Methods for producing equol-containing compositions

Abstract

To provide methods for producing equol-containing compositions substantially free of allergens.SOLUTION: Disclosed is a method for producing an equol-containing composition comprising the steps of: (1) processing an isoflavone with an enzyme that converts the isoflavone to an aglycon; (2) culturing an anaerobic microorganism in a culture medium containing the aglycon prepared in the step (1) to convert the aglycon to equol by the anaerobic microorganism; and (3) collecting the equol-containing composition containing equol formed in the step (2) from the culture medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing an equol-containing composition. [Background technology]

[0002] Soybeans contain many soy isoflavones, which have female hormone (estrogen) and antioxidant effects, and it has been shown that consuming isoflavones has a preventive effect against breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, etc. (Non-patent documents 1 to 6).

[0003] On the other hand, soybeans are listed as one of the five major allergenic foods in Japan, and soybean allergies are known to be caused by allergenic proteins in the storage proteins contained in soybeans. As of 1992, 16 types of soybean allergen proteins had been identified, and the major allergens with the highest frequency of allergic reactions are thought to be Gly m Bd 30K, Gly m Bd 60K (7S globulin α-subunit), and Gly m Bd 28K. (Non-patented Reference 7) For this reason, soybean processed foods with reduced soybean allergens have been developed (Patent Documents 1 to 3).

[0004] Recent research has revealed that when soybeans are powdered and the isoflavones are measured, 12 types of isoflavones are present, as shown in Table 1, of which daidzin, malonyldaidzin, genistin, and malonylgenistin account for the majority (Non-Patent Document 8).

[0005] [Table 1]

[0006] Furthermore, it has been found that the active component of soy isoflavones, which is important for exerting the above-mentioned female hormone and antioxidant effects, is mainly derived from equol, which is produced from daidzein via dihydrodaidzein when daidzin is metabolized in the body, as described below. Specifically, daidzin, the main isoflavone in soybeans, exists in the soybean as a glycoside bound to a sugar. When this glycoside enters the human or animal body, it is enzymatically converted via dihydrodaidzein to O-desmethylangolensin or equol by the action of digestive enzymes or β-glucosidase, an enzyme produced by intestinal bacteria, and equol in particular is known to have high estrogenic activity (Non-Patent Documents 9 and 10).

[0007] [ka]

[0008] However, even when people eat soybeans, their ability to metabolize the daidzein contained in soybeans into the active ingredient equol varies from person to person, and it has been shown that only about 50% of Japanese people and about 30% of Westerners are able to metabolize it (Non-Patent Documents 11 and 12). Therefore, so that people who cannot metabolize it can directly ingest the active ingredient equol, fermented soybean products containing equol have been developed, and some of these products have reduced soybean allergens (Patent Documents 4 to 6). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Publication No. 7-236427 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-220301 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-274900 [Patent Document 4] Patent No. 5030790 [Patent Document 5] Japanese Patent Publication No. 2012-228252 [Patent Document 6] Japanese Patent Publication No. 2012-161323

Non-licensed literature

[0010]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patented document 9

Non-patent document 10

[0011] For people who cannot metabolize daidzein to the active ingredient equol, it is preferable to ingest the active ingredient, equol, directly. However, although fermented products containing equol with reduced allergen content have been developed, no product that does not induce allergies and is essentially allergen-free has yet been developed. An object of the present invention is to provide a method for producing an equol-containing composition that is substantially free of allergens. [Means for solving the problem]

[0012] Based on the above findings, the inventors further investigated methods for obtaining an equol-containing composition that is substantially free of allergens, and discovered that this could be achieved by treating isoflavones with a specific enzyme and then fermenting it with a specific microorganism, leading to the completion of the present invention.

[0013] [1] A method for producing an equol-containing composition, comprising: (1) treating isoflavones derived from legume plants with an enzyme that converts the isoflavones to aglycones; (2) culturing anaerobic microorganisms in a medium containing the aglycones obtained in step (1), and converting the aglycones into equol using the anaerobic microorganisms; and (3) recovering an equol-containing composition containing the equol produced in step (2) from the medium. [2] The method according to [1], wherein the legume is one or more species selected from the group consisting of soybean, kudzu, red clover, and licorice. [3] The method according to [1] or [2], wherein the plant body of the legume family is a soybean hypocotyl. [4] The method according to any one of [1] to [3], wherein the isoflavones are aglycone glycosides. [5] The method according to [4], wherein the aglycone glycoside is one or more selected from the group consisting of daidzin, acetyldaidzin, malonyldaidzin, and succinyldaidzin. [6] The anaerobic microorganism is Asaccharobacter celatus ) Possess 16S rDNA with 95% or more identity to the 16S rDNA of DSM 18785 The method according to any one of [1] to [5], wherein the microorganism is [7] The anaerobic microorganism is a microorganism classified into the genus Asaccharobacter, Slackia, or Adlercreutzia. The method according to any one of [1] to [6]. [8] The method according to any one of [1] to [7], wherein the enzyme is β-glucosidase or pectinase. [9] A cosmetic preparation comprising an equol-containing composition produced by the method according to any one of [1] to [8].

[10] A pharmaceutical comprising an equol-containing composition produced by the method according to any one of [1] to [8].

[11] A food product comprising an equol-containing composition produced by the method according to any one of [1] to [8]. [Effects of the Invention]

[0014] According to the present invention, a method for producing an equol-containing composition that is substantially free of allergens can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is an image (photograph substituting a drawing) showing the results of allergen detection using FASTKIT Slim in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below by showing specific embodiments, but it goes without saying that the present invention is not limited to the specific embodiments exemplified.

[0017] The present invention provides (1) treating isoflavones with an enzyme that converts the isoflavones into aglycones; (2) culturing anaerobic microorganisms in a medium containing the aglycone obtained in step (1), and converting the aglycone into equol by the anaerobic microorganisms; and (3) recovering the equol-containing composition containing the equol produced in step (2) from the medium; The present invention relates to a method for producing an equol-containing composition, comprising:

[0018] The method for producing an equol-containing composition according to the present invention comprises: (1) an "enzyme treatment step" in which isoflavones are treated with an enzyme that converts the isoflavones to aglycones; (2) a "fermentation step" in which anaerobic microorganisms are cultured in a medium containing the aglycones obtained in step (1) and the aglycones are converted to equol by the anaerobic microorganisms; and (3) a "recovery step" in which the equol-containing composition containing the equol produced in step (2) is recovered from the medium.

[0019] The method for producing the equol-containing composition according to the present invention includes the above steps (1) to (3), but may also include other steps. Other steps include, for example, a step of preparing isoflavones, which serve as substrates for the enzyme treatment in the enzyme treatment step described below, by crushing, extracting, and refining resins from legumes such as soybean, kudzu, red clover, and licorice. For example, this can be carried out by a conventional method, such as the method for producing isoflavone compounds using soybean germ as a raw material disclosed in Japanese Patent Laid-Open Publication No. 11-263786. This step can be carried out before the enzyme treatment step described below.

[0020] <1. Enzyme treatment process> The enzyme treatment step of the present invention is a step of treating isoflavones with an enzyme that converts the isoflavones into aglycones.

[0021] <1-1. Isoflavones> The isoflavones of the present invention are glycosides of flavonoids with an isoflavone skeleton. The source material is not particularly limited, but they can be obtained from legumes such as soybean, kudzu, red clover, and licorice. Among these, soybeans, which contain a high content of daidzein as an aglycone, are preferred, and soybean hypocotyls are particularly preferred.

[0022] The isoflavones of the present invention are not particularly limited, and examples thereof include any derivatives such as acetylated glycosides of aglycones, malonylated glycosides of aglycones, and succinylated glycosides of aglycones. Examples of glycosides of aglycons include daidzin, etc. Examples of acetylated glycosides of aglycons include acetyldaidzin, etc. Examples of malonylated glycosides of aglycons include malonyldaidzin, etc. Examples of succinylated glycosides of aglycons include succinyldaidzin, etc. The isoflavones of the present invention may contain one or more of these. Among these, daidzin is preferred. In this specification, isoflavones refer to any derivatives such as the above-mentioned glycosides in which sugar is bound to an aglycone, and do not include derivatives without sugar bound, i.e., aglycones themselves.

[0023] The isoflavones used in the enzyme treatment step of the present invention may be purified or unpurified. Soybean hypocotyls may be used as they are, or may be unpurified and contain other components. Commercially available isoflavones include, for example, crushed soybean hypocotyls (manufactured by Fuji Oil Co., Ltd.) and isoflavone-rich extract powder (manufactured by Tama Biochemical Co., Ltd.).

[0024] <1-2. Aglycone> The aglycone of the present invention is not particularly limited, but examples thereof include daidzein, 6-hydroxydaidzein, and dihydrodaidzein. The aglycone of the present invention may contain one or more of these. Among these, daidzein is preferred.

[0025] <1-3. Types of enzymes> The enzyme that acts on isoflavones in the enzymatic treatment step of the present invention to convert the isoflavones to aglycones is not particularly limited as long as it has the activity of cleaving the bond between the aglycone and the sugar in any derivative such as the aglycone glycoside described above. From the viewpoint of equol production, however, it is preferably an enzyme within the enzyme code EC3, more preferably an enzyme within EC3.2, even more preferably an enzyme within EC3.2.1, and particularly preferably β-glucosidase (EC 3.2.1.21) or pectinase (EC 3.2.1.15), and from the viewpoint of productivity, it is most preferably pectinase.

[0026] <1-4. Enzyme treatment> <1-4-1. Isoflavone content> In the enzyme treatment step of the present invention, the content of isoflavones relative to the total amount of enzyme treatment solution is not particularly limited, but from the viewpoint of the production concentration of equol, it is preferably 0.1 g / L or more, more preferably 0.5 g / L or more, and even more preferably 1.0 g / L or more in terms of isoflavones. On the other hand, from the viewpoint of enzyme activity, it is preferably 10 g / L or less, more preferably 5 g / L or less, and even more preferably 3 g / L or less.

[0027] <1-4-2. Enzyme concentration> In the enzyme treatment step of the present invention, the content of the enzyme relative to the total amount of the enzyme treatment solution is not particularly limited as long as the enzyme treatment proceeds efficiently, but the content of the enzyme is preferably 0.01% by mass or more and 10% by mass or less, more preferably 0.05% by mass or more and 5% by mass or less, and even more preferably 0.1% by mass or more and 1% by mass or less.

[0028] <1-4-3. Enzyme treatment temperature> In the enzyme treatment step of the present invention, the temperature of the enzyme treatment solution is not particularly limited as long as the enzyme activity is maintained and the enzyme treatment proceeds efficiently, but in order to activate the enzyme and perform rapid enzyme treatment, the temperature is preferably 10°C or higher, more preferably 30°C or higher, and even more preferably 40°C or higher. On the other hand, from the viewpoint of maintaining the enzyme activity, the temperature is preferably 80°C or lower, more preferably 70°C or lower, and even more preferably 60°C or lower.

[0029] <1-4-4. pH during enzyme treatment> In the enzyme treatment step of the present invention, the pH of the enzyme treatment solution is not particularly limited as long as it maintains enzyme activity and the enzyme treatment proceeds efficiently, but it is preferably between 2 and 10, more preferably between 3 and 8, and even more preferably between 4 and 7.

[0030] <1-4-5. Enzyme treatment time> In the enzymatic treatment step of the present invention, the time for the enzymatic treatment is not particularly limited as long as the enzymatic activity is maintained and the enzymatic treatment proceeds efficiently, but from the viewpoint of enzymatic activity, it is preferably 1 hour or more, more preferably 4 hours or more, and even more preferably 15 hours or more, while from the viewpoint of productivity, it is preferably 72 hours or less, more preferably 48 hours or less, and even more preferably 36 hours or less.

[0031] <2. Fermentation process> The fermentation step of the present invention is a step of culturing anaerobic microorganisms in a medium containing the aglycone obtained in the enzyme treatment step, and converting the aglycone into equol by the anaerobic microorganisms. The medium for culturing the anaerobic microorganisms may contain other components, including isoflavones that were not converted from isoflavones to aglycones in the enzyme treatment step.

[0032] <2-1. Aglycone concentration> The aglycone in the fermentation step of the present invention is an aglycone converted from an isoflavone by an enzyme in the enzyme treatment step. In the fermentation process of the present invention, the concentration of the aglycone is not particularly limited, but in order to increase the concentration of equol produced, it is preferably 0.1 g / L or more, more preferably 0.5 g / L or more, and even more preferably 1.0 g / L or more as isoflavones. On the other hand, from the viewpoint of enzyme activity, it is preferably 10 g / L or less, more preferably 5 g / L or less, and even more preferably 3 g / L or less.

[0033] <2-2. Anaerobic microorganisms> The anaerobic microorganisms used in the present invention are not particularly limited as long as they can be cultured in a medium containing the aglycone and can ferment equol from the aglycone. However, bacteria classified in the family Coriobacteriaceae, bacteria classified in the family Streptococcaceae, or related bacteria are preferred as equol-producing microorganisms. Examples of such a method include:

[0034] Furthermore, anaerobic microorganisms classified into genera selected from the following group can be exemplified as microorganisms capable of producing equol: ·Coriobacterium genus Adlercreutzia genus ·Asaccharobacter genus ·Atopobium genus Collinsella genus Cryptobacterium genus Denitrobacterium spp. Eggerthella genus Enterorhabdus genus Gordonibacter genus ·Olsenella genus ·Genus Paraeggerthella Slackia genus Lactococcus genus

[0035] Microorganisms selected from those classified into these genera that utilize aglycone to produce equol through anaerobic fermentation are preferred microorganisms in the present invention.

[0036] Among the microorganisms classified into the above genera, it is preferable to use Asaccharobacter celatus DSM 18785 or a closely related microorganism having a 16S rDNA sequence that is 95% or more, preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more identical to the 16S rDNA (16S rRNA gene sequence) of Asaccharobacter celatus DSM 18785. The 16S rRNA gene sequence of Asaccharobacter celatus DSM 18785 is SEQ ID NO: 1.

[0037] Furthermore, among the above microorganisms, microorganisms belonging to the genera Asaccharobacter, Slackia, and Adlercreutzia are useful in the present invention. It is more preferable as a microorganism capable of producing equol.

[0038] The production of equol by a microorganism can be confirmed by quantifying daidzein, dihydrodaidzein, equol, etc. in the culture. These quantifications can be performed by those skilled in the art based on the descriptions in, for example, WO2012 / 033150, JP2012-135217A, JP2012-135218A, JP2012-135219A, etc.

[0039] Additionally, the following anaerobic microorganisms can be utilized: Adlercreutzia equolifaciens DSM 19450 Enterorhabdus mucosicola DSM 19490 Slackia isoflavoniconvertens HE8(DSM 22006) Slackia sp. TM-30 FERM P-20729 Eggerthella sp. KCCM-10490 Asaccharobacter celatus DSM 18785 Lactococcus garvieae DSM 6783

[0040] Among the above microorganisms, Asaccharobacter celatus DSM 18785 strain, Adlercreutzia equolifaciens DSM 19450 strain, or Slackia isoflavoniconvertens DSM 22006 strain, or related bacteria having properties as species similar to these bacteria, are It can be mentioned as a more preferred anaerobic microorganism.

[0041] The above anaerobic microorganisms can be obtained from the depository institutions indicated by their accession numbers. Each accession number indicates that the anaerobic microorganism has been deposited at the following depository institution: FERM International Patent Organism Depositary (IPOD) http: / / unit.aist.go.jp / pod / ci / index.html DSM German Collection of Microorganisms and Cell Cultures (DSMZ) http: / / www.dsmz.de / KCCM Korean Culture Center of Microorganisms

[0042] <2-3. Culture and fermentation conditions> The anaerobic microorganisms used in the present invention are cultured in a medium in contact with aglycone under conditions suitable for the production of equol. Conditions suitable for producing the equol-containing composition of the present invention refer to conditions that maintain the survival and activity of anaerobic microorganisms capable of producing equol. More specifically, these conditions refer to conditions that maintain gas-phase conditions (anaerobic conditions) that allow the survival of anaerobic microorganisms and provide nutrients to support the activity and growth of the anaerobic microorganisms. Various medium compositions suitable for the survival of anaerobic microorganisms are known. Therefore, those skilled in the art can select an appropriate medium composition for the anaerobic microorganisms capable of producing equol described above. For example, the GAM bouillon medium manufactured by Nissui Pharmaceutical Co., Ltd. and the BHI medium manufactured by Difco, which were used in the examples, can be used.

[0043] The medium used in the present invention can contain, for example, water-soluble organic compounds as a carbon source, such as sorbose, fructose, glucose, and organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, and formic acid.

[0044] The concentration of the organic matter added to the medium as a carbon source can be adjusted appropriately to efficiently grow anaerobic microorganisms in the medium. Generally, excess or deficiency can be avoided by selecting the amount added from the range of 0.1 to 10 wt / vol%.

[0045] In addition to the above carbon sources, a nitrogen source can be added to the medium. In the present invention, various nitrogen compounds that can be used in conventional fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are ammonium salts and nitrates. Preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder, etc. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate. More preferred nitrogen sources are arginine, citrulline, ornithine, lysine, yeast extract, and peptones.

[0046] In addition to carbon and nitrogen sources, other organic or inorganic substances suitable for culturing obligately anaerobic microorganisms can also be added to the medium. For example, the growth and activity of obligately anaerobic microorganisms can sometimes be enhanced by adding cofactors such as vitamins or inorganic compounds such as various salts to the medium. For example, the following inorganic compounds, vitamins, and microbial growth cofactors derived from animals and plants can be mentioned:

[0047] Examples of inorganic compounds include potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, alum, sodium molybdate, potassium chloride, boric acid, nickel chloride, sodium tungstate, sodium selenate, and ferrous ammonium sulfate.

[0048] Examples of vitamins include biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, vitamin B12, thiooctoic acid, and p-aminobenzoic acid. In addition, it may be beneficial to add hemin, a porphyrin compound.

[0049] Methods for producing a culture medium by adding these inorganic compounds, vitamins, or growth cofactors are known. The medium can be liquid, semi-solid, or solid. In the method for producing equol of the present invention, the preferred form of the medium is a liquid medium.

[0050] The anaerobic microorganisms used in the present invention can be cultured according to known microbial culture methods. For industrial production, a continuous fermentation system capable of continuously supplying the medium and substrate gas and equipped with a mechanism for recovering the culture is suitable.

[0051] In culturing the anaerobic microorganisms used in the present invention, it is necessary to prevent oxygen from entering the continuous culture system. A commonly used culture tank can be used as the culture vessel. Culture tanks that can also be used to cultivate anaerobic microorganisms are commercially available. An anaerobic atmosphere can be created by replacing the oxygen that enters the culture tank with an inert gas such as nitrogen or hydrogen gas.

[0052] For example, an anaerobic culture jar can be used as a bioreactor for culturing anaerobic microorganisms. The anaerobic culture jar is an airtight container made of metal, glass, or synthetic resin, and its interior can be isolated from atmospheric oxygen. Furthermore, the anaerobic culture jar can be equipped with a mechanism for removing molecular oxygen contained in the space inside the anaerobic culture jar and in the culture solution. For example, the inside of the anaerobic culture jar can be maintained in an anaerobic state by connecting a vacuum pump that suctions the inside of the anaerobic culture jar and supplying gases other than oxygen.

[0053] In the present invention, additional functions can be added to the culture tank. For example, in addition to commonly used agitation tanks, bubble column and draft tube culture tanks can also be used. The mixed gas blown into the liquid medium disperses the microorganisms, allowing sufficient contact between the microorganisms and the medium. Alternatively, microorganisms can be grown in a biotrickling filter, which is filled with highly breathable slag, other ceramic inorganic fillers, or organic synthetic materials such as polypropylene, while dripping water and allowing gas to pass through. Furthermore, the microorganisms used can be immobilized in carrageenan gel, alginate gel, acrylamide gel, chitin, cellulose, agar, or the like by standard methods.

[0054] In the fermentation process of the present invention, the combination of gases constituting the gas phase consisting of the above-mentioned substrate gases is not particularly limited, and one or more gases selected from hydrogen, carbon dioxide, nitrogen, etc. can be used as constituent components. It is preferable that the gas phase contains hydrogen as a constituent component.

[0055] In the above case, the partial pressure percentage concentration of hydrogen in the gas phase of the fermentation process is preferably 2 to 100%. For example, the partial pressure percentage concentration is preferably 2%, 4%, 6%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, or a concentration range with a lower limit (greater than or equal to) and an upper limit (less than or equal to) of a partial pressure percentage concentration selected from these.

[0056] Furthermore, in order to efficiently produce equol, the rate of aeration of the mixed gas that constitutes the gas phase into the culture tank is preferably 0.01 to 2.0 V / V / M gas volume / liquid volume / min.

[0057] The anaerobic microorganisms used in the present invention are generally anaerobic microorganisms capable of producing equol at temperatures around 37°C (30 to 42°C).

[0058] In the present invention, the pressure conditions for culturing anaerobic microorganisms are not particularly limited as long as they are conditions under which the microorganisms can grow, but preferred pressure conditions include a range of 0.02 to 0.2 MPa.

[0059] For sufficient growth of the microorganisms, the pH of the culture is preferably 3.0 to 8.0, more preferably 4.5 to 7.5. To increase the amount of equol recovered, the temperature of the culture tank is not particularly limited, but 37°C is a preferred temperature.

[0060] <3. Recovery process> The recovery step of the present invention is a step of recovering the equol-containing composition containing the equol produced in the fermentation step from the medium.

[0061] Those skilled in the art can use any known method to separate the produced anaerobic microorganisms from the culture solution. A preferred separation method is to use a hollow fiber ultrafiltration or microfiltration membrane with filtration and concentration capabilities. Furthermore, to achieve a filtration rate sufficient for separating the microorganisms from the culture solution, a membrane with an appropriate molecular weight cutoff can be selected depending on the microorganism used. The produced equol can be recovered from the culture medium by any means known to those skilled in the art. For example, equol can be recovered from the culture solution separated from the culture vessel through an ultrafiltration membrane. Methods for equol purification are known. For example, the culture can be centrifuged to remove the bacterial cells, the supernatant concentrated under reduced pressure, and then extracted with 70% ethanol or methanol. The extract can be further purified by silica gel chromatography, crystallization, or other procedures. Styrene-divinylbenzene adsorption resins such as Amberlite and Sepabeads can also be used.

[0062] In the recovery step of the present invention, to efficiently recover equol, the amount of fresh medium supplied to the culture tank is preferably such that the dilution rate of the culture in the culture tank is 0.04 to 2 / hr, more preferably 0.08 to 1 / hr.

[0063] <4. Equol-containing composition> The equol-containing composition obtained by the production method of the present invention can be provided for various uses, such as cosmetics, pharmaceuticals, foods, etc. When taken as a food, drink, or pharmaceutical, it can help prevent breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, etc.

[0064] <5. Cosmetics> When the equol-containing composition of the present invention is used as a cosmetic ingredient, it can be formulated into various desired cosmetic forms, including liquids such as aqueous solutions, lotions, sprays, suspensions, and emulsions; solids such as powders, granules, and blocks; semisolids such as creams and pastes; and gels. Such cosmetics are useful as a variety of cosmetic products, including face washes, emulsions, creams, gels, essences (serums), packs, and masks; makeup products such as foundations and lipsticks; oral cosmetics; fragrance cosmetics; hair cosmetics; and body cosmetics. Cosmetics containing the equol-containing composition obtained by the present invention are used as whitening cosmetics, acne-fighting cosmetics, and wrinkle-fighting cosmetics. Cosmetics containing the equol-containing composition of the present invention can be produced according to conventional methods. The amount, method, and timing of incorporation into the cosmetics can be selected as appropriate. Furthermore, the composition can be packaged in appropriate containers such as bottles, bags, cans, spray cans, spray containers, boxes, and packs, as needed. When the equol-containing composition of the present invention is used as a cosmetic ingredient, the content of the equol-containing composition relative to the total amount of the cosmetic is not particularly limited as long as the desired effects of the present invention are achieved, but is typically 0.00005 to 10% by mass, preferably 0.0001 to 5% by mass, and more preferably 0.0001 to 2% by mass in terms of equol.

[0065] <6. Pharmaceuticals> When the equol-containing composition of the present invention is used as a pharmaceutical ingredient, the dosage form can be selected depending on the disease to be prevented or treated, the pharmaceutical usage form, administration route, etc. Examples include tablets, coated tablets, pills, capsules, granules, powders, liquids, suspensions, emulsions, syrups, injections, suppositories, infusions, decoctions, tinctures, etc. These various preparations may be prepared in the usual manner by adding fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, colorants, flavorings, solubilizers, suspending agents, coating agents, etc. to the main drug as needed. The pharmaceutical preparations may be formulated using known adjuvants that are commonly used in the pharmaceutical formulation field, such as coloring agents, preservatives, perfumes, flavoring agents, sweeteners, and other pharmaceuticals. The equol-containing composition of the present invention can be used for the prevention and treatment of breast cancer, prostate cancer, osteoporosis, heart disease, and menopausal disorders.

[0066] When the equol-containing composition of the present invention is used as a raw material for a pharmaceutical product, the content of the equol-containing composition relative to the total amount of the pharmaceutical product is not particularly limited as long as the desired effects of the present invention are achieved, but is typically 0.001 to 30% by mass, preferably 0.01 to 20% by mass, and more preferably 0.1 to 10% by mass of equol. The dosage of the pharmaceutical product containing the equol-containing composition can be appropriately determined depending on the patient's age, weight, severity of symptoms, etc.

[0067] <7.Food> When the equol-containing composition of the present invention is used as a food ingredient, it can be used not only as a general food, but also as a food for specified health uses, a nutritional supplement, a functional food, a food for the sick, a food additive, etc. Examples of food forms that may be used include soft drinks, milk, pudding, jelly, candy, chewing gum, gummy candy, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cake, cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing, etc.

[0068] The equol-containing composition of the present invention can be composed primarily of water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. Proteins include, for example, animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, and meat protein, as well as their hydrolysates and butter. Carbohydrates include sugars, modified starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), and dietary fiber. Lipids include, for example, vegetable oils such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils, and interesterified oils. Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these ingredients may be used in combination, and synthetic products and / or foods containing large amounts of these ingredients may also be used.

[0069] Foods containing the equol-containing composition of the present invention can be produced according to conventional methods. The amount, method, and timing of incorporation into the food can be selected as appropriate. Furthermore, the composition can be packaged in suitable containers such as bottles, bags, cans, boxes, and packs, as needed. When the equol-containing composition of the present invention is used as a food ingredient, the content of the equol-containing composition relative to the total weight of the food is not particularly limited as long as the desired effect of the present invention is achieved, but is typically 0.01 to 10 mass% in terms of equol, preferably 0.05 to 5 mass%, and more preferably 0.1 to 2 mass%. [Example]

[0070] The present invention will be described in more detail below with reference to specific examples, but the present invention is not limited to these examples.

[0071] [Example 1] <1. Preparation of isoflavones> In this example, soybean hypocotyls (manufactured by Fuji Oil Co., Ltd.) were ground using a Waring blender for 1 minute three times (total of 3 minutes) and used as isoflavones.

[0072] <2. Enzyme treatment> The enzyme treatment solution was prepared using 270 g of soybean hypocotyls (Fuji Oil) as isoflavones, 2.7 g of Pectinase G Amano as an enzyme, and 1180 g of deionized water to make a total of 1450 g, and the pH was adjusted to 4 to 7. The enzyme treatment was carried out at 50°C for 20 to 24 hours with stirring.

[0073] <3. Fermentation by anaerobic microorganisms> (seed culture) 5.9 g of GAM bouillon medium (manufactured by Nissui Pharmaceutical) and 1.2 g of L-arginine hydrochloride were dissolved in 100 mL of pure water, and 20 mL of each was dispensed into 18 mm test tubes for culturing anaerobic bacteria (manufactured by Sanshin Kogyo) while nitrogen gas was passed through them. The tubes were then fitted with butyl rubber stoppers and plastic caps and sterilized at 115°C for 15 minutes. This medium was inoculated with Asaccharobacter celatus DSM 18785 strain, which had been frozen and stored at -80°C, and the gas phase was irradiated with hydrogen gas through a sterile filter for 2 minutes. After the above replacement, shaking culture was carried out at 37°C and 250 spm for 16 hours.

[0074] (fermentation) Similar to the above seed culture, GAM bouillon medium supplemented with L-arginine hydrochloride was sterilized in an autoclave at 115°C for 15 minutes, cooled to room temperature, and then inoculated with the above seed culture. The gas phase was aseptically replaced with hydrogen gas for at least 2 minutes, and then the medium was cultured with shaking at 37°C and 250 spm for fermentation.

[0075] <4. Recovery of equol-containing composition> After the fermentation, the culture solution was centrifuged at 3000 rpm for 10 minutes and filtered through a 0.45 μm MF membrane to remove insoluble matter, and the equol-containing composition solution was recovered. Equol-containing material (EQ-N) was obtained by lye processing.

[0076] 5. Allergen detection The obtained equol-containing composition was compared with FASTKIT Slim Soybean (manufactured by Nippon Meat Packers Central Research Institute). ) was used to detect allergens. The specific method followed the FATKIT Slim manual. Using 2 g of EQ-N as the sample, 38 mL of the kit's extraction buffer was added. After mixing for 30 to 60 minutes, extraction was performed three times. The mixture was then centrifuged at 3000 × g and 4°C for 20 minutes. The supernatant was filtered through filter paper and diluted 10-fold with the kit's dilution buffer to prepare the measurement sample. Measurements were performed by dropping 100 μL onto a test strip, allowing it to develop for 15 minutes, and then visually inspecting the sample.

[0077] [Comparative Example 1] Allergens were detected in the same manner as above for crushed soybean hypocotyls that had not been subjected to the enzyme treatment and fermentation.

[0078] <6.Results> When an allergen is present, a band appears at the position indicated by the arrow in the test strip shown in Figure 1. However, in Example 1, in which the equol-containing composition was recovered after enzyme treatment and fermentation, no band indicating the presence of an allergen was detected. On the other hand, in Comparative Example 1, in which the composition was not subjected to enzyme treatment or fermentation, no band indicating the presence of an allergen was detected. It was. From the above, it was found that by carrying out the above-described enzyme treatment and fermentation of the present invention, an equol-containing composition that is substantially free of allergens can be obtained. [Industrial Applicability]

[0079] By ingesting the equol-containing composition produced by the method of the present invention as a food, drink, medicine, etc., breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, etc. can be prevented.

Claims

[Claim 1] (1) treating a legume plant containing isoflavones with an enzyme that converts the isoflavones into aglycones to obtain an enzyme-treated solution, and fermenting the enzyme-treated solution containing the aglycones with an anaerobic microorganism to convert the aglycones into equol; and (2) recovering the equol-containing composition containing the equol produced in step (1) from the medium; A method for producing an equol-containing composition that is substantially free of allergens, comprising:

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