Method for producing equol and 5-hydroxyequol, and fermented product

Abstract

This method for producing equol and 5-hydroxyequol uses a microorganism capable of producing the equol from a daidzein and producing the 5-hydroxyequol from a genistein. The daidzein and the genistein are present at the start of culturing the microorganism. The molar transformation efficiency from the genistein into the 5-hydroxyequol is 0.150% / h or more. The molar concentration ratio of the genistein with respect to the daidzein at the start of the culturing is not less than 1.300 or not more than 0.700. The daidzein is daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof. The genistein is genistein, malonyldaidzin, acetyldaidzin, genistein, dihydrogenistein, or a combination thereof.

Description

Method for producing equol and 5-hydroxyequol, and fermented products

[0001] The present disclosure relates to methods for producing equol and 5-hydroxyequol, and fermented products containing 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, isoflavones contained in soybeans mainly include glycosides condensed with sugars, as well as isoflavone aglycones (hereinafter also referred to as aglycones) that are not condensed with sugars. Examples of glycoside-form isoflavones include daidzin, glycitin, and genistin. When these glycosides enter the human or animal body, they are converted into the aglycones daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or enzymes produced by intestinal bacteria (such as β-glucosidase). Of these, 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 object of the present disclosure is to provide at least a method for producing equol and 5-hydroxyequol, and a fermented product containing 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.

[13] 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 0.150% / h or more, the molar concentration ratio of the genisteins to the daidzeins at the start of culturing is 1.300 or more or 0.700 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.

[14] 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 1.300 or more or 0.700 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.

[15] A method for producing an oral composition, comprising the step of incorporating equol and 5-hydroxyequol into a base material, wherein the equol and 5-hydroxyequol are produced by the method described in any one of [1] to

[14] .

[16] A method for producing a composition for a jelly drink, comprising the step of incorporating equol, 5-hydroxyequol, collagen peptide, and agar, wherein the equol and 5-hydroxyequol are produced by the method described in any one of [1] to

[14] .

[17] A method for producing a composition for a jelly drink according to

[16] , wherein the equol is 0.01% to 0.05% by weight based on the total weight of the composition.

[18] A fermented product of a composition containing daidzeins and genisteins, wherein the fermented product contains equol and 5-hydroxyequol, 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 5-hydroxyequol content in the fermented product is 0.3 mg / g or more per 1 g of dry weight, the daidzeins are daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins are genistin, malonyldaidzin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof.

[19] An oral composition comprising the fermented product of

[18] and a base material.

[20] A composition for a jelly drink, comprising the fermented product of

[18] , a collagen peptide, and agar.

[21] The composition for a jelly drink according to

[20] , wherein the equol content is 0.01% by weight to 0.05% by weight based on the total weight of the composition.

[0011] The present disclosure provides at least a method for producing equol and 5-hydroxyequol, and a fermented product containing 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] 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. The microorganisms are not particularly limited as 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, the microorganisms may be introduced with genes encoding enzymes involved in equol production and 5-hydroxyequol production (e.g., daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR), etc.). The above microorganisms may also have the ability to produce ornithine from arginine. Microorganisms capable of producing equol from daidzeins and 5-hydroxyequol from genisteins may be known microorganisms or microorganisms obtained by conventional screening 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, and microorganisms belonging to the genus Collinsella. Microorganisms belonging to the genus Enterococcus, Enterorhabdus, Eubacterium, and 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, microorganisms belonging to the genus Slackia, Streptococcus Examples of suitable microorganisms include microorganisms belonging to the genus Lactococcus, microorganisms belonging to the genus Veillonella, microorganisms belonging to the genus Lactococcus, microorganisms belonging to the genus Hugonella, and microorganisms belonging to the genus Senegalmassilia, as well as microorganisms obtained by screening these microorganisms using, as an indicator, the ability to produce equol from daidzeins and / or the ability to produce 5-hydroxyequol from genisteins.Furthermore, genes encoding the enzymes involved in equol production and 5-hydroxyequol production can be obtained, for example, from microorganisms classified into the above-mentioned genera, etc. Examples of the above-mentioned microorganisms that also have the ability to produce ornithine from arginine include lactic acid bacteria such as those of the genus Lactococcus and Streptococcus, and more specifically, Lactococcus garvieae.

[0015] More specific examples of the microorganisms are as follows: Microorganisms belonging to the genus Adlercreutzia equolifaciens, such as Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain and Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain; Bacteroides ovatus E-23-15 strain (FERM Microorganisms belonging to the genus Bacteroides ovatus, such as Bifidobacterium breve ATCC 15700 strain; Microorganisms belonging to the genus Bifidobacterium breve, such as Bifidobacterium breve ATCC 15700 strain; Microorganisms belonging to the genus Bifidobacterium longum, such as Bifidobacterium longum NITE BP-02621 (ATCC BAA-999; BB536 strain); Clostridium sp. Microorganisms belonging to the genus Clostridium, such as Eggerthella sp. strain HGH136 (ATCC BAA-442); Microorganisms belonging to the genus Eggerthella, such as Eggerthella sp. Julong 732 strain (KCCM-10490), Eggerthella sp. strain YY7918 (note that the YY7918 strain is managed by the Gifu Prefectural Institute of Biotechnology, a public institution, and can be obtained from there), and Eggerthella sp. strain D1; Microorganisms belonging to the genus Enterococcus faecalis, such as the 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 the Eubacterium sp. D2 strain; - Finegoldia magna Microorganisms belonging to the genus Finegoldia magna, such as the Lactobacillus fermentum EPI3 strain; Microorganisms belonging to the genus Lactobacillus fermentum, such as the Lactobacillus fermentum DPPMA114 strain (DSM 23757); Microorganisms belonging to the genus Lactobacillus intestinalis, such as the Lactobacillus intestinalis KTCT13676BP strain; Lactobacillus mucosae 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 the genus Lactobacillus paracasei, such as the Lactobacillus plantarum DPPMA24W strain (DSM 23756) and the Lactobacillus plantarum DPPMASL33 strain (DSM 23756). Microorganisms belonging to the genus Lactobacillus plantarum, such as Lactobacillus sp. 23755;- Microorganisms belonging to the genus Lactobacillus, such as Lactobacillus rhamnosus DPPMAZ1 strain and Lactobacillus rhamnosus INIA P540 strain; - Microorganisms belonging to the genus Lactobacillus, such as Lactobacillus sp. Niu-O16 strain; - Lactococcus garvieae 20-92 strain (FERM Microorganisms belonging to the genus Lactococcus garvieae, such as Lactococcus sp. BP-10036; Microorganisms belonging to the genus Paraeggerthella, such as Paraeggerthella sp. SNR40-432 strain; 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 the genus Saccharomyces cerevisiae; Microorganisms belonging to the genus Saccharomyces cerevisiae, such as Saccharomyces cerevisiae ST18 strain (NITE P-300); Microorganisms belonging to the genus Saccharomyces cerevisiae, such as Saccharomyces cerevisiae DSM 24851 strain; Microorganisms belonging to the genus Saccharomyces cerevisiae, such as Saccharomyces cerevisiae DSM 22006 strain; Microorganisms belonging to the species Bacillus isoflavonica;- Microorganisms belonging to the genus Slackia, such as Slackia sp. FJK1 strain (NITE P-1562), Slackia sp. NATTS strain (FERM BP-11231), Slackia sp. YIT11861 strain (FERM BP-11231), Slackia sp. TM-30 strain; - Streptococcus constellatus E-23-17 strain (FERM Microorganisms belonging to the genus Streptococcus constellatus, such as Streptococcus intermedius A6G-225 strain (FERM BP-6436); Microorganisms belonging to the genus Streptococcus intermedius, such as Streptococcus intermedius A6G-225 strain (FERM BP-6437); Microorganisms belonging to the genus Veillonella, such as Veillonella sp. EP strain; Hugonella massiliensis DSM Microorganisms belonging to Hugonella massiliensis, such as strain 101782T;Microorganisms belonging to the genus Senegalmassilia faecalis, such as Senegalmassilia faecalis KGMB 04484T strain (KCTC 15721). Microorganisms also include those possessing 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 bred from strains of each microorganism or substantially equivalent strains thereof by mutation treatment, genetic recombination, selection of natural mutants, etc. may also be used. Furthermore, one or more types of microorganisms may be used. The above microorganisms can be obtained from the depository institution indicated by the deposit number. Each accession number indicates that the microorganism has been deposited at the following depository institution: FERM 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.org.uk / de / KCCM Korean Culture Center of Microorganisms KCTC Korean Collection for Type Cultures 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 of soybeans or soybean hypocotyls, 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 microorganism capable of producing equol from daidzeins and 5-hydroxyequol from genisteins may be cultured under conditions in which daidzeins and genisteins are present at the start of culture, using a composition containing daidzeins and genisteins as raw materials.

[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 may be a culture medium containing daidzeins and genisteins.

[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 when the microorganism is cultured. Furthermore, arginine may be added to the composition containing daidzeins and genisteins when the microorganism is cultured. The amount of arginine added to the composition containing daidzeins and genisteins is not particularly limited, but may be 0.30g / L or more, 0.40g / L or more, 0.50g / L or more, 0.60g / L or more, 0.70g / L or more, 0.80g / L or more, 0.90g / L or more, 1.00g / L or more, 1.10g / L or more, 1.20g / L or more, 1.30g / L or more, 1.40g / L or more, 1.50g / L or more, 1.60g / L or more, 1.70g / L or more, 1.80g / L or more, 1.90g / L or more, 2.00g / L or more, It may be 2.10 g / L or more, 2.20 g / L or more, 2.30 g / L or more, 2.40 g / L or more, 2.50 g / L or more, 2.60 g / L or more, 2.70 g / L or more, 2.80 g / L or more, 2.90 g / L or more, 3.00 g / L or more, 3.10 g / L or more, 3.20 g / L or more, 3.30 g / L or more, 3.40 g / L or more, 3.50 g / L or more, 3.60 g / L or more, 3.70 g / L or more, 3.80 g / L or more, 3.90 g / L or more, 4.00 g / L or more, 4.50 g / L or more, or 5.00 g / L or more. 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 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 amount of cyclodextrin added to a composition containing daidzeins and genisteins 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 , 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 preferable to set it to the solubility of dextrin at the culture temperature or less. 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, for example, 0.150 (% / h) or greater, or 0.200 (% / h) or greater. Furthermore, "the molar conversion efficiency of genisteins to 5-hydroxyequol is 0.150 (% / h) or greater" may mean initiating 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 0.150 (% / 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, for example, 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 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 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, 1.90 It may be 0 (% / h) or more, 1.950 (% / h) or more, 2.000 (% / h) or more, 2.050 (% / h) or more, 2.100 (% / h) or more, 2.150 (% / h) or more, or 2.200 (% / 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, 1.600 (% / h) or more, 1.650 (% / h) or more, 1.70 It may be 0 (% / 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, 2.000 (% / h) or more, 2.050 (% / h) or more, 2.100 (% / h) or more, 2.150 (% / h) or more, or 2.200 (% / h) or more, or it 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, 0.750 or less, 0.740 or less, 0.730 or less, 0.720 or less, 0.710 or less, 0.700 or less, 0.690 or less, 0.680 or less, 0.670 or less, 0.660 or less, 0.650 or less, 0.640 or less, 0.630 or less, 0.620 or less, 0.610 or less, 0.600 or less, 0.590 or less, 0. 580 or less, 0.570 or less, 0.560 or less, 0.550 or less, 0.540 or less, 0.530 or less, 0.520 or less, 0.510 or less, 0.500 or less, 0.490 or less, 0 .480 or less, 0.470 or less, 0.460 or less, 0.450 or less, 0.440 or less, 0.430 or less, 0.420 or less, 0.410 or less, 0.400 or less, 0.390 or less, 0 .. 380 or less, 0.370 or less, 0.360 or less, 0.350 or less, 0.340 or less, 0.330 or less, 0.320 or less, 0.310 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 , 0.090 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, for example, it may be 0.0001 to 0.900, 0.0005 to 0.800, 0.001 to 0.750, 0.001 to 0.700, 0.002 to 0.600, or 0.002 to 0.700.

[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, 0.60 mM or more, 0.70 mM or more, 0.80 mM or more, 0.90 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, 29.00 mM or more, 30.00 mM or more, 3 .50 mM or more, 8.00 mM or more, 8.50 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, for example, it may be 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, 0.60 mM to 40.00 mM, 1.00 mM to 40.00 mM, 0.70 mM to 30.00 mM, 0.80 mM to 30.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, 0.90 mM to 20.00 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, 0.60 mM or more, 0.70 mM or more, 0.80 mM or more, 0.90 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, .50 mM or more, 8.00 mM or more, 8.50 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, 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, 0.60 mM to 40.00 mM, 1.00 mM to 40.00 mM, 0.70 mM to 30.00 mM, 1.50 mM to 20.00 mM, The concentration may be between 0.80 mM and 30.00 mM, between 2.00 mM and 30.00 mM, between 0.90 mM and 20.00 mM, between 2.50 mM and 20.00 mM, between 1.00 mM and 10.00 mM, between 3.00 mM and 10.00 mM, between 1.50 mM and 5.00 mM, or between 3.50 mM and 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.Total content of equol and 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, 0.7 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 mg / g or more, 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 ornithine content per gram of dry weight in the resulting fermented product is 5 mg / g to 30 mg / g, preferably 7 mg / g to 20 mg / g, and more preferably 8 mg / g to 15 mg / g. The genisteins content per gram of dry weight in the resulting fermented product is 0.02 mg / g to 2.5 mg / g, preferably 0.05 mg / g to 2 mg / g. The glyciteins content per gram of dry weight in the resulting fermented product is 0.1 mg / g to 8.5 mg / g, preferably 2.5 mg / g to 6.5 mg / g. The daidzeins content per gram of dry weight in the resulting fermented product is 0.1 mg / g to 30 mg / g, preferably 0.1 mg / g to 1.5 mg / g.

[0047] The resulting fermented product is preferably substantially free of allergens. Examples of allergens include Gym4, Gm30k, Gm28k, 7S globulin, oleosin, and trypsin inhibitor. "Substantially free" means that no bands are detected by visual inspection of a test strip using, for example, FASTKIT Slim Soybean (manufactured by Nippon Meat Packers Central Research Institute) or Allergen Eye® Immunochromatography (manufactured by Prima Meat Packers Co., Ltd.).

[0048] 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, and may be, for example, 30.00 or less, 20.00 or less, 10.00 or less, 9.00 or less, 8.00 or less, 7.00 or less, 6.00 or less, 5.00 or less, 4.00 or less, 3.00 or less, 2.00 or less, 1.00 or less, or 0.50 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, 0.03 to 8.00, 0.04 to 7.00, 0.05 to 6.00, 0.06 to 5.00, 0.07 to 4.00, 0.08 to 3.00, 0.09 to 2.00, 0.10 to 1.00, or 0.20 to 0.50.

[0049] 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, and may be, for example, 30.00 or less, 20.00 or less, 10.00 or less, 9.00 or less, 8.00 or less, 7.00 or less, 6.00 or less, 5.00 or less, 4.00 or less, 3.00 or less, 2.00 or less, 1.00 or less, or 0.50 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, 0.03 to 8.00, 0.04 to 7.00, 0.05 to 6.00, 0.06 to 5.00, 0.07 to 4.00, 0.08 to 3.00, 0.09 to 2.00, 0.10 to 1.00, or 0.20 to 0.50.

[0050] 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.

[0051] The production method may include a step of purifying and concentrating the equol and 5-hydroxyequol from the resulting composition or fermentation product containing equol and 5-hydroxyequol. Examples of purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization or removal of polymeric substances by microfiltration (MF) or ultrafiltration (UF) or the like; recovery or removal of water-insoluble solids by centrifugal sedimentation or filtration; extraction with organic solvents or ionic liquids; and adsorption or decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon, or derivatized silica gel, either directly or in a column. Examples of concentration treatments in the concentration step include concentration using various evaporators (vacuum or atmospheric pressure, centrifugal thin film evaporators, falling thin film evaporators, wiper thin film evaporators, etc.), reverse osmosis membranes, ion exchange membranes, etc.

[0052] Furthermore, compositions or fermented products containing equol and 5-hydroxyequol can be used as is, or can be powdered using batch drying methods such as fluidized bed drying, freeze drying, and agitation drying (using a Nauta mixer, conical dryer, or ribbon mixer dryer), or continuous drying methods such as fluidized bed drying, drum dryer, and spray drying. The methods described in the above paragraphs may be performed before or after this powdering. Excipients such as lactose, dextrin, and cornstarch may also be added during powdering. After drying, the product can be pulverized using a mortar, homogenizer, Waring blender, jet mill, bead mill, ball mill, or the like, or sieved or sized to produce a powder more suitable for powder processing. The compositions or fermented products containing equol and 5-hydroxyequol obtained in this manner, as well as the purified compositions or fermented products, 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.

[0053] Equol has antiestrogenic effects (preventive effects against breast cancer, prostate cancer, etc.), and is effective in preventing and treating (estrogenic effects) the following symptoms associated with menopause in middle-aged and elderly women: hot flashes, sweating, chills in the lower back and limbs, shortness of breath, numbness in the limbs, loss of sensation in the limbs, difficulty falling asleep at night, waking up easily after falling asleep at night, excitability, nervousness, worrying about trivial matters, dizziness, nausea, fatigue, stiff shoulders, lower back pain, pain in the joints of the limbs, headache, heart palpitations, a feeling of ants crawling on the skin, and rough skin. 5-Hydroxyequol has the above-mentioned estrogen-like effects and 3-β-hydroxysteroid dehydrogenase inhibitory activity. In the present disclosure, the use may be therapeutic or non-therapeutic. It may also be preventive. The above-mentioned symptoms may be divided into "symptoms caused by disease" and "symptoms caused by something other than disease." For example, in the case of foods and beverages (including supplements) and health foods and beverages, they may target "symptoms caused by something other than disease" and may target healthy individuals. Furthermore, regarding effects on "dyslipidemia and hypertension," health foods and beverages may "help maintain lipids and blood pressure at healthy levels." Other estrogen-like effects may also "help maintain levels similar to those of healthy individuals" in health foods and beverages.

[0054] Furthermore, the present invention may also include the use of a composition or fermented product containing equol and 5-hydroxyequol to produce a functional food composition or health food composition that alleviates symptoms such as indefinite complaints or menopausal symptoms in healthy middle-aged or elderly women. The composition or fermented product containing equol and 5-hydroxyequol may be obtained by the production method of the present disclosure. Furthermore, for example, the composition or fermented product containing equol and 5-hydroxyequol may contain equol and 5-hydroxyequol, 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 5-hydroxyequol content in the fermented product is 0.3 mg / g or more per 1 g of dry weight, the daidzeins are daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins are genistin, malonyldaidzin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof. The total content of equol and 5-hydroxyequol in the composition or fermented product, the content of 5-hydroxyequol in the composition or fermented product, and the equol content are not particularly limited, and any appropriate combination of the numerical values ​​or numerical ranges disclosed herein may be used.

[0055] As described above, a composition containing equol and 5-hydroxyequol or a fermented product containing equol and 5-hydroxyequol can be obtained by the method for producing equol and 5-hydroxyequol or the method for producing a fermented product containing equol and 5-hydroxyequol. For convenience, the following description will focus on the fermented product, but the description is equally applicable to the composition.

[0056] Fermented products containing equol and 5-hydroxyequol (hereinafter simply referred to as fermented products) may contain acids such as citric acid, succinic acid, fumaric acid, lactic acid, gluconic acid, acetic acid, malic acid, orotic acid, ascorbic acid, benzoic acid, hydrochloric acid, sulfuric acid, and phosphoric acid; or salts or chelates of these acids. The salts are not particularly limited and may be alkali metal salts, alkaline earth metal salts, etc. The chelates are also not particularly limited. The content of the acids, salts, or chelates of these acids in the fermented product may be 0.7 to 20 wt%, 0.8 to 17 wt%, 0.8 to 14 wt%, 0.8 to 11 wt%, or 0.8 to 9 wt%, based on 100 wt% of the fermented product. The fermented product may also contain alkali metal hydroxides or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, and magnesium hydroxide; alkali metal carbonates or alkaline earth metal carbonates such as sodium carbonate, sodium bicarbonate, calcium carbonate, and calcium bicarbonate.

[0057] The pH of the solution or suspension of 50 g of the dried fermented product in 1 L of water may be 4 to 9, or 5 to 8.

[0058] The fermented product may contain substances produced during the fermentation process, such as 5-hydroxydehydroequol and ornithine, or substances originally contained in the raw materials. For example, if ornithine is desired in the fermented product, it is recommended to use a microorganism capable of producing equol from daidzeins, 5-hydroxyequol from genisteins, and ornithine from arginine. Furthermore, it is recommended to include arginine in the composition containing the raw materials, daidzeins and genisteins. In this case, the ornithine content can be adjusted depending on the amount of arginine present. Microorganisms capable of producing equol from daidzeins, 5-hydroxyequol from genisteins, and ornithine from arginine can be obtained by screening the above-mentioned microorganisms using standard methods, using the abilities of producing equol from daidzeins, 5-hydroxyequol from genisteins, and ornithine from arginine as indicators.

[0059] For example, the present invention may be a fermented product of a composition containing daidzeins and genisteins, the fermented product containing equol and 5-hydroxyequol, the total content of equol and 5-hydroxyequol in the fermented product being 0.5 mg / g or more and 100.0 mg / g or less per 1 g of dry weight, the 5-hydroxyequol content in the fermented product being 0.3 mg / g or more per 1 g of dry weight, the daidzeins being daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof, and the genisteins being genistin, malonyldaidzin, acetylgenistin, genistein, dihydrogenistein, or a combination thereof. The total content of equol and 5-hydroxyequol in the fermented product, the 5-hydroxyequol content, and the equol content in the fermented product are not particularly limited, and any appropriate combination of the numerical values ​​or numerical ranges disclosed herein may be used.

[0060] The fermented product can be used as it is, or as a base material or containing other ingredients, to be made into cosmetics, quasi-drugs, medical supplies, sanitary products, pharmaceuticals, food and beverages (including supplements), etc.

[0061] Oral compositions, such as pharmaceutical compositions and foods and beverages (including supplements), are described below, but are not limited to these. Compositions or fermented products containing equol and 5-hydroxyequol, as well as purified equol and 5-hydroxyequol, can be used in oral compositions. The present disclosure can provide, for example, a method for producing an oral composition. Specifically, this method is a method for producing an oral composition that includes a step of incorporating equol and 5-hydroxyequol into a base material, and the equol and 5-hydroxyequol are produced by the method disclosed herein. However, this method is not limited to oral compositions, and by appropriately modifying the base material, etc., it can also be applied to transdermal compositions and infusion compositions.

[0062] The amounts of equol and 5-hydroxyequol contained in the oral composition are not particularly limited as long as they are within a range that achieves the intended effect, and may be selected appropriately depending on the form of the composition, the number of times it is taken, the health condition of the subject, and other factors. Furthermore, any laws, regulations, or guidelines may be followed. For example, the amounts of equol, 5-hydroxyequol, equol, and 5-hydroxyequol vary depending on the form of the composition, the number of times it is taken, the purpose, and other factors, but may be selected appropriately from a range of approximately 0.000001% by weight to 20.0% by weight relative to the total weight of the composition. Furthermore, the amounts of the base material or other components described below are also not particularly limited as long as they are within a range that achieves the intended effect, and may be selected appropriately depending on the form of the composition, the number of times it is taken, the health condition of the subject, and other factors. Furthermore, any laws, regulations, or guidelines may be followed.

[0063] Here, the base material or other components are not particularly limited, but examples include the following: amino acids, such as BCAA (valine, leucine, isoleucine), alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, serine, γ-aminobutyric acid (GABA), hydroxyproline, ornithine, and citrulline.

[0064] Herbal medicines, such as ginseng (dried root of Panax ginseng C.A. Mey., ginseng of the Araliaceae family), yellow jing (rhizome of Polygonatum sibiricum Red., lily family), and Equidae (Equus asinus, donkey of the Equidae family). L.), Goji berries (dried mature fruits of the Lycium berry of the Solanaceae family), Hangbi (made by removing the internal organs of a Viper viper, peeling the skin, stretching it out and drying it), Yamayak (dried tuberous roots of the Chinese yam of the Dioscorea family), Jujube (dried mature fruits of the Jujube jujube of the Rhamnaceae family and plants of the same genus), Mulberry berries (dried mature fruits of the Solanaceae Makuwa tree), Safflower (dried corolla of the Carthamus tinctorius, Asteraceae family) dried), cinnamon bark, turmeric (dried tuberous roots of turmeric, ginger family, and yellow turmeric), sour jujube kernel (dried mature seeds of Jujube jujube, Rhamnaceae family), longan (pulp of longan, Sapindaceae family), dried tangerine peel (dried peel of Citrus unshiu, Rutaceae family), hippocampus (dried viscera of Cordyceps sinensis and spotted hippocampus, Syngmantaceae family), cordyceps sinensis (fruit body and sclerotium of Cordyceps sinensis (Berk.) Sacc., fenugreek, legume family), and Cornus chinensis (dried fruit of Cornus unshiu, Cornaceae family) can be used. It can also be used as a mixture of herbal medicines according to a Chinese herbal prescription. Depending on the form and dosage form, these herbal medicines can be crushed or powdered, or extracted with alcohol or hot water, and the extract can be dried and powdered to be used as an extract powder.

[0065] Vitamins, for example, vitamin B1s such as thiamine hydrochloride, thiamine nitrate, bisthiamine nitrate, dicethiamine hydrochloride, dibenzoylthiamine, dibenzoylthiamine hydrochloride, thiamine cetyl sulfate, thiamine thiocyanate, thiamine naphthalene-1,5-disulfonate, and thiamine lauryl sulfate, vitamin B2s such as riboflavin and riboflavin sodium phosphate, vitamin B6s such as pyridoxine hydrochloride and pyridoxal phosphate, vitamin B12s such as hydroxocobalamin hydrochloride, hydroxocobalamin acetate, cyanocobalamin, and hydroxocobalamin, folic acid, biotin, panthenol, pantothenic acids such as calcium pantothenate and sodium pantothenate, nicotinic acids such as nicotinic acid and nicotinamide, ascorbic acid, sodium ascorbate, calcium ascorbate, ascorbic acid, niacin, niacin, niacinamide, niacin ... powders of vitamin C such as corbic acid stearate, vitamin P such as methylhesperidin and rutin, powders of vitamin D such as vitamin D1, vitamin D2, vitamin D3, vitamin D4, powders of vitamin K such as vitamin K1, vitamin K2, vitamin K3, vitamin K4, vitamin K5, vitamin K6, vitamin K7, powders of carotenoid compounds such as palm oil carotene, Dunaliella carotene, cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, β-apo-8'-carotenal, bixin, and lycopene, powders of vitamin E such as d-α-tocopherol, d-β-tocopherol, d-γ-tocopherol, d-σ-tocopherol, and tocotrienol, and powders of vitamin F such as linoleic acid, linolenic acid, and arachidonic acid.

[0066] Minerals. Examples include the 12 minerals (calcium, magnesium, iron, zinc, selenium, chromium, manganese, copper, molybdenum, potassium, phosphorus, and iodine) published by the Ministry of Health, Labor and Welfare in 2000 as the 6th revised nutritional requirement for Japanese people. Such minerals may be inorganic or organic. The organic mineral in the present invention may be, for example, a mineral in which a metal such as zinc or copper is chemically bonded to an amino acid, which is a protein component, or to an organic compound.

[0067] Organic minerals include commercially available products such as zinc yeast, magnesium yeast, iron yeast, selenium yeast, chromium yeast, manganese yeast, copper yeast, molybdenum yeast, and iodine yeast, in which minerals are incorporated into the yeast cells, and heme iron obtained by enzymatically treating hemoglobin, followed by ultrafiltration or isoprecipitation, and then drying. Inorganic minerals include commercially available natural calcium and magnesium products suitable for use in foods, such as dolomite, eggshell calcium, coral calcium, sea urchin shell calcium, petrified seaweed calcium, pearl calcium, cow bone calcium, fish bone powder calcium, fish scale calcium, and milk calcium. For example, dolomite is obtained by crushing and heat-sterilizing dolomite ore, followed by grinding. Since the calcium / magnesium ratio is 2:1, the use of dolomite allows for the production of compositions suitable for calcium absorption.

[0068] Herbs, such as milk thistle extract, turmeric extract, mulberry leaf extract, gymnema sylvestre extract, ginkgo leaf extract, Eleuthero extract, blueberry extract, St. John's wort extract, saw palmetto fruit extract, pumpkin seed extract, sweet tea extract, perilla seed extract, perilla leaf extract, rooibos tea extract, Job's tears extract, echinacea extract, melilot extract, pueraria mirifica powder, chaste tree extract, pomegranate seed extract, Garcinia cambogia extract, Citrus aurantium extract, clove extract, Centella asiatica extract, grape seed extract, red wine extract, millet extract, horsetail extract, agaricus mushroom extract, maitake mushroom extract, garlic extract, aloe vera extract, and propolis extract. These herbs can be obtained by crushing fruits, seeds, leaves, etc., or extracts extracted from fruits, seeds, leaves, etc. with an aqueous ethanol solution, separating and concentrating the extracts, and then adding additives such as excipients as necessary and drying them. Commercially available products can be used.

[0069] Plant sterols, red koji powder, soybeans, soybean hypocotyls, fermented soybeans, fermented soybean hypocotyls, red clover, kudzu, soybean extract, soybean hypocotyl extract, fermented soybean extract, fermented soybean hypocotyl extract, red clover extract, kudzu extract, glucosamine, chondroitin sulfate-containing mucopolysaccharide protein, collagen peptide, chitosan, silk peptide, inositol, protein, bifidobacteria powder, lactic acid bacteria powder, brewer's yeast, chlorella, spirulina, royal jelly powder, green tea polyphenols, red wine polyphenols, apple polyphenols, grape seed polyphenols, grape leaf polyphenols, oolong tea polyphenols, perilla polyphenols, buckwheat polyphenols Examples of such extracts include alcohol, cocoa polyphenols, coffee polyphenols, sweet tea polyphenols, perilla extract, cocoa extract, buckwheat extract, citrus peel extract, blueberry extract, green tea extract, coffee extract, apple extract, evening primrose extract, oolong tea extract, grape seed extract, grape leaf extract, eucalyptus extract, guava extract, sweet tea extract, lychee seed extract, cranberry extract, banana juice, apple juice, grape juice, strawberry juice, melon juice, apple juice, mango juice, muscat juice, white grape juice, lemon juice, lime juice, mandarin orange juice, grapefruit juice, and yuzu juice, and commercially available products can be used.

[0070] Wheat flour, soft flour, butter, eggs, dextrin, wheat starch, rice starch, corn starch, potato starch, pregelatinized starch, partially pregelatinized starch, soybean flour, glutinous rice flour, matcha green tea, black sesame, gluten, honey, skim milk powder, baking soda, maltose, reduced lactose, reduced maltose, sorbitol, mannitol, erythritol, xylitol, white sugar, glucose, brown sugar, guar gum, xanthan gum, sodium alginate, gum arabic, tragacanth gum, pullulan, agar, gelatin, soybean dietary fiber, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethylethyl cellulose, cellulose acetate phthalate, Examples of suitable hydroxyethyl cellulose, hydroxypropyl starch, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, carboxymethyl starch sodium, hardened vegetable oil, vegetable oil powder, carnauba wax, cocoa butter powder, sucrose fatty acid ester, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, silicon dioxide, talc, aluminum silicate, calcium hydrogen phosphate, salt, potassium chloride, calcium lactate, acesulfame K, aspartame, advantame, neotame, licorice extract, stevia, stevia extract, sucralose, thaumatin, and swingle extract.

[0071] Examples of the gallic acid include gallic acid derivatives (e.g., methyl, ethyl, propyl, and butyl gallate), alkali metal salts of gallic acid such as sodium and potassium, chlorogenic acid, ellagic acid, tannic acid, tannins (e.g., proanthocyanidins, gallotannins, and ellagitannins), flavonoids (e.g., flavones, flavonols, flavanones, flavanonols, isoflavones, anthocyanins, and flavanols (catechin, epicatechin, gallocatechin, epigallocatechin gallate, epigallocatechin gallate, and the like)), chalcones, and aurones.

[0072] When the oral composition is a food or beverage composition, it may contain an acid as long as it does not impair the flavor. Examples of acids include, but are not limited to, citric acid, succinic acid, fumaric acid, lactic acid, gluconic acid, acetic acid, malic acid, orotic acid, ascorbic acid, benzoic acid, hydrochloric acid, sulfuric acid, and phosphoric acid; and salts or chelates of these acids. The salts are not particularly limited and may be alkali metal salts, alkaline earth metal salts, etc. The chelates are also not particularly limited. The composition may also contain alkali metal hydroxides or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, and magnesium hydroxide; or alkali metal carbonates or alkaline earth metal carbonates such as sodium carbonate, sodium bicarbonate, calcium carbonate, and calcium bicarbonate. The content of the acid, salt or chelate of this acid in the food or beverage composition may be 0.5 to 14 wt %, 0.6 to 12 wt %, 0.7 to 10 wt %, 0.8 to 8 wt %, or 0.8 to 6 wt %, when the food or beverage composition is taken as 100 wt %.

[0073] For example, the pH of a food or beverage composition is typically 4 to 9, or may be 5 to 8. If the composition is liquid, it can be measured as is. If the composition is not liquid, it can be dried and 50 g of the dried product can be dissolved or suspended in 1 L of water and measured. The pH of the food or beverage composition can be adjusted using a pH adjuster. Examples of pH adjusters include, but are not limited to, acids such as citric acid, succinic acid, fumaric acid, lactic acid, gluconic acid, acetic acid, malic acid, orotic acid, ascorbic acid, benzoic acid, hydrochloric acid, sulfuric acid, and phosphoric acid; salts or chelates of these acids; alkali metal hydroxides or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, and magnesium hydroxide; alkali metal carbonates or alkaline earth metal carbonates such as sodium carbonate, sodium bicarbonate, calcium carbonate, and calcium bicarbonate. The salts of the aforementioned acids are not particularly limited and may be alkali metal salts, alkaline earth metal salts, etc. The chelates of the aforementioned acids are also not particularly limited.

[0074] Specific examples of food and beverage compositions include, but are not limited to, soft drinks, carbonated drinks, fruit juice drinks, vegetable drinks, tea drinks, coffee drinks, dairy drinks, soy milk drinks, fermented drinks, yogurt, cocoa drinks, sports drinks, nutritional supplements, energy bars, low-calorie drinks, alcoholic drinks, non-alcoholic drinks, jelly drinks, frozen drinks, smoothies, herbal teas, candies, starch syrup, frozen desserts, candy tablets, supplements, creamy foods, seasonings, retort pouch foods, processed meat foods, paste foods, processed egg foods, canned foods, frozen desserts, soy processed foods, nutritional supplements, confectioneries (cookies, biscuits, chocolate confectioneries, chips, cakes, gum, candies, gummy candies, steamed buns, yokan, puddings, jellies, yogurt, ice cream, sherbet, nut bars, fruit snacks, macarons, etc.), bread, noodles, rice dishes, cereal foods, soups (powdered, freeze-dried), miso soup (powdered, freeze-dried), etc. Furthermore, in the case of a beverage composition, any known base material can be used without any particular limitation. For example, in addition to flavoring agents, fragrances, and acidulants (anhydrous citric acid, etc.), stabilizers (pectin, soybean polysaccharides, etc.), thickeners (tamarind gum, etc.), emulsifiers (sucrose fatty acid esters, etc.), milk (cow's milk, skim milk powder, etc.), pH adjusters, etc. may be optionally used.

[0075] The amount of equol and 5-hydroxyequol, even when contained in a food or beverage composition, is not particularly limited as long as the desired effect is achieved, and may be selected appropriately depending on the form of the composition, the number of times of ingestion, the health condition of the subject, and other factors. Furthermore, any laws, regulations, or guidelines may be followed. For example, the amount of equol, 5-hydroxyequol, equol, and 5-hydroxyequol will vary depending on the form of the composition, the number of times of ingestion, the purpose, and other factors, but may be selected appropriately from a range of approximately 0.000001 wt % to 20.0 wt % relative to the total weight of the composition. Furthermore, the amount of the base material or other components is also not particularly limited as long as the desired effect is achieved, and may be selected appropriately depending on the form of the composition, the number of times of ingestion, the health condition of the subject, and other factors. Furthermore, any laws, regulations, or guidelines may be followed.

[0076] For example, when the food or beverage composition is a jelly drink, the present disclosure provides a method for producing a jelly drink composition, specifically comprising the step of incorporating equol and 5-hydroxyequol into a base material, wherein the equol and 5-hydroxyequol are produced by the method disclosed herein. For example, the base material may be selected from gelling agents such as konjac flour, carrageenan, gelatin, pectin, agar, alginic acid, locust bean gum, and xanthan gum, as well as other ingredients, depending on the intended purpose. For example, a jelly drink composition containing collagen peptide, agar, equol, and 5-hydroxyequol may contain approximately 0.02% to 25% by weight of collagen peptide and approximately 0.005% to 2.0% by weight of equol, or may contain approximately 2% to 3% by weight of collagen peptide and approximately 0.01% to 0.05% by weight of equol, based on the total weight of the jelly drink composition. In the case of a jelly drink, the pH may be 2.5 to 4.0. The pH may be adjusted using a pH adjuster. In this case, the pH adjuster may be an organic acid such as phytic acid, citric acid, gluconic acid, succinic acid, acetic acid, tartaric acid, lactic acid, fumaric acid, or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid, or an acidic fruit juice such as lemon juice or apple juice.

[0077] For example, when the food or beverage composition is sparkling water, the present disclosure provides a method for producing a sparkling water composition, which includes the step of incorporating equol and 5-hydroxyequol into a base material, wherein the equol and 5-hydroxyequol are produced by the production method of the present disclosure. For example, the base material may be selected from liquids such as water or carbonated water, various flavorings, pH adjusters, and other ingredients according to the purpose.

[0078] As described above, the oral composition may contain the fermented product of the present disclosure. Specifically, the oral composition contains a fermented product containing equol and 5-hydroxyequol and a base material. In this case, the blending ratio of the fermented product is not particularly limited and is appropriately determined depending on the equol content in the oral composition and the form of the oral composition. As an example, the proportion of the fermented product (dry product) contained in the oral composition is 1 to 90% by weight, preferably 20 to 85% by weight, and preferably 30 to 80% by weight.

[0079] The oral composition containing a fermented product may be a food or beverage composition containing a fermented product. When the food or beverage composition is a jelly drink, it may specifically be a jelly drink composition containing a fermented product containing collagen peptide, agar, and equol and 5-hydroxyequol. In this case, the collagen peptide may be contained in an amount of approximately 0.02% to 25% by weight and the equol content may be approximately 0.005 to 2.0% by weight, or the collagen peptide may be contained in an amount of approximately 2% to 3% by weight and the equol content may be approximately 0.01 to 0.05% by weight, based on the total weight of the jelly drink.

[0080] For example, the concentration of equol in a food or beverage composition containing a fermented product is 0.005 to 10.0 wt%, preferably 0.1 to 7.0 wt%, and more preferably 0.3 to 3.0 wt%. For example, the concentration of 5-hydroxyequol in a food or beverage composition containing a fermented product is 0.005 to 10.0 wt%, preferably 0.1 to 7.0 wt%, and more preferably 0.3 to 3.0 wt%. For example, the concentration of ornithine in a food or beverage composition containing a fermented product is 0.008 to 30.0 wt%, preferably 0.15 to 20 wt%. For example, the concentration of daidzeins in a food or beverage composition containing a fermented product is 0.001 to 2.0 wt%, preferably 0.2 to 0.6 wt%. For example, the concentration of genisteins in a food or beverage composition containing a fermented product is 0.001 to 2.0 wt%, preferably 0.1 to 0.6 wt%. For example, the concentration of glyciteins in a food or drink composition containing a fermented product is 0.001 to 2.0% by weight, preferably 0.2 to 0.6% by weight.

[0081] Hereinafter, the oral composition will be described in the form of a solid preparation, but is not limited thereto. The form or shape of the solid preparation is not particularly limited, and includes any form or shape known to those skilled in the art, such as tablets, granules, films, and sheets.

[0082] In addition to equol and 5-hydroxyequol, the oral composition may optionally contain other pharmaceutically or food sanitation-acceptable ingredients, such as excipients, excipient aids, disintegrants, surfactants, lubricants, acidulants, sweeteners, flavorings, flavorings, colorants, stabilizers, fluidizers, and binders. These optional ingredients include, for example, those listed in the Pharmaceutical Additives Dictionary (Yakuji Nipposha) and the Japanese Pharmacopoeia, as well as designated or existing additives under Article 10 of the Food Sanitation Act, natural flavorings, and additives listed in the list of general food and beverage additives. There are no particular restrictions on the type of optional ingredient. Furthermore, as long as the desired effects of the present disclosure are achieved, there are no particular restrictions on the proportion of the optional ingredient in the oral composition, which can be determined appropriately by those skilled in the art. Examples of excipients include any sugar alcohol or sugar known to those skilled in the art. Representative examples of sugar alcohols or sugars include mannitol, erythritol, xylitol, trehalose, lactose, maltose, maltitol, glucose, sucrose, fructose, mannose, and sorbitol. Further, preferred examples include mannitol, erythritol, xylitol, trehalose, and lactose. While one type of sugar or sugar alcohol may be used, two or more appropriately selected compounds may also be used. Examples of disintegrants and excipient aids include starch and inorganic excipients. Examples of starches include corn starch, potato starch, waxy corn starch, partially pregelatinized starch, and pregelatinized starch. Examples of inorganic excipients include light anhydrous silicic acid, hydrated silicon dioxide, anhydrous calcium phosphate, anhydrous calcium hydrogen phosphate, aluminum metasilicate, calcium silicate, magnesium silicate, and magnesium oxide.

[0083] The oral composition of the present disclosure can be produced by any method or means known to those skilled in the art. For example, the oral composition of the present disclosure can be produced by simultaneously mixing the various components contained in the oral composition. Alternatively, the composition can be produced by various granulation processes. The granulation method is not particularly limited, and the composition can be produced by dry granulation or wet granulation. Dry granulation involves mixing the various component powders contained in the oral composition, either directly or with an appropriate binder, forming small masses under high pressure, and then appropriately crushing and granulating them. Specific examples of dry granulation include crushing granulation and roll compression. Wet granulation is a method of forming a complex by dispersing and drying each component in the presence of water. Specific examples of wet granulation include spray methods such as spray drying, tumbling granulation, agitation granulation, and fluidized bed granulation, freeze drying, and kneading granulation. The composition can be produced by any of these methods known to those skilled in the art. When manufacturing using a wet granulation process, the oral composition of the present disclosure may be manufactured in a single granulation step using all of the components contained in the oral composition together, or they may be added and mixed in multiple wet granulation steps. It should be noted that which one or two of the components contained in the oral composition to use in the multiple wet granulation steps of the above manufacturing method can be appropriately determined by those skilled in the art depending on the type and amount of each component. Furthermore, in each granulation step, various conditions such as the spray rate, inlet air temperature, exhaust temperature, and air volume can be appropriately determined by those skilled in the art depending on the type and amount of each component. In each granulation step, examples of the spray medium include solvents acceptable for pharmaceuticals and foods, such as water, ethanol, methanol, and acetone. Alternatively, examples of the spray medium include an aqueous solution in which less than 10% of the components of the oral composition are dissolved, with water or such an aqueous solution being particularly preferred. The various optional components described above that may be optionally contained in the oral composition of the present disclosure can be added appropriately in each granulation step. Alternatively, a separate wet granulation step may be carried out, at which stage these optional ingredients may be added and mixed.

[0084] All prior art documents cited herein are hereby incorporated by reference.

[0085] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to these examples.

[0086] In each example, the amount of isoflavones was determined by the following method.

[0087] (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.

[0088] (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.

[0089] (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)

[0090] (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.

[0091] (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.

[0092] (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.

[0093] 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.

[0094] 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.

[0095] (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.

[0096]

[0097]

[0098] 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.

[0099]

[0100] 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 0.150% / h or higher, the molar concentration ratio of the genisteins to the daidzeins at the start of culturing is 1.300 or higher or 0.700 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. 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 1.300 or more or 0.700 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.

3. A method for producing an oral composition, comprising the step of incorporating equol and 5-hydroxyequol into a base material, wherein the equol and 5-hydroxyequol are produced by the method of claim 1 or 2.

4. A method for producing a composition for a jelly drink, comprising the step of incorporating equol, 5-hydroxyequol, collagen peptide, and agar, wherein the equol and 5-hydroxyequol are produced by the method of claim 1 or 2.

5. A method for producing a jelly drink composition according to claim 4, wherein the equol content is 0.01 to 0.05% by weight based on the total weight of the composition.

6. A fermented product of a composition containing daidzeins and genisteins, wherein the fermented product contains equol and 5-hydroxyequol, 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 5-hydroxyequol content in the fermented product is 0.3 mg / g or more 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.

7. An oral composition comprising the fermented product of claim 6 and a substrate.

8. A composition for a jelly drink, comprising the fermented product according to claim 6, collagen peptide, and agar.

9. A jelly drink composition according to claim 8, wherein the equol content is 0.01% by weight to 0.05% by weight based on the total weight of the composition.

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