Method for producing equol

Abstract

To provide a method for efficiently producing equol using a microbe, consuming less energy compared to traditional methods.SOLUTION: A method for producing equol includes the step for fermenting at least one equol precursor, selected from the group consisting of daidzein glycosides, daidzein, and dihydrodaidzein, with microbes capable of assimilating the equol precursor to synthesize equol. The medium used for fermentation is sterilized under the following conditions: i) temperature: 70°C or higher and lower than 80°C, duration: 60 minutes or longer, ii) temperature: 80°C or higher and lower than 90°C, duration: 30 minutes or longer, or iii) temperature: 90°C or higher and lower than 100°C, duration: 10 minutes or longer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing equol using a microorganism capable of producing equol, particularly an anaerobic microorganism. [Background technology]

[0002] Isoflavones, which are found in large quantities in legumes such as soybeans and kudzu, are a type of polyphenol and a flavonoid whose basic structure is isoflavone. Recent research has revealed that isoflavones have female hormone (estrogen) and antioxidant effects, and that ingesting isoflavones has a preventive effect against breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, and other conditions. For example, in soybeans, isoflavones exist as glycosides covalently bound to sugars, such as daidzin, glycitin, and genistin, and only small amounts of aglycones exist. Some of these glycosides are further malonylated or acetylated. When these glycosides enter the human or animal body, they are converted to daidzein, glycitein, and genistein, respectively, by the action of digestive enzymes or β-glucosidase, an enzyme produced by intestinal bacteria. Furthermore, it is known that daidzein is enzymatically converted by the action of intestinal bacteria to dihydrodaidzein, and then to O-desmethylangolensin (O-DMA) or equol.

[0003] Of these metabolic products, equol is known to have the highest estrogenic activity. However, in humans, there are individual differences in isoflavone metabolism, and as mentioned above, only a few people possess intestinal bacteria capable of fermenting daidzein to produce equol, with the rate of such bacteria being approximately 50% in Japanese and approximately 30% in Westerners. Therefore, there has been a problem in that people who do not possess equol-producing bacteria are unable to produce equol in their bodies even if they ingest legume foods such as soybeans.

[0004] To overcome these problems, attempts have been made to produce equol ex vivo using anaerobic microorganisms such as lactic acid bacteria (Patent Documents 1 to 4).

[0005] When culturing microorganisms, the raw medium is sterilized. The minimum sterilization temperature and time required by the Japanese Pharmacopoeia is 115°C for 30 minutes, or 121°C for 15 minutes, or 126°C for 10 minutes, and generally, the medium is sterilized and cultured under these conditions. Because the temperature is over 100°C, it must be carried out in a pressure-resistant facility. In addition, because the medium components that are not heat stable at high temperatures decompose, it was necessary to add excessive amounts of components. After sterilization, it is necessary to cool it down to the culture temperature, so the higher the temperature, the more energy wasted. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2006-204296 A. [Patent Document 2] Special Publication No. 2006-504409. [Patent Document 3] JP 2008-61584 A. [Patent Document 4] JP 2010-104241 A. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of these circumstances, and its object is to provide a method for producing equol using microorganisms that reduces the amount of energy used compared to conventional methods and enables efficient production of equol. [Means for solving the problem]

[0008] The inventors discovered that sterilization at a lower temperature can suppress the decomposition of medium components, increase the rate of equol production, and also reduce the amounts of components required, and thus devised the following invention.

[0009] <1> A method for producing equol, comprising a step of fermenting at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein with a microorganism capable of assimilating the equol raw material to produce equol, Before the fermentation step, a medium containing the equol raw material is i) Temperature: 70°C or higher and lower than 80°C, time: 60 minutes or longer, or ii) Temperature: 80°C or higher and lower than 90°C, time: 30 minutes or longer, or iii) Temperature: 90°C or higher and lower than 100°C, Time: 10 minutes or longer, The above method, further comprising a step of sterilizing the mixture under the conditions of

[0010] <2> the above <1> In the above, sterilization is preferably carried out at i) a temperature of 70° C. or higher and lower than 80° C. for a period of 60 minutes or longer, preferably 60 minutes or longer and 16 hours or shorter, and more preferably 60 minutes or longer and 5 hours or shorter. <3> the above <1> In the above, sterilization is preferably carried out at ii) a temperature of 80° C. or higher and lower than 90° C. for a period of 30 minutes or longer, preferably 30 minutes or longer and 16 hours or shorter, and more preferably 30 minutes or longer and 5 hours or shorter. <4> the above <1> In the above, sterilization is preferably carried out at iii) a temperature of 90° C. or higher and lower than 100° C. for a period of 10 minutes or longer, preferably 10 minutes or longer and 10 hours or shorter, and more preferably 10 minutes or longer and 2 hours or shorter.

[0011] <5> the above <1> ~ <4> In any of the above, the particle size of the equol raw material is 1 mm or less, preferably 250 μm or less, and more preferably 100 μm or less. <6> the above <1> ~ <5> In any of the above, the fermentation process may be carried out under anaerobic conditions consisting of one or more gases including hydrogen. Effect of the Invention

[0012] The present invention provides a method for producing equol using microorganisms that uses less energy than conventional methods and can produce equol more efficiently. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides a method for producing equol, which comprises a step of fermenting at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein with a microorganism capable of assimilating the equol raw material to produce equol. The method comprises the steps of: preparing a medium containing an equol raw material before the fermentation step; i) Temperature: 70°C or higher and lower than 80°C, time: 60 minutes or longer, or ii) Temperature: 80°C or higher and lower than 90°C, time: 30 minutes or longer, or iii) Temperature: 90°C or higher and lower than 100°C, Time: 10 minutes or longer, The method includes a step of sterilizing the mixture under the conditions of

[0014] Conventionally, the sterilization process was carried out under temperature and time conditions such as 115°C for 30 minutes, 121°C for 15 minutes, or 126°C for 10 minutes. In contrast, the present invention is carried out under lower temperature conditions than conventional ones, and therefore can provide a method for producing equol more efficiently with less energy consumption than conventional methods. In addition, as described below, the sterilization conditions of the present invention can improve the productivity of equol. Furthermore, the present invention makes it possible to produce equol in large quantities at low cost, making it possible to supply equol to more people. It is believed that equol can be used to prevent breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, etc., when taken as is as a food or drink or medicine. The present invention will be described in detail below.

[0015] As described above, the method for producing equol of the present invention has a sterilization step and a fermentation step. The method of the present invention may include steps other than the fermentation step and the sterilization step, such as, but not limited to, a step of preparing an equol raw material, a pre-culture step, and a step of recovering the obtained equol.

[0016] <Sterilization process> The sterilization step is a step of sterilizing the medium containing the equol raw material before the fermentation step. The medium used in the sterilization step contains the equol raw material and may also contain a carbon source, a nitrogen source, etc., which will be described later. Note that the medium used in the sterilization step does not contain microorganisms capable of assimilating the equol raw material to produce equol.

[0017] The sterilization process should be carried out under the following conditions, which are sufficient to sterilize microorganisms incapable of producing equol that are present in the culture medium containing the equol raw material, to the extent that they do not affect the proliferation and production of equol by microorganisms capable of producing equol in the subsequent fermentation process, and which are sufficient to prevent components in the culture medium that affect the proliferation and metabolism of the microorganisms from being decomposed by the applied heat. i) Temperature: 70° C. or higher and lower than 80° C.; Time: 60 minutes or longer, preferably 60 minutes or longer and 16 hours or shorter, more preferably 60 minutes or longer and 5 hours or shorter; or ii) temperature: 80° C. or higher and lower than 90° C.; time: 30 minutes or longer, preferably 30 minutes or longer and 16 hours or shorter, more preferably 30 minutes or longer and 5 hours or shorter; or iii) Temperature: 90° C. or higher and lower than 100° C., time: 10 minutes or longer, preferably 10 minutes or longer and 10 hours or shorter, more preferably 10 minutes or longer and 2 hours or shorter. The sterilization step may be carried out in the tank in which the fermentation step is carried out, or in a separate device. In the case of heating for a long period of time, it is preferable to use a device with an automatic temperature control function.

[0018] <<Equol raw material>> In the method of the present invention, at least one equol source selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein is used as a raw material. The equol raw material may take any form as long as it is literally used as an equol raw material. The equol raw material may be in any form as long as it contains at least one selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein, for example, daidzein glycoside itself, daidzein itself, or dihydrodaidzein itself, or a substance containing them, such as soybeans, processed soybeans, soybean hypocotyls, processed soybean hypocotyls, such as soybean extracts, soybean hypocotyl extracts, and purified soybean hypocotyl extracts, and specifically, commercially available isoflavones.

[0019] Since the equol raw material is difficult to dissolve in water and / or for reaction efficiency in the fermentation process, the particle size is preferably small, for example, preferably 1 mm or less, more preferably 250 μm or less, and even more preferably 100 μm or less, but is not particularly limited to these particle sizes. It is desirable that the particle size becomes the above particle size after suspension in the medium.

[0020] <<Culture medium>> The medium is not particularly limited as long as it is suitable for fermenting equol in the fermentation step described below, and for example, the medium used in the Examples can be used. Furthermore, if the equol-producing bacterium used has nutritional requirements or characteristics, suitable nutritional components can be added thereto.

[0021] 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.

[0022] The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted in order to efficiently grow microorganisms, particularly anaerobic microorganisms, in the medium.

[0023] The medium may be supplemented with a nitrogen source. 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.

[0024] 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.

[0025] 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 Ferrous Ammonium Sulfate

[0026] 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.

[0027] 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.

[0028] The medium of the present invention may contain an antifoaming agent, preferably soybean oil, more preferably soybean oil with vitamin E.

[0029] <<Pre-culture step>> The method of the present invention may also include a step prior to the fermentation step, separate from the sterilization step, of culturing microorganisms capable of assimilating the equol raw material to produce equol under conditions suitable for sufficient proliferation without loss of equol-producing ability (also referred to as a "pre-culture step"; in this specification, "the ability to assimilate the equol raw material to produce equol" may sometimes be simply referred to as "equol-producing ability"). The conditions suitable for sufficient proliferation without loss of equol-producing ability refer to conditions under which the survival and activity of microorganisms capable of synthesizing equol, such as anaerobic microorganisms, can be maintained. More specifically, these conditions refer to conditions under which gas-phase conditions (anaerobic conditions) in which anaerobic microorganisms can survive are maintained and nutrients are provided to support the activity and proliferation of the anaerobic microorganisms. Various medium compositions suitable for the survival of microorganisms, for example anaerobic microorganisms, are known. Therefore, for microorganisms capable of producing equol, such as anaerobic microorganisms, described below, those skilled in the art can select an appropriate medium composition. For example, a medium having the same components as the medium used in the subsequent fermentation step, a medium used in the examples, etc., can be used, but is not limited to these.

[0030] In the method of the present application, the microorganisms described below, particularly anaerobic microorganisms, can be cultured according to known microbial culture methods. For industrial production, a continuous fermentation system capable of continuously supplying the medium and substrate gas and having a mechanism for recovering the culture can be used.

[0031] In the method of the present application, when anaerobic microorganisms are used, it is advisable to prevent oxygen from being mixed into the fermenter. A commonly used fermenter can be used as is. In addition, in the method of the present application, since the sterilization step is carried out without exceeding 100°C, which is the boiling point of water, the first type of pressure vessel is not necessary. Any device having a jacket or internal coil for heating the contents can be suitably used. An anaerobic atmosphere can be created by replacing the oxygen that enters the fermentation tank with an inert gas such as nitrogen.

[0032] <Fermentation process> The method of the present invention comprises a fermentation step after the pasteurization step and, if present, after the pre-culture step. The fermentation step is a step in which the above-mentioned equol raw material is fermented by a microorganism capable of producing equol. As described above, the fermentation step may be carried out in the tank used for the sterilization step, or may be carried out using a separate device.

[0033] <<Microorganisms>> The fermentation step in the method of the present invention uses a microorganism capable of producing equol. The microorganism used in the method of the present invention is not particularly limited as long as it is a microorganism capable of producing equol from the above-mentioned equol raw material. The microorganism may be an anaerobic microorganism, which can produce equol at a temperature of, for example, about 37° C. (e.g., 30 to 42° C.).

[0034] The equol-producing 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.

[0035] 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:

[0036] [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

[0037] Microorganisms capable of producing equol include, but are not limited to, those classified into the following genera: Genus Adlercreutzia Bacteroides genus Bifidobacterium Clostridium Genus Eggerthella Enterococcus Genus Enterorhabdus Eubacterium genus Genus Finegoldia Lactobacillus Lactococcus Genus Paraeggerthella Pediococcus genus Genus Proteus Genus Sharpea Genus Slackia Streptococcus Genus Veillonella

[0038] Specific examples of microorganisms capable of producing equol include, but are not limited to, the following microorganisms. Adlercreutzia 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 Lactococcus sp. Paraeggerthella sp. Pediococcus pentosaceus Proteus mirabilis Sharpea azabuensis Slackia equolifaciens Slackia isoflavoniconvertens Slackia sp. Streptococcus constellatus Streptococcus intermedius Veillonella sp.

[0039] Among the above-mentioned microorganisms, for example, microorganisms classified into the family Eggerthellaceae, microorganisms classified into the family Bifidobacteriaceae, microorganisms classified into the family Clostridiaceae, microorganisms classified into the family Coriobacteriaceae, microorganisms classified into the family Enterococcaceae, microorganisms classified into the family Eubacteriaceae, microorganisms classified into the family Morganellaceae, microorganisms classified into the family Peptoniphilaceae, microorganisms classified into the family Lactobacillaceae, microorganisms classified into the family Streptococcus, microorganisms classified into the family Veillonellaceae, and microorganisms related thereto can be mentioned. Preferably, the microorganism is classified into the genera Adlercreutzia, Bacteroides, Bifidobacterium, Clostridium, Coriobacterium, Egasella, Enterococcus, Eubacterium, Finegordia, Lactobacillus, Lactococcus, Paraegasella, Pediococcus, Proteus, Chapaea, Slaakia, Streptococcus, Veillonea, or related microorganisms thereof.More preferably, Adrechlautia aequorifaciens subsp. ceratus, Adrechlautia aequorifaciens subsp. aequorifaciens, Bacteroides obatus, Bifidobacterium breve, Bifidobacterium longum, Clostridium sp., Egassella sp., Enterococcus faecalis, Enterococcus faecium, Enterohabdus mucosicola, Eubacterium sp., Finegordia magna, Lactobacillus fermentum, Lactobacillus intestina Lactobacillus subtilis, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sp., Lactococcus garvieae, Lactococcus sp., Paraegasella sp., Pediococcus pentosaceus, Proteus mirabilis, Sharpea azabuensis, Slachia aequorifaciens, Slachia isoflavonic convertens, Slachia sp., Streptococcus constellatus, Streptococcus intermedius, Veillonea sp.

[0040] Among the above-mentioned microorganisms, any of the microorganisms described below or related microorganisms having similar species properties to these microorganisms can be given as more preferred 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 strain Lactobacillus mucosae EPI2 strain Lactobacillus paracasei JS1 strain Lactobacillus plantarum DPPMA24W strain Lactobacillus plantarum DPPMASL33 strain Lactobacillus rhamnosus strain DPPMAAZ1 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 2200 6 stocks 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.

[0041] The above microorganisms can be obtained from the depository institutions indicated by their accession numbers. Each accession number indicates that the microorganism has been deposited in 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

[0042] <<Gas phase in fermentation process>> The method of the present invention, particularly the fermentation step of the method of the present invention, is not limited as long as the conditions are such that the equol-producing bacteria can grow and produce equol. When the conditions are anaerobic conditions, it is preferable to carry out the fermentation under anaerobic conditions, for example, with one or more types of gases including hydrogen. In this case, the gas constituting the gas phase is not particularly limited as long as it is one or more types of gases including hydrogen, but it is preferable to have hydrogen and one or more types of gases other than hydrogen. The gas other than hydrogen is not particularly limited as long as it is a gas that can achieve anaerobic conditions, and examples of the gas include, but are not limited to, carbon dioxide, nitrogen, carbon monoxide, etc.

[0043] In order to efficiently recover equol, the amount of the mixed gas constituting the gas phase passed through the fermentation tank is preferably 0.001 to 2.0 V / V / M gas volume / liquid volume / min.

[0044] In the present invention, the temperature of the fermenter is not particularly limited, but it is preferably a temperature at which the above-mentioned microorganisms can exert their equol-producing ability, for example, 30°C to 40°C, preferably 33°C to 38°C.

[0045] In the present invention, 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 pressurized conditions. Preferred pressurization conditions include, but are not limited to, a pressure of 0.2 MPa or less.

[0046] 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, if necessary. Heat drying can be performed using, for example, a rotary drum dryer, spray drying can be performed using, for example, a spray dryer, and freeze drying can be performed using a freeze dryer. Any dryer that can dry a liquid may be used for the drying method. The dried fermentation culture may be subjected to a pulverization treatment, if necessary. EXAMPLES

[0047] 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 10 mL 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and sterilized at 121°C for 15 minutes with a butyl rubber stopper and a plastic cap 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, and then the medium was shake-cultured at 37°C and 200 spm for 18 hours to prepare a preculture solution.

[0048] [Table 1]

[0049] (main culture) <Sterilization process> Daidzein 1.2 g / L and L-arginine 3 g / L were added to the composition shown in Table 1, and 15 L of the medium was adjusted to pH 6.9. The medium was placed in a 30 L fermenter and sterilized under each sterilization condition. <Fermentation process> The above pre-culture solution was inoculated into this medium, and anaerobic fermentation was carried out at 37° C. The equol concentration in the main culture solution obtained after the fermentation process was analyzed by HPLC.

[0050] The results are shown in Table 2. Example 1 confirmed that the lower the sterilization temperature, the higher the equol production capacity.

[0051] [Table 2]

[0052] Example 2 (preculture) The medium, with the composition shown in Table 1 and adjusted to pH 6.9, was dispensed into 10 mL 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and sterilized at 121°C for 15 minutes with a butyl rubber stopper and a plastic cap attached while replacing the gas phase with nitrogen. Slackia equlifaciens DSM 24851 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, and then the medium was shake-cultured at 37°C and 200 spm for 36 hours to prepare a preculture solution.

[0053] (main culture) <Sterilization process> Daidzein 1.2 g / L and L-arginine 3 g / L were added to the composition shown in Table 1, and 15 L of the medium was adjusted to pH 6.9. The medium was placed in a 30 L fermenter and sterilized under each sterilization condition. <Fermentation process> The above pre-culture solution was inoculated into this medium, and anaerobic fermentation was carried out at 37° C. The equol concentration in the main culture solution obtained after the fermentation process was analyzed by HPLC.

[0054] The results are shown in Table 3. Example 2 confirmed that the lower the sterilization temperature, the higher the equol production ability.

[0055] [Table 3]

[0056] Example 3 (preculture) The medium with the composition shown in Table 1 and adjusted to pH 6.5 was dispensed into 10 mL of 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and the tubes were fitted with butyl rubber stoppers and plugs while the gas phase was replaced with nitrogen. The medium was fitted with a stick cap and sterilized at 121°C for 15 minutes. Lactococcus sp. DCL strain was inoculated into the medium, and the gas phase was replaced with hydrogen gas passed through a sterile filter for at least 2 minutes. After that, the medium was cultured with shaking at 37°C and 200 spm for 24 hours to prepare a preculture solution.

[0057] (main culture) <Sterilization process> 15 L of medium containing 70 g / L powdered soybean hypocotyl, 3 g / L L-arginine, and 2 g / L soybean oil containing vitamin E and adjusted to pH 6.5 was placed in a 30 L fermenter, and the medium was sterilized under each sterilization condition. <Fermentation process> The above pre-culture solution was inoculated into this medium, and anaerobic fermentation was carried out at 37° C. The equol concentration in the main culture solution obtained after the fermentation process was analyzed by HPLC.

[0058] The results are shown in Table 4. Example 3 confirmed that a lower sterilization temperature increased the amount of equol produced.

[0059] [Table 4]

[0060] Example 4 (preculture) The medium, with the composition shown in Table 5 and adjusted to pH 6.9, was dispensed into 10 mL 18 mm test tubes for culturing anaerobic microorganisms (manufactured by Sanshin Kogyo Co., Ltd.), and sterilized at 121°C for 15 minutes with a butyl rubber stopper and a plastic cap 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 2 minutes or more, and then the medium was shake-cultured at 37°C and 200 spm for 36 hours to prepare a preculture solution.

[0061] [Table 5]

[0062] (main culture) <Sterilization process> Daidzein 1.2 g / L and L-arginine 3 g / L were added to the composition shown in Table 5, and 1 L of the medium was adjusted to pH 6.9 and placed in a 2 L fermenter, and the medium was sterilized under each sterilization condition. <Fermentation process> The above pre-culture solution was inoculated into this medium, and anaerobic fermentation was carried out at 37° C. The equol concentration in the main culture solution obtained after the fermentation process was analyzed by HPLC. The results are shown in Table 6. Although the medium components were reduced from Example 1, it was confirmed that the lower the sterilization temperature, the higher the equol-producing ability and that equivalent productivity was maintained.

[0063] [Table 6]

Claims

1. A method for producing equol, comprising the step of fermenting soybean hypocotyls with a microorganism capable of assimilating the soybean hypocotyls to produce equol, Before the fermentation step, the medium containing the soybean hypocotyl i) Temperature: 70°C or higher and lower than 80°C, time: 60 minutes or longer, or ii) Temperature: 80°C or higher but lower than 90°C, time: 30 minutes or longer, or iii) Temperature: 90°C or higher and lower than 100°C, Time: 10 minutes or longer; The above method, further comprising a step of sterilizing the mixture under the conditions of

2. The method according to claim 1, wherein the sterilization is carried out at i) a temperature of 70°C or higher and lower than 80°C for 60 minutes or longer.

3. The method according to claim 1, wherein the sterilization is carried out at ii) a temperature of 80°C or higher and lower than 90°C for 30 minutes or longer.

4. The method according to claim 1, wherein the sterilization is carried out at iii) a temperature of 90°C or higher and lower than 100°C for 10 minutes or longer.

5. 5. The method according to any one of claims 1 to 4, wherein the fermentation step is carried out under anaerobic conditions consisting of one or more gases including hydrogen.