at pH 4.7. The same was true for 57 of the isolated strains. Sixteen strains grew at pH 4.7 but could not grow at pH 4.5. Only two strains, TN19-3 and TN32-3, were able to grow at pH 4.5.

[0031] (Low pH survival test) TN19-3 and TN32-3, which showed acid resistance in the low pH growth test, and the Miyagino strain as a control were precultured at 40 °C on TSB agar medium. The obtained cells were suspended in TSB liquid medium, and 10 6It was adjusted to CFU / mL. Specifically, the suspension of bacterial cells in TSB liquid medium was adjusted to a MacFarland turbidity of 1, and this was serially diluted 1 / 300. The MacFarland turbidity was determined by visual comparison with a standard solution. 100 μL of the suspension with the adjusted concentration was dropped into 4.9 mL of TSB liquid medium adjusted to pH 2.8, stirred, and then statically cultured at 40°C. After 1, 6, 24, and 48 hours from the start of the culture, 100 μL was collected after stirring the culture broth. The culture broth was serially diluted, and 100 μL of the diluted culture broth and the undiluted culture broth were individually spread on TSB agar medium and cultured at 40°C for 24 hours.

[0032] The number of colonies obtained after the culture was counted, and the colony-forming units (CFU / mL) per unit volume of the culture broth were calculated based on the dilution ratio. The percentage of the value (CFU / mL) thus calculated with respect to the colony-forming units (CFU / mL) per unit volume at the initial stage of the culture at low pH was calculated as the survival rate (%). Since the suspension was diluted 50-fold at the start of the culture at low pH, the colony-forming units per unit volume at the initial stage of the culture were 2×10 4 CFU / mL. The number of samples for one strain was 3. The average value and standard deviation of the results for each strain are shown in Figure 2.

[0033] As shown in Figure 2, the survival rate of the Miyagino strain decreased to about 75% after 1 hour, about 30% after 6 hours, and less than about 10% after 24 hours and 48 hours.

[0034] On the other hand, the survival rate of TN19-3 was about 100% after 1 hour, still about 73% after 6 hours, and maintained a high value exceeding 60% even after 24 hours and 48 hours.

[0035] Also, the survival rate of TN32-3 showed a survival rate of about 100% until 6 hours, and maintained a high value exceeding about 80% after 24 hours and exceeding 60% after 48 hours.

[0036] (Crude PGA extraction) The isolated strains shown in Fig. 3 and the Miyagino strain (shown as M-1 in the figure) were precultured at 40 °C on a TSB agar medium. The obtained bacterial cells were suspended in sterilized distilled water and adjusted to a McFarland turbidity of 0.5. The McFarland turbidity was determined by visual comparison with a standard solution.

[0037] 180 μL of sterilized distilled water was added to 20 μL of this suspension for dilution, and this diluted solution was dropped onto 10 g of boiled soybeans placed in a 50 mL tube. It was mixed by inversion to wet the whole soybeans. This was statically cultured at 40 °C for 24 hours.

[0038] 13 mL of PBS was dropped into the 50 mL tube after culturing, and it was stirred with a sterilized loop so as not to crush the beans, and the sticky substances were peeled off and dissolved from the beans. It was centrifuged at 4500 rpm for 5 minutes, and the supernatant was collected. The collected supernatant was centrifuged at 6000 rpm at 4 °C for 20 minutes. 10 mL of the supernatant was collected, 25 mL of 99.5% ethanol was added, and it was stored cooled at -20 °C for 30 minutes. It was centrifuged at 6000 rpm at 4 °C for 10 minutes, the supernatant was discarded, and the precipitate was dried to obtain crude PGA powder. The mass of the obtained crude PGA powder was measured. The average value of the numerical values obtained by repeating the same experiment three times for one strain was shown in Fig. 3 as the result.

[0039] The Miyagino strain showed a crude PGA amount of 0.55 g, and only TN32-3 exceeded this, with its crude PGA amount being 0.64 g. TN19-3, which showed high acid resistance together with TN32-3, was lower than the Miyagino strain in terms of the crude PGA amount, being 0.19 g.

[0040] (Bacterial identification) For the TN19-3 strain and the TN32-3 strain, 16S rRNA analysis was performed. Specifically, the sequence of the V3-V4 region of 16S rRNA was amplified by the PCR method, and the nucleotide sequence was determined by a sequencer. When the assembled sequence was input into a BLAST homology search, it showed more than 98% homology with many Bacillus subtilis.

[0041] In addition, the draft genomic sequence by short reads was decoded by DNBSEQ. When the obtained data was analyzed using SpeciesFinder at the Center for Genomic Epidemiology, it was determined to be Bacillus subtilis.

[0042] Furthermore, when the draft genome was analyzed by MLST, it was determined to be ST4 of Bacillus subtilis.

[0043] Based on the above results, strains TN19-3 and TN32-3 were identified as Bacillus subtilis.

[0044] (Summary) As described above, two strains, TN19-3 and TN32-3, which can grow at pH 4.5 and show a high survival rate over a long period even at pH 2.8, were isolated from Momijibafu. In particular, strain TN32-3 had a high crude PGA production amount.

Industrial Applicability

[0045] The Bacillus subtilis of the present invention is suitably used, for example, in foods, particularly soybean-containing foods.

Claims

1. A Bacillus subtilis that can grow at pH 4.5 and can survive at pH 2.

8.

2. Using 20 μL of a cell suspension having a turbidity of 0.5 McFarland, by static culture at 40°C for 24 hours, producing 0.5 g or more of crude polyglutamic acid from 10 g of boiled soybeans, The Bacillus subtilis according to Claim 1.

3. A Bacillus subtilis that is the TN19-3 strain (accession number: NITE AP-04010) or the TN32-3 strain (accession number: NITE AP-04011).

4. A food containing the Bacillus subtilis according to any one of Claims 1 to 3.

5. A food containing the Bacillus subtilis according to any one of Claims 1 to 3 and soybeans.

6. A fermented product of the Bacillus subtilis according to any one of Claims 1 to 3.

7. A fermented product of soybeans with the Bacillus subtilis according to any one of Claims 1 to 3.

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