Production method for inclusion compound in which equol is included in cyclodextrin

Abstract

To provide an equol inclusion compound improved in solubility into water, an equol-absorptive composition containing the equol inclusion compound, a production method for the inclusion compound, and a production method for the equol-absorptive composition containing the inclusion compound.SOLUTION: The problem above is solved by an inclusion compound in which equol is included in cyclodextrin and an equol-absorptive composition containing an equol inclusion compound in which equol is included in cyclodextrin. Production methods for the inclusion compound or the equol-absorptive composition include a step of adding cyclodextrin to a fermentation medium, when producing equol by fermentation from a material by utilizing microorganisms that produce the equol from at least one kind of equol materials selected from the group consisting of daidzein glucoside, daidzein, and dihydrodaidzein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an inclusion complex in which equol is included in cyclodextrin, and an equol absorbing composition containing the inclusion complex. The present invention also relates to a method for producing the inclusion complex, and a method for producing the equol absorbing composition. [Background technology]

[0002] Equol has female hormone (estrogen) and antioxidant properties, and is expected to have preventive effects against breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, and menopausal disorders, and is also sold as a health food to improve these diseases. Equol sold as a health food is provided as a tablet, and the dosage is about 3 to 4 tablets or 1000 to 4000 mg per day. The size of the tablet varies depending on the provider, but when the dosage per tablet is large, the tablet becomes large and some people find it difficult to swallow the tablet when taking it. In addition, the dosage is also large, which is also a problem.

[0003] Therefore, there is a demand for tablets or the like containing equol that can improve the absorption or transfer of equol into the blood, reduce the dosage per tablet, and reduce the size of the tablet. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 9-309902 [Patent Document 2] Patent Publication No. 2012-77087 [Patent Document 3] Patent Publication 2010-24240 [Patent Document 4] Patent Publication 2010-187647 [Patent Document 5] Patent 5851686 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide an equol inclusion complex having improved solubility in water in order to improve the absorption or transfer of equol into the blood, and an equol absorbing composition containing the equol inclusion complex. In particular, an object of the present invention is to provide an equol inclusion complex and an equol absorbing composition containing the equol inclusion complex that can increase the blood equol concentration when orally ingested via food or the like. Another object of the present invention is to provide a method for producing an equol inclusion complex having improved solubility in water, and a method for producing an equol absorbing composition containing an equol inclusion complex, other than or in addition to the above object. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors have discovered the following invention. <1> An inclusion complex in which equol is enclosed within cyclodextrin. <2> An equol absorbing composition comprising an equol inclusion complex in which equol is included in cyclodextrin. <3> the above <2> In the above, the composition is preferably one selected from the group consisting of a food composition and a pharmaceutical composition. <4> the above <2> or <3> In the present study, the solubility of equol or the equol inclusion complex in water was The dissolution property is preferably 0.5 mg / mL or more, more preferably 1.0 mg / mL or more, and even more preferably 1.5 mg / mL or more.

[0007] <5> the above <1> ~ <4> In any one of the above, the cyclodextrin may be one or more selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof. Preferably, the cyclodextrin is one or more selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, glycosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin. More preferably, the cyclodextrin is one or more selected from the group consisting of β-cyclodextrin, glycosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin. Most preferably, the cyclodextrin is β-cyclodextrin. <6> The content of cyclodextrin is preferably 0.5 to 5000 parts by weight, more preferably 1 to 100 parts by weight, and even more preferably 3 to 10 parts by weight, per part by weight of equol.

[0008] <7> A method for producing an inclusion complex in which equol is fermented from at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein using a microorganism that converts the raw material into equol, the method comprising the step of adding cyclodextrin to a fermentation medium to obtain an inclusion complex in which equol is encapsulated in cyclodextrin.

[0009] <8> A method for producing an equol-absorbing composition, which comprises the step of adding cyclodextrin to a fermentation medium when fermenting at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein to produce equol using a microorganism that converts the raw material into equol, thereby obtaining an equol-absorbing composition containing an inclusion complex in which equol is encapsulated in cyclodextrin. <9> the above <8> It is preferable to further include a step of adding an orally ingestible substance to obtain an equol absorbing composition containing an equol inclusion complex in which equol is included in cyclodextrin.

[0010] <10> the above <7> ~ <9> In any one of the above, a step of adding cyclodextrin may be further included after the fermentation production step. <11> the above <8> ~ <10> In any one of the above, the composition may be one selected from the group consisting of a food composition and a pharmaceutical composition. <12> the above <7> ~ <11> In any one of the above, the microorganism that converts the equol raw material into equol is preferably an anaerobic microorganism.

[0011] <13> the above <7> ~ <12> In any one of the above, the anaerobic microorganism is at least one selected from the group consisting of microorganisms belonging to the genus Adlercreutzia. It is better. <14> the above <7> ~ <13> In any one of the above, the solubility of equol or the equol inclusion complex in water is preferably 0.5 mg / mL or more, more preferably 1.0 mg / mL or more, and even more preferably 1.5 mg / mL or more.

[0012] <15> the above <7> ~ <14> In any one of the above, the cyclodextrin is preferably one or more selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof, and preferably, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, glycosyl- The cyclodextrin is preferably one or more selected from the group consisting of β-cyclodextrin, maltosyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin, more preferably one or more selected from the group consisting of β-cyclodextrin, glycosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin, and most preferably β-cyclodextrin. <16> the above <7> ~ <15> In any of the above, 0.5 to 5000 parts by weight, preferably 1 to 100 parts by weight, and more preferably 3 to 10 parts by weight of cyclodextrin is added per 1 part by weight of equol. Effect of the Invention

[0013] The present invention can provide an equol inclusion complex having improved solubility in water to improve the absorption or transfer of equol into the blood, and an equol absorbing composition containing the equol inclusion complex. In particular, the present invention can provide an equol inclusion complex that can increase the blood equol concentration when orally ingested via food or the like, and an equol absorbing composition containing the equol inclusion complex. Furthermore, the present invention can provide a method for producing an equol inclusion complex and a method for producing an equol absorbing composition containing an equol inclusion complex, which have improved solubility in water in addition to or in addition to the above-mentioned effects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present application provides an equol inclusion complex, an equol absorbing composition containing the equol inclusion complex, as well as a method for producing the equol inclusion complex and a method for producing the equol absorbing composition. <Equol inclusion complex> The present application provides an equol inclusion complex, i.e., an inclusion complex in which equol is included in cyclodextrin.

[0015] Here, the cyclodextrin may be one or more selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof. Preferably, the cyclodextrin is one or more selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, glycosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin. More preferably, the cyclodextrin is one or more selected from the group consisting of β-cyclodextrin, glycosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin. Most preferably, the cyclodextrin is a β-cyclodextrin.

[0016] The inclusion complex of the present invention contains 0.5 to 5000 parts by weight, preferably 1 to 100 parts by weight, and more preferably 3 to 10 parts by weight of cyclodextrin per part by weight of equol.

[0017] <Equol Absorbing Composition Containing Equol Inclusion Complex> The present application also provides an equol absorbing composition containing an equol inclusion complex, that is, an equol absorbing composition containing an equol inclusion complex in which equol is included in cyclodextrin. Cyclodextrin and equol inclusion complexes have the same definitions as above. The equol absorbing composition may contain, in addition to the equol inclusion complex, unincluded cyclodextrin, unincluded equol, and other components. Examples of other components include, but are not limited to, medium components and metabolites thereof used in the production method described below, fermentation products or parts thereof obtained by fermentation described below, sugars other than daidzein glycosides, enzymes used to process glycosides, isoflavones other than daidzein (including glycosides and isoflavone metabolites), orally acceptable substances described below, etc. In addition, cyclodextrin and other excipients, etc., which are added after the end of fermentation, may be included.

[0018] In the equol absorbing composition of the present application, the solubility of equol or the equol inclusion complex in water is preferably 0.5 mg / mL or more, more preferably 1.0 mg / mL or more, and even more preferably 1.5 mg / mL or more, and the upper limit of the solubility is preferably 11 mg / mL.

[0019] The equol-absorbing composition of the present application is preferably one selected from the group consisting of food compositions such as health food compositions, and pharmaceutical compositions, but is not limited thereto. The form of the equol absorbing composition of the present application is not particularly limited, but in consideration of oral ingestion, examples of the form of oral preparations include powders, tablets, coated tablets, granules, capsules, and the like.

[0020] The equol inclusion complex of the present application or the equol absorbing composition containing said inclusion complex has high water absorbency and solubility of equol. Therefore, when made into an oral preparation such as a tablet, coated tablet, or capsule, even if the amount of equol per tablet is reduced, it can be absorbed at a desired concentration when ingested. Therefore, by reducing the amount of equol per tablet, the size of the tablet can be reduced.

[0021] When the equol inclusion complex of the present application or an equol absorbing composition containing said inclusion complex is provided as a food composition, it can be provided as an ingredient for foods, beverages (including supplements), etc. Furthermore, when the equol inclusion complex of the present application or an equol absorbing composition containing said inclusion complex is provided as a food composition, it can be used as a general food, as well as a food for specified health uses, a nutritional supplement, a functional food, a food for the sick, a food additive, etc. Examples of the form of food that contains the equol-containing food composition of the present invention include, but are not limited to, soft drinks, milk, pudding, jelly, candy, gum, gummy candy, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cake, cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing, supplements, beverages with specified health benefits, and nutritional drinks.

[0022] The food composition may contain orally acceptable substances, such as, but not limited to, water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, and the like. Examples of proteins include, but are not limited to, animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, and meat protein, as well as hydrolysates thereof, butter, etc. Examples of carbohydrates include, but are not limited to, sugars, modified starch (dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), dietary fiber, and the like.

[0023] Examples of lipids include animal fats and oils such as lard, fish oil, and their fractionated oils, hydrogenated oils, and interesterified oils; vegetable fats and oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, and their fractionated oils, hydrogenated oils, and interesterified oils; but are not limited to these. Not done. Examples of vitamins include, but are not limited to, 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, but are not limited to, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, whey minerals, and the like. These ingredients may be used in combination of two or more kinds, and synthetic products and / or foods rich in these may also be used.

[0024] The blending ratio of the equol absorbing composition in these foods can be appropriately set depending on the type of food, the equol content, the age and sex of the person taking it, the expected effect, etc. One example is a ratio of 0.01 to 100 g, preferably 0.1 to 10 g, and more preferably 0.5 to 5 g of equol or equol inclusion complex per 100 g of food, but is not limited to these. The daily intake of a food containing an equol-containing food composition varies depending on the equol content in the composition, the age and weight of the person taking it, the number of times it is taken, etc., but an example is an amount of the composition equivalent to 0.01 to 10 g per day for an adult.

[0025] <Method for producing the equol inclusion complex> and <Method for producing the equol absorbing composition containing the equol inclusion complex> The present application provides a method for producing the above-mentioned equol inclusion complex. The present application also provides a method for producing an equol absorbing composition containing the equol inclusion complex. For example, the above-mentioned equol inclusion complex can be obtained by the following method. In other words, when fermenting and producing equol from at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein using a microorganism that converts the equol raw material into equol, an equol inclusion complex can be obtained by including a step of adding cyclodextrin to the fermentation medium.

[0026] For example, an equol absorbing composition containing an equol inclusion complex can be obtained by the following method. In other words, when fermenting and producing equol from at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein using a microorganism that converts the equol raw material into equol, a step of adding cyclodextrin to the fermentation medium can be included to obtain an equol absorption composition that contains an equol inclusion complex in which equol is encapsulated in cyclodextrin. In addition to adding cyclodextrin to the "fermentation medium", it may also be added after the fermentation production process.

[0027] <<Equol raw material>> The equol raw material used in the production method of the present invention may be in any form so long as it can be literally used as a raw material for equol. The equol raw material may be at least one selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein, and may be in any form, such as daidzein glycoside itself, daidzein itself, or dihydrodaidzein itself, or may be a substance containing them, such as soybeans, processed soybeans, soybean hypocotyls, processed soybean hypocotyls, such as soybean hypocotyl extracts, specifically commercially available isoflavones.

[0028] <<Microorganisms that utilize equol>> The equol-assimilating microorganism used in the production method of the present invention is not particularly limited as long as it produces equol from the above-mentioned equol raw material. Examples of microorganisms that assimilate equol include anaerobic microorganisms and lactic acid bacteria. The equol assimilation ability 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, JP 2012-135217, JP 2012-135218, JP 2012-135219, etc. An example of these quantification methods is shown below.

[0029] For example, ethyl acetate is added to the culture solution, which is vigorously stirred and then centrifuged to remove the ethyl acetate layer. If necessary, the same procedure can be performed several times on the same culture solution, and the ethyl acetate layers can be combined to obtain an equol extract. This extract is concentrated and dried under reduced pressure using an evaporator, and dissolved in methanol. This is then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, which can then be used as a high-performance liquid chromatography measurement sample. Examples of high-performance liquid chromatography conditions include, but are not limited to, the following:

[0030] [High performance liquid chromatography conditions] Column: Phenomenox Luna 5uC18, 2.0mm x 150mm (Shimadzu GLC) Mobile phase: Water / methanol [55:45, v / v] Flow rate: 0.2mL / min Column temperature: 40℃ Detection: UV280nm Retention times: dihydrodaidzein 13.8 min, daidzein 19.6 min, glycitein 22.5 min, equol 25.6 min, genistein 35.0 min

[0031] Microorganisms that assimilate equol include, but are not limited to, microorganisms classified into the following genera: Genus Adlercreutzia Bacteroides genus Bifidobacterium Clostridium Genus Eggerthella Enterococcus Genus Enterorhabdus Eubacterium genus Genus Finegoldia Lactobacillus genus Lactococcus Genus Paraeggerthella Pediococcus genus Genus Proteus Genus Sharpea Genus Slackia Streptococcus Genus Veillonella

[0032] Specific examples of microorganisms that assimilate equol include, but are not limited to, the following microorganisms. Adlercreutzia aequorifaciens subsp. ceratus equolifaciens subsp. celatus) Adlercreutzia equolifaciens subsp. equolifaciens Bacteroides ovatus Bifidobacterium breve Bifidobacterium longum Clostridium sp. Eggerthella sp. Enterococcus faecalis Enterococcus faecium Enterorhabdus mucosicola Eubacterium sp. Finegoldia magna Lactobacillus fermentum Lactobacillus mucosae Lactobacillus paracasei Lactobacillus plantarum Lactobacillus rhamnosus Lactobacillus sp. Lactococcus garvieae Paraeggerthella sp. Pediococcus pentosaceus Proteus mirabilis Sharpea azabuensis Slackia equolifaciens Slackia isoflavoniconvertens Slackia sp. Streptococcus constellatus Streptococcus intermedius Veillonella sp.

[0033] Among the above-mentioned microorganisms, for example, microorganisms classified into the family Eggerthellaceae, Microorganisms classified in the Bifidobacteriaceae family, microorganisms classified in the Clostridiaceae family, microorganisms classified in the Coriobacteriaceae family, microorganisms classified in the Enterococcaceae family, microorganisms classified in the Eubacteriaceae family, microorganisms classified in the Morganellaceae family, microorganisms classified in the Peptoniphilaceae family, microorganisms classified in the Lactobacillaceae family, microorganisms classified in the Streptococcus ceae family, microorganisms classified in the Veillonellaceae family, or microorganisms related to these. Preferred are the genus Adlercreutzia, the genus Bacteroides, The microorganism may be classified into the genera Bifidobacterium, Clostridium, Coriobacterium, Egasella, Enterococcus, Eubacterium, Finegordia, Lactobacillus, Paraegasella, Pediococcus, Proteus, Chapea, Slacia, Streptococcus, Veillonea, or related microorganisms thereof. More preferably, the microorganism may be classified into Adrecrautia aequorifaciens subsp. ceratus, Adrecrautia aequorifaciens subsp. aequorifaciens, Bacteroides obatus, Bifidobacterium breve, Bifidobacterium longum, Clostridium sp., Egasella sp., Enterococcus faecalis, Enterococcus faecium, Enterohabdus mucosicola, Eubacterium spp., Enterococcus faecium, Enterohabdus mucosicola, Eubacterium spp., Enterococcus faecalis, Enterococcus faecium, Enterohabdus mucosicola, Eubacterium spp., Enterococcus faecium ... Bacterium sp., Finegordia magna, Lactobacillus fermentum, Lactobacillus intestinalis, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sp., Lactococcus garvieae, Paraegasella sp., Pediococcus pentosaceus, Proteus mirabilis, Sharpea azabuensis, Slachia aequorifaciens, Slachia isoflavonic convertens, Slachia sp., Streptococcus constellatus, Streptococcus intermedius, Veillonea sp.

[0034] Among the above-mentioned microorganisms, any of the microorganisms described below or related microorganisms having similar species properties to these microorganisms can be mentioned as more preferred anaerobic microorganisms. Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain Bacteroides ovatus strain E-23-15 Bifidibacterium breve ATCC 15700 strain Bifidobacterium longum BB536 strain Clostridium sp. HGH136 strain Eggerthella sp. Julong 732 strain Eggerthella sp. YY7918 strain Eggerthella sp. D1 strain Enterococcus faecalis INIA P333 strain Enterococcus faecium strain EPI1 Enterohabdus mucosicola Mt1B8 strain Eubacterium sp. strain D2 Finegoldia magna EPI3 strain Lactobacillus fermentum DPPMA114 strain Lactobacillus intestinalis KTCT13676BP KK Lactobacillus mucosae EPI2 strain Lactobacillus paracasei JS1 strain Lactobacillus plantarum DPPMA24W strain Lactobacillus plantarum DPPMASL33 strain Lactobacillus rhamnosus DPPMAAZ1 strain Lactobacillus rhamnosus INIA P540 strain Lactobacillus sp. Niu-O16 strain Lactococcus garvieae strain 20-92 Paraeggerthella sp. strain SNR40-432 Pediococcus pentosaceus strain CS1 Proteus mirabilis LH-52 strain Sharpea azabuensis ST18 strain Slackia equolifaciens strain DSM 24851 Slackia isoflavoniconvertens DSM 22006 strain Slackia sp. FJK1 strain Slackia sp. NATTS strain Slackia sp. YIT11861 strain Slackia sp. TM-30 strain Streptococcus constellatus strain E-23-17 Streptococcus intermedius A6G-225 strain Veillonella sp. EP strain.

[0035] 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 institutions: 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

[0036] In the present invention, anaerobic microorganisms capable of producing equol are cultured under conditions suitable for equol production. In the present invention, conditions suitable for equol production refer to conditions under which the survival and activity of anaerobic microorganisms capable of producing equol are maintained. More specifically, gas phase conditions (anaerobic conditions) in which anaerobic microorganisms can survive are maintained, and nutrients are provided 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 shown above. For example, BHI medium manufactured by Difco or the medium used in the Examples can be used, but these Not limited to.

[0037] Water-soluble organic substances can be added to the medium used in the present invention as a carbon source. Examples of water-soluble organic substances include, but are not limited to, the following compounds: sugars such as sorbose, fructose, and glucose; Alcohols such as methanol; Organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, etc., or their salts.

[0038] The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted in order to efficiently grow anaerobic microorganisms in the medium. 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. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate. On the other hand, preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder, etc. More preferred organic nitrogen sources are arginine, cysteine, cystine, citrulline, lysine, yeast extract, and peptones.

[0039] Furthermore, in addition to the carbon and nitrogen sources, other organic or inorganic substances suitable for the production of equol can be added to the medium. For example, the growth and activity of anaerobic microorganisms can be enhanced by adding cofactors such as vitamins and 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.

[0040] Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium Sulfate Folic Acid Manganese Sulfate Pyridoxine Sodium Chloride Thiamine Cobalt chloride Riboflavin Calcium chloride Nicotinic acid Zinc Sulfate Pantothenic Acid Copper Sulfate Vitamin B12 Alum Thioctic acid Sodium molybdate p-aminobenzoic acid Potassium chloride Boric acid etc. Nickel chloride Sodium Tungstate Sodium Selenite Ammonium Ferrous Sulfate

[0041] A conventionally known method can be used to produce a culture solution by adding these inorganic compounds, vitamins, or growth cofactors. The medium can be liquid, semi-solid, or solid. In the present invention, the preferred form of the medium is a liquid medium. The medium of the present invention can contain dextrins. By culturing anaerobic microorganisms in a medium containing dextrins, a liquid containing equol and dextrins can be prepared without contacting the culture with dextrins after the culture. Dextrins can be added to the medium before or during the culture of the microorganism.

[0042] In the method of the present application, the anaerobic microorganism can be cultured according to a known method for culturing a microorganism. For industrial production, a continuous fermentation system capable of continuously supplying the medium and substrate gas and having a mechanism for recovering the culture can be used.

[0043] When anaerobic microorganisms are used in the production of food compositions containing equol inclusion complexes, it is advisable to prevent oxygen from entering the culture vessel. A commonly used fermentation tank can be used as the culture vessel. An anaerobic atmosphere can be created by replacing the oxygen that enters the fermentation tank with an inert gas such as nitrogen.

[0044] Depending on the shape of the fermentation tank, an agitator or the like can be used to thoroughly agitate the medium. By agitating the culture in the fermentation tank, the opportunities for the medium components and substrate gas to come into contact with the anaerobic microorganisms can be increased, optimizing the efficiency of equol production. The substrate gas can also be supplied as nanobubbles.

[0045] In the method of the present application, equol may be produced in a closed system such as a bottle or test tube sealed with a rubber stopper without ventilation. It may also be produced in a mixed gas phase consisting of one or more gases including hydrogen. When produced in a mixed gas phase, the hydrogen concentration is not particularly limited.

[0046] When the method of the present application is carried out in a mixed gas phase, the combination of gases constituting the gas phase is not particularly limited, and in addition to hydrogen, one or more gases selected from carbon dioxide, nitrogen, etc. can be used as constituent components. When aeration is performed to efficiently produce equol, the aeration rate of the mixed gas constituting the gas phase into the fermentation tank is preferably 0.01 to 2.0 V / V / M (volume of gas / volume of liquid / minute).

[0047] In the method of the present application, the microorganism may be cultured under normal pressure, but when pressurized, the pressurization conditions are not particularly limited as long as the microorganism can grow under the conditions. Preferred pressurization conditions include, but are not limited to, a range of 0.02 to 0.2 MPa.

[0048] To increase the amount of equol produced, the temperature of the fermenter is not particularly limited, but is preferably 30°C to 40°C, and more preferably 33°C to 38°C. The fermentation time can be appropriately set depending on the amount of equol produced, the amount of isoflavones remaining, etc. Examples of the fermentation time include, but are not limited to, 8 to 120 hours, preferably 12 to 72 hours, and particularly preferably 16 to 60 hours.

[0049] The fermentation culture obtained by the culture method of the present invention can be used in a solid form by heat drying, spray drying, or freeze drying, as necessary. Heat drying or spray drying can be performed, for example, using a spray dryer. Freeze drying can be performed using a freeze dryer. The fermentation culture that has been heat dried, spray dried, or freeze dried can be subjected to a powdering process, as necessary. EXAMPLES

[0050] The present invention will be described below based on examples, but the scope of the present invention is not limited to the following examples. <Example 1> <<Preculture>> The medium, with the composition shown in Table 1 and adjusted to pH 6.9, was dispensed into 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.) in 10 mL portions, and sterilized at 115°C for 15 minutes with butyl rubber stoppers and plastic caps attached while replacing the gas phase with nitrogen. Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain was inoculated into this medium, and the gas phase was replaced with hydrogen gas passed through a sterile filter for more than 2 minutes, after which it was shake-cultured at 37°C and 200 spm for 18 hours to prepare a preculture solution.

[0051] [Table 1]

[0052] <<Preparation of Equol>> The medium was adjusted to pH 6.9 by adding 3.3 g / L daidzein, 15 g / L β-cyclodextrin, and 10 g / L L-arginine to the composition shown in Table 1, and 20 mL of the medium was dispensed into 100 mL vials (Maruem), fitted with butyl rubber stoppers (Maruem), and sterilized at 115°C for 15 minutes by replacing the gas phase with nitrogen. 0.4 mL of the preculture solution prepared in Example 1 was inoculated into this medium, and the gas phase was replaced with hydrogen gas passed through a sterile filter for 2 minutes or more, after which it was cultured with shaking at 37°C and 200 spm for 48 hours. After 48 hours, 2. 8g / L was produced.

[0053] <<Preparation of equol inclusion complex>> The culture medium prepared in "Preparation of Equol" above was heated at 80°C for 10 minutes. It was then centrifuged at 6000 rpm for 10 minutes, and the supernatant was filtered through a 0.2 μm membrane to remove bacteria. The resulting bacteria-removed liquid was freeze-dried to obtain dried product A-1, which contains a β-cyclodextrin inclusion complex of equol.

[0054] <<Solubility test>> A solubility test was carried out on the dried product A-1 containing the equol inclusion complex obtained in the above <<Preparation of equol inclusion complex>>. As a control, a solubility test was also carried out on equol (manufactured by LC Laboratories) alone. Specifically, the solubility test was carried out under the following conditions. 300 mg of dried material A-1 containing equol inclusion complex was weighed out, 10 mL of water was added, and the mixture was stirred at room temperature for 2 hours. This liquid was filtered through a 0.2 μm filter to obtain solution B-1 of dried material A-1 containing equol inclusion complex. The equol concentration of this solution B-1 was analyzed. In addition, 10 mg of equol (LC Laboratories) was weighed out and the same procedure was carried out to obtain equol. A solution C-1 containing only equol was obtained. The concentration of equol dissolved in this solution C-1 was analyzed. As a result, the equol concentration in solution B-1 was 1.9 mg / mL, while the equol concentration in solution C-1 was 0.1 mg / mL, indicating that the equol inclusion complex A-1 has higher solubility than equol alone.

[0055] <<Pharmacokinetics (PK)>> Pharmacokinetics (PK) of the equol complex A-1 obtained in "Preparation of equol complex" was performed using 6-week-old Crl:CD(SD) rats by oral administration. As a control, equol (LC Laboratories) alone was also administered. Pharmacokinetics (PK) was performed. Specifically, the dosage was 100 mg / kg of equol complex A-1. The dose of equol (LC Laboratories) was 100 mg / kg for equol complex A-1. The dose was set at 6.3 mg / kg, which is considered to be equivalent. The test substance was administered orally once to 3 animals per group. The test substance was suspended in a 0.5% CMC aqueous solution, and the administration volume was 10 mL / kg.

[0056] PK was measured using plasma, and blood was collected at 0 min, 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h after the experiment. After the 24-h blood collection, the animals were euthanized. No deaths or toxic symptoms were observed in this study. Furthermore, no abnormalities were found in the gross observation of the organs and lymph nodes in the cranial, thoracic and abdominal cavities at autopsy 24 hours after administration. The obtained PK results are shown in Table 2. In Table 2, Cmax: maximum blood concentration, Tmax: time to reach Cmax, and AUC0-24: area are shown. From Table 2, it can be seen that the Cmax (maximum blood concentration) of equol inclusion complex A-1 is more than twice as high as the Cmax (maximum blood concentration) of equol alone. It can also be seen that the AUC0-24: area of ​​equol inclusion complex A-1 is higher than the AUC0-24: area of ​​equol alone. These results show that the equol inclusion complex of the present invention has good equol absorbability.

[0057] [Table 2]

[0058] <Example 2> <<Preparation of Equol 2>> Soybean hypocotyls (manufactured by Fuji Oil) were ground for 1 minute x 5 times using a Waring blender. 27 g of this ground material was added with 0.27 g of pectinase G Amano (manufactured by Amano Enzyme) and 118 g of deionized water, and the pH was maintained at 4 to 7, and enzyme treatment was performed while stirring at 50 ° C. Each nutrient source and L-arginine were added to obtain the composition shown in Table 1 at 10 g / L, and the pH was adjusted to 6.8 to 7.0. Then, 20 mL of this preparation was dispensed into 100 mL vials (manufactured by Maruemu), butyl rubber stoppers (manufactured by Maruemu), and the gas phase was replaced with nitrogen and sterilized at 115 ° C. for 15 minutes. 0.4 mL of preculture solution prepared in the same manner as in Example 1 was inoculated into this medium, and the gas phase was replaced with hydrogen gas passed through a sterile filter for 2 minutes or more, and then shaking culture was performed at 37 ° C. and 200 spm for 48 hours. After 48 hours, HPLC analysis confirmed the production of equol. This culture solution was heated at 80°C for 10 minutes and then freeze-dried to obtain fermented soybean hypocotyl powder containing equol.

[0059] <<Preparation of equol inclusion complex 2>> 10 mL of 15 g / L aqueous solution of β-cyclodextrin was added to 1 g of the fermented soybean hypocotyl powder prepared in <<Preparation of Equol 2>> above, and mixed thoroughly. The mixture was then centrifuged at 6000 rpm for 10 minutes, and the supernatant was filtered through a 0.2 μm membrane. The filtrate was freeze-dried to obtain a powder, yielding dried product A-2 containing an equol inclusion complex. Furthermore, Milli-Q water was added to 2 g of the fermented soybean hypocotyl powder prepared in <<Preparation of Equol 2>> above, and the same procedure was repeated to obtain a dried product D-1.

[0060] <<Solubility test 2>> The dried product A-2 containing the equol inclusion complex obtained in <<Preparation 2 of the equol inclusion complex>> above was dissolved in Milli-Q water to obtain a solution B-2. As a control, the dried product D-1 was also dissolved in Milli-Q water to obtain solution C-2. The amount of Milli-Q water added was equal to the amount required to return the dried product obtained in <<Preparation of Equol Inclusion Complex 2>> to its original liquid volume. The equol concentrations in Solutions B-2 and C-2 were measured by HPLC. As a result, the equol concentration in Solution B-2 was 0.28 mg / mL, while the equol concentration in Solution C-2 was 0.02 mg / mL. It can be seen that the equol inclusion complex A-2 has higher solubility than the non-inclusion complex D-1.

Claims

1. A method for producing a ... A method for producing an equol inclusion complex in which equol is included in β-cyclodextrin.

2. The method of claim 1, comprising the step of mixing equol with a β-cyclodextrin solution to obtain the solution containing equol and β-cyclodextrin.

3. The manufacturing method described in claim 2, wherein in the mixing step, β-cyclodextrin is mixed in an amount of 0.5 to 5,000 parts by weight per 1 part by weight of equol.

4. A method for producing a composition according to claim 2 or 3, wherein in the mixing step, 0.5 to 10 parts by weight of β-cyclodextrin are mixed per 1 part by weight of equol.

5. A method for producing the equol described in any one of claims 1 to 4, wherein the equol is produced by culturing an equol-producing microorganism in a culture medium containing at least one selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein.

6. A method for producing a composition according to any one of claims 1 to 5, further comprising a step of filtering the solution containing equol and β-cyclodextrin prior to the drying step.

7. The manufacturing method described in claim 5, wherein the microorganism that produces the equol is an anaerobic microorganism.

8. The microorganism that produces equol is selected from the group consisting of Adrechlautia aequorifaciens subsp. seratus, Adrechlautia aequorifaciens subsp. aequorifaciens, Bacteroides obatus, Bifidobacterium breve, Bifidobacterium longum, Clostridium sp., Egasella sp., Enterococcus faecalis, Enterococcus faecium, Enterohabdus mucosicola, Eubacterium sp., Finegordia magna, and Lactobacillus. fermentum, Lactobacillus intestinalis, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sp., Lactococcus garvieae, Paraegasella sp., Pediococcus pentosaceus, Proteus mirabilis, Sharpea azabuensis, Slaqia aequorifaciens, Slaqia isoflavonic convertens, Slaqia sp., Streptococcus constellatus, Streptococcus intermedius, and at least one selected from the group consisting of Veillonea sp.

9. A manufacturing method described in any one of claims 1 to 8, wherein the drying step involves a heat drying process, a spray drying process, or a freeze drying process.

10. A manufacturing method described in any one of claims 1 to 9, further comprising a step of powderizing the dried material obtained by the drying step.