WO2017033925A1 - Butyric acid-producing bacterium - Google Patents

Butyric acid-producing bacterium Download PDF

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WO2017033925A1
WO2017033925A1 PCT/JP2016/074497 JP2016074497W WO2017033925A1 WO 2017033925 A1 WO2017033925 A1 WO 2017033925A1 JP 2016074497 W JP2016074497 W JP 2016074497W WO 2017033925 A1 WO2017033925 A1 WO 2017033925A1
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butyric acid
acid
producing bacterium
yit
strain
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PCT/JP2016/074497
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Japanese (ja)
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敏彦 高田
久代　明
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株式会社ヤクルト本社
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Priority to JP2017536440A priority Critical patent/JP6712598B2/en
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium

Definitions

  • the present invention relates to a new butyric acid-producing bacterium and a butyric acid production enhancer containing the same.
  • Short-chain fatty acids produced by intestinal bacteria lower the pH in the intestine, suppress the growth of harmful bacteria, and are rapidly absorbed from the intestinal epithelium to be used as a host energy source via the portal vein Yes.
  • butyric acid is the most important energy source of intestinal epithelial cells, and is considered to have beneficial effects on the host, such as promoting mucin secretion via the vagus nerve.
  • Blautia coccoides group and Clostridium leptum ⁇ ⁇ subgroup which are the most dominant bacteria in the human intestine, contain many bacterial species that produce butyric acid and play an important role in maintaining the intestinal environment of the host.
  • a decrease in Blautia coccoides group and Clostridium leptum subgroup and a concomitant decrease in the butyric acid concentration in the intestinal tract have been reported (Non-patent Document 1).
  • Faecalibacterium prausnitzii a major butyrate-producing bacterium belonging to Clostridium leptum subgroup, induces the expression of anti-inflammatory cytokine IL-10 in an experimental system using cultured cells, and is administered to inflammatory bowel disease model mice. Is also attracting attention as one of the next-generation probiotic candidates (Non-Patent Document 2).
  • Vermeiren J. et al. 2012, FEMS Microbiol. Ecol. Sokol, H. et al. 2008, Proc Natl Acad Sci USA.
  • bacteria belonging to Clostridium leptum subgroup are bacteria that cannot produce butyric acid in a medium without acetic acid or that also produce formic acid together with butyric acid. It is known that the amount of short-chain fatty acids such as acetic acid and butyric acid is lower in patients with inflammatory bowel disease than in healthy individuals. Is useful in view of the beneficial effects of butyric acid in the intestine.
  • formic acid is corrosive and is also known as a mitochondrial toxin that can inhibit cytochrome c oxidase (Patent Document 1), and it is not preferable to ingest bacteria that produce formic acid.
  • an object of the present invention is to provide a new butyric acid-producing bacterium that produces butyric acid even in the absence of acetic acid and does not produce formic acid, and a butyric acid production enhancer containing the bacterium.
  • the present inventor tried to isolate a new butyric acid-producing bacterium from the sample library owned by the applicant, and as a result, produced butyric acid even in the absence of acetic acid, and further produced Clostridium leptum subgroup, which does not produce formic acid.
  • a new butyric acid-producing bacterium belonging to the present invention was found and the present invention was completed.
  • the present invention provides the following [1] to [7].
  • [1] A butyric acid-producing bacterium belonging to Clostridium leptum subgroup, which produces butyric acid and does not produce formic acid in a medium without acetic acid.
  • the number of bacteria is 1.0 ⁇ 10 6 cells inoculated into 2 mL of acetic acid-free YCG medium, and the amount of butyric acid produced when anaerobically cultured at 37 ° C. for 72 hours is 10 mM or more, and the amount of formic acid produced is 1 mM.
  • a butyric acid production enhancer comprising the butyric acid-producing bacterium according to any one of [1] to [4].
  • a composition for food, beverage, medicine or feed containing the butyric acid-producing bacterium according to any one of [1] to [4].
  • the butyric acid-producing bacterium of the present invention is a new bacterial species, and produces butyric acid even in the absence of acetic acid, and does not produce formic acid. Therefore, it is highly safe and is used as a butyric acid production enhancer in the intestine. It is useful as a product, medicine, and feed composition.
  • the butyric acid-producing bacterium of the present invention is a bacterium that produces butyric acid and does not produce formic acid in a medium without acetic acid, and belongs to the C. leptum subgroup.
  • the butyric acid-producing bacterium belonging to the conventional C. leptum subgroup is a bacterium that cannot produce butyric acid in the absence of acetic acid or that produces formic acid together with butyric acid
  • the butyric acid-producing bacterium of the present invention is a new one. It is a new bacterial species with characteristics.
  • the absence of acetic acid means that acetic acid is not present, or even if it is present, only a trace amount of 1 mM or less is present.
  • not producing formic acid means that formic acid is not produced at all or even when produced, it is less than 1 mM.
  • the characteristics of the butyric acid-producing bacterium of the present invention are inoculated with 2 mL of YCG (yeast extract-casitone glucose) medium in the absence of acetic acid at 1.0 ⁇ 10 6 cells and anaerobically cultured at 37 ° C. for 72 hours.
  • the butyric acid production amount is preferably 10 mM or more, and the formic acid production amount is preferably less than 1 mM, the butyric acid production amount under the conditions is 10 to 30 mM, and the formic acid production amount is less than 0.7 mM. More preferably.
  • the strain frozen stock solution is preferably a solution in which the cells are suspended in a 10% by mass skim milk medium or 20% by mass glycerol-added Mueller Hinton medium.
  • the measurement of the concentration of organic acids such as butyric acid and formic acid in the culture solution is not particularly limited as long as the organic acid concentration can be measured. For example, it can be measured by an HPLC system for organic acid analysis.
  • the butyric acid-producing bacterium of the present invention produces butyric acid even in a medium containing acetic acid.
  • butyric acid-producing bacterium of the present invention examples include Butyricicoccus sp. YIT 12787 (NITE BP-02106), Butyricicoccus sp. YIT 12788 (NITE BP-02095) and their mutants.
  • YIT 12787 and YIT 12788 are registered in the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation on August 20, 2015 and July 31, 2015. Deposited each day.
  • the 16S rRNA of YIT 12787 was SEQ ID NO: 3
  • the 16S rRNA of YIT 12788 was SEQ ID NO: 4.
  • Examples of the mutant strains of YIT 12787 and YIT 12788 in the present invention include mutants having a homology of 16S rRNA of 99% or more with SEQ ID NO: 3 and / or SEQ ID NO: 4. In the present invention, homology refers to the identity of base sequences.
  • the homology between YIT 12787 and YIT 12788 and related species was examined, the homology between YIT 12787 and Eubacterium desmolans ATCC 43058 T was 7.5 to 18.9%, and YIT 12787 and Butyricicoccus pullicaecorum 25 The homology with ⁇ 3 T was 10.3-14.3%. Further, the homology between YIT 12788 and Eubacterium desmolans ATCC 43058 T was 7.9 to 17.8%, and the homology between YIT 12788 and Butyricicoccus pullicaecorum 25-3 T was 10.7 to 16.5%. . Therefore, the butyric acid-producing bacterium of the present invention has a DNA homology of 20% or less with the related species Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T.
  • the phylogenetic tree of the butyric acid-producing bacterium of the present invention is as shown in FIG. 1, which is a new bacterium species belonging to C. leptum subgroup.
  • the butyric acid-producing bacteria of the present invention include bacteria having the following properties (1) and (2).
  • (1) The homology between the base sequence of 16S rRNA and SEQ ID NO: 3 and / or SEQ ID NO: 4 is 99% or more.
  • DNA homology with the related species Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T is 20% or less.
  • the butyric acid-producing bacterium of the present invention has the following mycological properties. As shown in Examples below, the butyric acid-producing bacterium of the present invention is different from Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T in sugar fermentation properties and enzyme activity properties. It can be seen that this is a new species belonging to the leptum subgroup.
  • the butyric acid-producing bacterium of the present invention is inoculated and cultured with various sample libraries and microorganisms collected from nature, and the grown colonies are fished and measured for the ability to produce butyric acid and formic acid in a medium without acetic acid. Can be isolated.
  • the confirmation of the butyric acid-producing bacterium of the present invention can be confirmed by organic acid analysis, sequence analysis, or the like.
  • the butyric acid-producing bacterium of the present invention can be proliferated and subcultured by anaerobic culture using a GAM medium or PY (peptone yeast extract) medium prepared in an anaerobic state, for example.
  • GAM medium or PY (peptone yeast extract) medium prepared in an anaerobic state, for example.
  • the butyric acid-producing bacterium of the present invention has no safety problem, produces butyric acid in the absence of acetic acid, and does not produce formic acid. Therefore, the composition containing the butyric acid-producing bacterium of the present invention is useful as a composition for foods and drinks, medicines or feeds.
  • the composition is useful as a butyric acid production enhancer because it produces butyric acid in the intestines of animals including humans.
  • butyric acid is not only used as an energy source for colonic mucosal epithelial cells, but also has an epithelial cell growth promoting action, an anti-inflammatory action, and an intestinal motility enhancing action, as well as colon cancer and ulcerative colitis. Therefore, the composition of the present invention is particularly useful as a pharmaceutical, food, drink or feed having these physiological activities.
  • composition of the present invention preferably contains 10 4 cfu to 10 14 cfu of butyric acid-producing bacteria as viable bacteria.
  • composition of the present invention can be in a form suitable for each of foods and drinks, medicines and feeds.
  • a medicine for example, it can be mixed with a solid or liquid non-toxic pharmaceutical carrier to form a conventional pharmaceutical preparation.
  • preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means on the preparation.
  • non-toxic pharmaceutical carrier examples include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like.
  • conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, excipients and the like can be appropriately added as necessary.
  • an additive that can be used as a food or drink is appropriately used, and it may be formed into a form suitable for food, that is, a granule, a granule, a tablet, a capsule, a paste, or the like, using a conventional means.
  • the types of food and drink include, for example, processed meat foods such as ham and sausage, processed fishery foods such as kamaboko and chikuwa, foods such as bread, confectionery, butter and powdered milk, water, fruit juice, milk, soft drinks and tea drinks. And the like. The same applies to feed.
  • composition of the present invention When the composition of the present invention is administered to animals including humans, it is preferably administered orally or enterally from the viewpoint of producing butyric acid in the intestine, and its dosage is as a butyric acid producing bacterium per day.
  • the number of viable bacteria is preferably 1.0 ⁇ 10 4 cfu or more, and more preferably 1.0 ⁇ 10 8 cfu to 1.0 ⁇ 10 12 cfu.
  • Example 1 Isolation of Butyrate-Producing Bacteria Two types (A, B) of samples were selected from the sample library owned by the applicant, and YCFA-M medium (YCFA supplemented with 0.5% mucin (acetic acid 20 mM, propionic acid) was selected. 1 mL of 5 mM) medium) was inoculated with 10 ⁇ L of a 10-fold diluted solution of each sample. After anaerobic culture at 37 ° C. for 8 hours, 10 ⁇ L of the supernatant after centrifugation at 4,000 G for 5 minutes was inoculated into 2 mL of new YCFA-M medium.
  • YCFA-M medium YCFA supplemented with 0.5% mucin (acetic acid 20 mM, propionic acid) was selected. 1 mL of 5 mM) medium
  • mucin acetic acid 20 mM, propionic acid
  • Example 2 Butyric acid and formic acid producing ability of strain A and strain B (1) Comparison of butyric acid and formic acid producing ability with known strains For strain A and strain B, including bacteria belonging to C. leptum subgroup in a medium without acetic acid The amount of butyric acid produced was compared with 18 known butyric acid producing bacteria (Table 1).
  • the supernatant after centrifugation was filtered through a 0.45 ⁇ m filter, and the amount of organic acid in the filtrate was measured using an organic acid analysis HPLC system described later.
  • organic acid analysis HPLC system There are 9 types of standard substances: succinic acid, sodium formate, sodium acetate, sodium propionate, sodium isobutyrate, n-sodium butyrate, sodium isovalerate, n-sodium valerate and lithium lactate (all Kanto Chemical Co., Ltd.)
  • Using a mixed aqueous solution of HPLC grade manufactured by the company quantification was performed by a two-point (0.01 and 0.2 ⁇ mol / 10 ⁇ L) absolute calibration curve method.
  • Table 2 shows the results of the organic acid producing ability of strain A and strain B using various media.
  • Strains A and B produced butyric acid in excess of 10 mM in the M2GSC medium without acetic acid, while the amount of formic acid produced was less than 1 mM and did not produce formic acid. Further, 20 mM or more of butyric acid was produced in the M2GSC + SCFA and YCFA medium supplemented with acetic acid. In the M2GSC + SCFA and YCFA media, the concentration of acetic acid after culturing was significantly lower than the initial content (Table 2), so it was considered that butyric acid was produced using acetic acid in the media. .
  • Example 3 16S rRNA sequence analysis of strain A and strain B
  • DNA was extracted by the bead phenol method. That is, the operation of suspending 200 ⁇ L of the bacterial solution of strain A and strain B in 1.0 mL of PBS, centrifuging at 15,000 rpm, and discarding the supernatant was repeated three times. The obtained pellet was first suspended in 450 ⁇ L of Extraction Buffer (100 mM Tris-HCl, 40 mM EDTA, pH 9.0) and 50 ⁇ L of 10% SDS, 0.3 g of glass beads (diameter 0.1 mm) and 500 ⁇ L of TE saturated.
  • Extraction Buffer 100 mM Tris-HCl, 40 mM EDTA, pH 9.0
  • Phenol was added and shaken vigorously for 30 seconds with FastPrep FP120 (power level 5.0). After centrifugation at 15,000 rpm for 10 minutes, isopropanol precipitation was performed, and the obtained DNA was dissolved in 1,000 ⁇ L of TE buffer. The obtained DNA solution was diluted 10 times with purified water to obtain a template DNA solution. Using the 8F and 15R primers shown in Table 3 for the template DNA solution, after reacting at 95 ° C for 5 minutes, 95 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute 30 seconds, 35 cycles The PCR reaction was performed, followed by a reaction at 72 ° C. for 5 minutes.
  • the PCR product was purified using High Pure PCR Product Purification Kit (Roche) and subjected to a sequencing reaction using BigDye Terminator v3.1 Cycle Sequence Kit (Applied Biosystems). The obtained sequence was subjected to a BLAST search of the Japan DNA Data Bank (DDBJ) and collated with a sequence database of known bacterial species. Furthermore, phylogenetic analysis was performed on the sequence of the isolates by the neighbor joining (NJ) method using Clustal W, and a phylogenetic tree was created using the Tree-View program.
  • DDBJ Japan DNA Data Bank
  • NJ neighbor joining
  • the created phylogenetic tree is shown in FIG.
  • the strain A was 1,495 bp (SEQ ID NO: 3) and the strain B was 1,500 bp (SEQ ID NO: 4). Both strains had 99% or more homology with each other.
  • strain A and strain B belonged to C. leptum subgroup and were considered to be closely related to Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T , which are known bacterial species.
  • strain A and Eubacterium desmolans ATCC 43058 T were 94.9%, and the homology between Butyricicoccus pullicaecorum 25-3 T was 96.3%.
  • the homology between strain B and Eubacterium desmolans ATCC 43058 T was 95.0%, and the homology between Butyricicoccus pullicaecorum 25-3 T was 96.3%.
  • the homology of 16S rRNA is 99% or more, but since strain A and strain B have less than 99% homology with known strains, new strains belonging to C. leptum subgroup It was thought that.
  • Strain A was transformed into Butyricicoccus sp.
  • YIT 12787 strain B was transformed into Butyricicoccus sp. It was named YIT 12788 and the properties of these strains belonging to the new strain were confirmed.
  • Example 4 Various biochemical properties
  • strains used were Eubacterium desmolans ATCC 43058 T (E. desmolans) and Butyricicoccus pullicaecorum 25-3 T (B. pulllicaecorum). The result was determined visually after 30 hours inoculation with the bacterial solution.
  • the results of the sugar fermentation property test are shown in Table 4.
  • YIT 12787 and YIT 12788 were positive for glucose utilization. All of E. desmolans were negative.
  • B.pullicaecorum assimilation of glucose, saccharose, salicin and xylose and hydrolysis of esculin were positive.
  • YIT 12787 and YIT 12788 can be distinguished from E. desmolans by the availability of glucose and the presence or absence of alkaline phosphatase, esterase (C4) and leucine allylamidase activity. Met. Moreover, it was distinguishable from B.pullicaecorum by the availability of saccharose, salicin and xylose, the hydrolytic ability of esculin, and the presence or absence of esterase (C4) activity.
  • the cells after fixation were washed 3 times with 5 mM EDTA solution, resuspended in 6 mL of the same solution, 1 mL each of 600 U / mL achromopeptidase and 50 mg / mL lysozyme were added, and the cells were lysed at 37 ° C. for 2 hours. .
  • 0.6 mL of SDS solution was added and treated at 60 ° C. for 10-30 minutes. Further, 150 ⁇ L of 10 mg / mL proteinase K was added and treated at 65 ° C.
  • a sequence library was prepared using TruSeq DNA PCR-Free Sample Prep LS Kit (Illumina) and DNA Shearing System M220 (Covaris) according to the protocol provided by Illumina.
  • the prepared sequence library was subjected to a 250 bp ⁇ 2 paired end sequence by the next-generation sequencer MiSeq (Illumina), the base sequence was decoded, and GC% was calculated based on the obtained base sequence result.
  • the GC content of the bacterial DNA was 53.9% for YIT 12787, 54.1% for YIT 12788, 54.3% for E. desmolans, and 54.0% for B.pullicaecorum. It was.
  • YIT 12787 and YIT 12788 have the following common biochemical properties.
  • Example 5 Properties of New Isolate One strain (C) was selected from the sample library owned by the applicant, and strain C was isolated by the method described in Example 1.
  • strain C and YIT 12787 had 99.1%
  • strain C and YIT 12788 had 99.3% homology of 16S rRNA. Both were 99% or more.
  • the homology between strain C and E. desmolans and B. pulllicaecorum was measured by the method of Example 4 (4)
  • the homology between strain C and E. desmolans was 8.8-18.
  • the homology between strain C and B.pullicaecorum was 10.6 to 15.5%. Therefore, the DNA-DNA homology between strain C and E. desmolans and B. pulllicaecorum was both 20% or less.
  • strain C The ability of strain C to produce butyric acid and formic acid was measured by the method of Example 2 (1). As a result, the amount of butyric acid produced by strain C was 13.3 mM, and the amount of formic acid produced was 0.0 mM. Therefore, the strain C in which the homology of YIT 12787 and YIT 12788 with 16S rRNA is 99% or more and the DNA homology with E. desmolans and B. pulllicaecorum is less than 20% is 1.
  • C. leptum subgroup in which 0 ⁇ 10 6 cells are inoculated into 2 mL of acetic acid-free YCG medium and anaerobic culture at 37 ° C. for 72 hours has a butyric acid production of 10 mM or more and a formic acid production of less than 1 mM It was found to be a butyric acid-producing bacterium belonging to
  • Example 6 Manufacture of tablets Various ingredients were mixed according to the formulation shown in Table 7 below, granulated, dried and sized, and then tableted to produce tablets.
  • a live cell of YIT 12787 was produced by freeze-drying (including 10 10 cells / g of live cell).

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Abstract

Provided is a novel butyric acid-producing bacterium that belongs to the Clostridium leptum subgroup, and, in a butyric acid-free culture medium, produces butyric acid but does not produce formic acid.

Description

酪酸産生菌Butyric acid producing bacteria
　本発明は、新たな酪酸産生菌及びそれを含有する酪酸産生増強剤に関する。 The present invention relates to a new butyric acid-producing bacterium and a butyric acid production enhancer containing the same.
　腸内細菌が産生する短鎖脂肪酸は、腸内のｐＨを下げ、有害菌の増殖を抑制し、また腸管上皮から速やかに吸収されることにより、門脈を経て宿主のエネルギー源として利用されている。特に酪酸は、腸上皮細胞の最も重要なエネルギー源であり、迷走神経を介してムチンの分泌を促進するなど、宿主に対して有益な作用を有すると考えられている。 Short-chain fatty acids produced by intestinal bacteria lower the pH in the intestine, suppress the growth of harmful bacteria, and are rapidly absorbed from the intestinal epithelium to be used as a host energy source via the portal vein Yes. In particular, butyric acid is the most important energy source of intestinal epithelial cells, and is considered to have beneficial effects on the host, such as promoting mucin secretion via the vagus nerve.
　ヒト腸内最優勢菌群であるBlautia coccoides group及びClostridium leptum subgroupは、酪酸を産生する細菌種を数多く含んでおり、宿主の腸管環境維持に重要な役割を担っている。炎症性腸疾患患者では、Blautia coccoides group及びClostridium leptum subgroupの減少、それに伴う腸管内の酪酸濃度の減少が報告されている（非特許文献１）。さらに、Clostridium leptum subgroupに属する主要な酪酸産生菌種であるFaecalibacterium prausnitziiは、培養細胞を用いた実験系において抗炎症性サイトカインＩＬ－１０の発現を誘導し、炎症性腸疾患モデルマウスへの投与によっても抗炎症反応を示すことから、次世代プロバイオティクス候補の一つとして注目されている（非特許文献２）。 Blautia coccoides group and Clostridium leptum ヒ ト subgroup, which are the most dominant bacteria in the human intestine, contain many bacterial species that produce butyric acid and play an important role in maintaining the intestinal environment of the host. In patients with inflammatory bowel disease, a decrease in Blautia coccoides group and Clostridium leptum subgroup and a concomitant decrease in the butyric acid concentration in the intestinal tract have been reported (Non-patent Document 1). Furthermore, Faecalibacterium prausnitzii, a major butyrate-producing bacterium belonging to Clostridium leptum subgroup, induces the expression of anti-inflammatory cytokine IL-10 in an experimental system using cultured cells, and is administered to inflammatory bowel disease model mice. Is also attracting attention as one of the next-generation probiotic candidates (Non-Patent Document 2).
特表２００９－５２５１４１号公報Special table 2009-525141
　しかしながら、従来知られているClostridium leptum subgroupに属する菌は、酢酸非存在の培地中では酪酸を産生することができないか、又は酪酸とともにギ酸も産生する菌であった。
　炎症性腸疾患患者では健常人と比較して酢酸、酪酸といった短鎖脂肪酸の量が低下することが知られており、そのような疾病の患者に対して、酢酸非存在でも酪酸を産生する菌を投与することは、腸内での酪酸の有益作用を考慮すると有用である。
　また、ギ酸は、腐食性を有するほか、チトクロムｃオキシダーゼを阻害しうるミトコンドリア毒素としても知られており（特許文献１）、ギ酸を産生する菌を摂取することは好ましくない。 However, conventionally known bacteria belonging to Clostridium leptum subgroup are bacteria that cannot produce butyric acid in a medium without acetic acid or that also produce formic acid together with butyric acid.
It is known that the amount of short-chain fatty acids such as acetic acid and butyric acid is lower in patients with inflammatory bowel disease than in healthy individuals. Is useful in view of the beneficial effects of butyric acid in the intestine.
In addition, formic acid is corrosive and is also known as a mitochondrial toxin that can inhibit cytochrome c oxidase (Patent Document 1), and it is not preferable to ingest bacteria that produce formic acid.
　従って、本発明の課題は、酢酸非存在であっても酪酸を産生し、さらに、ギ酸を産生しない新たな酪酸産生菌及びその菌を含有する酪酸産生増強剤を提供することにある。 Therefore, an object of the present invention is to provide a new butyric acid-producing bacterium that produces butyric acid even in the absence of acetic acid and does not produce formic acid, and a butyric acid production enhancer containing the bacterium.
　そこで本発明者は、出願人が保有するサンプルライブラリーから、新たな酪酸産生菌の分離を試みた結果、酢酸非存在であっても酪酸を産生し、さらに、ギ酸を産生しないClostridium leptum subgroupに属する新たな酪酸産生菌を見出し、本発明を完成した。 Therefore, the present inventor tried to isolate a new butyric acid-producing bacterium from the sample library owned by the applicant, and as a result, produced butyric acid even in the absence of acetic acid, and further produced Clostridium leptum subgroup, which does not produce formic acid. A new butyric acid-producing bacterium belonging to the present invention was found and the present invention was completed.
　すなわち、本発明は、次の〔１〕～〔７〕を提供するものである。
〔１〕酢酸非存在の培地において、酪酸を産生し、かつギ酸を産生しない、Clostridium leptum subgroupに属する酪酸産生菌。
〔２〕菌数１．０×１０6 ｃｅｌｌｓを酢酸非存在のＹＣＧ培地２ｍＬに接種し、３７℃で７２時間嫌気培養した際の酪酸産生量が、１０ｍＭ以上であり、かつギ酸産生量が１ｍＭ未満である〔１〕記載の酪酸産生菌。
〔３〕下記の（１）及び（２）の性質を有する〔１〕又は〔２〕記載の酪酸産生菌。
　（１）１６Ｓ　ｒＲＮＡの塩基配列と配列番号３及び／又は配列番号４との相同性が９９％以上
　（２）近縁種であるEubacterium desmolans  ＡＴＣＣ ４３０５８T及びButyricicoccus pullicaecorum　２５－３TとのＤＮＡの相同性が２０％以下
〔４〕ＮＩＴＥ　ＢＰ－０２１０６として寄託されたＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ．ＹＩＴ １２７８７、ＮＩＴＥ　ＢＰ－０２０９５として寄託されたＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８８又はそれらの変異株である〔１〕～〔３〕のいずれかに記載の酪酸産生菌。
〔５〕〔１〕～〔４〕のいずれかに記載の酪酸産生菌を含有することを特徴とする酪酸産生増強剤。
〔６〕〔１〕～〔４〕のいずれかに記載の酪酸産生菌を含有する飲食品、医薬又は飼料用組成物。
〔７〕〔１〕～〔４〕のいずれかに記載の酪酸産生菌の飲食品、医薬又は飼料用組成物製造のための使用。 That is, the present invention provides the following [1] to [7].
[1] A butyric acid-producing bacterium belonging to Clostridium leptum subgroup, which produces butyric acid and does not produce formic acid in a medium without acetic acid.
[2] The number of bacteria is 1.0 × 10 6 cells inoculated into 2 mL of acetic acid-free YCG medium, and the amount of butyric acid produced when anaerobically cultured at 37 ° C. for 72 hours is 10 mM or more, and the amount of formic acid produced is 1 mM. The butyric acid-producing bacterium according to [1], which is less than
[3] The butyric acid-producing bacterium according to [1] or [2], which has the following properties (1) and (2).
(1) The homology between the base sequence of 16S rRNA and SEQ ID NO: 3 and / or SEQ ID NO: 4 is 99% or more. (2) DNA of the closely related species Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T Homology 20% or less [4] Butyricococcus sp. Deposited as NITE BP-02106 Butyricicoccus sp. Deposited as YIT 12787, NITE BP-02095. The butyric acid-producing bacterium according to any one of [1] to [3], which is YIT 12788 or a mutant thereof.
[5] A butyric acid production enhancer comprising the butyric acid-producing bacterium according to any one of [1] to [4].
[6] A composition for food, beverage, medicine or feed containing the butyric acid-producing bacterium according to any one of [1] to [4].
[7] Use of the butyric acid-producing bacterium according to any one of [1] to [4] for producing a composition for food, beverage, medicine or feed.
　本発明の酪酸産生菌は、新菌種であり、酢酸非存在であっても、酪酸を産生し、さらにギ酸を産生しないことから、安全性が高く、腸内での酪酸産生増強剤として飲食品、医薬、飼料組成物として有用である。 The butyric acid-producing bacterium of the present invention is a new bacterial species, and produces butyric acid even in the absence of acetic acid, and does not produce formic acid. Therefore, it is highly safe and is used as a butyric acid production enhancer in the intestine. It is useful as a product, medicine, and feed composition.
Ｂｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８７及びＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８８を含むC. leptum subgroupの系統樹を示す。Butyricicoccus sp. YIT 12787 and Butyricicoccus sp. A phylogenetic tree of C. leptum subgroup containing YIT 12788 is shown.
　本発明の酪酸産生菌は、酢酸非存在の培地において、酪酸を産生し、かつギ酸を産生しない菌であり、C.leptum subgroupに属する。
　前述のように、従来のC.leptum subgroupに属する酪酸産生菌は、酢酸非存在で酪酸を産生することができないか、又は酪酸とともにギ酸を産生する菌であり、本発明の酪酸産生菌は新しい特性を有する新菌種である。
　ここで、酢酸非存在とは、酢酸が存在しないか、存在する場合でも１ｍＭ以下の微量しか存在しないことを意味する。また、ギ酸を産生しないとは、ギ酸を全く産生しないか、産生する場合でも１ｍＭ未満であることを意味する。 The butyric acid-producing bacterium of the present invention is a bacterium that produces butyric acid and does not produce formic acid in a medium without acetic acid, and belongs to the C. leptum subgroup.
As described above, the butyric acid-producing bacterium belonging to the conventional C. leptum subgroup is a bacterium that cannot produce butyric acid in the absence of acetic acid or that produces formic acid together with butyric acid, and the butyric acid-producing bacterium of the present invention is a new one. It is a new bacterial species with characteristics.
Here, the absence of acetic acid means that acetic acid is not present, or even if it is present, only a trace amount of 1 mM or less is present. Moreover, not producing formic acid means that formic acid is not produced at all or even when produced, it is less than 1 mM.
　本発明の酪酸産生菌の特性は、より詳細には、菌数１．０×１０6 ｃｅｌｌｓを酢酸非存在のＹＣＧ（yeast extract-casitone glucose）培地２ｍＬに接種し、３７℃で７２時間嫌気培養した際の酪酸産生量が、１０ｍＭ以上であり、かつギ酸産生量が１ｍＭ未満であるのが好ましく、当該条件下の酪酸産生量が１０～３０ｍＭであり、かつギ酸産生量が０．７ｍＭ未満であるのがより好ましい。
　ここで菌は、菌数１．０×１０9 ｃｅｌｌｓ／ｍＬの菌株凍結保存液を融解して１μＬ程度使用することが好ましい。また、菌株凍結保存液は、菌体を１０質量％スキムミルク培地又は２０質量％グリセロール添加ミューラーヒントン培地に懸濁した溶液が好ましい。
　培養液中の酪酸、ギ酸等の有機酸濃度の測定は、有機酸濃度が測定可能な方法であれば特に限定されないが、例えば有機酸分析用ＨＰＬＣシステムで測定することができる。 More specifically, the characteristics of the butyric acid-producing bacterium of the present invention are inoculated with 2 mL of YCG (yeast extract-casitone glucose) medium in the absence of acetic acid at 1.0 × 10 6 cells and anaerobically cultured at 37 ° C. for 72 hours. The butyric acid production amount is preferably 10 mM or more, and the formic acid production amount is preferably less than 1 mM, the butyric acid production amount under the conditions is 10 to 30 mM, and the formic acid production amount is less than 0.7 mM. More preferably.
Here, it is preferable to use about 1 μL of bacteria by thawing a frozen stock solution of 1.0 × 10 9 cells / mL strain. The strain frozen stock solution is preferably a solution in which the cells are suspended in a 10% by mass skim milk medium or 20% by mass glycerol-added Mueller Hinton medium.
The measurement of the concentration of organic acids such as butyric acid and formic acid in the culture solution is not particularly limited as long as the organic acid concentration can be measured. For example, it can be measured by an HPLC system for organic acid analysis.
　また、本発明の酪酸産生菌は、酢酸を含有する培地でも酪酸を産生する。 Moreover, the butyric acid-producing bacterium of the present invention produces butyric acid even in a medium containing acetic acid.
　本発明の酪酸産生菌の具体例としては、Ｂｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８７（ＮＩＴＥ　ＢＰ－０２１０６）、Ｂｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８８（ＮＩＴＥ　ＢＰ－０２０９５）及びこれらの変異株が挙げられる。
　ＹＩＴ １２７８７及びＹＩＴ １２７８８は、〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８　１２２号室　独立行政法人製品評価技術基盤機構　特許微生物寄託センターに２０１５年８月２０日及び２０１５年７月３１日にそれぞれ寄託した。 Specific examples of the butyric acid-producing bacterium of the present invention include Butyricicoccus sp. YIT 12787 (NITE BP-02106), Butyricicoccus sp. YIT 12788 (NITE BP-02095) and their mutants.
YIT 12787 and YIT 12788 are registered in the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation on August 20, 2015 and July 31, 2015. Deposited each day.
　ＹＩＴ １２７８７及びＹＩＴ １２７８８について１６Ｓ　ｒＲＮＡシークエンス解析を行ったところ、ＹＩＴ １２７８７の１６Ｓ　ｒＲＮＡは配列番号３であり、ＹＩＴ １２７８８の１６Ｓ　ｒＲＮＡは配列番号４であった。本発明におけるＹＩＴ １２７８７及びＹＩＴ １２７８８の変異株としては、１６Ｓ　ｒＲＮＡの相同性が配列番号３及び／又は配列番号４との間で９９％以上の変異株が挙げられる。本発明において相同性とは、塩基配列の同一性をいう。 When 16S rRNA sequence analysis was performed on YIT 12787 and YIT 12788, the 16S rRNA of YIT 12787 was SEQ ID NO: 3, and the 16S rRNA of YIT 12788 was SEQ ID NO: 4. Examples of the mutant strains of YIT 12787 and YIT 12788 in the present invention include mutants having a homology of 16S rRNA of 99% or more with SEQ ID NO: 3 and / or SEQ ID NO: 4. In the present invention, homology refers to the identity of base sequences.
　ＹＩＴ １２７８７及びＹＩＴ １２７８８と近縁種とのＤＮＡの相同性を検討したところ、ＹＩＴ １２７８７とEubacterium desmolans ＡＴＣＣ ４３０５８Tとの相同性は７．５～１８．９％であり、ＹＩＴ １２７８７とButyricicoccus pullicaecorum ２５-３Tとの相同性は１０．３～１４．３％であった。また、ＹＩＴ １２７８８とEubacteriumdesmolans ＡＴＣＣ ４３０５８Tとの相同性は７．９～１７．８％であり、ＹＩＴ １２７８８とButyricicoccus pullicaecorum ２５-３Tとの相同性は１０．７～１６．５％であった。従って、本発明の酪酸産生菌は、近縁種であるEubacterium desmolans ＡＴＣＣ ４３０５８T及びButyricicoccus pullicaecorum ２５-３TとのＤＮＡの相同性が２０％以下である。 When the homology of DNA between YIT 12787 and YIT 12788 and related species was examined, the homology between YIT 12787 and Eubacterium desmolans ATCC 43058 T was 7.5 to 18.9%, and YIT 12787 and Butyricicoccus pullicaecorum 25 The homology with −3 T was 10.3-14.3%. Further, the homology between YIT 12788 and Eubacterium desmolans ATCC 43058 T was 7.9 to 17.8%, and the homology between YIT 12788 and Butyricicoccus pullicaecorum 25-3 T was 10.7 to 16.5%. . Therefore, the butyric acid-producing bacterium of the present invention has a DNA homology of 20% or less with the related species Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T.
　かかる１６Ｓ　ｒＲＮＡの解析結果から、本発明の酪酸産生菌の系統樹は図１のとおりであり、C. leptum subgroupに属する新菌種である。また、本発明の酪酸産生菌には、下記（１）及び（２）の性質を有する菌が含まれる。
　（１）１６Ｓ　ｒＲＮＡの塩基配列と配列番号３及び／又は配列番号４との相同性が９９％以上。
　（２）近縁種であるEubacterium desmolans ＡＴＣＣ ４３０５８T及びButyricicoccus pullicaecorum ２５-３TとのＤＮＡの相同性が２０％以下。 From the analysis result of 16S rRNA, the phylogenetic tree of the butyric acid-producing bacterium of the present invention is as shown in FIG. 1, which is a new bacterium species belonging to C. leptum subgroup. In addition, the butyric acid-producing bacteria of the present invention include bacteria having the following properties (1) and (2).
(1) The homology between the base sequence of 16S rRNA and SEQ ID NO: 3 and / or SEQ ID NO: 4 is 99% or more.
(2) DNA homology with the related species Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T is 20% or less.
　また、本発明の酪酸産生菌は、以下に示す菌学的性質を有する。後記実施例に示すとおり、近縁種であるEubacterium desmolans ＡＴＣＣ ４３０５８T及びButyricicoccus pullicaecorum ２５-３Tとは糖発酵性状と酵素活性性状で異なっていることからも、本発明の酪酸産生菌が、C. leptum subgroupに属する新菌種であることがわかる。 Moreover, the butyric acid-producing bacterium of the present invention has the following mycological properties. As shown in Examples below, the butyric acid-producing bacterium of the present invention is different from Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T in sugar fermentation properties and enzyme activity properties. It can be seen that this is a new species belonging to the leptum subgroup.
（１）偏性嫌気性
（２）グラム陽性球菌
（３）胞子を形成しない
（４）運動性なし
（５）カタラーゼ陰性
（６）硫化水素陰性
（７）Ｇ＋Ｃ含量が５３．９％（ＹＩＴ １２７８７）、５４．１％（ＹＩＴ １２７８８）
（８）下記の糖発酵性状を有する
　　グルコース：＋
　　乳糖：－
　　サッカロース：－
　　マルトース：－
　　サリシン：－
　　キシロース：－
　　アラビノース：－
　　ゼラチン加水分解：－
　　エスクリン加水分解：－
　　グリセロール：－
　　セロビオース：－
　　マンノース：－
　　メレチトース：－
　　ラフィノース：－
　　ラムノース：－
　　トレハロース：－
（９）下記の酵素活性性状を有する
　　アルカリフォスファターゼ：＋
　　エステラーゼ（Ｃ４）：－
　　エステラーゼリパーゼ（Ｃ８）：－
　　リパーゼ（Ｃ１４）：－
　　ロイシンアリルアミダーゼ：＋
　　バリンアリルアミダーゼ：－
　　シスチンアリルアミダーゼ：－
　　トリプシン：－
　　α－キモトリプシン：－
　　酸性フォスファターゼ：＋
　　ナフトール－AS－BI－フォスフォヒドロラーゼ：－
　　α－ガラクトシダーゼ：－
　　β－ガラクトシダーゼ：－
　　β－グルクロニダーゼ：－
　　α－グルコシダーゼ：－
　　β－グルコシダーゼ：－
　　Ｎ－アセチル－β－グルコサミニダーゼ：－
　　α－マンノシダーゼ：－
　　α－フコシダーゼ：－ (1) Obligate anaerobic (2) Gram-positive cocci (3) No spore formation (4) No motility (5) Catalase negative (6) Hydrogen sulfide negative (7) G + C content 53.9% (YIT 12787 ), 54.1% (YIT 12788)
(8) Glucose having the following sugar fermentation properties: +
lactose:-
Sucrose:-
Maltose:-
Salicin:-
Xylose:-
Arabinose:-
Gelatin hydrolysis:-
Esculin hydrolysis:-
Glycerol:-
Cellobiose:-
Mannose:-
Meletetose:-
Raffinose:-
Rhamnose:-
Trehalose:-
(9) Alkaline phosphatase having the following enzyme activity: +
Esterase (C4):-
Esterase lipase (C8):-
Lipase (C14):-
Leucine allylamidase: +
Valine allylamidase:-
Cystine allylamidase:-
Trypsin:-
α-chymotrypsin:-
Acid phosphatase: +
Naphthol-AS-BI-phosphohydrolase:-
α-Galactosidase:-
β-galactosidase:-
β-glucuronidase:-
α-Glucosidase:-
β-Glucosidase:-
N-acetyl-β-glucosaminidase:-
α-Mannosidase:-
α-fucosidase:-
　本発明の酪酸産生菌は、各種サンプルライブラリーや自然界から収集してきた微生物等を接種して培養し、生育したコロニーを釣菌して酢酸非存在の培地にて酪酸及びギ酸の産生能を測定することにより単離することができる。なお、本発明の酪酸産生菌の確認は、有機酸分析、シークエンス解析等により確認できる。 The butyric acid-producing bacterium of the present invention is inoculated and cultured with various sample libraries and microorganisms collected from nature, and the grown colonies are fished and measured for the ability to produce butyric acid and formic acid in a medium without acetic acid. Can be isolated. The confirmation of the butyric acid-producing bacterium of the present invention can be confirmed by organic acid analysis, sequence analysis, or the like.
　本発明の酪酸産生菌は、例えば、嫌気状態で作製したＧＡＭ培地やＰＹ（ペプトン・イーストエクストラクト）培地を用いて嫌気培養することにより、増殖、継代することができる。 The butyric acid-producing bacterium of the present invention can be proliferated and subcultured by anaerobic culture using a GAM medium or PY (peptone yeast extract) medium prepared in an anaerobic state, for example.
　本発明の酪酸産生菌は、安全性の問題がなく、酢酸非存在で酪酸を産生し、かつギ酸を産生しない。従って、本発明の酪酸産生菌を含有する組成物は、飲食品、医薬又は飼料用組成物として有用である。当該組成物は、ヒトを含む動物の腸内で酪酸を産生することから酪酸産生増強剤として有用である。酪酸は、前述の如く、大腸粘膜上皮細胞のエネルギー源として利用されるだけでなく、上皮細胞の増殖促進作用、抗炎症作用、腸管の運動亢進作用を有し、また大腸癌や潰瘍性大腸炎の予防治療、エネルギー代謝調節作用を有することから、本発明の組成物は、これらの生理活性を有する医薬、飲食品、飼料として特に有用である。 The butyric acid-producing bacterium of the present invention has no safety problem, produces butyric acid in the absence of acetic acid, and does not produce formic acid. Therefore, the composition containing the butyric acid-producing bacterium of the present invention is useful as a composition for foods and drinks, medicines or feeds. The composition is useful as a butyric acid production enhancer because it produces butyric acid in the intestines of animals including humans. As mentioned above, butyric acid is not only used as an energy source for colonic mucosal epithelial cells, but also has an epithelial cell growth promoting action, an anti-inflammatory action, and an intestinal motility enhancing action, as well as colon cancer and ulcerative colitis. Therefore, the composition of the present invention is particularly useful as a pharmaceutical, food, drink or feed having these physiological activities.
　本発明の組成物中には、酪酸産生菌を生菌として１０4 ｃｆｕ～１０14 ｃｆｕ含有するのが好ましい。 The composition of the present invention preferably contains 10 4 cfu to 10 14 cfu of butyric acid-producing bacteria as viable bacteria.
　本発明の組成物は、飲食品、医薬又は飼料のそれぞれに適した形態とすることができる。医薬とする場合には、例えば、固体又は液体の医薬用無毒性担体と混合して、慣用の医薬品製剤の形態とすることができる。このような製剤としては、例えば、錠剤、顆粒剤、散剤、カプセル剤等の固形剤、溶液剤、懸濁剤、乳剤等の液剤、凍結乾燥製剤等が挙げられる。これらの製剤は製剤上の常套手段により調製することができる。上記の医薬用無毒性担体としては、例えば、グルコース、乳糖、ショ糖、澱粉、マンニトール、デキストリン、脂肪酸グリセリド、ポリエチレングリコール、ヒドロキシエチルデンプン、エチレングリコール、ポリオキシエチレンソルビタン脂肪酸エステル、アミノ酸、ゼラチン、アルブミン、水、生理食塩水等が挙げられる。また、必要に応じて、安定化剤、湿潤剤、乳化剤、結合剤、等張化剤、賦形剤等の慣用の添加剤を適宜添加することもできる。 The composition of the present invention can be in a form suitable for each of foods and drinks, medicines and feeds. In the case of a medicine, for example, it can be mixed with a solid or liquid non-toxic pharmaceutical carrier to form a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means on the preparation. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, excipients and the like can be appropriately added as necessary.
　また、飲食品とする場合は、固形状、液状等のいずれの形態とすることもできる。飲食品とする場合は、そのまま、又は種々の栄養成分と共に含有せしめればよい。具体的には、飲食品として使用可能な添加剤を適宜使用し、慣用の手段を用いて食用に適した形態、すなわち、顆粒状、粒状、錠剤、カプセル、ペースト等に成形すればよい。飲食品の種類としては、例えば、ハム、ソーセージ等の食肉加工食品、かまぼこ、ちくわ等の水産加工食品、パン、菓子、バター、粉乳等の食品や、水、果汁、牛乳、清涼飲料、茶飲料等の飲料が挙げられる。また飼料とする場合も同様である。 Moreover, when it is set as food and drink, it can be in any form such as solid or liquid. What is necessary is just to make it contain with a various nutrient component as it is when setting it as food-drinks. Specifically, an additive that can be used as a food or drink is appropriately used, and it may be formed into a form suitable for food, that is, a granule, a granule, a tablet, a capsule, a paste, or the like, using a conventional means. The types of food and drink include, for example, processed meat foods such as ham and sausage, processed fishery foods such as kamaboko and chikuwa, foods such as bread, confectionery, butter and powdered milk, water, fruit juice, milk, soft drinks and tea drinks. And the like. The same applies to feed.
　本発明の組成物は、ヒトを含む動物に投与する場合、腸内で酪酸を産生させる点から、経口的又は経腸的に投与するのが好ましく、その投与量は１日あたり酪酸産生菌として生菌数で１．０×１０4 ｃｆｕ以上が好ましく、さらに１．０×１０8 ｃｆｕ～１．０×１０12 ｃｆｕがより好ましい。 When the composition of the present invention is administered to animals including humans, it is preferably administered orally or enterally from the viewpoint of producing butyric acid in the intestine, and its dosage is as a butyric acid producing bacterium per day. The number of viable bacteria is preferably 1.0 × 10 4 cfu or more, and more preferably 1.0 × 10 8 cfu to 1.0 × 10 12 cfu.
　次に実施例を挙げて本発明を詳細に説明する。 Next, the present invention will be described in detail with reference to examples.
実施例１　酪酸産生菌の分離
　出願人が保有するサンプルライブラリーより２種（Ａ、Ｂ）のサンプルを選択し、ＹＣＦＡ－Ｍ培地（ムチンを０．５％添加したＹＣＦＡ（酢酸２０ｍＭ、プロピオン酸５ｍＭ）培地）１ｍＬに各サンプルの１０倍希釈液を１０μＬ接種し、３７℃、８時間嫌気培養後、４，０００Ｇ、５分間遠心後の上清１０μＬを新しいＹＣＦＡ－Ｍ培地２ｍＬに接種した。この操作を７回繰り返し、培養後の菌液を１０-6まで１０倍段階希釈した後、１０-4～１０-6の各希釈液を１％グルコース添加変法ＧＡＭ寒天培地に塗抹し、３７℃、２～３日間嫌気培養した。培養後のコロニーを形態別に分類し、形態ごとに８割以上のコロニー数を釣菌し、１％グルコース添加ＧＡＭブロスを用いて増菌した。３回の単コロニー分離を行った後、－８０℃に保存した。
　その結果、２菌株を単離し、それぞれ菌株Ａ及び菌株Ｂと仮称した。 Example 1 Isolation of Butyrate-Producing Bacteria Two types (A, B) of samples were selected from the sample library owned by the applicant, and YCFA-M medium (YCFA supplemented with 0.5% mucin (acetic acid 20 mM, propionic acid) was selected. 1 mL of 5 mM) medium) was inoculated with 10 μL of a 10-fold diluted solution of each sample. After anaerobic culture at 37 ° C. for 8 hours, 10 μL of the supernatant after centrifugation at 4,000 G for 5 minutes was inoculated into 2 mL of new YCFA-M medium. This operation was repeated 7 times after bacterial solution 10-fold serially diluted up to 10 -6 after culture, smeared 10-4 of each dilution to 10 -6 to 1% glucose added variant GAM agar culture medium, 37 Anaerobic culture was performed at 2 ° C. for 2-3 days. The colonies after the culture were classified according to the form, and the number of colonies of 80% or more was picked for each form, and enriched using 1% glucose-added GAM broth. After performing single colony separation three times, it was stored at −80 ° C.
As a result, two strains were isolated and temporarily named strain A and strain B, respectively.
実施例２　菌株Ａ及び菌株Ｂの酪酸及びギ酸産生能
（１）既知菌株との酪酸及びギ酸産生能の比較
　菌株Ａ及び菌株Ｂについて、酢酸非存在の培地におけるC. leptum subgroupに属する菌を含む既知の酪酸産生菌１８菌株（表１）との酪酸産生量の比較を行った。
　菌株Ａ、菌株Ｂ及び既知の酪酸産生菌１８菌株の凍結保存液（菌体を１０％スキムミルク培地又は２０％グリセロール添加ミューラーヒントン培地に懸濁した溶液）（菌数：１．０×１０9 ｃｅｌｌｓ／ｍＬ）を融解し、その１μＬ（菌数：１．０×１０6 ｃｅｌｌｓ）を酢酸非存在のＹＣＧ（yeast extract-casitone glucose）培地（当該培地にはギ酸、酢酸、酪酸等の有機酸は存在せず、０．０ｍＭである）２ｍＬに接種し、３７℃で７２時間嫌気培養した後、上清に１％濃度になるよう過塩素酸を添加し、一晩４℃に静置した。遠心後の上清を０．４５μｍのフィルターでろ過し、ろ液中の有機酸量を後述する有機酸分析用ＨＰＬＣシステムを用いて測定した。なお、標準物質にはコハク酸、蟻酸ナトリウム、酢酸ナトリウム、プロピオン酸ナトリウム、イソ酪酸ナトリウム、ｎ－酪酸ナトリウム、イソ吉草酸ナトリウム、ｎ－吉草酸ナトリウム及び乳酸リチウムの計９種類（全て関東化学株式会社製のＨＰＬＣグレード）の混合水溶液を用い、２点（０．０１及び０．２μｍｏｌ／１０μＬ）絶対検量線法で定量した。 Example 2 Butyric acid and formic acid producing ability of strain A and strain B (1) Comparison of butyric acid and formic acid producing ability with known strains For strain A and strain B, including bacteria belonging to C. leptum subgroup in a medium without acetic acid The amount of butyric acid produced was compared with 18 known butyric acid producing bacteria (Table 1).
Cryopreservation solution of strain A, strain B and 18 known butyric acid producing bacteria (solution in which cells are suspended in Mueller Hinton medium containing 10% skim milk medium or 20% glycerol) (Number of bacteria: 1.0 × 10 9 cells) 1 mL (bacterial count: 1.0 × 10 6 cells) of YCG (yeast extract-casitone glucose) medium (acetic acid-free organic acids such as formic acid, acetic acid and butyric acid) After inoculating 2 mL (not present and 0.0 mM) and anaerobically culturing at 37 ° C. for 72 hours, perchloric acid was added to the supernatant to a concentration of 1% and left at 4 ° C. overnight. The supernatant after centrifugation was filtered through a 0.45 μm filter, and the amount of organic acid in the filtrate was measured using an organic acid analysis HPLC system described later. There are 9 types of standard substances: succinic acid, sodium formate, sodium acetate, sodium propionate, sodium isobutyrate, n-sodium butyrate, sodium isovalerate, n-sodium valerate and lithium lactate (all Kanto Chemical Co., Ltd.) Using a mixed aqueous solution of HPLC grade manufactured by the company, quantification was performed by a two-point (0.01 and 0.2 μmol / 10 μL) absolute calibration curve method.
有機酸分析用HPLCシステム
　分析機器：２６９５アライアンスシステム、反応ポンプ、４３２電気伝導度測定器、カラム温度コントロールシステム（以上Ｗａｔｅｒｓ）
　カラム：有機酸分析用カラムShodex　ＫＣ－８１１（昭和電工）
　溶離液：１５ｍＭ過塩素酸＋７％アセトニトリル
　ｐＨ調整剤：１５ｍＭ過塩素酸＋７％アセトニトリル＋６０ｍＭトリスヒドロキシメチルアミノメタン
　カラム温度：４２℃
　セル温度：４５℃
　流速：１ｍＬ／ｍｉｎ HPLC system for organic acid analysis Analytical instruments: 2695 Alliance system, reaction pump, 432 conductivity meter, column temperature control system (Waters)
Column: Organic acid analysis column Shodex KC-811 (Showa Denko)
Eluent: 15 mM perchloric acid + 7% acetonitrile pH adjuster: 15 mM perchloric acid + 7% acetonitrile + 60 mM trishydroxymethylaminomethane Column temperature: 42 ° C.
Cell temperature: 45 ° C
Flow rate: 1 mL / min
　結果を表１に示す。菌株Ａ及び菌株Ｂの酪酸産生量は、１０ｍＭ以上であった。一方、Roseburia intestinalis ＤＳＭ １４６１０T及びClostridium butyricum ＪＣＭ １３９１Tの２菌株を除く１６菌株では、酢酸非存在の培地では酪酸の産生量は非常に少ないか又は産生しなかった。
　また、酪酸を１０ｍＭ以上産生する既知の２菌株については、腐食性や毒性があり、生体内で産生されるのが好ましくないギ酸を１ｍＭ以上産生した。一方、菌株Ａ及び菌株Ｂは、酢酸非存在の培地ではギ酸の産生量が１ｍＭ未満であり、ギ酸を産生しなかった。 The results are shown in Table 1. The amount of butyric acid produced by strain A and strain B was 10 mM or more. On the other hand, in 16 strains except 2 strains of Roseburia intestinalis DSM 14610 T and Clostridium butyricum JCM 1391 T , butyric acid was produced in a small amount or not in a medium without acetic acid.
Moreover, about 2 known strains producing butyric acid at 10 mM or more, formic acid was produced at 1 mM or more which is corrosive and toxic and is not preferable to be produced in vivo. On the other hand, strain A and strain B produced formic acid in a medium without acetic acid in an amount of less than 1 mM and did not produce formic acid.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（２）各種培地での菌株Ａ及び菌株Ｂの有機酸産生能
　菌株Ａ及び菌株ＢをＭ２ＧＳＣ（ギ酸０．９ｍＭ、酢酸０．９ｍＭ、イソ吉草酸２．３ｍＭ）、Ｍ２ＧＳＣ＋ＳＣＦＡ（ギ酸０．６ｍＭ、酢酸２０ｍＭ、プロピオン酸６ｍＭ、イソ吉草酸１．８ｍＭ）及びＹＣＦＡ（酢酸２０ｍＭ、プロピオン酸５ｍＭ）の各培地２ｍＬに対して、５×１０7ｃｅｌｌｓの菌数を接種し、それぞれ１週間、３７℃で培養後（培養後の菌数；５×１０9ｃｅｌｌｓ／ｍＬ）、実施例２の（１）記載の有機酸分析方法を用いて培養液中の酪酸濃度を測定した。 (2) Organic acid production ability of strain A and strain B in various media Strains A and B were prepared using M2GSC (0.9 mM formic acid, 0.9 mM acetic acid, 2.3 mM isovaleric acid), M2GSC + SCFA (0.6 mM formic acid, Acetic acid 20 mM, propionic acid 6 mM, isovaleric acid 1.8 mM) and YCFA (acetic acid 20 mM, propionic acid 5 mM) 2 mL of each medium were inoculated with 5 × 10 7 cells, each at 37 ° C. for 1 week. Then, the butyric acid concentration in the culture broth was measured using the organic acid analysis method described in Example 1 (1) (after culturing (number of bacteria after culturing; 5 × 10 9 cells / mL)).
　各種培地を用いた菌株Ａ及び菌株Ｂの有機酸産生能の結果を表２に示した。菌株Ａ及び菌株Ｂは、酢酸非存在のＭ２ＧＳＣ培地では、１０ｍＭを超える酪酸を産生する一方で、ギ酸の産生量は１ｍＭ未満であり、ギ酸を産生しなかった。また、酢酸を添加したＭ２ＧＳＣ＋ＳＣＦＡ及びＹＣＦＡ培地では２０ｍＭ以上の酪酸を産生した。なお、Ｍ２ＧＳＣ＋ＳＣＦＡ及びＹＣＦＡ培地では、培養後の酢酸の濃度が当初含有量よりも大幅に低下した（表２）ことから、培地中の酢酸を利用して酪酸を産生しているものと考えられた。 Table 2 shows the results of the organic acid producing ability of strain A and strain B using various media. Strains A and B produced butyric acid in excess of 10 mM in the M2GSC medium without acetic acid, while the amount of formic acid produced was less than 1 mM and did not produce formic acid. Further, 20 mM or more of butyric acid was produced in the M2GSC + SCFA and YCFA medium supplemented with acetic acid. In the M2GSC + SCFA and YCFA media, the concentration of acetic acid after culturing was significantly lower than the initial content (Table 2), so it was considered that butyric acid was produced using acetic acid in the media. .
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
実施例３　菌株Ａ及び菌株Ｂの１６Ｓ　ｒＲＮＡシークエンス解析
　菌株Ａ及び菌株Ｂについて、ビーズフェノール法によりＤＮＡを抽出した。すなわち、菌株Ａ及び菌株Ｂの菌液２００μＬを１．０ｍＬのＰＢＳに懸濁し、１５，０００ｒｐｍで遠心分離後上清を捨てるという操作を３回繰り返した。得られたペレットをまず４５０μＬのExtraction Buffer（１００ｍＭ　Ｔｒｉｓ－ＨＣｌ、４０ｍＭ　ＥＤＴＡ、ｐＨ９．０）と５０μＬの１０％ＳＤＳに浮遊し、０．３ｇのガラスビーズ（直径０．１ｍｍ）と５００μＬのＴＥ飽和フェノールを加えてFastPrep　ＦＰ１２０（パワーレベル５．０）により３０秒間激しく振とうした。１５，０００ｒｐｍで１０分間遠心した後、イソプロパノール沈殿を行って、得られたＤＮＡを１，０００μＬのＴＥバッファーに溶解した。得られたＤＮＡ溶液を精製水で１０倍希釈し、鋳型ＤＮＡ溶液とした。
　鋳型ＤＮＡ溶液に対して、表３に示す８Ｆ及び１５Ｒプライマーを用いて、９５℃５分で反応後、９５℃３０秒、５５℃３０秒、７２℃１分３０秒を１サイクルとして、３５サイクルのＰＣＲ反応を行い、その後７２℃５分反応を行った。ＰＣＲ産物をHigh Pure PCR Product Purification Kit(Roche)を用いて精製し、BigDye Terminator v3.1 Cycle Sequence Kit(Applied Biosystems)によるシークエンス反応に供した。得られた配列は日本ＤＮＡデータバンク（ＤＤＢＪ）のＢＬＡＳＴ検索に供し、既知菌種の配列データベースと照合した。さらに、Clustal  Ｗを用いた近隣結合（ＮＪ）法で分離株の配列を系統解析し、Tree-Viewプログラムを用いて系統樹を作成した。 Example 3 16S rRNA sequence analysis of strain A and strain B For strain A and strain B, DNA was extracted by the bead phenol method. That is, the operation of suspending 200 μL of the bacterial solution of strain A and strain B in 1.0 mL of PBS, centrifuging at 15,000 rpm, and discarding the supernatant was repeated three times. The obtained pellet was first suspended in 450 μL of Extraction Buffer (100 mM Tris-HCl, 40 mM EDTA, pH 9.0) and 50 μL of 10% SDS, 0.3 g of glass beads (diameter 0.1 mm) and 500 μL of TE saturated. Phenol was added and shaken vigorously for 30 seconds with FastPrep FP120 (power level 5.0). After centrifugation at 15,000 rpm for 10 minutes, isopropanol precipitation was performed, and the obtained DNA was dissolved in 1,000 μL of TE buffer. The obtained DNA solution was diluted 10 times with purified water to obtain a template DNA solution.
Using the 8F and 15R primers shown in Table 3 for the template DNA solution, after reacting at 95 ° C for 5 minutes, 95 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute 30 seconds, 35 cycles The PCR reaction was performed, followed by a reaction at 72 ° C. for 5 minutes. The PCR product was purified using High Pure PCR Product Purification Kit (Roche) and subjected to a sequencing reaction using BigDye Terminator v3.1 Cycle Sequence Kit (Applied Biosystems). The obtained sequence was subjected to a BLAST search of the Japan DNA Data Bank (DDBJ) and collated with a sequence database of known bacterial species. Furthermore, phylogenetic analysis was performed on the sequence of the isolates by the neighbor joining (NJ) method using Clustal W, and a phylogenetic tree was created using the Tree-View program.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　作成した系統樹を図１に示す。
　１６Ｓ　ｒＲＮＡシークエンス解析及び系統解析の結果、菌株Ａは１，４９５ ｂｐ（配列番号３）、菌株Ｂは１，５００ ｂｐ（配列番号４）であった。また、両菌株はお互いに９９％以上の相同性を有していた。
　系統解析の結果、菌株Ａ及び菌株Ｂは、C. leptum subgroupに属し、既知菌種であるEubacterium desmolans ＡＴＣＣ ４３０５８T 及びButyricicoccus pullicaecorum ２５－３T と近縁であると考えられた。また、菌株ＡとEubacterium desmolans ＡＴＣＣ ４３０５８Tとの相同性は９４．９％、Butyricicoccus pullicaecorum ２５－３Tとの相同性は９６．３％であった。菌株ＢとEubacterium desmolans ＡＴＣＣ ４３０５８Tとの相同性は９５．０％、Butyricicoccus pullicaecorum ２５－３Tとの相同性は９６．３％であった。
　同菌種であれば１６Ｓ　ｒＲＮＡの相同性は９９％以上となるが、菌株Ａ及び菌株Ｂは、既知菌種との相同性が９９％未満であるため、C. leptum subgroupに属する新菌種であると考えられた。菌株ＡをＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ．　ＹＩＴ １２７８７、菌株ＢをＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ．　ＹＩＴ １２７８８と命名し、さらに新菌種に属するこれら菌株の性質を確認した。 The created phylogenetic tree is shown in FIG.
As a result of 16S rRNA sequence analysis and phylogenetic analysis, the strain A was 1,495 bp (SEQ ID NO: 3) and the strain B was 1,500 bp (SEQ ID NO: 4). Both strains had 99% or more homology with each other.
As a result of phylogenetic analysis, strain A and strain B belonged to C. leptum subgroup and were considered to be closely related to Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T , which are known bacterial species. The homology between strain A and Eubacterium desmolans ATCC 43058 T was 94.9%, and the homology between Butyricicoccus pullicaecorum 25-3 T was 96.3%. The homology between strain B and Eubacterium desmolans ATCC 43058 T was 95.0%, and the homology between Butyricicoccus pullicaecorum 25-3 T was 96.3%.
If the same strain, the homology of 16S rRNA is 99% or more, but since strain A and strain B have less than 99% homology with known strains, new strains belonging to C. leptum subgroup It was thought that. Strain A was transformed into Butyricicoccus sp. YIT 12787, strain B was transformed into Butyricicoccus sp. It was named YIT 12788 and the properties of these strains belonging to the new strain were confirmed.
実施例４　各種生化学的性状
（１）糖発酵性状試験
　アピケンキ（シスメックス・ビオメリュー）を用い、添付説明書に従って実施した。使用菌株は、ＹＩＴ １２７８７、ＹＩＴ １２７８８のほか、近縁種であるEubacterium desmolans ＡＴＣＣ ４３０５８T(E.desmolans)及びButyricicoccus pullicaecorum ２５－３T (B.pullicaecorum)を用いた。結果判定は、菌液接種３０時間後に目視により行った。
　糖発酵性状試験の結果を表４に示す。ＹＩＴ １２７８７及びＹＩＴ １２７８８はグルコース資化性が陽性であった。E.desmolansでは、すべて陰性であった。B.pullicaecorumでは、グルコース、サッカロース、サリシン及びキシロースの資化性、エスクリンの加水分解が陽性であった。 Example 4 Various biochemical properties (1) Sugar fermentation property test Using Apikenki (Sysmex Biomelieu), it was carried out according to the attached instructions. In addition to YIT 12787 and YIT 12788, strains used were Eubacterium desmolans ATCC 43058 T (E. desmolans) and Butyricicoccus pullicaecorum 25-3 T (B. pulllicaecorum). The result was determined visually after 30 hours inoculation with the bacterial solution.
The results of the sugar fermentation property test are shown in Table 4. YIT 12787 and YIT 12788 were positive for glucose utilization. All of E. desmolans were negative. In B.pullicaecorum, assimilation of glucose, saccharose, salicin and xylose and hydrolysis of esculin were positive.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（２）酵素活性性状試験
　アピザイム（シスメックス・ビオメリュー）を用い、添付説明書に従って実施した。使用菌株は、ＹＩＴ １２７８７、ＹＩＴ １２７８８のほか、近縁種であるE.desmolans及びB.pullicaecorumを用いた。結果判定は、菌液接種５時間後に色調の変化を目視により５段階で評価した。
　酵素活性試験の結果を表５に示す。表中の数値が３以上を陽性反応とした。ＹＩＴ １２７８７及びＹＩＴ １２７８８はアルカリフォスファターゼ、ロイシンアリルアミダーゼ及び酸性フォスファターゼ活性が陽性であった。E.desmolansでは、エステラーゼ（Ｃ４）及び酸性フォスファターゼ活性が、B.pullicaecorumでは、アルカリフォスファターゼ、エステラーゼ（Ｃ４）、ロイシンアリルアミダーゼ及び酸性フォスファターゼ活性が陽性であった。 (2) Enzyme activity property test Using Apizza Im (Sysmex Biomelieu), it was carried out according to the attached instructions. In addition to YIT 12787 and YIT 12788, closely related species, E. desmolans and B. pulllicaecorum, were used. As a result, the change in color tone was visually evaluated in 5 stages 5 hours after inoculation with the bacterial solution.
The results of the enzyme activity test are shown in Table 5. A value of 3 or more in the table was regarded as a positive reaction. YIT 12787 and YIT 12788 were positive for alkaline phosphatase, leucine allylamidase and acid phosphatase activities. In E. desmolans, esterase (C4) and acid phosphatase activities were positive, and in B. pulllicaecorum, alkaline phosphatase, esterase (C4), leucine allylamidase and acid phosphatase activities were positive.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　前述の（１）及び（２）の結果より、ＹＩＴ １２７８７及びＹＩＴ １２７８８は、E.desmolansとは、グルコースの資化性、並びにアルカリフォスファターゼ、エステラーゼ（Ｃ４）及びロイシンアリルアミダーゼ活性の有無で鑑別可能であった。また、B.pullicaecorumとは、サッカロース、サリシン及びキシロースの資化性、並びにエスクリンの加水分解能、エステラーゼ（Ｃ４）活性の有無で鑑別可能であった。 From the results of (1) and (2) above, YIT 12787 and YIT 12788 can be distinguished from E. desmolans by the availability of glucose and the presence or absence of alkaline phosphatase, esterase (C4) and leucine allylamidase activity. Met. Moreover, it was distinguishable from B.pullicaecorum by the availability of saccharose, salicin and xylose, the hydrolytic ability of esculin, and the presence or absence of esterase (C4) activity.
（３）菌体ＤＮＡのＧＣ含量測定
　ＹＣＦＡＧ培地（ＹＣＦＡに１％グルコースを添加した培地、ＹＩＴ １２７８７、ＹＩＴ １２７８８及びB.pullicaecorumの培養に使用）又は１％イノシトール添加ＧＡＭ培地（E.desmolansの培養に使用）に、１×１０9ｃｅｌｌｓ／ｍＬの各菌株液を培地量に対して１／１０００量接種し、１３～１８時間培養し、対数増殖期の菌体1～2ｇを回収し、直ちに終濃度１％のパラホルムアルデヒド-ＰＢＳ溶液で２日間固定した。固定後の菌体を５ｍＭ ＥＤＴＡ溶液で３回洗浄した後、６ｍＬの同溶液に再懸濁し、６００Ｕ/ｍＬアクロモペプチダーゼ及び５０ｍｇ/ｍＬリゾチームをそれぞれ１ｍＬずつ添加して３７℃、２時間溶菌した。溶菌後の試料にＳＤＳ溶液を０．６ｍＬを加えて６０℃で１０－３０分処理した。さらに１０ｍｇ/ｍＬ プロテイナーゼＫを１５０μＬ加えて６５℃、６時間処理を行った後、フェノール/クロロホルム/イソアミルアルコール（２５：２４：１）を２０ｍＬ加えて、３０分間振とうした。８，０００Ｇ、１５分間遠心後の上清を回収し、クロロホルム/イソアミルアルコール（２４：１）を１６ｍＬ加え、３０分間振とう後、８，０００Ｇ、１５分間遠心して上清を回収した。クロロホルム/イソアミルアルコール処理を再度行い、回収した上清からエタノール処理により粗ＤＮＡを得た。粗ＤＮＡをTEバッファー適量に溶解し、ＲＮａｓｅ溶液（８０℃、５分間加熱処理した１ｍｇ/ｍＬのＲＮａｓｅＡ　１ｍＬにＲＮａｓｅＴ１　４μＬを加えたもの）をＤＮＡ溶液量に対して１/２０量加え、３７℃、１時間処理を行い、フェノール/クロロホルム/イソアミルアルコール（２５：２４：１）及びクロロホルム/イソアミルアルコール（２４：１）処理による除蛋白後、エタノール処理を行い、精製ＤＮＡ溶液を調製した。調製したＤＮＡサンプルについて、２６０ｎｍと２８０ｎｍの吸光度の確認、及びＱｕａｎｔ－ｉＴ ＰｉｃｏＧｒｅｅｎ ｄｓＤＮＡ Ａｓｓａｙ Ｋｉｔ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いた濃度測定を行った。上記のＤＮＡサンプルについて、ＴｒｕＳｅｑ　ＤＮＡ　ＰＣＲ-Ｆｒｅｅ　Ｓａｍｐｌｅ　Ｐｒｅｐ　ＬＳ　Ｋｉｔ（Ｉｌｌｕｍｉｎａ）及びＤＮＡ　Ｓｈｅａｒｉｎｇ　システム　Ｍ２２０（Ｃｏｖａｒｉｓ）を用い、Ｉｌｌｕｍｉｎａより提供されたプロトコルに従ってシーケンスライブラリの調製を行った。調製したシーケンスライブラリを、次世代シーケンサーＭｉＳｅｑ（Ｉｌｌｕｍｉｎａ）による２５０ｂｐ×２のペアエンドシーケンスに供試し、塩基配列の解読を行い、得られた塩基配列の結果を基にＧＣ％を算出した。
　その結果、菌体ＤＮＡのＧＣ含量は、ＹＩＴ １２７８７が５３．９％、ＹＩＴ １２７８８が５４．１％、近縁種のE.desmolansが５４．３％、B.pullicaecorumが５４．０％であった。 (3) Measurement of GC content of bacterial DNA YCFAG medium (medium supplemented with 1% glucose in YCFA, used for cultivation of YIT 12787, YIT 12788 and B.pullicaecorum) or GAM medium supplemented with 1% inositol (culture of E. desmolans 1 × 10 9 cells / mL of each strain solution was inoculated in 1/1000 volume of the medium volume, cultured for 13-18 hours, and 1-2 g of cells in logarithmic growth phase were collected and immediately It was fixed with a paraformaldehyde-PBS solution having a final concentration of 1% for 2 days. The cells after fixation were washed 3 times with 5 mM EDTA solution, resuspended in 6 mL of the same solution, 1 mL each of 600 U / mL achromopeptidase and 50 mg / mL lysozyme were added, and the cells were lysed at 37 ° C. for 2 hours. . To the sample after lysis, 0.6 mL of SDS solution was added and treated at 60 ° C. for 10-30 minutes. Further, 150 μL of 10 mg / mL proteinase K was added and treated at 65 ° C. for 6 hours, and then 20 mL of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added and shaken for 30 minutes. The supernatant after centrifugation at 8,000 G for 15 minutes was recovered, 16 mL of chloroform / isoamyl alcohol (24: 1) was added, shaken for 30 minutes, and then centrifuged at 8,000 G for 15 minutes to recover the supernatant. Chloroform / isoamyl alcohol treatment was performed again, and crude DNA was obtained from the collected supernatant by ethanol treatment. Dissolve the crude DNA in an appropriate amount of TE buffer, add RNase solution (1 mg / mL RNase A heated at 80 ° C. for 5 minutes, 4 mL of RNase T1) to the DNA solution, and add 1/20 amount to 37 ° C. After treatment for 1 hour, deproteinization by treatment with phenol / chloroform / isoamyl alcohol (25: 24: 1) and chloroform / isoamyl alcohol (24: 1) was followed by ethanol treatment to prepare a purified DNA solution. About the prepared DNA sample, the light absorbency of 260 nm and 280 nm was confirmed, and the density | concentration measurement was performed using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). For the above DNA sample, a sequence library was prepared using TruSeq DNA PCR-Free Sample Prep LS Kit (Illumina) and DNA Shearing System M220 (Covaris) according to the protocol provided by Illumina. The prepared sequence library was subjected to a 250 bp × 2 paired end sequence by the next-generation sequencer MiSeq (Illumina), the base sequence was decoded, and GC% was calculated based on the obtained base sequence result.
As a result, the GC content of the bacterial DNA was 53.9% for YIT 12787, 54.1% for YIT 12788, 54.3% for E. desmolans, and 54.0% for B.pullicaecorum. It was.
（４）E.desmolans及びB.pullicaecorumとのＤＮＡ-ＤＮＡ相同性
　ＹＩＴ １２７８７及びＹＩＴ １２７８８と、近縁種であるE.desmolans及びB.pullicaecorumとの、ＤＮＡ-ＤＮＡ相同性を確認するために、実施例４の（３）の方法で得たＤＮＡサンプルをマイクロプレートに固定し、フォトビオチンで標識したＤＮＡとのハイブリダイゼーション反応を行い、反応後の蛍光強度の数値から、各菌株間のＤＮＡ－ＤＮＡ相同性を算出した。
　結果を表６に示す。ＹＩＴ １２７８７とＹＩＴ １２７８８間の相同性は６７．３－７０．９％であり、同一菌種（亜種を含む）であることが示された。一方、ＹＩＴ １２７８７及びＹＩＴ １２７８８と近縁種であるE.desmolans及びB.pullicaecorumとのＤＮＡの相同性は、いずれも２０％未満であり、別菌種であることが示された。よって、ＤＮＡ－ＤＮＡ相同性の結果からもＹＩＴ １２７８７及びＹＩＴ １２７８８が新菌種であることが示された。 (4) DNA-DNA homology with E. desmolans and B. pulllicaecorum To confirm DNA-DNA homology between YIT 12787 and YIT 12788 and E. desmolans and B. pulllicaecorum, which are closely related species, The DNA sample obtained by the method of Example 4 (3) was immobilized on a microplate and subjected to a hybridization reaction with DNA labeled with photobiotin. From the values of fluorescence intensity after the reaction, the DNA- DNA homology was calculated.
The results are shown in Table 6. The homology between YIT 12787 and YIT 12788 was 67.3-70.9%, indicating the same bacterial species (including subspecies). On the other hand, the DNA homology between YIT 12787 and YIT 12788 and E. desmolans and B. pulllicaecorum, which are closely related species, was less than 20%, indicating that they were different species. Therefore, the results of DNA-DNA homology also showed that YIT 12787 and YIT 12788 are new strains.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（５）その他の生化学的性状
　ＹＩＴ １２７８７及びＹＩＴ １２７８８は、以下の共通の生化学的性状を有する。
　　（ｉ）偏性嫌気性
　　（ii）グラム陽性球菌
　　（iii）胞子を形成しない
　　（iv）運動性なし
　　（ｖ）カタラーゼ陰性
　　（vi）硫化水素陰性 (5) Other biochemical properties YIT 12787 and YIT 12788 have the following common biochemical properties.
(I) Obligate anaerobic (ii) Gram-positive cocci (iii) No spore formation (iv) No motility (v) Catalase negative (vi) Hydrogen sulfide negative
実施例５　新たな単離株の性質
　出願人が保有するサンプルライブラリーより１種（Ｃ）のサンプルを選択し、実施例１に記載の方法で菌株Ｃを単離した。
　菌株Ｃの１６Ｓ　ｒＲＮＡシークエンス解析を実施例３の方法で行ったところ、菌株ＣとＹＩＴ １２７８７とは９９．１％、菌株ＣとＹＩＴ １２７８８とは９９．３％の１６Ｓ　ｒＲＮＡの相同性を有しており、ともに９９％以上であった。
　また、菌株ＣのE.desmolans及びB.pullicaecorumとのＤＮＡ-ＤＮＡ相同性を実施例４の（４）の方法で測定したところ、菌株ＣとE.desmolansとの相同性は８．８～１８．９％であり、菌株ＣとB.pullicaecorumとの相同性は１０．６～１５．５％であった。よって、菌株ＣとE.desmolans及びB.pullicaecorumとのＤＮＡ-ＤＮＡ相同性はともに２０％以下であった。 Example 5 Properties of New Isolate One strain (C) was selected from the sample library owned by the applicant, and strain C was isolated by the method described in Example 1.
When the 16S rRNA sequence analysis of strain C was carried out by the method of Example 3, strain C and YIT 12787 had 99.1%, and strain C and YIT 12788 had 99.3% homology of 16S rRNA. Both were 99% or more.
Further, when the DNA-DNA homology of strain C with E. desmolans and B. pulllicaecorum was measured by the method of Example 4 (4), the homology between strain C and E. desmolans was 8.8-18. The homology between strain C and B.pullicaecorum was 10.6 to 15.5%. Therefore, the DNA-DNA homology between strain C and E. desmolans and B. pulllicaecorum was both 20% or less.
　菌株Ｃの酪酸及びギ酸の産生能を実施例２の（１）の方法で測定した。その結果、菌株Ｃの酪酸産生量は１３．３ｍＭであり、ギ酸の産生量は０．０ｍＭであった。
　よって、ＹＩＴ １２７８７及びＹＩＴ １２７８８の１６Ｓ　ｒＲＮＡとの相同性が９９％以上であり、また、E.desmolans及びB.pullicaecorumとのＤＮＡの相同性が２０％未満である菌株Ｃは、菌数１．０×１０6ｃｅｌｌｓを酢酸非存在のＹＣＧ培地２ｍＬに接種し、３７℃で７２時間嫌気培養した際の酪酸産生量が１０ｍＭ以上であり、かつギ酸産生量が１ｍＭ未満である、C.leptum subgroupに属する酪酸産生菌であることが分かった。 The ability of strain C to produce butyric acid and formic acid was measured by the method of Example 2 (1). As a result, the amount of butyric acid produced by strain C was 13.3 mM, and the amount of formic acid produced was 0.0 mM.
Therefore, the strain C in which the homology of YIT 12787 and YIT 12788 with 16S rRNA is 99% or more and the DNA homology with E. desmolans and B. pulllicaecorum is less than 20% is 1. C. leptum subgroup in which 0 × 10 6 cells are inoculated into 2 mL of acetic acid-free YCG medium and anaerobic culture at 37 ° C. for 72 hours has a butyric acid production of 10 mM or more and a formic acid production of less than 1 mM It was found to be a butyric acid-producing bacterium belonging to
実施例６　錠剤の製造
　下記表７の処方で各種成分を混合して造粒・乾燥・整粒した後に、打錠して錠剤を製造した。 Example 6 Manufacture of tablets Various ingredients were mixed according to the formulation shown in Table 7 below, granulated, dried and sized, and then tableted to produce tablets.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
１）ＹＩＴ １２７８７の生菌体を凍結乾燥して製造した（生菌体１０10個／ｇを含む）。 1) A live cell of YIT 12787 was produced by freeze-drying (including 10 10 cells / g of live cell).

Claims (7)

  1. 　酢酸非存在の培地において、酪酸を産生し、かつギ酸を産生しない、Clostridium leptum subgroupに属する酪酸産生菌。 A butyric acid-producing bacterium belonging to Clostridium ept leptum subgroup that produces butyric acid and does not produce formic acid in a medium without acetic acid.
  2. 　菌数１．０×１０6 ｃｅｌｌｓを酢酸非存在のＹＣＧ培地２ｍＬに接種し、３７℃で７２時間嫌気培養した際の酪酸産生量が、１０ｍＭ以上であり、かつギ酸産生量が１ｍＭ未満である請求項１記載の酪酸産生菌。 Butyrate production is 10 mM or more and formic acid production is less than 1 mM when inoculating 2 mL of YCG medium without acetic acid with 1.0 × 10 6 cells of bacteria and anaerobically culturing at 37 ° C. for 72 hours The butyric acid-producing bacterium according to claim 1.
  3. 　下記の（１）及び（２）の性質を有する請求項１又は２記載の酪酸産生菌。
    　（１）１６Ｓ　ｒＲＮＡの塩基配列と配列番号３及び／又は配列番号４との相同性が９９％以上
    　（２）近縁種であるEubacterium desmolans  ＡＴＣＣ ４３０５８T及びButyricicoccus pullicaecorum　２５－３TとのＤＮＡの相同性が２０％以下     The butyric acid-producing bacterium according to claim 1 or 2, which has the following properties (1) and (2).
    (1) The homology between the base sequence of 16S rRNA and SEQ ID NO: 3 and / or SEQ ID NO: 4 is 99% or more. (2) DNA of the closely related species Eubacterium desmolans ATCC 43058 T and Butyricicoccus pullicaecorum 25-3 T Less than 20% homology
  4. 　ＮＩＴＥ　ＢＰ－０２１０６として寄託されたＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８７、ＮＩＴＥ　ＢＰ－０２０９５として寄託されたＢｕｔｙｒｉｃｉｃｏｃｃｕｓ　ｓｐ． ＹＩＴ １２７８８又はそれらの変異株である請求項１～３のいずれか１項記載の酪酸産生菌。 Butyricicoccus sp. Deposited as NITE BP-02106 YIT 12787, Butyricicoccus sp. Deposited as NITE BP-02095. The butyric acid-producing bacterium according to any one of claims 1 to 3, which is YIT 12788 or a mutant thereof.
  5. 　請求項１～４のいずれか１項記載の酪酸産生菌を含有することを特徴とする酪酸産生増強剤。 A butyric acid production enhancer comprising the butyric acid-producing bacterium according to any one of claims 1 to 4.
  6. 　請求項１～４のいずれか１項記載の酪酸産生菌を含有する飲食品、医薬又は飼料用組成物。 A food, beverage, pharmaceutical or feed composition containing the butyric acid-producing bacterium according to any one of claims 1 to 4.
  7. 　請求項１～４のいずれか１項記載の酪酸産生菌の飲食品、医薬又は飼料用組成物製造のための使用。 Use of the butyric acid-producing bacterium according to any one of claims 1 to 4 for producing a composition for food, drink, medicine or feed.
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