JPWO2020013027A1 - 8-Method for producing prenylnaringenin - Google Patents
8-Method for producing prenylnaringenin Download PDFInfo
- Publication number
- JPWO2020013027A1 JPWO2020013027A1 JP2020530128A JP2020530128A JPWO2020013027A1 JP WO2020013027 A1 JPWO2020013027 A1 JP WO2020013027A1 JP 2020530128 A JP2020530128 A JP 2020530128A JP 2020530128 A JP2020530128 A JP 2020530128A JP WO2020013027 A1 JPWO2020013027 A1 JP WO2020013027A1
- Authority
- JP
- Japan
- Prior art keywords
- prenylnaringenin
- isoxanthohumol
- microorganisms
- blautia
- brautia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 244000005700 microbiome Species 0.000 claims abstract description 60
- YHWNASRGLKJRJJ-UHFFFAOYSA-N sophoraflavanone B Natural products C1C(=O)C2=C(O)C(CC=C(C)C)=C(O)C=C2OC1C1=CC=C(O)C=C1 YHWNASRGLKJRJJ-UHFFFAOYSA-N 0.000 claims abstract description 43
- YKGCBLWILMDSAV-GOSISDBHSA-N Isoxanthohumol Natural products O(C)c1c2C(=O)C[C@H](c3ccc(O)cc3)Oc2c(C/C=C(\C)/C)c(O)c1 YKGCBLWILMDSAV-GOSISDBHSA-N 0.000 claims abstract description 31
- YKGCBLWILMDSAV-SFHVURJKSA-N isoxanthohumol Chemical compound C1([C@H]2OC=3C(CC=C(C)C)=C(O)C=C(C=3C(=O)C2)OC)=CC=C(O)C=C1 YKGCBLWILMDSAV-SFHVURJKSA-N 0.000 claims abstract description 30
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- 241001603665 Blautia hominis Species 0.000 abstract description 12
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 17
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- 229930008679 prenylflavonoid Natural products 0.000 description 5
- 239000008346 aqueous phase Substances 0.000 description 4
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- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 3
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- -1 Calcium Chloride Nicotinic Acid Zinc Sulfate Chemical compound 0.000 description 3
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- 244000025221 Humulus lupulus Species 0.000 description 3
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- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
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- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 3
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 2
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 125000001844 prenyl group Chemical group [H]C([*])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 239000000047 product Substances 0.000 description 2
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- YOMBUJAFGMOIGS-UHFFFAOYSA-N 2-fluoro-1-phenylethanone Chemical compound FCC(=O)C1=CC=CC=C1 YOMBUJAFGMOIGS-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
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- WKLPMEPGENRPIL-UHFFFAOYSA-K [Cl-].[Na+].N1=C(C)C(O)=C(CO)C(CO)=C1.S(=O)(=O)([O-])[O-].[Mn+2] Chemical compound [Cl-].[Na+].N1=C(C)C(O)=C(CO)C(CO)=C1.S(=O)(=O)([O-])[O-].[Mn+2] WKLPMEPGENRPIL-UHFFFAOYSA-K 0.000 description 1
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- 239000003463 adsorbent Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
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- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
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- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- JCXJVPUVTGWSNB-UHFFFAOYSA-N nitrogen dioxide Inorganic materials O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
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- 235000008209 xanthohumol Nutrition 0.000 description 1
Classifications
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本発明の課題は、８−プレニルナリンゲニンの新規製造方法の提供であり、該課題を、イソキサントフモールを含有する溶液において、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物及びブラウティア・ホミニス（Blautia hominis）に属する微生物からなる群から選択される一又はそれ以上の微生物に、イソキサントフモールから８−プレニルナリンゲニンを産生させる工程を含む、８−プレニルナリンゲニンの製造方法で解決する。An object of the present invention is to provide a new method for producing 8-prenylnaringenin, which is a problem in which it has an ability to produce 8-prenylnaringenin from isoxanthohumol in a solution containing isoxanthohumol. Isoxanthohumol is one or more microorganisms selected from the group consisting of microorganisms belonging to Brautia coccoides, microorganisms belonging to Blautia schinkii, and microorganisms belonging to Blautia hominis. The solution is a method for producing 8-prenylnaringenin, which comprises the step of producing 8-prenylnaringenin from.
Description
本発明は、８−プレニルナリンゲニンの製造方法に関する。 The present invention relates to a method for producing 8-prenylnaringenin.
ホップ（Humulus lupulus L.）は、アサ科の多年草で、雌雄異株の蔓性植物である。雌株の毬花は、ビールに苦みなどを付与する原料を含み、さらに、キサントフモール、イソキサントフモール、８−プレニルナリンゲニンなどのプレニル化フラボノイドなどの有効な成分を含んでいる。これらプレニル化フラボノイドは様々な生理活性をもつことが報告されており、医薬や食品素材として注目されている。特に８−プレニルナリンゲニンは、エストロゲン活性（非特許文献１）、血管新生抑制（特許文献１）、廃用性筋萎縮抑制（特許文献２）といった生理活性を示すことが報告されている。 Hops (Humulus lupulus L.) are perennials of the family Cannabaceae and are dioecious vines. The female strain of hops contains a raw material that imparts bitterness to beer, and further contains effective components such as xanthohumol, isoxanthohumol, and prenylated flavonoids such as 8-prenylnaringenin. It has been reported that these prenylated flavonoids have various physiological activities, and are attracting attention as pharmaceuticals and food materials. In particular, 8-prenylnaringenin has been reported to exhibit physiological activities such as estrogen activity (Non-Patent Document 1), angiogenesis suppression (Patent Document 1), and disuse muscle atrophy suppression (Patent Document 2).
しかし、８−プレニルナリンゲニンはホップ毬花には極微量で存在する（非特許文献２）ため、天然抽出物での回収は困難である。したがって、ナリンゲニンのプレニル化により８−プレニルナリンゲニンを生産する合成戦略が開発されている。
例えば、ナリンゲニンを直接Ｃ−プレニル化する合成法、フロロアセトフェノンから出発する合成法が報告されている。しかし、いずれも反応の選択性が低く、低収率である。
また、ユウロピウム（ＩＩＩ）触媒を使ったクライゼン転移による製造方法も報告されている。該方法は効率的であるものの、小規模の製造方法としてしか報告されていない（非特許文献３）。
また、クララ（Sophora flavescens）由来の８−プレニルトランスフェラーゼがナリンゲニンの８位に特異的にプレニル化する活性を有することが知られている（非特許文献４）。しかし、該反応にはプレニル基の供給が必要であり、８−プレニルナリンゲニンの生産には当量のプレニル基を供給しなければならない。However, since 8-prenyl naringenin is present in a very small amount in hop flowers (Non-Patent Document 2), it is difficult to recover it with a natural extract. Therefore, synthetic strategies have been developed to produce 8-prenylnaringenin by prenylation of naringenin.
For example, a synthetic method for directly converting naringenin to C-prenyl, a synthetic method starting from fluoroacetophenone has been reported. However, all of them have low reaction selectivity and low yield.
In addition, a production method by Claisen transition using a europium (III) catalyst has also been reported. Although the method is efficient, it has only been reported as a small-scale manufacturing method (Non-Patent Document 3).
Further, it is known that 8-prenyltransferase derived from Sophora flavescens has an activity of specifically prenylating at the 8-position of naringenin (Non-Patent Document 4). However, the reaction requires the supply of prenyl groups, and the production of 8-prenylnaringenin must be supplied with an equivalent amount of prenyl groups.
一方、イソキサントフモールを出発物質とした脱メチル化反応による８−プレニルナリンゲニンの生産に関しても報告があり、微生物変換による製法として、ユーバクテリウム・リモスム（Eubacterium limosum）ＡＴＣＣ ８４８６株又はペプトストレプトコッカス・プロダクタス（Peptostreptococcus productus）ＡＴＣＣ ２７３４０株によって、イソキサントフモールを脱メチル化して８−プレニルナリンゲニンを産生する製造方法が報告されている（特許文献３）。 On the other hand, there is also a report on the production of 8-prenylnaringenin by demethylation reaction using isoxanthohumol as a starting material, and as a manufacturing method by microbial conversion, Eubacterium limosum ATCC 8486 strain or Peptstreptococcus -A production method for producing 8-prenylnaringenin by demethylating isoxanthohumol by the productus (Peptostreptococcus productus) ATCC 27340 strain has been reported (Patent Document 3).
本発明は、このような状況に鑑みてなされたものであり、８−プレニルナリンゲニンの新規製造方法の提供を課題とする。 The present invention has been made in view of such a situation, and an object of the present invention is to provide a new method for producing 8-prenylnaringenin.
本発明者らは、上記課題を解決するために鋭意研究を行った結果、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物と、ブラウティア・シンキ（Blautia schinkii）に属する微生物と、ブラウティア・ホミニス（Blautia hominis）に属する微生物とが、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有することを見出し、本発明を完成させた。本発明は以下のとおりである。 As a result of diligent research to solve the above problems, the present inventors have found that microorganisms belonging to Brautia coccoides, microorganisms belonging to Blautia schinkii, and Blautia hominis. ), And found that it has the ability to produce 8-prenylnaringenin from isoxanthohumol, and completed the present invention. The present invention is as follows.
〔１〕イソキサントフモールを含有する溶液において、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、及びブラウティア・ホミニス（Blautia hominis）に属する微生物からなる群から選択される一又はそれ以上の微生物に、イソキサントフモールから８−プレニルナリンゲニンを産生させる工程を含む、８−プレニルナリンゲニンの製造方法。
〔２〕前記ブラウティア・コッコイデス（Blautia coccoides）に属する微生物が、ブラウティア・コッコイデス（Blautia coccoides）ＪＣＭ １３９５株である、〔１〕に記載の製造方法。
〔３〕前記ブラウティア・シンキ（Blautia schinkii）に属する微生物が、ブラウティア・シンキ（Blautia schinkii）ＪＣＭ １４６５７株である、〔１〕又は〔２〕に記載の製造方法。
〔４〕前記ブラウティア・ホミニス（Blautia hominis）に属する微生物が、ブラウティア・ホミニス（Blautia hominis）ＪＣＭ ３２２７６株である、請求項１〜３のいずれか一項に記載の製造方法。[1] Microorganisms belonging to Brautia coccoides, which have the ability to produce 8-prenylnaringenin from isoxanthohumol in a solution containing isoxanthohumol, and microorganisms belonging to Blautia schinkii. A method for producing 8-prenylnaringenin, which comprises a step of causing one or more microorganisms selected from the group consisting of microorganisms belonging to Blautia hominis to produce 8-prenylnaringenin from isoxanthohumol. ..
[2] The production method according to [1], wherein the microorganism belonging to the Brautia coccoides is the Blautia coccoides JCM 1395 strain.
[3] The production method according to [1] or [2], wherein the microorganism belonging to the Brautia schinkii is the Brautia schinkii JCM 14657 strain.
[4] The production method according to any one of claims 1 to 3, wherein the microorganism belonging to the Brautia hominis is the Brautia hominis JCM 32276 strain.
本発明によれば、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する微生物を用いた、８−プレニルナリンゲニンの効率的な製造方法が提供できる。該微生物は、具体的には、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、及びブラウティア・ホミニス（Blautia hominis）に属する微生物からなる群から選択される一又はそれ以上の微生物である。
本発明により得られる８−プレニルナリンゲニンを、化粧品、医薬部外品、医療用品、衛生用品、医薬品、飲食品（サプリメントを含む。）等に用い、ヒトを含む対象がそれを使用又は摂取等することにより、８−プレニルナリンゲニンによる公知の効果を簡便に得ることができる。According to the present invention, it is possible to provide an efficient method for producing 8-prenylnaringenin using a microorganism capable of producing 8-prenylnaringenin from isoxanthohumol. Specifically, the microorganism is selected from the group consisting of a microorganism belonging to Brautia coccoides, a microorganism belonging to Blautia schinkii, and a microorganism belonging to Blautia hominis. Or more microorganisms.
The 8-prenylnaringenin obtained by the present invention is used in cosmetics, quasi-drugs, medical products, hygiene products, pharmaceuticals, foods and drinks (including supplements), etc., and is used or ingested by subjects including humans. Thereby, the known effect of 8-prenylnaringenin can be easily obtained.
本発明に係る８−プレニルナリンゲニンの製造方法は、イソキサントフモールを含有する溶液において、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、及びブラウティア・ホミニス（Blautia hominis）に属する微生物からなる群から選択される一又はそれ以上の微生物に、イソキサントフモールから８−プレニルナリンゲニンを産生させる工程を含む。 The method for producing 8-prenylnaringenin according to the present invention is a microorganism belonging to Blautia coccoides, which has an ability to produce 8-prenylnaringenin from isoxanthohumol in a solution containing isoxanthohumol. A step of causing one or more microorganisms selected from the group consisting of microorganisms belonging to Brautia schinkii and microorganisms belonging to Blautia hominis to produce 8-prenyl naringenin from isoxanthohumol. including.
（イソキサントフモールを含有する溶液）
イソキサントフモールを含有する溶液とは、該溶液において、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、及びブラウティア・ホミニス（Blautia hominis）に属する微生物からなる群から選択される一又はそれ以上の微生物に、イソキサントフモールから８−プレニルナリンゲニンを産生させることができるものであれば特に制限されない。好ましくは培地であり、より好ましくは後述する「培地、及び培養による８−プレニルナリンゲニンの産生」欄に記載した培地である。(Solution containing isoxanthohumol)
The solution containing isoxanthohumol is a microorganism belonging to Blautia coccoides, which has an ability to produce 8-prenylnaringenin from isoxanthohumol in the solution. Particularly limited as long as one or more microorganisms selected from the group consisting of the microorganisms belonging to the group and the microorganisms belonging to Blautia hominis can produce 8-prenylnaringenin from isoxanthohumol. Not done. It is preferably a medium, and more preferably the medium described in the “Medium and production of 8-prenylnaringenin by culture” column described later.
該溶液へイソキサントフモールを添加する場合には、８−プレニルナリンゲニンの産生前に添加しても、その途中で添加してもよく、また、一括添加、逐次添加、連続添加でもよい。溶液中のイソキサントフモールの含有量は、通常５ｍｇ／Ｌ以上である。 When isoxanthohumol is added to the solution, it may be added before the production of 8-prenylnaringenin, in the middle of the production, or may be added in a batch, sequentially, or continuously. The content of isoxanthohumol in the solution is usually 5 mg / L or more.
（微生物）
本発明における微生物は、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する限り、特に限定されない。
具体的には、ブラウティア（Blautia）属に属する微生物が挙げられる。
その中でも、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、ブラウティア・ホミニス（Blautia hominis）に属する微生物等が好ましい。
また、その中でも、ブラウティア・コッコイデス（Blautia coccoides）JCM 1395株やブラウティア・シンキ（Blautia schinkii）JCM 14657株、ブラウティア・ホミニス（Blautia hominis）JCM 32276株等がより好ましい。
本発明において、微生物は１種でも２種以上を用いてもよく、１株でも２株以上を用いてもよい。(Microorganisms)
The microorganism in the present invention is not particularly limited as long as it has the ability to produce 8-prenylnaringenin from isoxanthohumol.
Specific examples include microorganisms belonging to the genus Blautia.
Among them, microorganisms belonging to Brautia coccoides, microorganisms belonging to Blautia schinkii, microorganisms belonging to Blautia hominis and the like are preferable.
Among them, Blautia coccoides JCM 1395 strain, Blautia schinkii JCM 14657 strain, Blautia hominis JCM 32276 strain and the like are more preferable.
In the present invention, one type or two or more types of microorganisms may be used, or one strain or two or more strains may be used.
ＪＣＭ番号が付与された細菌は、Japan Collection of Microorganisms（国立研究開発法人理化学研究所バイオリソースセンター微生物材料開発室、郵便番号：305-0074、住所：茨城県つくば市高野台3-1-1）から入手することができる。 Bacteria with JCM numbers are available from Japan Collection of Microorganisms (Japan Collection of Microorganisms, RIKEN BioResource Center, Japan Collection of Microorganisms, Postal Code: 305-0074, Address: 3-1-1 Koyadai, Tsukuba City, Ibaraki Prefecture) You can get it.
（培地、及び培養による８−プレニルナリンゲニンの産生）
本工程では、前記溶液が培地であることが好ましい。該培地は特に限定されないが、たとえば日水製薬社製のＧＡＭ培地、変法ＧＡＭ培地、ブレインハートインフュージョン培地、Ｏｘｏｉｄ社製Ａｎａｅｒｏｂｅ Ｂａｓａｌ Ｂｒｏｔｈ（ＡＢＢ）培地などを使用することができる。(Production of 8-prenylnaringenin by medium and culture)
In this step, it is preferable that the solution is a medium. The medium is not particularly limited, and for example, a GAM medium manufactured by Nissui Pharmaceutical Co., Ltd., a modified GAM medium, a Brain Heart infusion medium, an Anaerobe Basal Bros (ABB) medium manufactured by Oxoid, and the like can be used.
培地に加える炭素源としての有機物の濃度は、効率的に培地中の嫌気性微生物を発育させるために適宜調節することができる。一般的には、０．１〜１０ｗｔ／ｖｏｌ％の範囲から添加量を選択することによって、過不足を避けることができる。 The concentration of organic matter added to the medium as a carbon source can be appropriately adjusted in order to efficiently grow anaerobic microorganisms in the medium. Generally, excess or deficiency can be avoided by selecting the addition amount from the range of 0.1 to 10 wt / vol%.
上記の炭素源に加えて、培地には、窒素源が加えることができる。本発明において、窒素源としては通常の発酵に用いうる各種の窒素化合物を用いることができる。好ましい無機窒素源は、たとえば、アンモニウム塩、及び硝酸塩である。好ましい有機窒素源は、たとえば、アミノ酸類、酵母エキス、ペプトン類、肉エキス、肝臓エキス、消化血清末などである。より好ましい無機窒素源は、硫安、塩化アンモニウム、リン酸アンモニウム、リン酸水素アンモニウム、硝酸カリウム及び硝酸ソーダである。より好ましい窒素源は酵母エキス、ペプトン類である。 In addition to the carbon sources mentioned above, a nitrogen source can be added to the medium. In the present invention, various nitrogen compounds that can be used for ordinary fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are, for example, ammonium salts and nitrates. Preferred organic nitrogen sources are, for example, amino acids, yeast extracts, peptones, meat extracts, liver extracts, digested serum powder and the like. More preferred sources of inorganic nitrogen are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate and sodium nitrate. More preferred nitrogen sources are yeast extract and peptones.
さらに、炭素源や窒素源に加えて、嫌気性微生物の培養に適した他の有機物あるいは無機物を培地に加えることもできる。たとえば、ビタミンなどの補因子や各種の塩類等の無機化合物を培地に加えることによって、嫌気性微生物の増殖や活性を増強できる場合もある。たとえば無機化合物、ビタミン類、動植物由来の微生物増殖補助因子として以下のものを挙げることができる。
無機化合物 ビタミン類
リン酸二水素カリウム ビオチン
硫酸マグネシウム 葉酸
硫酸マンガン ピリドキシン
塩化ナトリウム チアミン
塩化コバルト リボフラビン
塩化カルシウム ニコチン酸
硫酸亜鉛 パントテン酸
硫酸銅 ビタミンB12
明ばん チオオクト酸
モリブデン酸ソーダ ｐ−アミノ安息香酸
塩化カリウム
ホウ酸等
塩化ニッケル
タングステン酸ナトリウム
セレン酸ナトリウム
硫酸第一鉄アンモニウムFurthermore, in addition to carbon and nitrogen sources, other organic or inorganic substances suitable for culturing anaerobic microorganisms can be added to the medium. For example, the growth and activity of anaerobic microorganisms may be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. For example, the following can be mentioned as microbial growth cofactors derived from inorganic compounds, vitamins, and animals and plants.
Inorganic Compounds Vitamins Potassium Dihydrogen Phosphate Biotin Biotin Magnesium Sulfate Folic Acid Manganese Sulfate Pyridoxin Sodium Chloride Thiamine Cobalt Chloride Riboflavin Calcium Chloride Nicotinic Acid Zinc Sulfate Pantothenic Acid Copper Sulfate Vitamin B12
Akira thiooctate sodium molybdate p-aminobenzoic acid potassium chloride boric acid, etc. Nickel chloride sodium tungstate sodium selenite ammonium ferrous sulfate
培養中の気相、水相としては、空気又は酸素を含まないことが好ましく、たとえば、窒素及び又は水素を任意の比率で含むことや、窒素及び／又は二酸化炭素を任意の比率で含むことが挙げられ、水素を含む気相や水相であることが好ましい。
培地中の気相や水相をこのような環境にする方法は特に制限されないが、たとえば、培養前に上記ガスで気相を置換する、培養中も培養器の底部から供給する、培養器の気相部に供給する、培養前に上記ガスで水相をバブリングするなどの方法をとることが出来る。The gas phase and aqueous phase during cultivation preferably do not contain air or oxygen, and for example, nitrogen and / or hydrogen may be contained in an arbitrary ratio, or nitrogen and / or carbon dioxide may be contained in an arbitrary ratio. It is preferable that it is a gas phase or an aqueous phase containing hydrogen.
The method for setting the gas phase or aqueous phase in the medium to such an environment is not particularly limited. For example, the gas phase is replaced with the above gas before culturing, and the gas phase is supplied from the bottom of the incubator during culturing. A method such as supplying to the gas phase part or bubbling the aqueous phase with the above gas before culturing can be taken.
培養温度は、２０℃〜４５℃、より好ましくは２５℃〜４０℃、さらに好ましくは３０℃〜３７℃である。
培養器の加圧条件は、生育できる条件であれば特に制限されるものではないが、０．００１〜１ＭＰａの範囲、好ましくは０．０１〜０．５ＭＰａを挙げることができる。
培養時間としては、通常８〜３３６時間、好ましくは２４〜１６８時間を挙げることが出来る。The culture temperature is 20 ° C. to 45 ° C., more preferably 25 ° C. to 40 ° C., and even more preferably 30 ° C. to 37 ° C.
The pressurizing conditions of the incubator are not particularly limited as long as they can grow, but may be in the range of 0.001 to 1 MPa, preferably 0.01 to 0.5 MPa.
The culturing time is usually 8 to 336 hours, preferably 24 to 168 hours.
本発明におけるブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、ブラウティア・ホミニス（Blautia hominis）に属する微生物は、公知の微生物培養方法に従って培養することが出来る。工業的な製造には、培地やガスを連続的に供給することができ、かつ培養物を回収するための機構を備えた連続培養システムが好適である。 The microorganism belonging to Blautia coccoides, the microorganism belonging to Blautia schinkii, and the microorganism belonging to Blautia hominis in the present invention can be cultured according to a known microbial culture method. For industrial production, a continuous culture system capable of continuously supplying a medium or gas and having a mechanism for recovering the culture is suitable.
微生物の培養においては、培養システム内への酸素の混入を防ぐことが必要である。そのための培養器は、通常の嫌気的培養に持ちられる培養槽がそのまま利用できる。微生物の培養にも利用することができる培養タンクは市販されている。培養槽内に混入する酸素を、窒素などの不活性気体などで置換することにより、嫌気的な雰囲気を作ることができる。
たとえば、嫌気培養ジャーを、上記微生物を培養するためのバイオリアクターとすることができる。嫌気培養ジャーは、金属、ガラス、あるいは合成樹脂製の気密容器で構成され、内部を大気中の酸素から遮断することができる。In culturing microorganisms, it is necessary to prevent oxygen from being mixed into the culture system. As the incubator for that purpose, a culture tank that can be used for normal anaerobic culture can be used as it is. Culture tanks that can also be used for culturing microorganisms are commercially available. An anaerobic atmosphere can be created by substituting the oxygen mixed in the culture tank with an inert gas such as nitrogen.
For example, the anaerobic culture jar can be a bioreactor for culturing the above-mentioned microorganisms. The anaerobic culture jar is composed of an airtight container made of metal, glass, or synthetic resin, and can shield the inside from oxygen in the atmosphere.
（その他の工程）
本発明は、以下の工程を含んでもよい。
本発明は、例えば、得られた８−プレニルナリンゲニンを定量する工程を含んでもよい。定量方法は常法に従うことができる。たとえば、培養液の一部を採取して適宜希釈し、よく撹拌した後、ポリテロラフルオロエチレン（ＰＴＦＥ）膜などの膜を使用して濾過し、不溶物を除去したものを高速液体クロマトグラフィー定量することなどが挙げられる。(Other processes)
The present invention may include the following steps.
The present invention may include, for example, the step of quantifying the obtained 8-prenylnaringenin. The quantification method can follow a conventional method. For example, a part of the culture solution is collected, diluted appropriately, stirred well, and then filtered using a membrane such as a polyterolafluoroethylene (PTFE) membrane to remove insoluble matter, which is quantified by high performance liquid chromatography. And so on.
また、本発明は、上記工程で得られた８−プレニルナリンゲニンを回収する工程を含んでもよい。当該回収工程は、精製工程、濃縮工程等であってよい。精製工程における精製処理としては、熱などによる微生物の殺菌；精密濾過（ＭＦ）、限外濾過（ＵＦ）などによる除菌；固形物、高分子物質の除去；有機溶媒やイオン性液体などによる抽出；疎水性吸着剤、イオン交換樹脂、活性炭カラム等を用いた吸着、脱色といった処理を行うことができる。また、濃縮工程における濃縮処理としては、エバポレーター、逆浸透膜等による濃縮が挙げられる。
さらに、８−プレニルナリンゲニンを含む溶液は、凍結乾燥、噴霧乾燥などにより粉末化することができる。粉末化において、ラクトース、デキストリン、コーンスターチ等の賦形剤を添加することもできる。The present invention may also include a step of recovering 8-prenylnaringenin obtained in the above step. The recovery step may be a purification step, a concentration step, or the like. The refining process in the refining process includes sterilization of microorganisms by heat, etc .; sterilization by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and polymer substances; extraction by organic solvent, ionic liquid, etc. It is possible to perform treatments such as adsorption and decolorization using a hydrophobic adsorbent, an ion exchange resin, an activated carbon column and the like. In addition, as the concentration treatment in the concentration step, concentration by an evaporator, a reverse osmosis membrane or the like can be mentioned.
Further, the solution containing 8-prenylnaringenin can be pulverized by freeze-drying, spray-drying or the like. Excipients such as lactose, dextrin and cornstarch can also be added in the pulverization.
以下、具体的な実施例により本発明をさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to specific examples, but the present invention is not limited to these examples.
［実施例１］
ＡＢＢ培地（Ｏｘｏｉｄ社製）に、イソキサントフモール（ナカライテスク社製）を添加した後、加熱滅菌し、気相をＮ２：ＣＯ２：Ｈ２（８０％／１０％／１０％）ガスで置換したものを基本培地とした。最終濃度５ｍｇ／Ｌのイソキサントフモールを含む培地に、ブラウティア・コッコイデス（Blautia coccoides）JCM 1395株、ブラウティア・シンキ（Blautia schinkii）JCM 14657株、又はブラウティア・ホミニス（Blautia hominis）JCM 32276株を植菌し、３７℃で嫌気的に培養した。培養終了後、培養液５ｍＬに対して等量の酢酸エチル（１．５％ギ酸）でプレニルフラボノイド類を抽出し、得られた酢酸エチル相を回収後、乾固させた。このようにして得た乾固物をメタノール０．５ｍＬに再溶解し、ＨＰＬＣによりプレニルフラボノイド類の定量分析を行った。
ＨＰＬＣは以下に記載の条件で行った。LKT Laboratories社製のプレニルフラボノイド類を標品として用い、DMSOに溶解して用いた。[Example 1]
Isoxanthohumol (manufactured by Nacalai Tesque) is added to ABB medium (manufactured by Oxoid), then heat sterilized, and the gas phase is N 2 : CO 2 : H 2 (80% / 10% / 10%) gas. The medium replaced with was used as the basal medium. Blautia coccoides JCM 1395 strain, Blautia schinkii JCM 14657 strain, or Blautia hominis JCM 32276 strain was planted in a medium containing isoxanthohumol at a final concentration of 5 mg / L. The fungus was cultivated anaerobically at 37 ° C. After completion of the culture, prenylflavonoids were extracted with an equal amount of ethyl acetate (1.5% formic acid) in 5 mL of the culture solution, and the obtained ethyl acetate phase was recovered and dried. The dry matter thus obtained was redissolved in 0.5 mL of methanol, and quantitative analysis of prenylflavonoids was performed by HPLC.
HPLC was performed under the conditions described below. Prenylflavonoids manufactured by LKT Laboratories were used as a standard and dissolved in DMSO.
ＨＰＬＣ条件：
カラム：Ｉｎｅｒｔｓｉｌ ＯＤＳ‐３（２５０×４．６ｍｍ）（ＧＬ Ｓｃｉｅｎｃｅ社製）
溶離液：Ａ液（水／ギ酸＝９９／１）、Ｂ液（アセトニトリル／ギ酸＝９９／１）、およびＢ液２０％〜７０％のグラジェント
流速：１．０ｍＬ／ｍｉｎ
カラム温度：４０℃
検出：２９０ｎｍHPLC conditions:
Column: Inertsil ODS-3 (250 x 4.6 mm) (manufactured by GL Sciences)
Eluent: Solution A (water / formic acid = 99/1), Solution B (acetonitrile / formic acid = 99/1), and solution B 20% to 70% gradient flow rate: 1.0 mL / min
Column temperature: 40 ° C
Detection: 290 nm
その結果、１週間の培養により、表１に示すとおりブラウティア・コッコイデス（Blautia coccoides）JCM 1395株を用いた場合は、添加したイソキサントフモールの９６．９％が８−プレニルナリンゲニンに変換され、ブラウティア・シンキ（Blautia schinkii）JCM 14657株を用いた場合には、添加したイソキサントフモールの７．９％が８−プレニルナリンゲニンに変換され、ブラウティア・ホミニス（Blautia hominis）JCM 32276株を用いた場合には、添加したイソキサントフモールの９８．８％が８−プレニルナリンゲニンに変換された。 As a result, after 1 week of culturing, 96.9% of the added isoxanthohumol was converted to 8-prenyl naringenin when the Blautia coccoides JCM 1395 strain was used as shown in Table 1. When the Blautia schinkii JCM 14657 strain was used, 7.9% of the added isoxanthohumol was converted to 8-prenyl naringenin, and the Blautia hominis JCM 32276 strain was used. In some cases, 98.8% of the added isoxanthohumol was converted to 8-prenylnaringenin.
本発明によれば、イソキサントフモールから８−プレニルナリンゲニンを産生する能力を有する、ブラウティア・コッコイデス（Blautia coccoides）に属する微生物、ブラウティア・シンキ（Blautia schinkii）に属する微生物、及びブラウティア・ホミニス（Blautia hominis）に属する微生物からなる群から選択される一又はそれ以上の微生物を用いることによって、効率的に８−プレニルナリンゲニンを製造することができる。
本発明により得られる８−プレニルナリンゲニンを、化粧品、医薬部外品、医療用品、衛生用品、医薬品、飲食品（サプリメントを含む。）等に用い、ヒトを含む対象がそれを使用又は摂取等することにより、８−プレニルナリンゲニンによる公知の効果を簡便に得ることができる。According to the present invention, a microorganism belonging to Brautia coccoides, a microorganism belonging to Blautia schinkii, and a microorganism belonging to Blautia schinkii, which have an ability to produce 8-prenylnaringenin from isoxanthohumol, and Blautia hominis. By using one or more microorganisms selected from the group consisting of microorganisms belonging to hominis), 8-prenylnaringenin can be efficiently produced.
The 8-prenylnaringenin obtained by the present invention is used in cosmetics, quasi-drugs, medical products, hygiene products, pharmaceuticals, foods and drinks (including supplements), etc., and is used or ingested by subjects including humans. Thereby, the known effect of 8-prenylnaringenin can be easily obtained.
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| JP2010513359A (en) * | 2006-12-22 | 2010-04-30 | ブロシュ ローラント ザウル | Use of a composition comprising minerals and, in some cases, acetic acid producing bacteria and / or butyric acid producing bacteria, to avoid or reduce gas generation in the large intestine of mammals and abdominal symptoms resulting therefrom |
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| JP2010513359A (en) * | 2006-12-22 | 2010-04-30 | ブロシュ ローラント ザウル | Use of a composition comprising minerals and, in some cases, acetic acid producing bacteria and / or butyric acid producing bacteria, to avoid or reduce gas generation in the large intestine of mammals and abdominal symptoms resulting therefrom |
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