JP2017192331A - Method for producing urolithins - Google Patents

Abstract

An object of the present invention is to provide a method for producing urolithins more efficiently than before. A urolithin-producing step in which urolithins-producing microorganisms produce urolithins from ellagic acid by fermentation in a medium containing ellagic acid and cyclodextrin, and a recovery step of recovering the urolithins produced in the production step. A method for producing urolithins, comprising: [Selection figure] None

Description

  The present invention relates to a method for producing urolithins.

  Urolithins represented by urolithin A and urolithin C are known as ellagic acid metabolites derived from ellagitannins contained in pomegranate, raspberry, blackberry, cloudberry, strawberry, walnut and the like. It is known that ellagitannins are classified into hydrolyzable tannins, and when ingested, they are hydrolyzed and converted to ellagic acid. Such ellagitannins and ellagic acid have very low intestinal absorption in the body, but when they are ingested, they are known to be further metabolized by the human colonic microflora and converted to urolithins.

  Regarding the production of urolithins in vivo, it was reported in a study using rats that urolithin is produced as an in vivo metabolite of ellagitannin by ingesting ellagitannins such as geraniin and then analyzing urine urine in urine. (Non-Patent Document 1). Further, in Non-Patent Document 2, in humans, urolithin analogs are detected in urine after ingestion of pomegranate extract containing ellagitannin mainly including punicalagin, and urolithin A and urolithin C are the main urolithins. It has been reported. In particular, it has been reported that urolithin A has various effectiveness such as an antioxidant action (Non-patent Document 3), an anti-inflammatory action (Non-Patent Document 4), and an anti-glycation action (Non-Patent Document 5). On the other hand, Non-patent Document 6 reports that urolithin M3 and urolithin M4, which are metabolites derived from urolithin C and methylated in the liver and the like, are detected as one of urinary metabolites. Thus, urolithins are one of the most important compounds in the living body, and it is required to add urolithins to cosmetics, foods, raw materials for pharmaceuticals, processed products thereof and the like.

  As a method for synthesizing these urolithins, 2-bromo-5-methoxybenzoic acid is used as a starting material to obtain 2-bromo-5-hydroxybenzoic acid by demethylation and react with resorcinol to obtain urolithin A. Have been reported (Non-Patent Document 7). However, it was not suitable for the purpose of using urolithins as food.

  Recently, Gordonibacter urolithinfaciens has been isolated and identified as an intestinal bacterium that produces urolithin C, a kind of urolithin, from ellagic acid, and a method for producing urolithin C by fermenting ellagic acid using this intestinal bacterium has been reported. (Patent Document 1, Non-Patent Document 8). However, the accumulated concentration of urolithin C in the fermentation broth is about 2 mg / L, which is not suitable for such purpose.

International Publication No. 2014/147280 Pamphlet

J. Agric. Food Chem. 56, 393-400 (2008) Mol. Nutr. Food Res. 58, 1199-1211 (2014) Biosci. Biotechnol. Biochem. 76, 395-399 (2012) J. Agric. Food Chem. 60, 8866-8876 (2012) Mol. Nutr. Food Res. 55, S35-S43 (2011) Planta Med. 77, 1110-1115 (2011) J. Agric. Food Chem. 56, 393-400 (2008) Food Funct. 5 (8), 1779-1784 (2014)

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method for producing urolithins more efficiently than conventional methods.

  As a result of diligent research to solve the above problems, the present inventors have found that cyclodextrins coexist in the medium when urolithins are produced by fermentation using urolithin-producing microorganisms using ellagic acid as a raw material. The inventors have found that the production efficiency of urolithins is remarkably increased, and have completed the present invention. That is, the present invention is as follows.

<1>
A urolithin production step for fermenting urolithins from ellagic acid to a urolithin-producing microorganism in a medium containing ellagic acid and cyclodextrin, and a recovery step for recovering the urolithins produced in the production step Manufacturing method.
<2>
The production method according to <1>, wherein the cyclodextrin is one or more selected from the group consisting of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. <3>
The production method according to <1> or <2>, wherein the urolithins are urolithin C.
<4>
The production method according to any one of <1> to <3>, wherein the urolithin-producing microorganism is one or more selected from the group consisting of Bacteroides bacteria and Gordonibacter bacteria.

The present invention provides a method for producing urolithins with significantly higher production efficiency of urolithins than the conventional method of producing urolithins from ellagic acid, which is a raw material, in the absence of cyclodextrin using a urolithin-producing microorganism. be able to.
Urolithins are substances that exhibit effects such as antioxidant, anti-inflammatory and anti-glycation, and can be expected to prevent skin aging and wrinkle prevention, whitening effect, metabolic syndrome, diabetes and other symptoms. Using the urolithins obtained by the production method of the present invention, cosmetics, external quasi-drugs, external medical supplies, external hygiene products, external pharmaceuticals, foods, supplements, and the like can be provided.

  The present invention includes a urolithin production step in which a urolithin-producing microorganism produces urolithin from ellagic acid by fermentation in a medium containing ellagic acid and cyclodextrin, and a recovery step of recovering urolithin produced in the production step, Is a method for producing urolithins. Other steps may be included.

<1. Urolithin production process>
The urolithin production step in the production method of the present invention is a step in which urolithins are produced from ellagic acid by fermentation in a medium containing ellagic acid and cyclodextrin. Other steps may be included.

(Urolithins)
Although the urolithins of this invention are not specifically limited, The structure is a substance represented by following General formula (1). In addition, as shown in Table 1, urolithins are represented by R1-R6 in the chemical formula, and urolithin A, urolithin B, urolithin C, urolithin D, urolithin E, urolithin M3, urolithin M4, urolithin M5, urolithin M6, urolithin M7, and Examples include isouroritin A. Any one kind or two or more kinds of urolithins may be used.
Among these, urolithin C is preferable because production efficiency is remarkably increased by the production method of the present invention.

(Elagic acid)
The fermentation raw material in the present invention is ellagic acid. For example, it can be prepared as follows.
It can be prepared by extracting ellagitannins such as punicalagin and geraniin from plants and hydrolyzing them into ellagic acid, or by extracting ellagic acid. At this time, the kind of plant is not particularly limited, and examples thereof include pomegranate, raspberry, blackberry, cloudberry, boysenberry, strawberry, walnut, and ginger. Of these, pomegranate, boysenberry and genokosho are preferred, and pomegranate is more preferred because of its high content of ellagitannin and / or ellagic acid.

Any one of these plants may be used, or two or more thereof may be used in combination. Moreover, the extraction method and extraction conditions from this plant are not specifically limited, What is necessary is just to follow a conventional method. For example, a known extraction method such as water extraction, hot water extraction, hot water extraction, alcohol extraction, supercritical extraction or the like can be used.
When performing solvent extraction, the solvent may be, for example, water; alcohols such as lower alcohols such as methanol and ethanol, and polyhydric alcohols such as propylene glycol and 1,3-butylene glycol (anhydrous or hydrated). ); Ketones such as acetone; esters such as diethyl ether, dioxane, acetonitrile, and ethyl acetate; xylene; and the like, preferably water and ethanol. Any one of these solvents may be used, or two or more thereof may be used in combination.
The method for hydrolyzing the extracted ellagitannin such as punicalagin to ellagic acid is not particularly limited, and examples thereof include a method of hydrolyzing with an acid, an enzyme, and a microorganism.

The obtained ellagic acid can be used as it is, but it may be dried to be powdered for storage. Moreover, you may refine | purify, a concentration process, etc. to the obtained ellagic acid as needed. As the purification treatment, treatment such as filtration or adsorption or decolorization using an ion exchange resin or activated carbon column can be performed. As the concentration treatment, a conventional method such as an evaporator can be used.
In addition, the obtained ellagic acid (or purified product or concentrate) is subjected to lyophilization and pulverized, and excipients such as dextrin, corn starch, gum arabic and the like are added and pulverized by spray drying. You may powder according to well-known methods, such as a method. Further, it may be used after being dissolved in pure water, ethanol or the like as required.

If ellagic acid is contained, commercially available ellagic acid can also be used. For example, pomegranate extract, pomegranate concentrated fruit juice, pomegranate juice and the like can be used, but are not limited thereto.
The amount and concentration of ellagic acid during fermentation are not particularly limited. It can be set as appropriate according to the amount of fermentation broth and the amount of urolithins to be produced.

(Cyclodextrin)
In the production method of the present invention, the medium containing ellagic acid which is a fermentation raw material contains cyclodextrin.
Cyclodextrin is a kind of cyclic oligosaccharide having a cyclic structure in which several molecules of D-glucose are bonded by α (1 → 4) glucoside bond. For example, it is known that 5 or more glucoses are bound, α-cyclodextrin (cyclohexaamylose) bound with 6 glucoses, β-cyclodextrin (cycloheptaamylose) bound with 7 glucoses, and 8 bound. γ-cyclodextrin (cyclooctaamylose) can be mentioned. Further, examples of the cyclodextrin derivative include chemically modified cyclodextrins such as hydroxypropylation, acetylation, triacetylation, and methylation. The kind of cyclodextrin in this invention is not restrict | limited as long as a desired effect is show | played, Any 1 type may be used and 2 or more types may be used together.

  Among these, in the production method of the present invention, since the production efficiency of urolithins is further increased, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin are preferable, and β-cyclodextrin and γ-cyclodextrin are preferable. It is more preferable that

In the production method of the present invention, the cyclodextrin content in the medium containing ellagic acid, which is a fermentation raw material, increases the production efficiency of urolithins. It is not less than twice and not more than 40 times, preferably not less than 0.5 and not more than 20 times, more preferably not less than 1 and not more than 5 times.
Further, in any case of α-cyclodextrin simple substance, β-cyclodextrin simple substance, and γ-cyclodextrin simple substance, the molar ratio to ellagic acid is usually 0.1 to 40 times, preferably 0.5 to 20 times. It is not more than double, more preferably not less than 1 and not more than 5 times.

  The origin of cyclodextrin is not particularly limited, and may be a natural product or a synthetic product. There is no restriction | limiting in particular also about the extraction method and a refinement | purification method, What is necessary is just to follow a well-known method. Commercially available products may also be used, for example, cyclodextrins manufactured by Cyclochem or Shimizu Minato Sugar Co., Ltd.

(Urolithin-producing microorganism)
Although the urolithin-producing microorganism in the production method of the present invention is not particularly limited, it is preferably a bacterium belonging to the genus Bacteroides or Gordonibacter, more preferably a bacterium belonging to the genus Bacteroides uniformis, Gordonibacter urolithinfaciens, because of the higher production efficiency of urolithins 1 or more selected from the group consisting of bacteria belonging to Gordonibacter pamelaeae, more preferably from the group consisting of Bacteroides uniformis HGB5B146 (NITE BP-02193) strain, Gordonibacter urolithinfaciens strain DSM27213, and Gordonibacter pamelaeae strain DSM19378 One or more selected.

The Bacteroides uniformis HGB5B146 (NITE BP-02193) strain was established on January 20, 2016 by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (Kazusa Kamashizu 2-5, Kisarazu City, Chiba Prefecture 292-0818) 8 Room 122) was deposited internationally under the Budapest Treaty with the deposit number of NITE BP-02193.
The Gordonibacter urolithinfaciens DSM27213 strain and the Gordonibacter pamelaeae DSM19378 strain are deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, and those skilled in the art can obtain them at the time of filing this application.

  In the production method of the present invention, only one of these microorganisms may be used, or two or more thereof may be used in combination. Further, it may be a strain substantially equivalent to the specific strain listed above. The substantially equivalent strain refers to a strain having the same high urolithin-producing ability as the same strain. In addition, a substantially equivalent strain further has a homology of 98% or more, preferably 99% or more, more preferably 100%, with the base sequence of the 16S rRNA gene of the 16S rRNA gene. And preferably has the same mycological properties as the same strain. Furthermore, a strain bred from the same strain or a strain substantially equivalent thereto by mutation treatment, gene recombination, selection of a natural mutant strain, or the like may be used.

(fermentation)
In the production method of the present invention, the urolithin-producing microorganism produces urolithin by fermentation from ellagic acid as a fermentation raw material in a medium containing ellagic acid and cyclodextrin under conditions suitable for the production of urolithin.
Conditions suitable for the production of urolithins refer to conditions under which the survival and activity of urolithin-producing microorganisms are maintained. More specifically, it means that a gas phase condition capable of survival of the urolithin-producing microorganism is maintained, and nutrients are provided to support its activity and growth. Various medium compositions suitable for the survival of the urolithin-producing microorganism are known. Therefore, those skilled in the art can select an appropriate medium composition according to the urolithin-producing microorganism. For example, ANAEROBE BASAL BROTH (ABB) medium manufactured by OXIO can be used.

Moreover, a water-soluble organic substance can be added to a culture medium as a carbon source. Examples of water-soluble organic substances include the following compounds. The following compounds can be listed as water-soluble organic substances. That is, saccharides such as sorbose, fructose, glucose and glucose; alcohols such as methanol; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid and formic acid.
The concentration of the organic substance added to the medium as the carbon source can be adjusted as appropriate in order to efficiently grow it. Generally, excess and deficiency can be avoided by selecting the addition amount from the range of 0.1 to 10 wt / vol%.

In addition to the above carbon source, a nitrogen source is added to the medium. As the nitrogen source, various nitrogen compounds that can be used for ordinary fermentation can be used. Preferred inorganic nitrogen sources are ammonium salts and nitrates. More preferred are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, triammonium citrate, potassium nitrate and sodium nitrate.
On the other hand, preferred organic nitrogen sources are amino acids, yeast extracts, peptones (eg, polypeptone N), meat extracts (eg, Lab-Remco powder, bouillon, etc.), liver extracts, digested serum powder, and the like. More preferable organic nitrogen sources are arginine, cysteine, citrulline, lysine, yeast extract, and peptones (for example, polypeptone N).

  Furthermore, in addition to a carbon source and a nitrogen source, other organic substances or inorganic substances can be added to the medium. For example, growth and activity may be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. Examples of microorganism growth cofactors derived from animals and plants such as inorganic compounds and vitamins include the following.

Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium sulfate Folic acid Manganese sulfate Pyridoxine Sodium chloride Thiamine Cobalt Riboflavin Calcium chloride Nicotinic acid Zinc sulfate Pantothenic acid Copper sulfate Vitamin B12
Alum thiooctanoic acid sodium molybdate p-aminobenzoic acid potassium chloride boric acid nickel chloride sodium tungstate sodium selenate sodium ferrous sulfate sodium acetate trihydrate magnesium sulfate heptahydrate manganese sulfate tetrahydrate

  A method for producing a culture solution by adding growth cofactors derived from animals and plants such as these inorganic compounds and vitamins is known. The medium can be liquid, semi-solid, or solid. A preferred medium form is a liquid medium.

In the present invention, the urolithin-producing microorganism can be cultured according to a known culture method. In industrial production, a continuous fermentation system capable of continuously supplying a medium and a substrate gas and having a mechanism for collecting a culture is preferable.
As the incubator, a commonly used culture tank can be used as it is. Culture tanks that can also be used for culturing the microorganisms are commercially available. An anaerobic atmosphere can also be created by replacing oxygen mixed in the culture tank with an inert gas such as nitrogen or a substrate gas.

In the culture of the strain, an additional function can be given to the culture tank. For example, in addition to a commonly used stirring and mixing tank, a bubble column type or draft tube type culture tank can also be used. The above strain is released and dispersed by the mixed gas blown into the liquid medium, and the strain and the medium can be sufficiently brought into contact with each other. Biotrickling filter (biotrickling
filter)), the above strains are inhabited while dripping moisture in a highly breathable slag, other ceramic-based inorganic fillers, or packed layers of organic synthetic materials such as polypropylene, and gas is passed there. It can also be cultured. Furthermore, the strain to be used can be used by immobilizing it on carrageenan gel, alginic acid gel, acrylamide gel, chitin, cellulose, agar or the like by a conventional method.

  Depending on the shape of the culture tank, a stirrer or the like can be used to sufficiently stir the medium. By agitating the culture in the culture tank, it is possible to increase the chance of contacting the medium components and the substrate gas with the anaerobic microorganisms and optimize the production efficiency of urolithins. The substrate gas can also be supplied as nanobubbles.

For sufficient growth of the above strain, the pH of the culture is preferably 5.0 to 8.5, more preferably 6.0 to 8.0, and even more preferably 6.5 to 6.8. Further, the temperature of the culture tank is not particularly limited, but is preferably 20 ° C. to 42 ° C., more preferably 25 ° C. to 40 ° C., and further preferably 30 ° C. to 37 ° C. in order to increase the recovered amount of urolithins. A temperature of ° C can be mentioned.
Fermentation time can be suitably set according to the production amount of urolithins, the residual amount of ellagic acid, and the like. Usually, it can be exemplified by 8 to 340 hours, preferably 12 to 240 hours, more preferably 16 to 170 hours, but is not limited thereto.
In order to efficiently recover urolithins, the medium can be continuously supplied. The amount of fresh medium supplied to the culture tank is preferably 0.04 to 2 / hr, more preferably 0.08 to 1 / hr, as a dilution ratio in the culture in the culture tank.

  Moreover, it is preferable to culture | cultivate the said strain | stump | stock under the gaseous phase which consists of 1 or more types of gas containing hydrogen. The hydrogen concentration is not particularly limited. A combination of gases constituting the gas phase is not particularly limited, and one or more kinds of gases selected from carbon dioxide, nitrogen and the like in addition to hydrogen can be used as a constituent component. In order to efficiently recover urolithins, the aeration amount of the mixed gas constituting the gas phase to the culture tank is 0.01 to 2.0 V / V / M gas amount / liquid amount / minute. preferable.

  The pressurizing conditions for culturing the above strain are not particularly limited as long as the conditions allow growth. Preferred pressure conditions include, but are not limited to, a range of 0.02 to 0.2 MPa.

The culture purified solution obtained by fermentation contains a high concentration of urolithins. The content of urolithins in the culture solution varies depending on the strain used, fermentation conditions, etc., but at least in a total amount of 1.05 liters of the culture solution, usually 0.005 to 20 g, preferably 0.1 to 15 g, Preferably it is 1-10g.
The fact that urolithins were obtained by fermentation can be confirmed by quantifying ellagic acid, urolithin analogs and the like in the culture. These quantifications can be performed based on, for example, the description in Patent Document 1. An example is shown below.

For example, ethyl acetate to which an acid such as formic acid is added as necessary is added to the culture solution, and the mixture is vigorously stirred and then centrifuged to remove the ethyl acetate layer. If necessary, the same operation can be performed several times on the same culture solution, and the ethyl acetate layer can be combined to obtain a urolithin extract. The extract is concentrated under reduced pressure using an evaporator or the like, dried to dryness, and dissolved in methanol. This is filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane, and the insoluble matter is removed to obtain a sample for high performance liquid chromatography. Examples of conditions for high performance liquid chromatography include, but are not limited to, the following.

[High-performance liquid chromatography conditions]
Column: Inertsil ODS-3 (250 × 4.6 mm) (manufactured by GL Science)
Eluent: Water / acetonitrile / acetic acid = 74/25/1
Flow rate: 1.0 mL / min
Column temperature: 40 ° C
Detection: 305nm

<2. Recovery process>
The recovery step in the production method of the present invention is a step of recovering the urolithin produced in the urolithin production step. Other steps may be included.

(Recovery)
In the production method of the present invention, the produced urolithins can be obtained by solid-liquid separation of the obtained culture solution containing urolithins by filtration or centrifugation and collecting the liquid phase. As one method of solid-liquid separation, for example, filtration with a filter paper or an ultrafiltration membrane or a centrifugal separator such as a spar decanter can be mentioned.

  The urolithins obtained by the production method of the present invention can be used in a solid state by heat drying treatment, spray drying treatment or freeze drying treatment, if necessary. The heat drying process or the spray drying process can be performed using, for example, a spray drying apparatus. The freeze-drying process can be performed using a freeze-drying apparatus. The fermentation culture subjected to the heat drying treatment, spray drying treatment or freeze drying treatment may be subjected to a pulverization treatment as necessary.

  The urolithins that have been heat-dried or spray-dried or freeze-dried by such a method can contain 1 to 50% by weight of urolithins in a total amount in the dry powder. For example, 2 points selected from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% It is preferably a mass percent concentration indicated by a concentration range in which the mass percent concentration is a lower limit (˜ or higher or higher) and an upper limit (˜less than or lower).

<3. Uses of urolithins>
The urolithins obtained by the production method of the present invention can be used as cosmetics, quasi-drugs for external use, external medical supplies, external hygiene products, external pharmaceuticals, foods, supplements, and the like. By the action of urolithins, effects such as antioxidant, anti-inflammatory and anti-glycation can be expected.

  The urolithins obtained by the production method of the present invention may be present in a form containing only urolithins as components, but in addition to urolithins, known excipients, fragrances, colorants, emulsifiers, stabilizers , Thickener, enzyme, antiseptic, antibacterial agent, lubricant, surfactant, disintegrant, disintegration inhibitor, binder, absorption enhancer, adsorbent, moisturizer, solubilizer, preservative, flavoring agent In addition, a sweetener, an ultraviolet absorber, and the like may be blended as necessary within a range that does not impair the above effects.

(Cosmetics)
When the urolithins obtained by the production method of the present invention are used as cosmetic materials, liquids such as aqueous solutions, lotions, sprays, suspensions and emulsions; solids such as powders, granules and blocks; creams and pastes It can be prepared into cosmetics of various desired dosage forms such as a semi-solid form such as a gel. Such cosmetics include face wash, milky lotion, cream, gel, essence (beauty serum), basic cosmetics such as pack and mask, makeup cosmetics such as foundation and lipstick, oral cosmetics, aromatic cosmetics, hair cosmetics, body cosmetics, etc. Useful as various cosmetics.

  When used as a raw material for cosmetics, it can be produced according to conventional methods. Moreover, the compounding quantity to a foodstuff, a compounding method, and a compounding time can be selected suitably. Furthermore, it can be enclosed in appropriate containers, such as a bottle, a bag, a can, a box, and a pack, as needed.

  When used as a raw material for cosmetics, the content of urolithins relative to the total amount of cosmetics is not particularly limited as long as the above effects are exerted, but with respect to 100 g of cosmetics, the total amount in terms of dry weight, preferably 0.001 to 20 g, Preferably it is 0.01-10g, More preferably, it is 0.1-5g.

When used as a cosmetic material, known ingredients that are usually used can be added as appropriate.
For example, anionic surfactants (fatty acid soaps, sulfonate type anionic surfactants, sulfate ester type anionic surfactants, phosphate ester type anionic surfactants, acylmethyl taurine salts, monoalkyl phosphates , Acyl glutamate, isethionate, etc.), cationic surfactant (amine salt type cationic surfactant, quaternary ammonium type cationic surfactant (tetraalkylammonium type, pyridinium type)), nonionic Surfactant (glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, tetraoleic acid polyoxyethylene sorbit, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene Recall, polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyglycerin fatty acid ester, etc.), amphoteric surfactant (imidazoline type, betaine type, amino acid type) , Natural surfactants such as fluorosurfactants and silicone surfactants, synthetic surfactants,
Sodium alginate, propylene glycol alginate, gum arabic, xanthan gum, pectin, tragacanth, sodium carboxymethylcellulose, methylcellulose, carboxyvinyl polymer, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, cationized cellulose, cationized dextran, cationized dextrin, chitosan, Water-soluble polymers such as cationized vinylpyrrolidone polymer, N, N-dimethyl-3,5-methylenepiperidinium chloride polymer, milk protein, soybean protein, gelatin, egg protein, sodium caseinate, whey protein,
Ginkgo biloba, pearl oyster, carrot, shikoppi, caika, inchko, yashajitsu, licorice fraction, gokahi, sempukuka, hikai, yuzuuriha, chamomile, maronier, escin, terminaria, ruscogenin, butcher bloom, cola, guarana, mate, coffee, Cacao, Plectrantus, Tandin, Bisnaga, Silymarin, Leucocyanin, Hypericum Grass, Bear Goose, Perilla, Ogon, Keigai, Rosemary, Sage, Thyme, Artemisia, Paramecium, Prunus , Plant components such as Toki, Senkyu, Chimbu, Kanokosou, Bakushi, Spruce, Glaze, safflower, camellia, Bukkuri, mint
Succinic acid, fumaric acid, citric acid, pyruvic acid, glucuronic acid, 2-hydroxybutyric acid, lactic acid, malic acid, tartaric acid, tartronic acid, methyl pyruvate, ethyl pyruvate, vitamin A acid, vitamin C derivative, vitamin D, vitamin E, active ingredient such as oligopeptide, tranexamic acid ester, polyhydric alcohol, amino acid, mucopolysaccharide, protein, biological extract,
Moisturizers such as fermented metabolites, polysaccharides, plant extracts, phospholipids, ceramides,
Fats and oils (soybean oil, nutka oil, jojoba oil, avocado oil, almond oil, cacao oil, olive oil, sesame oil, persic oil, castor oil, coconut oil, mink oil, beef fat, pork fat, and other natural fats and oils Hydrogenated hardened oil and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid glyceride, diglyceride etc.), waxes (carnauba wax, whale wax, beeswax, lanolin etc.), hydrocarbons (liquid paraffin, Petrolatum, paraffin, microcrystalline wax, ceresin, squalane, pristane, etc.), higher fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, lanolinic acid, isostearic acid, etc. ), Higher alcohols (lauryl alcohol, Alcohol, stearyl alcohol, oleyl alcohol, cholesterol, 2-hexyldecanol, etc.), esters (cetyl octoate, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, Decyl oleate, Colesteel isostearate, etc., essential oils (mint oil, jasmine oil, cypress brain oil, cypress oil, spruce oil, ryu oil, turpentine oil, cinnamon oil, bergamot oil, mandarin oil, shibu oil, pine Oil, Lavender oil, Bay oil, Clove oil, Hiba oil, Rose oil, Eucalyptus oil, Lemon oil, Peppermint oil, Thyme oil, Rose oil, Sage oil, Menthol, Cineol, Eugenol, Citral, Citronellal, Borne Lumpur, linalool, geraniol, camphor, thymol, spilanthol, pinene, limonene, terpene compounds), fats and oils such as silicone oils (emollient component),
Sodium carbonate, sodium bicarbonate, sodium sesquicarbonate, borax, sodium sulfate, sodium sulfide, sodium nitrate, sodium thiosulfate, sodium polyphosphate, sodium phosphate, potassium chloride, potassium sulfide, calcium oxide, magnesium oxide, calcium carbonate, Inorganic salts such as magnesium carbonate,
Inorganic acids such as boric acid, metasilicic acid, silicic anhydride,
Yellow No. 4, Blue No. 1, Yellow No. 202, pigments such as chlorophyll, riboflavin, safflower, crocin, anthraquinone,
Fragrances,
Polymers such as acrylic resin, styrene resin, epoxy resin, nylon, polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate resin, polytetrafluoroethane, copolymers of these polymers, silicic acid, calcium silicate, natural aluminum silicate , Fine powders such as synthetic aluminum silicate, zeolite, titanium oxide, talc, kaolin, mica, bentonite,
Sulfur, Yunohana, mineral sand, mica powder, neutral white clay, rice cake, fungicide, preservative,
And other ingredients necessary for the preparation.

(External quasi-drugs, external medical supplies, external hygiene products, external pharmaceutical products)
When the urolithins obtained by the production method of the present invention are used as materials for quasi-drugs for external use, external-use medical supplies, external-use hygiene products, or external-use drugs, aqueous solutions, lotions, spray solutions, suspensions and emulsions Liquids such as powders, solids such as granules and blocks, semisolids such as creams and pastes, and gels.

  Specifically, basic cosmetics such as ointments, poultices, lotions, emulsions, creams, ointments, lotions, oils, packs, facial cleansers and skin cleansers, shampoos, rinses, hair treatments, hair creams, pomades, hair Makeup cosmetics such as sprays, hair styling, perms, hair nicks, hair dyes, hair growth and hair nourishment, foundations, white powder, funny, lipstick, blusher, eye shadow, eyeliner, mascara, eyebrows, eyelashes, Cosmetics for finishing such as nail polishes, perfumes, bath preparations, toothpastes, mouth fresheners, gargles, liquid odors / deodorants, sanitary products, sanitary cotton, wet tissue, and the like.

  These external quasi-drugs, external medical products, external hygiene products, and external pharmaceutical products are in accordance with conventional methods, and if necessary, fillers, extenders, excipients, binders, moisturizers, disintegrants, surface active agents. It can be produced using known adjuvants that can be usually used in the art, such as agents, lubricants, colorants, flavoring agents, solubilizing agents, suspending agents, and coating agents. In addition, these may contain coloring agents, preservatives, fragrances, flavors, sweeteners, and other external quasi-drugs, external medical supplies, external hygiene products, and external pharmaceutical products.

  When used as a material for external quasi-drugs, external medical supplies, external hygiene products, or external pharmaceuticals, it usually contains various known ingredients and additives used for them depending on the purpose. be able to. For example, higher alcohols, silicones, waxes, oils, alcohols, esters, metal soaps, gums, water-soluble polymer compounds, surfactants, vitamins, amino acids, animal, plant and herbal extracts Microbial culture metabolites such as and extracts, yeast extracts, pigments, astringents, bactericides / disinfectants, fragrances, pigments / colorants, sweeteners, hormones, sequestering agents, pH adjusters, chelating agents, antiseptics / preventions Bi-agent, refresher, stabilizer, emulsifier, animal / vegetable protein and its degradation product, animal / plant polysaccharide and its degradation product, animal / plant glycoprotein and its degradation product, blood flow promoter, anti-inflammatory Can contain agents, antiallergic agents, cell activators, keratolytic agents, wound treatment agents, foam enhancers, thickeners, oral preparations, deodorants / deodorants, bitterings, seasonings, enzymes, etc. .

  The aqueous solution is preferably applied to humans, but can be applied to animals other than humans as long as the above effects are exhibited.

  When used as a raw material for quasi-drugs, external-use medical supplies, external-use hygiene products, or external-use drugs, the content of urolithins relative to the total amount of each is not particularly limited as long as the above effects are exhibited, but each is 100 g. On the other hand, the total amount in terms of dry weight is preferably 0.001 to 20 g, more preferably 0.01 to 10 g, and still more preferably 0.1 to 5 g.

(Food)
When the urolithins obtained by the production method of the present invention are used as food materials, in addition to general foods, they can be used as foods for specified health use, dietary supplements, functional foods, foods for the sick, food additives, etc. . As food forms, soft drinks containing the aqueous solution, milk, pudding, jelly, candy, gum, gummi, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cakes, cream puffs, ham, meat sauce , Curry, stew, cheese, butter, dressing and the like.

  The urolithins can use water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like as main components. Examples of the protein include whole milk powder, skim milk powder, partially skim milk powder, casein, soybean protein, chicken egg protein, meat protein, and other animal and vegetable proteins, and their hydrolysates and butter. Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of lipids include lard, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oil, and vegetable oils such as transesterified oil. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these components may be used in combination, or a synthetic product and / or a food containing a large amount thereof may be used.

  When used as a food material, it can be produced according to a conventional method. Moreover, the compounding quantity to a foodstuff, a compounding method, and a compounding time can be selected suitably. Furthermore, it can be enclosed in appropriate containers, such as a bottle, a bag, a can, a box, and a pack, as needed.

  When used as a food material, the content of urolithins relative to the total amount of food is not particularly limited as long as the above effect is exhibited, but is the total amount in terms of dry weight, preferably 0.001 to 20 g, relative to 100 g of food. More preferably, it is 0.01-10g, More preferably, it is 0.1-5g.

(supplement)
A supplement is one of the food categories consisting of dietary supplements, and in this specification refers to a functional auxiliary substance capable of providing antioxidant, anti-inflammatory, anti-glycation and the like.
When the urolithin obtained by the production method of the present invention is used as a supplement material, it can be in any form of a solid, a gel, and a liquid. Examples of supplements include various processed foods and drinks, powders, tablets, pills, capsules, jellies, granules, and the like.

  When used as a supplement material, excipients such as dextrin, preservatives such as vitamin C, flavoring agents such as vanillin, pigments such as safflower pigment, monosaccharides, oligosaccharides and polysaccharides (eg, glucose, fructose, Sucrose, saccharose, and saccharides containing them), acidulant, flavor, fats and oils, emulsifier, whole milk powder, or agar additives. These components may be used in combination of two or more, and may contain many synthetic products and / or these.

  When used as a supplement material, it can be produced according to conventional methods. Moreover, the compounding quantity to a supplement, a compounding method, and a compounding time can be selected suitably. Furthermore, it can be enclosed in appropriate containers, such as a bottle, a bag, a can, a box, and a pack, as needed.

  When used as a supplement material, the content of urolithins relative to the total amount of the supplement is not particularly limited as long as the above effect is exhibited, but the total amount in terms of dry weight with respect to 100 g of the supplement, preferably 0.001 to 20 g, More preferably, it is 0.01-10g, More preferably, it is 0.1-5g.

(display)
Cosmetics, topical quasi-drugs, external medical supplies, external hygiene products, external medicines, foods, supplements, etc. using urolithins obtained by the production method of the present invention are antioxidant, anti-inflammatory, anti-glycation, etc. Therefore, it can be sold as a product with a display indicating that the product is used.

  The “display” means all acts for informing the consumer of the use, and if it is a display that can recall and analogize the use, the purpose of the display, the content of the display, the display Regardless of the target object / medium, etc., all fall under the “display” of the present invention. However, it is preferable to display in such an expression that the consumer can directly recognize the application. Specifically, the act of describing the use in the product or product packaging related to cosmetics, external quasi-drugs, external medical supplies, external hygiene products, external medicines, foods, supplements, etc. We transfer thing which listed use and display for the act of importing, transfer or delivery, importing, advertisement about product, price list or transaction document or display or distribute or describe these For example, an act provided by an electromagnetic (Internet, etc.) method by describing the above-mentioned use in the content information.

  On the other hand, the display is preferably a display approved by the government or the like (for example, a display which is approved based on various systems determined by the government and is performed in a mode based on such approval). Display on advertising materials at sales sites such as catalogs, pamphlets, and POPs, and other documents is preferable.

  In addition, for example, as long as it is a food that uses urolithins obtained by the production method of the present invention, examples of indications as health foods, functional foods, special-purpose foods, nutritional functional foods, quasi drugs, etc. Other indications approved by the Ministry of Health, Labor and Welfare, for example, foods for specified health use, indications approved under a similar system, can be exemplified. Examples of the latter can include a display as a food for specified health use, a display as a condition specific food for specified health use, a display that affects the structure and function of the body, a display for reducing disease risk, etc. Speaking of the health promotion law enforcement regulations (April 30, 2003 Ministry of Health, Labor and Welfare Ordinance No. 86) labeling as food for specified health use (especially labeling the use of health), and similar The display can be listed as a typical example.

  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.

[Examples 1-1 to 1-3: Production of urolithins using Gordonibacter urolithinfaciens strain DSM27213 in the presence of α-cyclodextrin]
Final concentration of 1 g / L ellagic acid (manufactured by SIGMA) and 1.2, 2.4, or 5 times the molar ratio of ellagic acid (Examples 1-1 and 1 respectively) -2 and 1-3) ABB medium (Oxoid) 10 mL each containing α-cyclodextrin is sterilized by heating in an autoclave at 121 ° C. for 15 minutes, and then the gas phase is N ₂ : CO ₂ : H _2. The gas was replaced with (80/10/10) gas. Gordonibacter urolithinfaciens strain DSM27213 was inoculated into this medium and cultured anaerobically at 37 ° C. for 1 week to produce urolithins.
After completion of the culture, 5 mL of the culture solution was extracted with an equal amount of ethyl acetate, and the resulting ethyl acetate phase was concentrated to dryness under reduced pressure. The obtained dried product was redissolved in 0.5 mL of methanol. Among the produced urolithins, quantitative analysis was performed by HPLC on urolithin C as a target. HPLC was performed under the conditions described below.

[Comparative Example 1]
Comparative example 1 was obtained by adding no α-cyclodextrin as a control.

HPLC analysis conditions:
Column: Inertsil ODS-3 (250 × 4.6 mm) (manufactured by GL Science)
Eluent: Water / acetonitrile / acetic acid = 74/25/1
Flow rate: 1.0 mL / min
Column temperature: 40 ° C
Detection: 305nm

  Table 2 shows the accumulated concentration and yield of urolithin C produced in the medium. Under any condition, urolithin C was produced at a higher accumulation concentration and yield than Comparative Example 1 in which α-cyclodextrin was not added. In addition, urolithin C manufactured by DALTON PHARMA was used as a standard and dissolved in DMSO.

[Examples 2-1 to 2-3: Production of urolithins using Bacteroides uniformis HGB5B146 (NITE BP-02193) strain in the presence of α-cyclodextrin]
Examples 2-1 to 2-3 were the same as Examples 1-1 to 1-3 except that the Gordonibacter urolithinfaciens DSM27213 strain was changed to the Bacteroides uniformis HGB5B146 (NITE BP-02193) strain.
[Comparative Example 2]
Comparative Example 2 was prepared by adding a substance without α-cyclodextrin as a control.

The accumulated concentration and yield of urolithin C produced in the medium are shown in Table 3. Under any condition, urolithin C was produced at a higher accumulation concentration and yield than Comparative Example 2 in which α-cyclodextrin was not added.

[Examples 3-1 to 3-3: Production of urolithins using Gordonibacter urolithinfaciens strain DSM27213 in the presence of β-cyclodextrin]
Examples 3-1 to 3-3 were the same as Examples 1-1 to 1-3 except that α-cyclodextrin was changed to β-cyclodextrin.
[Comparative Example 3]
Comparative Example 3 was prepared by adding no β-cyclodextrin as a control.

Table 4 shows the accumulated concentration and yield of urolithin C produced in the medium. Under any condition, urolithin C was produced at a higher accumulation concentration and yield than Comparative Example 3 in which β-cyclodextrin was not added.

[Examples 4-1 to 4-3: Production of urolithins using Bacteroides uniformis HGB5B146 (NITE BP-02193) strain in the presence of β-cyclodextrin]
Examples 4-1 to 4-3 were made in the same manner as Examples 3-1 to 3-3 except that Gordonibacter urolithinfaciens DSM27213 strain was changed to Bacteroides uniformis HGB5B146 (NITE BP-02193) strain.
[Comparative Example 4]
Comparative Example 4 was prepared by adding no β-cyclodextrin as a control.

Table 5 shows the accumulated concentration and yield of urolithin C produced in the medium. Under any condition, urolithin C was produced at a higher accumulation concentration and yield than Comparative Example 4 in which β-cyclodextrin was not added.

[Examples 5-1 to 5-3: Production of urolithins using Gordonibacter urolithinfaciens strain DSM27213 in the presence of γ-cyclodextrin]
Examples 5-1 to 5-3 were the same as Examples 1-1 to 1-3 except that α-cyclodextrin was changed to γ-cyclodextrin.
[Comparative Example 5]
Comparative Example 5 was prepared by adding no γ-cyclodextrin as a control.

  Table 6 shows the accumulated concentration and yield of urolithin C produced in the medium. Under any condition, urolithin C was produced at a higher accumulation concentration and yield than Comparative Example 5 in which γ-cyclodextrin was not added.

[Examples 6-1 to 6-3: Production of urolithins using Bacteroides uniformis HGB5B146 (NITE BP-02193) strain in the presence of γ-cyclodextrin]
Examples 6-1 to 6-3 were the same as Examples 5-1 to 5-3 except that the Gordonibacter urolithinfaciens DSM27213 strain was changed to the Bacteroides uniformis HGB5B146 (NITE BP-02193) strain.
[Comparative Example 6]
Comparative Example 6 was designated as a control to which γ-cyclodextrin was not added.

Table 7 shows the accumulated concentration and yield of urolithin C produced in the medium. Under either condition, urolithin C was produced at a higher accumulation concentration and yield than Comparative Example 6 in which γ-cyclodextrin was not added.

  By using urolithins obtained by the production method of the present invention as cosmetics, foods or drinks, pharmaceuticals, etc., skin aging and wrinkle prevention, whitening effect, metabolic syndrome, diabetes and other symptoms can be expected.

Claims (4)

  A urolithin production step for fermenting urolithins from ellagic acid to a urolithin-producing microorganism in a medium containing ellagic acid and cyclodextrin, and a recovery step for recovering the urolithins produced in the production step Manufacturing method.   The production method according to claim 1, wherein the cyclodextrin is at least one selected from the group consisting of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.   The production method according to claim 1 or 2, wherein the urolithin is urolithin C.   The production method according to any one of claims 1 to 3, wherein the urolithin-producing microorganism is one or more selected from the group consisting of Bacteroides bacteria and Gordonibacter bacteria.