JP2023105251A - Methods for producing equol-containing compositions - Google Patents

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

The present invention relates to a method for producing an equol-containing composition.

Soybeans contain a lot of soy isoflavones, and these isoflavones have female hormone effects (estrogen) and antioxidant effects. It has been clarified that it has a preventive effect against hypercholesterolemia, heart disease, menopausal disorder, etc. (Non-Patent Documents 1 to 6).

On the other hand, soybean is listed as one of the five major allergenic foods in Japan, and it is known that soybean allergy is caused by an allergen protein in the storage protein contained in soybean. As of 1992, 16 types of soybean allergen proteins have been identified, but the major allergens with high incidence of allergy are Gly m Bd 30K, Gly m Bd 60K (7S globulin α-subunit) and Gly m Bd 28K. It is believed that (Non-Patent Document 7). Therefore, soybean processed foods with reduced soybean allergens have been developed (Patent Documents 1 to 3).

A recent study revealed that when soybeans are powdered as they are and the isoflavones are measured, there are 12 types of isoflavones shown in Table 1, of which daidzin, malonyl daidzin, genistin, and malonyl genistin account for the majority. (Non-Patent Document 8).

Furthermore, the important active substance in these soy isoflavones for exhibiting the above female hormone action and antioxidant action is equol, which is produced from daidzein through dihydrodaidzein as described below when daidzin is metabolized in the body. It has been found that the main In other words, daidzin, which is the main isoflavone in soybeans, exists in the form of glycosides bound to sugars in soybeans. It is enzymatically converted to O-desmethylangolensin or equol via dihydrodaidzein by the action of β-glucosidase, which is an enzyme produced by bacteria, and equol in particular is known to have high estrogenic activity. (Non-Patent Documents 9 and 10).

However, even if people eat soybeans, there are individual differences in the ability to metabolize the daidzein contained in soybeans into effective equol. It has also become clear that this is not possible (Non-Patent Documents 11 and 12). Therefore, fermented soybeans containing equol have been developed so that people who cannot metabolize them can directly ingest equol, which is an active ingredient. (Patent Documents 4 to 6).

JP-A-7-236427 Japanese Patent Application Laid-Open No. 2008-220301 Japanese Patent Application Laid-Open No. 2003-274900 Patent No. 5030790 JP 2012-228252 A JP 2012-161323 A

Adlercreutz, H., The Lancet Oncol., 3, 364-373 (2002) Duncan, A. M. et al., Best Pract. Res. Clin. Endocrinol. Metab., 17, 253-271 (2003) Wu, A. H. et al., Carcinogenesis, 23, 1491-1496 (2002) Yamamoto, S. et al., J. Natl. Cancer Institute, 95, 906-913 (2003) Onozawa, M. et al., Jpn. J. Cancer Res., 90, 393-398 (1999) Ridges, L. et al., Asia Pac. J. Clin. Nutr., 10, 204-211 (2001) Tadashi Ogawa, Hideaki Tsuji, Noriko Bando (1992) Journal of Soybean Protein Nutrition Society Vol.13:86-91 Japan Food Research Laboratories, No.42 Sep., 1-2 (2005) Schmitt, E. et al., Toxicol. In Vitro, 15, 433-439 (2001) Sathyamoorthy, N. and Wang, T. T., Eur. J. Cancer, 33, 2384-2389 (1997) Arai, Y. et al., J. Epidemiol., 10, 127-135 (2000) Setchell, K. D. et al., J. Nutr., 133, 1027-1035 (2003)

For those who cannot metabolize daidzein to the effective equol, it is desirable to directly ingest the active ingredient equol. However, although equol-containing fermented products with reduced allergens have been developed, substantially allergen-free products that do not induce allergies have not 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.

The present inventors have made further studies based on the above findings, and have investigated a method for obtaining an equol-containing composition that is substantially free of allergens. The present invention was completed based on the idea that this can be achieved by fermentation. That is, the present invention is as shown below.

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

According to the present invention, it is possible to provide a method for producing an equol-containing composition substantially free of allergens.

Fig. 10 is an image showing the result of allergen detection using FASTKIT Slim in the present invention (photograph substituting for drawing).

Hereinafter, the present invention will be described in detail while showing specific embodiments, but it goes without saying that the present invention is not limited to the specific embodiments illustrated.

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

The method for producing an equol-containing composition according to the present invention includes (1) an "enzyme treatment step" of treating isoflavones with an enzyme that converts the isoflavones into aglycones, and (2) the (3) containing the equol produced in the step (2); and a "recovery step" of recovering the equol-containing composition from the medium.

The method for producing an equol-containing composition according to the present invention includes the above steps (1) to (3), but may include other steps.
As other steps, for example, legumes such as soybean, kudzu, red clover, and licorice are pulverized, extracted, and resin-purified to prepare isoflavones that serve as substrates for enzyme treatment in the enzyme treatment step described later. a step of performing. For example, conventional methods such as the method for producing isoflavone compounds using soybean germ as a raw material disclosed in JP-A-11-263786 can be used. This step can be performed before the enzymatic treatment step described below.

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

<1-1. Isoflavones>
The isoflavones of the present invention are flavonoid glycosides having isoflavone as a basic skeleton. The raw material is not particularly limited, but it can be obtained from, for example, leguminous plants such as soybean, kudzu, red clover, and licorice. Among these, soybeans having a high daidzein content as the aglycone are preferred, and soybean hypocotyls are more preferred.

The isoflavones of the present invention are not particularly limited, but any derivatives such as aglycone acetylated glycosides, aglycon malonylated glycosides, and aglycon succinylated glycosides can be mentioned.
Glycosides of aglycones include, for example, daidzin. Acetyl daidzin etc. can be mentioned as an acetylated glycoside of aglycon. Examples of malonyl glycosides of aglycones include malonyl daidzin. Examples of succinylated glycosides of aglycones include succinyldaidine and the like.
The isoflavones of the present invention may contain one or more of these. Among these, daidzin is preferred.
In the present specification, isoflavones refer to any derivatives of the above glycosides in which a sugar is bound to an aglycone, and do not include aglycones themselves, that is, without sugars.

The isoflavones used in the enzymatic treatment step of the present invention may or may not be purified. Soybean hypocotyls may be used as they are, or may contain other components without being refined. Commercially available ones include, for example, pulverized soybean hypocotyl (manufactured by Fuji Oil Co., Ltd.), isoflavone-rich extract powder (manufactured by Tama Biochemical Co., Ltd.), and the like.

<1-2. Aglycon>
The aglycone of the present invention is not particularly limited, but examples thereof include daidzein, 6-hydroxydaidzein, dihydrodaidzein and the like.
The aglycone of the present invention may contain one of these, or may contain a plurality of them. Among these, daidzein is preferred.

<1-3. Enzyme type>
The enzyme that acts on the isoflavones in the enzymatic treatment step of the present invention to convert the isoflavones into the aglycones cleaves the bond between the aglycones and sugars in any derivatives such as the above-described aglycon glycosides. Although it is not particularly limited as long as it has activity, from the viewpoint of equol production, the enzyme number is preferably an enzyme included in EC3, more preferably an enzyme included in EC3.2, and even more preferably an enzyme included in EC3.2.1. Enzymes included, particularly preferably β-glucosidase (EC 3.2.1.21) and pectinase (EC 3.2.1.15), most preferably pectinase from the viewpoint of productivity.

<1-4. Enzyme treatment>
<1-4-1. Content of isoflavones>
In the enzyme treatment step of the present invention, the content of isoflavones relative to the total amount of the enzyme-treated solution is not particularly limited, but is preferably 0.1 g/L or more as isoflavones from the viewpoint of equol production concentration. It is more preferably 5 g/L or more, and even more preferably 1.0 g/L or more. 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.

<1-4-2. Enzyme Concentration>
In the enzymatic treatment step of the present invention, the content of the enzyme with respect to the total amount of the enzymatic treatment solution is not particularly limited as long as the enzymatic treatment proceeds efficiently. , more preferably 0.05% by mass or more and 5% by mass or less, and more preferably 0.1% by mass or more and 1% by mass or less.

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

<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 the enzyme activity is maintained and the enzyme treatment proceeds efficiently. is more preferable, and 4 or more and 7 or less is even more preferable.

<1-4-5. Enzyme treatment time>
In the enzymatic treatment step of the present invention, the enzymatic treatment time is not particularly limited as long as the enzymatic activity is maintained and the enzymatic treatment proceeds efficiently. and more preferably 15 hours or more. On the other hand, from the viewpoint of productivity, the time is preferably 72 hours or less, more preferably 48 hours or less, and even more preferably 36 hours or less.

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

<2-1. Aglycon concentration>
The aglycone in the fermentation step of the present invention is an aglycone converted from isoflavones by an enzyme in the enzymatic treatment step.
In the fermentation process of the present invention, the concentration of the aglycone is not particularly limited, but in order to further increase the concentration of equol produced, it is preferably 0.1 g/L or more as isoflavones, such as 0.5 g. /L or more, more preferably 1.0 g/L or more. 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.

<2-2. Anaerobic microorganisms>
The anaerobic microorganism used in the present invention is not particularly limited as long as it can be cultured in a medium containing the aglycone and can ferment equol from the aglycone. , Streptococccaceae, or related fungi thereof can be exemplified as those having equol-producing ability.

Also, anaerobic microorganisms classified into genera selected from the following group can be exemplified as those having equol-producing ability.
・Coriobacterium genus ・Adlercreutzia genus ・Asaccharobacter genus ・Atopobium genus ・Collinsella genus ・Cryptobacterium genus ・Denitrobacterium Genus Denitrobacterium, Genus Eggerthella, Genus Enterorhabdus, Genus Gordonibacter, Genus Olsenella, Genus Paraeggerthella, Genus Slackia, Genus Lactococcus ) genus

Microorganisms that are selected from microorganisms classified into these genera and produce equol by anaerobic fermentation using aglycones are preferable microorganisms in the present invention.

Among the microorganisms classified into the above genera, the 16S rDNA of Asaccharobacter celatus DSM 18785 or the closely related Asaccharobacter celatus DSM 18785 (16S rRNA It is preferred to use microorganisms carrying 16S rDNA that has 95% or more, preferably 97% or more, more preferably 98% or more, even more preferably 99% or more identity with the gene sequence). The gene sequence for the 16S rRNA of Asaccharobacter celatus DSM 18785 is SEQ ID NO:1.

Furthermore, among the microorganisms described above, microorganisms belonging to the genera Asaccharobacter, Slackia, and Adlercreutzia are more preferable as microorganisms having equol-producing ability in the present invention.

The production of equol by microorganisms 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 pamphlet, JP-A-2012-135217, JP-A-2012-135218, and JP-A-2012-135219. .

In addition, the following anaerobic microorganisms can be used.
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

Among the above microorganisms, Asaccharobacter celatus DSM 18785 strain, Adlercreutzia equolifaciens DSM 19450 strain, or Slackia isoflavoniconvertens DSM 22006 strain , or related fungi having properties as species similar to those of these fungi can be mentioned as more preferred anaerobic microorganisms.

The above anaerobic microorganisms can be obtained from the depository indicated by the deposit number. Each accession number indicates that the anaerobic microorganism has been deposited with the following depositary institution.
FERM Patent Organism Depositary; 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

<2-3. Culture/fermentation conditions>
The anaerobic microorganism used in the present invention is brought into contact with the aglycon in a medium and cultured under conditions suitable for equol production.
Conditions suitable for producing the equol-containing composition of the present invention refer to those in which the survival and activity of anaerobic microorganisms having equol-producing activity are maintained. More specifically, it means maintaining gas phase conditions (anaerobic conditions) that allow the survival of anaerobic microorganisms and providing nutrients for supporting the activity and growth of the anaerobic microorganisms. Various media compositions suitable for the survival of anaerobic microorganisms are known. Therefore, those skilled in the art can select an appropriate medium composition for the above-described anaerobic microorganisms having equol-producing ability. For example, GAM bouillon medium manufactured by Nissui Pharmaceutical Co., Ltd., BHI medium manufactured by Difco, etc. used in the examples can be used.

For example, a water-soluble organic substance can be added as a carbon source to the medium used in the present invention. Examples of water-soluble organic substances include compounds such as sorbose, fructose, glucose, and organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, and formic acid.

The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted for efficient growth of anaerobic microorganisms in the medium. In general, excess or deficiency can be avoided by selecting the addition amount from the range of 0.1 to 10 wt/vol%.

In addition to the carbon sources mentioned above, a nitrogen source can be added to the medium. In the present invention, various nitrogen compounds that can be used in normal 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 extracts, peptones, meat extracts, liver extracts, digested serum powder and the like. 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.

Furthermore, in addition to carbon sources and nitrogen sources, other organic or inorganic substances suitable for culturing obligately anaerobic microorganisms can be added to the medium. For example, the addition of cofactors such as vitamins and inorganic compounds such as various salts to the medium may sometimes enhance the growth and activity of obligate anaerobic microorganisms. Examples of inorganic compounds, vitamins, and microbial growth cofactors derived from animals and plants include the following.

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, ferrous ammonium sulfate.

Examples of vitamins include biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, vitamin B12, thiooctic acid, and p-aminobenzoic acid. Additionally, the addition of hemin, which is a porphyrin compound, may be beneficial.

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

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

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

For example, an anaerobic jar can be a bioreactor for culturing anaerobic microorganisms. An anaerobic culture jar is composed of an airtight container made of metal, glass, or synthetic resin, and can isolate the inside from oxygen in the atmosphere. Furthermore, the anaerobic culture jar can be provided with a mechanism for removing molecular oxygen contained in the space inside the anaerobic culture jar and the culture solution. For example, the inside of the anaerobic culture jar can be maintained in an anaerobic state by connecting a vacuum pump for sucking the inside of the anaerobic culture jar, sucking the inside of the jar, and supplying a gas other than oxygen.

In the present invention, the fermenter can be given additional functions. For example, in addition to the generally used agitation mixing tank, a bubble column type or draft tube type culture tank can be used. Microorganisms are liberated and dispersed by the gas mixture blown into the liquid medium, allowing sufficient contact between the microorganisms and the medium. In addition, microorganisms are allowed to inhabit a highly air permeable slag such as a biotrickling filter, other ceramic-based inorganic fillings, or filling layers of organic synthetic substances such as polypropylene while dripping water. Cultivation can also be performed while gas is aerated there. Furthermore, the microorganisms to be used can be immobilized on carrageenan gel, alginic acid gel, acrylamide gel, chitin, cellulose, agar, etc. by a conventional method.

In the fermentation process of the present invention, the combination of gases constituting the gas phase consisting of the above substrate gases is not particularly limited, and one or more gases selected from hydrogen, carbon dioxide, nitrogen, etc. are used as constituents. It can be used as The gas phase preferably contains hydrogen as a constituent.

In the above case, the partial pressure percent concentration of hydrogen is preferably 2 to 100% in the gas phase of the fermentation process. For example, the lower limit (“~ It is preferably a partial pressure percent concentration indicated by a concentration range that is greater than or equal to or higher than) and an upper limit (less than or equal to or lower than).

In addition, in order to efficiently produce equol, it is preferable that the gas mixture constituting the gas phase passes through the culture tank at a rate of 0.01 to 2.0 V/V/M gas volume/liquid volume/minute. preferable.

The anaerobic microorganisms used in the present invention are usually anaerobic microorganisms capable of producing equol at temperatures around 37° C. (30-42° C.).

In the present invention, the pressurization conditions for culturing anaerobic microorganisms are not particularly limited as long as they are conditions under which the microorganisms can grow. A range can be mentioned.

The pH of the culture is preferably 3.0 to 8.0, more preferably 4.5 to 7.5, for sufficient growth of microorganisms. In order to increase the amount of equol recovered, the temperature of the culture tank is not particularly limited, but a preferred temperature is 37°C.

<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.

A person skilled in the art can use any method known to separate the produced anaerobic microorganisms and the culture product. A preferred separation method is a method using a hollow fiber type ultrafiltration or microfiltration membrane having filtration performance and concentration performance. In addition, in order to obtain a filtration rate sufficient for separating microorganisms from the produced liquid, a membrane having an appropriate cut-off molecular weight may be selected according to the microorganisms used. The equol produced can be recovered from the medium by any means known to those of skill in the art. For example, equol can be recovered from the culture fluid separated from the culture tank through an ultrafiltration membrane. Methods for purifying equol are known. For example, the culture is centrifuged to remove the cells, and the supernatant is concentrated under reduced pressure, dried and then extracted with 70% ethanol or methanol. The extract can be further purified by an operation such as silica gel chromatography or crystallization. Adsorption resins of styrene-divinylbenzene such as Amberlite and Sepabeads can also be used.

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

<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, and foods. Breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorder, etc. can be prevented by ingesting it as food, drink, pharmaceuticals, or the like.

<5. Cosmetics>
When the equol-containing composition of the present invention is used as a cosmetic material, the equol-containing composition may be in liquid form such as aqueous solution, lotion, spray liquid, suspension and emulsion; solid form such as powder, granule and block; Semi-solid forms such as creams and pastes; and cosmetics in various desired dosage forms such as gel forms can be prepared. Such cosmetics include facial cleansers, emulsions, creams, gels, essences, basic cosmetics such as face packs and masks, makeup cosmetics such as foundations and lipsticks, oral cosmetics, aromatic cosmetics, and hair cosmetics. It is useful as various cosmetics such as skin care products and body cosmetics. Cosmetics containing the equol-containing composition obtained by the present invention are used as whitening cosmetics, acne-improving cosmetics, and wrinkle-improving cosmetics.
Cosmetics containing the equol-containing composition of the present invention can be produced by conventional methods. In addition, the blending amount, blending method, and blending time in the cosmetic can be appropriately selected. Furthermore, if necessary, it can be enclosed in an appropriate container such as a bottle, bag, can, spray can, spray container, box, pack, or the like.
When the equol-containing composition of the present invention is used as a raw material for cosmetics, the content of the equol-containing composition relative to the total amount of the cosmetic is not particularly limited as long as the desired effect of the present invention is exhibited. It is 0.00005 to 10% by mass, preferably 0.0001 to 5% by mass, more preferably 0.0001 to 2% by mass.

<6. Pharmaceuticals>
When the equol-containing composition of the present invention is used as a raw material for pharmaceuticals, its dosage form can be selected according to the disease to be prevented or treated, the mode of use of the pharmaceutical, the route of administration, and the like. Examples include tablets, coated tablets, pills, capsules, granules, powders, liquids, suspensions, emulsions, syrups, injections, suppositories, infusions, decoctions, tinctures and the like. These various formulations may be added to the main drug in accordance with conventional methods, if necessary, with fillers, extenders, excipients, binders, moisturizing agents, disintegrants, surfactants, lubricants, coloring agents, and flavoring agents. , solubilizing agents, suspending agents, coating agents, and other known auxiliary agents that can be commonly used in the technical field of pharmaceutical preparation. In addition, coloring agents, preservatives, flavoring agents, flavoring agents, sweetening agents, etc., and other pharmaceutical agents may be contained in the pharmaceutical formulation.
The equol-containing composition of the present invention can be used for prevention and treatment of breast cancer, prostate cancer, osteoporosis, heart disease, and menopause.

When the equol-containing composition of the present invention is used as a raw material for pharmaceuticals, the content of the equol-containing composition relative to the total amount of the drug is not particularly limited as long as the desired effect of the present invention is exhibited. 001 to 30% by mass, preferably 0.01 to 20% by mass, more preferably 0.1 to 10% by mass. In addition, the dose of the drug containing the equol-containing composition can be appropriately set according to the patient's age, body weight, severity of symptoms, and the like.

<7. Food>
When the equol-containing composition of the present invention is used as a food material, it can be used as food for specified health uses, dietary supplement, functional food, food for the sick, food additive, etc., in addition to general food. Examples of food forms include soft drinks, milk, puddings, jellies, candies, gums, gummies, yogurts, chocolates, soups, cookies, snacks, ice creams, popsicles, breads and cakes containing the equol-containing composition of the present invention. , cream puff, ham, meat sauce, curry, stew, cheese, butter, dressing, and the like.

The equol-containing composition of the present invention can use water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like as main ingredients. Examples of proteins include animal and plant proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soybean protein, chicken egg protein and meat protein, hydrolysates thereof, and butter. Carbohydrates include saccharides, processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Lipids include, for example, vegetable oils such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils, and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. , folic acid, etc. Minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Organic acids include, for example, malic acid, citric acid, lactic acid, and tartaric acid. These ingredients may be used in combination of two or more, and synthetic products and/or foods containing these in large amounts may be used.

Foods containing the equol-containing composition of the present invention can be produced by conventional methods. In addition, the amount to be added to the food, the method of blending, and the timing of blending can be appropriately selected. Furthermore, if necessary, it can be enclosed in an appropriate container such as a bottle, bag, can, box, pack, or the like.
When the equol-containing composition of the present invention is used as a food material, the content of the equol-containing composition relative to the total amount of the food is not particularly limited as long as the desired effect of the present invention is exhibited. 01 to 10% by mass, preferably 0.05 to 5% by mass, more preferably 0.1 to 2% by mass.

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

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

<2. Enzyme treatment>
As the enzyme-treated solution, 270 g of soybean hypocotyls (manufactured by Fuji Oil Co., Ltd.) as isoflavones, 2.7 g of pectinase G Amano as enzymes, and 1180 g of deionized water were used to make a total of 1450 g, and the pH was 4 to 7. was prepared by adjusting to Enzymatic treatment was carried out at 50° C. for 20-24 hours with stirring.

<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 are dissolved in 100 mL of pure water, and 20 mL each is divided into 18 mm test tubes for anaerobic bacteria culture (manufactured by Sanshin Kogyo) while passing nitrogen gas. The mixture was poured, covered with a butyl rubber stopper and a plastic cap, and sterilized at 115°C for 15 minutes.
Asaccharobacter celatus DSM 18785 strain that had been frozen at -80°C was inoculated into this medium, and the gas phase was replaced with hydrogen gas through a sterile filter for 2 minutes or longer. , 250 spm for 16 hours.

(fermentation)
GAM bouillon medium supplemented with L-arginine hydrochloride is sterilized in an autoclave at 115° C. for 15 minutes in the same manner as the seed culture, cooled to room temperature, inoculated with the seed culture, and the gas phase is hydrogen gas. After aseptically substituting with for 2 minutes or more, culture was performed with shaking at 37° C. and 250 spm for fermentation.

<4. Recovery of equol-containing composition>
The fermented culture solution was centrifuged at 3000 rpm for 10 minutes and filtered through a 0.45 μm MF membrane to remove insoluble matter to recover an equol-containing composition solution. An equol-containing material (EQ-N) was obtained by drying (spray drying) the composition liquid.

<5. Detection of allergen>
The equol-containing composition thus obtained was subjected to allergen detection using FASTKIT slim soybeans (manufactured by Nippon Ham Central Research Institute).
The specific method followed the FATKIT slim manual. Using 2 g of EQ-N as a sample, add 38 mL of extraction buffer in the kit, mix for 30 to 60 minutes, extract 3 times, then centrifuge at 3000 x g, 4 ° C., 20 minutes, The supernatant was filtered with filter paper, diluted 10-fold with the dilution buffer in the kit, and used as a measurement sample. The measurement was carried out by dropping 100 μL onto the test strip, developing it for 15 minutes, and then making a visual judgment.

[Comparative Example 1]
Allergens were detected in the same manner as described above for the crushed soybean hypocotyls without the enzyme treatment and fermentation.

<6. Results>
When an allergen is present, a band appears at the position indicated by the arrow in the test strip shown in FIG. The indicated band was not detected. On the other hand, a band indicating the presence of the allergen was detected in Comparative Example 1, which did not undergo enzyme treatment and fermentation.
From the above, it was found that an equol-containing composition substantially free of allergens can be obtained by performing the enzyme treatment and fermentation according to the present invention.

Breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorder, and the like can be prevented by ingesting the equol-containing composition produced by the method of the present invention as food, drink, pharmaceuticals, or the like.

Claims (1)

(1) A leguminous plant body containing isoflavones is treated with an enzyme that converts the isoflavones into aglycones to obtain an enzyme-treated solution, and the enzyme-treated solution containing the aglycones is fermented with anaerobic microorganisms. , converting the aglycone to equol, and
(2) recovering the equol-containing composition containing the equol produced in the step (1) from the fermentation liquid obtained by the fermentation;
A method for producing an equol-containing composition that is substantially free of allergens (However, after washing the soybeans, steaming them with high-pressure steam at 130°C for 20 minutes, and applying the softened soybeans to Bacillus natto or Aspergillus oryzae for one day. In this process, daidzin, a glycoside, is converted to daidzein by β-glucosidase produced by Bacillus natto or Aspergillus oryzae. 20905), except for the method of producing a soybean food material with a high equol content, in which the culture solution is applied and fermentation is advanced for one day.).