Method for producing functional substance

Abstract

To provide a technique for producing a predetermined demethylated polyphenol in which at least a methyl group in a methoxy group is eliminated from a predetermined polyphenol having a methoxy group in the side chain thereof.SOLUTION: Provided is a method for producing esculetin. In a technique for producing esculetin from scoparone, for example, the method comprises the step of, causing a microorganism belonging to the genus Blautia and / or a microorganism belonging to the genus Eubacterium, having the ability to produce esculetin from scoparone in a solution containing scoparone.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a functional substance. In particular, the present disclosure relates to a method for producing esculetin, 4-acetylresorcinol, or eriodictyol. [Background technology]

[0002] Some polyphenols are used as active ingredients in cosmetics, medicines, and health foods, such as esculetin, 4-acetylresorcinol, and eriodictyol.

[0003] Esculetin is incorporated into cosmetics, anti-inflammatory skin preparations for external use, anti-obesity agents, etc. (Patent Documents 1 to 3). It has been reported that esculetin can be obtained from horse chestnut by ethanol extraction or hot water extraction (Patent Document 3), and by reacting a solution containing 6-hydroxy-7-methoxycoumarin and 1,2-dichloroethane with aluminum chloride (Patent Document 4).

[0004] 4-Acetylresorcinol is a useful compound as a synthetic intermediate for 2,4-dihydroxy-3-propylacetophenone, a raw material for allergy treatment drugs, and as a raw material for photosensitive materials and sunscreen cosmetics. It has been reported that 4-acetylresorcinol can be obtained by reacting resorcinol with acetic acid in the presence of a protonic acid catalyst while removing water (Patent Document 5).

[0005] Eriodictyol promotes activation of Akt, which mediates 67LR-dependent cell death induction pathway It has been reported that this enhances the anti-cancer effect of EGCG and shows a synergistic effect in the inhibition of body fat accumulation and prevention of lipid metabolism disorders by green tea (Non-Patent Document 1). Eriodictyol is produced by extracting hesperetin from Blautia sp. MRG-PMF1. It has been reported that it is produced by a strain (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2007-161727 A [Patent Document 2] JP 2006-28094 A [Patent Document 3] JP 2009-292811 A [Patent Document 4] JP 2003-261559 A [Patent Document 5] Japanese Patent Application Publication No. 08-225484 [Non-patent literature]

[0007] [Non-Patent Document 1] Journal of the Japanese Society of Nutrition and Food Science Vol. 72 No. 5 205-210 (2019) [Non-Patent Document 2] J. Agric. Food Chem., 62, 12377-12383 (2014) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present disclosure is to provide at least a technique for producing a specific demethylated polyphenol in which a methyl group has been removed from a specific polyphenol having a methoxy group on a side chain. [Means for solving the problem]

[0009] <1> Ability to produce esculetin from scoparone in a solution containing scoparone A method for producing esculetin, comprising a step of causing a microorganism belonging to the genus Blautia and / or a microorganism belonging to the genus Eubacterium, which has the formula: <2> In a solution containing paeonol, a microorganism belonging to the genus Blautia having the ability to produce 4-acetylresorcinol from paeonol was subjected to the treatment with paeonol. A method for producing 4-acetylresorcinol, comprising the step of producing 4-acetylresorcinol from 4-acetylresorcinol. <3> In a solution containing hesperetin, a microorganism belonging to the genus Eubacterium having the ability to produce eriodictyol from hesperetin was subjected to hesperetin synthesis. A method for producing eriodictyol, comprising a step of producing eriodictyol from eriodictyol. <4> The microorganism belonging to the genus Blautia is Blautia sp. DC 3652 (NITE BP-02924) strain; <1> The manufacturing method described in <5> The microorganism belonging to the genus Eubacterium is Eubacterium lysate. It is a microorganism belonging to the genus Eubacterium limosum. <1> or <4> The manufacture of method. <6> The microorganisms belonging to Eubacterium limosum are The strain is Eubacterium limosum JCM 6421. <5> The manufacturing method described in Construction method. <7> The microorganism belonging to the genus Blautia is Blautia sp. DC 3652 (NITE BP-02924) strain; <2> The manufacturing method described in <8> The microorganism belonging to the genus Eubacterium is Eubacterium lysate. It is a microorganism belonging to the genus Eubacterium limosum. <3> The manufacturing method described in <9> The microorganisms belonging to Eubacterium limosum are The strain is Eubacterium limosum JCM 6421. <8> The manufacturing method described in Construction method. Effect of the Invention

[0010] The present disclosure can at least provide an effect of providing a technique for producing a specific demethylated polyphenol in which a methyl group has been removed from a specific polyphenol having a methoxy group in a side chain, and can also provide an effect of providing a microorganism useful therefor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Each configuration and combination thereof in each embodiment is merely an example, and addition, omission, substitution, and other modifications of the configuration are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited to the embodiments, but is limited only by the scope of the claims.

[0012] Esculetin is also called 6,7-dihydroxycoumarin. 4-Acetylresorcinol is sometimes called 2,4-dihydroxyacetophenone, 2',4'-dihydroxyacetophenone, resacetophenone, 2,4-DHAP, etc. Eriodictyol is also known as (S)-3',4',5,7-tetrahydroxyflavanone.

[0013] In this disclosure, the microorganisms to which the JCM number is assigned are listed in the Japan Collection of Microorganisms (RIKEN BioResource Center, Microorganism Materials Development Division, zip code 014-01500). It can be obtained from: (Address: 3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture, 305-0074)

[0014] <Method of producing esculetin> One aspect of the present disclosure is to detect esculetin from scoparone in a solution containing scoparone. A microorganism belonging to the genus Blautia and / or Euba having the ability to produce A microorganism belonging to the genus Eubacterium was made to produce esculetin from scoparone. The method for producing esculetin includes the step of: Scoparone has two methoxy groups in the side chain. When the methyl group of one of the methoxy groups is eliminated, scopoletin or isoscopoletin is produced, and when the methyl groups of the other methoxy group are eliminated, esculetin is produced. In this embodiment, however, if esculetin is produced, scopoletin or isoscopoletin may be produced, or both may be produced.

[0015] (Microorganisms capable of producing esculetin from scoparone) The microorganism having the ability to produce esculetin from scoparone used in this embodiment is a microorganism belonging to the genus Blautia having the ability to produce esculetin from scoparone. and / or microorganisms belonging to the genus Eubacterium. In addition, microorganisms belonging to the genus Blautia can be read as bacteria belonging to the genus Blautia, and microorganisms belonging to the genus Eubacterium can be read as bacteria belonging to the genus Blautia. , can be read as bacteria belonging to the genus Eubacterium.

[0016] As for microorganisms belonging to the genus Blautia, esculetin is extracted from scoparone. As long as it has the ability to produce the enzyme, the enzyme may be, but is not limited to, for example, a microorganism belonging to Blautia prodacta (e.g., JCM 1471 strain, etc.), a microorganism belonging to Blautia coccoides (e.g., JCM 1395 strain, etc.), a microorganism belonging to Blautia spp. Examples of suitable microorganisms include those belonging to the genus Blautia schinki (e.g., JCM 14657 strain, etc.), and those belonging to the genus Blautia hominis (e.g., JCM 32276 strain, etc.). In addition to the above, Blautia sp. DC 3652 (NITE BP-02924) strain, Blautia sp. DC 3653 (NITE BP-02629) strain, Blautia Blautia sp. DC 3654 (NITE BP-02925) strain, etc. , Blautia sp. DC 3652 (NITE BP-02924).

[0017] Blautia sp. DC 3652 (NITE BP-02924) strain was Based on the treaty, on March 20, 2019, the National Institute of Technology and Evaluation (NITE) It was deposited internationally at the Depository Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, 292-0818) and assigned the accession number NITE BP-02924. Blautia sp. DC 3653 (NITE BP-02629) strain was On the date, it was deposited in Japan at the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) and was assigned the accession number NITE P-02629. The data was transferred to an international deposit under the Budapest Treaty on December 27, 2018, and assigned the accession number NITE BP-02629. Blautia sp. DC 3654 (NITE BP-02925) strain was Based on the treaty, on March 20, 2019, the National Institute of Technology and Evaluation (NITE) It was deposited at the International Depository Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) and assigned the accession number NITE BP-02925.

[0018] Taking Blautia prodacta JCM 1471 strain as an example, in this embodiment, the Blautia prodacta JCM 1471 strain is not limited to the deposited strain, and may be a strain substantially equivalent to the deposited strain. The substantially equivalent strain refers to a strain belonging to the same genus or species as the deposited strain, and having the ability to produce esculetin from scoparone. Furthermore, the substantially equivalent strain refers to a strain whose 16S rRNA gene base sequence has a homology of 98.5% or more, preferably 98.7% or more, more preferably 99% or more, and even more preferably 100% to the 16S rRNA gene base sequence of the deposited strain. Furthermore, the deposited strain has the ability to produce esculetin from scoparone. As long as the strain is capable of being cultivated, it may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, or the like. The same is true for the other previously mentioned deposited strains belonging to the genus Blautia which have the ability to produce esculetin from scoparone.

[0019] Microorganisms belonging to the genus Eubacterium include scoparon to esculentum. As long as it has the ability to produce retin, the microorganism is not particularly limited, but for example, a microorganism belonging to Eubacterium limosum (e.g., JCM 6421 strain, JCM 6501 strain, JCM 9978 strain, etc. Preferred is Eubacterium limosum JCM 6421 strain.

[0020] Let us use Eubacterium limosum JCM 6421 as an example. In this embodiment, the Eubacterium limosum JCM 6421 strain is The deposited strain is not limited to the deposited strain, and may be a strain substantially equivalent to the deposited strain. The substantially equivalent strain refers to a strain belonging to the same genus or species as the deposited strain and having the ability to produce esculetin from scoparone. The substantially equivalent strain is a strain whose 16S rRNA gene base sequence has a homology of 98.5% or more, preferably 98.7% or more, more preferably 99% or more, and even more preferably 100% to the 16S rRNA gene base sequence of the deposited strain. Furthermore, the deposited strain may be a strain bred from the deposited strain or a substantially equivalent strain by mutation treatment, genetic recombination, selection of natural mutants, or the like, so long as it has the ability to produce esculetin from scoparone. The same is true for the other previously mentioned deposited strains belonging to the genus Eubacterium which have the ability to produce esculetin from scoparone.

[0021] In this embodiment, one or more kinds of the microorganisms may be used, and one or more strains of the microorganisms may be used.

[0022] (Residual form of a microorganism capable of producing esculetin from scoparone) In this embodiment, the microorganism belonging to the genus Blautia and / or the microorganism belonging to the genus Eubacterium that has the ability to produce esculetin from scoparone includes resting forms thereof. The resting body refers to a microbial body that is not growing after removing medium components from a cultured microorganism by a procedure such as centrifugation, washing with a salt solution or buffer, and suspending in the same liquid as the washing liquid. In this embodiment, the resting body refers to a microbial body that has at least a metabolic system capable of producing esculetin from scoparone. The microorganism is a bacterium belonging to the genus Blautia and / or Eubacterium scoparone that has the ability to produce esculetin from scoparone. Since the bacterium belongs to the genus Eubacterium, the stationary form is a resting bacterial body. Examples of salt solutions include physiological saline, etc. Examples of buffer solutions include phosphate buffer, Tris-HCl buffer, citrate-phosphate buffer, citrate buffer, MOPS buffer, acetate buffer, glycine buffer, etc. The pH and concentration of each can be appropriately adjusted according to a conventional method.

[0023] (Solution containing scoparone) In this embodiment, the solution containing scoparone is not particularly limited as long as it allows the microorganism to produce esculetin from scoparone in the solution. A medium is preferable, and a medium described in the section "Medium and production of esculetin by culture" below is more preferable. When the microorganism is stationary, the above-mentioned salt solution or buffer solution is preferable. In addition, the term "medium" as used herein refers to a solution in which microorganisms can grow, including a minimal medium, and does not refer to a solution in which microorganisms cannot grow, such as the above-mentioned salt solution or buffer solution. does not include.

[0024] When scoparone is added to the solution, it may be added before or during the production of esculetin, and may be added all at once, gradually, or continuously. The content of scoparone in the solution is usually 0.001 g / L or more, preferably 0.01 g / L or more, more preferably 0.05 g / L or more, and usually 100 g / L or less, preferably 20 g / L or less, more preferably 10 g / L or less.

[0025] (Culture medium and production of esculetin by culture) In the step of producing esculetin from scoparone, the solution is preferably a medium. The medium is not particularly limited, but examples of the medium that can be used include ANAEROBE BASAL BROTH (ABB medium) manufactured by Oxoid, Wilkins-Chalgren Anaerobe Broth (CM0643) manufactured by Oxoid, GAM medium, modified GAM medium, and brain heart infusion medium manufactured by Nissui Pharmaceutical Co., Ltd.

[0026] Water-soluble organic matter can also be added to the medium as a carbon source. Examples of water-soluble organic matter include the following compounds: sugars such as glucose, arabinose, sorbitol, fructose, mannose, sucrose, trehalose, and xylose; alcohols such as glycerol; and organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, and fumaric acid.

[0027] The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted for efficient growth. In general, the amount added can be selected from the range of 0.1 to 10 wt / vol%.

[0028] In addition to the carbon sources, a nitrogen source may be added to the medium, and various nitrogen compounds that can be used in normal fermentation may be used as the nitrogen source. Preferred inorganic nitrogen sources include ammonium salts and nitrates, and more preferred are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate. Examples of organic nitrogen sources include amino acids, yeast extract, peptones (e.g., polypeptone N, soybean peptone, etc.), meat extracts (e.g., Ehrlich bonito extract, Lab-Remco powder, bouillon, etc.), seafood extracts, liver extracts, digested serum powder, fish oil, etc.

[0029] Furthermore, in addition to carbon and nitrogen sources, the growth and activity may be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. For example, the following microbial growth cofactors derived from plants and animals, such as inorganic compounds, vitamins, and fatty acids, can be mentioned.

[0030] Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium Sulfate Folic Acid Manganese Sulfate Pyridoxine Sodium Chloride Thiamine Cobalt chloride Riboflavin Calcium chloride Nicotinic acid Zinc Sulfate Pantothenic Acid Copper Sulfate Vitamin B12 Alum Thioctic acid Sodium molybdate p-aminobenzoic acid Potassium chloride Vitamin K Boric acid etc. Nickel chloride Sodium Tungstate Sodium Selenite Ammonium Ferrous Sulfate Sodium Acetate Trihydrate Magnesium sulfate heptahydrate Manganese sulfate tetrahydrate

[0031] Furthermore, adding reducing agents such as cysteine, cystine, sodium sulfide, sulfite, ascorbic acid, glutathione, thioglycolic acid, and rutin, and enzymes that decompose reactive oxygen species such as catalase and superoxide mutase to the medium may be preferable in this case, since this may improve the growth of the microorganism.

[0032] The gas phase and aqueous phase during the culture preferably do not contain air and / or oxygen, and may, for example, contain nitrogen and / or hydrogen at any ratio, or nitrogen and / or carbon dioxide at any ratio, and are preferably gas phases and aqueous phases containing hydrogen. The ratio of hydrogen in the gas phase is usually 0.5 vol% or more, preferably 10 vol% or more, since this promotes the production of esculetin. is 1.0 vol% or more, more preferably 2.0 vol% or more, while it is usually 100 vol% or less, preferably It is preferably 20 vol % or less, and more preferably 10 vol % or less.

[0033] The method for creating such an environment in the gas phase and aqueous phase during culture is not particularly limited, but examples of the method include replacing the gas phase with the gas before culture, supplying the gas from the bottom of the culture vessel and / or to the gas phase of the culture vessel during culture, and bubbling the aqueous phase with the gas before culture. As the hydrogen, hydrogen gas may be used as it is. Alternatively, a source of hydrogen such as formic acid and / or a salt thereof may be added to the culture medium, and hydrogen may be generated during culture by the action of the microorganism.

[0034] The amount of ventilation is preferably 0.005 to 2 vvm, and more preferably 0.05 to 0.5 vvm. The mixed gas can also be supplied in the form of nanobubbles. The culture temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, and further preferably 30°C to 37°C. The pressurization conditions for the culture vessel are not particularly limited as long as they allow growth, but are preferably in the range of 0.001 to 1 MPa, and more preferably 0.01 to 0.5 MPa. The culture time is preferably 8 to 340 hours, more preferably 12 to 170 hours, and further preferably 16 to 120 hours.

[0035] Furthermore, the production of esculetin can be promoted by adding a surfactant, an adsorbent, an inclusion compound, or the like to the culture medium, which is preferable in this case. An example of a surfactant is Tween 80, which can be added at a concentration of about 0.001 g / L to about 10 g / L. Examples of the adsorbent include cellulose and its derivatives; dextrin; hydrophobic adsorbents such as the Diaion HP series and Sepabeads series manufactured by Mitsubishi Chemical Corporation; and the Amberlite XAD series manufactured by Organo Corporation.

[0036] Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and their analogues, such as methyl-β-cyclodextrin and trimethyl-β- Examples of the inclusion compounds include cyclodextrin, hydroxypropyl-β-cyclodextrin, etc. Among these, γ-cyclodextrin is sometimes the most effective, and is therefore preferred in that case. In addition, the coexistence of two or more types of inclusion compounds can further promote the production of esculetin, and is therefore preferred in that case. The amount of addition, in terms of the total molar ratio when scoparone is taken as 1, is usually 0.1 or more, preferably 0.5 or more, more preferably 1.0 or more, while it is usually 5.0 or less, preferably 2.5 or less, more preferably 2.0 or less.

[0037] (Production of esculetin by resting cells of a microorganism capable of producing esculetin from scoparone) When the microorganism is in a resting state, the solution is preferably a salt solution or a buffer solution described in the section "Resting state of a microorganism capable of producing esculetin from scoparone" instead of the medium. For other conditions, the descriptions in the above section "Medium and production of esculetin by culture" are used.

[0038] (Other processes) This embodiment may include, for example, a step of quantifying the obtained esculetin. The method for quantifying the amount of esculetin may be a conventional method. For example, a part of the culture medium may be sampled, appropriately diluted, thoroughly stirred, and then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, followed by quantifying the amount of esculetin by high performance liquid chromatography.

[0039] This embodiment may also include a step of recovering the obtained esculetin. The recovery step includes a purification step, a concentration step, and the like. Purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), and the like; removal of solids and polymeric substances; extraction with organic solvents, ionic liquids, and the like; and adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, and the like. Concentration treatments in the concentration step include concentration using an evaporator, a reverse osmosis membrane, and the like. Furthermore, the obtained solution containing esculetin can be powdered by freeze-drying, spray-drying, etc. In the powdering process, an excipient such as lactose, dextrin, corn starch, etc. can also be added.

[0040] <Method of producing 4-acetylresorcinol> One aspect of the present disclosure is a microorganism belonging to the genus Blautia having the ability to produce 4-acetylresorcinol from paeonol in a solution containing paeonol. The method for producing 4-acetylresorcinol includes a step of producing 4-acetylresorcinol from paeonol.

[0041] (Microorganism capable of producing 4-acetylresorcinol from paeonol) The microorganism capable of producing 4-acetylresorcinol from paeonol used in this embodiment is a microorganism belonging to the genus Blautia that is capable of producing 4-acetylresorcinol from paeonol. In addition, microorganisms belonging to the genus Blautia can be read as bacteria belonging to the genus Blautia.

[0042] Microorganisms belonging to the genus Blautia are known to produce 4-acetylglucosamine from paeonol. As long as it has the ability to produce resorcinol, the microorganism is not particularly limited, and examples thereof include microorganisms belonging to Blautia prodacta (e.g., JCM 1471 strain, etc.), microorganisms belonging to Blautia coccoides (e.g., JCM 1395 strain, etc.), Microorganisms belonging to Blautia schinki (e.g., JCM 14657 strain, etc.), Examples of such microorganisms include those belonging to Blautia hominis (e.g., JCM 32276 strain, etc.). In addition to the above, Blautia sp. DC 3652 (NITE BP-02924) strain, Blautia sp. DC 3653 (NITE BP-02629) strain, Blautia Blautia sp. DC 3654 (NITE BP-02925) strain, etc. , Blautia sp. DC 3652 (NITE BP-02924).

[0043] Blautia sp. DC 3652 (NITE BP-02924) strain, Blautia The deposit information for Blautia sp. DC 3653 (NITE BP-02629) and Blautia sp. DC 3654 (NITE BP-02925) is as described above.

[0044] Taking Blautia prodacta JCM 1471 strain as an example, in this embodiment, the Blautia prodacta JCM 1471 strain is not limited to the deposited strain, and may be a strain substantially equivalent to the deposited strain. The substantially equivalent strain refers to a strain belonging to the same genus or species as the deposited strain and having the ability to produce 4-acetylresorcinol from paeonol. The substantially equivalent strain is a strain whose 16S rRNA gene base sequence has a homology of 98.5% or more, preferably 98.7% or more, more preferably 99% or more, and even more preferably 100% to the 16S rRNA gene base sequence of the deposited strain. Furthermore, the deposited strain may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc., so long as it has the ability to produce 4-acetylresorcinol from paeonol. The same is true for the other previously mentioned deposited strains belonging to the genus Blautia which have the ability to produce 4-acetylresorcinol from paeonol.

[0045] In this embodiment, one or more kinds of the microorganisms may be used, and one or more strains of the microorganisms may be used.

[0046] (Residual form of a microorganism capable of producing 4-acetylresorcinol from paeonol) In this embodiment, the microorganism belonging to the genus Blautia and having the ability to produce 4-acetylresorcinol from paeonol includes its resting form. For details of the microbial cell, the description in the above section "Microbial cell having the ability to produce esculetin from scoparone" is incorporated herein by reference for the case of a microbial cell having the ability to produce 4-acetylresorcinol from paeonol.

[0047] (Solution containing Paeonol) For details of the solution containing paeonol in this embodiment, the description in the "Solution containing scoparone" section above is incorporated by reference in the case of producing 4-acetylresorcinol from paeonol.

[0048] (Culture medium and production of 4-acetylresorcinol by culture) For details of the process for producing 4-acetylresorcinol from paeonol, the description in the section "Culture medium and production of esculetin by culture" is incorporated herein by reference for the case of producing 4-acetylresorcinol from paeonol.

[0049] (Production of 4-acetylresorcinol by resting cells of microorganisms capable of producing 4-acetylresorcinol from paeonol) When the microorganism is a stationary body of a microorganism having the ability to produce 4-acetylresorcinol from paeonol, the solution is The salt solutions and buffer solutions described in the section entitled "Static Formation of Microorganisms Having the Compound" are preferred. For other conditions, the description in the above section "Culture medium and production of esculetin by culture" is used for producing 4-acetylresorcinol from paeonol.

[0050] (Other processes) This embodiment may include, for example, a step of quantifying the obtained 4-acetylresorcinol. For details of the step of quantifying the obtained 4-acetylresorcinol, the description in the "Other steps" section of the "Method for producing esculetin" according to the embodiment is incorporated herein by reference as the step of quantifying the obtained 4-acetylresorcinol.

[0051] This embodiment may also include a step of recovering the obtained 4-acetylresorcinol. For details of the step of recovering the obtained 4-acetylresorcinol, the description in the "Other steps" section of the "Method for producing esculetin" according to the embodiment is incorporated herein by reference as the step of recovering the obtained 4-acetylresorcinol.

[0052] <Method of producing eriodictyol> One aspect of the present disclosure is a microorganism belonging to the genus Eubacterium that has the ability to produce eriodictyol from hesperetin in a solution containing hesperetin. The method for producing eriodictyol includes a step of producing eriodictyol from hesperetin.

[0053] Furthermore, in a solution containing hesperetin, the microorganism capable of producing eriodictyol from hesperetin may be a microorganism belonging to the genus Blautia and / or a microorganism belonging to the genus Eubacterium that has the ability to produce eriodictyol from hesperetin. In addition, microorganisms belonging to the genus Blautia can be read as bacteria belonging to the genus Blautia, and microorganisms belonging to the genus Eubacterium can be read as bacteria belonging to the genus Blautia. , can be read as bacteria belonging to the genus Eubacterium.

[0054] Microorganisms belonging to the genus Blautia include hesperetin, erythrophorus, and erythrophorus. As long as the microorganism has the ability to produce aldehyde, it is not particularly limited. For example, microorganisms belonging to Blautia prodacta (e.g., JCM 1471 strain, etc.), microorganisms belonging to Blautia coccoides (e.g., JCM 1395 strain, etc.), Examples of suitable microorganisms include those belonging to the genus Blautia schinki (for example, JCM 14657 strain, etc.), and those belonging to the genus Blautia hominis (for example, JCM 32276 strain, etc.). In addition to the above, Blautia sp. DC 3652 (NITE BP-02924) strain, Blautia sp. DC 3653 (NITE BP-02629) strain, Blautia Blautia sp. DC 3654 (NITE BP-02925) strain, etc. , Blautia sp. DC 3652 (NITE BP-02924).

[0055] Blautia sp. DC 3652 (NITE BP-02924) strain, Blautia The deposit information for Blautia sp. DC 3653 (NITE BP-02629) and Blautia sp. DC 3654 (NITE BP-02925) is as described above.

[0056] Taking Blautia prodacta JCM 1471 strain as an example, in this embodiment, the Blautia prodacta JCM 1471 strain is not limited to the deposited strain, but may be a strain substantially equivalent to the deposited strain. A substantially equivalent strain refers to a strain belonging to the same genus or species as the deposited strain, and having the ability to produce eriodictyol from hesperetin. In addition, a substantially equivalent strain refers to a strain having a 16S rRNA gene sequence similar to that of the deposited strain, and having the ability to produce eriodictyol from hesperetin. The nucleotide sequence of the 16S rRNA gene of the deposited strain has a homology of 98.5% or more, preferably 98.7% or more, more preferably 99% or more, and even more preferably 100% to the nucleotide sequence of the 16S rRNA gene of the deposited strain. Furthermore, the deposited strain may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, or the like, so long as it has the ability to produce eriodictyol from hesperetin. The same is true for the other previously mentioned deposited strains belonging to the genus Blautia which have the ability to produce eriodictyol from hesperetin.

[0057] Microorganisms belonging to the genus Eubacterium include hesperetin, erythropoietin, and erythropoietin. As long as it has the ability to produce odctyols, it is not particularly limited, but examples thereof include microorganisms belonging to Eubacterium limosum (e.g., JCM 6421 strain, JCM 6501 strain, JCM 9978 strain, etc.), etc. Preferably, it is Eubacterium limosum JCM 6421 strain.

[0058] Let us use Eubacterium limosum JCM 6421 as an example. In this embodiment, the Eubacterium limosum JCM 6421 strain is The deposited strain is not limited to the deposited strain, and may be a strain substantially equivalent to the deposited strain. A substantially equivalent strain refers to a strain belonging to the same genus or species as the deposited strain and having the ability to produce eriodictyol from hesperetin. A substantially equivalent strain refers to a strain whose 16S rRNA gene base sequence has a homology of 98.5% or more, preferably 98.7% or more, more preferably 99% or more, and even more preferably 100% to the 16S rRNA gene base sequence of the deposited strain. Furthermore, the deposited strain may be a strain bred from the deposited strain or a substantially equivalent strain by mutation treatment, genetic recombination, selection of natural mutants, or the like, so long as it has the ability to produce eriodictyol from hesperetin. The same is true for the other previously mentioned deposited strains belonging to the genus Eubacterium which have the ability to produce eriodictyol from hesperetin.

[0059] In this embodiment, one or more kinds of the microorganisms may be used, and one or more strains of the microorganisms may be used.

[0060] (Residual form of a microorganism capable of producing eriodictyol from hesperetin) In this embodiment, the microorganism belonging to the genus Blautia and / or the microorganism belonging to the genus Eubacterium that has the ability to produce eriodictyol from hesperetin includes resting forms thereof. For details of the microbial cell, the description in the above section "Microbial cell capable of producing esculetin from scoparone" is incorporated herein by reference for the case of a microbial cell capable of producing eriodictyol from hesperetin.

[0061] (Solution containing hesperetin) For details of the solution containing hesperetin in this embodiment, the description in the section "Solution containing scoparone" above is used by reference in the case of producing eriodictyol from hesperetin.

[0062] (Culture medium and production of eriodictyol by culture) For details of the process of producing eriodictyol from hesperetin, the description in the section "Culture medium and production of esculetin by culture" is incorporated herein by reference for the case of producing eriodictyol from hesperetin.

[0063] (Eriodictyol production by stationary cells of microorganisms capable of producing eriodictyol from hesperetin) (Production of riodictyol) When the microorganism is a resting state of a microorganism having the ability to produce eriodictyol from hesperetin, the solution is preferably a salt solution or buffer solution as described in the section "Resting state of a microorganism having the ability to produce esculetin from scoparone." As for other conditions, the description in the above section "Medium and production of esculetin by culture" is used for the production of eriodictyol from hesperetin.

[0064] (Other processes) This embodiment may include, for example, a step of quantifying the obtained eriodictyol. For details of the step of quantifying the obtained eriodictyol, the description in the "Other steps" section of the "Method for producing esculetin" according to the embodiment is used as the step of quantifying the obtained eriodictyol.

[0065] In addition, this embodiment may include a step of recovering the obtained eriodictyol. For details of the step of recovering the obtained eriodictyol, the description in the "Other steps" section of the "Method for producing esculetin" according to the embodiment is used as the step of recovering the obtained eriodictyol. EXAMPLES

[0066] Examples are set forth below, but none of the examples are to be construed in a limiting sense.

[0067] <Example 1> Wilkins-Chalgren Anaerobe broth (Thermo Fisher Scientific) was filled with Scoparon (later As described above, when the microorganism used was Blautia sp. DC 3652 (NITE BP-02924) strain, the final concentration was 50 mg / L, and when the microorganism used was Eubacterium limosum JCM 6421 strain, the final concentration was 100 mg / L. After that, the mixture was sterilized by heating and aerated. The fermentation medium was prepared by replacing the air phase with N2:CO2:H2 (80%:10%:10%) gas. The fermentation medium was used with Blautia sp. DC 3652 (NITE BP-02924) strain, which is a microorganism capable of producing esculetin from scoparone, or Eu Eubacterium limosum JCM 6421 strain was inoculated and incubated at 37°C for 72 hours. After incubation, the culture was diluted with 3 volumes of ethanol per 5 mL of culture medium, and the mixture was filtered with a 0.45 μm filter. After filtering the mixture, the filtrate was subjected to quantitative analysis of esculetin by HPLC under the following conditions.

[0068] (HPLC conditions) Column: Inertsil ODS-3 (4.6 mm x 25 cm, 5 μm) Eluent A: H2O / Formic acid (99 / 1) Eluent B: Acetonitrile / Formic acid (99 / 1) Gradient conditions: Solution B 0-5 min (20%) → 10 min (70%) → 30 min (70%) Column temperature: 40℃ Flow rate: 1.0 mL / min Detection: UV at 340 nm

[0069] As a result of the cultivation, Blautia sp. DC 3652 (NITE BP-02924) strain was identified. When Eubacterium limosum JCM 6421 was used, 6.1% of scoparone was converted to esculetin, whereas when Eubacterium limosum JCM 6421 was used, 14.4% of scoparone was converted to esculetin.

[0070] <Example 2> A modified GAM medium (manufactured by Nissui Pharmaceutical) was added with paeonol (final concentration 50 mg / L), sterilized by heating, and the gas phase was replaced with N2:CO2:H2 (80%:10%:10%) gas to be used as the fermentation medium. Blautia sp. DC 3652 (NITE BP-02924) strain, which is a microorganism capable of producing 4-acetylresorcinol from paeonol, was inoculated into the fermentation medium and cultured anaerobically at 37°C for 72 hours. After the culture, the culture solution was diluted with 3 volumes of ethanol per 5 mL, filtered through a 0.45 μm filter, and the filtrate was used for quantitative analysis of 4-acetylresorcinol by HPLC under the following conditions.

[0071] (HPLC conditions) Column: Inertsil ODS-3 (4.6 mm x 25 cm, 5 μm) Eluent A: H2O / Formic acid (99 / 1) Eluent B: Acetonitrile / Formic acid (99 / 1) Gradient conditions: Solution B 0-5 min (20%) → 10 min (70%) → 30 min (70%) Column temperature: 40℃ Flow rate: 1.0 mL / min Detection: UV at 280 nm

[0072] As a result of the incubation, 2.2% of paeonol was converted to 4-acetylresorcinol.

[0073] <Example 3> Wilkins-Chalgren Anaerobe broth (Thermo Fisher Scientific) was added with hesperetin ( As described below, when Blautia sp. DC 3652 (NITE BP-02924) strain was used as the microorganism, a final concentration of 250 mg / L was added, and when Eubacterium limosum JCM 6421 strain was used, a final concentration of 100 mg / L was added, followed by heat sterilization and replacement of the gas phase with N2:CO2:H2 (80%:10%:10%) gas to prepare the fermentation medium. Blautia sp. DC 3652 (NITE BP-02924) strain was added as the microorganism capable of producing eriodictyol from hesperetin to the fermentation medium. or Eubacterium limosum JCM 6421 strain was inoculated and incubated at 37°C. After incubation, the culture was diluted with 3 volumes of ethanol per 5 mL of culture solution, filtered through a 0.45 μm filter, and the filtrate was subjected to HPLC under the following conditions to detect eriodictyol. Quantitative analysis of the samples was performed.

[0074] (HPLC conditions) Column: Inertsil ODS-3 (4.6 mm x 25 cm, 5 μm) Eluent A: H2O / Formic acid (99 / 1) Eluent B: Acetonitrile / Formic acid (99 / 1) Gradient conditions: Solution B 0-5 min (20%) → 10 min (50%) → 30 min (50%) Column temperature: 40℃ Flow rate: 1.0 mL / min Detection: UV at 280 nm

[0075] As a result of the cultivation, Blautia sp. DC 3652 (NITE BP-02924) strain was identified. When used, 39.9% of hesperetin was converted to eriodictyol. When Eubacterium limosum JCM 6421 strain was used, hesperetin of which 10.1% was converted to eriodictyol.

Claims

1. In a solution containing paeonol, a microorganism belonging to the genus Blautia having the ability to produce 4-acetylresorcinol from paeonol was subjected to the A method for producing 4-acetylresorcinol, comprising the step of producing 4-acetylresorcinol from a strain of the fungus.

2. In a solution containing hesperetin, a microorganism belonging to the genus Eubacterium capable of producing eriodictyol from hesperetin was found to produce eriodictyol from hesperetin. A method for producing eriodictyol, comprising a step of producing eriodictyol from.

3. 2. The method according to claim 1, wherein the microorganism belonging to the genus Blautia is Blautia sp. DC 3652 (NITE BP-02924) strain.

4. The microorganism belonging to the genus Eubacterium is Eubacterium limos.

3. The method according to claim 2, wherein the microorganism is Eubacterium limosum.

5. The microorganism belonging to Eubacterium limosum is Eubacterium limosum.

5. The method according to claim 4, wherein the Eubacterium limosum JCM 6421 strain is used. method.