Method for producing equol and 5-hydroxyequol

Abstract

Provided is a method for producing equol and 5-hydroxyequol using a microorganism having a capability of producing equol from a daidzein compound and a capability of producing 5-hydroxyequol from a genistein compound, wherein the daidzein compound and the genistein compound are present at the start of the culture of the microorganism, the molar conversion efficiency of the genistein compound to 5-hydroxyequol is 0.200% / h or more, the molar concentration ratio of the genistein compound to the daidzein compound at the start of the culture is 1.500 or more or 0.500 or less, the daidzein compound is selected from daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, and combinations thereof, and the genistein compound is selected from genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, and combinations thereof.

Description

Method for producing equol and 5-hydroxyequol

[0001] The present disclosure relates to methods for producing equol and 5-hydroxyequol.

[0002] Isoflavones, which are abundant in legumes such as soybeans and kudzu, are a type of polyphenol, a flavonoid with an isoflavone as its basic structure. Recent studies have revealed that isoflavones have female hormone (estrogen) and antioxidant effects, and that ingesting isoflavones has preventive effects against breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, etc. (Non-Patent Documents 1 to 6).

[0003] Furthermore, soybeans contain isoflavones that are primarily glycosides condensed with sugars, with very small amounts of non-condensed isoflavone aglycones (hereinafter also referred to as aglycones). Examples of glycoside-form isoflavones include daidzin, glycitin, and genistin. Upon entering the human or animal body, these glycosides are converted into the aglycones daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or enzymes produced by intestinal bacteria (e.g., β-glucosidase). Daidzein is known to be enzymatically converted to equol via dihydrodaidzein (also referred to as dihydrodaidzein) by the action of intestinal bacteria. Similarly, it is known that genistein is converted into dihydrogenistein (also called dihydrogenistein) and 5-hydroxyequol by the action of some intestinal bacteria (Non-Patent Document 7).

[0004] Equol is known to have the highest estrogenic activity of these metabolites (Non-Patent Documents 8 and 9), but 5-hydroxyequol also has estrogen-like activity and has been reported to have 3-β-hydroxysteroid dehydrogenase inhibitory activity (Patent Document 1). By inhibiting the biosynthesis of aldosterone and glucocorticoids, it is expected to be used in the prevention or treatment of excesses of these hormones.

[0005] However, isoflavone metabolism differs from person to person, and as mentioned above, only a small proportion of people possess intestinal bacteria capable of fermenting daidzein to produce equol, with the prevalence being approximately 50% in Japanese and approximately 30% in Westerners (Non-Patent Documents 10 and 11). It is believed that 5-hydroxyequol is produced by enzymes possessed by equol-producing bacteria (Non-Patent Document 7). Furthermore, the enzymes involved in the synthesis of equol from daidzein possessed by equol-producing bacteria are daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR), and the genes encoding these enzymes are organized as clusters with similar sequences and genetic structures even among different equol-producing strains of bacteria, such as those from the genera Slacka, Eggsella, and Lactococcus (Non-Patent Document 12). Therefore, Slackia isoflavonicconvertens (DSM 22006), which contains the enzymes involved in equol production, and intestinal lactic acid bacteria into which genes encoding the enzymes involved in equol production have been introduced, can simultaneously produce equol and 5-hydroxyequol (Non-Patent Document 12).

[0006] People who do not have intestinal bacteria that produce equol or 5-hydroxyequol are unable to produce equol or 5-hydroxyequol in their bodies, even if they ingest foods containing daidzein or genistein from soybeans, etc. To solve these problems, attempts have been made in recent years to produce equol using anaerobic microorganisms such as lactic acid bacteria and intestinal bacteria (Patent Documents 2 to 5), but no such efforts have been reported for 5-hydroxyequol.

[0007] Japanese Patent Application Laid-Open No. 2003-81875 Japanese Patent Application Laid-Open No. 2006-204296 Japanese Patent Application Laid-Open No. 2006-504409 Japanese Patent Application Laid-Open No. 2008-61584 Japanese Patent Application Laid-Open No. 2010-104241

[0008] Adlercreutz, H. , The Lancet Oncol. , 3, 364-373 (2002) Duncan, A. M. et al. , Best Pract. Res. Clin. Endocrinol. Metab. , 17, 253-271 (2003) Wu, A. H. et al. , Carcinogenesis, 23, 1491-1496 (2002) Yamamoto, S. et al. , J. Natl. Cancer Inst. , 95, 906-913 (2003) Onozawa, M. et al. , Jpn. J. Cancer Res. , 90, 393-398 (1999) Ridges, L. et al. , Asia Pac. J. Clin. Nutr. , 10, 204-211 (2001) Matthies, A. et al. , Appl. Environ. Microbiol. , 74(15), 4847-52 (2008) Schmitt, E. et al. , Toxicol. In Vitro, 15, 433-439 (2001) Sathyamoorthy, N. and Wang, T. T. , Eur. J. Cancer, 33, 2384-2389 (1997) Arai, Y. et al. , J. Epidemiol. , 10, 127-135 (2000) Setchell, K. D. et al. , J. Nutr. , 133, 1027-1035 (2003) International Journal of Food Microbiology 360 (2021) 109328

[0009] An objective of the present disclosure is to provide at least a method for producing equol and 5-hydroxyequol.

[0010] The gist of the present disclosure relates to the following: [1] A method for producing equol and 5-hydroxyequol using a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins, wherein daidzeins and genisteins are present at the start of culturing the microorganism, the molar conversion efficiency of the genisteins to 5-hydroxyequol is 0.200% / h or more, the molar concentration ratio of the genisteins to the daidzeins at the start of culturing is 1.500 or more or 0.500 or less, the daidzeins are daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins are genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof. [2] The production method according to [1], wherein the molar conversion efficiency of the genisteins to 5-hydroxyequol is 0.250% / h or more. [3] The production method according to [1] or [2], wherein the molar concentration ratio of the genisteins to the daidzeins is 1.800 or more or 0.400 or less. [4] The production method according to any one of [1] to [3], wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to the genus Adreklautia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Slackia, and microorganisms belonging to the genus Lactococcus. [5] The production method according to [4], wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to Adreklautia aequorifaciens, microorganisms belonging to Slackia aequorifaciens, and microorganisms belonging to Lactococcus garvieae. [6] The production method according to any one of [1] to [5], wherein β-cyclodextrin is further present at the start of culturing the microorganism. [7] The method according to any one of [1] to [6], wherein arginine is further present at the start of culturing the microorganism.[8] A method for producing a fermented product containing equol and 5-hydroxyequol by culturing a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins, wherein daidzeins and genisteins are present at the start of culturing the microorganism, the molar concentration ratio of the genisteins to the daidzeins at the start of culturing is 1.500 or more or 0.500 or less, the total content of equol and 5-hydroxyequol in the fermented product is 0.5 mg / g or more and 100.0 mg / g or less per 1 g of dry weight, the daidzeins are daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins are genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof. [9] The method according to [8], wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to the genus Adreklautia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Slackia, and microorganisms belonging to the genus Lactococcus.

[10] The method according to [9], wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to Adreklautia aequorifaciens, microorganisms belonging to Slackia aequorifaciens, and microorganisms belonging to Lactococcus garvieae.

[11] The method according to any one of [8] to

[10] , wherein β-cyclodextrin is further present at the start of culturing the microorganism.

[12] The method according to any one of [8] to

[11] , wherein arginine is further present at the start of culturing the microorganism.

[0011] The present disclosure provides at least a method for producing equol and 5-hydroxyequol. Furthermore, the resulting equol and 5-hydroxyequol can be used in cosmetics, quasi-drugs, medical supplies, hygiene products, pharmaceuticals, food and beverages (including supplements), etc., and subjects, including humans, can easily obtain the known effects of equol and 5-hydroxyequol by using or ingesting them.

[0012] FIG. 1 is a graph showing the relationship between the molar concentration ratio of genisteins to daidzeins at the start of culture and the molar conversion efficiency of genisteins to 5-hydroxyequol.

[0013] In the present disclosure, unless otherwise specified, the expressions "XX to YY" or "XX to YY" representing a range of values ​​mean a range of values ​​including the lower and upper limits, which are the endpoints. When values ​​are described in stages, each value or each upper and lower limit of each value can be combined in any combination.

[0014] <Method for Producing Equol and 5-Hydroxyequol> Microorganisms may be used for production. There are no particular limitations on the microorganisms, so long as they have the ability to produce equol (hereinafter also referred to as EQ) from daidzeins and the ability to produce 5-hydroxyequol (hereinafter also referred to as 5HEQ) from genisteins. Furthermore, microorganisms into which genes encoding the enzymes involved in equol production and 5-hydroxyequol production (e.g., daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR), etc.) have been introduced may also be used. Microorganisms capable of producing equol from daidzeins and 5-hydroxyequol from genisteins may be known microorganisms or microorganisms obtained by screening using conventional methods.For example, microorganisms belonging to the genus Coriobacterium, microorganisms belonging to the genus Adlercreutzia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Atopobium, microorganisms belonging to the genus Bacteroides, microorganisms belonging to the genus Bifidobacterium, microorganisms belonging to the genus Clostridium, Microorganisms that cause bacterial infection, microorganisms belonging to the genus Collinsella, microorganisms belonging to the genus Cryptobacterium, microorganisms belonging to the genus Denitrobacterium, microorganisms belonging to the genus Eggerthella, microorganisms belonging to the genus Enterococcus, microorganisms belonging to the genus Enterorhabdus, microorganisms belonging to the genus Eubacterium Organisms, microorganisms belonging to the genus Finegoldia, microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Gordonibacter, microorganisms belonging to the genus Olsenella, microorganisms belonging to the genus Paraeggerthella, microorganisms belonging to the genus Pediococcus, microorganisms belonging to the genus Proteus, microorganisms belonging to the genus Sharpea Examples of suitable microorganisms include those belonging to the genus Slackia, those belonging to the genus Streptococcus, those belonging to the genus Veillonella, and those belonging to the genus Lactococcus, as well as those obtained by screening these microorganisms using standard methods to measure the ability to produce equol from daidzeins and / or the ability to produce 5-hydroxyequol from genisteins. Genes encoding the enzymes involved in equol production and 5-hydroxyequol production can be obtained from microorganisms classified into the above genera, for example.

[0015] More specific microorganisms are as follows: - Microorganisms belonging to Adrecreutzia aequolifaciens subsp. ceratus, such as Adrecreutzia aequolifaciens subsp. ceratus strain DSM 18785, and microorganisms belonging to Adrecreutzia aequolifaciens subsp. ceratus, such as Adrecreutzia aequolifaciens subsp. ceratus strain DSM 18785, and microorganisms belonging to Adrecreutzia aequolifaciens subsp. ceratus, such as Adrecreutzia aequolifaciens subsp. ceratus strain DSM 19450; - Microorganisms belonging to Bacteroides ovatus, such as Bacteroides ovatus E-23-15 strain (FERM BP-6435); - Microorganisms belonging to Bifidobacterium breve, such as Bifidobacterium breve ATCC 15700 strain; - Bifidobacterium longum NITE BP-02621 (ATCC BAA-999; BB536 strain); - Microorganisms belonging to the genus Clostridium, such as Clostridium sp. HGH136 strain (ATCC BAA-442); - Eggerthella sp. Julong 732 strain (KCCM-10490), Eggerthella sp. YY7918 strain (note that the YY7918 strain is managed by the Gifu Prefectural Institute of Biotechnology, a public institution, and can be obtained from there), Eggerthella sp. Microorganisms belonging to the genus Eggerthella, such as Eggerthella sp. D1 strain;- Microorganisms belonging to the genus Enterococcus faecalis, such as the Enterococcus faecalis INIA P333 strain; - Microorganisms belonging to the genus Enterococcus faecium, such as the Enterococcus faecium EPI1 strain; - Microorganisms belonging to the genus Enterohabdus mucosicola, such as the Enterohabdus mucosicola Mt1B8 strain (DSM 19490); - Microorganisms belonging to the genus Eubacterium, such as Eubacterium sp. D2 strain; - Microorganisms belonging to Finegoldia magna, such as Finegoldia magna EPI3 strain; - Microorganisms belonging to Lactobacillus fermentum, such as Lactobacillus fermentum DPPMA114 strain (DSM 23757); - Lactobacillus intestinalis Microorganisms belonging to the genus Lactobacillus intestinalis, such as the Lactobacillus intestinalis KTCT13676BP strain; Microorganisms belonging to the genus Lactobacillus mucosae, such as the Lactobacillus mucosae EPI2 strain; Microorganisms belonging to the genus Lactobacillus paracasei, such as the Lactobacillus paracasei JS1 strain;- Microorganisms belonging to Lactobacillus plantarum, such as Lactobacillus plantarum DPPMA24W strain (DSM 23756) and Lactobacillus plantarum DPPMASL33 strain (DSM 23755); - Lactobacillus rhamnosus DPPMAAZ1 strain, Lactobacillus rhamnosus INIA Microorganisms belonging to the genus Lactobacillus, such as the P540 strain; Microorganisms belonging to the genus Lactobacillus, such as the Lactobacillus sp. Niu-O16 strain; Microorganisms belonging to the genus Lactococcus, such as the Lactococcus garvieae 20-92 strain (FERM BP-10036); Paraeggerthella sp. sp.) strain SNR40-432; - Microorganisms belonging to Pediococcus pentosaceus, such as Pediococcus pentosaceus CS1 strain; - Microorganisms belonging to Proteus mirabilis, such as Proteus mirabilis LH-52 strain; - Microorganisms belonging to Sharpea azabuensis, such as Sharpea azabuensis ST18 strain (NITE P-300); azabuensis); Microorganisms belonging to the genus Slackia aequolifaciens, such as Slackia aequolifaciens DSM 24851 strain;- Microorganisms belonging to Slackia isoflavonicconvertens, such as Slackia isoflavonicconvertens DSM 22006 strain; - Slackia sp. FJK1 strain (NITE P-1562), Slackia sp. NATTS strain (FERM BP-11231), Slackia sp. YIT11861 strain (FERM BP-11231), Slackia sp. Microorganisms belonging to the genus Slackia, such as Streptococcus sp. TM-30 strain; Microorganisms belonging to Streptococcus constellatus, such as Streptococcus constellatus E-23-17 strain (FERM BP-6436); Microorganisms belonging to Streptococcus intermedius, such as Streptococcus intermedius A6G-225 strain (FERM BP-6437); Veillonella sp. Microorganisms belonging to the genus Veillonella, such as the Veillonella sp. EP strain, are also included. Microorganisms having a 16S rDNA with a base sequence identical to the base sequence of the 16S rDNA (16S rRNA gene) of these microorganisms by 95% or more, preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more. Strains may be bred from strains of each microorganism or substantially equivalent strains by mutation treatment, genetic recombination, selection of natural mutants, or the like. One or more species of microorganisms may be used. The microorganisms can be obtained from the depository institutions indicated by the deposit number. Each accession number indicates that the microorganism has been deposited at the following depository institution: FERM International Patent Biological Depository;International Patent Organization 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 ATCC American Type Culture Collection NITE National Institute of Technology and Evaluation Patent Microorganism Deposit Center (NPMD);

[0016] In the method for producing equol and 5-hydroxyequol, genisteins such as genistin, malonylgenistin, acetylgenistin, genistein, and dihydrogenistein are preferably used as raw materials for 5-hydroxyequol. Daidzeins such as daidzin, malonyldaidzin, acetyldaidzin, daidzein, and dihydrodaidzein are preferably used as raw materials for equol. The raw materials are not particularly limited as long as they contain genisteins or daidzeins. For example, the raw materials may contain glyciteins such as glycitin, malonylglycitin, acetylglycitin, glycitein, and dihydroglycitein in addition to genisteins and daidzeins. The raw materials may be, for example, soybeans or soybean hypocotyls, either as they are or after defatting or deproteinization. Extracts of soybeans or soybean hypocotyls, or purified extracts thereof, may also be used. Tempeh, alfalfa, or extracts thereof may also be used. Furthermore, if the microorganism has an enzyme that converts glycosides to aglycones, the raw material containing daidzeins and genisteins can be used as is. If the microorganism does not have an enzyme that converts glycosides to aglycones, the raw material containing daidzeins and genisteins can be used after converting the glycosides to aglycones by, for example, subjecting the raw material to an enzymatic treatment.

[0017] The method for producing equol and 5-hydroxyequol is not particularly limited, and any conventionally known method may be used. For example, a method may be used in which a microorganism capable of producing equol from daidzeins and 5-hydroxyequol from genisteins is cultured under conditions in which daidzeins and genisteins are present at the start of culture (e.g., in a composition containing daidzeins and genisteins).

[0018] The composition containing daidzeins and genisteins is not particularly limited as long as it can cause a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins to produce equol from daidzeins and 5-hydroxyequol from genisteins. The composition containing daidzeins and genisteins may contain components other than daidzeins and genisteins, as long as it can cause a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins to produce equol from daidzeins and 5-hydroxyequol from genisteins. The composition containing daidzeins and genisteins is preferably a culture medium.

[0019] Anaerobic microorganisms are cultured anaerobically, and aerobic microorganisms are cultured aerobically. For example, in the case of anaerobic microorganisms, gas phase conditions (anaerobic conditions) that allow their survival can be maintained, and nutrients can be provided to support the activity and growth of the anaerobic microorganisms. Various medium compositions and gas phase conditions suitable for the survival of anaerobic microorganisms and the production of equol and / or 5-hydroxyequol are known. In other words, appropriate medium compositions and gas phase configuration conditions can be selected for each microorganism. Media containing various nutrients such as Anaerobe Basal Broth (ABB) medium, GAM bouillon medium, modified GAM medium, and BHI medium can be used. These media may also contain the following various components as appropriate:

[0020] The medium may contain water-soluble organic substances such as sugars (e.g., sorbose, fructose, glucose, etc.) and organic acids (e.g., valeric acid, butyric acid, propionic acid, acetic acid, and formic acid). It may also contain inorganic nitrogen sources (e.g., ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium hydrogen phosphate); nitrates (e.g., potassium nitrate and sodium nitrate); amino acids (e.g., arginine, citrulline, ornithine, and lysine); and organic nitrogen sources (e.g., yeast extract, peptones, meat extract, liver extract, and digested serum powder). It may also contain vitamins (e.g., biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, vitamin B12, thiooctoic acid, and p-aminobenzoic acid), as well as the porphyrin compound hemin.

[0021] Furthermore, arginine may be present at the start of culturing the microorganism. Furthermore, arginine may be further contained in the composition containing daidzeins and genisteins at the start of culturing the microorganism. The content of arginine in the composition containing daidzeins and genisteins at the start of culturing is not particularly limited, but may be 0.30 g / L or more, 0.40 g / L or more, 0.50 g / L or more, 0.60 g / L or more, 0.70 g / L or more, 0.80 g / L or more, 0.90 g / L or more, 1.00 g / L or more, 1.10 g / L or more, 1.20 g / L or more, 1.30 g / L or more, 1.40 g / L or more, 1.50 g / L or more, 1.60 g / L or more, 1.70 g / L or more, 1.80 g / L or more, 1.90 g / L or more, 2.00 g / L or more. or greater, 2.10 g / L or greater, 2.20 g / L or greater, 2.30 g / L or greater, 2.40 g / L or greater, 2.50 g / L or greater, 2.60 g / L or greater, 2.70 g / L or greater, 2.80 g / L or greater, 2.90 g / L or greater, 3.00 g / L or greater, 3.10 g / L or greater, 3.20 g / L or greater, 3.30 g / L or greater, 3.40 g / L or greater, 3.50 g / L or greater, 3.60 g / L or greater, 3.70 g / L or greater, 3.80 g / L or greater, 3.90 g / L or greater, 4.00 g / L or greater, 4.50 g / L or greater, or 5.00 g / L or greater. The upper limit is not particularly limited, but may be 10.00 g / L or less, 9.00 g / L or less, 8.00 g / L or less, or 7.00 g / L or less. Specifically, the upper limit may be, for example, 0.30 g / L to 10.00 g / L, 0.40 g / L to 9.00 g / L, 0.50 g / L to 8.00 g / L, or 0.60 g / L to 7.00 g / L.

[0022] Furthermore, cyclodextrin may be present at the start of culturing the microorganism. Furthermore, cyclodextrin may be further added to the composition containing daidzeins and genisteins at the start of culturing the microorganism. Examples of cyclodextrin include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof such as glycosyl-cyclodextrin, maltosyl-cyclodextrin, hydroxypropyl-cyclodextrin, and methyl-cyclodextrin. One type of cyclodextrin may be used, or two or more types may be used. The content of cyclodextrin in the composition containing daidzeins and genisteins at the start of culture is not particularly limited, and may be 0.01 g / L or more, 0.05 g / L or more, 0.1 g / L or more, 0.2 g / L or more, 0.3 g / L or more, 0.4 g / L or more, 0.5 g / L or more, 0.6 g / L or more, 0.7 g / L or more, 0.8 g / L or more, 0.9 g / L or more, 1.0 g / L or more, 1.5 g / L or more, 2.0 g / L or more, 2.5 g / L or more, 3.0 g / L or more, 3.5 g / L or more, 4.0 g / L or more, 4.5 g / L or more, 5.0 g / L or more, 5.5 g / L or more, The upper limit of the dextrin concentration may be 6.0 g / L or more, 6.5 g / L or more, 7.0 g / L or more, 7.5 g / L or more, 8.0 g / L or more, 8.5 g / L or more, 9.0 g / L or more, 9.5 g / L or more, 10.0 g / L or more, 10.5 g / L or more, 11.0 g / L or more, 11.5 g / L or more, 12.0 g / L or more, 12.5 g / L or more, 13.0 g / L or more, 13.5 g / L or more, 14.0 g / L or more, 14.5 g / L or more, 15.0 g / L or more, 15.5 g / L or more, 16.0 g / L or more, 16.5 g / L or more, 17.0 g / L or more, or 17.5 g / L or more. The upper limit is not particularly limited, but it is preferably set to be equal to or lower than the solubility of dextrin at the culture temperature. For example, in the case of β-cyclodextrin, the concentration should be 30.5 g / L or less.

[0023] The medium can be liquid, semi-solid, or solid, and the content of each component in the medium can be selected appropriately to match the growth of the microorganism and the production of the target substance.

[0024] In culturing anaerobic microorganisms, the combination of gases supplied to the aqueous phase or constituting the gas phase is not particularly limited, and a mixed gas containing one or more constituents selected from hydrogen, carbon dioxide, nitrogen, etc. is preferably used. The mixed gas preferably contains hydrogen as a constituent. The method for creating an environment suitable for culturing in the gas phase or aqueous phase during culturing is not particularly limited, but may include, for example, replacing the gas phase with the mixed gas before culturing, supplying the mixed gas from the bottom of the culturing device during culturing, supplying the mixed gas to the gas phase of the culturing device, or bubbling the aqueous phase with the mixed gas before culturing. Hydrogen may be used as is, or a hydrogen precursor such as formic acid may be added to the culture medium to generate hydrogen during culturing by the action of the microorganisms.

[0025] When hydrogen is included as a constituent, the partial pressure percentage of hydrogen in the mixed gas may be 0.1% to 100%. For example, the partial pressure percentage may be 0.1%, 1%, 2%, 4%, 6%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, or a concentration range with a lower and upper limit of two partial pressure percentages selected from these. Specifically, the partial pressure percentage of hydrogen in the mixed gas may be, for example, 0.1% to 100%, 1% to 80%, 2% to 50%, 4% to 40%, 6% to 30%, or 10% to 20%.

[0026] When a gas mixture is passed through the culture apparatus during culture, the amount of gas mixture passed through is not particularly limited, but may be adjusted to about 0.005 V / V / M (gas volume / liquid volume / minute) to 2.0 V / V / M (gas volume / liquid volume / minute). The pressure conditions are not particularly limited as long as they allow the microorganism to grow and produce substances, but may be in the range of 0.01 MPa to 0.5 MPa.

[0027] The culture temperature and culture pH are not particularly limited, but may be set to a temperature and pH that are suitable for the growth and substance production of each microorganism. Generally, the temperature is set to about 30°C to 42°C, but may also be set to, for example, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, or 34°C or higher, or 42°C or lower, 41°C or lower, 40°C or lower, or 39°C or lower. Specifically, the temperature may be, for example, 30°C to 42°C, 31°C to 41°C, 32°C to 40°C, 33°C to 39°C, or 34°C to 39°C. The pH may be 3.0 to 9.0, but may also be, for example, 3.0 or more, 4.0 or more, 5.0 or more, 6.0 or more, 6.1 or more, 6.2 or more, 6.3 or more, 6.4 or more, 6.5 or more, 6.6 or more, 6.7 or more, or 6.8 or more, or 9.0 or less, 8.5 or less, 8.4 or less, 8.3 or less, 8.2 or less, 8.1 or less, 7.5 or less, or 7.0 or less. Specifically, for example, the pH may be 3.0 to 9.0, 4.0 to 8.5, 5.0 to 8.4, 6.0 to 8.3, 6.1 to 8.2, or 6.2 to 8.1.

[0028] The culture time is not particularly limited, but is usually about 6 to 340 hours, and may be, for example, 6 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 25 hours or more, 30 hours or more, 35 hours or more, 40 hours or more, or 45 hours or more. The upper limit may be set appropriately depending on the purpose, and may be 340 hours or less, 300 hours or less, 250 hours or less, 200 hours or less, 150 hours or less, 140 hours or less, 130 hours or less, 120 hours or less, 110 hours or less, 100 hours or less, 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 75 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, or 25 hours or less. Specifically, for example, it may be 6 hours to 340 hours, 8 hours to 300 hours, 9 hours to 250 hours, 10 hours to 200 hours, 11 hours to 150 hours, 12 hours to 140 hours, 13 hours to 130 hours, 14 hours to 120 hours, 15 hours to 110 hours, 16 hours to 100 hours, 17 hours to 95 hours, 18 hours to 90 hours, 19 hours to 85 hours, 20 hours to 80 hours, 25 hours to 75 hours, 30 hours to 70 hours, 35 hours to 65 hours, 40 hours to 60 hours, or 45 hours to 55 hours.

[0029] The molar conversion rate of genisteins to 5-hydroxyequol may be 10.0% or more. Alternatively, "the molar conversion rate of genisteins to 5-hydroxyequol is 10.0% or more" may mean initiating the cultivation of a microorganism capable of producing equol from daidzein and 5-hydroxyequol from genistein in the presence of daidzeins and genisteins, and culturing the microorganism until the molar conversion rate of genisteins to 5-hydroxyequol reaches at least 10.0%. The molar conversion rate of genisteins to 5-hydroxyequol is 10.0% or more, 11.0% or more, 12.0% or more, 13.0% or more, 14.0% or more, 15.0% or more, 16.0% or more, 18.0% or more, 20.0% or more, 22.0% or more, 24.0% or more, 26.0% or more, 28.0% or more, 30.0% or more, 32.0% or more, 34.0% or more, 36.0% or more, 38.0% or more, 40.0% or more, 42.0% or more, 44.0% or more, 46.0% or more, 48.0% or more, It may be 50.0% or more, 52.0% or more, 54.0% or more, 56.0% or more, 58.0% or more, 60.0% or more, 62.0% or more, 64.0% or more, 66.0% or more, 68.0% or more, 70.0% or more, 72.0% or more, 74.0% or more, 76.0% or more, 78.0% or more, or 80.0% or more, and the upper limit is not particularly limited, but may be 100.0% or less, 98.0% or less, 96.0% or less, 94.0% or less, 92.0% or less, or 90.0% or less. Specifically, it may be, for example, 10.0% to 100.0%, 11.0% to 98.0%, 12.0% to 96.0%, 13.0% to 94.0%, 14.0% to 92.0%, or 15.0% to 90.0%.

[0030] The molar conversion rate of genisteins to 5-hydroxyequol may be less than 10.0% at 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, or 10 hours after the start of the culture. The molar conversion rate of genisteins to 5-hydroxyequol at 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, or 10 hours from the start of culture may be 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, or 1.0% or less. The molar conversion rate of genisteins to 5-hydroxyequol at 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, or 10 hours from the start of culture may be 0%.

[0031] The molar conversion rate of daidzeins to equol may be 10.0% or more. Alternatively, "the molar conversion rate of daidzeins to equol is 10.0% or more" may mean initiating the cultivation of a microorganism capable of producing equol from daidzein and 5-hydroxyequol from genistein in the presence of daidzeins and genisteins, and culturing the microorganism until the molar conversion rate of daidzeins to equol reaches at least 10.0%. The molar conversion rate of daidzeins to equol is 10.0% or more, 11.0% or more, 12.0% or more, 13.0% or more, 14.0% or more, 15.0% or more, 16.0% or more, 18.0% or more, 20.0% or more, 22.0% or more, 24.0% or more, 26.0% or more, 28.0% or more, 30.0% or more, 32.0% or more, 34.0% or more, 36.0% or more, 38.0% or more, 40.0% or more, 42.0% or more, 44.0% or more, 46.0% or more, 48.0% or more, 50.0% or more, 51.0% or more, 52.0% or more, 53.0% or more, 54.0% or more, 55.0% or more, 56.0% or more, 57.0% or more, 58.0% or more, 59.0% or more, 60.0% or more, 61.0% or more, 62.0% or more, 63.0% or more, 64.0% or more, 65.0% or more, 66.0% or more, 67.0% or more, 68.0% or more, 69.0% or more, 70.0% or more, 71.0% or more, 72.0% or more, 73.0% or more, 74.0% or more, 75.0% or more, 76.0% or more, 77.0% or more, 78.0% or more, 79.0% or more, 80.0% or more, 81.0% or more, %, 52.0% or more, 54.0% or more, 56.0% or more, 58.0% or more, 60.0% or more, 62.0% or more, 64.0% or more, 66.0% or more, 68.0% or more, 70.0% or more, 72.0% or more, 74.0% or more, 76.0% or more, 78.0% or more, or 80.0% or more, and the upper limit is not particularly limited, but may be 100.0% or less, 98.0% or less, 96.0% or less, 94.0% or less, 92.0% or less, or 90.0% or less. Specifically, it may be, for example, 10.0% to 100.0%, 11.0% to 98.0%, 12.0% to 96.0%, 13.0% to 94.0%, 14.0% to 92.0%, or 15.0% to 90.0%.

[0032] The molar conversion rate of daidzeins to equol may be less than 10.0% at 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, or 10 hours after the start of culture. The molar conversion rate of daidzeins to equol at 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, or 10 hours from the start of culture may be 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, or 1.0% or less. The molar conversion rate of daidzeins to equol at 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, or 10 hours from the start of culture may be 0%.

[0033] The molar conversion efficiency of genisteins to 5-hydroxyequol may be 0.200 (% / h) or more. Furthermore, "the molar conversion efficiency of genisteins to 5-hydroxyequol is 0.200 (% / h) or more" may mean initiating the cultivation of a microorganism capable of producing equol from daidzein and 5-hydroxyequol from genistein in the presence of daidzeins and genisteins, and culturing the microorganism until the molar conversion efficiency of genisteins to 5-hydroxyequol reaches at least 0.200 (% / h). The molar conversion efficiency is the value (unit: % / h) obtained by dividing the molar conversion rate (%) by the time required for cultivation (unit: "h", also referred to as cultivation time).

[0034] The molar conversion efficiency of genisteins to 5-hydroxyequol was 0.120 (% / h) or more, 0.130 (% / h) or more, 0.140 (% / h) or more, 0.150 (% / h) or more, 0.160 (% / h) or more, 0.170 (% / h) or more, 0.180 (% / h) or more, 0.190 (% / h) or more, 0.200 (% / h) or more, 0.210 (% / h) or more, 0.220 (% / h) or more, 0.230 (% / h) or more, 0.250 ( % / h) or more, 0.300 (% / h) or more, 0.350 (% / h) or more, 0.400 (% / h) or more, 0.450 (% / h) or more, 0.500 (% / h) or more, 0.550 (% / h) or more, 0.600 (% / h) or more Above, 0.650 (% / h) or more, 0.700 (% / h) or more, 0.750 (% / h) or more, 0.800 (% / h) or more, 0.850 (% / h) or more, 0.900 (% / h) or more, 0.950 (% / h) or more, 1.00 0 (% / h) or more, 1.050 (% / h) or more, 1.100 (% / h) or more, 1.150 (% / h) or more, 1.200 (% / h) or more, 1.250 (% / h) or more, 1.300 (% / h) or more, 1.350 (% / h) ) or more, 1.400 (% / h) or more, 1.450 (% / h) or more, 1.500 (% / h) or more, 1.550 (% / h) or more, 1.600 (% / h) or more, 1.650 (% / h) or more, 1.700 (% / h) or more, 1. It may be 750 (% / h) or more, 1.800 (% / h) or more, 1.850 (% / h) or more, 1.900 (% / h) or more, 1.950 (% / h) or more, or 2.000 (% / h) or more, and the upper limit is not particularly limited, but may be 10.000 (% / h) or less, 9.000 (% / h) or less, 8.000 (% / h) or less, 7.000 (% / h) or less, 6.000 (% / h) or less, or 5.000 (% / h) or less. Specifically, it may be, for example, 0.120 (% / h) to 10.000 (% / h), 0.130 (% / h) to 9.000 (% / h), 0.140 (% / h) to 8.000 (% / h), 0.150 (% / h) to 7.000 (% / h), 0.160 (% / h) to 6.000 (% / h), or 0.170 (% / h) to 5.000 (% / h).

[0035] The molar conversion efficiency of daidzeins to equol may be 0.200 (% / h) or more. Alternatively, "the molar conversion efficiency of daidzeins to equol is 0.200 (% / h) or more" may mean initiating the cultivation of a microorganism capable of producing equol from daidzein and 5-hydroxyequol from genistein in the presence of daidzeins and genisteins, and culturing the microorganism until the molar conversion efficiency of daidzeins to equol reaches at least 0.200 (% / h).The molar conversion efficiency of daidzein to equol is 0.120 (% / h) or more, 0.130 (% / h) or more, 0.140 (% / h) or more, 0.150 (% / h) or more, 0.160 (% / h) or more, 0.170 (% / h) or more, 0.180 (% / h) or more, 0.190 (% / h) or more, 0.200 (% / h) or more, 0.210 (% / h) or more, 0.220 (% / h) or more, 0.230 (% / h) or more, 0.250 (% / h) or more. , 0.300 (% / h) or more, 0.350 (% / h) or more, 0.400 (% / h) or more, 0.450 (% / h) or more, 0.500 (% / h) or more, 0.550 (% / h) or more, 0.600 (% / h) or more, 0.6 50 (% / h) or more, 0.700 (% / h) or more, 0.750 (% / h) or more, 0.800 (% / h) or more, 0.850 (% / h) or more, 0.900 (% / h) or more, 0.950 (% / h) or more, 1.000 (% / h) or more h) or more, 1.050 (% / h) or more, 1.100 (% / h) or more, 1.150 (% / h) or more, 1.200 (% / h) or more, 1.250 (% / h) or more, 1.300 (% / h) or more, 1.350 (% / h) or more , 1.400 (% / h) or more, 1.450 (% / h) or more, 1.500 (% / h) or more, 1.550 (% / h) or more, 1.600 (% / h) or more, 1.650 (% / h) or more, 1.700 (% / h) or more, 1.75 It may be 0 (% / h) or more, 1.800 (% / h) or more, 1.850 (% / h) or more, 1.900 (% / h) or more, 1.950 (% / h) or more, or 2.000 (% / h) or more, and the upper limit is not particularly limited, but may be 10.000 (% / h) or less, 9.000 (% / h) or less, 8.000 (% / h) or less, 7.000 (% / h) or less, 6.000 (% / h) or less, or 5.000 (% / h) or less. Specifically, it may be, for example, 0.120 (% / h) to 10.000 (% / h), 0.130 (% / h) to 9.000 (% / h), 0.140 (% / h) to 8.000 (% / h), 0.150 (% / h) to 7.000 (% / h), 0.160 (% / h) to 6.000 (% / h), or 0.170 (% / h) to 5.000 (% / h).

[0036] The molar conversion efficiency of genisteins to 5-hydroxyequol is 0.120 (% / h) or more, 0.130 (% / h) or more, 0.140 (% / h) or more, 0.150 (% / h) or more, 0.160 (% / h) or more, 0.170 (% / h) or more, 0.180 (% / h) or more, 0.190 (% / h) or more, 0.200 (% / h) or more, 0.210 (% / h) or more, 0.220 (% / h) or more, 0.230 (% / h) or more, 0.250 (% / h) or more, 0.300 (% / h) or more, 0.350 (% / h) or more, 0.400 (% / h) or more, 0.450 (% / h) or more / h) or more, 0.500 (% / h) or more, 0.550 (% / h) or more, 0.600 (% / h) or more, 0.650 (% / h) or more, 0.700 (% / h) or more, 0.750 (% / h) or more, 0.800 (% / h) or more, 0.850 (% / h) or more, 0.900 (% / h) or more, 0.950 (% / h) or more, 1.000 (% / h) or more, 1.050 (% / h) or more, 1.100 (% / h) or more, 1.150 (% / h) or more, 1.200 (% / h) or more, 1.250 (% / h) or more, 1.300 (% / h) or more, 1.350 (% / h) or more, 1.400 (% / h) or more, 1.450 (% / h) or more, 1.500 (% / h) or more, 1.550 (% / h) or more, 1.600 (% / h) or more, 1.650 (% / h) or more, 1.700 (% / h) or more, 1.750 (% / h) or more, 1.800 (% / h) or more, 1.850 (% / h) or more, 1.900 (% / h) or more, 1.950 (% / h) or more, or 2.000 (% / h) or more, the molar conversion efficiency of daidzeins to equol is 0.120 (% / h) or more, 0.130 (% / h) or more, 0.140 (% / h) or more, 0.150 (% / h) or more, 0.160 (% / h) or more, 0.170 (% / h) or more, 0.180 (% / h) or more, 0.190 (% / h) or more, 0.200 (% / h) or more, 0.210 (% / h) or more, 0.220 (% / h) or more, 0.230 (% / h) or more, 0.250 (% / h) or more, 0.300 ( % / h) or more, 0.350 (% / h) or more, 0.400 (% / h) or more, 0.450 (% / h) or more, 0.500 (% / h) or more, 0.550 (% / h) or more, 0.600 (% / h) or more, 0.650 (% / h) or more, 0.700 (% / h) or more, 0.750 (% / h) or more,0.800 (% / h) or more, 0.850 (% / h) or more, 0.900 (% / h) or more, 0.950 (% / h) or more, 1.000 (% / h) or more, 1.050 (% / h) or more, 1.100 (% / h) or more, 1.150 (% / h) or more, 1 .200 (% / h) or more, 1.250 (% / h) or more, 1.300 (% / h) or more, 1.350 (% / h) or more, 1.400 (% / h) or more, 1.450 (% / h) or more, 1.500 (% / h) or more, 1.550 (% / h) or more, 1.6 The RH may be 1.00 (% / h) or more, 1.650 (% / h) or more, 1.700 (% / h) or more, 1.750 (% / h) or more, 1.800 (% / h) or more, 1.850 (% / h) or more, 1.900 (% / h) or more, 1.950 (% / h) or more, or 2.000 (% / h), and may be 10.000 (% / h) or less, 9.000 (% / h) or less, 8.000 (% / h) or less, 7.000 (% / h) or less, 6.000 (% / h) or less, or 5.000 (% / h) or less. Specifically, it may be, for example, 0.120 (% / h) to 10.000 (% / h), 0.130 (% / h) to 9.000 (% / h), 0.140 (% / h) to 8.000 (% / h), 0.150 (% / h) to 7.000 (% / h), 0.160 (% / h) to 6.000 (% / h), or 0.170 (% / h) to 5.000 (% / h).

[0037] The molar concentration ratio of genisteins to daidzeins at the start of culture (or fermentation), i.e., the value obtained by dividing the "molar concentration of genisteins" by the "molar concentration of daidzeins," may be 1.000 or more, 1.100 or more, 1.200 or more, 1.300 or more, 1.400 or more, 1.500 or more, 1.600 or more, 1.700 or more, 1.800 or more, 1.900 or more, 2.000 or more, 3.000 or more, 4.000 or more, or 5.000 or more, or may be 1,000,000 or less, 500,000 or less, 100,000 or less, 50,000 or less, 40,000 or less, 30,000 or less, 20,000 or less, 15,000 or less, 10,000 or less, 7,000 or less, or 5,000 or less. Specifically, it may be, for example, 1.000 to 1000.000, 1.100 to 500.000, 1.200 to 100.00, 1.300 to 100.00, 1.400 to 50.000, 1.500 to 40.000, 1.600 to 30.000, 1.700 to 20.000, 1.800 to 15.000, 1.900 to 10.000, 2.000 to 7.000, or 3.000 to 5.000. Or, 0.900 or less, 0.800 or less, 0.700 or less, 0.600 or less, 0.500 or less, 0.400 or less, 0.350 or less, 0.300 or less, 0.250 or less, 0.200 or less, 0.180 or less, 0.170 or less, 0.160 or less, 0.150 or less, 0.140 or less, 0.130 or less, 0.120 or less, 0.110 or less, 0.100 or less, 0 It may be 0.090 or less, 0.080 or less, 0.070 or less, 0.060 or less, 0.050 or less, 0.040 or less, 0.030 or less, 0.020 or less, 0.010 or less, 0.006 or less, 0.005 or less, 0.004 or less, or 0.003 or less, or 0.0001 or more, 0.0005 or more, 0.001 or more, or 0.002 or more. Specifically, it may be, for example, 0.0001 to 0.900, 0.0005 to 0.800, 0.001 to 0.700, or 0.002 to 0.600.

[0038] The total content of daidzeins and genisteins in the composition containing daidzeins and genisteins at the start of culturing (or fermentation) is not particularly limited. For example, it may be 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.50 mM or more, 1.00 mM or more, 1.50 mM or more, 2.00 mM or more, 2.50 mM or more, 3.00 mM or more, 3.50 mM or more, 4.00 mM or more, 4.50 mM or more, 5.00 mM or more, 5.50 mM or more, 6.00 mM or more, 6.50 mM or more, 7.00 mM or more, 7.50 mM or more, 8.0 ... . 50 mM or more, 9.00 mM or more, 9.50 mM or more, 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, or 20.00 mM or less. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, or 2.50 mM to 20.00 mM.

[0039] The content of daidzein in the composition containing daidzeins and genisteins at the time of starting the culture (or fermentation) is not particularly limited. For example, the content of daidzein may be 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.50 mM or more, 1.00 mM or more, 1.50 mM or more, 2.00 mM or more, 2.50 mM or more, 3.00 mM or more, 3.50 mM or more, 4.00 mM or more, 4.50 mM or more, 5.00 mM or more, 5.50 mM or more, 6.00 mM or more, 6.50 mM or more, 7.00 mM or more, 7.50 mM or more, 8.0 ... . 50 mM or more, 9.00 mM or more, 9.50 mM or more, 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, or 20.00 mM or less. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, or 2.50 mM to 20.00 mM.

[0040] The content of genistein in the composition containing daidzeins and genisteins at the time of starting the culture (or fermentation) is not particularly limited. For example, it may be 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.50 mM or more, 1.00 mM or more, 1.50 mM or more, 2.00 mM or more, 2.50 mM or more, 3.00 mM or more, 3.50 mM or more, 4.00 mM or more, 4.50 mM or more, 5.00 mM or more, 5.50 mM or more, 6.00 mM or more, 6.50 mM or more, 7.00 mM or more, 7.50 mM or more, 8.00 mM or more, 8.50 mM or more, 9.00 mM or more, 10.00 mM or more, 11.00 mM or more, 12.00 mM or more, 13.00 mM or more, 14.00 mM or more, 15.00 mM or more, 16.00 mM or more, 17.00 mM or more, 18.00 mM or more, 19.00 mM or more, 20.00 mM or more, 21.00 mM or more, 22.00 mM or more, 23.00 mM or more, 24.00 mM or more, 25.00 mM or more, 26.00 mM or more, 27.00 mM or more, 28.00 mM or more, 29.00 mM or more, 30.00 mM or more, 31.00 mM or more, 32.00 mM or more, 33.00 mM or more, 34.00 mM or more, 35.00 mM or more, 36.00 . 50 mM or more, 9.00 mM or more, 9.50 mM or more, 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, 20.00 mM or less, 10.00 mM or less, 5.00 mM or less, or 1.00 mM or less. The lower limit and upper limit may be any combination thereof that does not contradict each other. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, 2.50 mM to 20.00 mM, 3.00 mM to 10.00 mM, or 3.50 mM to 5.00 mM.

[0041] The total content of equol and 5-hydroxyequol in the culture (or fermentation) at the end of the culture (or fermentation) is not particularly limited. For example, it may be 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.50 mM or more, 1.00 mM or more, 1.50 mM or more, 2.00 mM or more, 2.50 mM or more, 3.00 mM or more, 3.50 mM or more, 4.00 mM or more, 4.50 mM or more, 5.00 mM or more, 5.50 mM or more, 6.00 mM or more, 6.50 mM or more, 7.00 mM or more, 7.50 mM or more, 8.00 mM or more, 8.50 mM or more, 9.00 mM or more, 10.00 mM or more, 11.00 mM or more, 12.00 mM or more, 13.00 mM or more, 14.00 mM or more, 15.00 mM or more, 16.00 mM or more, 17.00 mM or more, 18.00 mM or more, 19.00 mM or more, 20.00 mM or more, 21.00 mM or more, 22.00 mM or more, 23.00 mM or more, 24.00 mM or more, 25.00 mM or more, 26.00 mM or more, 27.00 mM or more, 28.00 mM or more, 29.00 mM or more, 30.00 mM or more, 31.00 mM or more, 32.00 mM or more, 33.00 mM or more, 34.00 mM or more, 35.00 mM or more, 36.0 . 50 mM or more, 9.00 mM or more, 9.50 mM or more, 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, or 20.00 mM or less. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, or 2.50 mM to 20.00 mM.

[0042] The equol content in the culture (or fermented product) at the end of the culture (or fermentation) is not particularly limited, and may be, for example, 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.50 mM or more, 1.00 mM or more, 1.50 mM or more, 2.00 mM or more, 2.50 mM or more, 3.00 mM or more, 3.50 mM or more, 4.00 mM or more, 4.50 mM or more, 5.00 mM or more, 5.50 mM or more, 6.00 mM or more, 6.50 mM or more, 7.00 mM or more, 7.50 mM or more, 8.00 mM or more, 8.50 mM or more, 9.00 mM or more, 10.00 mM or more, 11.00 mM or more, 12.00 mM or more, 13.00 mM or more, 14.00 mM or more, 15.00 mM or more, 16.00 mM or more, 17.00 mM or more, 18.00 mM or more, 19.00 mM or more, 20.00 mM or more, 21.00 mM or more, 22.00 mM or more, 23.00 mM or more, 24.00 mM or more, 25.00 mM or more, 26.00 mM or more, 27.00 mM or more, 28.00 mM or more, 29.00 mM or more, 30.00 mM or more, 31.00 mM or more, 32.00 mM or more, 33.00 mM or more, 34.00 mM or more, 35.00 mM or more, 36.00 mM or more, 3 . 50 mM or more, 9.00 mM or more, 9.50 mM or more, 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, or 20.00 mM or less. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, or 2.50 mM to 20.00 mM.

[0043] The content of 5-hydroxyequol in the culture (or fermentation) at the end of the culture (or fermentation) is not particularly limited. For example, it may be 0.01 mM or more, 0.02 mM or more, 0.05 mM or more, 0.10 mM or more, 0.50 mM or more, 1.00 mM or more, 1.50 mM or more, 2.00 mM or more, 2.50 mM or more, 3.00 mM or more, 3.50 mM or more, 4.00 mM or more, 4.50 mM or more, 5.00 mM or more, 5.50 mM or more, 6.00 mM or more, 6.50 mM or more, 7.00 mM or more, 7.50 mM or more, 8.00 mM or more, 8.50 mM or more, 9.00 mM or more, 10.00 mM or more, 11.00 mM or more, 12.00 mM or more, 13.00 mM or more, 14.00 mM or more, 15.00 mM or more, 16.00 mM or more, 17.00 mM or more, 18.00 mM or more, 19.00 mM or more, 20.00 mM or more, 21.00 mM or more, 22.00 mM or more, 23.00 mM or more, 24.00 mM or more, 25.00 mM or more, 26.00 mM or more, 27.00 mM or more, 28.00 mM or more, 29.00 mM or more, 30.00 mM or more, 31.00 mM or more, 32.00 mM or more, 33.00 mM or more, 34.00 mM or more, 35.00 mM or more, 36.00 mM or more, . 50 mM or more, 9.00 mM or more, 9.50 mM or more, 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, 20.00 mM or less, or 10.00 mM or less. The lower limit and upper limit may be any compatible combination thereof. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, 2.50 mM to 20.00 mM, or 3.00 mM to 10.00 mM.

[0044] By fermenting daidzeins and genisteins with a microorganism capable of producing equol from daidzeins and 5-hydroxyequol from genisteins, a fermented product containing equol and 5-hydroxyequol at any desired value and ratio per gram of dry weight can be obtained.

[0045] The values ​​of equol and 5-hydroxyequol per 1 g of dry weight in the obtained fermented product are, for example, as follows: Equol: 0.1 mg / g or more, 0.2 mg / g or more, 0.3 mg / g or more, 0.4 mg / g or more, 0.5 mg / g or more, 0.6 mg / g or more, 0.7 mg / g or more, 0.8 mg / g or more, 0.9 mg / g or more, 1.0 mg / g or more, 1.1 mg / g or more, 1.2 mg / g or more, 1.3 mg / g or more, 1.4 mg / g or more, 1.5 mg / g or more, 1.6 mg / g or more, 1.7 mg / g or more, 1.8 mg / g or more, 1.9 mg / g or more, 2.0 mg / g or more, 2.1 mg / g or more, 2.2 mg / g or more, 2.3 mg / g or more, 2.4 mg / g or more, 2.5 mg / g or more, 2.6 mg / g or more, 2.7 mg / g or more mg / g or more, 2.8 mg / g or more, 2.9 mg / g or more, 3.0 mg / g or more, 3.5 mg / g or more, 4.0 mg / g or more, 4.5 mg / g or more, 5.0 mg / g or more, 6.0 mg / g or more, 7.0 mg / g or more, 8.0 mg / g or more, 9.0 mg / g or more, 10.0 mg / g or more, 11.0 mg / g or more, 12.0 mg / g or more, 13.0 mg / g or more, 14.0 mg / g or more, 15.0 mg / g or more, 16.0 mg / g or more, 17.0 mg / g or more, 18.0 mg / g or more, 19.0 mg / g or more, 20.0 mg / g or more, 25.0 mg / g or more, or 30.0 mg / g or more. The upper limit is not particularly limited, and may be, for example, 500.0 mg / g or less, 400.0 mg / g or less, 300.0 mg / g or less, 200.0 mg / g or less, 100.0 mg / g or less, or 50.0 mg / g or less. Specifically, it may be, for example, 0.1 mg / g to 500.0 mg / g, 0.2 mg / g to 400.0 mg / g, 0.3 mg / g to 300.0 mg / g, 0.4 mg / g to 200.0 mg / g, 0.5 mg / g to 100.0 mg / g, or 0.6 mg / g to 50.0 mg / g.5-Hydroxyequol: 0.1 mg / g or more, 0.2 mg / g or more, 0.3 mg / g or more, 0.4 mg / g or more, 0.5 mg / g or more, 0.6 mg / g or more, 0.7mg / g or more, 0.8mg / g or more, 0.9mg / g or more, 1.0mg / g or more, 1.1mg / g or more, 1.2mg / g or more, 1.3 mg / g or more, 1.4 mg / g or more, 1.5 mg / g or more, 1.6 mg / g or more, 1.7 mg / g or more, 1.8 mg / g or more, 1.9 mg / g or more, 2 .0 mg / g or more, 2.1 mg / g or more, 2.2 mg / g or more, 2.3 mg / g or more, 2.4 mg / g or more, 2.5 mg / g or more, 2.6 mg / g or more. It may be 2.7 mg / g or more, 2.8 mg / g or more, 2.9 mg / g or more, 3.0 mg / g or more, 3.5 mg / g or more, 4.0 mg / g or more, 4.5 mg / g or more, 5.0 mg / g or more, 6.0 mg / g or more, 7.0 mg / g or more, 8.0 mg / g or more, 9.0 mg / g or more, 10.0 mg / g or more, 11.0 mg / g or more, 12.0 mg / g or more, 13.0 mg / g or more, 14.0 mg / g or more, 15.0 mg / g or more, 16.0 mg / g or more, 17.0 mg / g or more, 18.0 mg / g or more, 19.0 mg / g or more, 20.0 mg / g or more, 25.0 mg / g or more, or 30.0 mg / g or more. The upper limit is not particularly limited, and may be, for example, 500.0 mg / g or less, 400.0 mg / g or less, 300.0 mg / g or less, 200.0 mg / g or less, 100.0 mg / g or less, or 50.0 mg / g or less. Specifically, it may be, for example, 0.1 mg / g to 500.0 mg / g, 0.2 mg / g to 400.0 mg / g, 0.3 mg / g to 300.0 mg / g, 0.4 mg / g to 200.0 mg / g, 0.5 mg / g to 100.0 mg / g, or 0.6 mg / g to 50.0 mg / g.

[0046] The ratio of the 5-hydroxyequol content per 1 g of dry weight to the equol content per 1 g of dry weight in the resulting fermented product, i.e., the ratio of 5-hydroxyequol (mg / g) divided by equol (mg / g), may be, for example, 0.001 or more, 0.005 or more, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.10 or more, 0.20 or more, 0.30 or more, 0.40 or more, 0.50 or more, 0.60 or more, 0.70 or more, 0.80 or more, 0.90 or more, 1.00 or more, 1.50 or more, 2.00 or more, 3.00 or more, 4.00 or more, or 5.00 or more. The upper limit is not particularly limited, but may be, for example, 30.00 or less, 20.00 or less, 10.00 or less, 9.00 or less, or 8.00 or less. Specifically, it may be, for example, 0.001 to 30.00, 0.005 to 20.00, 0.01 to 10.00, 0.02 to 9.00, or 0.03 to 8.00.

[0047] The ratio of the molar concentration of 5-hydroxyequol to the molar concentration of equol in the resulting fermented product, i.e., the ratio of 5-hydroxyequol (mmol / L) divided by equol (mmol / L), may be, for example, 0.001 or more, 0.005 or more, 0.010 or more, 0.020 or more, 0.030 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.10 or more, 0.20 or more, 0.30 or more, 0.40 or more, 0.50 or more, 0.60 or more, 0.70 or more, 0.80 or more, 0.90 or more, 1.00 or more, 1.50 or more, 2.00 or more, 3.00 or more, 4.00 or more, or 5.00 or more. The upper limit is not particularly limited, but may be, for example, 30.00 or less, 20.00 or less, 10.00 or less, 9.00 or less, or 8.00 or less. Specifically, it may be, for example, 0.001 to 30.00, 0.005 to 20.00, 0.01 to 10.00, 0.02 to 9.00, or 0.03 to 8.00.

[0048] The production method may include a step of quantifying the obtained equol and 5-hydroxyequol. The quantification method can be a conventional method. For example, a portion of the culture medium is sampled and appropriately diluted, dissolved in an organic solvent, or extracted with an organic solvent, thoroughly stirred, and then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, followed by quantification by high-performance liquid chromatography.

[0049] The production method may include a step of purifying and a step of concentrating the obtained equol and 5-hydroxyequol. Examples of purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), or the like; removal of solids and polymeric substances; extraction with organic solvents or ionic liquids; and adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, or the like. Concentration treatments in the concentration step include concentration using an evaporator or reverse osmosis membrane. Furthermore, solutions containing equol and 5-hydroxyequol can be powdered by freeze-drying, spray-drying, or the like. For powderization, excipients such as lactose, dextrin, and cornstarch can also be added. The obtained equol and 5-hydroxyequol can be used in cosmetics, quasi-drugs, medical supplies, hygiene products, pharmaceuticals, foods and beverages (including supplements), and the like, and the known effects of equol and 5-hydroxyequol can be easily obtained by consumption or ingestion by subjects, including humans.

[0050] All prior art documents cited herein are hereby incorporated by reference.

[0051] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to these examples.

[0052] In each example, the amount of isoflavones was determined by the following method.

[0053] (Quantitative Determination of Isoflavone Concentration) 2 g of the culture solution is added with 70% aqueous ethanol to make a final volume of 50 mL, solubilized by ultrasonication, and then filtered through a 0.45 μm filter, and the filtrate is analyzed by HPLC.

[0054] (Calculation method for isoflavone concentration) The concentration of daidzin is determined from a one-point calibration curve using daidzin (Fujifilm Wako Pure Chemical Industries, Ltd., product number 046-27743) as the standard. The concentrations of other isoflavones are calculated using the quantitative coefficients for daidzin described in WO2017 / 170995: malonyldaidzin (1.444), malonylglycitin (1.351), malonylgenistin (1.095), acetyldaidzin (1.094), acetylglycitin (1.197), and acetylgenistin (1.064), as well as the quantitative coefficients obtained from Daicel Corporation: glycitin (1.199), genistin (0.840), daidzin (0.665), glycitein (0.789), dihydrodaidzein (3.432), and dihydrogenistein (8.364). The concentrations of isoflavone aglycones and metabolites were calculated from a single-point calibration curve using daidzein (manufactured by LC Laboratories, product number D-2946), glycitein (manufactured by Fujifilm Wako Pure Chemical Industries, product number 076-04703), genistein (manufactured by Fujifilm Wako Pure Chemical Industries, product number 073-05531), dihydrodaidzein (manufactured by Toronto Research Chemicals, product number D449000), dihydrogenistein (synthesized by Nagara Science), equol (manufactured by Daicel, product number 100152-001G), and 5-hydroxyequol (manufactured by Toronto Research Chemicals, product number H825750) as standards.

[0055] (HPLC analysis of isoflavones) The following method is used based on the Ministry of Health, Labor and Welfare Notification (Shokuan-hatsu No. 0823001). Column: YMC-Pack ODS-AM, inner diameter 4.6 mm, length 250 mm (YMC Co., Ltd.) Column temperature: 25°C Mobile phase: (Solution A) acetonitrile / water / acetic acid = 15 / 85 / 0.1 (v / v / v) (Solution B) acetonitrile / water / acetic acid = 35 / 65 / 0.1 (v / v / v) Gradient conditions: Solution A Solution B 0 min 100 0 50 min 0 100 55 min 0 100 56 min 100 0 65 min 100 0 Flow rate: 1.0 mL / min Detector: UV spectrophotometer (254 nm) Injection volume: 10 μL Equipment used: HPLC (Shimadzu Corporation)

[0056] (Preculture) ABB medium (Anaerobe Basal Broth: manufactured by Thermo Fisher Scientific) was used as the preculture medium, and the strains shown in Table 2 were inoculated. The gas phase was replaced with hydrogen gas passed through a sterile filter, and then the preculture was carried out at 37°C and 200 spm for 24 hours with shaking to obtain a preculture solution. The SI strain was cultured in the same manner except that Wilkins-Challgreen (abbreviated as WC) medium was used.

[0057] (Main Culture) (Examples 1-3) 0.2 g / L of pectinase G Amano (Amano Enzyme) was added to 100 g / L of soybean hypocotyls (containing isoflavone glycosides such as daidzin, glycitin, and genistin), and the mixture was stirred overnight at 50°C to liberate sugars from the isoflavone glycosides and convert them into aglycones. To this mixture, yeast extract (containing arginine) was added as a nutrient, and β-cyclodextrin (β-CD) was added as an additive. The pH at the start of main culture was adjusted to 6.50 to 7.10, resulting in the medium composition at the start of main culture shown in Table 1.

[0058] (Comparative Examples 1-3, Examples 4-12) Daidzein, genistein, and ABB medium and arginine as nutrients, and β-CD as an additive as needed, were added, and the pH at the start of main culture was adjusted to 6.50 to 7.10, to give the medium composition at the start of main culture shown in Table 1.

[0059] Example 13: 0.2 g / L of pectinase G Amano (Amano Enzyme) was added to 100 g / L of soybean hypocotyls (containing isoflavone glycosides such as daidzin, glycitin, and genistin), and the mixture was incubated overnight at 50°C with stirring to liberate sugars from the isoflavone glycosides and convert them into aglycones. Arginine was added as a nutrient, and the pH was adjusted to 6.50 to 7.10 at the start of main culture, providing the medium composition at the start of main culture shown in Table 1.

[0060] The main culture medium thus prepared in Examples 1-13 and Comparative Examples 1-3 was dispensed in 15 mL aliquots into 100 mL vials, sealed with butyl rubber stoppers, and purged with nitrogen gas. The vials were then autoclaved at 121°C for 15 minutes. The preculture solution was inoculated into the main culture medium (inoculation rate 1%), and the gas phase was purged with hydrogen gas passed through a sterile filter. Shaking culture was then carried out at 37°C and 250 rpm for the culture time listed in Table 1. After culture, isoflavones were analyzed.

[0061] (Examples 14-15) Daidzein, genistein, and nutrients such as ABB medium yeast extract and arginine, as well as β-CD as an additive if necessary, were added to the medium. The pH at the start of the main culture was adjusted to 6.50 to 7.10, and the medium composition at the start of the main culture shown in Table 1 was obtained. Each of the main culture media prepared in this manner was dispensed in 1 L aliquots into 2 L mini jars. After gas substitution with nitrogen gas, the jars were autoclaved at 121°C for 15 minutes. The preculture solution was inoculated into the main culture medium (inoculation rate 1%), and agitated culture was carried out at 37°C and 500 rpm while aerating anaerobic gas through a sterile filter for the culture time listed in Table 1. After the culture, isoflavones were analyzed. The results are shown in Table 1. Table 2 lists the details of the strains used and their abbreviations.

[0062]

[0063]

[0064] Using these techniques, fermentation of daidzein and genistein compounds with microorganisms capable of producing equol from daidzeins and 5-hydroxyequol from genisteins can produce fermented products containing equol and 5-hydroxyequol at any desired values ​​and ratios per gram of dry weight, as shown in Table 3. Since the activity of daidzein reductase (DZNR), an enzyme involved in equol production, is known to be inhibited by equol ( Applied and Environmental Microbiology, 2016, Vol. 82, No. 7, 1992-2002 ), it is speculated that a similar inhibition occurs with 5-hydroxyequol. Therefore, although the reason is unclear, it is speculated that the conversion of genistein compounds to 5-hydroxyequol is likely to be inhibited when daidzeins and genisteins are present at approximately equimolar concentrations. It is presumed that as the daidzeins and genisteins move away from the vicinity of equimolar concentrations, the environment becomes more conducive to conversion of genisteins to 5-hydroxyequol.

[0065]

[0066] According to the present disclosure, it is possible to efficiently produce equol and 5-hydroxyequol, and the resulting equol and 5-hydroxyequol can be used in cosmetics, quasi-drugs, medical supplies, hygiene products, pharmaceuticals, food and beverages (including supplements), etc., and by using or ingesting them, subjects including humans can easily obtain the known effects of equol and 5-hydroxyequol.

Claims

1. A method for producing equol and 5-hydroxyequol using a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins, wherein daidzeins and genisteins are present at the start of culturing the microorganism, the molar conversion efficiency of the genisteins to 5-hydroxyequol is 0.200% / h or higher, the molar concentration ratio of the genisteins to the daidzeins at the start of culturing is 1.500 or higher or 0.500 or lower, the daidzeins are daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins are genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof.

2. The production method according to claim 1, wherein the molar conversion efficiency of the genisteins to 5-hydroxyequol is 0.250% / h or more.

3. The method according to claim 1 or 2, wherein the molar concentration ratio of the genisteins to the daidzeins is 1.800 or more or 0.400 or less.

4. The method of claim 1 or 2, wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to the genus Adrecrauzia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Slackia, and microorganisms belonging to the genus Lactococcus.

5. The method according to claim 4, wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to Adrecrautia aequorifaciens, microorganisms belonging to Slacchia aequorifaciens, and microorganisms belonging to Lactococcus garvieae.

6. The method according to claim 1 or 2, wherein β-cyclodextrin is further present at the start of culturing the microorganism.

7. The method of claim 1 or 2, wherein arginine is also present at the start of culturing the microorganism.

8. A method for producing a fermented product containing equol and 5-hydroxyequol by culturing a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins, wherein daidzeins and genisteins are present at the start of culturing the microorganism, the molar concentration ratio of the genisteins to the daidzeins at the start of culturing is 1.500 or more or 0.500 or less, the total content of equol and 5-hydroxyequol in the fermented product is 0.5 mg / g or more and 100.0 mg / g or less per g of dry weight, the daidzeins are daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins are genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof.

9. The method according to claim 8, wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to the genus Adrecrauzia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Slackia, and microorganisms belonging to the genus Lactococcus.

10. The method according to claim 9, wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to Adrecrautia aequorifaciens, microorganisms belonging to Slacchia aequorifaciens, and microorganisms belonging to Lactococcus garvieae.

11. The method of claim 8 or 9, wherein β-cyclodextrin is also present at the start of culturing the microorganism.

12. The method of claim 8 or 9, wherein arginine is also present at the start of culturing the microorganism.

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