WO2019189388A1

WO2019189388A1 - Method for culturing equol-producing bacteria

Info

Publication number: WO2019189388A1
Application number: PCT/JP2019/013241
Authority: WO
Inventors: 佐藤　直; 薫森山; 辻　浩和
Original assignee: 株式会社ヤクルト本社
Priority date: 2018-03-27
Filing date: 2019-03-27
Publication date: 2019-10-03
Also published as: JP7433215B2; JPWO2019189388A1

Abstract

Provided is a culturing method for efficiently proliferating equol-producing bacteria belonging to Coriobacteriaceae. The present invention relates to a method for culturing equol -producing bacteria belonging to Coriobacteriaceae in an arginine-containing culture medium, wherein either a) or b) or both are carried out for culturing: a) adjusting the initial pH to a weakly acidic level; and b) mixing a lactic acid bacteria.

Description

Method for culturing equol-producing bacteria

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

Isoflavones, abundant in soy foods, are known as functional ingredients that are effective in improving menopause such as indefinite complaints, preventing osteoporosis, preventing hyperlipidemia and arteriosclerosis, and preventing breast cancer and prostate cancer. ing. Recent research has revealed that one of the isoflavones, Daidzein, is metabolized by the intestinal bacteria in the body into equol, which has a stronger estrogen action and antioxidant action. Therefore, it is attracting attention as one of the main active ingredients that exert the above-mentioned action.

Production of daidzein to equol in the body is not uniformly performed in all humans, but there are individual differences in the production ability, and it has been reported that 30 to 50% of humans have the ability to produce equol. (Non-Patent Document 1). Accordingly, intestinal bacteria having the ability to produce equol have been vigorously searched, and as microorganisms having the ability to produce equol, Bacteroides obatas, Streptococcus intermedias, Streptococcus constellatus (Patent Document 1), Lactococcus garbier (patent document 2), Bifidobacterium addresscentis TM-1 strain, Bifidobacterium breve JCM 1273 (patent document 3), Propionibacterium frownerecki, Bifidobacterium Lactis, Lactobacillus acidophilus, Lactococcus lactis, Enterococcus faecium, Lactobacillus casei, Lactobacillus salivaius (Patent Document 4) have been reported. Furthermore, it is known that microorganisms having high equol-producing ability are concentrated in Coriobacteriaceae, such as Adroleicia equolifaciens, Srackia isoflavonicombense, Srackia aequifiens. , Slackia spp. TM-30 strain, Eggerella sp. Y7918, SNU-Julong 732 (Non-Patent Document 2), and gram positive battery do03 (Non-Patent Document 3) have been reported. In addition, the present applicant, as a microorganism having an extremely high ability to convert equol from daidzein, is a bacterium belonging to the genus Sluckia, Slacia sp. YIT 11861 (FERM BP-11231) is found (Patent Document 5). Ecole-producing bacteria belonging to the Coriobacteriaceae, including this strain, can be used as new probiotics to help prevent the above diseases by efficiently converting daidzein to equol in the intestine due to its high activity. There is expected. Furthermore, it can be used for efficient production of equol.

In order to use such coriobacteria family equol-producing bacteria for the production of probiotics and equol, it is first necessary to establish a method for obtaining a large amount of the cells. In general, in order to obtain microbial cells in high yield, culture using a liquid medium is preferable. However, coriobacteriaceae, in particular, the echol-producing bacteria belonging to the genus Surakchia have a low growth potential in liquid media, and many of them are cultured in agar media. In addition, the cholorobacteriaceae-producing equol-producing bacteria, including the genus Sracchia, take approximately 3 days, sometimes as long as 7 days, to significantly limit their industrial applicability. .

International Publication No. 99/7392 International Publication No. 2005/42 JP 2006-204296 A JP-T-2006-504409 Japanese Patent No. 5631862

The present invention relates to providing a culture method for efficiently growing equol-producing bacteria of the Coriobacteriaceae family.

As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have the ability to produce an equol-producing bacterium belonging to the family Coriobacteriaceae in a medium containing arginine by cultivating the initial pH to be weakly acidic, or by culturing mixed with lactic acid bacteria. The present invention was 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 family Coriobacteriaceae in a medium containing arginine, wherein one or both of the following a) and b) are performed.
a) Adjusting the initial pH to slightly acidic and cultivating b) Mixing and culturing lactic acid bacteria 2) The method of 1), wherein the arginine content in the medium is 0.4 to 2.5 w / v%.
3) The method according to 1) or 2), wherein the initial pH is pH 5.5 to 6.5.
4) The method according to any one of 1) to 3), wherein the lactic acid bacterium is at least one 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 Sracchia.
6) Ecole producing bacterium is Slackia sp. The method according to any one of 1) to 5), which is a YIT 11861 (FERM BP-11231) strain.
7) Lactic acid bacteria are Lactobacillus casei YIT 9029 strain (FERM BP-1366), Lactobacillus acidophilus YIT 0198 (JCM1028), Lactococcus lactis YIT 2027 (FERM BP-6224), and Streptococcus thermophilus YIT 200 YIT 200 -The method of any one of 1) to 6), which is one or more selected from 7538).

According to the present invention, equol-producing bacteria belonging to the Coriobacteriaceae family 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 development and research as probiotics of equol-producing bacteria belonging to the family Coriobacteria.

The figure which shows the growth property (influence of arginine) of YIT 11861 strain. The figure which shows the growth property (influence of the initial pH of an arginine containing medium) of YIT 11861 stock | strain. The figure which shows the growth property (examination of the arginine density | concentration of an arginine containing culture medium) of YIT 11861 strain. The figure which shows the growth property (comparison of pH conditions in stirring culture) of YIT 11861 strain.

In the present invention, an equol-producing bacterium belonging to the Coriobacteriaceae family (hereinafter referred to as “equol-producing bacterium”) is a coriobacteria family that produces equol by assimilating daidzeins (daidzein glycoside, daidzein, dihydrodaidzein). The microorganism is not particularly limited as long as it is a microorganism belonging to the genus No. 3, and examples include microorganisms belonging to the genus Adrecrotia, Egacera, and Thrackia, and more specifically, Adrecrotia equolifaciens, Eggerthella sp. Y7918, Slackia isoflavonicon vertence, Slackia equolifaciens, Slackia spp. TM-30 strain, Slackia sp. YIT 11861 etc. are mentioned. Of these, a bacterium belonging to the genus Slackia is preferable, and more preferably, Slaccia sp. It is YIT 11861 stock (FERM BP-11231).

In the present invention, the arginine contained in the medium may be L-form, D-form, or a mixture thereof, but L-arginine is preferred. Arginine may take the form of a salt. Examples of the salt include hydrochloride, glutamate, citrate and the like, preferably hydrochloride.
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% at the lower limit, and 10.0 w / v% at the upper limit. Content defined by any combination of the lower limit and the upper limit selected from any one of 0 w / v%, 2.5 w / v%, 2.1 w / v%, or 1.7 w / v% 0.4 to 2.5 w / v%, preferably 0.8 to 2.1 w / v%, more preferably 1.2 to 1.7 w / v%. When an arginine salt is used, the arginine content 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 broth medium manufactured by Nissui Pharmaceutical Co., Ltd., a modified GAM broth 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. Examples of water-soluble organic substances include glucose, galactose, fructose, arabinose, xylose, mannose, rhamnose, ribose, sorbose, trehalose, cellobiose, lactose, maltose, sucrose, raffinose, melibiose, melezitose, lactulose, glycogen, erythritol, sorbitol, Adonitol, mannitol, inositol, lactitol, galactooligosaccharide, fructooligosaccharide, inulin, soluble starch and other sugars, as well as pyruvic acid, malic acid, succinic acid, lactic acid, valeric acid, isovaleric acid, isobutyric acid, butyric acid, propionic acid and Examples 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 equol-producing bacteria in the medium. In general, the addition amount can be selected from the range of 0.05 to 5 w / v%.

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

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

In the present invention, the culture of equol-producing bacteria belonging to the Coriobacteriaceae includes, as one aspect, a) culturing by adjusting the initial pH to slightly acidic.
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 a pH condition defined by an arbitrary combination of the lower limit and the upper limit, and refers to a 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 equol-producing bacteria in the above medium may be, for example, 0.01 to 50 v / v%, preferably 0.1 to 5 v / v%, more preferably about 0.5 v / v%.

Culture is performed 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, preferably nitrogen gas, and nitrogen gas and carbon dioxide. It is more preferable to use a mixed gas of hydrogen and hydrogen. The mixed gas preferably has a hydrogen gas content of 0.1 to 80%, more preferably 1 to 10%. For example, three kinds of mixed gases (nitrogen: carbon dioxide gas: hydrogen = 88%: 5%: 7%) can be used.

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

The culture may be stationary culture, but is preferably agitated, more preferably at an agitation speed of about 50 to 650 rpm.

The culture of equol-producing bacteria belonging to the family Coriobacteria in the present invention includes, in addition to the above-described aspect a) or as another aspect, b) lactic acid bacteria mixed and cultured.
Examples of the lactic acid bacteria used herein include Lactobacillus casei (L. casei), Lactobacillus acidophilus (L. acidophilus), Lactobacillus plantarum (L. plantarum), Lactobacillus buchneri (L. buchneri), L. gallinarum, L. amylovorus, L. brevis, L. rhamnosus, L. kefir, L. kefir Lactobacillus cruvatus (L. curvatus), Lactobacillus zeae (L. zeae), Lactobacillus helveticus (L. helveticus), Lact L. salivarius, L. gasseri, Lactobacillus fermentum, L. reuteri, L. cripatus, L. crispatus, Lactobacillus delbrukki Subspecies. Bulgaricus (L. delbrueckii subsp. Bulgaricus), Lactobacillus delbrukii Subspecies. Lactobacillus genus Lactobacillus such as L. delbrueckii subsp. Delbrueckii, Lactobacillus johnsonii, Streptococcus thermophilus, Streptococcus genus Streptococcus Lactococcus lactis subsp. Lactis, Lactococcus lactis subspecies. Examples include bacteria of the genus Lactococcus such as Lactococcus lactis subsp. Cremoris, among which Lactobacillus casei, L. acidophilus, 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), Lactobacillus casei YIT 10003 (FERM BP-7707). Among them, Lactobacillus casei YIT 9029 (FERM BP-1366) is more preferable. Preferred examples of Lactococcus lactis include Lactococcus lactis YIT 2027 (FERM BP-6224). The Streptococcus thermophilus is preferably Streptococcus thermophilus YIT 2001 (FERM BP-7538). These strains are deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (currently the National Institute for Product Evaluation Technology Patent Biological Depositary). As the Lactobacillus acidophilus, Preferably, Lactobacillus acidophilus YIT 0198 (JCM1028) is mentioned. This strain is deposited with the Microbial Materials Development Office (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 inoculated with the above-mentioned equol-producing bacteria and cultured.
The seeding amount of lactic acid bacteria may be, for example, 0.01 to 50 v / v%, preferably 0.05 to 5 v / v%, more preferably about 0.5 v / v%.

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

(A) Preparation of Inoculum Bacteria In an anaerobic glove box, 1.0 W / v% glucose-added modified GAM (Gifanaanaerobic medium) plate medium (Nissui Pharmaceutical) 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 “NATSS strain”) was inoculated and cultured at 37 ° C. for 24 hours. The grown colonies were suspended in 2 mL of modified GAM medium (Nissui Pharmaceutical) with 1.0 w / v% glucose added per plate to prepare an inoculum.

(B) Evaluation of Proliferation OD ₆₀₀ at 0 hours and 24 hours after the start of culture was measured, and ΔOD ₆₀₀ from 0 hours was obtained as an index of proliferation.

(C) NATTS strain / Lactic acid bacteria count The 200 μL culture solution was mixed with 400 μL RNAlater ^™ (Ambion) and allowed to stand at room temperature for 10 minutes. Thereafter, the mixture was centrifuged at 12,000 rpm for 5 minutes at 4 ° C., the supernatant was removed by decantation, and then stored at −80 ° C. until RNA extraction. After thawing the cryopreserved pellet, the conventional 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 number of NATTS strains was measured by the method. The primers used are shown in Table 1.
The number of lactic acid bacteria is determined using the above RNA as a template, and the standard method [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. 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- 5451 (2010)]. Table 2 shows the primers used.

Test Example 1 Effect of Arginine Addition (1) Arginine hydrochloride was added to the modified GAM medium so that the final concentration was 1.0 w / v% (0.8 w / v% when converted to the amount of arginine in the free form). Then, after autoclaving at 115 ° C. for 15 minutes, anaerobic substitution was performed for 2 days or more in advance in an anaerobic glove box (COY LABORATORY PRODUCTS) to prepare a test medium. As the anaerobic gas, a mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used.
The bacterial solution prepared according to the above (A) was inoculated into 4 mL of test medium dispensed in a test tube with an aluminum cap, and cultured at 37 ° C. for 24 hours in an anaerobic glove box. At that time, a modified GAM medium (containing 0.1% arginine) without addition of arginine hydrochloride was used as a control. The proliferation of the microbial 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 by about 2.6 times compared to the case of no addition (FIG. 1).

Test Example 2 Examination of initial pH and arginine concentration (1) Effect of initial pH Modified method in which arginine hydrochloride was added at 1.0 w / v% (0.8% w / v% in terms of free arginine amount) For GAM medium (containing 0.1% arginine) or modified GAM medium not containing arginine hydrochloride, the initial pH was adjusted to 5.0, 5.5, 6.0 and 6.5 using hydrochloric acid. After autoclaving at 115 ° C. for 15 minutes, the test medium was prepared by anaerobic replacement for 2 days or more in advance in an anaerobic glove box (COY LABORATORY PRODUCTS). As the anaerobic gas, a mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used. At that time, a modified GAM medium without pH adjustment without adding arginine hydrochloride was used as a control.
Into 4 mL of test medium dispensed in a test tube with an aluminum cap, 1/200 of the bacterial solution prepared according to the above (A) was inoculated, 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, the ΔOD ₆₀₀ value increased by 1.2, 1.3 and 1.1 times compared to the medium without pH adjustment (pH 6.9) at the initial pH of 5.5, 6.0 and 6.5, respectively. (FIG. 2). In particular, the highest ΔOD ₆₀₀ value was exhibited when the initial pH was 6.0. On the other hand, growth was remarkably suppressed at pH 5.0. On the other hand, under the condition where arginine hydrochloride was not added, there was almost no difference in ΔOD ₆₀₀ value compared to the control.

(2) Examination of Arginine Concentration Arginine hydrochloride has final concentrations of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 w / v% (in terms of free arginine, 0 .4, 0.8, 1.2, 1.7, 2.1, and 2.5 w / v%) to the modified GAM medium, and the initial pH is set to 5.5 or 6.0 with hydrochloric acid. Then, after autoclaving at 115 ° C. for 15 minutes, anaerobic substitution was performed 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 mixed gas of nitrogen, carbon dioxide and hydrogen (nitrogen: carbon dioxide: hydrogen = 88%: 5%: 7%) was used.
Into 4 mL of test medium dispensed in a test tube with an aluminum cap, 1/200 of the bacterial solution prepared according to the above (A) was inoculated, 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, a significant growth-promoting effect was observed when the arginine hydrochloride concentration was 0.5 w / v% or higher compared to the case where no addition was made (FIG. 3), and the initial pH 5.5 and 6.0 conditions were 2.0%. The effect of addition was the highest.

Test Example 3 Effect of Agitation Modified GAM medium (initial pH 5.5 or 6.0) supplemented with 2.0 w / v% arginine hydrochloride (1.7 w / v% in terms of free arginine amount) 200 mL After autoclaving (using 500 ml Kolben) at 115 ° C. for 15 minutes, anaerobic substitution was carried out for 2 days or more in advance in an anaerobic glove box (COY LABORATORY PRODUCTS) to prepare a test medium. As the anaerobic gas, a 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 in an anaerobic glove box at 37 ° C. for 24 hours while rotating the stirrer at a stirring speed of 750 rpm.
In addition, a culture medium having the same composition as that described above and subjected to static culture without rotation was used as a control. The rotational speed of the stirring culture was set to a speed at which the gas phase gas can be diffused throughout the medium and can be stably rotated for 24 hours. The proliferation of the cells was evaluated according to the method described in (B) above.

As a result, in both pH conditions, the growth was improved by performing the agitation culture as compared with the stationary culture (FIG. 4). When the initial pH 5.5 and 6.0 were compared under stirring conditions, the pH 5.5 condition showed a higher ΔOD ₆₀₀ value than the pH 6.0, compared to static culture with the same medium composition at pH 5.5. Increased about 1.7 times.

Test Example 4 Mixed culture with lactic acid bacteria (1) Lactic acid strain acidophilus YIT0198, L. casei YIT9029 (LcS)
・ Lac. lactis ss. lactis YIT2027,
・ St. thermophilus YIT2001

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

(3) Results acidophilus YIT0198, Lac. lactis YIT2027, St. When Thermophilus YIT 2001 and LcS were mixed and cultured, the number of NATTS strains after 24 hours of culture was higher than that of NATTS strain alone, When acidophilus YIT0198 and LcS were mixed and cultured, it was significantly higher (Table 2, p <0.05). In particular, the number of NATTS strains when mixed with LcS was the highest, and was almost the same as the number of bacteria when cultured in an optimal medium established so far (Table 3). In addition, the number of LcS bacteria was also significantly higher when mixed culture with NATTS strain compared to single culture (Table 3, p <0.05).

Claims

A method for culturing equol-producing bacteria belonging to the Coriobacteriaceae in a medium containing arginine, wherein one or both of the following a) and b) are performed.
a) Adjust the initial pH to slightly acidic and culture b) Mix and culture lactic acid bacteria
The method according to claim 1, wherein the content of arginine in the medium is 0.4 to 2.5 w / v%.
The method according to claim 1 or 2, wherein the initial pH is from 5.5 to 6.5.
The method according to any one of claims 1 to 3, wherein the lactic acid bacterium is at least one selected from Lactobacillus casei, Lactobacillus acidophilus, Lactococcus 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 Sracchia.
Ecole producing bacterium is Slackia sp. The method according to any one of claims 1 to 5, wherein the strain is YIT 11861 (FERM BP-11231).
Lactic acid bacteria are Lactobacillus casei YIT 9029 strain (FERM BP-1366), Lactobacillus acidophilus YIT 0198 (JCM1028), Lactococcus lactis YIT 2027 (FERM BP-6224) and Streptococcus thermophilus YITB IBFP38 FIT38F The method according to any one of claims 1 to 6, which is one or more selected from the group consisting of