Method for producing equol and 5-hydroxyequol

Abstract

To provide a method for producing equol and 5-hydroxyequol.SOLUTION: A method for producing equol and 5-hydroxyequol using a microorganism having a capability of producing equol from a predetermined daidzein compound and a capability of producing 5-hydroxyequol from a predetermined genistein compound, wherein the daidzein compound and the genistein compound are present at the start of the culture of the microorganism, the molar conversion efficiency of the genistein compound to 5-hydroxyequol is 0.200% / h or more, and the molar concentration ratio of the genistein compound to the daidzein compound at the start of the culture is 1.500 or more or 0.500 or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[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] The isoflavones contained in soybeans mainly contain glycosides, which are condensed with sugars, but also contain small amounts of isoflavone aglycones (hereinafter referred to as aglycones), which are not condensed with sugars. Examples of glycoside-type isoflavones include daidzin, glycitin, and genistin. When these glycosides enter the human or animal body, they are converted into their aglycones, daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or enzymes produced by intestinal bacteria (such as β-glucosidase). It is known that daidzein is enzymatically converted to equol via dihydrodaidzein (also called dihydrodaidzein) by the action of intestinal bacteria. Similarly, it is known that genistein is also converted to 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 estrogenic 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 varies from person to person, and as mentioned above, only a small number 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). The genes encoding these enzymes are organized as clusters with similar sequences and gene structures even among different equol-producing strains of bacteria, such as those from the genera Slackia, Egasella, 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 address these issues, 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. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-81875 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-204296 [Patent Document 3] Special Publication No. 2006-504409 [Patent Document 4] Japanese Patent Application Laid-Open No. 2008-61584 [Patent Document 5] Japanese Patent Application Laid-Open No. 2010-104241 [Non-patent literature]

[0008] [Non-Patent Document 1] Adlercreutz, H., The Lancet Oncol., 3, 364-373 (2002) [Non-patent document 2] Duncan, AM et al., Best Pract. Res. Clin. Endocrinol. Metab., 17, 253-271 (2003) [Non-patent document 3] Wu, AH et al., Carcinogenesis, 23, 1491-1496 (2002) [Non-patent document 4] Yamamoto, S. et al., J. Natl. Cancer Inst., 95,906-913 (2003) [Non-patent document 5] Onozawa, M. et al., Jpn. J. Cancer Res., 90, 393-398 (1999) [Non-patent document 6] Ridges, L. et al., Asia Pac. J. Clin. Nutr., 10, 204-211 (2001) [Non-Patent Document 7] Matthies, A. et al., Appl. Environ. Microbiol., 74(15), 4847-52(2008) [Non-patent document 8] Schmitt, E. et al., Toxicol. In Vitro, 15, 433-439 (2001) [Non-Patent Document 9] Sathyamoorthy, N. and Wang, TT, Eur. J. Cancer, 33, 2384-2389 (1997) [Non-Patent Document 10] Arai, Y. et al., J. Epidemiol., 10, 127-135 (2000) [Non-Patent Document 11] Setchell, KD et al., J. Nutr., 133, 1027-1035 (2003) [Non-Patent Document 12] International Journal of Food Microbiology 360 (2021) 109328 Summary of the Invention [Problem to be solved by the invention]

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

[0010] 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, 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; a molar concentration ratio of the genisteins to the daidzeins at the start of the culture is 1.500 or more or 0.500 or less; the daidzein-class compound is daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof; The method for producing the genisteins is genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein or a combination thereof. [2] The method of producing according to [1], wherein the molar conversion efficiency of the genisteins to 5-hydroxyequol is 0.250% / h or more. [3] The 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 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 Adrecratia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Slackia, and microorganisms belonging to the genus Lactococcus. [5] The method according to [4], wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to Adrecrautia aequorifaciens, microorganisms belonging to Slackia aequorifaciens, and microorganisms belonging to Lactococcus garvieae. [6] The 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] By culturing microorganisms that have the ability to produce equol from daidzein and 5-hydroxyequol from genistein, A method for producing a fermented product containing equol and 5-hydroxyequol, comprising: daidzeins and genisteins are present at the start of culturing the microorganism; a molar concentration ratio of the genisteins to the daidzeins at the start of the culture 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 daidzein-class compound is daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof; The method, wherein 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 Adreclautia, 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 Adrecrautia 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. [Effects of the Invention]

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

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

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

[0014] <Method of producing equol and 5-hydroxyequol> Microorganisms are preferably 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 those into which genes encoding the enzymes involved in equol production and 5-hydroxyequol production (e.g., daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR), etc.) have been introduced. Microorganisms that have the ability to produce equol from daidzeins and 5-hydroxyequol from genisteins can be publicly known or microorganisms that can be obtained by screening using conventional methods. For example, microorganisms belonging to the genus Coriobacterium, microorganisms belonging to the genus Adlercreutzia, microorganisms belonging to the genus Asaccharobacter, microorganisms belonging to the genus Atopobium, microorganisms belonging to the genus Bacteroides, microorganisms belonging to the genus Bifidibacterium, microorganisms belonging to the genus Clostridium, microorganisms belonging to the genus Collinsella, microorganisms belonging to the genus Cryptobacterium, microorganisms belonging to the genus Denitrobacterium, microorganisms belonging to the genus Eggerthella, microorganisms belonging to the genus Enterococcus, microorganisms belonging to the genus Enterorhabdus, and microorganisms belonging to the genus Eubacterium. Examples of organisms include microorganisms belonging to the genus Finegoldia, microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Gordonibacter, microorganisms belonging to the genus Olsenella, microorganisms belonging to the genus Paraeggerthella, microorganisms belonging to the genus Pediococcus, microorganisms belonging to the genus Proteus, microorganisms belonging to the genus Sharpea, microorganisms belonging to the genus Slackia, microorganisms belonging to the genus Streptococcus, microorganisms belonging to the genus Veillonella, and microorganisms belonging to the genus Lactococcus, as well as microorganisms obtained by screening these microorganisms using the ability to produce equol from daidzeins and / or the ability to produce 5-hydroxyequol from genisteins as indicators. Genes encoding the enzymes involved in the production of 5-hydroxyequol and 5-hydroxyequol can be obtained, for example, from microorganisms classified into the above genera.

[0015] More specific microorganisms are as follows: Adlercreutzia equolifaciens subsp. celatus DSM microorganisms belonging to Adlercreutzia equolifaciens subsp. ceratus, such as strain 18785, and microorganisms belonging to Adlercreutzia equolifaciens subsp. equolifaciens, such as strain DSM 19450; Microorganisms belonging to the genus Bacteroides ovatus, such as Bacteroides ovatus strain E-23-15 (FERM BP-6435); Microorganisms belonging to the genus Bifidibacterium breve, such as Bifidibacterium breve ATCC 15700 strain; Bifidobacterium longum NITE BP-02621 (ATCC BAA-999; strain BB536) and other microorganisms belonging to the genus Bifidobacterium longum; Microorganisms belonging to the genus Clostridium, such as Clostridium sp. strain HGH136 (ATCC BAA-442); Microorganisms belonging to the genus Eggerthella, such as Eggerthella sp. Julong 732 strain (KCCM-10490), Eggerthella sp. YY7918 strain (the YY7918 strain is managed by the Gifu Prefectural Institute of Biotechnology, a public institution, and is available from there), and Eggerthella sp. D1 strain; Enterococcus faecalis, such as Enterococcus faecalis INIA P333 strain faecalis); Microorganisms belonging to the genus Enterococcus faecium, such as the Enterococcus faecium EPI1 strain; Microorganisms belonging to the genus Enterohabdus mucosicola, such as Enterohabdus mucosicola strain Mt1B8 (DSM 19490); Microorganisms belonging to the genus Eubacterium, such as Eubacterium sp. strain D2; - Microorganisms belonging to the genus Finegoldia magna, such as Finegoldia magna strain EPI3; - Microorganisms belonging to the genus Lactobacillus fermentum, such as Lactobacillus fermentum strain DPPMA114 (DSM 23757); - Microorganisms belonging to the genus Lactobacillus intestinalis, such as the Lactobacillus intestinalis KTCT13676BP strain; - Microorganisms belonging to the Lactobacillus mucosae family, such as the Lactobacillus mucosae EPI2 strain; - Microorganisms belonging to Lactobacillus paracasei, such as Lactobacillus paracasei strain JS1; - Microorganisms belonging to the genus Lactobacillus plantarum, such as Lactobacillus plantarum strain DPPMA24W (DSM 23756) and Lactobacillus plantarum strain DPPMASL33 (DSM 23755); - Microorganisms belonging to the genus Lactobacillus rhamnosus, such as the Lactobacillus rhamnosus DPPMAAZ1 strain and the Lactobacillus rhamnosus INIA P540 strain; Microorganisms belonging to the genus Lactobacillus, such as Lactobacillus sp. Niu-O16 strain; - Microorganisms belonging to the genus Lactococcus garvieae, such as Lactococcus garvieae strain 20-92 (FERM BP-10036); Microorganisms belonging to the genus Paraeggerthella, such as Paraeggerthella sp. strain SNR40-432; Microorganisms belonging to the genus Pediococcus pentosaceus, such as the Pediococcus pentosaceus CS1 strain; - Microorganisms belonging to the genus Proteus mirabilis, such as Proteus mirabilis strain LH-52; Microorganisms belonging to the genus Sharpea azabuensis, such as Sharpea azabuensis strain ST18 (NITE P-300); Microorganisms belonging to the genus Slackia equolifaciens, such as Slackia equolifaciens strain DSM 24851; Microorganisms belonging to the genus Slackia, such as Slackia isoflavoniconvertens strain DSM 22006; Microorganisms belonging to the genus Slackia, such as Slackia sp. strain FJK1 (NITE P-1562), Slackia sp. strain NATTS (FERM BP-11231), Slackia sp. strain YIT11861 (FERM BP-11231), and Slackia sp. strain TM-30; Microorganisms belonging to the genus Streptococcus constellatus, such as Streptococcus constellatus strain E-23-17 (FERM BP-6436); Microorganisms belonging to the genus Streptococcus intermedius, such as Streptococcus intermedius strain A6G-225 (FERM BP-6437); Veillonella sp. EP strain and other Veillonella species A microorganism belonging to the genus Veillonella. Further examples include microorganisms having 16S rDNA with a base sequence that is 95% or more, preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more identical to the base sequence of the 16S rDNA (16S rRNA gene) of these microorganisms. 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 institutions indicated by the accession numbers. 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.htmlDSM German Collection of Microorganisms and Cell Cultures (DSMZ) http: / / www.dsmz.de / KCCM Korean Culture Center of Microorganisms ATCC American Type Culture Collection NITE National Institute of Technology and Evaluation Patent Microorganism Deposit Center (NPMD)

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

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

[0018] The composition containing daidzeins and genisteins is not particularly limited as long as it can cause a microorganism having the ability to produce equol from daidzeins and the ability to produce 5-hydroxyequol from genisteins to produce equol from daidzeins and 5-hydroxyequol from genisteins. The composition containing daidzeins and genisteins 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 is capable of producing 5-hydroxyequol from daidzeins. The composition containing daidzeins and genisteins is preferably a medium.

[0019] Anaerobic microorganisms are cultured anaerobically, and aerobic microorganisms are cultured aerobically. For example, in the case of anaerobic microorganisms, gas phase conditions (anaerobic conditions) that allow their survival are maintained, and nutrients are 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), GAM bouillon, modified GAM, and BHI media are suitable. These media may also contain the following components as appropriate:

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

[0021] Furthermore, arginine may be present at the start of culturing the microorganism.Furthermore, arginine may be contained in the composition containing daidzeins and genisteins at the start of culturing the microorganism.The content of arginine in the composition containing daidzeins and genisteins at the start of culturing is not particularly limited, but may be 0.30g / 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, 1.10g / L or more, 1.20g / 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, 1.9 ... / 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, 2.0 0g / L or more, 2.10g / L or more, 2.20g / L or more, 2.30g / L or more, 2.40g / L or more, 2.50g / L or more, 2.60g / L or more, 2.70g / L or more, 2. The concentration may be 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, it may be, for example, 0.30 g / L to 10.00 g / L, 0.40 g / L to 9.00 g / L, 0.50 g / L to 8.00 g / L, or 0.60 g / L to 7.00 g / L.

[0022] Furthermore, cyclodextrin may be present at the start of culturing the microorganism. Furthermore, cyclodextrin may be further contained in 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. At the start of culturing, daidzeins and genisteins may be further contained in the composition. Examples of cyclodextrin include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof such as glycosyl-cyclodextrin, maltosyl-cyclodextrin, hydroxypropyl-cyclodextrin, and methyl-cyclodextrin. One type of cyclodextrin may be used, or two or more types may be used. The content of cyclodextrin in the composition containing zeins and genisteins is not particularly limited, but 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, 1.5g / L or more, 2.0g / L or more, 2.5g / L or more, 3.0g / L or more, 3.5g / L or more, 4.0g / L or more, 4.5g / L or more, 5.0g / L or more, 5.5g / L or more , 6.0g / L or more, 6.5g / L or more, 7.0g / L or more, 7.5g / L or more, 8.0g / L or more, 8.5g / L or more, 9.0g / L or more, 9.5g / L or more, 10.0g / L or more The concentration may be 10.5 g / L or more, 11.0 g / L or more, 11.5 g / L or more, 12.0 g / L or more, 12.5 g / L or more, 13.0 g / L or more, 13.5 g / L or more, 14.0 g / L or more, 14.5 g / L or more, 15.0 g / L or more, 15.5 g / L or more, 16.0 g / L or more, 16.5 g / L or more, 17.0 g / L or more, or 17.5 g / L or more. The upper limit is not particularly limited, but it is preferably set to the solubility of the dextrin at the culture temperature or less. For example, in the case of β-cyclodextrin, it is preferably set to 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 examples of suitable methods include 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 through 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 device during cultivation, the gas mixture flow rate is not particularly limited, but is preferably adjusted to approximately 0.005 V / V / M (gas volume / liquid volume / min) to 2.0 V / V / M (gas volume / liquid volume / min). The pressure conditions are also not particularly limited as long as they allow the microorganisms to grow and produce substances, but should 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 selected to suit 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, the pH may be, for example, 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, the reaction time may be, for example, 6 to 340 hours, 8 to 300 hours, 9 to 250 hours, 10 to 200 hours, 11 to 150 hours, 12 to 140 hours, 13 to 130 hours, 14 to 120 hours, 15 to 110 hours, 16 to 100 hours, 17 to 95 hours, 18 to 90 hours, 19 to 85 hours, 20 to 80 hours, 25 to 75 hours, 30 to 70 hours, 35 to 65 hours, 40 to 60 hours, or 45 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 there is no particular upper limit, but it 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 is 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 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 cultivation 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 daidzein to equol was 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, % 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 there is no particular upper limit, but it 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 from genisteins to 5-hydroxyequol may be 0.200 (% / h) or more. In addition, "the molar conversion efficiency from genisteins to 5-hydroxyequol is 0.200 (% / h) or more" refers to the ability to produce equol from daidzein in the presence of daidzeins and genisteins, and the ability to convert genisteins to 5-hydroxyequol. Alternatively, it may mean initiating the cultivation of a microorganism capable of producing 5-hydroxyequol from genistein, and culturing the microorganism until the molar conversion efficiency of genisteins to 5-hydroxyequol reaches at least 0.200 (% / h). The molar conversion efficiency is the value (unit: "% / h") obtained by dividing the molar conversion rate (%) by the time required for cultivation (unit: "h", also referred to as the cultivation time).

[0034] The molar conversion efficiency of genisteins to 5-hydroxyequol was 0.120 (% / h) or more, 0.130 (% / h) or more, 0.140 (% / h) or more, 0.150 (% / h) or more, 0.160 (% / h) or more, 0.170 (% / h) or more, 0.180 (% / h) or more, 0.190 (% / h) or more, 0.200 (% / h) or more, 0.210 (% / h) or more, 0.220 (% / h) or more, 0.230 (% / h) or more, 0.250 ( % / h) or more, 0.300(% / h) or more, 0.350(% / h) or more, 0.400(% / h) or more, 0.450(% / h) or more, 0.500(% / h) or more, 0.550(% / h) or more, 0.600(% / h) or more Above, 0.650(% / h) or more, 0.700(% / h) or more, 0.750(% / h) or more, 0.800(% / h) or more, 0.850(% / h) or more, 0.900(% / h) or more, 0.950(% / h) or more, 1.00 0(% / h) or more, 1.050(% / h) or more, 1.100(% / h) or more, 1.150(% / h) or more, 1.200(% / h) or more, 1.250(% / h) or more, 1.300(% / h) or more, 1.350(% / h) ) or more, 1.400 (% / h) or more, 1.450 (% / h) or more, 1.500 (% / h) or more, 1.550 (% / h) or more, 1.600 (% / h) or more, 1.650 (% / h) or more, 1.700 (% / h) or more, 1. It may be 750 (% / h) or more, 1,800 (% / h) or more, 1,850 (% / h) or more, 1,900 (% / h) or more, 1,950 (% / h) or more, or 2,000 (% / h) or more, and the upper limit is not particularly limited, but may be 10,000 (% / h) or less, 9,000 (% / h) or less, 8,000 (% / h) or less, 7,000 (% / h) or less, 6,000 (% / h) or less, or 5,000 (% / h) or less. Specifically, it may be, for example, 0.120 (% / h) to 10,000 (% / h), 0.130 (% / h) to 9,000 (% / h), 0.140 (% / h) to 8,000 (% / h), 0.150 (% / h) to 7,000 (% / h), 0.160 (% / h) to 6,000 (% / h), or 0.170 (% / h) to 5,000 (% / h).

[0035] The molar conversion efficiency of daidzeins to equol may be 0.200 (% / h) or more. Alternatively, "the molar conversion efficiency of daidzeins to equol is 0.200 (% / h) or more" may mean initiating the cultivation of a microorganism capable of producing equol from daidzein and 5-hydroxyequol from genistein in the presence of daidzeins and genisteins, and culturing the microorganism until the molar conversion efficiency of daidzeins to equol reaches at least 0.200 (% / h). The molar conversion efficiency of daidzein to equol 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 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 Above, 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 It may be 0.400 (% / 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, 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), 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 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, 0.240 (% / h) or more, 0.250 (% / h) or more, 0.260 (% / h) or more, 0.270 (% / h) or more, 0.280 (% / h) or more, 0.290 (% / h) or more, 0.300 (% / h) or more, 0.310 (% / h) or more, 0.320 (% / h) or more, 0.330 (% / h) or more, 0.340 (% / 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, 1.2 00(% / 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, 1.60 It may be 0 (% / 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 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, 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) and above. It may be 6,000 (% / h), or 0.170 (% / h) to 5,000 (% / h).

[0037] The molar concentration ratio of genisteins to daidzeins at the start of culturing (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 100,000, 1,300 to 100,000, 1,400 to 50,000, 1,500 to 40,000, 1,600 to 30,000, 1,700 to 20,000, 1,800 to 15,000, 1,900 to 10,000, 2,000 to 7,000, or 3,000 to 5,000. Or, 0.900 or less, 0.800 or less, 0.700 or less, 0.600 or less, 0.500 or less, 0.400 or less, 0.350 or less, 0.300 or less, 0.250 or less, 0.200 or less, 0.180 or less, 0.170 or less, 0.160 or less, 0.150 or less, 0.140 or less, 0.130 or less, 0.120 or less, 0.110 or less, 0.100 or less, 0 It may be 0.090 or less, 0.080 or less, 0.070 or less, 0.060 or less, 0.050 or less, 0.040 or less, 0.030 or less, 0.020 or less, 0.010 or less, 0.006 or less, 0.005 or less, 0.004 or less, or 0.003 or less, or 0.0001 or more, 0.0005 or more, 0.001 or more, or 0.002 or more. Specifically, it may be, for example, 0.0001 to 0.900, 0.0005 to 0.800, 0.001 to 0.700, or 0.002 to 0.600.

[0038] The total content of daidzeins and genisteins in the composition containing daidzeins and genisteins at the start of culturing (or fermentation) is not particularly limited, 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, 8.0 ... or greater than 14.00 mM, 14.50 mM, 15.00 mM, 15.50 mM, 16.00 mM, 16.50 mM, or greater, or 17.00 mM. 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, 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, 10 ... 0.00mM or more, 1.50mM or more, 2.00mM or more, 2.50mM or more, 3.00mM or more, 3.50mM or more, 4.00mM or more, 4.50mM or more, 5.00mM or more, 5.50mM or more, 6.00mM or more, 6.50mM or more, 7.00mM or more, 7.50mM or more, 8.00mM or more, 8.50mM or more, 9.00mM or more, 9.50mM or more , 10.00 mM or more, 10.50 mM or more, 11.00 mM or more, 11.50 mM or more, 12.00 mM or more, 12.50 mM or more, 13.00 mM or more, 13.50 mM or more, 14.00 mM or more, 14.50 mM or more, 15.00 mM or more, 15.50 mM or more, 16.00 mM or more, 16.50 mM or more, or 17.00 mM or more. The upper limit is not particularly limited, and may be, for example, 400.00 mM or less, 300.00 mM or less, 200.00 mM or less, 100.00 mM or less, 50.00 mM or less, 40.00 mM or less, 30.00 mM or less, or 20.00 mM or less. Specifically, it may be, for example, 0.01 mM to 400.00 mM, 0.02 mM to 300.00 mM, 0.05 mM to 200.00 mM, 0.10 mM to 100.00 mM, 0.50 mM to 50.00 mM, 1.00 mM to 40.00 mM, 1.50 mM to 30.00 mM, 2.00 mM to 30.00 mM, or 2.50 mM to 20.00 mM.

[0040] The content of genistein in the composition containing daidzeins and genisteins at the time of starting the culture (or fermentation) is not particularly limited, 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, 8.0 ... or greater than 14.00 mM, 14.50 mM, 15.00 mM, 15.50 mM, 16.00 mM, 16.50 mM, or greater, or 17.00 mM. 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 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, 3.00 mM to 10.00 mM, or 3.50 mM to 5.00 mM.

[0041] The total content of equol and 5-hydroxyequol in the culture (or fermentation) at the end of the culture (or fermentation) is not particularly limited, 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, 8.0 ... The upper limit may be, but is not particularly limited to, 400.00 mM or less, 300.00 mM or less, 400.00 mM or less, 400.00 mM or less, 400.00 mM or less, 400.00 mM or less, 400.00 mM or less, 400.00 mM or less, 400.00 mM or more ... The concentration 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, 8.0 ... or greater than 14.00 mM, 14.50 mM, 15.00 mM, 15.50 mM, 16.00 mM, 16.50 mM, or greater, or 17.00 mM. 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, 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, 8.0 ... or greater than 14.00 mM, 14.50 mM, 15.00 mM, 15.50 mM, 16.00 mM, 16.50 mM, or greater, or 17.00 mM. The upper limit is not particularly limited either, 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] Daidzein and genistein compounds are fermented with microorganisms that have the ability to produce equol from daidzein compounds and 5-hydroxyequol from genistein compounds, resulting in the production of equol and 5-hydroxyequol per 1g of dry weight. Fermentates containing any value and ratio can be obtained by weight per unit volume.

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

[0046] The ratio of the equol content per 1 g of dry weight to the 5-hydroxyequol 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), is, for example, as follows: 0.001 or more, 0.005 or more, 0.01 or more, 0.02 or more, 0.03 or more, 0.0 The molecular weight may be 4 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.10 or more, 0.20 or more, 0.30 or more, 0.40 or more, 0.50 or more, 0.60 or more, 0.70 or more, 0.80 or more, 0.90 or more, 1.00 or more, 1.50 or more, 2.00 or more, 3.00 or more, 4.00 or more, or 5.00 or more. The upper limit is not particularly limited, but may be, for example, 30.00 or less, 20.00 or less, 10.00 or less, 9.00 or less, or 8.00 or less. Specifically, the molecular weight may be, for example, 0.001 to 30.00, 0.005 to 20.00, 0.01 to 10.00, 0.02 to 9.00, or 0.03 to 8.00.

[0047] The ratio of the molar concentration of 5-hydroxyequol to the molar concentration of equol in the resulting fermented product, i.e., the ratio of 5-hydroxyequol (mmol / L) divided by equol (mmol / L), is, for example, as follows: It may be 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, or 8.00 or less. Specifically, it may be, for example, 0.001 to 30.00, 0.005 to 20.00, 0.01 to 10.00, 0.02 to 9.00, or 0.03 to 8.00.

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

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

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

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

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

[0053] (Quantitative determination of isoflavone concentration) 2 g of the culture medium is mixed with 70% aqueous ethanol to make a final volume of 50 mL, solubilized by ultrasonication, and then filtered through a 0.45 μm filter. The filtrate is then analyzed by HPLC.

[0054] (How to calculate isoflavone concentration) The concentration of daidzin was 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 were calculated using the quantitative coefficients for daidzin (malonyldaidzin (1.444), malonylglycitin (1.351), malonylgenistin (1.095), acetyldaidzin (1.094), acetylglycitin (1.197), and acetylgenistin (1.064) described in WO2017 / 170995, and 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 (LC Laboratories, product number D-2946), glycitein (Fujifilm Wako Pure Chemical Industries, product number 076-04703), genistein (Fujifilm Wako Pure Chemical Industries, product number 073-05531), dihydrodaidzein (Tronto Research Chemicals, product number D449000), dihydrogenistein (Nagara Science Synthetic), equol (Daicel, product number 100152-001G), and 5-hydroxyequol (Tronto Research Chemicals, product number H825750) as standards.

[0055] (HPLC analysis of isoflavones) The following method is used based on the Ministry of Health, Labor and Welfare Notification (Shokuan-hatsu No. 0823001). Column: YMC-Pack ODS-AM Inner diameter 4.6 mm, length 250 mm (YMC Co., Ltd.) Column temperature: 25℃ 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 50min 0 100 55min 0 100 56min 100 0 65min 100 0 Flow rate: 1.0mL / min Detector: UV spectrophotometer (254 nm) Injection volume: 10μL Equipment used: HPLC (Shimadzu Corporation)

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

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

[0058] (Comparative Examples 1-3, Examples 4-12) Daidzein, genistein, and ABB medium and arginine as nutrients, as well as β-CD as an additive if necessary, were added, and 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.

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

[0060] The main culture medium thus prepared in Examples 1-13 and Comparative Examples 1-3 was added to 100 mL The vials were filled with 15 mL each, sealed with butyl rubber stoppers, and purged with nitrogen gas before being 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. The culture was then performed with shaking at 37°C and 250 rpm for the incubation times listed in Table 1. After cultivation, isoflavones were analyzed.

[0061] (Examples 14-15) Daidzein, genistein, and nutrients such as ABB medium yeast extract and arginine were added, and β-CD was added as an additive if necessary. 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 was as shown in Table 1. Each of the main culture media thus prepared in Examples 14 and 15 was dispensed in 1 L portions into 2 L mini jars, and after replacing the gas 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 agitation 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 cultivation, isoflavones were analyzed. The results are shown in Table 1. Table 2 shows the details of the strains used and their abbreviations.

[0062] [Table 1]

[0063] [Table 2]

[0064] Using these techniques, daidzein and genistein compounds can be fermented with microorganisms capable of producing equol from daidzein compounds and 5-hydroxyequol from genistein compounds, thereby producing fermented products containing equol and 5-hydroxyequol at any desired values ​​and ratios per gram of dry weight, as exemplified in Table 3. 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), suggesting that similar inhibition occurs with 5-hydroxyequol. Therefore, although the reason is unclear, it is speculated that when daidzein and genistein species are present at near-equal molar concentrations, the conversion of genistein species to 5-hydroxyequol is likely to be inhibited. Furthermore, as the concentrations of daidzein and genistein species move away from near-equal molar concentrations, it is speculated that the environment becomes more conducive to the conversion of genistein species to 5-hydroxyequol.

[0065] [Table 3] [Industrial Applicability]

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

Claims

[Claim 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, comprising: 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; a molar concentration ratio of the genisteins to the daidzeins at the start of the culture is 1.500 or more or 0.500 or less; the daidzein-class compound is daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, or a combination thereof; The method for producing the genisteins is genistin, malonylgenistin, acetylgenistin, genistein, dihydrogenistein or a combination thereof.

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