Method for purifying daidzin

Abstract

To provide a simplified method enabling purification of daidzin from a plant extract.SOLUTION: A method for purifying daidzin comprises suspending 10 g or more of a plant extract containing daidzin per 1 L of an aqueous ethanol solution of 30-90 vol.%, the plant extract containing 1-10 wt.% of genistein and 1-30 wt.% of glycitein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for purifying daidzin. [Background technology]

[0002] Isoflavones, which are found in large quantities in legumes such as soybeans and kudzu, are a type of polyphenol and a flavonoid with an isoflavone as its basic structure. Recent research has revealed that isoflavones have female hormone (estrogen) and antioxidant properties, and that consuming isoflavones has a preventive effect against breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, and other conditions. In soybeans, for example, isoflavones exist as glycosides covalently bound to sugars, such as daidzin, glycitin, and genistin, with only small amounts of aglycones present. These glycosides are also malonylated and acetylated. Once in the human or animal body, these glycosides are converted to daidzein, glycitein, and genistein, respectively, by digestive enzymes or the enzyme β-glucosidase produced by intestinal bacteria. Furthermore, intestinal bacteria convert daidzein via dihydrodaidzein to O-desmethylangolensin (O-DMA) or equol, and genistein via dihydrogenistein to 5-hydroxyequol.

[0003] Equol is known to have the highest estrogenic activity among isoflavones, and is known to be able to be produced from daidzein using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1 to 4). Conventionally, only chemically synthesized high-purity daidzeins that do not contain isoflavones other than daidzein have been available, and these have been unsuitable for food use. One method for obtaining highly purified equol (free of isoflavones other than daidzein) is to further purify the fermented product of soybean hypocotyl extract using ethanol extraction or column chromatography (Patent Documents 5 to 7). However, there are environmental concerns regarding the large amount of waste liquid generated during the purification of the fermented product and the use of volatile organic compounds during the purification of equol. In addition, equipment for column chromatography and explosion-proof equipment for handling hazardous materials such as volatile organic compounds are expensive. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-204296 [Patent Document 2] Special Publication No. 2006-504409 [Patent Document 3] Japanese Patent Application Laid-Open No. 2008-061584 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-104241 [Patent Document 5] Patent Publication No. 2021-106606 [Patent Document 6] Japanese Patent Publication No. 2022-037077 [Patent Document 7] Japanese Patent Publication No. 2022-044055 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present disclosure is to provide a simple method for purifying daidzin from plant extracts. An object of the present disclosure is to provide a daidzein-containing composition in which the contents of glyciteins and genisteins are each 0.1% by weight or less. [Means for solving the problem]

[0006] The present disclosure includes the following aspects. [1] A composition in which the content of glyciteins and genisteins is 0.1% by weight or less, respectively, relative to the total dry weight of the composition, and the content of naturally derived daidzeins relative to the total content of isoflavones is more than 99.5%. [2] A method for purifying daidzin, comprising suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution, wherein the plant extract contains 1 to 10% by weight of genisteins and 1 to 30% by weight of glyciteins. [Effects of the Invention]

[0007] According to the present disclosure, a simple method for purifying daidzin from a plant extract can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a chart showing the results of HPLC analysis of soybean hypocotyl extract crude purified product B. [Figure 2] 1 is a chart showing the results of HPLC analysis of the dried product obtained under test condition 6C. DETAILED DESCRIPTION OF THE INVENTION

[0009] An embodiment of the present disclosure will be described in detail below. The present disclosure is not limited to the following embodiment, and can be implemented by making appropriate modifications within the scope that does not impair the effects of the present disclosure. 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 within the scope of the present disclosure. The present disclosure is not limited by the embodiments, but is limited only by the claims. Each feature disclosed herein may be combined with any other feature disclosed herein. When a specific description given for one embodiment also applies to other embodiments, that description may be omitted in the other embodiments. In this disclosure, the expression "X to Y" regarding a numerical range means "X or more and Y or less." When a specific description given for one embodiment also applies to other embodiments, that description may be omitted in the other embodiments. Any numerical range or value inherently contains a range of error necessarily resulting from the standard deviation found in their respective testing measurements. Unless otherwise indicated, each individual value of a numerical range herein is incorporated herein as if it were individually recited herein. In this specification, the term "comprise" refers to containing a certain component as at least a part, and includes cases where the composition consists solely of that component.

[0010] [Terminology] <Isoflavones> In this specification, unless otherwise specified, isoflavones include daidzeins, genisteins, and glyciteins. In this specification, unless otherwise specified, daidzeins include daidzin, malonyldaidzin, acetyldaidzin, daidzein, dihydrodaidzein, and equol. In this specification, unless otherwise specified, the term "genisteins" includes genistin, malonylgenistin, acetylgenistin, genistein, and dihydrogenistein. In this specification, unless otherwise specified, the term "glyciteins" includes glycitin, malonylglycitin, acetylglycitin, glycitein, and dihydroglycitein.

[0011] <Soybean allergens> As used herein, soybean allergens are components derived from soybean seeds, soybean cotyledons, and soybean hypocotyls that can cause allergies in mammals, including humans, and include Gym4, Gm30K, Gm28K, 7S globulin mix (β-conglycine), oleosin, and trypsin inhibitor. <Soy allergen content detection method> An appropriate amount of the composition to be measured is added to an appropriate amount of extraction buffer (e.g., Tris HCl pH 7.5, containing 1 M EDTA, and containing an appropriate amount of protease inhibitor), and the mixture is stirred to extract the water-soluble components. The solids are then removed by filtration to obtain an extract. The total protein content of the resulting extract is measured using a Bio-Rad protein assay system. Furthermore, soybean allergens (Gym4, Gm30K, Gm28K, 7S globulin mix, oleosin, trypsin inhibitor) contained in the obtained extract are detected by Western blotting. The extract or proteins extracted from the extract are electrophoresed and blotted onto a hydrophobic membrane. To detect soy allergens on the membrane, antibodies that can detect Gym4, Gm30K, Gm28K, 7S globulin mix, oleosin, and trypsin inhibitor can be used.

[0012] <Water> In this specification, unless otherwise specified, "water" refers to purified water such as distilled water and ion-exchanged water, tap water, alkaline electrolyzed water, etc., but is not limited to these, and any water suitable for use in food production (in Japan, drinking water that complies with the Water Supply Act) can be used.

[0013] [First embodiment] <Daizin Refining Method> The method for purifying daidzin according to this embodiment is a method comprising suspending a plant extract containing daidzin in a 30 to 90% by volume aqueous ethanol solution.

[0014] According to the method for purifying daidzin of this embodiment, daidzin can be purified more simply and / or in a shorter time than conventional methods for purifying daidzin from plant extracts. For example, the method for purifying daidzin according to the present embodiment does not require a step of bacterial fermentation of a plant extract as a material, and therefore does not require disposal of the fermented product and / or an extraction and purification step using volatile organic compounds from the fermented product, etc. Therefore, the method for purifying daidzin according to the present embodiment is environmentally friendly and / or can be carried out relatively inexpensively without requiring an expensive production plant.

[0015] The purified product obtained by the daidzin purification method of this embodiment (also referred to as a "daidzin-containing composition" in this embodiment) tends to have a lower content and / or content ratio of components other than daidzin compared to purified products obtained by conventional methods for purifying daidzin. More specifically, the daidzin purification method of this embodiment makes it easy to obtain a daidzin-containing composition in which the content of glyciteins and genisteins is 0.1% by weight or less, respectively, relative to the total dry weight of the purified product. In one embodiment, the daidzin purification method of this embodiment makes it easy to obtain a daidzin-containing composition in which the content of naturally-derived daidzeins is greater than 99.5% of the total content of isoflavones contained in the composition. In one embodiment, the method for purifying daidzin according to the present embodiment is a method for efficiently removing isoflavones other than daidzin from a plant extract containing daidzin. The removed isoflavones include genisteins, glyciteins, and / or daidzein.

[0016] The method for purifying daidzin according to this embodiment may include one or more of the steps described below, or other steps, to the extent that it includes suspending a plant extract containing daidzin in a 30 to 90% by volume aqueous ethanol solution.

[0017] The purified product obtained by the daidzin purification method according to this embodiment tends to have a lower content and / or content rate of soybean allergens compared to purified products obtained by conventional methods for purifying daidzin. More specifically, the method for purifying daidzin according to this embodiment makes it easier to obtain a daidzin-containing composition that is substantially free of soybean allergens in the dried purified product.

[0018] Here, the content of soybean allergen in the daidzin-containing composition can be detected or measured by the "Method for detecting soybean allergen-containing composition" in the "Explanation of terms" above. "Substantially free of soybean allergens" means that the composition is substantially free of all soybean allergens, and may be below the detection limit using a "method for detecting a soybean allergen-containing composition," or may mean that the detection value using the method is not significantly different from that of a negative control.If the daidzin-containing composition is an oral composition or a component thereof, the content may be such that it does not induce soybean allergies in animals administered or ingested.

[0019] [Plant extract] The "plant" in the plant extract is not limited to a plant containing daidzin, but is preferably a legume, more preferably one or more of soybean, kudzu, red clover, alfalfa, and licorice, and even more preferably soybean, in consideration of the daidzin content. The plant may be a part separated from the plant. In consideration of the daidzin content, when the plant is soybean, soybean seeds are preferred, and soybean seed hypocotyls are even more preferred. Furthermore, the plant may be, for example, raw, or a processed product obtained by subjecting the whole or part of the plant to a heat treatment, drying treatment, steaming treatment, or the like. For example, when the plant is soybean seeds, processed products include tofu, fried tofu, soy milk, natto, soy sauce, miso, tempeh, etc. The plant is not limited by its place of origin, variety, etc., as long as the daidzin contained therein is not significantly lost.

[0020] Here, "soybean hypocotyl" refers to the part of soybean that becomes the young shoot and radicle upon germination, and is known to contain a large amount of daidzeins such as daidzein glycoside and daidzein. The soybean hypocotyl may also be one that has been subjected to a defatting treatment or a deproteinizing treatment. The shape of the soybean hypocotyl is not limited, and it may be in the form of powder, or in the form of pulverized or crushed granules or lumps.

[0021] The "plant extract" may be any of the above plants that have been subjected to any treatment for extracting isoflavones, and is not limited by the type of treatment. Examples of treatments include filtration, various column chromatography methods, crystallization, and centrifugation. The treatment may be, for example, the method for preparing a crude product described in "1. Preparation of purified raw material crude product A" or "3. Preparation of purified raw material crude product B" in the "Examples" section.

[0022] In one embodiment, the plant extract is preferably a soybean (soybean seed) extract, more preferably a soybean hypocotyl extract.

[0023] The plant extract is preferably a dried plant extract. The "dried plant extract" is not limited by the type of treatment, as long as it is obtained by subjecting the above-mentioned plant extract to any treatment for drying and / or solidification. For example, when the plant extract is in the form of a liquid (solution or suspension), a solid content may be obtained by centrifugation, followed by drying to obtain a dried product. The drying method may be selected appropriately by a person skilled in the art from among vacuum drying, or heat drying methods such as spray drying and drum drying.

[0024] The plant extract may be a commercially available product. For example, when the plant is a soybean hypocotyl, a soybean hypocotyl extract (Fujiflavone P40, manufactured by Fujicco Co., Ltd.) can be used. When extracted from a plant, it can be extracted with, for example, aqueous ethanol. If necessary, the extract may be purified using a resin, activated carbon, filtration, or the like, crystallized, and then subjected to solid-liquid separation and dried.

[0025] [Isoflavones] The isoflavones are as described above in the "Explanation of Terms."

[0026] [Method for detecting or measuring isoflavones] In this specification, the content of isoflavones can be measured by a method well known to those skilled in the art, for example, by high performance liquid chromatography (HPLC). Various conditions for the HPLC method can be appropriately determined by those skilled in the art, and examples thereof include the following conditions:

[0027] In one embodiment, in the method for purifying daidzin, the method for producing daidzein, and / or the method for producing equol according to this embodiment, the content and / or content ratio of isoflavones can be measured by high performance liquid chromatography under the following conditions.

[0028] (High-performance liquid chromatography condition 1) Column: YMC-Pack ODS-AM-303 (4.6 mm x 250 mm (YMC) Mobile phase: A: Acetonitrile / water / acetic acid mixture (15:85:0.1 V / V / V) B Acetonitrile / water / acetic acid mixture (35:65:0.1 V / V / V) Concentration gradient: A linear concentration gradient from A to B is performed over 50 minutes. Flow rate: 1.0mL / min Column temperature: 35℃ Measurement wavelength: 254nm Injection volume: 10μL Retention times: daidzin 10.3 minutes, genistin 16.9 minutes, glycitin 11.0 minutes, malonyldaidzin 17.8 minutes, malonylgenistin 24.5 minutes, malonylglycitin 18.3 minutes, acetyldaidzin 23.2 minutes, acetylgenistin 33.3 minutes, acetylglycitin 23.6 minutes, daidzein 30.3 minutes, genistein 42.9 minutes, glycitein 32.4 minutes, dihydrodaidzein 30.2 minutes, dihydrogenistein 38.9 minutes, dihydroglycitein 32.4 minutes

[0029] (High-performance liquid chromatography condition 2) Column: Phenomenox SYNERGI 4 μm, mPOLAR-RP, 4.6 mm x 150 mm (Shimadzu GLC) Mobile phase: water / methanol [55:45, v / v] Flow rate: 1.0mL / min Column temperature: 40℃ Measurement wavelength: UV 280 nm Injection volume: 10μL Retention times: dihydrodaidzein 8.8 minutes, daidzein 9.9 minutes, dihydroglycitein 10.6 minutes, glycitein 12.2 minutes, dihydrogenistein 12.5 minutes, genistein 16.5 minutes, equol 14.1 minutes

[0030] In one embodiment, the plant extract contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins. The content of genisteins in the plant extract is preferably 1 to 5% by weight, more preferably 1 to 2% by weight. The content of glyciteins in the plant extract is preferably 1 to 5% by weight, more preferably 1 to 2% by weight.

[0031] In one embodiment, the plant extract preferably contains 70% by weight or more of daidzin, more preferably 80% by weight or more, even more preferably 85% by weight or more, and even more preferably 88% by weight or more. When the plant extract contains 88% by weight or more of daidzin, it may contain 90% by weight or more.

[0032] In one embodiment, the plant extract preferably contains daidzein in an amount of 1% by weight or more, more preferably 1.5% by weight or more.

[0033] When the plant extract contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins, when the daidzin content is 70% by weight or more, and / or when the daidzein content is 1% by weight or more, the content and / or percentage of components other than daidzin in the resulting purified product is likely to be low, i.e., the content and / or percentage of components other than daidzin (including soybean allergens) in the resulting purified product is likely to be low, and / or the content and / or percentage of daidzin in the resulting purified product is likely to be high. Thus, a highly purified daidzin-containing composition can be easily obtained. The tendency for the content and / or content rate of components other than daidzin in the obtained purified product to be low may also mean that the removal rate of daidzein, genisteins, and / or glyciteins is likely to be high.

[0034] For example, by ensuring that the genistein content in the plant extract is 1 to 10% by weight and / or the glycitein content is 1 to 30% by weight, it becomes easier to adjust the daidzin content in the resulting purified product to 92% by weight or more, preferably 93% by weight or more, and more preferably more than 99.5% by weight.

[0035] [Ethanol aqueous solution suspension step] In this embodiment, the ethanol concentration of the aqueous ethanol solution in which the plant extract containing daidzin is suspended is 30 to 90% by volume, preferably 40 to 90% by volume, more preferably 40 to 80% by volume, and even more preferably 40 to 70% by volume.

[0036] Taking into consideration the ease of reducing the content and / or content rate of components other than daidzin and / or the yield of daidzin, the amount of plant extract suspended in the aqueous ethanol solution is preferably 1 g or more, more preferably 2 g or more, more preferably 3 g or more, more preferably 4 g or more, more preferably 4 to 20 g, more preferably 4 to 10 g, more preferably 6 to 10 g, and even more preferably 6 to 8 g per 300 mL of aqueous ethanol solution. Furthermore, taking into consideration the ease of reducing the content and / or percentage of components other than daidzin and / or the yield of daidzin, the amount of plant extract suspended in the aqueous ethanol solution is preferably 10 g or more, and more preferably 13 g or more, per 1 L of the aqueous ethanol solution. In one embodiment, the amount may be 10 to 50 g, preferably 10 to 40 g, more preferably 10 to 35 g, more preferably 13 to 35 g, more preferably 13 to 33 g, more preferably 20 to 33 g, and even more preferably 20 to 27 g, per 1 L of the aqueous ethanol solution.

[0037] By using 1 g or more (preferably 2 g or more, more preferably 3 g or more, and even more preferably 4 g or more) of plant extract per 300 mL of aqueous ethanol solution, or 10 g or more of plant extract per 1 L of aqueous ethanol solution, the plant extract becomes less soluble in the aqueous ethanol solution, i.e., the plant extract becomes easier to suspend in the aqueous ethanol solution. As used herein, "suspension" refers to a state in which at least a portion of the plant extract is in suspension, and also encompasses a state in which the remaining portion of the plant extract is in a dissolved state. For example, a dissolved state may refer to a state in which the solute passes through a 7 μm filter when the solution is filtered. By facilitating the suspension of the plant extract in the aqueous ethanol solution, the daidzin purification method tends to increase the yield of daidzin from the plant extract, and therefore tends to increase the content and / or content rate of daidzin in the purified product, because the daidzin suspended in the aqueous ethanol solution is contained in the purified product, as will be seen in the subsequent steps.

[0038] In one embodiment, the method for purifying daidzin according to this embodiment comprises suspending 1 g or more of a plant extract containing daidzin per 300 mL of a 30 to 90% by volume aqueous ethanol solution (the plant extract containing daidzin contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins). In one embodiment, the method for purifying daidzin according to this embodiment comprises suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution (the plant extract containing daidzin contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins).

[0039] When the ethanol concentration of the aqueous ethanol solution is 30 to 90% by volume, and / or when the amount of plant extract suspended per 300 mL of aqueous ethanol solution is 1 g or more (or when the amount of plant extract suspended per 1 L of aqueous ethanol solution is 10 g or more), the content and / or percentage of components other than daidzin in the obtained purified product is likely to be low, i.e., the content and / or percentage of components other than daidzin (including soybean allergens) in the obtained purified product is likely to be low, and / or the content and / or percentage of daidzin in the obtained purified product is likely to be high. Therefore, it becomes easier to obtain a highly purified daidzin-containing composition. The tendency for the content and / or content rate of components other than daidzin in the obtained purified product to be low may also mean that the removal rate of daidzein, genisteins, and / or glyciteins is likely to be high.

[0040] Here, the absolute amounts of the aqueous ethanol solution and the plant extract can be appropriately determined by a person skilled in the art depending on the amount of purified product to be obtained, the ease of operation, and / or the type or scale of purification equipment.

[0041] [Suspension maintenance step] In one embodiment, the method for purifying daidzin according to this embodiment comprises suspending a plant extract in an aqueous ethanol solution and maintaining the suspension at a temperature of 0°C or higher. That is, in one embodiment, the method for purifying daidzin according to this embodiment includes suspending a plant extract containing daidzin in a 30 to 90% by volume aqueous ethanol solution, and maintaining the suspension at a temperature of 0°C or higher. In one embodiment, the method for purifying daidzin according to this embodiment comprises suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution (the plant extract containing daidzin contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins), and maintaining the suspension at a temperature of 0°C or higher.

[0042] The maintenance temperature may be 0°C or higher, but considering the ease of reducing the content and / or content rate of components other than daidzin in the resulting purified product and / or the yield of daidzin, it is preferably 30°C or higher, more preferably 30 to 70°C, more preferably 30 to 60°C, even more preferably 40 to 60°C, and even more preferably 50 to 60°C. The temperature to be maintained can be appropriately determined within the range of 0°C or higher, taking into consideration the yield of daidzin and the removal rate of components other than daidzin. The maintenance temperature can be appropriately determined by a person skilled in the art within a range in which the suspension obtained in the "aqueous ethanol suspension step" can maintain the suspension state of the plant extract. In this case, it is preferable to determine the maintenance temperature taking into consideration the amount of the plant extract suspended in the aqueous ethanol solution in the "aqueous ethanol suspension step".

[0043] In one embodiment, the suspension of the plant extract in the aqueous ethanol solution is maintained at 0°C or higher for at least 1 hour, but is preferably maintained for at least 3 hours, considering the ease of reducing the content and / or percentage of components other than daidzin in the resulting purified product and / or the yield of daidzin. When the maintenance time is 3 hours or longer, it may be 5 hours or longer or 7 hours or longer, but is more preferably 10 hours or longer, more preferably 15 hours or longer, more preferably 20 hours or longer, and even more preferably 24 hours or longer. When the maintenance time is 3 hours or longer, it may be 20 to 72 hours, 20 to 48 hours, or 24 to 48 hours.

[0044] By maintaining a suspension of a plant extract in an aqueous ethanol solution at a temperature of 0°C or higher, or by maintaining a suspension of a plant extract in an aqueous ethanol solution at a temperature of 0°C or higher for 1 hour or more, the content and / or concentration of components other than daidzin in the resulting purified product is likely to be reduced, i.e., the content and / or concentration of components other than daidzin (including soybean allergens) in the resulting purified product is likely to be reduced, and / or the content and / or concentration of daidzin in the resulting purified product is likely to be increased. Thus, it becomes easier to obtain a highly purified daidzin-containing composition. The tendency for the content and / or content rate of components other than daidzin in the obtained purified product to be low may also mean that the removal rate of daidzein, genisteins, and / or glyciteins is likely to be high.

[0045] In one embodiment, maintaining the suspension at a temperature of 0°C or higher may be achieved by maintaining the suspension in an apparatus capable of maintaining the temperature at 0°C or higher. Preferably, the suspension is maintained while being stirred or shaken to prevent precipitation of insoluble components in the suspension. The stirring or shaking is not limited by the conditions or method as long as the insoluble components do not precipitate, and may be performed, for example, by shaking manually, using a stirrer and stir bar, using a roller stirrer, or using a rocking mixer. The strength of the shaking or stirring is not limited as long as the insoluble components in the suspension do not precipitate, and can be appropriately set by those skilled in the art, and may be, for example, 1000 rpm.

[0046] [Separation process] In one embodiment, the method for purifying daidzin according to this embodiment includes suspending a plant extract containing daidzin in a 30 to 90% by volume aqueous ethanol solution, maintaining the suspension at a temperature of 0°C or higher, and separating insoluble components (also referred to as insoluble matter or solid content) containing daidzin. In one embodiment, the method for purifying daidzin according to this embodiment includes suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution (the plant extract containing daidzin contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins), maintaining the suspension at a temperature of 0°C or higher, and separating insoluble components (also referred to as insoluble matter or solid content) containing daidzin.

[0047] Separation of the insoluble components may be carried out by methods well known to those skilled in the art, for example, by subjecting the suspension to filtration (filtration separation) and / or centrifugation (centrifugation or centrifugal filtration separation). In one embodiment, when separating the insoluble components by centrifugal filtration separation, the conditions for centrifugation and filtration are not limited as long as at least a portion of the insoluble components can be separated, and can be appropriately set by those skilled in the art. For example, centrifugation at 1000 rpm for 5 minutes and / or filtration through a 7 μm filter may be performed.

[0048] According to the method for purifying daidzin of this embodiment, the insoluble components contain daidzin at a high concentration, and therefore, by separating and recovering the insoluble components from the suspension, a purified product containing highly purified daidzin can be obtained.

[0049] [Drying process] In one embodiment, the method for purifying daidzin according to the present embodiment may include separating the insoluble components containing daidzin and then drying the insoluble components. That is, in one embodiment, the method for purifying daidzin according to this embodiment includes suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution (the plant extract containing daidzin contains 1 to 10% by weight of genisteins and / or 1 to 30% by weight of glyciteins), maintaining the suspension at a temperature of 0°C or higher, separating insoluble components (also referred to as insoluble matter or solid content) containing daidzin, and drying the separated insoluble components.

[0050] [Number of times processed] In one embodiment, the method for purifying daidzin according to this embodiment may be repeated two or more times. In each step of the second and subsequent purification methods, examples and preferred examples are as explained above. In each step of the second or subsequent purification method, each step may be performed under different conditions within the scope of this embodiment.

[0051] By repeating the process two or more times, the content and / or content ratio of components other than daidzin in the obtained purified product tends to be lower, i.e., the content and / or content ratio of components other than daidzin (including soybean allergens) in the obtained purified product tends to be lower, and / or the content and / or content ratio of daidzin in the obtained purified product tends to be higher, thereby making it easier to obtain a highly purified daidzin-containing composition. The tendency for the content and / or content rate of components other than daidzin in the obtained purified product to be low may also mean that the removal rate of daidzein, genisteins, and / or glyciteins is likely to be high.

[0052] The second or subsequent purification methods are methods in which the purified product obtained by the previous daidzin purification method is used as the plant extract. The purified product obtained by the previous daidzin purification method is the purified product separated and recovered by the above-mentioned separation step. It may also be a dried purified product.

[0053] In one embodiment, the method for purifying daidzin according to this embodiment is repeated twice.

[0054] The dried purified product may be subjected to a powdering treatment as needed to obtain a powder.

[0055] The insoluble components may be dried by a method well known to those skilled in the art, such as natural drying, heat drying, vacuum drying, or the like. In this embodiment, the dried insoluble component may be a purified product.

[0056] The dried insoluble components do not contain the aqueous ethanol solution used in the method for purifying daidzin in this embodiment, and are therefore particularly suitable when the purified product (daidzin-containing composition) is an oral composition, as described in the second embodiment.

[0057] In one embodiment, the purified product obtained by the daidzin purification method according to the present embodiment is a daidzin-containing composition in which the contents of glyciteins and genisteins are each 0.1 wt% or less relative to the total dry weight of the composition. In one embodiment, the purified product is a daidzin-containing composition containing isoflavones (including daidzeins), in which the content of naturally-derived daidzeins relative to the total isoflavone content is more than 99.5%. The purified product is preferably a "daidzin-containing composition" described in the "daidzein-containing composition" according to the second embodiment, and examples and suitable examples thereof are as described in the second embodiment.

[0058] In one embodiment, the method for purifying daidzin according to this embodiment is a method for obtaining a daidzin-containing composition in which the content of glyciteins and genisteins is 0.1% by weight or less, respectively, relative to the total dry weight of the composition. In one embodiment, the method for purifying daidzin according to this embodiment is a method for producing the "daidzin-containing composition" described in the "daidzein-containing composition" according to the second embodiment.

[0059] The purified product obtained by the daidzein purification method according to this embodiment can be used as a material for producing daidzein and / or equol, as described below in "Methods for producing daidzein and equol." The purified product obtained by the daidzin purification method according to this embodiment can also be used as an oral composition or as a material for the oral composition, as described in the "Oral composition" of the second embodiment.

[0060] <Methods of producing daidzein and equol> The method for producing daidzein and the method for producing equol according to this embodiment include the above-mentioned "method for purifying daidzin."

[0061] Examples and preferred examples of the method for purifying daidzin in the method for producing daidzein and the method for producing equol are as described in "Method for Purifying Daidzin."

[0062] The above-mentioned "daidzin purification method" allows for the production of a daidzin-containing composition containing highly purified daidzin. Therefore, by using the daidzin-containing composition as a material for the method for producing daidzein or equol, it becomes easier to obtain daidzein or equol with a reduced content and / or rate of undesired components. Furthermore, the above-mentioned "daidzin purification method" makes it easy to obtain a purified product in which soybean allergens are reduced compared to the raw material soybean extract or soybean hypocotyl extract, or which is free of soybean allergens. Therefore, by using the daidzin-containing composition obtained by the "daidzin purification method" as a raw material, it is possible to produce daidzein or equol that can be safely administered or easily ingested by subjects with soybean allergies.

[0063] From the viewpoint of making it easier to obtain daidzein or equol with a reduced content and / or content rate of undesired components, it is preferable that the daidzin-containing composition is a purified product obtained by separating insoluble components including daidzin and then drying.

[0064] Equol is known to have the highest estrogenic activity among isoflavones. The upper limit of safe daily intake of soy isoflavones as a food for specified health uses is set at 30 mg / day. Therefore, when preparing an equol-containing composition as a food for specified health uses, reducing the amount of isoflavones other than equol allows the subject to ingest more equol.

[0065] Therefore, it is preferable to produce daidzein and / or equol by a method including the above-mentioned "daidzin purification method," or to produce daidzein and / or equol using daidzin obtained by the above-mentioned "daidzin purification method" as a material, since this makes it easier to obtain daidzein or equol with a reduced content and / or concentration rate of undesired components.

[0066] Daidzin is known as a daidzein glycoside, and is known to be converted by bacteria in the animal intestine to equol via the intermediate metabolites daidzein and / or dihydrodaidzein. Therefore, in one embodiment, the method for producing daidzein comprises converting daidzin contained in the purified product (daidzin-containing composition) obtained by the above-mentioned "method for purifying daidzin" into daidzein. In one embodiment, the method for producing equol comprises converting daidzin contained in the purified product (daidzin-containing composition) obtained by the above-described "method for purifying daidzin" to equol. In one embodiment, the method for producing equol comprises converting daidzin to daidzein and converting daidzein to equol.

[0067] The conversion of daidzin to daidzein, the conversion of daidzin to equol, and the conversion of daidzein to equol can each be carried out by bacteria. The bacteria are not limited as long as they have the ability to assimilate daidzin, one of the equol raw materials, to produce daidzein, or have the ability to produce equol (also referred to as equol-producing ability). Bacteria capable of producing daidzein may be bacteria capable of directly converting the equol raw material into daidzein, or bacteria capable of converting the equol raw material into daidzein via an intermediate metabolite. The bacteria capable of producing equol may be bacteria capable of directly converting the equol raw material into equol, or bacteria capable of converting the equol raw material into equol via an intermediate metabolite such as daidzein. The bacteria may be a combination of multiple bacterial species that are capable of performing each of the steps required for the production of daidzein from equol raw materials, or for the production of equol from equol raw materials. In one embodiment, the bacteria are preferably anaerobic bacteria, more preferably lactic acid bacteria or bifidobacteria.

[0068] Specifically, bacteria known to those skilled in the art to have the ability to convert daidzin to daidzein or the ability to convert daidzin to equol, or bacteria screened by conventional methods, can be used. For example, bacteria belonging to the genus Lactococcus such as Lactococcus garvieae, bacteria belonging to the genus Streptococcus such as Streptococcus intermedius and Streptococcus constellatus, bacteria belonging to the genus Bacteroides such as Bacteroides ovatus, bacteria belonging to the genus Blautia such as Blautia producta, Blautia coccoides, and Blautia schinki, bacteria belonging to the genus Eubacterium such as Eubacterium rectale and Eubacterium siraeum, bacteria belonging to the genus Eggerthella sp. It is known that at least some bacteria belonging to the genus Egasella, such as Egasella sp., have the ability to produce daidzein and / or equol. Two or more of these bacteria may be used in combination. The bacterium may be, for example, a bacterium isolated by a person skilled in the art from a human fecal sample by a well-known method using the presence or absence of the ability to produce equol as an indicator.

[0069] In one embodiment, in the method for producing daidzein and the method for producing equol, converting daidzin into daidzein by bacteria or converting daidzin into equol by bacteria comprises culturing the bacteria in a medium suitable for the bacteria used.

[0070] The medium is not limited as long as it can culture bacteria, and may be, 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 manufactured by Nissui Pharmaceutical Co., Ltd., or a medium having the composition shown in Table 1 below. Additives that can be used for culturing bacteria may be added to the medium as appropriate. For example, water-soluble organic substances such as sugars such as glucose, arabinose, sorbitol, fructose, mannose, sucrose, trehalose, and xylose, alcohols such as glycerol, and organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, and fumaric acid may be added as carbon sources. Inorganic nitrogen sources such as ammonium salts and nitrates may also be added as nitrogen sources. Organic nitrogen sources such as amino acids, yeast extract, peptones (e.g., polypeptone N, soybean peptone), meat extracts (e.g., Ehrlich bonito extract, Lab-Remco powder, bouillon), seafood extract, liver extract, digested serum powder, and fish oil may also be added as nitrogen sources. Cofactors such as vitamins and / or inorganic compounds such as various salts may also be added. [Table 1]

[0071] Various conditions for bacterial culture (e.g., pH, aqueous and gas phase environments, temperature, pressure conditions, culture time) can be appropriately set by those skilled in the art to suit the bacteria used. For example, bacteria may be inoculated into a medium with a pH of 6 to 7, the gas phase replaced with hydrogen gas, and then cultured with shaking at 37°C.

[0072] In one embodiment, in the method for producing daidzein and the method for producing equol, converting daidzin to daidzein or converting daidzin to equol using bacteria may involve treating daidzin and / or daidzein with an enzyme produced by the bacteria, instead of converting them using the bacteria themselves. Examples of such enzymes include β-glucosidase, and the β-glucosidase may be aromases. The enzymes may be isolated by culturing bacteria or may be commercially available products.

[0073] In one embodiment, in the method for producing daidzein and the method for producing equol, converting daidzin into daidzein by bacteria or converting daidzin into equol by bacteria may include mixing and culturing enzyme-treated daidzin and / or daidzein with bacteria cultured in a medium suitable for the bacteria used. The cultured bacteria are mixed with enzyme-treated daidzin and / or daidzein and cultured, whereby the bacteria convert daidzin into daidzein or equol.

[0074] The various conditions (pH, aqueous and gaseous environments, temperature, pressure conditions, culture time, etc.) used when mixing the cultured bacteria with daidzin and / or daidzein and culturing them can be appropriately set by those skilled in the art to suit the bacteria used. For example, the bacteria may be inoculated into a medium at pH 6 to 7, aerated with a mixed gas of hydrogen and nitrogen, and cultured at 37°C. By culturing, a fermented product containing daidzein and / or equol is obtained.

[0075] The fermented product can be separated from the fermentation liquid and isoflavones by methods well known to those skilled in the art, and daidzein and / or equol can be recovered.

[0076] In one embodiment, a method for producing an oral composition is disclosed, which includes one or more of the methods described above in "Method for Purifying Daidzin" and "Method for Producing Daidzein and Method for Producing Equol." In one embodiment, the oral composition is the composition described in the "Oral Composition" of the second embodiment. In one embodiment, the method for producing an oral composition includes the method for producing equol described above in "Method for Producing Daidzein and Method for Producing Equol." This method for producing equol includes the "Method for Purifying Daidzein," and it is easy to obtain equol with a reduced content and / or content rate of undesired components. Therefore, by including this method for producing equol in the method for producing an oral composition, it is possible to reduce the content and / or content rate of isoflavones other than equol, and obtain an oral composition with a higher equol content. The obtained oral composition is suitably used as a food composition or a pharmaceutical composition, or as an ingredient thereof, as described in the "Oral composition" of the second embodiment.

[0077] In one embodiment, a method for producing a food composition or food, or a pharmaceutical composition or medicine is disclosed, which includes any one or more of the methods described above in the "Method for purifying daidzein" and "Method for producing daidzein and method for producing equol."

[0078] [Second embodiment] <Daidzein-containing composition> The composition of this embodiment is a composition in which the content of glyciteins and genisteins is 0.1% by weight or less, respectively, relative to the total dry weight of the composition, and the content of naturally derived daidzeins relative to the total content of isoflavones is more than 99.5%.

[0079] In one embodiment, the composition according to this embodiment is a daidzein-containing composition. The composition according to this embodiment is not limited to the range in which it contains daidzeins, and in one embodiment, it may be an isoflavone-containing composition that contains isoflavones other than daidzeins.

[0080] Since the content of glyciteins and genisteins in the composition is 0.1% by weight or less, respectively, relative to the total dry weight of the composition, the composition is likely to have a high content and / or content ratio of isoflavones other than glyciteins and genisteins, i.e., the composition is likely to contain isoflavones other than glyciteins and genisteins with a high degree of purification.

[0081] In one embodiment, the content of glyciteins is 0.1% by weight or less, preferably 0.0% by weight, based on the total dry weight of the composition. In one embodiment, the content of genisteins relative to the total dry weight of the composition is 0.1% by weight or less, preferably 0.0% by weight.

[0082] Here, in the compositions according to this embodiment (including daidzein-containing compositions, daidzin-containing compositions, daidzein-containing compositions, and equol-containing compositions), the content and / or content ratio of daidzeins and isoflavones other than daidzeins can be detected or measured by the high performance liquid chromatography method described in the "Method for detecting or measuring isoflavones" of the first embodiment.

[0083] The phrase "containing daidzeins derived from natural products" means that at least one of the daidzein compounds contained in the composition is derived from natural products. The composition is not limited to containing one or more daidzein compounds derived from natural products, and may contain one or more daidzein compounds other than those derived from natural products. For one daidzein compound, some may be derived from natural products and the rest may be derived from non-natural products. Furthermore, one or more isoflavone compounds other than daidzein may be derived from non-natural products. For one isoflavone compounds other than daidzein, some may be derived from natural products and the rest may be derived from non-natural products.

[0084] Here, "naturally derived" means that it is not obtained by chemical synthesis, or in other words, that it is obtained by extraction or purification from a natural product (e.g., a living organism such as an animal, plant, or fungus). "Non-naturally occurring" means that the compound is obtained by chemical synthesis. The chemical synthesis is not limited by the method. Although naturally occurring isoflavones and chemically synthesized isoflavones (including daidzeins) have the same chemical structure, they can be distinguished by analytical methods well known to those skilled in the art.

[0085] In one embodiment, the content of naturally derived daidzeins relative to the total content of isoflavones in the composition is preferably 99.6% or more, more preferably 99.7% or more, more preferably 99.8% or more, more preferably 99.9% or more, and even more preferably 100%.

[0086] In one embodiment, when the composition contains isoflavones other than daidzein, it is preferable that more than 99.5% of the isoflavones, including daidzein, are derived from natural sources, more preferably 99.6% or more, even more preferably 99.7% or more, even more preferably 99.8% or more, even more preferably 99.9% or more, and even more preferably 100%.

[0087] The composition may contain, as a component other than isoflavones, a component obtained by chemical synthesis (chemically synthesized component). In one embodiment, when the composition is used as an oral composition or as an ingredient for an oral composition, it is preferable that it does not contain any chemically synthesized components, as explained below.

[0088] In the composition according to this embodiment, the lower the content and / or content rate of chemically synthesized components, the more suitable it is for use in food applications. Specifically, for example, when the content of naturally occurring daidzeins relative to the total content of isoflavones is greater than 99.5%, when greater than 99.5% of the isoflavones, including daidzeins, are naturally occurring, when all of the daidzeins contained in the composition are naturally occurring, when all of the isoflavones contained in the composition are naturally occurring, or when the components contained in the composition do not contain chemically synthesized components (when all of the components contained in the composition are naturally occurring), it becomes easier to reduce or avoid the inclusion of impurities in the composition resulting from the chemical synthesis process.Naturally occurring isoflavones have a proven track record of safety in food applications. Compositions in which impurity contamination is reduced or avoided and / or which have a proven safety record are suitable for oral administration or ingestion, and therefore can be suitably used as oral compositions or as ingredients for oral compositions, as described in the "Oral Compositions" section of this embodiment.

[0089] In one embodiment, the natural product is preferably a plant, preferably a legume, more preferably one or more of soybean, kudzu, red clover, alfalfa, and licorice, more preferably soybean, more preferably soybean seed, and even more preferably soybean hypocotyl. The isoflavones contained in the composition may be derived from multiple different natural products. Here, the soybean hypocotyl is as described in the first embodiment.

[0090] In one embodiment, the daidzein-containing composition preferably has a reduced content and / or percentage of one or two soybean allergens2, more preferably has a reduced content and / or percentage of all soybean allergens, and more preferably is substantially free of all soybean allergens, compared to the plant extract, daidzin-containing composition, daidzein-containing composition, or daidzein-containing composition that are raw materials for its production. The reduced soybean allergens make it easier for subjects with soybean allergies to safely administer or ingest the isoflavone-containing composition when used as an oral composition.

[0091] The content of soybean allergens in the daidzein-containing composition can be detected or measured by the "Method for detecting soybean allergen-containing composition" in the "Explanation of terms" section above. "Substantially free of soybean allergens" means that the composition is substantially free of all soybean allergens, and may be below the detection limit using a "method for detecting a soybean allergen-containing composition," or may mean that the detection value using the method is not significantly different from that of a negative control.If the daidzin-containing composition is an oral composition or a component thereof, the content may be such that it does not induce soybean allergies in animals administered or ingested.

[0092] In one embodiment, the daidzein-containing composition according to this embodiment (preferably, the daidzin-containing composition) is a composition used for producing daidzein.

[0093] In one embodiment, the daidzein-containing composition according to this embodiment (preferably a daidzin-containing composition or a daidzein-containing composition) is a composition used for producing equol.

[0094] [Daizin-containing composition] In one embodiment, the daidzein-containing composition is preferably a daidzin-containing composition. When the daidzein-containing composition is a daidzin-containing composition, the daidzein-containing composition is preferably a daidzin-containing composition having a daidzin content of more than 99.5 wt % relative to the total dry weight of the composition. In one embodiment, the daidzin-containing composition preferably contains less than 0.1 wt % of daidzein relative to the total dry weight of the composition, and in one embodiment, the daidzin-containing composition preferably contains more than 99.5 wt % of daidzin and less than 0.1 wt % of daidzein.

[0095] In one embodiment, when the daidzein-containing composition is a daidzin-containing composition, it is preferably a composition prepared by a method including the "daidzin purification method" described in the first embodiment.

[0096] A composition containing highly purified daidzin, in which the daidzin content is more than 99.5 wt % based on the total dry weight of the composition, can be suitably used for producing daidzein. By using a daidzein-containing composition containing daidzein at a high degree of purification as a material, it becomes easier to obtain a daidzein-containing composition having a high daidzein content and / or content rate, i.e., a daidzein-containing composition containing daidzein at a high degree of purification. Furthermore, by using a daidzin-containing composition containing highly purified daidzin as a material, it becomes easier to obtain an equol-containing composition that has a high content and / or content rate of equol, i.e., contains equol at a high degree of purification.

[0097] Daidzein-containing compositions and equol-containing compositions made from daidzin can be prepared by methods well known to those skilled in the art, for example, by the method described in the "Methods for producing daidzein and equol" of the first embodiment.

[0098] [Daidzein-containing composition] In another embodiment, the daidzein-containing composition according to this embodiment is a daidzein-containing composition.

[0099] In one embodiment, the daidzein-containing composition contains 80% by weight or more, preferably 90% by weight or more, and more preferably more than 99.5% by weight of daidzein based on the total dry weight of the composition.

[0100] The daidzein-containing composition can be prepared by the method described below in "Method for preparing a daidzein-containing composition."

[0101] Daidzein is converted to equol by intestinal bacteria. By using a daidzein-containing composition containing highly purified daidzein as a starting material, it becomes easier to obtain an equol-containing composition that has a reduced content and / or percentage of undesired components and / or a high content and / or percentage of equol, i.e., a highly purified equol. Therefore, a daidzein-containing composition having a daidzein content of more than 80% by weight based on the total dry weight of the composition is suitable for use as a material for an equol-containing composition. An equol-containing composition using daidzein as a raw material can be prepared by methods well known to those skilled in the art, for example, by the method described in the "Methods for producing daidzein and equol" in the first embodiment.

[0102] [Equol-containing composition] In another embodiment, the daidzein-containing composition according to this embodiment is an equol-containing composition.

[0103] The equol-containing composition can be prepared by the method described below in "Method for preparing a daidzein-containing composition."

[0104] In an equol-containing composition, the content of genisteins and glyciteins is preferably less than 0.7 mg / g, more preferably 0.5 mg / g or less, even more preferably 0.3 mg / g or less, and even more preferably 0.0 mg / g or less, based on the total dry weight of the composition.

[0105] Equol is known to have the highest estrogenic activity among isoflavones. The upper limit of safe daily intake of soy isoflavones as a food for specified health uses is set at 30 mg / day. Therefore, when preparing an equol-containing composition as a food for specified health uses, reducing the content and / or content rate of isoflavones other than equol allows the subject to ingest more equol.

[0106] In one embodiment, the equol-containing composition preferably has a reduced content of saponins, and more preferably is substantially free of saponins, compared to the plant extract, daidzin-containing composition, and / or daidzein-containing composition that are the raw materials for its production. "Substantially free of saponins" means that the equol-containing composition is substantially free of all saponins detected by the method described below, and may be below the detection limit by the detection method described below, or may mean that the detection value by the method is not significantly different from a negative control. In one embodiment, the content of saponins may be a percentage (%) of the total content of isoflavones contained in the composition (i.e., calculated as "content of saponins / content of isoflavones x 100").

[0107] The content of saponins in the equol-containing composition can be detected or measured by the following method. The saponins referred to in this specification are saponins that can be detected or measured by the following methods.

[0108] (Method for detecting saponin content) The composition to be measured (equol-containing composition, saponins equivalent to 1 mg) is extracted with a mixture of 1 mL of water and 18 mL of methanol. The insoluble components are further extracted with 20 mL of methanol, and the extracts are mixed. The methanol is removed from this mixture by vacuum concentration. 10 mL of 10% hydrochloric acid in methanol is added, and the mixture is heated to reflux at 80°C for 2 hours. Ethanol is then added, and this is the sample for high-performance liquid chromatography analysis. Accurately weigh 10 mg of each of the standards of soyasapogenol A (molecular weight: 474.72) and soyasapogenol B (molecular weight: 458.73) into a 100 mL volumetric flask, dissolve in ethanol, and adjust to the specified volume to make the standard solution. To calculate the content of soybean saponin Ab (molecular weight: 1437.52) and soybean saponin Bb (molecular weight: 943.12), convert from the molecular weights of soyasapogenol A and soyasapogenol B.

[0109] The high performance liquid chromatography conditions are, for example, as follows. Column: Inertsil ODS-2 5 μm, φ2.0 mm × 150 mm (GL Sciences) Mobile phase: acetonitrile / 1% acetic acid water = 7:3 Flow rate: 0.2mL / min Column temperature: 40℃ Detection: UV 210nm Retention times: Sapogenol A 6.78 min, Sapogenol 14.13 min

[0110] <Method for preparing a daidzein-containing composition> The daidzein-containing composition according to this embodiment can be prepared by, for example, a cooling crystallization method or a water addition crystallization method, which are well known to those skilled in the art. For example, the cooling crystallization method is exemplified in "2. Purification test using crude purified product A as a raw material" in the "Examples" section of this specification, and involves suspending a plant extract in an aqueous ethanol solution, filtering the filtrate, cooling and maintaining the filtrate at 10°C, and separating and recovering the insoluble components by solid-liquid separation. The water addition crystallization method is exemplified in "2. Purification test using crude purified product A as a raw material" in the "Examples" section of this specification, and involves suspending a plant extract in an aqueous ethanol solution, filtering the filtrate, adding water to the filtrate, cooling the filtrate to 30°C, and maintaining the filtrate, and separating and recovering the insoluble components by solid-liquid separation. In the cooling crystallization method and the water addition crystallization method, the same steps may be repeated two or more times.

[0111] The daidzein-containing composition according to this embodiment is preferably prepared by, for example, the "method for purifying daidzin" described in the first embodiment or a method including the "method for purifying daidzin". According to the "method for purifying daidzin" (repulp aging method) described in the first embodiment, a daidzin-containing composition with a high degree of daidzin purification can be prepared more easily and / or in a shorter time than conventional methods such as the cooling crystallization method and water addition crystallization method. Examples and preferred examples of this method are as described in the first embodiment.

[0112] When the daidzein-containing composition of this embodiment is a daidzein-containing composition or an equol-containing composition, it is preferable to manufacture it using, for example, daidzin prepared by the cooling crystallization method, water-addition crystallization method, or the like, or daidzin prepared by the "daidzin purification method" described in the first embodiment. In particular, the "daidzin purification method" (repulp aging method) described in the first embodiment makes it easy to prepare a highly purified daidzin-containing composition simply and / or in a short time. Furthermore, as explained in the first embodiment, such a highly purified daidzin-containing composition has a reduced content and / or content rate of soybean allergens or is free of soybean allergens. Therefore, it is preferable to prepare a daidzein-containing composition or an equol-containing composition using daidzin prepared by the method according to the first embodiment as a raw material. By using highly purified daidzin or a daidzin-containing composition as a raw material, highly purified daidzein or a daidzein-containing composition, or highly purified equol or an equol-containing composition, can be easily obtained. Such highly purified daidzein or a daidzein-containing composition, or highly purified equol or an equol-containing composition, has a reduced soy allergen content and / or a reduced ratio of soy allergens, or is free of soy allergens. Furthermore, daidzein and / or equol produced using daidzin prepared by the "daidzin purification method" described in the first embodiment are made from a plant extract containing daidzin, and can be suitably used as an oral composition or as a material for an oral composition.

[0113] Daidzein can be prepared using daidzin as a raw material, or equol can be prepared using daidzin as a raw material by methods well known to those skilled in the art. For example, they can be prepared by the methods exemplified in the "Methods for producing daidzein and equol" described in the first embodiment. In one embodiment, the equol-containing composition of this embodiment is a composition prepared by the method described in the "Method for Producing Daidzein and Method for Producing Equol" in the first embodiment.

[0114] <Oral Composition> The daidzein-containing composition according to this embodiment may contain any additives as long as the effects of the invention are achieved. Non-limiting examples of additives include excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, diluents, preservatives, flavoring agents, and flavoring agents. There are no limitations on these additives as long as they are orally administered.

[0115] The daidzein-containing composition according to this embodiment can be suitably used as an oral composition or as an ingredient for an oral composition. In one embodiment, the daidzein-containing composition according to this embodiment is an oral composition.

[0116] When used orally, the daidzein-containing composition may be any of the above-mentioned daidzin-containing composition, daidzein-containing composition, and equol-containing composition. In one embodiment, the oral composition is an equol-containing composition.

[0117] The oral composition can be used as a pharmaceutical composition or a food composition.

[0118] The subject to which the food composition or a food containing it is ingested, or the subject to which the pharmaceutical composition or a medicine containing it is administered, is not limited as long as it is an animal in need thereof, and may be a human or a non-human animal. Non-human animal species may be, for example, monkeys, dogs, cats, horses, cows, pigs, sheep, goats, rabbits, guinea pigs, hamsters, mice, and / or rats, and are not limited by their use as livestock animals, pet animals, laboratory animals, etc., but are preferably mammals, and more preferably humans.

[0119] [Food composition] The food compositions can be used alone or in combination of two or more food compositions to prepare foods such as beverages, soups, processed meat products, processed vegetables, processed fruit products, seasonings, concentrated foods, and supplements (nutritional supplements, nutritional supplement drinks).

[0120] A food product according to one embodiment includes the food composition according to this embodiment. In one embodiment, the food product is a supplement comprising the food composition according to this embodiment.

[0121] Here, processed foods refer to natural food ingredients that have been processed and / or cooked, and include frozen foods, retort foods, canned foods, bottled foods, etc. The form of the food is not limited, but is preferably a form suitable for oral use, and from the viewpoint of ease of ingestion, it may be in a fluid form such as liquid, syrup, paste, gel, jelly, cream, emulsion, spray, mousse, lotion, etc., or in a solid form such as powder, granules, tablets, capsules, soft capsules, etc. Tablets may be any of orally disintegrating tablets, chewable tablets, effervescent tablets, dispersion tablets, and dissolving tablets.

[0122] The food composition may take any form as long as the effects of the invention can be obtained, for example, a drink, powder, paste, jelly, capsule, tablet, or the like. The food composition may be ingested alone or mixed with other foods and beverages. Non-limiting examples of other foods and beverages include oil-containing products such as edible oils, dressings, mayonnaise, and margarine; liquid foods such as soups, dairy drinks, soft drinks, tea drinks, alcoholic drinks, energy drinks, jelly drinks, and functional drinks; carbohydrate-containing foods such as rice dishes, noodles, and bread; processed livestock foods such as ham and sausage; processed seafood foods such as kamaboko, dried fish, and salted fish; processed vegetable foods such as pickles; semi-solid foods such as jelly and yogurt; fermented foods such as miso and fermented drinks; various confectioneries such as Western confectioneries, Japanese confectioneries, candies, chewing gum, gummy candies, frozen desserts, and frozen desserts; retort pouch products such as curry, thickened sweets, and Chinese soup; instant foods such as instant soup and instant miso soup, and microwaveable foods.

[0123] The food containing the food composition is preferably a functional food that has the effect of improving undesired symptoms and / or poor physical condition in a subject who ingests it (including functions related to maintaining and improving health). Examples of such functional foods in Japan include general foods, including nutritional supplements, health supplements, and nutritionally adjusted foods, as well as health functional foods (including nutritional functional foods, foods for specified health uses (Tokuho), and foods with functional claims), which are labeled according to national safety and efficacy standards.

[0124] The food composition or the food containing the same may be a food composition or food labeled with a function related to daidzeins. For example, when a food composition or a food made from the same is a food with nutrient functional properties, the food with nutrient functional properties may be a food that is labeled as containing one or more daidzeins.

[0125] Furthermore, when the food composition or a food made from the same is a food for specified health uses or a food with functional claims, the food for specified health uses or a food with functional claims may be a food that displays, for example, the following functions for daidzein. Helps maintain healthy bone composition in middle-aged and elderly women Maintains skin elasticity Helps maintain healthy skin Maintains skin moisture Maintains the skin's barrier function Relieve or help relieve symptoms associated with breast cancer Relieve or help relieve symptoms associated with prostate cancer Relieve or help relieve symptoms associated with osteoporosis Relieve or help relieve symptoms associated with high cholesterol Relieve or help relieve symptoms associated with heart disease

[0126] The food composition can be ingested by an individual at any frequency or interval as long as the effects of the invention are achieved.

[0127] In one embodiment, a method is disclosed for exerting the function of any of the above food compositions in a subject in need thereof, comprising ingesting the food composition to a subject. In one embodiment, the use of isoflavones to exert any of the above functions in a subject is disclosed. In one embodiment, the use of isoflavones for the production of a food composition or food product having any of the above functions is disclosed. In one embodiment, isoflavones are disclosed for use in any of the above functions in a subject.

[0128] Pharmaceutical Composition The pharmaceutical composition can be used alone or in combination of two or more food compositions to prepare a medicament. The pharmaceutical composition may be a composition that is a medicine itself, or a composition that is used as a material for medicines. The pharmaceutical composition may be a prescription drug, an over-the-counter drug, or a quasi-drug.

[0129] The pharmaceutical use may be any pharmaceutical use corresponding to the functions listed above in the "food composition" section, including, but not limited to, pharmaceuticals for preventing or treating dry skin, breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, and heart disease.

[0130] The pharmaceutical compositions may be formulated into one or more dosage forms. The pharmaceutical composition can be administered to an individual systemically or locally at any administration frequency or interval as long as the effects of the present invention are achieved. Non-limiting examples of administration routes include oral administration, intravenous administration, intramuscular administration, intrathecal administration, sublingual administration, rectal administration, ocular administration, otic administration, transauricular administration, and transdermal administration.

[0131] The pharmaceutical composition may take any form as long as the effects of the present invention can be obtained. For example, the pharmaceutical composition may take the form of tablets, granules, powders, capsules, emulsions, suspensions, syrups, injections or drip infusions (sterile solutions, suspensions, etc.), etc.

[0132] "Treatment" includes the reduction, alleviation, or relief of symptoms of a condition and / or disease, while "prevention" includes protection against future onset of a condition and / or disease and the inhibition of progression. Desirable therapeutic effects of treatment include alleviation of symptoms, improvement of direct or indirect pathological consequences of a disease, reduction in the rate of progression of worsening symptoms, recovery or alleviation of the disease state, and improvement in prognosis. Treatment and / or prevention include administering a pharmaceutical composition or medicament to a subject diagnosed with a condition and / or disease for the purpose of treatment and / or prevention.

[0133] In one embodiment, a method is disclosed for exerting a function of any of the above pharmaceutical compositions in a subject in need thereof (a pharmaceutical function corresponding to the functions listed in the "food composition" above), comprising administering the pharmaceutical composition to the subject. In one embodiment, the use of isoflavones to exert a function of any of the above pharmaceutical compositions in a subject (a pharmaceutical function corresponding to the functions listed in the above "food composition") is disclosed. In one embodiment, the pharmaceutical function is the prevention or treatment of osteoporosis or the prevention or treatment of dry skin. [Food and pharmaceutical preparation] The foods and medicines can be prepared by methods well known to those skilled in the art.

[0134] For example, in the case of tablets, the powder of the daidzein-containing composition according to the second embodiment can be made into tablets by adding an excipient and one or more of a lubricant or a flow agent in a conventional manner to enhance moldability, and then tableting can be carried out in a conventional manner.

[0135] For example, in the case of capsules, the daidzein-containing composition according to the second embodiment can be prepared by filling it, either directly or after diluting it with an excipient, into food or pharmaceutical capsules made of a plant base in a conventional manner.

[0136] For example, in the case of chewable tablets, flavorings may be added to correct the taste.

[0137] For example, in the case of granules, desired foods or medicines can be produced by adding an excipient and granulating in a conventional manner.

[0138] For example, in the case of drinks, jellies, etc., they can be prepared by dissolving or suspending them in water in a conventional manner.

[0139] A non-limiting list of exemplary embodiments and combinations of exemplary embodiments of the present disclosure are disclosed below. [1] A composition in which the content of glyciteins and genisteins is 0.1% by weight or less, respectively, relative to the total dry weight of the composition, and the content of naturally derived daidzeins relative to the total content of isoflavones is more than 99.5%. [2] The composition according to claim 1, wherein the naturally derived daidzein is soybean-derived daidzein. [3] The composition according to [1] or [2], wherein the daidzein is daidzin. [4] The composition according to [1] or [2], wherein the daidzein is equol. [5] The composition according to any one of [1] to [4], wherein the daidzein content is less than 0.1% by weight. [6] The composition according to any one of [3] to [5], which is used for producing daidzein. [7] The composition according to [4] or [5] for use in producing equol. [8] The composition according to any one of [1] to [7], which is for oral administration. [9] [8] A supplement comprising the composition described in.

[10] A method for purifying daidzin, comprising suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution, wherein the plant extract contains 1 to 10% by weight of genisteins and 1 to 30% by weight of glyciteins.

[11] The purification method according to

[10] , wherein the plant extract is a soybean hypocotyl extract.

[12] The purification method described in

[10] or

[11] , wherein the plant extract contains 85% by weight or more of daidzin.

[13] The purification method according to any one of

[10] to

[12] , which comprises maintaining a suspension of the plant extract in an aqueous ethanol solution at a temperature of 30°C or higher.

[14] A method for producing daidzein, comprising the purification method according to any one of

[10] to

[13] .

[15] A method for producing equol, including the purification methods of

[10] to

[13] .

[16] A method for producing a food composition, comprising the method for producing equol described in

[15] . [Example]

[0140] The present invention will be described below based on examples, but the scope of the present invention is not limited to the following examples. The measurement results in the examples are shown as values ​​rounded to the nearest whole number.

[0141] [Method for measuring isoflavones] In each test in the Examples, the content of isoflavones in the sample was measured as follows. Measurements were made under condition 1 in "1. Preparation of crude refined product A as a refined raw material" to "8. Purification test using crude refined product B as a raw material," and under condition 1 or 2 in "9. Equol fermentation test."

[0142] (High-performance liquid chromatography condition 1) Column: YMC-Pack ODS-AM-303 (4.6 mm x 250 mm (YMC) Mobile phase: A: Acetonitrile / water / acetic acid mixture (15:85:0.1 V / V / V) B Acetonitrile / water / acetic acid mixture (35:65:0.1 V / V / V) Concentration gradient: A linear concentration gradient from A to B was performed for 50 minutes. Flow rate: 1.0mL / min Column temperature: 35℃ Measurement wavelength: 254nm Injection volume: 10μL Retention times: daidzin 10.3 minutes, genistin 16.9 minutes, glycitin 11.0 minutes, malonyldaidzin 17.8 minutes, malonylgenistin 24.5 minutes, malonylglycitin 18.3 minutes, acetyldaidzin 23.2 minutes, acetylgenistin 33.3 minutes, acetylglycitin 23.6 minutes, daidzein 30.3 minutes, genistein 42.9 minutes, glycitein 32.4 minutes, dihydrodaidzein 30.2 minutes, dihydrogenistein 38.9 minutes, dihydroglycitein 32.4 minutes

[0143] (High-performance liquid chromatography condition 2) Column: Phenomenox SYNERGI 4 μm, mPOLAR-RP, 4.6 mm x 150 mm (Shimadzu GLC) Mobile phase: water / methanol [55:45, v / v] Flow rate: 1.0mL / min Column temperature: 40℃ Measurement wavelength: UV 280 nm Injection volume: 10μL Retention times: dihydrodaidzein 8.8 minutes, daidzein 9.9 minutes, dihydroglycitein 10.6 minutes, glycitein 12.2 minutes, dihydrogenistein 12.5 minutes, genistein 16.5 minutes, equol 14.1 minutes

[0144] [Method for detecting saponin content] The composition to be measured (equol-containing composition, saponins content equivalent to 1 mg) was extracted with a mixture of 1 mL of water and 18 mL of methanol. The insoluble matter was further extracted with 20 mL of methanol, and the extracts were mixed. The methanol was removed from this mixture by vacuum concentration. 10 mL of 10% hydrochloric acid in methanol was added, and the mixture was heated to reflux at 80°C for 2 hours. Ethanol was then added, and this was used as a sample for high-performance liquid chromatography analysis. 10 mg of each of the standards of soyasapogenol A (molecular weight: 474.72) and soyasapogenol B (molecular weight: 458.73) was accurately weighed into a 100 mL volumetric flask, dissolved in ethanol, and adjusted to the specified volume to prepare a standard solution. The contents of soybean saponin Ab (molecular weight: 1437.52) and soybean saponin Bb (molecular weight: 943.12) were calculated from the molecular weights of soyasapogenol A and soyasapogenol B. In the examples, the content of saponins is shown as a percentage (%) of the total content of isoflavones contained in the composition (i.e., calculated as "content of saponins / content of isoflavones x 100").

[0145] The high performance liquid chromatography conditions were, for example, as follows: Column: Inertsil ODS-2 5 μm, φ2.0 mm × 150 mm (GL Sciences) Mobile phase: acetonitrile / 1% acetic acid water = 7:3 Flow rate: 0.2mL / min Column temperature: 40℃ Detection: UV 210nm Retention times: Sapogenol A 6.78 min, Sapogenol B 14.13 min

[0146] 1. Preparation of Refined Raw Material Crude Product A 280 g of soybean hypocotyl extract (Fujiflavone P40 (daidzin content approximately 25% by weight or less), manufactured by Fujicco Co., Ltd.) was dissolved in 6.80 kg of 90% (V / V) aqueous ethanol solution at 50°C and filtered using a 7 μm filter. 6.53 kg of water was added to the resulting filtrate while stirring, and after cooling to 30°C, the mixture was stirred and aged for 24 hours. The insolubilized soybean hypocotyl extract was centrifuged (1000 rpm, 5 minutes) to obtain solids, which were then vacuum-dried at 60°C for 24 hours to obtain 29 g of soybean hypocotyl extract crude product A (hereinafter also referred to as isoflavone crude product A).

[0147] The resulting crude product A had a daidzin content of 88.3% by weight, a daidzein content of 2.2% by weight, a genistein content of 1.4% by weight, and a glycitein content of 1.8% by weight.

[0148] [2. Purification test using crude product A as raw material] Using crude product A as the raw material, a purification test was carried out using the following method.

[0149] Test condition 1A (cooling crystallization method): 4.0 g of crude product A was dissolved in 500 mL of 90% (V / V) aqueous ethanol at 50°C, filtered using a 7 μm filter, and the filtrate was cooled to 10°C over 12 hours and stirred at 1000 rpm at 10°C for 2 days. The precipitated crystals were collected by solid-liquid separation through filtration using a 7 μm filter and then vacuum-dried at 60°C for 24 hours to obtain a dried product.

[0150] Test condition 1B (water addition crystallization method): 4.0 g of crude product A was dissolved in 500 mL of 90% (V / V) aqueous ethanol at 50°C, filtered using a 7 μm filter, and 461 mL of water was added to the filtrate to make a 50% (V / V) aqueous ethanol solution. The solution was then cooled to 30°C over 6 hours and stirred at 1000 rpm at 30°C for 2 days. The precipitated crystals were separated by filtration using a 7 μm filter, recovered, and vacuum dried at 60°C for 24 hours to obtain a dried product.

[0151] Test condition 1C (repulp aging method): 4.0 g of crude product A was suspended in 300 mL of 50% (V / V) aqueous ethanol solution and stirred at 1000 rpm for 6 hours at 60° C. Insoluble matter was separated by filtration using a 7 μm filter, and the solid was recovered. The solid was then vacuum dried at 60° C. for 24 hours to obtain a dried product.

[0152] The contents of daidzin, daidzein, genisteins, and glyciteins were measured for the dried products obtained under each of the above test conditions. The composition of the obtained dried product, the daidzin yield, and the removal rates of daidzein, genisteins, and glyciteins are shown in Table 1. Here, the yield in Table 1 is a calculated value of what percentage of daidzin was recovered under each test condition relative to the content of daidzin in "crudely purified product A," and the removal rate is a calculated value of what percentage of the content of daidzin in "crudely purified product A" was removed under each test condition.

[0153] Compared with the cooling crystallization method and the water addition crystallization method, the repulp ripening method significantly increased the removal rates of daidzein, genisteins, and glyciteins. The repulp ripening method also significantly increased the yield of daidzin.

[0154] [Table 1]

[0155] 3. Preparation of Refined Raw Material Crude Product B 2.0 kg of soybean hypocotyl extract (isoflavones content 40% by volume (daidzin content approximately 60% by weight or less)) was dissolved in 55.0 kg of 90% (V / V) aqueous ethanol solution at 50°C, and the solution was filtered using a 7 μm filter. 52.0 kg of water was added to the resulting filtrate with stirring, and after cooling to 30°C, the mixture was stirred and aged for 24 hours. The insolubilized soybean hypocotyl extract was centrifuged (1000 rpm, 5 minutes) to obtain solids, which were then vacuum-dried at 60°C for 24 hours to obtain 234 g of soybean hypocotyl extract crude product B (hereinafter also referred to as isoflavone crude product B).

[0156] The resulting crude product B had a daidzin content of 90.3% by weight, a daidzein content of 1.9% by weight, a genistein content of 1.1% by weight, and a glycitein content of 1.4% by weight.

[0157] [4. Verification of ethanol concentration in the repulp maturation method] Using isoflavone crude product B as the raw material, repulping conditions (ethanol concentration) were investigated. 4.0 g of isoflavone crude product B was suspended in 300 mL of 30-100% (V / V) ethanol aqueous solution (Table 2) and stirred at 1000 rpm at 60°C for 6 hours. Insoluble matter was separated by filtration using a 7 μm filter, and the solid was recovered. The recovered material was then vacuum dried at 60°C for 24 hours to obtain a dried product.

[0158] The composition of the obtained dried product, the daidzin yield, and the removal rates of daidzein, genisteins, and glyciteins are shown in Table 2. The removal rate of daidzein was high at an ethanol concentration of 40% or more, the removal rate of genisteins was high at an ethanol concentration of 40% or more and 80% or less, and the removal rate of glyciteins was high at an ethanol concentration of 40% or more and 70% or less.

[0159] [Table 2]

[0160] [5. Verification of temperature in repulp maturation method] Using isoflavone crude product B as the raw material, the repulping conditions (temperature) were investigated. 4.0 g of isoflavone crude product B was suspended in 300 mL of 50% (V / V) aqueous ethanol solution and stirred at 1000 rpm for 6 hours at 30-60°C (Table 3). The insoluble matter was separated by filtration using a 7 μm filter, and the recovered solid was then vacuum dried at 60°C for 24 hours to obtain a dried product.

[0161] The composition of the obtained dried product, the daidzin yield, and the removal rates of daidzein, genisteins, and glyciteins are shown in Table 3. The higher the repulping temperature, the higher the removal rates of daidzein, genisteins, and glyciteins. Furthermore, even when the repulping temperature was low, the removal rate of isoflavones (daidzein, genisteins, and glyciteins) was comparable to that of the crystallization method, and the daidzin yield was extremely high. The repulp maturation method was found to be a much more efficient purification method than the crystallization method.

[0162] [Table 3]

[0163] [6. Verification of the amount of isoflavones used in the repulp maturation method] Using Isoflavone Crude Product B as the raw material, the repulping conditions (amount of isoflavones) were investigated. 4.0, 5.0, 6.0, or 8.0 g of Isoflavone Crude Product B (Table 4) was suspended in 300 mL of 50% (V / V) aqueous ethanol solution and stirred at 1,000 rpm for 6 hours at 60°C. The insoluble matter was separated by filtration using a 7 μm filter, recovered, and then vacuum-dried at 60°C for 24 hours to obtain a dried product.

[0164] The composition of the obtained dried product, the daidzin yield, and the removal rates of daidzein, genisteins, and glyciteins are shown in Table 4. The removal rates of daidzein, genisteins, and glyciteins did not change when 4.0 g or more and 8.0 g or less of isoflavone crude product B was used in 300 mL of 50% (V / V) ethanol aqueous solution. Even at 10.0 g, the removal rate was slightly lower, but still about 80%. The daidzin yield was higher when a larger amount of isoflavone crude product B was used and the concentration was higher.

[0165] [Table 4]

[0166] [7. Verification of mixing time in repulp maturation method] The repulping conditions (time) were investigated using Isoflavone Crude Product B as the raw material. 4.0 g of Isoflavone Crude Product B was suspended in 300 mL of 50% (V / V) aqueous ethanol solution and stirred at 1000 rpm at 60°C for each repulping time shown in Table 5. The insoluble matter was collected by solid-liquid separation through filtration using a 7 μm filter, and then vacuum dried at 60°C for 24 hours to obtain a dried product.

[0167] The composition of the obtained dried product, daidzin yield, and removal rates of daidzein, genisteins, and glyciteins are shown in Table 5. When the repulping time was 3 hours, the daidzin yield was low, but the removal rates of daidzein, genisteins, and glyciteins were high. When the repulping time was 5 hours or longer, the daidzin yield stabilized. Furthermore, when the repulping time was extended to 20 hours, the removal rates of daidzein, genisteins, and glyciteins tended to improve while maintaining the daidzin yield.

[0168] [Table 5]

[0169] [8. Purification test using crude product B as raw material] Using crude isoflavone product B as the raw material, a purification test was carried out using the following method.

[0170] Test condition 6A1 (cooling crystallization): 2.0 g of isoflavone crude product B was dissolved in 1500 mL of 90% (V / V) aqueous ethanol at 50°C. The solution was filtered through a 7 μm filter, and the filtrate was cooled to 10°C over 12 hours and stirred at 1000 rpm for 2 days. The precipitated solid was collected by filtration through a 7 μm filter for solid-liquid separation and then vacuum dried at 60°C for 24 hours to obtain 5.6 g of dried product. 4.0 g of this dried product was dissolved in 500 mL of 90% (V / V) aqueous ethanol at 50°C. The solution was filtered through a 7 μm filter, and the filtrate was cooled to 10°C over 12 hours and stirred at 1000 rpm for 2 days. The precipitated solid was collected by filtration through a 7 μm filter for solid-liquid separation and then vacuum dried at 60°C for 24 hours to obtain 2.0 g of dried product.

[0171] Test condition 6A2 (cooling crystallization): 1.0 g of the dried product obtained under test condition 6A1 (cooling crystallization) was dissolved in 125 mL of 90% (V / V) aqueous ethanol at 50°C and filtered using a 7 μm filter. The filtrate was cooled to 10°C over 12 hours and stirred at 1000 rpm for 2 days. The precipitated solid was collected by filtration using a 7 μm filter for solid-liquid separation and then vacuum-dried at 60°C for 24 hours to obtain a dried product.

[0172] Test condition 6B1 (water addition crystallization method): 2.0 g of crude isoflavone product B1 was dissolved in 1500 mL of 90% (V / V) aqueous ethanol at 50°C, filtered through a 7 μm filter, and 1383 mL of water was added to the filtrate to make a 50% (V / V) solution. The solution was then cooled to 30°C over 6 hours and stirred at 1000 rpm for 2 days. The precipitated solid was collected by filtration through a 7 μm filter for solid-liquid separation and then vacuum dried at 60°C for 24 hours to obtain 5.6 g of dried product. 4.0 g of this dried product was dissolved in 500 mL of 90% (V / V) aqueous ethanol at 50°C, filtered through a 7 μm filter, and 461 mL of water was added to the filtrate to make a 50% (V / V) solution. The solution was then cooled to 30°C over 6 hours and stirred at 1000 rpm for 2 days. The precipitated solid was separated into solid and liquid by filtering using a 7 μm filter, and then recovered. The solid was dried in vacuum at 60° C. for 24 hours to obtain 2.1 g of a dried product.

[0173] Test condition 6B2 (water addition crystallization): 1.0 g of the dried product obtained under test condition 6B1 (cooling crystallization) was dissolved in 125 mL of 90% (V / V) aqueous ethanol at 50°C, filtered using a 7 μm filter, and 115 mL of water was added to the filtrate to make it 50% (V / V). The solution was then cooled to 30°C over 6 hours and stirred at 1000 rpm for 2 days. The precipitated solid was collected by filtration using a 7 μm filter for solid-liquid separation and then vacuum dried at 60°C for 24 hours to obtain a dried product.

[0174] Test condition 6C (repulp aging method): 12.0 g of isoflavone crude product B was suspended in 450 mL of 50% (V / V) aqueous ethanol and stirred at 60°C for 24 hours at 1000 rpm. The insoluble matter was separated into solid and liquid by filtration using a 7 μm filter, and then collected. The mixture was then vacuum dried at 60°C for 24 hours to obtain 8.3 g of dried material. 4.0 g of this dried material was suspended in 160 mL of 50% (V / V) aqueous ethanol and stirred at 60°C for 24 hours at 1000 rpm. The insoluble matter was separated into solid and liquid by filtration using a 7 μm filter, and then collected. The mixture was then vacuum dried at 60°C for 24 hours to obtain 3.1 g of dried material.

[0175] The composition of the dried product, the daidzin yield, and the removal rates of daidzein, genisteins, and glyciteins are shown in Table 6. Compared to the cooling crystallization method and the water-addition crystallization method, the repulp aging method produced a significantly higher daidzin yield and significantly higher removal rates of its daidzein, genisteins, and glyciteins. Furthermore, with the cooling crystallization method and the water-addition crystallization method, even after three crystallizations, 0.1% or more of the daidzein, genisteins, and glyciteins remained. However, with the repulp aging method, even after two crystallizations, 0.0% of the daidzein, genisteins, and glyciteins remained.

[0176] [Table 6]

[0177] The HPLC chart of isoflavone crude product B is shown in Figure 1, and the HPLC chart of the dried product obtained under test condition 6C is shown in Figure 2. No isoflavones other than daidzin (daidzein, genisteins, or glyciteins) were detected in the dried product obtained under test condition 6C.

[0178] 9. Equol Fermentation Test An equol fermentation test was carried out using the following samples 1A to 1D. <Various raw materials> Sample 1A: Soybean hypocotyl extract (Fujiflavone P40, manufactured by Fujicco Co., Ltd.) Sample 1B: Soybean hypocotyl extract (isoflavone content 40% by weight) Sample 1C: Soybean hypocotyl extract (crude product B) Sample 1D: Dried product obtained under test condition 6C described in "8. Purification test using crude product B as raw material"

[0179] The soybean allergen content was measured as follows. An appropriate amount of the composition to be measured was added to an appropriate amount of extraction buffer (Tris HCl pH 7.5, containing 1 M EDTA, and containing an appropriate amount of protease inhibitor), and the mixture was stirred to extract the water-soluble components. The solids were then removed by filtration to obtain an extract. The total protein content of the resulting extract was measured using a Bio-Rad protein assay system. Furthermore, soybean allergens (Gym4, Gm30K, Gm28K, 7S globulin mix, oleosin, and trypsin inhibitor) contained in the extract were detected by Western blotting. Total proteins were electrophoresed and blotted onto a hydrophobic membrane. Antibodies capable of detecting Gym4, Gm30K, Gm28K, 7S globulin mix, oleosin, and trypsin inhibitor were used to detect soybean allergens on the membrane.

[0180] The composition of the raw materials used is shown in Table 7. [Table 7]

[0181] <Enzyme treatment> For each of Samples 1A to 1D, 2.5 g of each was weighed out in terms of isoflavones (daidzin, daidzein, genisteins, and glyciteins), and 50 mg of aromatase (manufactured by Amano Enzyme Inc.) was added as an enzyme, and 450 g of deionized water was used to adjust the pH to 4 to 7. The enzyme treatment was carried out at 50°C for 24 hours with stirring.

[0182] <Fermentation by anaerobic microorganisms> (seed culture) A medium with the composition shown in Table 8 and adjusted to pH 6.9 was dispensed into 10 mL 18 mm test tubes for anaerobic microbial culture (manufactured by Sanshin Kogyo Co., Ltd.). The tubes were capped with butyl rubber stoppers and plastic caps and sterilized at 121°C for 15 minutes while the gas phase was replaced with nitrogen. One loopful of Eggerthella sp. DC 3215 strain was inoculated into the medium, and the gas phase was replaced with sterile-filtered hydrogen gas for at least 2 minutes. The medium was then cultured with shaking at 37°C and 200 spm for 18 hours. This yielded a preculture solution.

[0183] [Table 8]

[0184] (main culture) To the enzyme-treated solution of each of Samples 1A to 1D (obtained by the above-mentioned "enzyme treatment"), 3 g of L-arginine and each component shown in Table 8 were added to the respective concentrations shown in Table 8, and 500 mL of each medium adjusted to pH 6.9 was heated at 121°C for 15 minutes and sterilized by high-pressure steam. Five tubes of the above pre-culture solution (obtained by the above "seed culture") were inoculated into 500 mL of this medium, and the gas phase was aerated with hydrogen / nitrogen gas at a ratio of 6:4. The culture was carried out at 37°C for 48 hours, and the concentrations of isoflavones and equol in the main culture solution were analyzed by HPLC.

[0185] <Recovery of equol-containing composition> After the above culture, the fermented liquid was dried (spray dried) to obtain a dried product containing equol.

[0186] The obtained fermentation liquid and dried product of the fermentation liquid (dried fermentation product) are shown in Table 9. By using the crude product (Sample 1C) or daidzin (Sample 1D) purified by the repulp aging method as the fermentation raw material, the fermentation liquid obtained had a higher equol content than when Samples 1A and 1B were used, and no detectable genisteins or glyciteins were found. These liquids also contained no saponins. When sample 1D was used, genisteins and glyciteins were not detected even in the dried fermented product, and the sample was negative for soybean allergens.

[0187] [Table 9]

Claims

1. the content of glyciteins and genisteins is 0.1% by weight or less, respectively, based on the total dry weight of the composition; The content of naturally derived daidzeins is more than 99.5% of the total isoflavones. composition.

2. 2. The composition according to claim 1, wherein the naturally-derived daidzein is derived from soybeans.

3. 3. The composition according to claim 1, wherein the daidzein is daidzin.

4. 3. The composition according to claim 1, wherein the daidzein is equol.

5. 3. The composition according to claim 1, wherein the daidzein content is less than 0.1% by weight.

6. The composition of claim 3 for use in producing daidzein.

7. The composition according to claim 3 for use in producing equol.

8. The composition of claim 4, which is for oral administration.

9. A supplement comprising the composition of claim 8.

10. The method comprises suspending 10 g or more of a plant extract containing daidzin per 1 L of a 30 to 90% by volume aqueous ethanol solution, The plant extract contains 1 to 10% by weight of genisteins and 1 to 30% by weight of glyciteins. Daidzin purification method.

11. The purification method according to claim 10, wherein the plant extract is a soybean hypocotyl extract.

12. The purification method according to claim 10 or 11, wherein the plant extract contains 85% by weight or more of daidzin.

13. 12. The method for purifying the plant extract according to claim 10 or 11, comprising maintaining a suspension of the plant extract in an aqueous ethanol solution at a temperature of 30°C or higher.

14. A method for producing daidzein, comprising the purification method according to claim 10 or 11.

15. A method for producing equol, comprising the purification method according to claim 10 or 11.

16. A method for producing a food composition, comprising the method for producing equol according to claim 15.

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