JP5851685B2 - 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 daidzein glycosides 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.

  Patent Literature 5 includes equol-producing bacteria, Escherichia genus, Raoultella genus, Lactobacillus genus, Pantoea genus, Saccharomyces genus, Enterococcus ccterus A mixed microorganism containing at least one non-equol-producing bacterium selected from microorganisms belonging to the genus (Serratia) is cultured in a medium containing daiseins to produce a daidzein metabolite. As a solubilizer for Daicein, (1) cyclodextrin or (2) an interface having a polyethylene glycol glycol chain and being soluble in water and ethanol and having an HLB value of 11 or more Sex agents are described to be added to the medium.

  As described above, in Patent Document 5, when a mixed microorganism containing an equol-producing bacterium and at least one non-equol-producing bacterium is cultured in a medium containing daidzeins, a surfactant such as Tween is used. It is described that equol production is increased by adding it to the medium. However, in patent document 5, only the photograph of a thin layer chromatography (TLC) is shown, only the qualitative result is shown and the density | concentration of the produced | generated equol is unknown. 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 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.C. 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) Unevenness having equol-producing ability by adding 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, to a medium containing isoflavones. This is a method for producing equol, in which a kind of anaerobic microorganism is allowed to act.

  (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) An equol-producing composition comprising isoflavones, a kind of obligate anaerobic microorganism having equol-producing ability, and a surfactant having a cloud point of 50 ° C. or higher.

  (6) The equol-producing composition according to (5), wherein the isoflavones 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 nonionic interface 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), wherein the composition is at least one selected from active agents.

  (9) A 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 method for producing equol of the present invention, the production amount of equol is increased by increasing the amount of isoflavones added in the medium as compared with the conventional method, and surprisingly, there is almost no foaming due to the surfactant. In addition, production in the fermenter and 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, equol productivity is higher than before, and there is little or no foaming due to a surfactant, so that the produced equol can be easily recovered.

  According to the food, food additive, and pharmaceutical of the present invention, for example, breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, and menopause are prevented or improved.

It is a figure explaining productivity of equol in comparative example 1 and Examples 1-3. It is a figure explaining productivity of equol in comparative example 1 and examples 4-6. It is a figure explaining productivity of equol in comparative example 2 and example 7.

  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, 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 is added to a medium containing isoflavones. This is a method for producing equol by allowing one kind of obligate anaerobic microorganism having equol-producing ability 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.
Moreover, 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.

  A daidzein glycoside is converted into the aforementioned daidzein, glycitein, or genistein by the action of a digestive enzyme or β-glucosidase, which is an enzyme produced by an intestinal bacterium, when entering the human or animal body.

  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) (http://unit.aist.go.jp/pod/ci/index.htmL)
ATCC: American Type Culture Collection
DSM: Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH

  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”), at least one selected from the group consisting of ester ether type nonionic surfactants consisting of polyoxyethylene sorbitan monooleate (also referred to as “Tween 80”) is used.

  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 this embodiment, an obligate anaerobic microorganism having equol-producing ability is brought into contact with isoflavones such as daidzein and isoflavone aglycone under conditions suitable for equol production and cultured. 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, an anaerobic culture jar can be maintained in an anaerobic state by agitating nitrogen, helium, hydrogen, carbon dioxide gas and the like 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 stirring the culture in the culture tank, it is possible to increase the chance of contacting the medium components, the substrate, and the 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 includes isoflavones, a kind of obligate anaerobic microorganism having equol-producing ability, and a cloud point of 50 ° C. or higher, preferably 60 ° C. or higher and 100 ° C. or lower, more preferably 70 ° C. And a surfactant at 100 ° C. or lower.

  In addition, isoflavones, a kind of obligate 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. Is the same as described above, and is not described here.

[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.

  Foods and drinks made by adding equol as a food, food additive, for example, health food, soft drinks, milk, pudding, jelly, rice cake, gum, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, Examples include bread, cake, cream puff, ham, meat sauce, curry, stew, cheese, butter, dressing and the like.

  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, 2.0mm x 150mm (Shimadzu GC)
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”.

Examples 1-6, Comparative Example 1:
5.9 g of GAM broth medium (manufactured by Nissui Pharmaceutical) and 1.21 g of L-arginine hydrochloride (1% arginine with respect to the medium) are dissolved in 100 mL of pure water, and 10.0 mL of anaerobic bacteria are cultured through carbon dioxide gas. The solution was dispensed into 100 mL vial bottle (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. ) Was replaced for 3 minutes or more, and then cultured with shaking at 37 ° C. and 200 spm for 16 hours.

  0.1 mL of this seed culture solution was inoculated in a GAM bouillon + 1% arginine medium prepared to have the composition shown in Table 2 below and previously sterilized at 115 ° C. for 15 minutes, and hydrogen / carbon dioxide (4: 1) , Vol / vol) and aseptically replaced for 3 minutes or more, and then cultured under shaking at 37 ° C. and 200 spm. After 9, 27, 79 hours, the culture solution was taken out, and the EQL produced by high performance liquid chromatography was quantified. The results are shown in FIGS.

  It was revealed that EQL production was remarkably promoted by the addition of Tween 80 and Tween 20.

Example 7, Comparative Example 2:
0.1 mL of this seed culture solution cultured in the same manner as in Example 1 was inoculated in a GAM bouillon + 1% arginine medium that had been prepared to have the composition shown in Table 3 and previously sterilized at 115 ° C. for 15 minutes. After aseptically replacing with / carbon dioxide (4: 1, vol / vol) for 3 minutes or more, shaking culture was performed at 37 ° C. and 200 spm. After 9, 27, 79 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 revealed that EQL production was promoted not only by the reagent daidzein but also by isoflavone aglycone when Tween 20 was added.

Example 8: Testing of foamability;
0.1 mL of this seed culture cultured in the same manner as in Example 1 was inoculated in a GAM bouillon + 1% arginine medium containing 35 g / L of isoflavone aglycone and 1 g / L of Tween 20 previously sterilized at 115 ° C. for 15 minutes, After aseptically substituting 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, 1.54 g / L of EQL was generated. As in Example 2, 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 Tween 20 was added, the foam immediately disappeared even when shaken, and the foam volume was 0 mL. On the other hand, the foam capacity of a standard 1 g / L aqueous solution of Tween 20 tested under similar conditions was 40 mL.

  It became clear that the problem of foaming due to the addition of Tween does not occur in this culture method.

(Reference example)
Change in DAI solubility with Tween addition:
Patent Document 5 describes that the amount of DAI dissolved increased by adding Tween, and therefore the amount of EQL generated also increased. In order to confirm this, the DAI solubility in this medium was measured. Reagent DAI 7.4 g / L, isoflavone aglycone 25 g / L, Tween 20 1 g / L added or not added 10 mL GAM bouillon + 1% arginine medium was 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 4. It was confirmed that both the reagent DAI and isoflavone aglycone had higher DAI solubility when not added, and the DAI concentration was lowered when Tween was added. It became clear that the increase in the EQL production amount due to the addition of Tween was not an effect due to the increase in DAI solubility.

  In the method for producing equol of the present invention, the amount of isoflavones in the medium is increased as compared with the conventional method, so that the amount of equol produced is increased as compared with the conventional method. In addition, it is easier to recover the produced equol than in the past.

  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 food additive and medicine 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.

Claims (7)

Ester ether type nonionic surfactant of the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monooleate in a medium containing isoflavones A method for producing equol, characterized by adding a surfactant which is at least one selected from the above, and allowing a single species of obligate anaerobic microorganisms having equol-producing ability to act,
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 capable of producing equol, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monooleate A surfactant that is at least one selected from the group consisting of ester ether type nonionic surfactants, 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.