JPWO2019189388A1 - Culturing method of equol-producing bacteria - Google Patents

Abstract

Provided is a culture method for efficiently growing an equol-producing bacterium belonging to the Coriobacterium family.
A method for culturing an equol-producing bacterium belonging to the Coriobacterium family in a medium containing arginine, wherein either or both of the following a) and b) are carried out.
a) Culturing after adjusting the initial pH to weakly acidic b) Mixing and culturing lactic acid bacteria

Description

The present invention relates to a culturing method for efficiently growing equol-producing bacteria.

Isoflavones, which are abundant in soybean foods, are known as functional ingredients that are effective in improving menopausal disorders such as indefinite complaints, preventing osteoporosis, preventing hyperlipidemia and arteriosclerosis, and preventing breast cancer and prostate cancer. ing. Recent studies have revealed that one of the isoflavones, Daidzein, is metabolized by intestinal bacteria in the body to equol, which has stronger estrogenic and antioxidant effects. It is attracting attention as one of the main active ingredients that exerts the above-mentioned action.

The production of daidzein to equol in the body is not uniform in all humans, and there are individual differences in the production ability, and it has been reported that 30 to 50% of humans have equol-producing ability. (Non-Patent Document 1). Therefore, the search for intestinal bacteria capable of producing equol has been energetically carried out, and as microorganisms capable of producing equol, Bacteroides obatas, Streptococcus intermedias, Streptococcus constellatas (Patent Document 1), Lactococcus galvier (Patent Document 2), Bifidobacterium Addresscentis TM-1 strain, Bifidobacterium Breve JCM 1273 (Patent Document 3), Propionibacterium frenderecki, Bifidobacterium. Lactobacillus, Lactobacillus acidophilus, Lactococcus lactis, Enterococcus festium, Lactobacillus casei, and Lactobacillus salivarius (Patent Document 4) have been reported. Furthermore, microorganisms with high equol-producing ability are known to be concentrated in the Coriobacteriaceae family, and are known to be concentrated in the Coriobacteriaceae family, such as Adrecroitia equolifaciens, Slackia isoflavoniconverten, and Slackia equolifacience. , Slackia spp. TM-30 strain, Eggerthella sp. Y7918, SNU-Julong732 (Non-Patent Document 2), and gram positive bacteria do03 (Non-Patent Document 3) have been reported. In addition, the applicant has identified as a microorganism having an extremely high ability to convert equol from daidzein, which is a bacterium belonging to the genus Slackia, Slackia sp. YIT 11861 (FERM BP-11231) has been found (Patent Document 5). Ecole-producing bacteria belonging to the Coriobacterium family, including the strain, are used as new probiotics that help prevent the above diseases by efficiently converting daidzein to ecole in the intestine due to their high activity. There is expected. Furthermore, it can be used for efficient production of equol.

In order to utilize such equol-producing bacteria of the Coriobacterium family for the production of probiotics and equol, it is first necessary to establish a method for obtaining a large amount of the bacterium. Generally, in order to obtain bacterial cells in a high yield, culture using a liquid medium is preferable. However, most of the Ecole-producing bacteria of the Coriobacterium family, especially the genus Slackia, are cultivated on an agar medium because of their low proliferation in a liquid medium. In addition, Ecole-producing bacteria of the Coriobacterium family, including Slackia spp., Take as long as approximately 3 days, sometimes 7 days, to grow, significantly limiting their industrial applicability. ..

International Publication No. 99/7392 International Publication No. 2005/42 Japanese Unexamined Patent Publication No. 2006-204296 Special Table 2006-504409 Japanese Patent No. 5631862

Proc Soc Exp Biol Med, Vol.217, No.3, p335-339 (1998) Appl Environ Microbiol, Vol.71, p214-219 (2005) J Biosci Bioeng, Vol.102, p247-250 (2006)

The present invention relates to providing a culture method for efficiently growing an equol-producing bacterium of the family Coriobacterium.

As a result of diligent studies to solve the above problems, the present inventors have cultivated the equol-producing bacteria of the Coriobacterium family in a medium containing arginine at a weakly acidic initial pH, or by culturing in a mixed culture with lactic acid bacteria. The present invention has been completed by finding that it can be efficiently propagated.

That is, the present invention relates to the following 1) to 7).
1) A method for culturing an equol-producing bacterium belonging to the Coriobacterium family in a medium containing arginine, wherein either or both of the following a) and b) are carried out.
a) Culturing after adjusting the initial pH to weak acidity b) Culturing by mixing lactic acid bacteria 2) The method of 1) in which the arginine content in the medium is 0.4 to 2.5 w / v%.
3) The method of 1) or 2) where the initial pH is pH 5.5-6.5.
4) The method according to any one of 1) to 3), wherein the lactic acid bacterium is one or more selected from Lactobacillus casei, Lactobacillus acidophilus, Lactococcus lactis and Streptococcus thermophilus.
5) The method according to any one of 1) to 4), wherein the equol-producing bacterium is a bacterium belonging to the genus Slackia.
6) Ecole-producing bacteria are Slackia sp. The method according to any one of 1) to 5), which is a strain of YIT 11861 (FERM BP-11231).
7) Lactobacillus casei YIT 9029 strain (FERM BP-1366), Lactobacillus acidophilus YIT 0198 (JCM1028), Lactobacillus lactis YIT 2027 (FERM BP-6224) and Streptococcus thermophilus YIT 200 One or more of the methods 1) to 6) selected from -7538).

According to the present invention, equol-producing bacteria belonging to the family Coriobacterium can be efficiently grown in a liquid medium, and a large amount of viable bacterial solution can be supplied over a long period of time. Therefore, the present invention is useful for the development and research as probiotics of equol-producing bacteria belonging to the family Coriobacterium.

The figure which shows the proliferativeness (effect of arginine) of YIT 11861 strain. The figure which shows the proliferativeness of the YIT 11861 strain (the influence of the initial pH of the arginine-containing medium). The figure which shows the proliferativeness of the YIT 11861 strain (examination of the arginine concentration of the arginine-containing medium). The figure which shows the proliferativeness (comparison of pH conditions in the stirring culture) of the YIT 11861 strain.

In the present invention, as the equol-producing bacterium belonging to the family Coriobacterium (hereinafter referred to as "equol-producing bacterium"), the family of coliobacterium that produces equol by assimilating daidzeins (daidzein glycoside, daidzein, dihydrodzein) The microorganisms belonging to the genus are not particularly limited, and examples thereof include microorganisms belonging to the genus Adrecroitia, the genus Eggerthella, the genus Slackia, and more specifically, Adrecroitia equolifaciens, Eggerthella sp. Y7918, Slackia isoflavoni conversion, Slackia equolifaciens, Slackia spp. TM-30 strain, Slackia sp. YIT 11861 and the like can be mentioned. Of these, bacteria of the genus Slackia are preferable, and more preferably, Slackia sp. YIT 11861 strain (FERM BP-11231).

In the present invention, the arginine contained in the medium may be L-form, D-form, or a mixture thereof, but is preferably L-arginine. Arginine may be in the form of a salt, and examples of the salt include hydrochloride, glutamic acid, citrate and the like, preferably hydrochloride.
The lower limit of the arginine content in the medium is selected from any one of 0.4 w / v%, 0.8 w / v% or 1.2 w / v%, and the upper limit is 10.0 w / v%, 5. Content specified by any combination of the lower and upper limits, selected from any one of 0w / v%, 2.5w / v%, 2.1w / v% or 1.7w / v%. It is 0.4 to 2.5 w / v%, preferably 0.8 to 2.1 w / v%, and more preferably 1.2 to 1.7 w / v%. When a salt of arginine is used, the content of arginine is an amount converted to free arginine.

As the medium used in the method of the present invention, a known medium suitable for the survival of anaerobic microorganisms can be used. The medium can be liquid, semi-solid or solid, but is preferably a liquid medium. For example, a GAM bouillon medium manufactured by Nissui Pharmaceutical Co., Ltd., a modified GAM bouillon medium, a BHI medium manufactured by Difco, or the like can be used.
For example, a water-soluble organic substance can be added to the medium used in the present invention as a carbon source. Water-soluble organic substances include glucose, galactose, fructose, arabinose, xylose, mannose, lambnorth, ribose, sorbose, trehalose, cellobiose, lactose, maltose, sucrose, raffinose, melibiose, meregitos, lactulose, glycogen, erythritol, sorbitol, In addition to sugars such as adonitol, mannose, inositol, lactose, galactooligosaccharide, fructo-oligosaccharide, inulin, and soluble glucose, pyruvate, malic acid, succinic acid, lactic acid, valeric acid, isovaleric acid, isobutyric acid, butyric acid, propionic acid Examples thereof include compounds such as organic acids such as acetic acid.

The concentration of organic matter added to the medium as a carbon source can be appropriately adjusted in order to efficiently grow the equol-producing bacteria in the medium. Generally, the addition amount can be selected from the range of 0.05 to 5 w / v%.

In addition to the above carbon sources, nitrogen sources can be added to the medium. Preferred inorganic nitrogen sources include ammonium salts and nitrates, and examples thereof include ammonium chloride, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, ammonium citrate, potassium nitrate, sodium nitrate and the like. Examples of the organic nitrogen source include amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder and the like, and preferably arginine, citrulline, ornithine, lysine, yeast extract, peptones and the like.

Further, in addition to the carbon source and the nitrogen source, other organic or inorganic substances suitable for culturing the equol-producing bacteria, for example, cofactors such as vitamins and inorganic compounds such as various salts can be added to the medium. Examples of the inorganic compound include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, amber, sodium molybdenate, potassium chloride, and the like. Examples thereof include borate, nickel chloride, sodium tungstate, sodium selenate, and ammonium ferrous sulfate.
Examples of vitamins include biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, vitamin B12, thiooctonic acid, and p-aminobenzoic acid.
It is also possible to add hemin, which is a porphyrin compound.

In the present invention, one aspect of culturing the equol-producing bacterium belonging to the family Coriobacterium is a) adjusting the initial pH to weakly acidic and culturing.
Here, "weakly acidic" means that the lower limit is selected from any one of pH 5.5, 5.7 or 6, and the upper limit is selected from any one of pH 6.5, 6.3 or 6. Refers to the pH condition defined by any combination of the lower limit and the upper limit, and refers to the condition of pH 5.5 to 6.5, more preferably pH 5.5 to 6.
The pH is adjusted by adding an acid such as hydrochloric acid or sulfuric acid.

The seeding amount of the equol-producing bacterium on the medium may be, for example, 0.01 to 50 v / v%, preferably 0.1 to 5 v / v%, and more preferably about 0.5 v / v%.

Culturing is carried out in an anaerobic atmosphere. Examples of the anaerobic gas constituting the anaerobic atmosphere include nitrogen gas alone, a mixed gas of nitrogen gas and carbon dioxide, or a mixed gas of nitrogen gas, carbon dioxide and hydrogen, but nitrogen gas is preferable, and nitrogen gas and carbon dioxide are preferable. It is more preferable to use a mixed gas of and hydrogen. The mixed gas preferably has a hydrogen gas ratio of 0.1 to 80%, more preferably 1 to 10%. For example, a three-kind mixed gas (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) can be used.

The culture temperature is preferably 25 to 42 ° C, more preferably 30 to 40 ° C, and even more preferably 35 to 39 ° C.
The culturing time is preferably 5 to 96 hours, more preferably 10 to 72 hours, and even more preferably 12 to 48 hours.

The culture may be static culture, but it is preferably stirred, and more preferably at a stirring speed of about 50 to 650 rpm.

In the present invention, the culturing of the equol-producing bacterium belonging to the family Coriobacterium includes, in addition to the above-mentioned aspect a), or as another embodiment, b) culturing a mixture of lactic acid bacteria.
Examples of the lactic acid bacteria used here include Lactobacillus casei (L. casei), Lactobacillus acidophilus (L. acidofilus), Lactobacillus plantarum (L. plantarum), Lactobacillus buchineri (L. buchneri), and the like. Lactobacillus gallinarum, L. amylovourus, L. brevis, L. rhamnosus, Lactobacillus kefir, L. kefir Lactobacillus cruvatus, Lactobacillus zeae, Lactobacillus herveticus, L. salivalius, L. gasseri, Lactobacillus fermentum, L. reuteri, Lactobacillus crispatus, Lactobacillus del Brucchi subspecies. Bulgarikas (L. delbruecchii subsp. Bulgaricus), Lactobacillus del Brucchi subspecies. Lactobacillus bacteria such as L. delbruecchii subsp. Delbruecchii, L. jonsoni, Streptococcus thermophilus, Streptococcus thermophilus, etc. Streptococcus thermophilus. Lactococcus lactis subspecies (Lactococcus lactis subspecies), Lactococcus lactis subspecies. Examples of bacteria of the genus Lactococcus such as Cremoris (Lactococcus lactis subsp. Cremoris) include Lactobacillus casei (L. casei), Lactobacillus acidophilus (L. acidofilus), and Lactococcus lactis (Lac. Lactis). , Streptococcus thermophilus is preferred. The Lactobacillus casei is preferably Lactobacillus casei YIT 9018 (FERM BP-665), Lactobacillus casei YIT 9029 (FERM BP-1366), and Lactobacillus casei YIT 10073 (FERM BP-7707). Of these, Lactobacillus casei YIT 9029 (FERM BP-1366) is more preferable. As the Lactococcus lactis, preferably, Lactococcus lactis YIT 2027 (FERM BP-6224) can be mentioned. As the Streptococcus thermophilus, preferably, Streptococcus thermophilus YIT 2001 (FERM BP-7538) can be mentioned. These strains have been deposited at the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (currently the Patent Organism Depositary Center of the National Institute of Technology and Evaluation). As the Lactobacillus acidophilus, preferably, Lactobacillus acidophilus YIT 0198 (JCM1028) can be mentioned. This strain has been deposited with the Japan Collection of Microorganisms (JCM). Such lactic acid bacteria may be used alone or in combination of two or more.

Lactic acid bacteria are pre-cultured in a medium such as MRS medium in advance, and the pre-culture is added to the medium in which the above-mentioned equol-producing bacteria are inoculated and cultured.
The seeding amount of the lactic acid bacterium may be, for example, 0.01 to 50 v / v%, preferably 0.05 to 5 v / v%, and more preferably about 0.5 v / v%.

The culture performed by mixing the equol-producing bacteria and the lactic acid bacteria is performed under anaerobic or slightly aerobic conditions, and the culture temperature is preferably 25 to 42 ° C, more preferably 30 to 40 ° C, and 35 to 39 ° C. Is more preferable.
The culturing time is preferably 5 to 96 hours, more preferably 10 to 72 hours, and even more preferably 12 to 48 hours.
The culture may be static culture, but may be performed at a stirring speed of about 50 to 650 rpm.

(A) Preparation of inoculated bacterial solution In an anaerobic glove box, 1.0 w / v% glucose-added modified GAM (Gifu anaerobic medium) plate medium (Nissui Pharmaceutical Co., Ltd.) was added to Slackia sp. 200 μL of a frozen strain (dispersion medium 20% glycerol solution) of YIT 11861 strain (hereinafter, also referred to as “NATTS strain”) was inoculated and cultured at 37 ° C. for 24 hours. The grown colonies were suspended in 2 mL of 1.0 w / v% glucose-added modified GAM medium (Nissui Pharmaceutical Co., Ltd.) per plate to prepare an inoculated bacterial solution.

_{(B) Evaluation of Proliferativeness OD 600} was measured 0 hours and 24 hours after the start of culturing, and ΔOD ₆₀₀ from 0 hours was obtained and used as an index of proliferation.

(C) Measurement of the number of NATTS strains and lactic acid bacteria 200 μL of the culture solution was ^{mixed with 400 μL of RNAlater TM} (Ambion) and allowed to stand at room temperature for 10 minutes. Then, the supernatant was decanted by centrifugation at 12,000 rpm at 4 ° C. for 5 minutes, and then stored at −80 ° C. until RNA extraction. After thawing the cryopreserved pellets, the standard method [1) Tsuji, H. et al. Isolation and characterization of the equol-producing bacterium Slackia sp. Strain NATTS. Arch Microbiol. 192, 279-87 (2010), 2) Matsuda, K et al. Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules. Appl Environ Microbiol. 75: 1961-1969 (2009)] The bacterial count of the NATTS strain was measured by the method. The primers used are as shown in Table 1.
The number of lactic acid bacteria is determined by using the above RNA as a template. [2) Matsuda, K. et al. Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules. Appl Environ Microbiol. 75, 1961-1969 (2009), 3) Kubota, H. et al. Detection of human intestinal catalase-negative, gram-positive cocci by rRNA-targeted reverse transcription-PCR. Appl Environ Microbiol. 76, 5440- It was measured according to 5451 (2010)]. The primers used are shown in Table 2.

Test Example 1 Effect of addition of arginine (1) Add arginine hydrochloride to the modified GAM medium so that the final concentration is 1.0 w / v% (0.8 w / v% when converted to the amount of free arginine). Then, after autoclaving at 115 ° C. for 15 minutes, anaerobic replacement was performed in advance in an anaerobic glove box (COY LABORATORY PRODUCTS) for 2 days or more to prepare a test medium. As the anaerobic gas, a three-kind mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used.
A 1/200 amount of the bacterial solution prepared according to (A) above was inoculated into 4 mL of the test medium dispensed into a test tube with an aluminum cap, and cultured in an anaerobic glove box at 37 ° C. for 24 hours. At that time, as a control, a modified GAM medium (containing 0.1% arginine) to which arginine hydrochloride was not added was used. The growth of the cells was evaluated according to the method described in (B) above.

(2) Results As a result of adding 1.0 w / v% arginine hydrochloride to the modified GAM medium, the ΔOD ₆₀₀ value increased about 2.6 times as compared with the case where it was not added (Fig. 1).

Test Example 2 Examination of initial pH and arginine concentration (1) Effect of initial pH A modified method in which 1.0 w / v% of arginine hydrochloride (0.8% w / v% when converted to the amount of free arginine) was added. For GAM medium (containing 0.1% arginine) or modified GAM medium without arginine hydrochloride, adjust the initial pH to 5.0, 5.5, 6.0 and 6.5 using hydrochloric acid. After autoclaving at 115 ° C. for 15 minutes, anaerobic replacement was performed in advance in an anaerobic glove box (COY LABORATORY PRODUCTS) for 2 days or more to prepare a test medium. As the anaerobic gas, a three-kind mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used. At that time, as a control, a modified GAM medium having no pH adjusted to which arginine hydrochloride was not added was used.
A 1/200 amount of the bacterial solution prepared according to (A) above was inoculated into 4 mL of the test medium dispensed into a test tube with an aluminum cap, and cultured in an anaerobic glove box at 37 ° C. for 24 hours. The proliferation of the cells was evaluated according to the method described in (B) above.

As a result, at the initial pH of 5.5, 6.0 and 6.5, the ΔOD ₆₀₀ value increased by 1.2, 1.3 and 1.1 times, respectively, as compared with the medium without pH adjustment (pH 6.9). (Fig. 2). Among them, the highest pH of ΔOD ₆₀₀ was shown when the initial pH was 6.0. On the other hand, at pH 5.0, proliferation was significantly suppressed. On the other hand, under the condition of no addition of arginine hydrochloride, there was almost no difference in the _{ΔOD 600 value as compared with the control.}

(2) Examination of arginine concentration The final concentration of arginine hydrochloride is 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 w / v% (when converted to free arginine, it is 0). .4, 0.8, 1.2, 1.7, 2.1 and 2.5 w / v%) were added to the modified GAM medium, and the initial pH was 5.5 or 6.0 with hydrochloric acid. After autoclaving at 115 ° C. for 15 minutes, the test medium was anaerobically replaced in an anaerobic glove box (COY LABORATORY PRODUCTS) for 2 days or more in advance to prepare a test medium. As the anaerobic gas, a three-kind mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used.
A 1/200 amount of the bacterial solution prepared according to (A) above was inoculated into 4 mL of the test medium dispensed into a test tube with an aluminum cap, and cultured in an anaerobic glove box at 37 ° C. for 24 hours. The proliferation of the cells was evaluated according to the method described in (B) above.

As a result, when the arginine hydrochloride concentration was 0.5 w / v% or more, a significant growth promoting effect was observed as compared with the case where no addition was added (Fig. 3), and the conditions of initial pH 5.5 and 6.0 were 2.0%. The addition effect was the highest.

Test Example 3 Effect of stirring 200 mL of modified GAM medium (initial pH 5.5 or 6.0) to which 2.0 w / v% of arginine hydrochloride (1.7 w / v% when converted to the amount of free arginine) was added. (Using 500 ml corben) was autoclaved at 115 ° C. for 15 minutes and then anaerobically replaced in an anaerobic glove box (COY LABORATORY PRODUCTS) for 2 days or more in advance to prepare a test medium. As the anaerobic gas, a three-kind mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used.
The test medium was inoculated with 1/200 of the bacterial solution prepared according to (A) above, and cultured at 37 ° C. for 24 hours in an anaerobic glove box while rotating the stirrer at a stirring speed of 750 rpm.
In addition, a medium having the same composition as described above was used as a control after being statically cultured without rotation. The rotation speed of the stirring culture was set to a speed at which the gas in the gas phase could be diffused throughout the medium and could be stably rotated for 24 hours. The proliferation of the cells was evaluated according to the method described in (B) above.

As a result, improvement in proliferation was observed by performing stirring culture as compared with static culture under both pH conditions (Fig. 4). When the initial pH 5.5 and 6.0 were compared under stirring conditions, the condition of pH 5.5 was higher than that of pH 6.0, and the ΔOD ₆₀₀ value was higher. At pH 5.5, compared with the static culture having the same medium composition. It increased about 1.7 times.

Test Example 4 Mixed culture with lactic acid bacteria (1) Lactic acid bacteria strain ・ L. acidofilus YIT0198, L .; casei YIT9029 (LcS)
・ Lac. lactis ss. lactis YIT2027,
・ St. thermophilus YIT 2001

(2) The inoculated bacterial solution of the NATTS strain was prepared in the same manner as in (A) above. For various lactic acid bacteria, microbank (Iwaki) frozen beads on which the cells were adsorbed were inoculated into 4 mL of MRS medium and aerobically cultured at 37 ° C. for 24 hours. The test medium was 0.5 w / v% glucose / 1.5 w / v% arginine hydrochloride added-modified GAM (pH unadjusted) 10 mL, which was saturated with nitrogen gas and sealed in a test tube with a butyl stopper. The test medium was inoculated with 0.5% of a combination of lactic acid bacteria and NATTS strain or each independently, and anaerobically cultured at 37 ° C. for 24 hours. An MRS medium was added instead of the lactic acid bacterium culture solution to the NATTS single culture, and a modified GAM medium was added instead of the NATTS strain culture solution to the lactic acid bacterium single culture.
After 24 hours of culturing, the number of NATTS strain and each lactic acid bacterium was measured in the culture broth by the method described in (C) above.

(3) Result L. acidofilus YIT0198, Lac. lactis YIT2027, St. When the thermophilus YIT 2001 and LcS were mixed and cultured, the number of NATTS strains 24 hours after the culture was higher than that of the NATTS strain alone culture, and in particular, L. When acidofilus YIT0198 and LcS were mixed and cultured, the levels were significantly higher (Table 2, p <0.05). In particular, the number of bacteria of the NATTS strain when mixed with LcS was the highest, which was almost the same as the number of bacteria when cultured in the optimum medium established so far (Table 3). In addition, the number of LcS bacteria was also significantly higher when co-cultured with the NATTS strain as compared with the single culture (Table 3, p <0.05).

Claims (7)

A method for culturing an equol-producing bacterium belonging to the Coriobacterium family in a medium containing arginine, wherein either or both of the following a) and b) are carried out.
a) Culturing after adjusting the initial pH to weakly acidic b) Mixing and culturing lactic acid bacteria The method according to claim 1, wherein the arginine content in the medium is 0.4 to 2.5 w / v%. The method according to claim 1 or 2, wherein the initial pH is pH 5.5 to 6.5. The method according to any one of claims 1 to 3, wherein the lactic acid bacterium is one or more selected from Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus lactis and Streptococcus thermophilus. The method according to any one of claims 1 to 4, wherein the equol-producing bacterium is a bacterium belonging to the genus Slackia. Ecole-producing bacteria are Slackia sp. The method according to any one of claims 1 to 5, which is a YIT 11861 strain (FERM BP-11231). Lactobacillus casei YIT 9029 strain (FERM BP-1366), Lactobacillus acidophilus YIT 0198 (JCM1028), Lactobacillus lactis YIT 2027 (FERM BP-6224) and Streptococcus thermophilus YIT 2001 The method according to any one of claims 1 to 6, which is one or more selected from).