WO2012033150A1 - Process for production of equol - Google Patents
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- WO2012033150A1 WO2012033150A1 PCT/JP2011/070430 JP2011070430W WO2012033150A1 WO 2012033150 A1 WO2012033150 A1 WO 2012033150A1 JP 2011070430 W JP2011070430 W JP 2011070430W WO 2012033150 A1 WO2012033150 A1 WO 2012033150A1
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- the present invention relates to a method for producing equol using an anaerobic microorganism having equol-producing ability.
- Isoflavones which are abundant in leguminous plants such as soybeans and kuzu, are a class of polyphenols and are flavonoids based on isoflavones. According to recent research, isoflavones have female hormonal action (estrogens) and antioxidant action, and by taking isoflavones, breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. Is known to have a preventive effect (Non-Patent Documents 1 to 6).
- Isoflavones exist, for example, in soybeans in the form of glycosides covalently linked to sugars, daidzin, glycitin, and genistin, but in very small amounts in the form of aglycone. Only. Some of these glycosides are further malonylated and acetylated. These glycosides enter the body of humans and animals by the action of digestive enzymes or ⁇ -glucosidase, which is an enzyme produced by intestinal bacteria, respectively, such as daidzein, glycitein, genistein and genistein. Become. Furthermore, it is known that daidzein is enzymatically converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by the action of intestinal bacteria. (FIG. 1).
- O-DMA O-desmethylangolensin
- Equol is known to have the highest estrogenic activity among these metabolites (Non-patent Documents 7 and 8).
- Non-patent Documents 7 and 8 there are individual differences in the metabolism of isoflavones, and as mentioned above, few people have enteric bacteria that have the ability to ferment daidzein and produce equol, and the ownership rate is about Japanese. It is clear that it is about 30% for Westerners and about 30% (Non-patent Documents 9 and 10). Therefore, there is a problem that a person who does not have an equol-producing bacterium cannot produce equol in the body even when taking legumes such as soybeans.
- Non-Patent Document 11 there was an example (Non-Patent Document 11) in which four types of anaerobic microorganisms were mixed and cultured in a hydrogen gas phase to try to produce equol, but mixed culture is suitable for practical production (industrialization). It was not.
- the inventors of the present invention have conducted intensive research.
- the inventors of the present invention have examined a suitable culture condition for equol production using bacteria classified into the family Coriobacteriaceae as an example of anaerobic microorganisms.
- the culture was carried out in the presence of a gas phase composed of a plurality of types of mixed gas, and daidzein was fermented to examine suitable culture conditions for equol production.
- the present invention provides the following [1] to [6].
- [1] A method for producing equol using an anaerobic microorganism having an equol-producing ability, comprising the following steps; (1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). .
- the anaerobic microorganism is a fungus classified into the family Coriobacteriaceae, a fungus classified into the Streptococcaceae family, or a related fungus thereof, [1] A process for producing equol according to any one of [3].
- the anaerobic microorganism is selected from the genus Coriobacterium, Adlercreutzia, Asaccharobacter, Atopobium, Collinsella, Cryptobacterium (Cryptobacterium), Denitrobacterium, Eggerthella, Enterorhabdus, Gordonibacter, Olsenella, Paraeggerthella, Slackia ( The method for producing equol according to any one of [1] to [3], wherein the bacterium is classified into any one of the genus Slackia) or the genus Lactococcus.
- the anaerobic microorganism is an Asaccharobacter celatus DSM 18785 strain, an Adlercreutzia equolifaciens DSM 19450 strain, or a Slackia isoflavoniconvertens.
- an efficient production method of equol using daidzein as a raw material utilizing anaerobic microbial fermentation can be realized, and industrial production technology of equol is provided. It is considered that breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. can be prevented by ingesting equol produced by the method of the present invention as it is as a food or drink or medicine. .
- the present invention relates to a method for producing equol using an anaerobic microorganism having an equol-producing ability including the following steps. (1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). .
- the combination of gases constituting the gas phase in the step (1) of the present invention is not particularly limited, and one or more gases selected from hydrogen, carbon dioxide, nitrogen, etc. can be used as a constituent component. It is.
- the gas phase preferably contains hydrogen as a constituent component.
- the mass percent concentration of hydrogen is 40 to 100% in the gas phase of step (1).
- the mass percent concentration 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 than” and an upper limit ( ⁇ less than or lower), and the mass of hydrogen. More preferably, the percent concentration is 100%.
- the aeration amount of the mixed gas constituting the gas phase to the culture tank is preferably 0.01 to 2.0 V / V / M gas amount / liquid amount / minute.
- the anaerobic microorganism used in the production of equol in the present invention is an anaerobic microorganism having an equol-producing ability at a temperature around 37 ° C. (30 to 42 ° C.).
- the pressurizing conditions for culturing anaerobic microorganisms are not particularly limited as long as the microorganisms can grow, but preferable pressurizing conditions include a range of 0.02 to 0.2 MPa. be able to.
- the present invention includes a step of bringing daidzein into contact with an anaerobic microorganism capable of producing equol.
- an anaerobic microorganism that produces equol can be used by fermenting daidzein.
- bacteria that are classified into the family Coriobacteriaceae, bacteria that are classified into the family Streptococcaceae, or related bacteria thereof are exemplified as having equol-producing ability. Can do.
- microorganisms classified into the genus selected from the following groups can be illustrated as having equol-producing ability.
- Cryptobacterium genus Denitrobacterium genus Egacea ( Eggerthella genus Enterorhabdus genus Gordonibacter genus Orsenella genus Paraeggerthella genus Slackia genus Lactococcus genus
- microorganisms selected from microorganisms classified into these genera and producing equol by anaerobic fermentation using daidzein is a preferable microorganism in the present invention.
- microorganisms belonging to the genus Asaccharobacter are preferable as microorganisms having equol-producing ability in the present invention.
- the production of equol by the microorganism can be confirmed by quantifying daidzein, dihydrodaidzein, and equol in the culture. More specifically, for example, the following microorganisms can be used as microorganisms having equol-producing ability in the present invention.
- One or more related bacteria having properties as species similar to those of these bacteria can be mentioned as more preferable anaerobic microorganisms.
- the anaerobic microorganisms can be obtained from the depository indicated by the deposit number. Each deposit number indicates that the anaerobic microorganism is deposited at the next depository.
- FERM Patent Organism Depositary International Patent Organism Depositary (IPOD) http://unit.aist.go.jp/pod/ci/index.html
- DSM German Collection of Microorganisms and Cell Cultures http://www.dsmz.de/ KCCM Korean Culture Center of Microorganisms
- an anaerobic microorganism having equol-producing ability is brought into contact with daidzein under conditions suitable for equol production and cultured.
- the conditions suitable for production of equol in the present invention means that the survival and activity of anaerobic microorganisms having equol production activity are maintained. More specifically, it means that gas phase conditions (anaerobic conditions) that allow anaerobic microorganisms to survive are maintained and nutrients are provided to support the activity and growth of the anaerobic microorganisms.
- gas phase conditions anaerobic conditions
- Various medium compositions suitable for the survival of anaerobic microorganisms are known.
- the GAM bouillon medium manufactured by Nissui Pharmaceutical Co., Ltd., the BHI medium manufactured by Difco, etc. used in the examples can be used.
- a water-soluble organic substance can be added as a carbon source to the medium used in the present invention.
- the following compounds can be listed as water-soluble organic substances.
- Sorbose Fructose Glucose Methanol Organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid
- the concentration of organic substances added to the medium as a carbon source can be adjusted as appropriate in order to efficiently develop anaerobic microorganisms in the medium. In general, excess and deficiency can be avoided by selecting the addition amount from the range of 0.1 to 10 wt / vol%.
- a nitrogen source is added to the medium.
- various nitrogen compounds that can be used for normal fermentation can be used as the nitrogen source.
- Preferred inorganic nitrogen sources are ammonium salts and nitrates.
- Preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder and the like. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate and sodium nitrate. More preferred nitrogen sources are arginine, citrulline, ornithine, lysine, yeast extract, and peptones.
- anaerobic microorganisms can be added to the medium.
- the growth and activity of anaerobic microorganisms can be enhanced by adding inorganic compounds such as cofactors such as vitamins and various salts to the medium.
- cofactors such as vitamins and various salts
- the following can be mentioned as microbial growth cofactors derived from inorganic compounds, vitamins and animals and plants.
- the medium can be liquid, semi-solid, or solid.
- a preferred medium form is a liquid medium.
- anaerobic microorganisms can be cultured according to a known microorganism culture method.
- a continuous culture system continuously culture system equipped with a mechanism for continuously supplying a medium and a substrate gas and recovering the culture is suitable.
- anaerobic microorganisms In the culture of anaerobic microorganisms, it is necessary to prevent oxygen from being mixed into the continuous culture system.
- a commonly used culture tank can be used as it is.
- Culture tanks that can also be used for culturing anaerobic microorganisms are commercially available.
- An anaerobic atmosphere can be created by replacing oxygen mixed in the culture tank with an inert gas such as nitrogen or a substrate gas.
- an anaerobic culture jar can be used as a bioreactor for culturing anaerobic microorganisms.
- the anaerobic culture jar is composed of an airtight container made of metal, glass, or synthetic resin, and can block the inside from oxygen in the atmosphere.
- the anaerobic culture jar can be equipped with a mechanism for removing molecular oxygen contained in the space inside the anaerobic culture jar or in the culture solution.
- a vacuum pump that sucks the inside of the anaerobic culture jar is connected and sucked, and a gas other than oxygen is supplied, so that the inside can be maintained in an anaerobic state.
- an additional function can be given to the culture tank.
- a bubble column type or draft tube type culture tank can also be used.
- the microorganisms are released and dispersed by the mixed gas blown into the liquid medium, and the microorganisms and the medium can be sufficiently brought into contact with each other.
- microorganisms inhabit while dripping moisture on a highly breathable slag like a biotrickling filter, other ceramic-based inorganic fillers, or packed layers of organic synthetic materials such as polypropylene, It can also be cultured while aeration of gas.
- the microorganisms to be used can be used by immobilizing them on carrageenan gel, alginic acid gel, acrylamide gel, chitin, cellulose, agar, etc. by a conventional method.
- a preferable separation method is a method using a hollow fiber type ultrafiltration or microfiltration membrane having filtration performance and concentration performance.
- a membrane having an appropriate fractional molecular weight may be selected according to the microorganism used. Equol can be recovered from the culture solution separated from the culture tank through the ultrafiltration membrane. Methods for purifying equol are known. For example, the cells are removed by centrifuging the culture, and the supernatant is concentrated under reduced pressure, dried and then extracted with 70% ethanol or methanol.
- the extract can be purified by further operations such as silica gel chromatography and crystallization.
- 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 to optimize the production efficiency of equol.
- the substrate gas can also be supplied as nanobubbles.
- the pH of the culture is preferably 3.0 to 8.0, more preferably 4.5 to 7.5.
- the temperature of the culture tank is not particularly limited, but 37 ° C. can be mentioned as a preferable temperature.
- the amount of fresh medium supplied to the culture tank is preferably such that the dilution rate in the culture in the culture tank is 0.04 to 2 / hr per hour. A more preferable dilution rate is 0.08 to 1 / hr.
- the equol obtained by the production method of the present invention can be provided as a medicine or a food or drink.
- substances generally used for the formulation eg, starch, dextrin, lactose, corn starch, inorganic salts, etc.
- dosage forms in the case of providing as pharmaceuticals include ampoules, tablets, capsules, granules, fine granules, powders, infusions, drinks and the like.
- the forms include health food, soft drinks, milk, pudding, jelly, rice cake, gum, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cakes, Examples include cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing and the like. It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
- Examples 1 to 7 Dissolve 5.9 g of GAM broth medium (manufactured by Nissui Pharmaceutical) and 0.5 g of L-arginine hydrochloride in 100 mL of pure water, and add 10 mL each to an anaerobic bacteria culture 18 mm test tube (manufactured by Sangen Industrial) through carbon dioxide. Dispensed, butyl rubber stopper and plastic cap, and sterilized at 115 ° C. for 15 minutes. This medium was inoculated with Asaccharobacter celatus DSM 18785, which had been stored frozen at -80 ° C, and the gas phase was maintained for 2 minutes with hydrogen / carbon dioxide [4: 1] through a sterile filter.
- shaking culture was performed at 37 ° C. and 160 spm for 16 hours. Inoculate 0.1 mL of this seed culture into the previously sterilized GAM bouillon + arginine medium, and then add 0.2 mL each of 20 mM daidzein (LKT) solution (dissolved in DMSO) sterilized with 0.45 ⁇ m PTFE membrane in advance. Then, the gas phase was aseptically replaced with gases having the respective compositions shown in Table 1 for 2 minutes or longer, and then cultured under shaking at 37 ° C. and 160 spm. After 24 hours, the culture solution was taken out, the turbidity (OD660) of the culture solution was measured, and equol was quantified by high performance liquid chromatography.
- LKT 20 mM daidzein
- Example 8 A GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL of this was dispensed into 100 mL volume vial (manufactured by Maruemu) through carbon dioxide gas, and a butyl rubber stopper was attached to make an aluminum cap. Sterilized at ° C for 15 minutes.
- 0.1 mL of a seed culture solution of Asaccharobacter celatus DSM 18785 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then filter-sterilized with a 0.45 ⁇ m PTFE membrane in advance 40
- LKT mM daidzein
- GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL each was dispensed into an 18 mm test tube for anaerobic bacteria culture (manufactured by Sangen Industry), with a butyl rubber stopper and a plastic cap at 115 ° C. Sterilized for 15 minutes.
- Examples 18 to 21 A GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL of this was dispensed into 100 mL volume vials (manufactured by Maruemu) through carbon dioxide gas, and a butyl rubber stopper was attached, and an aluminum cap was further added. Sterilized for 15 minutes. To this, 0.1 mL of seed culture solution of Adlercreutzia equolifaciens DSM 19450 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then pre-filter sterilized with a 0.45 ⁇ m PTFE membrane.
- equol can be produced with high efficiency using anaerobic microorganisms. It is considered that breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. can be prevented by ingesting equol produced by the method of the present invention as it is as a food or drink or medicine. .
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Abstract
The present inventors have made studies on cultivation conditions suitable for the production of equol, using bacteria that are classified into the family Coriobacteriaceae and the family Streptococcaceae as one example of microorganism groups. As a result, it is found that the equol production efficiency is dramatically increased when the concentration of a hydrogen gas in a gas phase in which fermentation is to be carried out is 40-100% (particularly 100%) by mass. Namely, the present inventors succeeded in the establishment of cultivation conditions suitable for the production of equol using an anaerobic microorganism.
Description
本発明は、エクオール生産能を有する嫌気性微生物によるエクオールの製造方法に関する。
The present invention relates to a method for producing equol using an anaerobic microorganism having equol-producing ability.
大豆、葛などのマメ科の植物に多く含まれているイソフラボン類はポリフェノールの分類のひとつであり、イソフラボンを基本骨格とするフラボノイドである。近年の調査により、イソフラボン類は女性ホルモン作用（エストロゲン）や抗酸化作用を有し、イソフラボン類を摂取することにより、乳癌、前立腺癌、骨粗しょう症、高コレステロール血症、心疾患、更年期障害などに対して予防効果があることが明らかとなっている（非特許文献１～６）。
イソフラボン類は、たとえば大豆内では、糖と共有結合した配糖体の形、ダイジン(daidzin)、グリシチン（glycitin）、ゲニスチン(genistin)として存在しており、アグリコンの形ではごく少量存在しているのみである。これら配糖体はさらにマロニル化、アセチル化されているものも存在している。これらの配糖体は、ヒトや動物の体内に入ると消化酵素又は腸内細菌の産生する酵素であるβグルコシダーゼ等の働きにより、それぞれダイゼイン（daidzein）、グリシテイン（glycitein）、ゲニステイン（genistein）となる。さらに、ダイゼインは腸内細菌の働きにより、ジヒドロダイゼイン（dihydrodaidzein）を経て、O-デスメチルアンゴレンシン（O-desmethylangolensin：O-DMA） 又はエクオール（equol）へと酵素的に変換されることが知られている（図１）。 Isoflavones, which are abundant in leguminous plants such as soybeans and kuzu, are a class of polyphenols and are flavonoids based on isoflavones. According to recent research, isoflavones have female hormonal action (estrogens) and antioxidant action, and by taking isoflavones, breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. Is known to have a preventive effect (Non-PatentDocuments 1 to 6).
Isoflavones exist, for example, in soybeans in the form of glycosides covalently linked to sugars, daidzin, glycitin, and genistin, but in very small amounts in the form of aglycone. Only. Some of these glycosides are further malonylated and acetylated. These glycosides enter the body of humans and animals by the action of digestive enzymes or β-glucosidase, which is an enzyme produced by intestinal bacteria, respectively, such as daidzein, glycitein, genistein and genistein. Become. Furthermore, it is known that daidzein is enzymatically converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by the action of intestinal bacteria. (FIG. 1).
イソフラボン類は、たとえば大豆内では、糖と共有結合した配糖体の形、ダイジン(daidzin)、グリシチン（glycitin）、ゲニスチン(genistin)として存在しており、アグリコンの形ではごく少量存在しているのみである。これら配糖体はさらにマロニル化、アセチル化されているものも存在している。これらの配糖体は、ヒトや動物の体内に入ると消化酵素又は腸内細菌の産生する酵素であるβグルコシダーゼ等の働きにより、それぞれダイゼイン（daidzein）、グリシテイン（glycitein）、ゲニステイン（genistein）となる。さらに、ダイゼインは腸内細菌の働きにより、ジヒドロダイゼイン（dihydrodaidzein）を経て、O-デスメチルアンゴレンシン（O-desmethylangolensin：O-DMA） 又はエクオール（equol）へと酵素的に変換されることが知られている（図１）。 Isoflavones, which are abundant in leguminous plants such as soybeans and kuzu, are a class of polyphenols and are flavonoids based on isoflavones. According to recent research, isoflavones have female hormonal action (estrogens) and antioxidant action, and by taking isoflavones, breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. Is known to have a preventive effect (Non-Patent
Isoflavones exist, for example, in soybeans in the form of glycosides covalently linked to sugars, daidzin, glycitin, and genistin, but in very small amounts in the form of aglycone. Only. Some of these glycosides are further malonylated and acetylated. These glycosides enter the body of humans and animals by the action of digestive enzymes or β-glucosidase, which is an enzyme produced by intestinal bacteria, respectively, such as daidzein, glycitein, genistein and genistein. Become. Furthermore, it is known that daidzein is enzymatically converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by the action of intestinal bacteria. (FIG. 1).
エクオールは、これらの代謝産物の中で最もエストロゲン活性が高いことが知られている（非特許文献７及び８）。しかしながら、人間の場合、イソフラボンの代謝には個人差があり、上記のようにダイゼインを発酵させてエクオールを産生する能力を有する腸内細菌を保有する人は少なく、その保有率は日本人で約５割、欧米人で約３割程度であることが明らかとなっている（非特許文献９及び１０）。そのため、エクオール産生菌を保有しない人は、大豆等のマメ科食物を摂取してもエクオールを体内で産生することができないという問題点が存在していた。
Equol is known to have the highest estrogenic activity among these metabolites (Non-patent Documents 7 and 8). However, in the case of humans, there are individual differences in the metabolism of isoflavones, and as mentioned above, few people have enteric bacteria that have the ability to ferment daidzein and produce equol, and the ownership rate is about Japanese. It is clear that it is about 30% for Westerners and about 30% (Non-patent Documents 9 and 10). Therefore, there is a problem that a person who does not have an equol-producing bacterium cannot produce equol in the body even when taking legumes such as soybeans.
これらの課題を克服するために、近年、乳酸菌等の嫌気性微生物を用いて体外的にエクオールを産生させる試みがなされている（特許文献１～４）。しかしながら、どのような発酵条件・発酵方法により、より効果的・実用的にエクオールが製造できるかについては、明らかとなっていなかった。たとえば、４種類の嫌気性微生物を水素気相下で混合培養し、エクオールの製造を試みた例（非特許文献１１）が存在していたが、混合培養では実用的生産（工業化）には適さないものであった。
In order to overcome these problems, attempts have been made in recent years to produce equol ex vivo using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1 to 4). However, it has not been clarified what fermentation conditions and fermentation methods can produce equol more effectively and practically. For example, there was an example (Non-Patent Document 11) in which four types of anaerobic microorganisms were mixed and cultured in a hydrogen gas phase to try to produce equol, but mixed culture is suitable for practical production (industrialization). It was not.
本発明は、このような状況に鑑みてなされたものであり、その目的は、嫌気性微生物の作用を利用して、より最適な気相条件によりダイゼインを発酵させ、従来よりも効果的にエクオールを製造できる方法を提供することにある。
The present invention has been made in view of such a situation, and an object thereof is to ferment daidzein under a more optimal gas phase condition by utilizing the action of anaerobic microorganisms, and to achieve equol more effectively than before. It is providing the method which can manufacture.
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。
本発明者らは、嫌気性微生物の一例としてコーリオバクテリアセアエ（Coriobacteriaceae）科に分類される菌を用い、エクオール製造の好適な培養条件の検討を行った。
具体的には、アサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株、アドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens） DSM 19450株、又はスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens） DSM 22006株を、複数種類の混合ガスからなる気相存在下で培養し、ダイゼインを発酵させ、エクオール製造における好適な培養条件の検討を行った。 In order to solve the above problems, the present inventors have conducted intensive research.
The inventors of the present invention have examined a suitable culture condition for equol production using bacteria classified into the family Coriobacteriaceae as an example of anaerobic microorganisms.
Specifically, Asaccharobacter celatus DSM 18785, Adlercreutzia equolifaciens DSM 19450, or Slackia isoflavoniconvertens DSM 22006 The culture was carried out in the presence of a gas phase composed of a plurality of types of mixed gas, and daidzein was fermented to examine suitable culture conditions for equol production.
本発明者らは、嫌気性微生物の一例としてコーリオバクテリアセアエ（Coriobacteriaceae）科に分類される菌を用い、エクオール製造の好適な培養条件の検討を行った。
具体的には、アサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株、アドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens） DSM 19450株、又はスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens） DSM 22006株を、複数種類の混合ガスからなる気相存在下で培養し、ダイゼインを発酵させ、エクオール製造における好適な培養条件の検討を行った。 In order to solve the above problems, the present inventors have conducted intensive research.
The inventors of the present invention have examined a suitable culture condition for equol production using bacteria classified into the family Coriobacteriaceae as an example of anaerobic microorganisms.
Specifically, Asaccharobacter celatus DSM 18785, Adlercreutzia equolifaciens DSM 19450, or Slackia isoflavoniconvertens DSM 22006 The culture was carried out in the presence of a gas phase composed of a plurality of types of mixed gas, and daidzein was fermented to examine suitable culture conditions for equol production.
その結果、発酵が行われる気相において水素ガスの質量パーセント濃度が４０～１００％である場合（特に１００％である場合）に、エクオールの製造効率が飛躍的に高まることが明らかとなり、水素にエクオール生成促進効果があることが判明した。
即ち、本発明者らは、嫌気性微生物を用いたエクオール製造における好適な培養条件の確立に成功し、これにより本発明を完成するに至った。 As a result, when the mass percent concentration of hydrogen gas is 40 to 100% (especially when it is 100%) in the gas phase in which fermentation is carried out, it becomes clear that the production efficiency of equol increases dramatically. It was found that equol production was promoted.
That is, the present inventors have succeeded in establishing suitable culture conditions in equol production using anaerobic microorganisms, thereby completing the present invention.
即ち、本発明者らは、嫌気性微生物を用いたエクオール製造における好適な培養条件の確立に成功し、これにより本発明を完成するに至った。 As a result, when the mass percent concentration of hydrogen gas is 40 to 100% (especially when it is 100%) in the gas phase in which fermentation is carried out, it becomes clear that the production efficiency of equol increases dramatically. It was found that equol production was promoted.
That is, the present inventors have succeeded in establishing suitable culture conditions in equol production using anaerobic microorganisms, thereby completing the present invention.
本発明は、より具体的には以下の〔１〕～〔６〕を提供するものである。
〔１〕次の工程を含むエクオール生産能を有する嫌気性微生物によるエクオールの製造方法；
（１）ダイゼインを、該嫌気性微生物により、水素を含む１種類以上の気体からなる気相下で発酵させる工程、及び
（２）工程（１）の嫌気性微生物が生産したエクオールを回収する工程。
〔２〕工程（１）の気相において、水素の質量パーセント濃度が４０～１００％であることを特徴とする、〔１〕に記載のエクオールの製造方法。
〔３〕工程（１）の気相において、水素の質量パーセント濃度が１００％であることを特徴とする、〔１〕に記載のエクオールの製造方法。
〔４〕前記嫌気性微生物がコーリオバクテリアセアエ（Coriobacteriaceae）科に分類される菌、ストレプトコッカセアエ（Streptococcaceae）科に分類される菌、又はこれらの類縁菌であることを特徴とする、〔１〕～〔３〕のいずれかに記載のエクオールの製造方法。
〔５〕前記嫌気性微生物が、コーリオバクテリウム（Coriobacterium）属、アドレクラウチア（Adlercreutzia）属、アサッカロバクター（Asaccharobacter）属、アトポビウム（Atopobium）属、コリンゼラ（Collinsella）属、クリプトバクテリウム（Cryptobacterium）属、デニトロバクテリウム（Denitrobacterium）属、エガセラ（Eggerthella）属、エンテロハブダス（Enterorhabdus）属、ゴードニバクター（Gordonibacter）属、オルセネラ（Olsenella）属、パラエゲセエラ（Paraeggerthella）属、スラッキア（Slackia）属、又はラクトコッカス(Lactococcus)属、のいずれかに分類される菌であることを特徴とする、〔１〕～〔３〕のいずれかに記載のエクオールの製造方法。
〔６〕前記嫌気性微生物が、アサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株、アドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens）DSM 19450株、又はスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens）DSM 22006株のいずれかであることを特徴とする、〔１〕～〔３〕のいずれかに記載のエクオールの製造方法。 More specifically, the present invention provides the following [1] to [6].
[1] A method for producing equol using an anaerobic microorganism having an equol-producing ability, comprising the following steps;
(1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). .
[2] The method for producing equol according to [1], wherein the mass percentage concentration of hydrogen is 40 to 100% in the gas phase of step (1).
[3] The method for producing equol according to [1], wherein the mass percentage concentration of hydrogen is 100% in the gas phase of step (1).
[4] The anaerobic microorganism is a fungus classified into the family Coriobacteriaceae, a fungus classified into the Streptococcaceae family, or a related fungus thereof, [1] A process for producing equol according to any one of [3].
[5] The anaerobic microorganism is selected from the genus Coriobacterium, Adlercreutzia, Asaccharobacter, Atopobium, Collinsella, Cryptobacterium (Cryptobacterium), Denitrobacterium, Eggerthella, Enterorhabdus, Gordonibacter, Olsenella, Paraeggerthella, Slackia ( The method for producing equol according to any one of [1] to [3], wherein the bacterium is classified into any one of the genus Slackia) or the genus Lactococcus.
[6] The anaerobic microorganism is an Asaccharobacter celatus DSM 18785 strain, an Adlercreutzia equolifaciens DSM 19450 strain, or a Slackia isoflavoniconvertens. The method for producing equol according to any one of [1] to [3], which is any one of DSM 22006 strains.
〔１〕次の工程を含むエクオール生産能を有する嫌気性微生物によるエクオールの製造方法；
（１）ダイゼインを、該嫌気性微生物により、水素を含む１種類以上の気体からなる気相下で発酵させる工程、及び
（２）工程（１）の嫌気性微生物が生産したエクオールを回収する工程。
〔２〕工程（１）の気相において、水素の質量パーセント濃度が４０～１００％であることを特徴とする、〔１〕に記載のエクオールの製造方法。
〔３〕工程（１）の気相において、水素の質量パーセント濃度が１００％であることを特徴とする、〔１〕に記載のエクオールの製造方法。
〔４〕前記嫌気性微生物がコーリオバクテリアセアエ（Coriobacteriaceae）科に分類される菌、ストレプトコッカセアエ（Streptococcaceae）科に分類される菌、又はこれらの類縁菌であることを特徴とする、〔１〕～〔３〕のいずれかに記載のエクオールの製造方法。
〔５〕前記嫌気性微生物が、コーリオバクテリウム（Coriobacterium）属、アドレクラウチア（Adlercreutzia）属、アサッカロバクター（Asaccharobacter）属、アトポビウム（Atopobium）属、コリンゼラ（Collinsella）属、クリプトバクテリウム（Cryptobacterium）属、デニトロバクテリウム（Denitrobacterium）属、エガセラ（Eggerthella）属、エンテロハブダス（Enterorhabdus）属、ゴードニバクター（Gordonibacter）属、オルセネラ（Olsenella）属、パラエゲセエラ（Paraeggerthella）属、スラッキア（Slackia）属、又はラクトコッカス(Lactococcus)属、のいずれかに分類される菌であることを特徴とする、〔１〕～〔３〕のいずれかに記載のエクオールの製造方法。
〔６〕前記嫌気性微生物が、アサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株、アドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens）DSM 19450株、又はスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens）DSM 22006株のいずれかであることを特徴とする、〔１〕～〔３〕のいずれかに記載のエクオールの製造方法。 More specifically, the present invention provides the following [1] to [6].
[1] A method for producing equol using an anaerobic microorganism having an equol-producing ability, comprising the following steps;
(1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). .
[2] The method for producing equol according to [1], wherein the mass percentage concentration of hydrogen is 40 to 100% in the gas phase of step (1).
[3] The method for producing equol according to [1], wherein the mass percentage concentration of hydrogen is 100% in the gas phase of step (1).
[4] The anaerobic microorganism is a fungus classified into the family Coriobacteriaceae, a fungus classified into the Streptococcaceae family, or a related fungus thereof, [1] A process for producing equol according to any one of [3].
[5] The anaerobic microorganism is selected from the genus Coriobacterium, Adlercreutzia, Asaccharobacter, Atopobium, Collinsella, Cryptobacterium (Cryptobacterium), Denitrobacterium, Eggerthella, Enterorhabdus, Gordonibacter, Olsenella, Paraeggerthella, Slackia ( The method for producing equol according to any one of [1] to [3], wherein the bacterium is classified into any one of the genus Slackia) or the genus Lactococcus.
[6] The anaerobic microorganism is an Asaccharobacter celatus DSM 18785 strain, an Adlercreutzia equolifaciens DSM 19450 strain, or a Slackia isoflavoniconvertens. The method for producing equol according to any one of [1] to [3], which is any one of DSM 22006 strains.
嫌気性微生物の代謝によってエクオールが製造されることは、学術的にはかねてより知られていたものの、エクオール製造に適した嫌気性微生物及び培養条件については、確立されていなかった。
Although it has been known from an academic viewpoint that equol is produced by metabolism of anaerobic microorganisms, anaerobic microorganisms and culture conditions suitable for equol production have not been established.
本発明によって、嫌気性微生物発酵を利用した、ダイゼインを原料とするエクオールの効率的な製造方法が実現でき、エクオールの工業的な製造技術が提供された。本発明の方法により製造されたエクオールを、飲食品又は医薬品等としてそのまま摂取することにより、乳癌、前立腺癌、骨粗しょう症、高コレステロール血症、心疾患、更年期障害等を予防できるものと考えられる。
According to the present invention, an efficient production method of equol using daidzein as a raw material utilizing anaerobic microbial fermentation can be realized, and industrial production technology of equol is provided. It is considered that breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. can be prevented by ingesting equol produced by the method of the present invention as it is as a food or drink or medicine. .
本発明は、次の工程を含むエクオール生産能を有する嫌気性微生物によるエクオールの製造方法に関する。
（１）ダイゼインを、該嫌気性微生物により、水素を含む１種類以上の気体からなる気相下で発酵させる工程、及び
（２）工程（１）の嫌気性微生物が生産したエクオールを回収する工程。 The present invention relates to a method for producing equol using an anaerobic microorganism having an equol-producing ability including the following steps.
(1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). .
（１）ダイゼインを、該嫌気性微生物により、水素を含む１種類以上の気体からなる気相下で発酵させる工程、及び
（２）工程（１）の嫌気性微生物が生産したエクオールを回収する工程。 The present invention relates to a method for producing equol using an anaerobic microorganism having an equol-producing ability including the following steps.
(1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). .
本方法発明の工程（１）の気相を構成する気体の組み合わせは特に制限されるものではなく、水素、二酸化炭素、窒素等から選択される１種類以上の気体を構成成分として用いることが可能である。当該気相においては、水素が構成成分として含まれていることが好ましい。
The combination of gases constituting the gas phase in the step (1) of the present invention is not particularly limited, and one or more gases selected from hydrogen, carbon dioxide, nitrogen, etc. can be used as a constituent component. It is. The gas phase preferably contains hydrogen as a constituent component.
上記の場合、工程（１）の気相において、水素の質量パーセント濃度が４０～１００％であることが好ましい。たとえば、４０％、４５％、５０％、５５％、６０％、６５％、７０％、７５％、８０％、８５％、９０％、９５％、１００％、又はこれらから選択される２点の質量パーセント濃度を下限（「～以上、又は、～より高い）及び上限（～以下、又は、～より低い）、とする濃度範囲により示される質量パーセント濃度であることが好ましい。また、水素の質量パーセント濃度が１００％であることがより好ましい。
In the above case, it is preferable that the mass percent concentration of hydrogen is 40 to 100% in the gas phase of step (1). For example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or two points selected from these The mass percent concentration 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 than” and an upper limit (˜less than or lower), and the mass of hydrogen. More preferably, the percent concentration is 100%.
また、効率よくエクオールを回収するためには、気相を構成する混合気体の培養槽への通気量は0.01～2.0 V/V/Mガス量/液量/分であることが好ましい。
Further, in order to efficiently recover equol, the aeration amount of the mixed gas constituting the gas phase to the culture tank is preferably 0.01 to 2.0 V / V / M gas amount / liquid amount / minute.
本発明でエクオールの製造に用いられる嫌気性微生物は、３７℃付近（30～42℃）の温度でエクオール生産能を有する嫌気性微生物である。
The anaerobic microorganism used in the production of equol in the present invention is an anaerobic microorganism having an equol-producing ability at a temperature around 37 ° C. (30 to 42 ° C.).
本発明において、嫌気性微生物を培養する際の加圧条件は、当該微生物が生育できる条件であれば特に限定されるものではないが、好ましい加圧条件としては、0.02～0.2MPaの範囲を挙げることができる。
In the present invention, the pressurizing conditions for culturing anaerobic microorganisms are not particularly limited as long as the microorganisms can grow, but preferable pressurizing conditions include a range of 0.02 to 0.2 MPa. be able to.
本発明は、ダイゼインを、エクオールの生産能を有する嫌気性微生物と接触させる工程を含む。本発明においては、ダイゼインを発酵させることにより、エクオールを生産する嫌気性微生物を用いることができる。
The present invention includes a step of bringing daidzein into contact with an anaerobic microorganism capable of producing equol. In the present invention, an anaerobic microorganism that produces equol can be used by fermenting daidzein.
本発明においては、コーリオバクテリアセアエ（Coriobacteriaceae）科に分類される菌、ストレプトコッカセアエ（Streptococcaceae）科に分類される菌、又はこれらの類縁菌を、エクオール生産能を有するものとして例示することができる。
In the present invention, bacteria that are classified into the family Coriobacteriaceae, bacteria that are classified into the family Streptococcaceae, or related bacteria thereof are exemplified as having equol-producing ability. Can do.
また、以下の群から選択される属に分類される微生物を、エクオール生産能を有するものとして例示することができる。
コーリオバクテリウム（Coriobacterium）属
アドレクラウチア（Adlercreutzia）属
アサッカロバクター（Asaccharobacter）属
アトポビウム（Atopobium）属
コリンゼラ（Collinsella）属
クリプトバクテリウム（Cryptobacterium）属
デニトロバクテリウム（Denitrobacterium）属
エガセラ（Eggerthella）属
エンテロハブダス（Enterorhabdus）属
ゴードニバクター（Gordonibacter）属
オルセネラ（Olsenella）属
パラエゲセエラ（Paraeggerthella）属
スラッキア（Slackia）属
ラクトコッカス(Lactococcus)属 Moreover, the microorganisms classified into the genus selected from the following groups can be illustrated as having equol-producing ability.
Coriobacterium genus Adlercreutzia genus Asaccharobacter genus Atopobium genus Collinsella genus Cryptobacterium genus Denitrobacterium genus Egacea ( Eggerthella genus Enterorhabdus genus Gordonibacter genus Orsenella genus Paraeggerthella genus Slackia genus Lactococcus genus
コーリオバクテリウム（Coriobacterium）属
アドレクラウチア（Adlercreutzia）属
アサッカロバクター（Asaccharobacter）属
アトポビウム（Atopobium）属
コリンゼラ（Collinsella）属
クリプトバクテリウム（Cryptobacterium）属
デニトロバクテリウム（Denitrobacterium）属
エガセラ（Eggerthella）属
エンテロハブダス（Enterorhabdus）属
ゴードニバクター（Gordonibacter）属
オルセネラ（Olsenella）属
パラエゲセエラ（Paraeggerthella）属
スラッキア（Slackia）属
ラクトコッカス(Lactococcus)属 Moreover, the microorganisms classified into the genus selected from the following groups can be illustrated as having equol-producing ability.
Coriobacterium genus Adlercreutzia genus Asaccharobacter genus Atopobium genus Collinsella genus Cryptobacterium genus Denitrobacterium genus Egacea ( Eggerthella genus Enterorhabdus genus Gordonibacter genus Orsenella genus Paraeggerthella genus Slackia genus Lactococcus genus
したがって、これらの属に分類された微生物から選択され、ダイゼインを利用して嫌気発酵によってエクオールを生成する微生物は、本発明における好ましい微生物である。中でも、アサッカロバクター（Asaccharobacter）属に属する微生物は、本発明におけるエクオール産生能を有する微生物として好ましい。微生物がエクオールを生成することは、培養物中のダイゼイン、ジヒドロダイゼイン、エクオールを定量することにより確認することができる。より具体的には、たとえば、以下の微生物を、本発明におけるエクオール産生能を有する微生物として利用することができる。
Adlercreutzia equolifaciens DSM 19450
Enterorhabdus mucosicola DSM 19490
Slackia isoflavoniconvertens HE8（DSM 22006）
Slackia sp. TM-30 FERM AP-20729号
Eggerthella sp. KCCM-10490
Asaccharobacter celatus DSM 18785
Lactococcus garvieae DSM 6783 Therefore, a microorganism selected from microorganisms classified into these genera and producing equol by anaerobic fermentation using daidzein is a preferable microorganism in the present invention. Among these, microorganisms belonging to the genus Asaccharobacter are preferable as microorganisms having equol-producing ability in the present invention. The production of equol by the microorganism can be confirmed by quantifying daidzein, dihydrodaidzein, and equol in the culture. More specifically, for example, the following microorganisms can be used as microorganisms having equol-producing ability in the present invention.
Adlercreutzia equolifaciens DSM 19450
Enterorhabdus mucosicola DSM 19490
Slackia isoflavoniconvertens HE8 (DSM 22006)
Slackia sp.TM-30 FERM AP-20729 Eggerthella sp.KCCM-10490
Asaccharobacter celatus DSM 18785
Lactococcus garvieae DSM 6783
Adlercreutzia equolifaciens DSM 19450
Enterorhabdus mucosicola DSM 19490
Slackia isoflavoniconvertens HE8（DSM 22006）
Slackia sp. TM-30 FERM AP-20729号
Eggerthella sp. KCCM-10490
Asaccharobacter celatus DSM 18785
Lactococcus garvieae DSM 6783 Therefore, a microorganism selected from microorganisms classified into these genera and producing equol by anaerobic fermentation using daidzein is a preferable microorganism in the present invention. Among these, microorganisms belonging to the genus Asaccharobacter are preferable as microorganisms having equol-producing ability in the present invention. The production of equol by the microorganism can be confirmed by quantifying daidzein, dihydrodaidzein, and equol in the culture. More specifically, for example, the following microorganisms can be used as microorganisms having equol-producing ability in the present invention.
Adlercreutzia equolifaciens DSM 19450
Enterorhabdus mucosicola DSM 19490
Slackia isoflavoniconvertens HE8 (DSM 22006)
Slackia sp.TM-30 FERM AP-20729 Eggerthella sp.KCCM-10490
Asaccharobacter celatus DSM 18785
Lactococcus garvieae DSM 6783
特に以下に記載するアサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株、アドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens）DSM 19450株、又はスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens）DSM 22006株のいずれか又はこれらの菌と同様の種としての性質を有する類縁の菌をより好ましい嫌気性微生物として挙げることができる。
In particular, Asaccharobacteractcelatus DSM 18785, Adlercreutzia equolifaciens DSM 19450, or Slackia イ ソ isoflavoniconvertens DSM 22006, which are listed below One or more related bacteria having properties as species similar to those of these bacteria can be mentioned as more preferable anaerobic microorganisms.
上記嫌気性微生物は、その寄託番号に示された寄託機関から入手することができる。各受託番号は、当該嫌気性微生物が、それぞれ次の寄託機関に寄託されていることを示す。
FERM 特許生物寄託センター；International Patent Organism Depositary (IPOD)
http://unit.aist.go.jp/pod/ci/index.html
DSM German Collection of Microorganisms and Cell Cultures (DSMZ)
http://www.dsmz.de/
KCCM Korean Culture Center of Microorganisms The anaerobic microorganisms can be obtained from the depository indicated by the deposit number. Each deposit number indicates that the anaerobic microorganism is deposited at the next depository.
FERM Patent Organism Depositary; International Patent Organism Depositary (IPOD)
http://unit.aist.go.jp/pod/ci/index.html
DSM German Collection of Microorganisms and Cell Cultures (DSMZ)
http://www.dsmz.de/
KCCM Korean Culture Center of Microorganisms
FERM 特許生物寄託センター；International Patent Organism Depositary (IPOD)
http://unit.aist.go.jp/pod/ci/index.html
DSM German Collection of Microorganisms and Cell Cultures (DSMZ)
http://www.dsmz.de/
KCCM Korean Culture Center of Microorganisms The anaerobic microorganisms can be obtained from the depository indicated by the deposit number. Each deposit number indicates that the anaerobic microorganism is deposited at the next depository.
FERM Patent Organism Depositary; International Patent Organism Depositary (IPOD)
http://unit.aist.go.jp/pod/ci/index.html
DSM German Collection of Microorganisms and Cell Cultures (DSMZ)
http://www.dsmz.de/
KCCM Korean Culture Center of Microorganisms
本発明においては、エクオール生産能を有する嫌気性微生物は、エクオールの生産に適した条件でダイゼインと接触させられ、培養される。本発明におけるエクオールの生産に適した条件とは、エクオール生成活性を持つ嫌気性微生物の生存と活動が維持されることを言う。より具体的には、嫌気性微生物の生存が可能な気相条件（嫌気性条件）が維持され、当該嫌気性微生物の活性と増殖を支持するための栄養素が与えられることを言う。嫌気性微生物の生存に適した種々の培地組成が公知である。したがって、先に示したエクオール生産能を有する嫌気性微生物について、当業者は、適切な培地組成を選択することができる。たとえば、実施例において用いた日水製薬社製のGAMブイヨン培地や、Difco社製のＢＨＩ培地等を使用することができる。
In the present invention, an anaerobic microorganism having equol-producing ability is brought into contact with daidzein under conditions suitable for equol production and cultured. The conditions suitable for production of equol in the present invention means that the survival and activity of anaerobic microorganisms having equol production activity are maintained. More specifically, it means that gas phase conditions (anaerobic conditions) that allow anaerobic microorganisms to survive are maintained and nutrients are provided to support the activity and growth of the anaerobic microorganisms. Various medium compositions suitable for the survival of anaerobic microorganisms are known. Therefore, those skilled in the art can select an appropriate medium composition for the anaerobic microorganisms having the equol-producing ability shown above. For example, the GAM bouillon medium manufactured by Nissui Pharmaceutical Co., Ltd., the BHI medium manufactured by Difco, etc. used in the examples can be used.
たとえば、本発明で用いられる培地には、水溶性の有機物を炭素源として加えることができる。水溶性の有機物として、以下の化合物を挙げることができる。
ソルボース
フラクトース
グルコース
メタノール
吉草酸、酪酸、プロピオン酸、酢酸、ギ酸など有機酸類 For example, a water-soluble organic substance can be added as a carbon source to the medium used in the present invention. The following compounds can be listed as water-soluble organic substances.
Sorbose Fructose Glucose Methanol Organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid
ソルボース
フラクトース
グルコース
メタノール
吉草酸、酪酸、プロピオン酸、酢酸、ギ酸など有機酸類 For example, a water-soluble organic substance can be added as a carbon source to the medium used in the present invention. The following compounds can be listed as water-soluble organic substances.
Sorbose Fructose Glucose Methanol Organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid
炭素源としての培地に加える有機物の濃度は、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 substances added to the medium as a carbon source can be adjusted as appropriate in order to efficiently develop anaerobic microorganisms in the medium. In general, excess and 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、窒素源が加えられる。本発明において、窒素原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、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アルギニン、シトルリン、オルニチン、リジン、酵母エキス、ペプトン類である。
In addition to the above carbon source, a nitrogen source is added to the medium. In the present invention, various nitrogen compounds that can be used for normal fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are ammonium salts and nitrates. Preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder and the like. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate and sodium nitrate. More preferred nitrogen sources are arginine, citrulline, ornithine, lysine, 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 the carbon source and nitrogen source, other organic or inorganic substances suitable for culturing anaerobic microorganisms can be added to the medium. For example, in some cases, the growth and activity of anaerobic microorganisms can be enhanced by adding inorganic compounds such as cofactors such as vitamins and 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 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 Ammonium ferrous sulfate
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 the carbon source and nitrogen source, other organic or inorganic substances suitable for culturing anaerobic microorganisms can be added to the medium. For example, in some cases, the growth and activity of anaerobic microorganisms can be enhanced by adding inorganic compounds such as cofactors such as vitamins and 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 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 Ammonium ferrous sulfate
これらの無機化合物やビタミン類、あるいは増殖補助因子を添加して培養液を製造する方法は公知である。培地は、液体、半固体、あるいは固体とすることができる。本発明のエクオールの製造方法において、好ましい培地の形態は、液体培地である。
A method for producing a culture solution by adding these inorganic compounds, vitamins, or growth cofactors is known. The medium can be liquid, semi-solid, or solid. In the equol production method of the present invention, a preferred medium form is a liquid medium.
本発明において、嫌気性微生物は、公知の微生物の培養方法にしたがって培養することができる。工業的な製造には、培地や基質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 (continuous fermentation system)が好適である。
In the present invention, anaerobic microorganisms can be cultured according to a known microorganism culture method. For industrial production, a continuous culture system (continuous culture system) equipped with a mechanism for continuously supplying a medium and a substrate gas and recovering the culture is suitable.
嫌気性微生物の培養においては、連続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。培養器は通常用いられる培養槽がそのまま利用できる。嫌気性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あるいは基質ガス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。
In the culture of anaerobic microorganisms, it is necessary to prevent oxygen from being mixed into the continuous culture system. As the incubator, a commonly used culture tank can be used as it is. Culture tanks that can also be used for culturing anaerobic microorganisms are commercially available. An anaerobic atmosphere can be created by replacing oxygen mixed in the culture tank with an inert gas such as nitrogen or a substrate gas.
たとえば、嫌気培養ジャー(anaerobic jar)を、嫌気性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。さらに、嫌気培養ジャーは、嫌気培養ジャー内部の空間や培養液中に含まれる分子状酸素を除去するための機構を備えることができる。たとえば、嫌気培養ジャー内部を吸引する真空ポンプを接続して吸引し、酸素以外の気体を供給することで、内部を嫌気状態に維持することができる。
For example, an anaerobic culture jar can be used as a bioreactor for culturing anaerobic microorganisms. The anaerobic culture jar is composed of an airtight container made of metal, glass, or synthetic resin, and can block the inside from oxygen in the atmosphere. Furthermore, the anaerobic culture jar can be equipped with a mechanism for removing molecular oxygen contained in the space inside the anaerobic culture jar or in the culture solution. For example, a vacuum pump that sucks the inside of the anaerobic culture jar is connected and sucked, and a gas other than oxygen is supplied, so that the inside can be maintained in an anaerobic state.
本発明においては、培養槽に付加的な機能を与えることができる。たとえば、通常使用される撹はん混合槽のほか、気泡塔型、ドラフトチューブ型の培養槽も利用できる。液体培地に吹き込まれる混合気体によって微生物は遊離分散され、微生物と培地を十分に接触させることができる。また、バイオトリックリングフィルター（biotrickling filter）のように通気性の高いスラグ、その他セラミック系の無機充てん物、あるいはポリプロピレン等の有機合成物質の充てん層に、水分を滴らせながら微生物を生息させ、そこにガスを通気しながら培養することもできる。さらに、使用する微生物は常法によりカラギーナンゲル、アルギン酸ゲル、アクリルアミドゲル、キチン、セルロース、寒天などに固定化して用いることもできる。
In the present invention, an additional function can be given to the culture tank. For example, in addition to a commonly used stirred mixing tank, a bubble column type or draft tube type culture tank can also be used. The microorganisms are released and dispersed by the mixed gas blown into the liquid medium, and the microorganisms and the medium can be sufficiently brought into contact with each other. In addition, microorganisms inhabit while dripping moisture on a highly breathable slag like a biotrickling filter, other ceramic-based inorganic fillers, or packed layers of organic synthetic materials such as polypropylene, It can also be cultured while aeration of gas. Furthermore, the microorganisms to be used can be used by immobilizing them on carrageenan gel, alginic acid gel, acrylamide gel, chitin, cellulose, agar, etc. by a conventional method.
当業者は、生成した嫌気性微生物と培養生成液を分離するため、公知の方法を用いることができる。好ましい分離の方法は、ろ過性能、濃縮性能を有するホローファイバー型限外ろ過あるいは精密ろ過膜を利用する方法である。また、微生物と該生成液の分離に十分なろ過速度を得るためには使用する微生物に応じて適当な分画分子量の膜を選択すれば良い。培養槽から限外ろ過膜を通して分離された培養液からエクオールを回収することができる。エクオールの精製方法は公知である。たとえば、培養物を遠心分離などで菌体を除き、上清を減圧下に濃縮、乾固後、70%エタノールあるいはメタノールで抽出する。抽出液をさらにシリカゲルクロマトグラフィーや晶析などの操作を行うことで精製できる。培養槽の形状によっては、培地を十分に撹はんするため、撹はん機等を利用することもできる。培養槽内の培養物を攪拌することによって、培地成分や基質ガスを嫌気性微生物に接触させる機会を増やして、エクオールの生成効率を最適化することができる。また基質ガスをナノバブルとして供給することもできる。
Those skilled in the art can use known methods to separate the produced anaerobic microorganisms from the culture product solution. A preferable separation method is a method using a hollow fiber type ultrafiltration or microfiltration membrane having filtration performance and concentration performance. In addition, in order to obtain a filtration rate sufficient for separating the microorganism and the product solution, a membrane having an appropriate fractional molecular weight may be selected according to the microorganism used. Equol can be recovered from the culture solution separated from the culture tank through the ultrafiltration membrane. Methods for purifying equol are known. For example, the cells are removed by centrifuging the culture, and the supernatant is concentrated under reduced pressure, dried and then extracted with 70% ethanol or methanol. The extract can be purified by further operations such as silica gel chromatography and crystallization. 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 to optimize the production efficiency of equol. The substrate gas can also be supplied as nanobubbles.
微生物の十分な生育のため、培養物のpHは、3.0～8.0が好ましく、4.5～7.5がより好ましい。また、エクオールの回収量を増加させるため、培養槽の温度は特に制限されるものではないが、37℃を好ましい温度として挙げることができる。
For the sufficient growth of microorganisms, the pH of the culture is preferably 3.0 to 8.0, more preferably 4.5 to 7.5. In order to increase the amount of equol recovered, the temperature of the culture tank is not particularly limited, but 37 ° C. can be mentioned as a preferable temperature.
効率よくエクオールを回収するため、培養槽に供給される新鮮な培地の量は、培養槽内の培養物における希釈率が時間当たり0.04～2/hrが好ましい。より好ましい希釈率は0.08～1/hrである。
In order to efficiently recover equol, the amount of fresh medium supplied to the culture tank is preferably such that the dilution rate in the culture in the culture tank is 0.04 to 2 / hr per hour. A more preferable dilution rate is 0.08 to 1 / hr.
本発明の製造方法により得られたエクオールは、医薬品又は飲食物として提供することが可能である。
エクオールを医薬品として提供する場合、その製剤化には、一般的に製剤化に用いられる物質（たとえば、デンプン、デキストリン、乳糖、コーンスターチ、無機塩類等）を用いることができる。医薬品として提供する場合の剤型としては、アンプル、錠剤、カプセル剤、顆粒剤、細粒剤、散剤、輸液、ドリンク剤等を例示することができる。 The equol obtained by the production method of the present invention can be provided as a medicine or a food or drink.
When equol is provided as a pharmaceutical product, substances generally used for the formulation (eg, starch, dextrin, lactose, corn starch, inorganic salts, etc.) can be used for the formulation. Examples of dosage forms in the case of providing as pharmaceuticals include ampoules, tablets, capsules, granules, fine granules, powders, infusions, drinks and the like.
エクオールを医薬品として提供する場合、その製剤化には、一般的に製剤化に用いられる物質（たとえば、デンプン、デキストリン、乳糖、コーンスターチ、無機塩類等）を用いることができる。医薬品として提供する場合の剤型としては、アンプル、錠剤、カプセル剤、顆粒剤、細粒剤、散剤、輸液、ドリンク剤等を例示することができる。 The equol obtained by the production method of the present invention can be provided as a medicine or a food or drink.
When equol is provided as a pharmaceutical product, substances generally used for the formulation (eg, starch, dextrin, lactose, corn starch, inorganic salts, etc.) can be used for the formulation. Examples of dosage forms in the case of providing as pharmaceuticals include ampoules, tablets, capsules, granules, fine granules, powders, infusions, drinks and the like.
エクオールを飲食物として提供する場合、その形態としては、健康食品、清涼飲料、ミルク、プリン、ゼリー、飴、ガム、ヨーグルト、チョコレート、スープ、クッキー、スナック菓子、アイスクリーム、アイスキャンデー、パン、ケーキ、シュークリーム、ハム、ミートソース、カレー、シチュー、チーズ、バター、ドレッシング等を例示することができる。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。 When providing equol as a food or drink, the forms include health food, soft drinks, milk, pudding, jelly, rice cake, gum, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cakes, Examples include cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing and the like.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。 When providing equol as a food or drink, the forms include health food, soft drinks, milk, pudding, jelly, rice cake, gum, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cakes, Examples include cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing and the like.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
以下、本発明を実施例によりさらに具体的に説明するが本発明はこれら実施例に制限されるものではない。
以下、各実施例においては、以下の方法により、濁度の測定、ダイゼイン、ジヒドロダイゼイン、エクオールの定量を行った。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Hereinafter, in each Example, turbidity was measured and daidzein, dihydrodaidzein, and equol were quantified by the following methods.
以下、各実施例においては、以下の方法により、濁度の測定、ダイゼイン、ジヒドロダイゼイン、エクオールの定量を行った。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Hereinafter, in each Example, turbidity was measured and daidzein, dihydrodaidzein, and equol were quantified by the following methods.
濁度の測定
培養液を純水で3倍に希釈した液の660 nmの濁度を測定し、3倍して元の培養液の濁度とした。 Measurement of turbidity The turbidity at 660 nm of a solution obtained by diluting the culture solution three times with pure water was measured, and three times as the turbidity of the original culture solution.
培養液を純水で3倍に希釈した液の660 nmの濁度を測定し、3倍して元の培養液の濁度とした。 Measurement of turbidity The turbidity at 660 nm of a solution obtained by diluting the culture solution three times with pure water was measured, and three times as the turbidity of the original culture solution.
ダイゼイン、ジヒドロダイゼイン、エクオールの定量
培養液0.5 mLに対し、酢酸エチル1.5 mLを加えて、激しく攪拌した後、3000 rpmで10秒遠心し、酢酸エチル層をパスツールピペットで可能な限り取り出した。同培養液に同様の操作をあと2回行い、それら酢酸エチル層を合わせてエクオール抽出液を得た。この抽出液をエバポレーターで減圧下に濃縮、乾固し、1.0 mLのメタノールに溶解させた。これを0.45 μmのPTFE膜でろ過し、不溶物を除去したものを高速液体クロマトグラフィー測定サンプルとした。 1.5 mL of ethyl acetate was added to 0.5 mL of the quantitative culture solution of daidzein, dihydrodaidzein, and equol, and after vigorous stirring, the mixture was centrifuged at 3000 rpm for 10 seconds, and the ethyl acetate layer was removed with a Pasteur pipette as much as possible. The same operation was performed twice more on the same culture solution, and these ethyl acetate layers were combined to obtain an equol extract. This extract was concentrated to dryness under reduced pressure using an evaporator, and dissolved in 1.0 mL of methanol. This was filtered through a 0.45 μm PTFE membrane to remove insolubles, and used as a sample for high performance liquid chromatography measurement.
培養液0.5 mLに対し、酢酸エチル1.5 mLを加えて、激しく攪拌した後、3000 rpmで10秒遠心し、酢酸エチル層をパスツールピペットで可能な限り取り出した。同培養液に同様の操作をあと2回行い、それら酢酸エチル層を合わせてエクオール抽出液を得た。この抽出液をエバポレーターで減圧下に濃縮、乾固し、1.0 mLのメタノールに溶解させた。これを0.45 μmのPTFE膜でろ過し、不溶物を除去したものを高速液体クロマトグラフィー測定サンプルとした。 1.5 mL of ethyl acetate was added to 0.5 mL of the quantitative culture solution of daidzein, dihydrodaidzein, and equol, and after vigorous stirring, the mixture was centrifuged at 3000 rpm for 10 seconds, and the ethyl acetate layer was removed with a Pasteur pipette as much as possible. The same operation was performed twice more on the same culture solution, and these ethyl acetate layers were combined to obtain an equol extract. This extract was concentrated to dryness under reduced pressure using an evaporator, and dissolved in 1.0 mL of methanol. This was filtered through a 0.45 μm PTFE membrane to remove insolubles, and used as a sample for high performance liquid chromatography measurement.
（高速液体クロマトグラフィー条件）
カラム：J’sphere ODS M-80, 2.0（直径）×250 mm（ワイエムシィ製）
移動相：水/アセトニトリル/酢酸［70：30：0.1, v/v］
流速：0.2 mL/min
カラム温度.：40 ℃
検出：UV280 nm
保持時間：ダイゼイン 14.1分、ジヒドロダイゼイン 15.1分、エクオール 26.3分 (High performance liquid chromatography conditions)
Column: J'sphere ODS M-80, 2.0 (diameter) x 250 mm (manufactured by YMC)
Mobile phase: Water / acetonitrile / acetic acid [70: 30: 0.1, v / v]
Flow rate: 0.2 mL / min
Column temperature: 40 ° C
Detection: UV280 nm
Retention time: daidzein 14.1 min, dihydrodaidzein 15.1 min, equol 26.3 min
カラム：J’sphere ODS M-80, 2.0（直径）×250 mm（ワイエムシィ製）
移動相：水/アセトニトリル/酢酸［70：30：0.1, v/v］
流速：0.2 mL/min
カラム温度.：40 ℃
検出：UV280 nm
保持時間：ダイゼイン 14.1分、ジヒドロダイゼイン 15.1分、エクオール 26.3分 (High performance liquid chromatography conditions)
Column: J'sphere ODS M-80, 2.0 (diameter) x 250 mm (manufactured by YMC)
Mobile phase: Water / acetonitrile / acetic acid [70: 30: 0.1, v / v]
Flow rate: 0.2 mL / min
Column temperature: 40 ° C
Detection: UV280 nm
Retention time: daidzein 14.1 min, dihydrodaidzein 15.1 min, equol 26.3 min
〔実施例１～７〕
GAMブイヨン培地（日水製薬製）5.9 gとL-アルギニン塩酸塩0.5 gを純水100 mLに溶かし、炭酸ガスを通じながら10 mLずつ嫌気性菌培養用18 mm試験管（三紳工業製）に分注し、ブチルゴム栓、プラスチックキャップをして115℃、15分間滅菌した。
この培地に-80℃で凍結保存していたアサッカロバクター・セラツス（Asaccharobacter celatus） DSM 18785株を植菌し、無菌フィルターを通した水素/炭酸ガス［4:1］で気相を2分間以上置換した後、37℃、160 spmで16時間振とう培養を行った。この種培養液0.1 mLを先の滅菌したGAMブイヨン＋アルギニン培地に植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した20 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.2 mLずつ添加し、気相を表1に示すそれぞれの組成のガスで無菌的に2分間以上置換した後、37℃、160 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、培養液の濁度（OD660）を測定し、また、高速液体クロマトグラフィーによりエクオールを定量した。
その結果、水素濃度が高いほど菌体の生育量、エクオール生成量ともに多いこと、炭酸ガス、窒素のみ、あるいは炭酸ガス/窒素混合ガスでは生育、エクオール生成とも極めて少ないことが明らかとなった（表１、図２）。 [Examples 1 to 7]
Dissolve 5.9 g of GAM broth medium (manufactured by Nissui Pharmaceutical) and 0.5 g of L-arginine hydrochloride in 100 mL of pure water, and add 10 mL each to an anaerobic bacteria culture 18 mm test tube (manufactured by Sangen Industrial) through carbon dioxide. Dispensed, butyl rubber stopper and plastic cap, and sterilized at 115 ° C. for 15 minutes.
This medium was inoculated with Asaccharobacter celatus DSM 18785, which had been stored frozen at -80 ° C, and the gas phase was maintained for 2 minutes with hydrogen / carbon dioxide [4: 1] through a sterile filter. After the above replacement, shaking culture was performed at 37 ° C. and 160 spm for 16 hours. Inoculate 0.1 mL of this seed culture into the previously sterilized GAM bouillon + arginine medium, and then add 0.2 mL each of 20 mM daidzein (LKT) solution (dissolved in DMSO) sterilized with 0.45 μm PTFE membrane in advance. Then, the gas phase was aseptically replaced with gases having the respective compositions shown in Table 1 for 2 minutes or longer, and then cultured under shaking at 37 ° C. and 160 spm. After 24 hours, the culture solution was taken out, the turbidity (OD660) of the culture solution was measured, and equol was quantified by high performance liquid chromatography.
As a result, it was found that the higher the hydrogen concentration, the greater the amount of microbial growth and equol production, and the extremely low growth and equol production with carbon dioxide, nitrogen alone, or carbon dioxide / nitrogen mixed gas (Table). 1, FIG. 2).
GAMブイヨン培地（日水製薬製）5.9 gとL-アルギニン塩酸塩0.5 gを純水100 mLに溶かし、炭酸ガスを通じながら10 mLずつ嫌気性菌培養用18 mm試験管（三紳工業製）に分注し、ブチルゴム栓、プラスチックキャップをして115℃、15分間滅菌した。
この培地に-80℃で凍結保存していたアサッカロバクター・セラツス（Asaccharobacter celatus） DSM 18785株を植菌し、無菌フィルターを通した水素/炭酸ガス［4:1］で気相を2分間以上置換した後、37℃、160 spmで16時間振とう培養を行った。この種培養液0.1 mLを先の滅菌したGAMブイヨン＋アルギニン培地に植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した20 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.2 mLずつ添加し、気相を表1に示すそれぞれの組成のガスで無菌的に2分間以上置換した後、37℃、160 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、培養液の濁度（OD660）を測定し、また、高速液体クロマトグラフィーによりエクオールを定量した。
その結果、水素濃度が高いほど菌体の生育量、エクオール生成量ともに多いこと、炭酸ガス、窒素のみ、あるいは炭酸ガス/窒素混合ガスでは生育、エクオール生成とも極めて少ないことが明らかとなった（表１、図２）。 [Examples 1 to 7]
Dissolve 5.9 g of GAM broth medium (manufactured by Nissui Pharmaceutical) and 0.5 g of L-arginine hydrochloride in 100 mL of pure water, and add 10 mL each to an anaerobic bacteria culture 18 mm test tube (manufactured by Sangen Industrial) through carbon dioxide. Dispensed, butyl rubber stopper and plastic cap, and sterilized at 115 ° C. for 15 minutes.
This medium was inoculated with Asaccharobacter celatus DSM 18785, which had been stored frozen at -80 ° C, and the gas phase was maintained for 2 minutes with hydrogen / carbon dioxide [4: 1] through a sterile filter. After the above replacement, shaking culture was performed at 37 ° C. and 160 spm for 16 hours. Inoculate 0.1 mL of this seed culture into the previously sterilized GAM bouillon + arginine medium, and then add 0.2 mL each of 20 mM daidzein (LKT) solution (dissolved in DMSO) sterilized with 0.45 μm PTFE membrane in advance. Then, the gas phase was aseptically replaced with gases having the respective compositions shown in Table 1 for 2 minutes or longer, and then cultured under shaking at 37 ° C. and 160 spm. After 24 hours, the culture solution was taken out, the turbidity (OD660) of the culture solution was measured, and equol was quantified by high performance liquid chromatography.
As a result, it was found that the higher the hydrogen concentration, the greater the amount of microbial growth and equol production, and the extremely low growth and equol production with carbon dioxide, nitrogen alone, or carbon dioxide / nitrogen mixed gas (Table). 1, FIG. 2).
〔実施例８〕
実施例１～７と同様にしてGAM＋アルギニン培地を調製し、この10 mLを、炭酸ガスを通じながら100 mL容バイヤルビン（マルエム製）に分注し、ブチルゴム栓をしてさらにアルミキャップをし、115℃、15分間滅菌した。
これに実施例１～７と同様の方法で培養したアサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株の種培養液を0.1 mL植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した40 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.5 mL添加し、気相を無菌的に水素/炭酸ガス［4:1］で3分間以上置換した後、37℃、200 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、高速液体クロマトグラフィーにより生じたエクオールを定量した。
その結果、1.87 mMのエクオールが生成し、またダイゼインは0.06 mMに減少していること、ジヒドロダイゼインは検出されないことが判明した。これらのことから、水素ガス供給の著しい効果が明らかとなった。また、本条件の培養においてダイゼインに対してエクオール収率が94%と極めて高いことも明らかになった。 Example 8
A GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL of this was dispensed into 100 mL volume vial (manufactured by Maruemu) through carbon dioxide gas, and a butyl rubber stopper was attached to make an aluminum cap. Sterilized at ° C for 15 minutes.
To this, 0.1 mL of a seed culture solution of Asaccharobacter celatus DSM 18785 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then filter-sterilized with a 0.45 μm PTFE membrane inadvance 40 Add 0.5 mL of mM daidzein (LKT) solution (dissolved in DMSO), and aseptically replace the gas phase with hydrogen / carbon dioxide [4: 1] for at least 3 minutes, then shake at 37 ° C and 200 spm. Culture was performed. After 24 hours, the culture solution was taken out and equol produced by high performance liquid chromatography was quantified.
As a result, 1.87 mM equol was produced, daidzein was reduced to 0.06 mM, and dihydrodaidzein was not detected. From these things, the remarkable effect of hydrogen gas supply became clear. It was also revealed that the equol yield was extremely high at 94% with respect to daidzein in the culture under these conditions.
実施例１～７と同様にしてGAM＋アルギニン培地を調製し、この10 mLを、炭酸ガスを通じながら100 mL容バイヤルビン（マルエム製）に分注し、ブチルゴム栓をしてさらにアルミキャップをし、115℃、15分間滅菌した。
これに実施例１～７と同様の方法で培養したアサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株の種培養液を0.1 mL植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した40 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.5 mL添加し、気相を無菌的に水素/炭酸ガス［4:1］で3分間以上置換した後、37℃、200 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、高速液体クロマトグラフィーにより生じたエクオールを定量した。
その結果、1.87 mMのエクオールが生成し、またダイゼインは0.06 mMに減少していること、ジヒドロダイゼインは検出されないことが判明した。これらのことから、水素ガス供給の著しい効果が明らかとなった。また、本条件の培養においてダイゼインに対してエクオール収率が94%と極めて高いことも明らかになった。 Example 8
A GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL of this was dispensed into 100 mL volume vial (manufactured by Maruemu) through carbon dioxide gas, and a butyl rubber stopper was attached to make an aluminum cap. Sterilized at ° C for 15 minutes.
To this, 0.1 mL of a seed culture solution of Asaccharobacter celatus DSM 18785 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then filter-sterilized with a 0.45 μm PTFE membrane in
As a result, 1.87 mM equol was produced, daidzein was reduced to 0.06 mM, and dihydrodaidzein was not detected. From these things, the remarkable effect of hydrogen gas supply became clear. It was also revealed that the equol yield was extremely high at 94% with respect to daidzein in the culture under these conditions.
〔実施例 ９～１７〕
実施例１～７と同様にしてGAM＋アルギニン培地を調製し、10 mLずつ嫌気性菌培養用18 mm試験管（三紳工業製）に分注し、ブチルゴム栓、プラスチックキャップをして115℃、15分間滅菌した。
これに実施例１～７と同様の方法で培養したアサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株の種培養液を0.1 mL植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した40 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.5 mL添加した。次いで、それぞれの試験管の気相を表２及び表３に示す組成のガスで無菌的に2分間以上置換した後、37℃、160 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、高速液体クロマトグラフィーによりエクオールを定量した。
その結果を表２及び表３に示す。水素濃度にほぼ比例してエクオール生成量が増加し、水素のみで置換した場合が最もエクオール濃度が高いことが分かった。他のガスの影響については、炭酸ガス、窒素では顕著な差異はない結果であった（図３）。 [Examples 9 to 17]
GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL each was dispensed into an 18 mm test tube for anaerobic bacteria culture (manufactured by Sangen Industry), with a butyl rubber stopper and a plastic cap at 115 ° C. Sterilized for 15 minutes.
To this, 0.1 mL of a seed culture solution of Asaccharobacter celatus DSM 18785 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then filter-sterilized with a 0.45 μm PTFE membrane inadvance 40 0.5 mL of mM daidzein (LKT) solution (dissolved in DMSO) was added. Next, the gas phase of each test tube was aseptically replaced with a gas having the composition shown in Tables 2 and 3 for 2 minutes or more, and then cultured under shaking at 37 ° C. and 160 spm. After 24 hours, the culture solution was taken out and equol was quantified by high performance liquid chromatography.
The results are shown in Tables 2 and 3. It was found that equol production increased almost in proportion to the hydrogen concentration, and the equol concentration was highest when only hydrogen was replaced. Regarding the effects of other gases, there was no significant difference between carbon dioxide and nitrogen (FIG. 3).
実施例１～７と同様にしてGAM＋アルギニン培地を調製し、10 mLずつ嫌気性菌培養用18 mm試験管（三紳工業製）に分注し、ブチルゴム栓、プラスチックキャップをして115℃、15分間滅菌した。
これに実施例１～７と同様の方法で培養したアサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株の種培養液を0.1 mL植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した40 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.5 mL添加した。次いで、それぞれの試験管の気相を表２及び表３に示す組成のガスで無菌的に2分間以上置換した後、37℃、160 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、高速液体クロマトグラフィーによりエクオールを定量した。
その結果を表２及び表３に示す。水素濃度にほぼ比例してエクオール生成量が増加し、水素のみで置換した場合が最もエクオール濃度が高いことが分かった。他のガスの影響については、炭酸ガス、窒素では顕著な差異はない結果であった（図３）。 [Examples 9 to 17]
GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL each was dispensed into an 18 mm test tube for anaerobic bacteria culture (manufactured by Sangen Industry), with a butyl rubber stopper and a plastic cap at 115 ° C. Sterilized for 15 minutes.
To this, 0.1 mL of a seed culture solution of Asaccharobacter celatus DSM 18785 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then filter-sterilized with a 0.45 μm PTFE membrane in
The results are shown in Tables 2 and 3. It was found that equol production increased almost in proportion to the hydrogen concentration, and the equol concentration was highest when only hydrogen was replaced. Regarding the effects of other gases, there was no significant difference between carbon dioxide and nitrogen (FIG. 3).
〔実施例 １８～２１〕
実施例１～７と同様にしてGAM＋アルギニン培地を調製し、この10 mLを炭酸ガスを通じながら100 mL容バイヤルビン（マルエム製）に分注し、ブチルゴム栓をしてさらにアルミキャップをし、115℃、15分間滅菌した。
これに実施例１～７と同様の方法で培養したアドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens） DSM 19450株の種培養液を0.1 mL植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した40 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.5 mL添加した。気相を表４ に示すそれぞれの組成のガスで無菌的に3分間以上置換した後、37℃、200 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、高速液体クロマトグラフィーによりエクオールを定量した。
その結果、表４に示すように水素にエクオール生成促進効果があることが判明した。 [Examples 18 to 21]
A GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL of this was dispensed into 100 mL volume vials (manufactured by Maruemu) through carbon dioxide gas, and a butyl rubber stopper was attached, and an aluminum cap was further added. Sterilized for 15 minutes.
To this, 0.1 mL of seed culture solution of Adlercreutzia equolifaciens DSM 19450 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then pre-filter sterilized with a 0.45 μm PTFE membrane. 0.5 mL of the 40 mM daidzein (LKT) solution (dissolved in DMSO) was added. The gas phase was aseptically replaced with gas having the respective compositions shown in Table 4 for 3 minutes or longer, and then cultured with shaking at 37 ° C. and 200 spm. After 24 hours, the culture solution was taken out and equol was quantified by high performance liquid chromatography.
As a result, it was found that hydrogen has an equol production promoting effect as shown in Table 4.
実施例１～７と同様にしてGAM＋アルギニン培地を調製し、この10 mLを炭酸ガスを通じながら100 mL容バイヤルビン（マルエム製）に分注し、ブチルゴム栓をしてさらにアルミキャップをし、115℃、15分間滅菌した。
これに実施例１～７と同様の方法で培養したアドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens） DSM 19450株の種培養液を0.1 mL植菌し、次いで予め0.45 μmのPTFE膜でフィルター滅菌した40 mMダイゼイン（LKT製）溶液（DMSOに溶解）を0.5 mL添加した。気相を表４ に示すそれぞれの組成のガスで無菌的に3分間以上置換した後、37℃、200 spmの条件で振とう培養を行った。24時間後、培養液を取り出し、高速液体クロマトグラフィーによりエクオールを定量した。
その結果、表４に示すように水素にエクオール生成促進効果があることが判明した。 [Examples 18 to 21]
A GAM + arginine medium was prepared in the same manner as in Examples 1 to 7, and 10 mL of this was dispensed into 100 mL volume vials (manufactured by Maruemu) through carbon dioxide gas, and a butyl rubber stopper was attached, and an aluminum cap was further added. Sterilized for 15 minutes.
To this, 0.1 mL of seed culture solution of Adlercreutzia equolifaciens DSM 19450 strain cultured in the same manner as in Examples 1 to 7 was inoculated, and then pre-filter sterilized with a 0.45 μm PTFE membrane. 0.5 mL of the 40 mM daidzein (LKT) solution (dissolved in DMSO) was added. The gas phase was aseptically replaced with gas having the respective compositions shown in Table 4 for 3 minutes or longer, and then cultured with shaking at 37 ° C. and 200 spm. After 24 hours, the culture solution was taken out and equol was quantified by high performance liquid chromatography.
As a result, it was found that hydrogen has an equol production promoting effect as shown in Table 4.
〔実施例 ２２～２５〕
実施例１～７と同様にしてスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens） DSM 22006株について気相を水素に置換する効果を検討した。その結果、表５に示すように水素にエクオール生成促進効果があることが判明した。 [Examples 22 to 25]
In the same manner as in Examples 1 to 7, the effect of substituting the gas phase with hydrogen for the Slackia isoflavoniconvertens DSM 22006 strain was examined. As a result, it was found that hydrogen has an equol production promoting effect as shown in Table 5.
実施例１～７と同様にしてスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens） DSM 22006株について気相を水素に置換する効果を検討した。その結果、表５に示すように水素にエクオール生成促進効果があることが判明した。 [Examples 22 to 25]
In the same manner as in Examples 1 to 7, the effect of substituting the gas phase with hydrogen for the Slackia isoflavoniconvertens DSM 22006 strain was examined. As a result, it was found that hydrogen has an equol production promoting effect as shown in Table 5.
本発明の方法によって、嫌気性微生物を利用してエクオールを高効率で製造することができる。
本発明の方法により製造されたエクオールを、飲食品又は医薬品等としてそのまま摂取することにより、乳癌、前立腺癌、骨粗しょう症、高コレステロール血症、心疾患、更年期障害等を予防できるものと考えられる。 By the method of the present invention, equol can be produced with high efficiency using anaerobic microorganisms.
It is considered that breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. can be prevented by ingesting equol produced by the method of the present invention as it is as a food or drink or medicine. .
本発明の方法により製造されたエクオールを、飲食品又は医薬品等としてそのまま摂取することにより、乳癌、前立腺癌、骨粗しょう症、高コレステロール血症、心疾患、更年期障害等を予防できるものと考えられる。 By the method of the present invention, equol can be produced with high efficiency using anaerobic microorganisms.
It is considered that breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. can be prevented by ingesting equol produced by the method of the present invention as it is as a food or drink or medicine. .
Claims (6)
- 次の工程を含むエクオール生産能を有する嫌気性微生物によるエクオールの製造方法；
（１）ダイゼインを、該嫌気性微生物により、水素を含む１種類以上の気体からなる気相下で発酵させる工程、及び
（２）工程（１）の嫌気性微生物が生産したエクオールを回収する工程。 A method for producing equol by an anaerobic microorganism having equol-producing ability, comprising the following steps;
(1) a step of fermenting daidzein with the anaerobic microorganism in a gas phase composed of one or more kinds of gas containing hydrogen; and (2) a step of recovering equol produced by the anaerobic microorganism in step (1). . - 工程（１）の気相において、水素の質量パーセント濃度が４０～１００％であることを特徴とする、請求項１に記載のエクオールの製造方法。 2. The method for producing equol according to claim 1, wherein the mass percentage concentration of hydrogen is 40 to 100% in the gas phase of step (1).
- 工程（１）の気相において、水素の質量パーセント濃度が１００％であることを特徴とする、請求項１に記載のエクオールの製造方法。 The method for producing equol according to claim 1, wherein the mass percent concentration of hydrogen is 100% in the gas phase of step (1).
- 前記嫌気性微生物がコーリオバクテリアセアエ（Coriobacteriaceae）科に分類される菌、ストレプトコッカセアエ（Streptococcaceae）科に分類される菌、又はこれらの類縁菌であることを特徴とする、請求項１～３のいずれかに記載のエクオールの製造方法。 The anaerobic microorganism is a bacterium classified into the family Coriobacteriaceae, a bacterium classified into the family Streptococcaceae, or a related bacterium. 4. The method for producing equol according to any one of 3 above.
- 前記嫌気性微生物が、コーリオバクテリウム（Coriobacterium）属、アドレクラウチア（Adlercreutzia）属、アサッカロバクター（Asaccharobacter）属、アトポビウム（Atopobium）属、コリンゼラ（Collinsella）属、クリプトバクテリウム（Cryptobacterium）属、デニトロバクテリウム（Denitrobacterium）属、エガセラ（Eggerthella）属、エンテロハブダス（Enterorhabdus）属、ゴードニバクター（Gordonibacter）属、オルセネラ（Olsenella）属、パラエゲセエラ（Paraeggerthella）属、スラッキア（Slackia）属、又はラクトコッカス(Lactococcus)属、のいずれかに分類される菌であることを特徴とする、請求項１～３のいずれかに記載のエクオールの製造方法。 The anaerobic microorganisms include the genus Coriobacterium, the genus Adlercreutzia, the genus Asaccharobacter, the genus Atopobium, the genus Collinsella, and the Cryptobacterium. Genus, Denitrobacterium genus, Eggerthella genus, Enterorhabdus genus, Gordonibacter genus, Olsenella genus, Paraeggerthella genus, Slackia genus The method for producing equol according to any one of claims 1 to 3, wherein the bacterium is classified into one of the genus Lactococcus.
- 前記嫌気性微生物が、アサッカロバクター・セラツス（Asaccharobacter celatus）DSM 18785株、アドレクラウチア・エクオーリファシエンス（Adlercreutzia equolifaciens）DSM 19450株、又はスラッキア・イソフラボニコンバーテンス（Slackia isoflavoniconvertens）DSM 22006株のいずれかであることを特徴とする、請求項１～３のいずれかに記載のエクオールの製造方法。 The anaerobic microorganism may be an Asaccharobacter atcelatus DSM78518785 strain, an Adlercreutzia facequolifaciens DSM 19450 strain, or a Slackia isoflavoniconvertens strain DSM 2006 DSM 2006 The method for producing equol according to any one of claims 1 to 3, wherein the method is any one of the following.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102633763A (en) * | 2012-04-11 | 2012-08-15 | 黑龙江大学 | Preparation method of (S)-equol |
| JP2014054234A (en) * | 2012-09-14 | 2014-03-27 | Daicel Corp | Composition containing ornithine and equol |
| CN104673722A (en) * | 2015-03-03 | 2015-06-03 | 河北农业大学 | Oxytolerant Prunella and application thereof in aerobic synthesis of dihydrosoybean isoflavone |
| JP2020010635A (en) * | 2018-07-18 | 2020-01-23 | 株式会社ダイセル | Method for producing functional substance |
| WO2023199929A1 (en) * | 2022-04-13 | 2023-10-19 | 株式会社ダイセル | Equol manufacturing method |
| US12070443B2 (en) | 2018-06-15 | 2024-08-27 | The Board Of Regents Of The University Of Texas System | Methods of treating and preventing breast cancer with S-equol |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102633763A (en) * | 2012-04-11 | 2012-08-15 | 黑龙江大学 | Preparation method of (S)-equol |
| JP2014054234A (en) * | 2012-09-14 | 2014-03-27 | Daicel Corp | Composition containing ornithine and equol |
| CN104673722A (en) * | 2015-03-03 | 2015-06-03 | 河北农业大学 | Oxytolerant Prunella and application thereof in aerobic synthesis of dihydrosoybean isoflavone |
| US12070443B2 (en) | 2018-06-15 | 2024-08-27 | The Board Of Regents Of The University Of Texas System | Methods of treating and preventing breast cancer with S-equol |
| JP2020010635A (en) * | 2018-07-18 | 2020-01-23 | 株式会社ダイセル | Method for producing functional substance |
| JP7376225B2 (en) | 2018-07-18 | 2023-11-08 | 株式会社ダイセル | Method for manufacturing functional substances |
| WO2023199929A1 (en) * | 2022-04-13 | 2023-10-19 | 株式会社ダイセル | Equol manufacturing method |
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| JP5916132B2 (en) | 2016-05-11 |
| JPWO2012033150A1 (en) | 2014-01-20 |
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