Method for producing equol

Abstract

To provide a method for producing equol using microorganisms, which can effectively produce equol at a lower hydrogen concentration and is safer than conventional methods.SOLUTION: Provided is a method for producing equol, comprising the step of fermenting at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein by a microorganism capable of assimilating the equol raw material to produce equol, in a gas phase consisting of one or more gases including hydrogen, the fermentation step being characterized in that using a fermenter with a culture liquid volume of 100 L or more, i) stirring is performed in the fermenter with an agitation power of 0.1 kW / kL or more, preferably 0.2 kW / kL or more, more preferably 0.4 kW / kL or more, and / or ii) the gas is introduced by using a sparger with a pore size of 2 mm or less, preferably 1 mm or less, more preferably 0.5 mm or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing equol using a microorganism capable of producing equol. [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). However, it has not been clear what fermentation conditions and methods would enable more effective and practical production of equol. For example, there is a case in which equol production was attempted by mixed culture of four types of anaerobic microorganisms in a hydrogen gas phase (Non-Patent Document 1), but mixed culture was not suitable for practical production (industrialization).

[0005] Since this is the culture of anaerobic microorganisms, static culture is usually used (Patent Document 5). Meanwhile, regarding a method for more effective and practically producing equol by changing fermentation conditions and methods, it has been reported that the production efficiency of equol is dramatically increased when the mass percent concentration of hydrogen gas in the gas phase in which fermentation is carried out is 40 to 100% (especially when it is 100%) (Patent Document 6). However, when hydrogen is ignited in the presence of oxygen, a violent explosion occurs. When mixed with air, the hydrogen concentration range in which an explosion occurs is a hydrogen concentration with a lower limit of 4.1% and an upper limit of 74.2%. In other words, the hydrogen concentration range in which the production efficiency of equol is dramatically increased is a hydrogen concentration at which a violent explosion may occur if hydrogen-containing gas leaks from the production equipment, and therefore, at the time of industrialization, very expensive production equipment with sufficient explosion prevention measures was required. [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. [Patent Document 5] WO2007 / 066655. [Patent Document 6] WO2012 / 033150. [Non-patent literature]

[0007] [Non-Patent Document 1] Decroos, K. et al., Arch Microbiol., 183, 45-55 (2005). Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of these circumstances, and its object is to provide a method for producing equol using microorganisms, which can effectively produce equol at a lower hydrogen concentration that is safer than conventional methods. [Means for solving the problem]

[0009] The present inventors have discovered that in a process 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 in a gas phase consisting of one or more gases including hydrogen, by using a fermenter with a culture liquid volume of 100 L or more and using conditions of a certain level of stirring power and / or a certain level of gas supply rate using a sparger, equol can be produced effectively at a lower gas phase hydrogen concentration than in the past, regardless of the type of bacteria. Specifically, the inventors have discovered the following invention.

[0010] <1> 1. A method for producing equol, comprising the steps of: fermenting at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein, in a gas phase containing one or more gases including hydrogen, by a microorganism capable of assimilating the equol raw material to produce equol, In the fermentation step, when a fermenter with a culture medium volume of 100 L or more is used, i) stirring the fermenter with a stirring power of 0.1 kW / kL or more, preferably 0.2 kW / kL or more, more preferably 0.4 kW / kL or more, and / or ii) The gas is introduced by using a sparger with a hole diameter of 2 mm or less, preferably with a hole diameter of 1 mm or less, more preferably with a hole diameter of 0.5 mm or less. The above method, characterized in that <2> the above <1> The hydrogen concentration of the gas is preferably 30% or less, more preferably 10% or less, and even more preferably 4% or less. Effect of the Invention

[0011] INDUSTRIAL APPLICABILITY The present invention provides a method for producing equol using microorganisms that can effectively produce equol at a lower hydrogen concentration that is safer than conventional methods. In particular, the present invention makes it possible to realize an efficient method for producing equol using daidzein as a raw material, utilizing microbial, particularly anaerobic microbial fermentation, at a safe low hydrogen concentration even on an industrial scale, and provides a technology for mass production of equol.

[0012] The method of the present invention makes it possible to mass-produce equol at low cost, allowing equol to be supplied to a greater number of people. It is believed that equol, when ingested as is as a food or drink or medicine, can prevent breast cancer, prostate cancer, osteoporosis, hypercholesterolemia, heart disease, menopausal disorders, etc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides a method for producing equol, comprising the steps of: fermenting at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein, in a gas phase containing one or more gases including hydrogen, by a microorganism capable of assimilating the equol raw material to produce equol, In the fermentation step, a fermenter having a culture liquid volume of 100 L or more is used, i) stirring the fermenter with a stirring power of 0.1 kW / kL or more, preferably 0.2 kW / kL or more, more preferably 0.4 kW / kL or more, and / or ii) The gas is introduced by using a sparger with a hole diameter of 2 mm or less, preferably with a hole diameter of 1 mm or less, more preferably with a hole diameter of 0.5 mm or less. The above method is characterized in that Although it has long been academically known that equol is produced through the metabolism of microorganisms, particularly anaerobic microorganisms, the culture conditions for anaerobic microorganisms that produce equol at safe low hydrogen concentrations even on an industrial scale had not been established, making the method of the present invention useful. The present invention will be described in detail below.

[0014] <Fermentation process> The present invention provides a method for producing equol, which comprises the above-mentioned fermentation step. The method of the present invention may include steps other than the fermentation step, such as, but not limited to, a step of preparing an equol raw material and a step of recovering the obtained equol. A specific example of a process other than the fermentation process is a sterilization process for sterilizing the microorganisms used in the fermentation, but is not limited thereto.

[0015] <<Equol raw material>> In the method of the present invention, particularly in the fermentation step of 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.

[0016] <<Microorganisms>> The method of the present invention, particularly the fermentation step in the method of the present invention, uses a microorganism that has the ability to assimilate an equol raw material and produce equol. Note that in this specification, "the ability to assimilate an equol raw material and produce equol" may sometimes be simply referred to as "equol-producing ability." The equol-utilizing 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.).

[0017] 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.

[0018] 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:

[0019] [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

[0020] 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

[0021] 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.

[0022] 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.

[0023] Among the above-mentioned microorganisms, any of the microorganisms described below or related microorganisms having similar species properties to these microorganisms can be mentioned as more preferred anaerobic microorganisms. Adlercreutzia equolifaciens subsp. celatus DSM 18785 strain Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450 strain Bacteroides ovatus strain E-23-15 Bifidibacterium breve ATCC 15700 strain Bifidobacterium longum BB536 strain Clostridium sp. HGH136 strain Eggerthella sp. Julong 732 strain Eggerthella sp. YY7918 strain Eggerthella sp. D1 strain Enterococcus faecalis INIA P333 strain Enterococcus faecium strain EPI1 Enterohabdus mucosicola Mt1B8 strain Eubacterium sp. strain D2 Finegoldia magna EPI3 strain Lactobacillus fermentum DPPMA114 strain Lactobacillus intestinalis KTCT13676BP 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 22006 strain Slackia sp. FJK1 strain Slackia sp. NATTS strain Slackia sp. YIT11861 strain Slackia sp. TM-30 strain Streptococcus constellatus strain E-23-17 Streptococcus intermedius A6G-225 strain Veillonella sp. EP strain.

[0024] The above anaerobic microorganisms can be obtained from the depository institutions indicated by their accession numbers. Each accession number indicates that the anaerobic microorganism has been deposited at the following depository institutions: FERM International Patent Organism Depositary (IPOD) http: / / unit.aist.go.jp / pod / ci / index.html DSM German Collection of Microorganisms and Cell Cultures (DSMZ) http: / / www.dsmz.de / KCCM Korean Culture Center of Microorganisms

[0025] In the present invention, anaerobic microorganisms capable of producing equol are cultured under conditions suitable for equol production. In the present invention, the conditions suitable for equol production refer to conditions under which the survival and activity of anaerobic microorganisms capable of producing equol are maintained. More specifically, gas phase conditions (anaerobic conditions) in which anaerobic microorganisms can survive are maintained, and nutrients are provided to support the activity and growth of the anaerobic microorganisms. Various medium compositions suitable for the survival of anaerobic microorganisms are known. Therefore, for the anaerobic microorganisms capable of producing equol shown above, those skilled in the art can select an appropriate medium composition. For example, BHI medium manufactured by Difco and the medium used in the examples can be used, but are not limited to these.

[0026] 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.

[0027] The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted in order to efficiently grow anaerobic microorganisms in the medium.

[0028] A nitrogen source can be added to the medium. In the present invention, various nitrogen compounds that can be used in normal fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are ammonium salts and nitrates. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate. On the other hand, preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder, and the like. More preferred organic nitrogen sources are arginine, cysteine, cystine, citrulline, lysine, yeast extract, and peptones.

[0029] 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.

[0030] 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

[0031] 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.

[0032] 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.

[0033] The medium of the present invention may contain an antifoaming agent, preferably soybean oil, more preferably soybean oil with vitamin E.

[0034] In the method of the present application, the microorganism, particularly the anaerobic microorganism, can be cultured according to a known method for culturing a microorganism. For industrial production, a continuous fermentation system capable of continuously supplying the medium and the substrate gas and having a mechanism for recovering the culture can be used.

[0035] In the method of the present invention, when anaerobic microorganisms are used, it is advisable to prevent oxygen from entering the fermentation tank. A commonly used fermentation tank can be used as is. An anaerobic atmosphere can be created by replacing oxygen that enters the fermentation tank with an inert gas such as nitrogen.

[0036] <<Gas phase in fermentation process>> The method of the present invention, particularly the fermentation step of the method of the present invention, is carried out in a gas phase consisting of one or more gases including hydrogen. The gas constituting the gas phase is not particularly limited as long as it is one or more gases including hydrogen, but it is preferable to have hydrogen and one or more gases other than hydrogen. Examples of gases other than hydrogen include, but are not particularly limited to, carbon dioxide, nitrogen, carbon monoxide, etc. The hydrogen concentration of the gas is preferably 30% or less, more preferably 10% or less, and even more preferably 4% or less.

[0037] <<Fermentation tank, stirring power, sparger hole diameter>> The size of the fermenter is not particularly limited as long as it can hold 100 L of culture medium or more. When the fermentation tank is an aeration agitation tank, the power of the agitator is not particularly limited as long as it is 0.1 kW / kL or more. When the gas is supplied using a sparger, the hole diameter of the sparger is not particularly limited.

[0038] When the fermentation tank is an aeration stirred tank, if the power of the stirrer is less than 0.1 kW / kL, the hole diameter of the sparger supplying the gas is 2 mm or less, preferably 1 mm or less, more preferably 0.5 mm or less.

[0039] When the fermenter is a bubble column without an agitator, the hole diameter of the sparger for supplying the gas is 2 mm or less, preferably 1 mm or less, more preferably 0.5 mm or less. If necessary, a draft tube can be installed.

[0040] Furthermore, 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.

[0041] 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.

[0042] 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.

[0043] The fermentation time can be appropriately set depending on the amount of equol produced, the amount of isoflavones remaining, etc. Examples of the fermentation time include, but are not limited to, 8 to 120 hours, preferably 12 to 72 hours, and particularly preferably 16 to 60 hours.

[0044] 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

[0045] 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 1) 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 preculture solution 1.

[0046] [Table 1]

[0047] (Preculture 2) 15 L of medium with the composition shown in Table 1 and adjusted to pH 6.9 was placed in a 30 L fermenter, heated at 121°C for 15 minutes and sterilized by high pressure steam. Preculture solution 1 was inoculated into this medium, gas was replaced with a hydrogen / nitrogen mixed gas, and cultured at 37°C for 18 hours to prepare preculture solution 2.

[0048] (main culture) Daidzein 1g / L and L-arginine 3g / L were added to the composition shown in Table 1, and 100L of medium adjusted to pH 6.9 was placed in a 200L fermenter and heated at 121°C for 15 minutes for high-pressure steam sterilization. Preculture solution 2 described above was inoculated into this medium, and gas was replaced with a mixed gas with the respective hydrogen / nitrogen ratio. The mixed gas was aerated through a sparger with the respective pore size while stirring at the respective power. Culture was carried out at 37°C for 18 hours, and the equol concentration in the main culture solution was analyzed by HPLC.

[0049] The results are shown in Table 2. From Example 1, it was confirmed that when the gas phase hydrogen concentration was 30% or less, the equol concentration was significantly improved by increasing the stirring power to 0.1 kW / kL or more. It was also confirmed that when no stirring power was applied, the equol concentration was improved by setting the sparger hole diameter to 2 mm or less.

[0050] [Table 2]

[0051] Example 2 (Preculture 1) 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 preculture solution 1.

[0052] (Preculture 2) 15 L of medium with the composition shown in Table 1 and adjusted to pH 6.9 was placed in a 30 L fermenter, heated at 121°C for 15 minutes and sterilized by high pressure steam. Preculture solution 1 was inoculated into this medium, gas was replaced with a hydrogen / nitrogen mixed gas, and cultured at 37°C for 18 hours to prepare preculture solution 2.

[0053] (Preculture 3) 100 L of medium with the composition shown in Table 1 and adjusted to pH 6.9 was placed in a 200 L fermenter, heated at 121°C for 15 minutes and sterilized by high pressure steam. Preculture solution 2 was inoculated into this medium, gas was replaced with a hydrogen / nitrogen mixed gas, and cultured at 37°C for 18 hours to prepare preculture solution 3.

[0054] (Main culture) Daidzein 1g / L and L-arginine 3g / L were added to the composition shown in Table 1, and 2000L of medium adjusted to pH 6.9 was placed in a 4000L fermenter and heated at 121°C for 15 minutes for high-pressure steam sterilization. Preculture solution 3 described above was inoculated into this medium, and gas was replaced with nitrogen mixed gas with a hydrogen concentration of 4%, and stirring was performed at each power while aerating the mixed gas through a sparger with a pore size of 3 mm. Cultivation was carried out at 37°C for 18 hours, and the equol concentration in the main culture solution was analyzed by HPLC.

[0055] The results are shown in Table 3. Example 2 confirmed that even if the size of the fermenter was increased, the equol concentration was significantly improved by setting the stirring power to 0.1 kW / kL or more.

[0056] [Table 3]

[0057] Example 3 (Preculture 1) 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. Eggerthella sp. DC 3215 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 preculture solution 1.

[0058] (Preculture 2) 15 L of medium with the composition shown in Table 1 and adjusted to pH 6.9 was placed in a 30 L fermenter, heated at 121°C for 15 minutes and sterilized by high pressure steam. Preculture solution 1 was inoculated into this medium, gas was replaced with a hydrogen / nitrogen mixed gas, and cultured at 37°C for 36 hours to prepare preculture solution 2.

[0059] (Main culture) Daidzein 0.5g / L and L-arginine 3g / L were added to the composition shown in Table 1, and 100L of medium adjusted to pH 6.9 was placed in a 200L fermenter and heated at 121°C for 15 minutes for high-pressure steam sterilization. Preculture solution 2 described above was inoculated into this medium, and gas was replaced with nitrogen mixed gas with a hydrogen concentration of 4%, and stirring was performed at each power while aerating the mixed gas through a sparger with a pore size of 3 mm. Cultivation was carried out at 37°C for 72 hours, and the equol concentration in the main culture solution was analyzed by HPLC.

[0060] The results are shown in Table 4. From Example 3, it was confirmed that equol was produced by setting the stirring power to 0.1 kW / kL or more.

[0061] [Table 4]

[0062] Example 4 (Preculture 1) The medium, with the composition shown in Table 1 and adjusted to pH 6.5, 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 butyl rubber stoppers and plastic caps attached while replacing the gas phase with nitrogen. Lactococcus sp. DCL 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, after which it was cultured with shaking at 37°C and 200 spm for 24 hours to prepare preculture solution 1.

[0063] (Preculture 2) 15 L of medium with the composition shown in Table 1 and adjusted to pH 6.5 was placed in a 30 L fermenter, heated at 121°C for 15 minutes and sterilized by high pressure steam. Preculture solution 1 was inoculated into this medium, gas was replaced with a hydrogen / nitrogen mixed gas, and cultured at 37°C for 24 hours to prepare preculture solution 2.

[0064] (main culture) 100 L of medium containing 50 g / L powdered soybean hypocotyl, 3 g / L L-arginine, and 2 g / L soybean oil containing vitamin E, adjusted to pH 6.5, was placed in a 200 L fermenter and sterilized by high pressure steam at 121°C for 15 minutes. The above pre-culture solution 2 was inoculated into this medium, and gas was replaced with a nitrogen mixed gas with a hydrogen concentration of 4%, and the mixed gas was aerated through a 3 mm sparger while stirring at each power. Cultivation was carried out at 37°C for 96 hours, and the equol concentration in the main culture solution was analyzed by HPLC.

[0065] The results are shown in Table 5. From Example 4, it was confirmed that equol was produced by setting the stirring power to 0.1 kW / kL or more.

[0066]

Table 5

Claims

[Claim 1] A method for producing equol, comprising the steps of: fermenting at least one equol raw material selected from the group consisting of daidzein glycosides, daidzein, and dihydrodaidzein using a microorganism that assimilates the equol raw material to produce equol in a gas phase consisting of one or more gases including hydrogen; In the fermentation process described above, a fermentation tank with a culture medium volume of 100 L or more is used. i) Stirring is performed in the fermentation tank with a stirring power of 0.1 kW / kL or more, and / or ii) The gas is introduced using a spurger with a pore size of 2 mm or less. The above method characterized by the following features.