JPWO2019189389A1 - Ecole manufacturing method - Google Patents

Abstract

Provided is a method for producing equol capable of efficiently producing equol.
A method for producing equol by culturing an equol-producing bacterium and a Lactobacillus casei YIT 9029 strain in a medium containing daidzein.

Description

The present invention relates to a method for efficiently producing equol using an equol-producing bacterium.

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 ovatus, 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 acidophilus, Lactobacillus lactis, Enterococcus festum, Lactobacillus casei, Lactobacillus salivarius (Patent Document 4), SNU-Julong732 (Non-Patent Document 2), Adrecloycia equolifaciens, Slackia isoflavoni-convertence, Slackia equolifaciens, Slackia spp. TM-30 strain, Eggerthella sp. Y7918 and gram positive bacteria do03 (Non-Patent Document 3) have been reported. In addition, the applicant has identified as a microorganism having a high ability to convert equol from daidzein, which is a bacterium belonging to the genus Slackia, Slackia sp. YIT 11861 (NATTS Co., Ltd.) has been found (Patent Document 5).

On the other hand, L. The YIT 9029 strain, which is a casei-shirota strain (LcS), is widely used in lactic acid bacteria beverages and fermented milk, and has been shown to have various physiological actions.
However, nothing is known about using the YIT 9029 strain in combination with an equol-producing bacterium.

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 method for producing equol capable of efficiently producing equol by microorganisms.

As a result of diligent studies to solve the above problems, the present inventors have found that equol can be efficiently produced by co-culturing equol-producing bacteria and Lactobacillus casei YIT 9029 strain in a medium containing daidzein. The heading, the present invention was completed.

That is, the present invention relates to the following 1) to 10).
1) A method for producing equol, in which an equol-producing bacterium and Lactobacillus casei YIT 9029 strain (FERM BP-1366) are cultivated in a medium containing daidzeins.
2) The method of 1), wherein the daidzeins are one or more selected from daidzein, daidzein glycosides and dihydrodaidzein.
3) The method of 1) or 2), wherein the medium containing daidzeins is a medium containing soymilk as a main component.
4) Any method of 1) to 3), wherein the equol-producing bacterium is a bacterium belonging to the Coriobacterium family.
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 YIT 11861 strain (FERM BP-1366).
7) A method for producing an equol-containing fermentation composition, in which an equol-producing bacterium and Lactobacillus casei YIT 9029 strain (FERM BP-1366) are cultivated in a medium containing daidzeins.
8) The method of 7), wherein the medium containing daidzeins is a medium containing soymilk as a main component.
9) An equol-containing fermentation composition obtained by culturing an equol-producing bacterium and a Lactobacillus casei YIT 9029 strain in a medium containing daidzeins.
10) The equol-containing fermentation composition of 9), wherein the medium containing daidzeins is a medium containing soymilk as a main component.

According to the present invention, equol or a fermentation composition containing equol can be efficiently produced.

The figure which shows the number of bacteria of NATTS strain after culturing. Concentration of product after culturing. The values in the figure are mean ± standard deviation (n = 3). Changes in the number of NATTS strains in co-culture of LcS and NATTS strains. The values in the figure are mean ± standard deviation (n = 3). Changes in equol concentration in co-culture of LcS and NATTS strains. The values in the figure are mean ± standard deviation (n = 3).

In the present invention, the daidzeins mean one or more selected from daidzein, daidzein glycosides and dihydrodaidzein, and the daidzein glycosides specifically include daidzin, malonyldaidzin, acetyldaidzin and the like. Can be mentioned.

In the present invention, equol refers to 7-hydroxy-3- (4'-hydroxyphenyl) -chroman, which is a major metabolite of daidzeins. There are enantiomers (R-equol and S-equol) in the equol, but the equol obtained by the present invention may be a racemate and contains only one of the enantiomers or one of the enantiomers in large quantities. It may be.

In the present invention, the equol-producing bacterium is not particularly limited as long as it is a microorganism that produces equol by assimilating dyzeins (Bifidobacterium, Dizein, dihydrodizein), and is not particularly limited, and is, for example, the genus Slackia, the genus Adrecloytia, and the like. Examples include microorganisms belonging to the genera Eggerthella, Bacteroides, Streptococcus, Lactococcus, Bifidobacterium, Propionibacterium, Lactobacillus, Enterococcus, etc., and more specifically, Slackia isoflavoniconverter. Tens, Slackia equolifaciens, Slackia spp. TM-30 strain, Slackia sp. YIT 11861 strain (also referred to herein as "NATTS strain"), Adrecroitia equolifaciens, Eggerthella sp. Y7918, 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 lactis, Lactobacillus acidophilus, Lactococcus lactis, Enterococcus fesium, Lactobacillus casei, Lactobacillus salivarius (Patent) Document 4) and the like can be mentioned.
Of these, Slackia bacterium belonging to the Coriobacterium family, Adrecroitia bacterium, and Eggerthella bacterium are preferable, and Slackia bacterium is more preferable. Specifically, for example, Slackia isoflavoni conversion, Slackia equolifaciens, Slackia spp. TM-30 strains are mentioned, more preferably Slackia sp. It is a YIT 11861 (FERM BP-11231) strain.

Lactobacillus casei YIT 9029 (FERM BP-1366) strain is L. Casei Shirota strain (also referred to as "LcS" in this specification), January 12, 1981, National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (currently, Incorporated Administrative Agency Product Evaluation Technology Infrastructure) It has been deposited at the National Institute of Advanced Industrial Science and Technology).

The method for producing equol of the present invention is carried out by culturing a mixture of equol-producing bacteria and Lactobacillus casei YIT 9029 strain in a medium containing daidzeins.
The medium containing daidzein can be prepared by adding daidzein or a solution in which daidzein is dispersed or dissolved to the medium. In this case, a solubilizer can be appropriately contained in the medium in order to increase the solubility of daidzeins in water. Further, the medium containing daidzein may be a raw material containing daidzein, for example, a medium containing soymilk as a main component (hereinafter, referred to as "soymilk medium").

The type and composition of the medium are not particularly limited as long as the microorganism of the present invention can be cultured. The medium is usually a liquid medium, but may be a solid medium. In addition, a milk-based medium (for example, milk, goat milk, sheep milk, soy milk and other animal and plant-derived liquid milk, non-fat dry milk, whole milk powder, or milk reduced from powdered milk or concentrated milk is used as it is or with water. It may be a diluted milk medium).
The pH of the medium (at the start of culturing) is usually 5.0 to 8.0, preferably 5.5 to 7.5, and more preferably 6.0 to 7.0.
Further, for example, a water-soluble organic substance can be added to the medium 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, butyric acid, isobutyric acid, propionic acid and Examples thereof include compounds such as organic acids such as acetic acid.
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, cysteine, citrulline, ornithine, lysine, yeast extract, peptones and the like. Be done. In particular, it is preferable to add arginine from the viewpoint of improving the growth of equol-producing bacteria. The arginine may be L-form, D-form, or a mixture thereof, but L-arginine is preferable. 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 arginine content in the medium is 0.4 to 2.5 w / v%, preferably 0.8 to 2.1 w / v%. When a salt of arginine is used, the content of arginine is an amount converted to free arginine.

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, and potassium chloride. Examples thereof include boric acid, 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.

As the soymilk medium, soymilk or soymilk products produced from soymilk (for example, low-fat soymilk (Fuji Oil Co., Ltd.), etc.) are used as they are or diluted as necessary to prepare a basal medium, and vitamin A, as appropriate. Examples include vitamins such as B vitamins, vitamin C, and vitamin E, and salts such as various peptides, yeast extract, tea extract, oleic acid, amino acids (arginine salt, cysteine salt, etc.), calcium, and magnesium. Be done.

The seeding amount of the equol-producing bacterium on the medium is, for example, 0.01 to 50 v / v%, preferably 0.1 to 5.0 v / v%, and more preferably 1.0 v / v%. The seeding amount of Lactobacillus casei YIT9029 is, for example, 0.01 to 5.0 v / v%, preferably 0.01 to 1.0 v / v%, and more preferably 0.1 v / v%. Be done.

The culturing temperature and culturing time may be such that daidzeins can be assimilated, and the culturing temperature is usually 25 to 42 ° C., preferably 30 to 40 ° C., and more preferably 35 to 39 ° C. The culture time is usually 1 to 7 days, preferably 1 to 3 days.
Further, the culture is usually carried out by static culture under anaerobic conditions using nitrogen gas, carbon dioxide gas, hydrogen gas or a mixed gas thereof, but stirring culture may be carried out.

After the culture is completed, the equol can be recovered, for example, by fractionating the cultured culture with an appropriate organic solvent. After that, if necessary, the solvent is removed and the product is purified by a known method to obtain equol.
In addition, for example, when culturing is performed using a soymilk medium or a milk medium containing daizeins, the culture can be used as it is or by mixing it with other food materials usually added to fermented soymilk foods to obtain equol. It can be a fermented composition to be contained.

The above fermentation composition can be provided as foods, pharmaceuticals, cosmetics and the like. For example, it can be a fermented soymilk food containing live bacteria such as beverages, kefir, and yogurt, and its forms include, for example, hard type, soft type, plain type, sweet type, fruit type, drink type, frozen type, and the like. Be done.

If necessary, such a fermentation composition may contain various materials that can be blended into foods, pharmaceuticals, cosmetics, etc., for example, optional components such as various sugars, thickeners, emulsifiers, and various vitamins. it can. Specifically, sugars such as sucrose, palatinose, trehalose, lactose, xylose, and malt sugar, sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, palatinit, reduced syrup, reduced maltose syrup, aspartame, somatin, sucralose, and acesulfam. High sweetness sweeteners such as K and stevia, various thickening (stabilizing) agents such as agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum, gellan gum, carboxymethyl cellulose, soybean polysaccharide, propylene glycol alginate, etc. Emulsifiers such as sugar fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitol fatty acid ester, lecithin, milk fat such as cream, butter, sour cream, acidulant such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, gluconic acid, etc. , Vitamin A, Vitamin B, Vitamin C, Vitamin E and other vitamins, calcium, magnesium, zinc, iron, manganese and other minerals, yogurt, berry, orange, carrageenan, perilla, citrus , Apple-based, mint-based, grape-based, apricot-based, pair, custard cream, peach, melon, banana, tropical, herb-based, tea, coffee-based and other flavors can be blended.

Example 1
(1) Strain used Ecole-producing strain: Slackia sp. YIT 11861 (hereinafter referred to as NATTS strain)
Lactobacillus acidophilus YIT 0198
Lactobacillus casei YIT 0180 ^T
Lactobacillus casei YIT 9029 (LcS)
Lactococcus lactis ss lactis YIT 2008 ^T
Lactococcus lactis ss lactis YIT 2027
Streptococcus thermophilus YIT 2037 ^T
Streptococcus thermophilus YIT 2001
Streptococcus thermophilus YIT 2021

(2) Medium used A modified GAM plate medium (Nissui Pharmaceutical Co., Ltd.) was used for culturing the NATTS strain, and an MRS medium (Becton Dickinson: BD) was used for culturing various lactic acid bacteria.
The soymilk medium had the composition shown in Table 1, and components other than cysteine hydrochloride and arginine hydrochloride were dissolved in purified water and heated at 100 ° C. for 30 minutes to degas. Blowing cooling the _{N 2} gas after the addition of cysteine hydrochloride, after aliquoted 12.6mL in test tubes, 115 ° C., and 20 minutes autoclaving. The medium was prepared by adding 1.4 mL of filter-sterilized 15 w / v% arginine hydrochloride before culturing.

(3) Culturing method For the NATTS strain, 200 μL of a cryopreserved solution (dispersion medium: 20% glycerol solution) of the NATTS strain was inoculated on a modified GAM plate medium (Nissui Pharmaceutical Co., Ltd.) in an anaerobic glove box, and the NATTS strain was inoculated at 37 ° C. for 24 hours. It was cultured. The grown colonies were suspended in 2 mL of a modified GAM liquid medium per plate to prepare an inoculum solution.
For various lactic acid bacteria, a cryopreserved strain (microbank: Iwaki) was inoculated into 4 mL of MRS medium and aerobically cultured at 37 ° C. for 24 hours to prepare a preculture solution.
The co-culture of the NATTS strain and the lactic acid bacterium was inoculated into the soymilk medium so that the NATTS strain was 1.0% and the lactic acid bacterium strain was 0.1%, and anaerobically cultured at 37 ° C. for 72 hours (n = 3 for each combination). (Implemented in). Nitrogen gas was used as the anaerobic gas.

(4) Method for measuring the number of bacteria of NATTS strain 1) Pretreatment of culture solution 800 μL of 1M Tris-HCl (pH 8.0) was added to 200 μL of the culture solution, and 38.3 mg of 1M Tris-HCl (pH 8.0) was added thereto. 150 μL of protease P “Amano” 3SD (Amano Enzyme) dissolved to / mL was added, and the cells were incubated at 45 ° C. for 3 minutes. The pellet was centrifuged at 20,400 × g for 5 minutes at 4 ° C., the supernatant was removed by pipetting, and the pellet was resuspended in 200 μL TE buffer (pH 8.0). 400 μL of RNA project bacterial reagent (QIAGEN) was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes, and then centrifuged at 20,400 × g for 5 minutes to remove the supernatant. The resulting precipitate was stored at −80 ° C. until RNA extraction.

2) Measurement of bacterial counts Previously reported from the above precipitation [1) 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- RNA was extracted according to 1969 (2009)], and the RT-qPCR method [2) Tsuji, H. et al. Isolation and characterization of the equol-producing bacterium Slackia sp. Strain NATTS. Arch Microbiol. 192, 279-87 (2010) )] To measure the number of NATTS strains. The primers used are shown in Table 2. In order to prepare the standard curve, the number of bacteria in the culture solution was adjusted based on the result of DAPI count of the pure culture solution, and the RNA extracted after the enzyme treatment was applied in the same manner as in 1) above was used.

(5) Analysis of isoflavone aglycone and its biotransforms in soymilk medium 0.5 mL of anaerobic soymilk medium 72 hours after culturing was transferred to a screw cap test tube, 2.5 mL of diethyl ether was added, and the mixture was vigorously mixed. The test tube was centrifuged at 1,000 xg for 10 minutes, the ether phase was collected in a new test tube, and then 2.5 mL of diethyl ether was added again to the remaining aqueous phase and mixed vigorously. The test tube was centrifuged at 1,000 × g for 10 minutes, the ether phase was collected in the above test tube, and then dried under a nitrogen stream by a block heater heated to 40 ° C. The dried extract was dissolved in 250 μL of 80 v / v% methanol and then filtered through a 0.45 μm filter. A liquid chromatograph mass spectrometer (LC / MS) was used to quantify daidzein and its derivatives in the filtrate. As a system, an alliance HPLC system (Nippon Waters) equipped with a mass spectrometer ZQ 4000 was used. Sample apply using CadenzaCD-C18 (inner diameter: 3.0 mm, length: 75 mm, particle size 3 μm, intact) for the separation column and 0.1% formic acid / acetonitrile mixed solution (70:30) for the mobile phase. The amount was 10 μL. Waters Emper 2 software (Japan Waters) was used for the analysis. The standard curve was prepared using a standard methanol solution of daidzein, equol, dihydrodaidzein (DHD) and o-desmethylangolencin (o-DMA). The quantification range of each substance was 10 to 1,000 ng / mL.
The amount of isoflavone aglycone contained as a glycoside in the soymilk medium is 0.2 M acetate buffer (pH 5) containing almond-derived β-glucosidase (100 U / mL, SIGMA) in 0.5 mL of uninoculated soymilk medium. 0.5 mL of .0) was added and reacted at 37 ° C. for 16 hours, and the concentration of free isoflavone aglycone was measured using 0.5 mL of the reaction solution in the same manner as described above.

(6) Results 1) By co-culturing the 8 strains of lactic acid bacteria tested with all the strains, the number of NATTS strains after culturing was significantly higher than that of the NATTS strain alone culture (Fig. 1-A, B). Among the strains used for co-culture, L. when combined with casei YIT 9029 strain (LcS), it has reached ¹⁰ 9 cells over the number of bacteria per 1 mL.

2) L. When casei YIT 0180 ^T , Lac lactis s lactis YIT 2008 ^T and Lac lactis s lactis YIT 2027 were co-cultured with the NATTS strain, the concentrations of daidzein and genistein in the culture medium increased (FIGS. 2-A, B). Of the above three strains, L. Ecole production was observed in the combination of casei YIT 0180T and NATTS strain, but no equol production was observed in other strains. Only when LcS and NATTS strains were combined, the daidzein concentration was low and the equol concentration was remarkably high as compared with other lactic acid bacteria. (Fig. 2). Moreover, when the uninoculated medium was treated with β-glucosidase (enzyme treatment), daidzein was produced, and it was confirmed that these were present in the medium as glycosides. From this, it was found that when the Lcs and NATTS strains were combined, the daidzein present in the medium was efficiently converted to equol.

Example 2 Changes in the number of NATTS strains and products over time in co-culture of NATTS strain and LcS (1) Culture method Inoculate soymilk medium so that NATTS strain is 1.0% and LcS is 0.1%. , The culture was carried out in the same manner as in Example 1 (3) (n = 3). A part of the culture solution was sampled 0, 24, 48, and 72 hours after the start of the culture.

(2) The number of NATTS strains and the concentrations of isoflavone aglycone and its metabolites in the culture medium were measured by the same methods as in Examples 1 (4) and (5).

(3) Results 1) In the NATTS strain single culture, a slight increase in the number of bacteria was observed in 24 hours of culture, but it decreased with time after that. On the other hand, in co-culture with LcS, the number of NATTS strains reached the maximum after 24 hours of culture, and remained almost constant until 72 hours thereafter (FIG. 3).

2) With the NATTS strain alone, there was no change in the daidzein concentration, and DHD and equol, which are metabolites of daidzein, were not detected (Fig. 4-A). On the other hand, in the LcS single culture, the production of daidzein was observed in 24 hours of culture, and the steady state was maintained until 72 hours of culture (Fig. 4-B). In the co-culture of the NATTS strain and LcS, the production of equol was observed in 24 hours of culture, and reached a steady state in 48 hours (Fig. 4-C). In addition, while the daidzein concentration was constantly low, the formation of DHD was observed within 24 hours of culturing, suggesting that it was converted to equol via DHD.

Claims (10)

A method for producing equol, in which an equol-producing bacterium and Lactobacillus casei YIT 9029 strain (FERM BP-1366) are cultivated in a medium containing daidzein. The method according to claim 1, wherein the daidzein is one or more selected from daidzein, daidzein glycoside and dihydrodaidzein. The method according to claim 1 or 2, wherein the medium containing daidzeins is a medium containing soymilk as a main component. The method according to any one of claims 1 to 3, wherein the equol-producing bacterium is a bacterium belonging to the Coriobacterium family. 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 YIT 11861 strain (FERM BP-11231). A method for producing an equol-containing fermentation composition, in which an equol-producing bacterium and Lactobacillus casei YIT 9029 strain (FERM BP-1366) are cultivated in a medium containing daidzeins. The method according to claim 7, wherein the medium containing daidzeins is a medium containing soymilk as a main component. An equol-containing fermentation composition obtained by culturing an equol-producing bacterium and Lactobacillus casei YIT 9029 strain (FERM BP-1366) in a medium containing daidzeins. The equol-containing fermentation composition according to claim 9, wherein the medium containing daidzeins is a medium containing soymilk as a main component.