JP7632853B2 - Composition for the production of 6,3'-dihydroxyequol - Google Patents

Description

The present disclosure relates to compositions for the production of 6,3'-dihydroxyequol.

Equol is known to have the highest estrogenic activity among the isoflavone metabolites contained in soybeans (Non-Patent Documents 1 and 2).
Similarly to equol, equol derivatives such as 5-hydroxyequol have been reported to have estrogen-like activity and 3β-hydroxysteroid dehydrogenase inhibitory activity (Patent Document 1). By inhibiting the biosynthesis of aldosterone and glucocorticoids, they are expected to be useful in the prevention or treatment of excesses of these hormones.
Furthermore, in a report on a method for synthesizing isoflavan derivatives using an o-quinone methide-based technique, it has been disclosed that 3'-hydroxyequol can be obtained by chemical synthesis (Non-Patent Document 3).

On the other hand, enzymes known as flavin-dependent oxidases are known. The substrate specificity and action of these enzymes vary depending on the type. For example, it has been reported that HpaB and HpaC possessed by the Pseudomonas aeruginosa PAO1 strain are active against hydroxystilbene (resveratrol). A method for producing hydroxystilbene by allowing a substrate compound to react with a protein that has 50% or more homology at the amino acid level with HpaB and HpaC derived from the Pseudomonas aeruginosa PAO1 strain has been reported (Patent Documents 2 and 3).

In addition, the inventors of the present application have developed a technology that can convert equol in an equol-containing composition to 3'-hydroxyequol or 6-hydroxyequol using a specific enzyme or microorganism (Patent Document 4).

JP 2003-81875 A JP 2015-130819 A JP 2017-074019 A JP 2020-092690 A

Adlercreutz, H., The Lancet Oncol., 3, 364-373 (2002) Duncan, A. M. et al., Best Pract. Res. Clin. Endocrinol. Metab., 17, 253-271 (2003) Tetrahedron Letters, 49(18), 2974-2978 (2008)

The objective of the present disclosure is to provide at least a composition for producing 6,3'-dihydroxyequol.

<1> An equol-containing composition,
The composition comprises a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.
<2> The composition according to <1>, wherein the equol-containing composition is equol, a fermented soybean germ extract, a fermented soybean hypocotyl, a fermented soybean, or an alfalfa fermented product.
<3> The composition described in <1> or <2>, wherein the first enzyme and the second enzyme are flavin-dependent oxidases, and the first microorganism and the second microorganism are microorganisms having equol oxidation activity.
<4> A method for producing 6,3'-dihydroxyequol, comprising the step of reacting an equol-containing composition with a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.
<5> The manufacturing method described in <4>, wherein the acting step is a step of culturing the first microorganism and the second microorganism in a medium containing the equol-containing composition to produce 6,3'-dihydroxyequol.
<6> The production method described in <4> or <5>, wherein the first enzyme and the second enzyme are flavin-dependent oxidases, and the first microorganism and the second microorganism are microorganisms having equol oxidation activity.
<7> A method for producing a food or beverage containing 6,3'-dihydroxyequol, comprising the steps of: allowing a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol, to act on an equol-containing composition to produce 6,3'-dihydroxyequol; and blending the 6,3'-dihydroxyequol with ingredients for the food or beverage.
<8> A method for producing a pharmaceutical product containing 6,3'-dihydroxyequol, comprising the steps of: allowing a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol, to act on an equol-containing composition to produce 6,3'-dihydroxyequol; and blending the 6,3'-dihydroxyequol with pharmaceutical materials.

The present disclosure may have at least the effect of providing a composition for producing 6,3'-dihydroxyequol.

HPLC chromatogram showing the results of Example 2, one embodiment of the present disclosure, in which 1 indicates (S)-equol, 2 indicates (S)-3'-hydroxyequol, 3 indicates (S)-6-hydroxyequol, and 4 indicates (S)-6,3'-dihydroxyequol. Graph showing the results of Example 3, one embodiment of the present disclosure. In the figure, open circles represent (S)-equol, closed triangles represent (S)-3-hydroxyequol, closed squares represent (S)-6-hydroxyequol, and closed circles represent (S)-6,3'-dihydroxyequol. A graph showing the time course of conversion of (S)-equol to (S)-3'-hydroxyequol by Escherichia coli expressing HpaBro _-3 , as described in Patent Document 4. In the figure, open circles represent (S)-equol and closed circles represent (S)-3'-hydroxyequol. A graph showing the time course of conversion of (S)-equol to (S)-6-hydroxyequol by Escherichia coli expressing HpaB _pl-1 , as described in Patent Document 4. In the figure, open circles represent (S)-equol and closed circles represent (S)-6-hydroxyequol.

The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible as appropriate without departing from the spirit of this disclosure. This disclosure is not limited to the embodiments, but is limited only by the scope of the claims.

The composition of the present disclosure is a concept that includes a mixture, regardless of whether the components are homogeneous or heterogeneous.

(Composition)
One aspect of the present disclosure is a composition comprising an equol-containing composition, a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.

There are no limitations on the embodiment of the equol-containing composition, so long as it is a composition in which 6,3'-dihydroxyequol is produced by a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.
Examples of the equol-containing composition include fermented products of legumes such as soybeans, kidney beans, broad beans, peanuts, chickpeas, kudzu, red clover, alfalfa, licorice, etc. In other words, if the legume is soybeans, the equol-containing composition is a fermented product of soybeans themselves (soybean fermented product).
In addition, the equol-containing composition may be a fermented product of a part of soybean, such as a fermented product of soybean germ parts (fermented soybean germ product) or a fermented product of soybean hypocotyl parts (fermented soybean hypocotyl product) in the case of soybean among the legumes. Furthermore, in the case of soybean germ fermentation, the equol-containing composition may be a fermented product obtained by fermenting an extract obtained from soybean germ parts (fermented soybean germ extract product).
The soybean fermented product is a fermented product obtained by fermenting whole soybeans, and is different from fermented products obtained by fermenting a part of soybeans, such as the soybean germ fermented product obtained by using a soybean germ portion, and the soybean hypocotyl fermented product obtained by using a soybean hypocotyl portion.

As an example of the fermentation method, in the case of obtaining a fermented product of soybeans themselves (soybean fermented product), soybeans themselves may be prepared according to a conventional method, and koji mold may be added thereto and fermented to obtain a fermented product. As for the preparation method, soybeans themselves may be prepared as they are, or soybeans may be prepared by subjecting them to a heating treatment, drying treatment, steaming treatment, etc. and then mashing them, or soybeans may be prepared by subjecting them to a heating treatment, drying treatment, steaming treatment, etc. after mashing them.
The fermented soybean germ product, the fermented soybean hypocotyl product, and the fermented soybean germ extract product can be obtained in a manner according to the usual methods.

The equol-containing composition may be one obtained by acting an enzyme on the isoflavones derived from the legume plant, or a fermented product obtained by fermentation using a microorganism. For example, soybeans may be used as isoflavone aglycones obtained by acting an enzyme such as β-glucosidase on soybean-derived isoflavones, or isoflavone aglycones obtained by fermentation using a microorganism having β-glucosidase activity on soybean-derived isoflavones.

The equol-containing composition may be equol. The equol may be (R)-equol or (S)-equol.

In this embodiment, the first enzyme capable of converting equol in the equol-containing composition to 3'-hydroxyequol is not limited as long as it can convert equol in the equol-containing composition to 3'-hydroxyequol.
Furthermore, the enzyme is also capable of hydroxylating the 3'-position when the substrate is 6-hydroxyequol rather than equol. That is, the enzyme is capable of converting equol in the equol-containing composition to 3'-hydroxyequol and of converting 6-hydroxyequol to 6,3'-dihydroxyequol.

The enzyme may be an enzyme produced by a microorganism, or an enzyme obtained without relying on microbial production (for example, an enzyme obtained by chemical synthesis).
In the case of an enzyme produced by a microorganism, the microorganism may be a microorganism that originally expresses the enzyme, or a microorganism that has been made to express the enzyme by known techniques such as genetic recombination.
When an enzyme produced by a microorganism is to be recovered, a known method can be used as the recovery method. For example, after culturing a microorganism, the microorganism is collected by a method such as filtration or centrifugation, washed with a buffer solution or physiological saline, and the enzyme can be recovered by, for example, physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (e.g., treatment with a cell wall-lytic enzyme such as lysozyme, etc.), or chemical treatment (e.g., contact treatment with a surfactant, etc.), either alone or in combination. The recovered enzyme may then be subjected to separation and purification treatment.

Examples of the first enzyme capable of converting equol in the equol-containing composition to 3'-hydroxyequol include oxidoreductases (dehydrogenases, cytochromes, catalases, oxidases, oxygenases (e.g., flavin-dependent oxidases, etc.), fatty acid desaturases, etc.), transferases (acyltransferases, phosphotransferases, aminotransferases, etc.), protein degrading enzymes (proteases), lipid degrading enzymes (lipases), carbohydrate degrading enzymes (amylase, lysozyme, β-galactosidase, etc.), phosphate degrading enzymes (nucleases, phosphatases, restriction enzymes), hydrolases (urease, lactonase, ATP hydrolase, etc.), lyases (carbonic hydratase, pyruvate decarboxylase, etc.), isomerases (racemsases, phosphoglycerate phosphomutase, glucose-6-phosphate isomerase, etc.), and synthetic enzymes (DNA ligase, aminoacyl-tRNA synthetases, acyl-CoA synthetases, carboxylases, etc.).
Preferably it is an oxygenase, more preferably a flavin-dependent oxidase.

Examples of flavin-dependent oxidases include those described in Patent Document 4, such as HpaB _pa (SEQ ID NO: 1) derived from Pseudomonas aeruginosa PAO1, HpaB ec (SEQ ID NO: 2) derived from Escherichia coli BL21 (DE3), HpaB _{pl -2} (SEQ ID NO: 4) and HpaB _pl-3 (SEQ ID NO: 5) derived from Photorhabdus luminescens sub sp. laumondii TTO1, and HpaB _ro-1 (SEQ ID NO: 6), HpaB _ro-2 (SEQ ID NO: 7), and HpaB _ro-3 (SEQ ID NO: 8) derived from Rhodococcus opacus B4.

Furthermore, examples of the flavin reductase that supplies reduced flavin to the flavin-dependent oxidase include those described in Patent Document 4, such as _HpaCpa (SEQ ID NO: 17) derived from Pseudomonas aeruginosa PAO1.

Furthermore, the amino acid sequence of HpaB _pa derived from Pseudomonas aeruginosa PAO1 may be an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more identity to the amino acid sequence represented by SEQ ID NO:1, as long as it is capable of converting equol in the equol-containing composition to 3'-hydroxyequol.
The same applies to each HpaB and HpaC _pa other than the above-mentioned HpaB _pa .

The first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol is not limited in its form as long as it can convert equol in the equol-containing composition to 3'-hydroxyequol. The microorganism is preferably a microorganism having equol oxidizing activity (equol oxygenase), and more preferably a microorganism expressing the flavin-dependent oxidase.
Furthermore, the microorganism is also capable of hydroxylating the 3'-position when the substrate is 6-hydroxyequol rather than equol. That is, the microorganism is capable of converting equol in the equol-containing composition to 3'-hydroxyequol and also of converting 6-hydroxyequol to 6,3'-dihydroxyequol.

The microorganism may be a microorganism that is originally capable of converting equol to 3'-hydroxyequol, or may be a microorganism that has been modified by known techniques such as genetic recombination so that it can convert equol to 3'-hydroxyequol. There are no limitations on the classification, genus, species, etc. of such a microorganism, but preferred examples include bacteria (such as Escherichia coli), fungi (such as yeast, filamentous fungi), etc.

In this embodiment, the second enzyme capable of converting equol in the equol-containing composition to 6-hydroxyequol is not limited in its embodiment, so long as it can convert equol in the equol-containing composition to 6-hydroxyequol.
Furthermore, this enzyme is also capable of hydroxylating the 6-position when the substrate is 3'-hydroxyequol rather than equol. That is, the enzyme is capable of converting equol in the equol-containing composition to 6-hydroxyequol, and also of converting 3'-hydroxyequol to 6,3'-dihydroxyequol.

The enzyme may be an enzyme produced by a microorganism, or an enzyme obtained without relying on microbial production (for example, an enzyme obtained by chemical synthesis).
In the case of an enzyme produced by a microorganism, the microorganism may be a microorganism that originally expresses the enzyme, or a microorganism that has been made to express the enzyme by known techniques such as genetic recombination.
When an enzyme produced by a microorganism is to be recovered, a known method can be used as the recovery method. For example, after culturing a microorganism, the microorganism is collected by a method such as filtration or centrifugation, washed with a buffer solution or physiological saline, and the enzyme can be recovered by, for example, physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (e.g., treatment with a cell wall-lytic enzyme such as lysozyme, etc.), or chemical treatment (e.g., contact treatment with a surfactant, etc.), either alone or in combination. The recovered enzyme may then be subjected to separation and purification treatment.

Examples of enzymes that can convert equol in the equol-containing composition to 6-hydroxyequol include oxidoreductases (dehydrogenases, cytochromes, catalases, oxidases, oxygenases (e.g., flavin-dependent oxidases, etc.), fatty acid desaturases, etc.), transferases (acyltransferases, phosphotransferases, aminotransferases, etc.), protein degrading enzymes (proteases), lipid degrading enzymes (lipases), carbohydrate degrading enzymes (amylase, lysozyme, β-galactosidase, etc.), phosphate degrading enzymes (nucleases, phosphatases, restriction enzymes), hydrolases (urease, lactonase, ATP hydrolase, etc.), lyases (carbonic hydratase, pyruvate decarboxylase, etc.), isomerases (racems, phosphoglycerate phosphomutase, glucose-6-phosphate isomerase, etc.), and synthetic enzymes (DNA ligase, aminoacyl-tRNA synthetases, acyl-CoA synthetases, carboxylases, etc.).
Preferably it is an oxygenase, more preferably a flavin-dependent oxidase.

Examples of flavin-dependent oxidase include those described in Patent Document 4, such as HpaB _pl-1 (SEQ ID NO: 3) derived from Photorhabdus luminescens subsp. laumondii TTO1.

Furthermore, examples of the flavin reductase that supplies reduced flavin to the flavin-dependent oxidase include those described in Patent Document 4, such as _HpaCpa (SEQ ID NO: 17) derived from Pseudomonas aeruginosa PAO1.

Furthermore, the amino acid sequence of HpaB _pl-1 derived from Photorhabdus luminescens sub sp. laumondii TTO1 may be an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more identity to the amino acid sequence shown in SEQ ID NO:3, as long as it is capable of converting equol in the equol-containing composition to 6-hydroxyequol.
The same is true for _HpaCpa .

The second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol is not limited in its form as long as it is capable of converting equol in the equol-containing composition to 6-hydroxyequol. The microorganism is preferably a microorganism having equol oxidizing activity (equol oxygenase), and more preferably a microorganism expressing the flavin-dependent oxidase.
Furthermore, the microorganism is also capable of hydroxylating the 6-position when the substrate is 3'-hydroxyequol rather than equol. That is, the microorganism is capable of converting equol in the equol-containing composition to 6-hydroxyequol and also of converting 3'-hydroxyequol to 6,3'-dihydroxyequol.

The microorganism may be a microorganism that is originally capable of converting equol to 6-hydroxyequol, or may be a microorganism that has been modified by known techniques such as genetic recombination so that it can convert equol to 6-hydroxyequol. There are no limitations on the classification, genus, species, etc. of such a microorganism, but preferred examples include bacteria (such as Escherichia coli), fungi (such as yeast, filamentous fungi), etc.

(Production method)
Another aspect of the present disclosure is a method for producing 6,3'-dihydroxyequol, comprising the step of reacting an equol-containing composition with a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.

In this embodiment, the above-mentioned explanations are incorporated with respect to the equol-containing composition, the first enzyme or first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and the second enzyme or second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.

In this embodiment, the step of subjecting the equol-containing composition to a first enzyme capable of converting equol in the equol-containing composition to 3'-hydroxyequol includes a step of reacting equol in the equol-containing composition with the enzyme in a solution containing the equol-containing composition.
Note that, although the process of allowing a first enzyme capable of converting equol in the equol-containing composition to act on the equol-containing composition to convert the equol in the equol-containing composition to 3'-hydroxyequol is described here, the same applies to the process of allowing a second enzyme capable of converting equol in the equol-containing composition to act on the equol-containing composition to convert the equol in the equol-containing composition to 6-hydroxyequol.

Examples of solutions containing the equol-containing composition include water, organic solvents, and two-phase mixtures of an organic solvent and an aqueous medium.
Examples of the organic solvent include ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane.
Examples of the aqueous medium include ethanol and acetone.

In addition, in order to increase the solubility of the substrate equol, an inclusion compound may be added to the solution.
Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and their analogues, such as methyl-β-cyclodextrin, trimethyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.
The amount of the inclusion compound added to the solution is, in terms of molar ratio, typically 0.1 equivalents or more, preferably 0.5 equivalents or more, and more preferably 1.0 equivalents or more relative to the equol in the equol-containing composition, and is typically 5.0 equivalents or less, preferably 2.5 equivalents or less, and more preferably 2.0 equivalents or less.

The reaction temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, and further preferably 30°C to 37°C.
The reaction time is preferably 8 to 340 hours, more preferably 12 to 170 hours, and even more preferably 16 to 120 hours.
The pH of the reaction solution is preferably 4 to 10, more preferably 6 to 8.

In this embodiment, when an enzyme produced by a microorganism is used as the first enzyme capable of converting equol in the equol-containing composition to 3'-hydroxyequol, the method may include a step of recovering the enzyme from the microorganism that produces the enzyme prior to the step of allowing the enzyme to act on the equol-containing composition, and may include a step of culturing the microorganism that produces the enzyme prior to that.

The step of culturing the microorganism that produces the enzyme is not particularly limited as long as it is carried out under conditions that allow the microorganism to produce the enzyme. For example, the conditions for culturing the microorganism in a medium containing an equol-containing composition, which will be described later, can be mentioned.

The process of recovering the enzyme from the microorganisms that produce the enzyme may include a process of collecting the microorganisms by a method such as filtration or centrifugation, and may further include a process of washing the microorganisms with a buffer solution or physiological saline. Furthermore, the process may include a process of performing a physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), a biochemical treatment (e.g., treatment with a cell wall-lytic enzyme such as lysozyme, etc.), or a chemical treatment (e.g., contact treatment with a surfactant, etc.), and these processes may be used alone or in combination to recover the enzyme. The process may also include a process of purifying or concentrating the enzyme.

In this embodiment, the step of subjecting the equol-containing composition to the action of a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol includes a step of causing the microorganism to convert equol in the equol-containing composition to 3'-hydroxyequol in a solution containing the equol-containing composition, for example, a step of culturing the microorganism in a medium containing the equol-containing composition and producing 3'-hydroxyequol by fermentation from equol in the equol-containing composition.
Note that, although the process of treating the equol-containing composition with a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol is described here, the same applies to the process of treating the equol-containing composition with a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.

A solution containing an equol-containing composition may be a solution in which the microorganisms can grow or not.

Examples of solutions in which the microorganisms can grow include culture media, which may be minimal or synthetic media. Commercially available media include, for example, ANAEROBE BASAL BROTH (ABB medium) manufactured by Oxoid, Wilkins-Chalgren Anaerobe Broth (CM0643) manufactured by Oxoid, GAM medium, modified GAM medium, brain heart infusion medium, LB medium, and the like manufactured by Nissui Pharmaceutical Co., Ltd.

Water-soluble organic matter can be added to the medium as a carbon source. Examples of water-soluble organic matter include sugars such as glucose, arabinose, sorbitol, fructose, mannose, sucrose, trehalose, xylose, galactose, starch, starch hydrolysates, molasses, and blackstrap molasses; natural carbohydrates such as wheat and corn; alcohols such as glycerol, methanol, and ethanol; hydrocarbons such as normal paraffin; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, fumaric acid, gluconic acid, pyruvic acid, and citric acid; and amino acids such as glycine, glutamine, and asparagine.
The concentration of the organic matter can be appropriately adjusted for efficient growth, and is generally in the range of 0.1 to 10 wt/vol %.

In addition to the carbon source, the medium may contain a nitrogen source, which may be any of various nitrogen compounds that can be used in normal fermentation.
Preferred inorganic nitrogen sources include ammonium salts, nitrates, urea, and the like, and more preferred are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, and sodium nitrate.
Examples of organic nitrogen sources include amino acids, milk casein, casamino acids, corn steep liquor, yeast extract, peptones (e.g., polypeptone N, soybean peptone, etc.), meat extracts (e.g., Ehrlich bonito extract, Lab-Remco powder, bouillon, etc.), seafood extracts, liver extracts, digestive serum powder, fish oil, etc.

In addition to carbon and nitrogen sources, growth and activity can sometimes be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. For example, the following are examples of microbial growth cofactors derived from plants and animals, such as inorganic compounds, vitamins, and fatty acids.

Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium sulfate Folic acid Manganese sulfate Pyridoxine Sodium chloride Thiamine Cobalt chloride Riboflavin Calcium chloride Nicotinic acid Zinc sulfate Pantothenic acid Copper sulfate Vitamin B12
Alum, Thiooctic acid, Sodium molybdate, p-aminobenzoic acid, Potassium chloride, Vitamin K
Boric acid, etc. Nickel chloride Sodium tungstate Sodium selenate Ammonium ferrous sulfate Sodium acetate trihydrate Magnesium sulfate heptahydrate Manganese sulfate tetrahydrate

In addition, growth may be improved by adding to the medium known reducing agents such as L-cysteine (hydrochloride), thioglycolic acid, ascorbic acid, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, sodium sulfide, sulfite, thioglycolic acid, and rutin, reducing activators such as L-cystine, and enzymes that break down reactive oxygen species such as catalase and superoxide mutase.

As a culture method, for example, conventional culture methods such as shaking culture, aeration and agitation culture, continuous culture, and fed-batch culture can be used.

The gas phase and aqueous phase during the culture preferably do not contain air or oxygen, and may, for example, contain nitrogen and/or hydrogen in any ratio, or nitrogen and/or carbon dioxide in any ratio. A gas phase and aqueous phase containing hydrogen are preferred. The proportion of hydrogen in the gas phase is usually 0.5% or more, preferably 1.0% or more, more preferably 2.0% or more, since oxidation of the 3' position is promoted, while it is usually 100% or less, preferably 20% or less, more preferably 10% or less.

There are no particular limitations on the method for creating such an environment for the gas phase and aqueous phase during culture, but examples of the method include replacing the gas phase with the gas before culture, supplying the gas from the bottom of the culture vessel and/or supplying the gas to the gas phase part of the culture vessel during culture, and bubbling the aqueous phase with the gas before culture. As the hydrogen, hydrogen gas may be used as it is. Alternatively, a hydrogen source such as formic acid and/or a salt thereof may be added to the culture medium, and hydrogen may be generated during culture by the action of the microorganisms.

The amount of ventilation is, for example, 0.005 to 2 vvm, and preferably 0.05 to 0.5 vvm. The mixed gas can also be supplied in the form of nanobubbles.
The culture temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, and even more preferably 30°C to 37°C.
The pressurization conditions for the culture vessel are not particularly limited as long as they allow growth, and are, for example, 0.001 to 1 MPa, preferably 0.01 to 0.5 MPa.
The culture time is usually 8 to 340 hours, preferably 12 to 170 hours, more preferably 16 to 120 hours.
The pH of the medium at the start of the culture is usually 4 to 10, preferably 6 to 8.

In addition, the addition of a surfactant, adsorbent, inclusion compound, etc. to the medium may promote the oxidation of the 3'-position.
Examples of surfactants include SDS, TritonX-100, Tween80, and Adekanol, and can be added at about 0.001 g/L to 10 g/L.
Examples of the adsorbent include cellulose and its derivatives; dextrin; hydrophobic adsorbents such as the Diaion HP series and Sepabeads series manufactured by Mitsubishi Chemical Corporation; and the Amberlite XAD series manufactured by Organo Corporation.
Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and their analogues, such as methyl-β-cyclodextrin, trimethyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.
The amount of the inclusion compound added is, in terms of molar ratio, usually 0.1 equivalents or more, preferably 0.5 equivalents or more, and more preferably 1.0 equivalents or more, relative to the equol in the equol-containing composition, and is usually 5.0 equivalents or less, preferably 2.5 equivalents or less, and more preferably 2.0 equivalents or less.

Examples of solutions in which the microorganisms cannot grow include salt solutions and buffer solutions. An example of a salt solution is physiological saline. Examples of buffer solutions include phosphate buffer, Tris-HCl buffer, citrate-phosphate buffer, citrate buffer, MOPS buffer, acetate buffer, and glycine buffer. The pH and concentration of the buffer can be appropriately adjusted according to conventional methods.

Such a solution in which the microorganism cannot grow can be used, for example, when the microorganism is in a stationary state. A stationary state refers to a microbial organism that is not growing and that is obtained by removing medium components from a cultured microorganism by centrifugation or other procedures, washing with a salt solution or buffer solution, and then suspending the microbial organism in the same liquid as the washing liquid. In this embodiment, the stationary state refers to a microbial organism that has at least a metabolic system capable of producing 3'-hydroxyequol from equol.

The case where the solution may be either one in which the microorganism can grow or one in which the microorganism cannot grow is, for example, a case where an immobilized microorganism is used as the microorganism, which is a microorganism immobilized by a known method such as a polyacrylamide gel method, a sulfur-containing polysaccharide gel method (carrageenan gel method), an alginic acid gel method, or an agar gel method.
In the present disclosure, such gels that fix microorganisms are also defined as solutions.

This embodiment may include, for example, a step of quantifying the obtained 6,3'-dihydroxyequol. The quantification method may follow a conventional method. For example, a portion of the culture medium may be sampled, appropriately diluted, thoroughly stirred, and then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, followed by quantifying the amount by high performance liquid chromatography.

This embodiment may also include a step of recovering the obtained 6,3'-dihydroxyequol. The recovery step includes a purification step, a concentration step, and the like. Purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), and the like; removal of solids and polymeric substances; extraction with an organic solvent, an ionic liquid, and the like; and adsorption and decolorization using a hydrophobic adsorbent, an ion exchange resin, an activated carbon column, and the like. Concentration treatments in the concentration step include concentration using an evaporator, a reverse osmosis membrane, and the like.
Furthermore, a solution containing 6,3'-dihydroxyequol can be powdered by freeze-drying, spray-drying, etc. In the powdering process, an excipient such as lactose, dextrin, corn starch, etc. can also be added.

Another aspect of the present disclosure is a method for producing a food or beverage containing 6,3'-dihydroxyequol, comprising the steps of: reacting an equol-containing composition with a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol, thereby producing 6,3'-dihydroxyequol; and blending the 6,3'-dihydroxyequol with ingredients for the food or beverage.

The process for producing 6,3'-dihydroxyequol is the same as that described above. In this embodiment, it is preferable to include a process for recovering the produced 6,3'-dihydroxyequol after the process for producing 6,3'-dihydroxyequol, and to combine the recovered 6,3'-dihydroxyequol with ingredients for a food or beverage.

The ingredients for food and beverages can be any ingredients commonly used for food and beverages, and there are no particular restrictions on the time of mixing.

The food and drink products produced may contain water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. as main components.
Examples of proteins include animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, and meat protein, as well as hydrolysates thereof, and butter.
Examples of carbohydrates include sugars, modified starches (dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fibers.
Examples of lipids include vegetable oils and fats such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, fish oil, fractionated oils thereof, hydrogenated oils, and interesterified oils.
Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid.
Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals.
Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid.
These components may be used in combination of two or more kinds, or may be synthetic products.

Specific examples of the foods and beverages to be manufactured include general foods and beverages, as well as foods for specified health uses, nutritional supplements, functional foods, and foods for the sick. There are no particular limitations on the form of these foods and beverages, but specific examples include main dishes such as bread and noodles; side dishes such as cheese, ham, sausages, and seafood products; beverages such as fruit juice drinks, carbonated drinks, and milk drinks; luxury items such as cookies, cakes, jellies, puddings, candies, and yogurt; and supplements such as capsules (soft capsules and hard capsules), tablets, granules, powders, jellies, liposome preparations, and nutritional drinks.

The amount of 6,3'-dihydroxyequol contained in the total amount of the food or beverage produced is not particularly limited, but is preferably an amount that allows the effects of 6,3'-dihydroxyequol to be obtained when the food or beverage is ingested. The amount of 6,3'-dihydroxyequol contained in the total amount of the food or beverage is usually 0.0001 to 50% by mass, preferably 0.001 to 50% by mass, and more preferably 0.01 to 50% by mass.

When the food or beverage is a supplement, it may be in the form of a solid, gel, or liquid, for example, various processed food or beverage products, powders, tablets, pills, capsules, jellies, granules, etc.

The supplement may also contain additives such as excipients such as dextrin, preservatives such as vitamin C, flavorings such as vanillin, colorants such as safflower color, monosaccharides, oligosaccharides and polysaccharides (e.g., glucose, fructose, sucrose, saccharose, and carbohydrates containing these), acidulants, flavorings, oils and fats, emulsifiers, whole milk powder, or agar. Two or more of these ingredients may be used in combination, or may be synthetic products.

Another aspect of the present disclosure is a method for producing a pharmaceutical product containing 6,3'-dihydroxyequol, comprising the steps of: treating an equol-containing composition with a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol, thereby producing 6,3'-dihydroxyequol; and combining the 6,3'-dihydroxyequol with pharmaceutical ingredients.

The process for producing 6,3'-dihydroxyequol is the same as that described above. In this embodiment, it is preferable to include a process for recovering the produced 6,3'-dihydroxyequol after the process for producing 6,3'-dihydroxyequol, and to combine the recovered 6,3'-dihydroxyequol with a pharmaceutical ingredient.

Pharmaceutical raw materials may be any commonly used pharmaceutical raw materials, and there are no particular restrictions on the timing of their addition.

The pharmaceuticals produced can be used as pharmaceuticals for preventing or treating diseases that can be prevented or treated by ingestion or administration of 6,3'-dihydroxyequol. The dosage form can be selected according to the disease to be prevented or treated, the mode of use of the pharmaceutical, the route of administration, etc. Examples include pharmaceuticals for internal use such as tablets, granules, powders, capsules, soft capsules, and syrups; pharmaceuticals for external use such as emulsions, suspensions, ointments, creams, lotions, gels, sprays, patches, poultices, liniments, aerosols, ointments, packs, inhalants, and suppositories; and injections. These various preparations can be formulated according to conventional methods using known auxiliary agents that can be normally used in the pharmaceutical formulation technology field, such as fillers, bulking agents, excipients, binders, moisturizing agents, disintegrants, surfactants, lubricants, colorants, flavorings, dissolving agents, suspending agents, and coating agents, as necessary for the main drug. In addition, the medicine may contain coloring agents, preservatives, fragrances, flavorings, sweeteners, and other medicines.

The amount of 6,3'-dihydroxyequol contained in the total amount of the pharmaceutical is not particularly limited, but is preferably an amount that allows the desired effect of 6,3'-dihydroxyequol to be obtained when the pharmaceutical is ingested or administered. The amount of 6,3'-dihydroxyequol contained in the total amount of the pharmaceutical is usually 0.0001 to 50% by mass, preferably 0.001 to 50% by mass, and more preferably 0.01 to 50% by mass.

In the present disclosure, the information on the base sequence of the flavin-dependent oxidase gene, the base sequence of the flavin reductase gene that supplies reduced flavin to the flavin-dependent oxidase, and the amino acid sequences of the enzymes encoded by each of them are as follows:
SEQ ID NO: 1 is the amino acid sequence of the enzyme _HpaBpa encoded by the gene _hpaBpa (ORF No. PA4091) of flavin-dependent oxidase derived from Pseudomonas aeruginosa PAO1.
SEQ ID NO: 2 is the amino acid sequence of the enzyme _HpaBec encoded by the gene _hpaBpa (ORF No. B21_04188) of flavin-dependent oxidase derived from Escherichia coli BL21 (DE3).
SEQ ID NO: 3 is the amino acid sequence of the enzyme HpaB pl _-1 encoded by the flavin-dependent oxidase gene hpaB _pl-1 (ORF No. plu0246) derived from Photorhabdus luminescens subsp. laumondii TTO1.
SEQ ID NO: 4 is the amino acid sequence of the enzyme HpaB _pl-2 encoded by the flavin-dependent oxidase gene (ORF No. plu0975) derived from Photorhabdus luminescens subsp. laumondii TTO1.
SEQ ID NO:5 is the amino acid sequence of the enzyme HpaB _pl-3 encoded by the flavin-dependent oxidase gene (ORF No. plu4027) derived from Photorhabdus luminescens subsp. laumondii TTO1.
SEQ ID NO: 6 is the amino acid sequence of the enzyme HpaB _ro-1 encoded by the flavin-dependent oxidase gene (ORF No. ROP_20940) derived from Rhodococcus opacus B4.
SEQ ID NO: 7 is the amino acid sequence of the enzyme HpaB _ro-2 encoded by the flavin-dependent oxidase gene (ORF No. ROP_22410) derived from Rhodococcus opacus B4.
SEQ ID NO: 8 is the amino acid sequence of the enzyme HpaB _ro-3 encoded by the flavin-dependent oxidase gene (ORF No. ROP_37410) derived from Rhodococcus opacus B4.
SEQ ID NO: 17 is the amino acid sequence of the enzyme _HpaCpa derived from Pseudomonas aeruginosa PAO1 and encoded by the gene (ORF no. PA4092) of flavin reductase that supplies reduced flavin to flavin-dependent oxidase.

SEQ ID NO:9 is the nucleotide sequence of the gene _hpaBpa , which is a flavin-dependent oxidase gene derived from Pseudomonas aeruginosa PAO1 and corresponds to ORF No. PA4091.
SEQ ID NO: 10 is the nucleotide sequence of the gene _hpaBec , which is a flavin-dependent oxidase gene derived from Escherichia coli BL21 (DE3) and corresponds to ORF No. B21_04188.
SEQ ID NO:11 is the nucleotide sequence of the gene hpaB _pl-1 , which is a flavin-dependent oxidase gene derived from Photorhabdus luminescens subsp. laumondii TTO1 and corresponds to ORF No. plu0246.
SEQ ID NO: 12 is the nucleotide sequence of the gene hpaB _pl-2 , which is a flavin-dependent oxidase gene derived from Photorhabdus luminescens subsp. laumondii TTO1 and corresponds to ORF No. plu0975.
SEQ ID NO: 13 is the nucleotide sequence of the gene hpaB _pl-3 , which is a flavin-dependent oxidase gene derived from Photorhabdus luminescens subsp. laumondii TTO1 and corresponds to ORF No. plu4027.
SEQ ID NO: 14 is the nucleotide sequence of the gene hpaB _ro-1, which is a flavin-dependent oxidase gene derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_20940.
SEQ ID NO: 15 is the nucleotide sequence of the gene hpaB _ro-2, which is a flavin-dependent oxidase gene derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_22410.
SEQ ID NO: 16 is the nucleotide sequence of the gene hpaB _ro-3, which is a flavin-dependent oxidase gene derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_37410.
SEQ ID NO: 18 is the nucleotide sequence of the gene _hpaCpa , which is derived from Pseudomonas aeruginosa PAO1 and is a gene for flavin reductase that supplies reduced flavin to flavin-dependent oxidase, and corresponds to ORF no. PA4092.

Examples are described below, but none of the examples should be construed as limiting.

[Example 1] Construction of recombinant E. coli expressing flavin-dependent oxidase
Two genes encoding flavin-dependent oxidases were used. Specifically, the gene hpaB _pl-1 (corresponding to ORF No. plu0246, the amino acid sequence of which is shown in SEQ ID NO: 3) derived from Photorhabdus luminescens sub sp. laumondii TTO1 and the gene hpaB _ro-3 (corresponding to ORF No. ROP_37410, the amino acid sequence of which is shown in SEQ ID NO: 8) derived from Rhodococcus opacus B4 were synthesized.
hpaB _pl-1 was amplified by PCR using primers of SEQ ID NOs: 19 and 20, then digested with restriction enzymes PciI and BamHI, and ligated to pETDuet-1 vector (Novagen) to prepare pETDhpaB _pl-1 .
For hpaB _ro-3 , primers of SEQ ID NOs: 21 and 22 were used to amplify the gene by PCR, followed by cleavage with restriction enzymes NdeI and MunI, and then ligated to the pETDuet-1 vector to prepare pETDhpaB _ro-3 .

In addition, a gene encoding a flavin reductase that supplies reduced flavin to the flavin-dependent oxidase was also used. Specifically, the gene _hpaCpa (corresponding to ORF no. PA4092; the amino acid sequence is shown in SEQ ID NO: 17) derived from Pseudomonas aeruginosa PAO1 was ligated to the pCDFDuet-1 vector (Novagen) to form the plasmid _pCDFDhpaCpa .

Furthermore, pETDhpaB _pl-1 and pETDhpaB _ro-3 were each introduced into Escherichia coli BL21 Star (DE3) together with pCDFDhpaC _pa by the heat shock method. When _{pETDhpaB pl-1} was introduced, the plasmid pGro7 (Takarabio) to which a gene encoding a chaperonin was linked was also introduced to promote soluble expression of the protein.

[Example 2] Analysis of the reaction between flavin-dependent oxidase and (S)-equol
E. coli carrying pETDhpaB _pl-1 , pCDFDhpaC _pa and pGro7 were cultured and expression induced as follows. The recombinant E. coli was inoculated into LB medium (tryptone 1%, yeast extract 0.5%, NaCl 1% (pH 7.0)) containing 50 μg/ml ampicillin, 50 μg/ml streptomycin, 30 μg/ml chloramphenicol and 4 mg/ml arabinose, and cultured at 30°C for 6 hours. After 6 hours, 1 mM isopropyl-β-D-thiogalactopyranoside was added and cultured at 15°C for another 15 hours to induce gene expression. After harvesting, the bacteria were washed with 50 mM potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol, and the collected bacteria were used for the reaction.

E. coli carrying pETDhpaB _ro-3 and pCDFDhpaC _pa were cultured and expression induced as follows. The recombinant E. coli was inoculated into LB medium containing 50 μg/ml ampicillin and 50 μg/ml streptomycin and cultured at 30°C for 6 hours. After 6 hours, 1 mM isopropyl-β-D-thiogalactopyranoside was added and cultured at 25°C for an additional 15 hours to induce gene expression. After harvesting, the bacteria were washed with 50 mM potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol, and the collected bacteria were used in the reaction.

For the reaction, 250 g/l (final concentration, wet cell weight) of E. coli cells carrying pETDhpaB _pl-1 , pCDFDhpaC _pa , and pGro7, and 125 g/l (final concentration, wet cell weight) of E. coli cells carrying pETDhpaB _ro-3 and pCDFDhpaC _pa were added simultaneously, and 20 ml of a reaction solution containing 10 mM (S)-equol, 4% (v/v), 4% (v/v) of dimethyl sulfoxide, 10% (v/v) of glycerol, 1.5% (v/v) of Tween 80, and 200 mM potassium phosphate buffer (pH 7.5) was prepared and shaken at 30°C.

After the reaction, 5 μl of 5N HCl, 250 μl of H ₂ O, and 500 μl of methanol were added to 250 μl of the above reaction solution to prepare a sample for HPLC analysis. HPLC analysis was performed using a Shimadzu device (Prominence, LC-20 system) and a Waters column (XTerra MS C18 IS, column length 4.6 × 20 mm, particle size 3.5 μm). The developing solvent was 0.1% formic acid solution (solution A) and methanol (solution B). After flowing at 5% solution B from 0 to 3 minutes at a flow rate of 0.5 ml/min, the concentration of solution B was increased linearly from 3 to 4 minutes to 40%, from 4 to 14 minutes to 80%, and from 14 to 15 minutes to 100%. Furthermore, after flowing 100% solution B from 15 to 18 minutes, solution B was decreased linearly to 5% from 18 to 19 minutes, and then solution B was increased to 5% from 19 to 22 minutes to elute the sample.

As a result, after 4 hours of reaction, three peaks were detected in addition to the peak of the substrate (S)-equol (retention time 14.5 minutes, peak 1) in HPLC analysis (Figure 1). As described in Patent Document 4, HpaB _ro-3 is known to have the activity of hydroxylating the 3' position of (S)-equol, and the peak at 13.6 minutes (peak 2) coincided with the retention time of (S)-3'-hydroxyequol. As described in Patent Document 4, HpaB _pl-1 is known to have the activity of hydroxylating the 6th position of (S)-equol, and the peak at 13.2 minutes (peak 3) coincided with the retention time of (S)-6-hydroxyequol. Furthermore, a peak corresponding to a new conversion product was detected at a retention time of 12.5 minutes (peak 4).

Analysis of this conversion product by LC-MS suggested that it was a dihydroxylated form of (S)-equol. Furthermore, when the conversion product was purified and analyzed by NMR, it was identified as (S)-6,3'-dihydroxyequol, hydroxylated at the 6- and 3'-positions. The results of the NMR and LC-MS analyses are shown below.

¹ H NMR (400 MHz, methanol-d ₄ ): δ=2.78 (m, 2H, H-4), 2.96 (m, 1H, H-3), 3.81 (m, 1H, H-2), 4.12 (m, 1H, H-2), 6.24 (s, 1H, H-8), 6.47 (s, 1H, H-5), 6.57 (dd, J=8.1, 1.9 Hz, 1H, H-6'), 6.67 (d, J=1.9 Hz, 1H, H-2'), 6.71 (d, J=8.1 Hz, 1H, H-5'); ¹³ C NMR (400 MHz, methanol-d ₄ ): δ=148.7 (C-8a), 146.4 (C-3'), 145.4 (C-7), 145.2 (C-4'), 140.1 (C-6), 134.9 (C-1'), 119.6 (C-6'), 116.5 (C-5), 116.5 (C-5'), 115.4 (C-2'), 113.8 (C-4a), 104.4 (C-8), 72.1 (C-2), 39.8 (C-3), 33.2 (C-4); MS (ESI) (m/z): calculated for C ₁₅ H ₁₄ O ₅ [MH] ^- : 273.076; found: 273.068.

Example 3: Production of (S)-6,3'-dihydroxyequol on a flask scale Using the method shown in Example 2, we attempted to produce (S)-6,3'-dihydroxyequol on a flask scale. As a result, (S)-equol was converted to (S)-6,3'-dihydroxyequol via (S)-3'-hydroxyequol or (S)-6-hydroxyequol (Figure 2). The amount of (S)-6,3'-dihydroxyequol produced reached 8.7 mM (2.4 g/l) in 24 hours. Furthermore, no by-products were detected (Figure 1). From the above, it was demonstrated that (S)-6,3'-dihydroxyequol can be efficiently produced on a flask scale.

In Patent Document 4, when E. coli expressing HpaB _ro-3 was reacted with (S)-equol, (S)-3'-hydroxyequol was produced (Figure 3), and when E. coli expressing HpaB _pl-1 was reacted with (S)-equol, (S)-6-hydroxyequol was produced (Figure 4), but (S)-6,3'-dihydroxyequol was not produced by either enzyme alone. In this study, (S)-6,3'-dihydroxyequol was produced by simultaneously reacting E. coli expressing HpaB _ro-3 and E. coli expressing HpaB _pl-1 with (S)-equol. Although the substrate specificity of enzymes is generally strict, the results of this study indicate that HpaB _ro-3 also acts on (S)-6-hydroxyequol, and that HpaB _pl-1 also acts on (S)-3'-hydroxyequol. By utilizing this unexpectedly broad substrate specificity, it became possible to produce (S)-6,3'-dihydroxyequol. Furthermore, when only one of the enzymes reacts with (S)-equol, the substrate (S)-equol remains (Figures 3 and 4), resulting in waste of raw material. On the other hand, when E. coli expressing HpaB _ro-3 and E. coli expressing HpaB _pl-1 are reacted with (S)-equol simultaneously, all of the (S)-equol is converted to (S)-6,3'-dihydroxyequol without any remaining (S)-equol (Figure 1), making this reaction an efficient and groundbreaking synthetic method that eliminates waste of raw materials.

Claims (10)

An equol-containing composition,
A composition comprising a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol,
the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, the enzyme being capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The composition, wherein the second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO:3, and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol (excluding embodiments in which the first enzyme and/or the second enzyme is cytochrome P450). The composition according to claim 1, wherein the equol-containing composition is equol, a fermented soybean germ extract, a fermented soybean hypocotyl, a fermented soybean, or an alfalfa fermented product. the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol,
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO:8, the enzyme being an enzyme that can convert the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The composition according to claim 1 or 2, wherein the second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO: 3 , and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol. A method for producing 6,3'-dihydroxyequol, comprising the step of reacting an equol-containing composition with a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol,
the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, the enzyme being capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:3, and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol (excluding embodiments in which the first enzyme and/or the second enzyme is cytochrome P450). The method according to claim 4 , wherein the affecting step is a step of culturing the first microorganism and the second microorganism in a medium containing the equol-containing composition to produce 6,3'-dihydroxyequol. the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol,
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO:8, the enzyme being an enzyme that can convert the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The production method according to claim 4 or 5, wherein the second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO: 3 , and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol. A method for producing a food or beverage containing 6,3'-dihydroxyequol, comprising the steps of: allowing a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol, to act on an equol-containing composition to produce 6,3'-dihydroxyequol; and blending the 6,3'-dihydroxyequol with ingredients for the food or beverage,
the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, the enzyme being capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:3, and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol (excluding embodiments in which the first enzyme and/or the second enzyme is cytochrome P450). the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol,
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO:8, the enzyme being an enzyme that can convert the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The method according to claim 7, wherein the second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO: 3 , and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol. A method for producing a pharmaceutical product containing 6,3'-dihydroxyequol, comprising the steps of: treating an equol-containing composition with a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3'-hydroxyequol, and a second enzyme or a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol, thereby producing 6,3'-dihydroxyequol; and blending the 6,3'-dihydroxyequol with a pharmaceutical material,
the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 and 4 to 8, the enzyme being capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:3, and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol (excluding embodiments in which the first enzyme and/or the second enzyme is cytochrome P450). the first enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:8, capable of converting the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol,
the second enzyme is an enzyme having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:3, capable of converting the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol,
the first microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO:8, the enzyme being an enzyme that can convert the equol to the 3'-hydroxyequol and the 6-hydroxyequol to the 6,3'-dihydroxyequol;
The method according to claim 9, wherein the second microorganism is a microorganism that expresses an enzyme having an amino acid sequence that has 90% or more identity to the amino acid sequence represented by SEQ ID NO: 3 , and the enzyme is an enzyme that can convert the equol to the 6-hydroxyequol and the 3'-hydroxyequol to the 6,3'-dihydroxyequol.

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