JP5851687B2 - Equol production method, equol production composition, food, food additive and pharmaceutical - Google Patents

Description

  The present invention relates to a method for producing equol, an equol-producing composition, a food using the same, a food additive, and a pharmaceutical product.

  Isoflavones, which are abundant in leguminous plants such as soybeans and kuzu, are a class of polyphenols and are flavonoids based on isoflavones. According to recent research, isoflavones have female hormonal action (estrogens) and antioxidant action, and by taking isoflavones, breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. It has become clear that there is a preventive effect (see Non-Patent Document 1 to Non-Patent Document 6).

  In addition, isoflavones contained in soybeans mainly contain dicein glycosides covalently bonded to sugar, and contain a very small amount of isoflavone aglycone. Examples of the dicein glycoside include daidzin, glycitin, and genistin.

  Here, some daidzein glycosides are malonylated and acetylated, and these glycosides are digestive enzymes or enzymes produced by intestinal bacteria when they enter the human or animal body. By the action of a certain β-glucosidase or the like, it becomes daidzein, glycitein, and genistein, respectively.

  Furthermore, it is known that daidzein is enzymatically converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by the action of intestinal bacteria. It has been.

  Equol is known to have the highest estrogenic activity among these metabolites (Non-patent Documents 7 and 8). However, in the case of humans, the metabolism of isoflavones varies from person to person, and few people have enteric bacteria that have the ability to ferment daidzein and produce equol as described above. It is clear that it is about 30% for Westerners and about 30% (Non-patent Documents 9 and 10). Therefore, people who do not have equol-producing bacteria cannot produce equol in their bodies even if they take legumes such as soybeans.

  Thus, in recent years, attempts have been made to produce equol ex vivo using anaerobic microorganisms such as lactic acid bacteria (see, for example, Patent Document 1 to Patent Document 4). However, it has not been clarified what fermentation conditions and fermentation methods can produce equol more effectively and practically. Moreover, all produced | generated equol density | concentration was low and could not be mass-produced.

  In addition, clathrate compounds represented by cyclodextrin are used to stabilize unstable compounds, alleviate food bitterness, deodorize, and solubilize poorly soluble compounds using the clathrate compounds. . In recent years, in the production of biological compounds, a production method in which cyclodextrin is added as a raw material solubilizer to a culture medium has been proposed (see, for example, Patent Document 5 to Patent Document 7).

  Patent Document 8 discloses equol-producing bacteria, Escherichia genus, Raoultella genus, Lactobacillus genus, Pantoea genus, Saccharomyces genus, Enterococcus cc. And a mixed microorganism comprising at least one equol non-producing bacterium selected from the microorganisms belonging to the genus Serratia, and culturing in a medium containing daiseins to produce daidzein metabolites. As a solubilizer of Daicein, which is (1) cyclodextrin or (2) polyethylene glycol glycol chain, water and ethanol are soluble, and HLB value is 11 or more A surfactant have been described to be added to the medium.

  As described above, in Patent Document 8, when culturing a mixed microorganism containing equol-producing bacteria and at least one non-equol-producing bacterium in a medium containing daidzeins, β-cyclodextrin or polyethylene glycol group is used. It is described that the production amount of equol is increased by adding a surfactant having a call chain to the medium, but the effect when the cyclodextrins and the surfactant are added simultaneously is not described. In general, it is extremely difficult to obtain a highly reproducible result by mixing and culturing two or more kinds of microorganisms.

  Further, in the production of microorganisms, when a nonionic surfactant such as Tween is added to the medium, the foaming is usually severe during and after the culture, making it difficult to perform the culture operation and recover the product. It was known.

JP 2006-204296 A JP-T-2006-504409 JP 2008-61584 A JP 2010-104241 A Japanese Patent No. 3549594 Japanese Patent No. 3135740 JP 2000-295999 A JP 2010-187647 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 Inst., 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) 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)

  An object of the present invention is to provide a method for producing equol at a level that can be industrially implemented by culturing a single type of microorganism. An equol-producing composition is also provided.

  As a result of intensive studies to solve the above problems, the present invention has been reached as follows.

  (1) In a medium containing isoflavones, β-cyclodextrin, β-cyclodextrin derivative or γ-cyclodextrin and a cloud point of 50 ° C. or higher, preferably 60 ° C. or higher and 100 ° C. or lower, more preferably 70 ° C. or higher and 100 ° C. This is a method for producing equol, in which the following surfactant is added to act one kind of obligate anaerobic microorganism having equol-producing ability.

  (2) The equol production method according to (1), wherein the isoflavones are at least one selected from the group consisting of isoflavones, isoflavone aglycones, daidzein glycosides, daidzein, and dihydrodaidzein.

  (3) The obligate anaerobic microorganisms include the genus Adlercreutzia, the genus Asaccharobacter, the genus Eggerella, the genus Eubacterium, the genus Lactococcus, The method for producing equol according to (1) or (2) above, wherein the equol is a single species selected from the group consisting of (Sharpea), Streptococcus, Slackia, and Bacteroides. .

  (4) The surfactant is an ester ether type non-ion of the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate The method for producing equol according to any one of (1) to (3), wherein the equol is at least one selected from surfactants.

  (5) Isoflavones, a kind of obligate anaerobic microorganism having equol-producing ability, β-cyclodextrin or β-cyclodextrin derivative or γ-cyclodextrin, and a kind of obligately anaerobic microorganism having equol-producing ability And a surfactant having a cloud point of 50 ° C. or higher, preferably 60 ° C. or higher and 100 ° C. or lower, more preferably 70 ° C. or higher and 100 ° C. or lower.

  (6) The isoflavones are equol-producing compositions as described in (5) above, which are at least one selected from the group consisting of isoflavones, isoflavone aglycones, daidzein glycosides, daidzein, and dihydrodaidzein.

  (7) The obligate anaerobic microorganisms include the genus Adlercreutzia, the genus Asaccharobacter, the genus Eggerthella, the genus Eubacterium, the genus Lactococcus, The equol-producing composition according to (5) or (6) above, which is a single species selected from the group consisting of (Sharpea), Streptococcus, Strackia, and Bacteroides. .

  (8) The surfactant is an ester ether type non-ion of the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate The equol-producing composition according to any one of (5) to (7) above, which is at least one selected from surfactants.

  (9) A food or food additive comprising the equol-producing composition according to (5) to (8) above and equol.

  (10) A pharmaceutical comprising the equol-producing composition according to (5) to (8) above and equol.

  According to the equol production method of the present invention, the equol production amount is increased as compared with the conventional method. Surprisingly, there is little or no foaming due to the surfactant, the production in the fermenter and the recovery of the produced equol are easy, and the equol yield is increased as compared with the prior art.

  According to the equol-producing composition of the present invention, the equol production amount is increased as compared with the conventional case. In addition, there is little or no foaming due to the surfactant, and it is easy to recover the produced equol.

  According to the food, food additive, and pharmaceutical of the present invention, symptoms associated with estrogen deficiency, such as breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder and the like are prevented or improved.

It is a figure explaining the productivity of equol in Examples 1-2 and Comparative Examples 1-4.

  The equol production method, equol-producing composition, food, food additive, and pharmaceutical in the embodiment of the present invention will be described below.

[Method of manufacturing equol]
In the method for producing equol in the present embodiment, the medium containing isoflavones contains β-cyclodextrin, β-cyclodextrin derivative or γ-cyclodextrin and a cloud point of 50 ° C or higher, preferably 60 ° C or higher and 100 ° C or lower. More preferably, it is a production method in which a surfactant having a temperature of 70 ° C. or more and 100 ° C. or less is added, and a kind of obligate anaerobic microorganism having equol production ability is allowed to act.

  The isoflavones in the present embodiment are mainly obtained from legumes such as soybeans, kudzu, red clover, licorice, and are selected from the group consisting of isoflavones, isoflavone aglycones, daidzein glycosides, daidzein, and dihydrodaidzein. At least one kind.

  The isoflavones can be used as they are, or can be converted into isoflavone aglycones by the action of an enzyme such as β-glucosidase or a microorganism, and used for the main culture. Isoflavone aglycone is obtained, for example, by adding koji mold to soybean germ, fermenting it, and extracting it from the obtained fermented soybean germ.

  Isoflavone aglycone is mainly composed of daidzein, glycitein, and genistein.

  When the daidzein glycoside enters the human or animal body, it becomes the above-described daidzein, glycitein, or genistein, respectively, by the action of digestive enzymes or β-glucosidase that is an enzyme produced by intestinal bacteria.

  In addition, daidzein is enzymatically converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by the action of intestinal bacteria.

  The isoflavones in the present embodiment are preferably isoflavone, isoflavone aglycone, daidzin, and daidzein. Here, as the isoflavone, for example, Fujiflavone P40 and P10 manufactured by Fujicco Corporation can be used. As the isoflavone aglycone, for example, AglyMax-30 "(manufactured by Nichimo Biotex) can be used.

  The obligate anaerobic microorganisms in the present embodiment include the genus Adlercreutzia, the genus Asaccharobacter, the genus Eggerella, the genus Eubacterium, the genus Lactococcus, It is a single species selected from the group consisting of the genus Sharpea, the genus Streptococcus, the genus Slackia, and the genus Bacteroides.

  As the obligate anaerobic microorganism in the present embodiment, microorganisms shown in Table 1 below are preferable.

  Further, in the present embodiment, Asaccharobacter ceratus DSM 18785 is more preferable as the obligate anaerobic microorganism that produces equol.

The anaerobic microorganisms can be obtained from the depository indicated by the deposit number. Each deposit number indicates that the anaerobic microorganism is deposited at the next depository.
FERM: International Patent Organism Depositary (IPOD) (https://unit.aist.go.jp/pod/ci/index.htmL)
ATCC: American Type Culture Collection
DSM: Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH

  In the present embodiment, among cyclodextrins, β-cyclodextrin (cycloheptamilode), β-cyclodextrin derivatives or γ-cyclodextrin is preferable.

  The addition amount of β-cyclodextrin or β-cyclodextrin derivative in the present embodiment is 0.5 mol to 5 mol times, preferably 1.0 mol to 3 mol times that of daidzein in the medium.

  Examples of the β-cyclodextrin derivative in the present embodiment include glucosyl-β-cyclodextrin (6-O-a-D-Glucosyl-b-cyclodextrin), maltosyl-β-cyclodextrin (6-O-a- D-Maltosyl-b-cyclodextrin), Hydroxypropyl-b-cyclodextrin, Methyl-b-cyclodextrin, Poly-b-cyclodextrin ) And the like.

  The surfactant in the present embodiment is a surfactant having a cloud point of 50 ° C. or higher, preferably 60 ° C. or higher and 100 ° C. or lower, more preferably 70 ° C. or higher and 100 ° C. or lower.

  The cloud point was measured in accordance with Japanese Industrial Standard (JIS) K2269 (crude point of crude oil and petroleum products and petroleum product cloud point test method). The above test method keeps the sample at least 14 ° C. above the expected cloud point and removes it by any suitable method such as rubbing with dry filter paper if moisture is present, making it completely transparent. Place this sample in a test tube, cool according to the regulations, and observe the inside of the test tube every time the temperature of the sample drops by 1 ° C. The temperature at which the cloudiness of the sample was first observed at the bottom of the sample is taken as the cloud point.

  Examples of the surfactant having the above characteristics include polyoxyethylene sorbitan monolaurate (also referred to as “Tween 20”), polyoxyethylene sorbitan monopalmitate (also referred to as “Tween 40”), polyoxyethylene sorbitan monostearate. (Also referred to as “Tween 60”), polyoxyethylene sorbitan monooleate (also referred to as “Tween 80”), and at least one selected from ester ether type nonionic surfactants.

  Furthermore, as said ester ether type nonionic surfactant, Tween 20, Tween 40, Tween 60, and Tween 80 are preferable, for example.

  The addition amount of the surfactant in the present embodiment is 0.01 to 1.0 mass / volume% (wt / vol%), preferably 0.05 to 0.5 mass / volume% (wt / vol%). ).

  In the present embodiment, an obligate anaerobic microorganism having an equol-producing ability is brought into contact with isoflavones such as daidzein and cultured under conditions suitable for equol production. Conditions suitable for equol production refer to conditions under which the survival and activity of obligate anaerobic microorganisms having equol production activity are maintained. More specifically, it means that gas phase conditions (anaerobic conditions) that allow survival of obligate anaerobic microorganisms are maintained, and nutrients are provided to support the activity and growth of obligate anaerobic microorganisms.

  Here, as a variety of medium compositions suitable for the survival of obligate anaerobic microorganisms, known culture compositions are used, and those skilled in the art appropriately select the medium composition for the anaerobic microorganisms having the equol-producing ability described above. Can do. As the medium of the present embodiment, for example, a GAM bouillon medium manufactured by Nissui Pharmaceutical Co., Ltd., a BHI medium manufactured by Difco Corporation, or the like is 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 sorbose, fructose, glucose, methanol, and organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, and formic acid.

  The concentration of the organic substance added to the medium as a carbon source can be adjusted as appropriate in order to efficiently grow anaerobic microorganisms in the medium. Generally, excess and deficiency can be avoided by selecting the addition amount from the range of 0.1 to 10 wt / vol%.

  In addition to the above carbon source, a nitrogen source is added to the medium. In the present embodiment, various nitrogen compounds that can be used for normal fermentation are 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 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 a carbon source and a nitrogen source, other organic substances or inorganic substances suitable for the culture of obligate anaerobic microorganisms can be added to the medium. For example, the growth and activity of obligate anaerobic microorganisms may be enhanced by adding inorganic compounds such as vitamins and other cofactors and various salts to the medium. For example, the following can be mentioned as microbial growth cofactors derived from inorganic compounds, vitamins and animals and plants.

  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, Examples include sodium tungstate, sodium selenate, and ferrous ammonium sulfate.

  Examples of vitamins include biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, vitamin B12, thiooctoic acid, and p-aminobenzoic acid.

  Methods for producing a culture solution by adding these inorganic compounds, vitamins, or growth cofactors are known. Therefore, a culture solution is prepared according to a manufacturing method as appropriate. The medium can be liquid, semi-solid, or solid. In the method for producing equol in the present embodiment, a preferred medium form is a liquid medium.

  In the present embodiment, the obligate anaerobic microorganism can be cultured according to a known microorganism culturing method. For industrial production, it is also possible to use a continuous fermentation system that can continuously supply a medium and a substrate gas and that has a mechanism for recovering the culture.

  In the culture of obligate anaerobic microorganisms, it is necessary to prevent oxygen from being mixed into the continuous culture system. As the incubator, a commonly used culture tank can be used as it is. Culture tanks that can also be used for the culture of anaerobic microorganisms are commercially available. By replacing oxygen mixed in the culture tank with an inert gas such as nitrogen or a substrate gas, an anaerobic atmosphere can be obtained. Can be made.

  For example, an anaerobic jar can be a bioreactor for culturing anaerobic microorganisms. The anaerobic culture jar is composed of an airtight container made of metal, glass, or synthetic resin, and can block the inside from oxygen in the atmosphere. Furthermore, the anaerobic culture jar can be equipped with a mechanism for removing molecular oxygen contained in the space inside the anaerobic culture jar or in the culture solution. For example, nitrogen, helium, hydrogen, carbon dioxide gas, or the like can be maintained in an anaerobic state by aeration and stirring from the bottom.

  In the present embodiment, an additional function can be given to the culture tank. For example, in addition to a commonly used stirring and mixing tank, a bubble column type or draft tube type culture tank can also be used. The microorganisms are released and dispersed by the mixed gas blown into the liquid medium, and the microorganisms and the medium can be sufficiently brought into contact with each other. In addition, microorganisms inhabit while dripping moisture on a highly breathable slag like a biotrickling filter, other ceramic-based inorganic fillers, or packed layers of organic synthetic materials such as polypropylene, It can also be cultured while aeration of gas. Furthermore, the microorganisms to be used can be used by immobilizing them on carrageenan gel, alginic acid gel, acrylamide gel, chitin, cellulose, agar, etc. by a conventional method.

  In this embodiment, a person skilled in the art can use a known method in order to separate the produced anaerobic microorganisms from the culture product solution. A preferable separation method is a method using a hollow fiber type ultrafiltration or microfiltration membrane having filtration performance and concentration performance. Further, in order to obtain a filtration rate sufficient for separating the microorganism and the product solution, a membrane having an appropriate fractional molecular weight may be selected according to the microorganism used. Equol can be recovered from the culture solution separated from the culture tank through the ultrafiltration membrane. A method for purifying the collected equol is known. For example, the cells are removed from the culture by centrifugation, the supernatant is concentrated under reduced pressure, dried, and then extracted with 70% ethanol or methanol. The extract can be purified by further operations such as silica gel chromatography and crystallization. Depending on the shape of the culture tank, a stirrer or the like can be used to sufficiently stir the medium. By agitating the culture in the culture tank, it is possible to increase the chance of contacting the medium components and necessary gas with the anaerobic microorganisms, and to optimize the production efficiency of equol. A single or mixed gas such as hydrogen, nitrogen, carbon dioxide, or the like can also be supplied as micro or nano bubbles.

  For sufficient growth of microorganisms, the pH of the culture is preferably 3.0 to 9.0, more preferably 5.5 to 8.5. In order to increase the amount of equol recovered, the temperature of the culture tank is not particularly limited, but 37 ° C. can be mentioned as a preferable temperature.

[Equol production composition]
The equol-producing composition in the present embodiment comprises: one kind of obligate anaerobic microorganism having equol-producing ability; β-cyclodextrin or β-cyclodextrin derivative or γ-cyclodextrin; And a surfactant having a cloud point of 50 ° C. or higher, preferably 60 ° C. or higher and 100 ° C. or lower, more preferably 70 ° C. or higher and 100 ° C. or lower.

  It should be noted that isoflavones, a kind of obligate anaerobic microorganism capable of producing equol, β-cyclodextrin, β-cyclodextrin derivative or γ-cyclodextrin, and a cloud point of 50 ° C. or higher, preferably 60 ° C. or higher 100 The surfactant having a temperature not higher than ° C., more preferably not lower than 70 ° C. and not higher than 100 ° C. is the same as described above, and thus description thereof is omitted here.

[Food, food additives, pharmaceuticals]
The food, food additive, or pharmaceutical in the present embodiment includes the equol-producing composition described above and equol.

  Furthermore, the food, food additive or pharmaceutical product in the present embodiment contains equol obtained by the production method of the present embodiment.

  When equol is provided as a pharmaceutical product, in addition to equol, substances generally used for formulation (eg, starch, dextrin, lactose, corn starch, inorganic salts, etc.) can be used for formulation. Examples of dosage forms in the case of providing as pharmaceuticals include ampoules, tablets, capsules, granules, fine granules, powders, infusions, drinks and the like.

  As a food and drink formed by adding equol as a food additive, for example, health food, soft drink, milk, pudding, jelly, rice cake, gum, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicle, bread, Examples include cake, cream puff, ham, meat sauce, curry, stew, cheese, butter and dressing.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

  Hereinafter, in each Example, daidzein, dihydrodaidzein, equol, glycitein, and genistein were quantified by the following method.

Quantification of daidzein, dihydrodaidzein, equol, glycitein, genistein:
To 0.5 mL of the culture solution, 1.5 mL of ethyl acetate was added and stirred vigorously, followed by centrifugation at 3000 rpm for 10 seconds, and the ethyl acetate layer was removed as much as possible with a Pasteur pipette. The same operation was performed twice more on the same culture solution, and these ethyl acetate layers were combined to obtain an equol extract. This extract was concentrated and dried under reduced pressure using an evaporator, and dissolved in 1.0 mL of methanol. This was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane, and the insoluble matter was removed to obtain a sample for high performance liquid chromatography measurement.

[High-performance liquid chromatography conditions]
Column: Phenomenox Luna 5uC18, 2.0 mm x 150 mm (Shimadzu GLC)
Mobile phase: water / methanol [55:45, v / v]
Flow rate: 0.2 mL / min
Column temperature: 40 ° C
Detection: UV280nm
Retention time: 13.8 minutes for dihydrodaidzein, 19.6 minutes for daidzein, 22.5 minutes for glycitein, 25.6 minutes for equol, and 35.0 minutes for genistein.

  Daidzein (hereinafter abbreviated as “DAI”) used in the following examples was purchased from LKT Laboratories, Inc. The product made by company was used. As isoflavone aglycone, AglyMax-30 manufactured by Nichimo Biotex was used. The composition thereof is 21.2% DAI, 9.8% glycitein, and 3.0% genistein (both are manufacturer analysis values, weight%), and equol is abbreviated as “EQL”. Β-cyclodextrin is also referred to as “β-CD” below.

Examples 1-2, Comparative Examples 1-4:
5.9 g of GAM broth medium (manufactured by Nissui Pharmaceutical) and 1.21 g of L-arginine hydrochloride (1% of arginine with respect to the medium) are dissolved in 100 mL of pure water, and 10.0 mL of anaerobic bacteria while passing carbon dioxide. The solution was dispensed into 100 mL vial bottle for culture (manufactured by LMM Co., Ltd.), sterilized at 115 ° C. for 15 minutes with a butyl rubber stopper and an aluminum cap. 0.2 mL of Asaccharobacter celatus DSM 18785 strain that had been stored frozen at −80 ° C. in this medium was inoculated, and hydrogen / carbon dioxide gas (4: 1, vol / vol) passed through a sterile filter. After replacing the gas phase for 3 minutes or more, shaking culture was performed at 37 ° C. and 200 spm for 16 hours.

  Prepare a medium in which DAI, cyclodextrin and Tween 20 were added to GAM bouillon + 1% arginine medium so as to have the composition shown in Table 2 (daidzein and cyclodextrin were added in equimolar amounts) at 115 ° C for 15 minutes. Sterilized. After inoculating them with 0.1 mL of this seed culture solution and aseptically replacing with hydrogen / carbon dioxide gas (4: 1, vol / vol) for 3 minutes or more, shake culture under conditions of 37 ° C. and 200 spm. went. After 26, 49 hours, the culture solution was taken out, and the EQL produced by high performance liquid chromatography was quantified. The results are shown in FIG.

  It was found that by adding β-cyclodextrin and Tween 20 at the same time, the amount of EQL generated was increased compared to the addition of each of them alone.

Examples 3-5, Comparative Examples 5-9:
A medium in which AglyMax-30, cyclodextrin and Tween 20 were added to a GAM bouillon + 1% arginine medium so as to have the composition shown in Table 3 was prepared (daidzein and cyclodextrin were added in equimolar amounts) at 115 ° C., 15 Sterilized for minutes. After inoculating 0.1 mL of each seed culture solution cultured in the same manner as in Example 1 and aseptically replacing with hydrogen / carbon dioxide gas (4: 1, vol / vol) for 3 minutes or more, 37 ° C., 200 spm The shaking culture was performed under the conditions described above. After 21, 50 hours, the culture solution was taken out, and the EQL produced by high performance liquid chromatography was quantified. The results are shown in Table 4.

  It was also clarified that isoflavone aglycone AglyMax-30 also promoted EQL production over reagent DAI when β-cyclodextrin and Tween 20 were added simultaneously, reaching 6.25 g / L. Thus, in this method, EQL can be accumulated at a very high concentration.

Examples 6-7, Comparative Examples 10-11: Use of β-cyclodextrin derivatives and Tween 20;
A medium in which AglyMax-30, β-cyclodextrin, glucosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, and Tween 20 were added to a GAM bouillon + 1% arginine medium to the composition shown in Table 5 was prepared (Daizein). And cyclodextrins were added in equimolar amounts) and sterilized at 115 ° C. for 15 minutes. After inoculating 0.1 mL of each seed culture solution cultured in the same manner as in Example 1 and aseptically replacing with hydrogen / carbon dioxide gas (4: 1, vol / vol) for 3 minutes or more, 37 ° C., 200 spm The shaking culture was performed under the conditions described above. After 50 hours, the culture solution was taken out, and the EQL produced by high performance liquid chromatography was quantified. The results are shown in Table 6.

  It was revealed that even when Tween 20 was added to glucosyl-β-cyclodextrin at the same time, the EQL generation concentration was increased, and that the EQL generation concentration was higher than the combination of β-cyclodextrin and Tween 20.

Example 12: Foamability test;
This species cultured in the same manner as in Example 1 in a GAM bouillon + 1% arginine medium containing 35 g / L of isoflavone aglycone, 32.9 g / L of β-cyclodextrin, and 1 g / L of Tween 20 previously sterilized at 115 ° C. for 15 minutes. After inoculating 0.1 mL of the culture solution and aseptically replacing with hydrogen / carbon dioxide gas (4: 1, vol / vol) for 3 minutes or more, shaking culture was performed at 37 ° C. and 200 spm. After 76 hours, the culture solution was taken out and equol produced by high performance liquid chromatography was quantified. As a result, 5.83 g / L of EQL was generated. As in Examples 6 to 9, Aglymax-30 manufactured by Nichimo Biotex was used as the isoflavone aglycone.

  50 mL of this culture solution was placed in a 100 mL stoppered graduated cylinder, and the foam volume was measured with reference to 7.2 of the biodegradation test method of JIS K 3363 (1990) synthetic detergent. That is, 50 mL of the culture solution to which Tween 20 was added was placed in a stoppered test tube, shaken up and down 50 times (about twice per second), and allowed to stand for 30 seconds to measure the foam volume. As a result, in the culture solution to which β-cyclodextrin and Tween 20 were added, the foam immediately disappeared even when shaken, and the foam volume was 0 mL. On the other hand, the foam capacity of the standard Tween 20 1 g / L aqueous solution tested under the same conditions was 40 mL.

  It became clear that the problem of foaming due to simultaneous addition of β-cyclodextrin and Tween 20 does not occur in this culture method.

(Reference example)
Changes in DAI solubility due to simultaneous addition of β-cyclodextrin and Tween 20:
Japanese Patent Application Laid-Open No. 2010-187647 describes that the amount of DAI dissolved increases due to the addition of Tween, and hence the amount of EQL generated also increases. In order to confirm this, the DAI solubility in this medium was measured. Reagent DAI 7.4 g / L with or without Tween 20 1 g / L or isoflavone aglycone Aglymax-30 25 g / L, b-cyclodextrin 22.7 g / L, 10 mL GAM bouillon + 1% arginine medium Prepared and sterilized at 115 ° C. for 15 minutes. After cooling to room temperature, the medium was taken out, the supernatant was separated by centrifugation, and DAI in the supernatant was quantified by high performance liquid chromatography.

  The results are shown in Table 7. It was confirmed that both the reagent DAI and isoflavone aglycone had the highest solubility when β-cyclodextrin was added, and that the DAI concentration was lowered when Tween was added. It was revealed that the increase in EQL production by adding β-cyclodextrin and Tween 20 simultaneously is not simply an effect of increasing DAI solubility.

  In the method for producing equol of the present invention, the amount of isoflavones added in the medium is increased as compared to the conventional method, and the production of equol is greatly increased. The yield is greatly increased compared to the conventional method.

  According to the equol-producing composition of the present invention, the equol productivity is higher than before, and the equol produced is easier to recover than the conventional.

  According to the foods, food additives, and pharmaceuticals of the present invention, symptoms associated with estrogen deficiency, such as breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, climacteric disorder, etc. are prevented or improved. .

Claims (7)

In a medium containing isoflavones, β-cyclodextrin, β-cyclodextrin derivative or γ-cyclodextrin, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate and polyoxy Adding at least one surfactant selected from the group consisting of ester ether type nonionic surfactants consisting of ethylene sorbitan monooleate and allowing a single species of obligate anaerobic microorganisms capable of producing equol to act A process for producing equol characterized by
The anaerobic microorganisms are A sucker Arthrobacter (Asaccharobacter) genus method.   The method for producing equol according to claim 1, wherein the addition amount of the surfactant is 0.01 to 1.0 mass / volume%.   The method for producing equol according to claim 1 or 2, wherein the isoflavones are at least one selected from the group consisting of isoflavones, isoflavone aglycones, daidzein glycosides, daidzein, and dihydrodaidzein. Isoflavones, a single species of obligate anaerobic microorganism having equol-producing ability, β-cyclodextrin or β-cyclodextrin derivative or γ-cyclodextrin, and one kind of obligate anaerobic microorganism having equol-producing ability, At least one selected from the group consisting of ester ether type nonionic surfactants consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monooleate A surfactant, and
The anaerobic microorganisms equol-producing composition which is a A sucker Arthrobacter (Asaccharobacter) genus.   The equol-producing composition according to claim 4, wherein the isoflavones are at least one selected from the group consisting of isoflavones, isoflavone aglycones, daidzein glycosides, daidzein, and dihydrodaidzein.   A food or food additive comprising the equol-producing composition according to claim 4 or 5, and equol.   A pharmaceutical comprising the equol-producing composition according to claim 4 or 5, and equol.