JP4811760B2 - Enterobacteria and its utilization to improve equol production by utilization of daidzein - Google Patents

Description

  The present invention improves the ability to produce equol by assimilating daidzein-utilizing intestinal bacteria and strain-specific primers having the ability to assimilate daidzein to produce equol, or daidzein from human intestinal flora The present invention relates to intestinal bacteria and their use.

Patent Document 1 (International Publication WO99 / 07392) contains daidzein containing as an essential component Bacteiroides FERM BP-6435, Streptococcus FERM BP-6436 and Streptococcus FERM BP-6437, which are microorganisms that assimilate daidzein to produce equol. Things are listed.

  However, all of these microorganisms are microbial species that are rarely used as probiotics. Probiotics refers to the ability related to useful microorganisms that can improve the bacterial flora in the gastrointestinal tract and bring about beneficial effects on the host, and their ability to promote growth, and are recently attracting attention. So far, only Bifidobacterium and Lactobacillus have been used as probiotics.

  Cardiovascular disease preventive effect of soybean food [Non-patent document 1 = J. Nutr. 128, pp. 1589-1592 (1998)], suggesting the preventive effect of specific cancers such as breast cancer and prostate cancer [Non-Patent Document 2 = Nutr Cancer, 21, 113-131 (1994)]. Isoflavone is a functional component contained in soybean, and daidzein is one of the main components of isoflavone. It has been reported that this daidzein can be assimilated to equol, which has strong antioxidant and estrogenic effects, by the action of enteric bacteria [Non-patent Document 3 = Food Chem. Toxicol. 41, 631-636 (2003)]. However, no method for isolating such specific intestinal bacteria has been reported.

  However, recently, it was reported in a volunteer study that a person who took a soy food containing isoflavone had a significant effect on lowering plasma cholesterol and plasma triglycerides in a person with high equol-producing ability. [Non-Patent Document 4 = Ann. Nutr. Metab, 48, 67-78 (2004)]. In addition, there is a report that there is a correlation between low breast cancer risk and high urinary equol excretion [Non-patent Document 5 = Lancet, 350, 990-994 (1997)], and men's prostate cancer Has been reported to be low in people with high equol production ability [Non-patent Document 6 = Jpn. J. et al. Clin. Oncol. 32, pp. 296-300 (2002)], it is speculated that equol plays an important role in the prevention of heart disease and the prevention of breast and prostate cancer by isoflavones.

  Non-Patent Document 7 [Journal of Food Science Vol. 67, 3104-3113 (2002)] and Non-Patent Document 8 [Journal of Food Science Vol. 68, 623-631 (2002)] use isoflavones using Bifidobacterium. Is described. However, the bacteria disclosed in this document have not been reported on the effect on equol production when added to human feces, and the equol production ability is not mentioned at all. It can be said that there is no improvement effect. There is no mention of the use of the identified microorganisms as probiotics.

This inventor is nonpatent literature 9-11 [Nutr. Res. 22, 705-513 (2002); Nutr. Sci. Vitaminol. 48, 225-229 (2002); Microb. Ecol Health. Dis 16: 18-22 (2004)] reports the effects of equol on animal health and reports that the health has improved by actually increasing the ability to produce equol. However, no human intestinal bacterium has actually been reported that has an effect of improving the ability of assimilating daidzein to equol in the human intestinal flora. Since such enteric bacteria are useful as microorganisms having an effect of improving equol production ability, there is still a need in the art.
International Publication WO99 / 07392 Pamphlet J. et al. Nutr. 128, 1589-1592 (1998) Nutr Cancer, 21, 113-131 (1994) Food Chem. Toxicol. 41, 631-636 (2003) Ann. Nutr. Metab, 48, 67-78 (2004). Lancet, 350, 990-994 (1997) Jpn. J. et al. Clin. Oncol. 32, 296-300 (2002) Journal of Food Science 67, 3104-3113 (2002) Journal of Food Science 68, 623-631 (2002) Nutr. Res. Volume 22, pages 705-513 (2002) J. et al. Nutr. Sci. Vitaminol. 48, 225-229 (2002) Microb. Ecol Health. Dis 16: 18-22 (2004)

  Intestines with the ability to effectively improve the ability to assimilate daidzein and produce equol by utilizing daidzein in the coexistence with gut microbiota No endobacterium has been found so far. Therefore, an object of the present invention is to provide an intestinal bacterium having the ability to assimilate daidzein in the intestine to produce equol or improve the equol production of the entire intestinal flora.

Various reports reveal that equol is very low in production capacity in 40% to 60% of people, even if they consume soy products. For people with low equol-producing ability, increasing equol-producing ability is very important in order to more effectively exert the effect of preventing lifestyle-related diseases that are expected to involve isoflavones.

  Although it is conceivable to increase the equol production capacity of people with very low equol production ability by food, food ingredients that have high equol production capacity for people with very low equol production capacity have been found at present. There is a report that even if a person takes a long-term soybean product, the ability to produce equol does not change [Am. J. et al. Clin Nutr. 80, 692-699 (2003)], it is difficult to increase the ability to produce equol by simply ingesting ordinary food.

In order to change people with low equol-producing ability to people with higher equol-producing ability, Daidzein-utilizing ability to produce equol by assimilating daidzein or its derivatives (eg, glycosides such as daidzin) as a probiotic Intestinal bacteria having an ability to assimilate equol, such as Bifidobacterium, can be considered in which the ability to produce equol in the intestinal flora is enhanced by the action of the ingesting bacterium. A search has been made.

  However, by simply adding it to the faecal flora, daidzein is assimilated by adding daidzein to enteric bacteria and faecal flora that have the ability to assimilate daidzein and improve productivity. No intestinal bacterium has been found so far that improves equol production but does not have daidzein utilization. Accordingly, the present invention improves the productivity of producing equol by assimilating intestinal bacteria having the ability to assimilate daidzein and assimilating daidzein and specific primers and daidzein for detecting this bacterium. It is an object to provide enteric bacteria.

  As a result of investigating daidzein assimilation ability to assimilate daidzein or a derivative thereof and produce equol for many intestinal bacteria, the present inventors have the ability to assimilate daidzein efficiently by using an EG medium. Bacteria could be identified. And this bacterium actually improved the equol production capacity of the gut microbiota. In addition, we were able to design a species-specific primer for this bacterium. Furthermore, the above-described problems have been solved by finding an intestinal bacterium that does not have daidzein assimilation ability but significantly improves the ability to produce equol in the intestinal flora.

  Using this novel identification method, the present inventors have been able to produce an equol-producing genus Slackia (for example, the genus Slackia TM-30; National Institute of Advanced Industrial Science and Technology, December 2, 2017). Bifidobacterium adolescentis capable of producing equol (for example, Bifidobacterium adolescentis TMl strain; National Institute of Advanced Industrial Science and Technology, December 16, 2016) The deposit, accession number FERM P-20325 (reception number FERM AP-20325) and the Bifidobacterium breve JCM 1273 of the preservation strain of the microbial strain storage facility of RIKEN were found.

  The representative genus Slackia and Bifidobacterium adolescentis of the present invention are those isolated from feces of healthy persons, for example, by the method newly disclosed in the present invention. Therefore, this invention also includes this novel bacteria acquisition method.

The present invention has the ability to assimilate daidzein and produce equol, which has the ability to produce equol by assimilating daidzein in human intestinal bacteria (especially the genus Slackia TM-30) and intestinal flora. Intestinal bacteria (Bifidobacterium adolescentis, Bifidobacterium breve) having the ability to effectively improve the ability to produce equol, and compositions comprising this enteric bacterium, and the ability to assimilate daidzein to produce equol Provided is a rapid detection / quantitative measurement method using molecular biological techniques of human enteric bacteria. Also provided is a method for improving the ability to produce equol comprising the step of administering an enteric bacterium or composition of the invention to a subject in need of treatment.
(1) Ability to assimilate daidzein to produce equol, and has the ability to assimilate human enteric bacteria and species-specific primers or daizein from human intestinal flora to produce equol. Intestinal bacteria to improve.
(2) The intestinal bacterium according to item 1, wherein the intestinal bacterium belongs to the genus Slackia.
(3) The intestinal bacterium according to item 1, wherein the intestinal bacterium belongs to the genus Bifidobacterium.
(4) The intestinal bacterium according to item 3, wherein the intestinal bacterium belongs to the species Bifidobacterium adolescentis or Bifidobacterium breve.
(5) The intestinal bacterium according to item 1, wherein the intestinal bacterium is a species-specific primer of the intestinal bacterium and can be selectively detected by a part of the sequence shown in SEQ ID NO: 3.
(6) The enteric bacterium according to item 1, wherein the enteric bacterium has a daidzein assimilation ability of 1.0% or more measured after 24 hours using daidzein as a substrate.
(7) The intestinal bacterium according to item 1, wherein the enteric bacterium has a daidzein assimilation ability of 10% or more measured after 24 hours using daidzein as a substrate.
(8) The enteric bacterium according to item 1, wherein the enteric bacterium has a daidzein assimilation ability of 20% or more measured after 24 hours using daidzein as a substrate.
(9) The enteric bacterium according to item 1, wherein the enteric bacterium has a daidzein assimilation ability of 30% or more measured after 24 hours using daidzein as a substrate.
(10) The intestinal bacterium according to item 1, wherein the intestinal bacterium has an effect of imparting equol productivity to non-equol-producing human flora mice.
(11) The intestinal bacterium according to item 1, wherein the intestinal bacterium has an effect of improving equol productivity more than twice that of an equol-producing human flora mouse.
(12) The intestinal bacterium according to item 7 or 8, wherein the intestinal bacterium is of the genus Slackia.
(13) The above enterobacteria can be obtained at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary, Slacia TM-30 strain, receipt number FERM AP-20729 (reception date December 2, 2005), accession number. Bifidobacterium adoles centis strain TM-1 deposited as FERM P-20325 (receipt number FERM AP-20325) (receipt date: December 16, 2004), or a conserved strain of RIKEN Microbial System Storage Facility The intestinal bacterium according to item 1, which is Bifidobacterium breve JCM 1273.
(14) The enteric bacterium according to item 1, which is a bacterium that can be used as a probiotic.
(15) The intestinal bacterium according to item 1, further having an ability to assimilate starch or glycogen.
(16) A combination of enteric bacteria according to item 1, wherein the enteric bacteria included in the combination include bacteria belonging to the genus Slackia and bacteria belonging to the genus Bifidobacterium.
(17) The combination according to item 16, wherein the combination includes a combination of Slackia TM-30 strain and Bifidobacterium doles centis TM-1 strain.
(18) A composition comprising an enteric bacterium according to any one of items 1 to 14 or a combination according to any one of items 15 to 17 for improving the ability to produce equol.
(19) The composition according to item 18, which is a food or a medicine.
(20) The composition according to item 18, further comprising another active ingredient.
(21) The composition according to item 18, wherein the intestinal bacterium is provided by being dissolved in a liquid.
(22) The composition according to item 18, wherein the intestinal bacterium is provided in a solid form.
(23) The composition according to item 18, wherein the intestinal bacterium is provided by being freeze-dried.
(24) The composition according to item 18, further comprising a component that protects the intestinal bacteria from gastric juice (25) a method for improving the ability to produce equol, according to any one of items 1 to 14 Including the step of administering the enteric bacterium according to any one of claims 15 to 17 or the composition according to any one of items 18 to 24 to a subject in need of treatment. how to.
(26) A method for producing a bacterium for having daidzein assimilation ability for producing daequine to produce equol or for improving the equol production ability, wherein a sample expected to contain the bacterium is obtained by using an EG medium. A method comprising the step of culturing using
(27) A method for producing a human enteric bacterium having an ability to assimilate daidzein to produce equol, which enhances the ability to produce equol, or human enteric bacteria,
A) providing a sample containing human intestinal derived material or feces;
B) A step of culturing the sample at 37 ° C. using EG medium;
C) Brain Heart Infusion 37g, Agar 1g, L-Cysteine Hydrochloric Acid 0.5g, Sodium Carbonate 4g dissolved in 1 liter of distilled water with heating, and dispensed into a test tube while blowing oxygen-free carbon dioxide gas, and butyl rubber The sample was suspended and suspended in 0.2 ml of a reaction solution prepared by autoclaving sterilization so that the bacterial concentration in the sample was about 10 ⁹ / ml, and 1 μl of daidzein (20 mg / ml) was added. And a step of incubation at 37 ° C. for 24 hours in a simple anaerobic culture apparatus Aneropack;
D) a step of analyzing isoflavones in the reaction solution; and E) a method comprising selecting and producing enterobacteria having the ability to assimilate daidzein or its derivatives to produce equol.
(28) The method according to item 27, wherein the sample is derived from feces of a healthy person.
(29) A bacterium prepared by the method according to items 27 to 29.
(30) A method for producing a pharmaceutical comprising equol or improving equol productivity, wherein the intestinal bacterium according to any one of items 1 to 14 or any one of items 15 to 17 A method comprising: mixing the described combination with daidzein; and incubating the mixed mixture under conditions where the enteric bacteria assimilate.
(31) A step of mixing the enteric bacterium according to any one of items 1 to 14 or the combination according to any one of items 15 to 17 and daidzein, and the mixed mixture into the intestine A composition produced by a method comprising incubating under conditions in which bacteria are assimilated.
(32) A bacterium obtained by the method according to item 30.
(33) The intestinal bacterium according to any one of items 1 to 14, the bacterium obtained by the method according to item 30, or the any one of items 15 to 17 for producing a medicament containing equol Use of the combinations described.
(34) The intestinal bacterium according to any one of items 1 to 14, the bacterium obtained by the method according to item 30, or the items 15 to 17 for producing a medicament for improving equol production ability Use of a combination according to any one of the items.

  Accordingly, it will be appreciated that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description.

  The present invention provides for the first time a method for identifying daidzein-assimilating bacteria that assimilate daidzein and produces equol, thereby assimilating such bacteria and intestinal flora daidzein to produce equol. For the first time, it has become possible to identify and produce bacteria with improved ability. The Sackia spp. Or Bifidobacterium spp. That make up the equol by assimilating daidzein that could be identified using the present invention can significantly improve the equol-producing ability when added to the faecal flora. confirmed. The Bifidobacterium of the present invention can improve daidzein assimilating ability to assimilate daidzein in the gut microbiota and produce equol in a short period of time by orally ingesting or separately with the isoflavone-containing food. . In addition, by using the strain-specific primer of the genus Slackia of the present study that assimilate daidzein to produce equol, it enables high-speed detection of enteric bacteria having the ability to produce equol by molecular biological techniques. This will provide an epoch-making method for the detection of productive intestinal bacteria.

  Accordingly, the present invention provides enterobacteria useful as probiotics or health foods, medicines and the like. The present invention is effective in preventive medicine for female breast cancer, male prostate cancer and the like.

  The present invention will be described below. Throughout this specification, singular articles (eg, “a”, “an”, “the”, etc. in English, corresponding articles in other languages, adjectives, etc.) It should be understood that it also includes the plural concept. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  In the present specification, daidzein is a general name of 4 ′, 7-dihydroxy-3-didehydroisoflavan-4-one (4 ′, 7-dihydroxy-2,3-didehydroflavan-4-one). Represented by the following structure

Is an isoflavone having the structure

As used herein, a derivative of daidzein refers to a compound that can be converted to daidzein by hydrolysis or the like. As such derivatives, for example, glycosides can be mentioned, and typical examples include daidzin, malonyl daidzin, acetyl daidzin, etc. In the present specification, daidzin is 7- (Β-D-glucopyranosyloxy) -4′-hydroxy-2,3-didehydroisoflavan-4-one (7- (β
-D-glucopyrosyloxy) -4'-hydroxy-2,3-dehydrohydroflavan-4-One), represented by the following structure:

Have

  In this specification, equol is represented by the general name 7-hydroxy-3- (4-hydroxyphenyl) chroman and has the following structure:

Is a compound having the structure Ecole is known as an isoflavone with strong antioxidant and estrogenic effects.

  As used herein, “daidzein assimilation ability to assimilate daidzein to produce equol” refers to a cell that produces equol when exposed to daidzein or a derivative thereof (eg, a glycoside such as daidzin). Says ability.

  In the present specification, the daidzein assimilation ability of such bacteria is determined by measuring the equol concentration as follows, and the value is also referred to as daidzein assimilation ability value.

<Measurement method of daidzein utilization in vitro>
Sackia spp. Are cultured for 48 hours at 37 ° C. using EG medium. In 0.2 ml of the reaction solution, the bacteria of the genus Slackia are suspended so that the bacterial concentration is about 10 ⁹ / ml, 1 μl of daidzein (20 mg / ml) is added, and then a simple anaerobic culture apparatus Aneropack is used at 37 ° C., 24 Time incubation was performed.

  The EG medium used in the present specification is prepared as follows.

Composition: Horse meat leachate 930mL
Proteose pettone No 3 (Difco) 10g
Yeast extract (Difco) 5g
Na ₂ HPO ₄ 4g
Glucose 1.5g
L-cystine (preliminarily dissolved in a small amount of 0.1N HCl) 0.2 g
Antifoaming agent (Torre Silicon 10% solution) 5mL
L-cysteine / HCL / H2O 0.5 g
Horse blood 50mL
pH 7.6-7.8.

Production method: L-Cysteine · HCl · _{H 2} O and heating the components other than equine blood were dissolved, correct the pH, 115 ° C., cooled to 50 ° C. After 20 minutes sterilization, L- cysteine · HCl · _H 2 O Add horse blood and dispense into a petri dish to make a flat plate. The horse leachate uses 500 g of horse leachate per 1000 ml of medium.
The reactivity used in the daidzein assimilation method as used herein is prepared as follows: Brain Heart Infusion 37g, Agar 1g, L-cysteine-hydrochloric acid 0.5g, sodium carbonate 4g. Dissolve by heating in 1 liter of distilled water, dispense into an test tube while blowing oxygen-free carbon dioxide, plug a butyl rubber plug, and perform autoclave sterilization.

After completion of the reaction in the measurement test of the ability to assimilate daidzein and produce equol in the present specification, 4 times the amount of methanol and acetic acid mixed solution (methanol: acetic acid = 100:
Add 5) and vortex vigorously for 60 seconds. Subsequently, the supernatant obtained by centrifugation is filtered through a filter, and the filtrate is analyzed by HPLC equipped with an electrochemical detector to analyze isoflavones.

  The human intestinal bacterium of the present invention belongs to the genus Slackia or the genus Bifidobacterium. The genus Slackia is a kind of indigenous bacteria in the human intestine, and was previously classified into the genus Eubacterium. Bifidobacterium belonging to the genus Bifidobacterium is a genus of bacteria belonging to the family Actinomycesaceae and refers to a branched, bifurcated rod-like or spatula-like gonococcus. Bacteria belonging to the genus Bifidobacterium are major constituents of the intestinal flora of infants nurtured with breast milk, and lactic acid and acetic acid produced by degrading sugar in the digestive tract are considered to be useful for digestion.

  One of the human intestinal bacteria of the present invention was considered to be Bifidobacterium from the colony form of the BL medium. As used herein, “Bifidobacterium adolescentis” is a bacterial species belonging to the genus Bifidobacterium, and the properties used here are the sugar fermentation test method (isolation of the world's most anaerobic bacteria of enterobacteria). And identification: according to the method described in Sobunsha, 1984). On the other hand, the genus Slackia was not able to find clues for bacterial identification from colony morphology and microscopic observation. Therefore, Rapid ID32 A API (manufactured by Biomeryu Co., Ltd.) and Shinoda et al. Identification was performed by adopting a phylogenetic classification method by genetic analysis [Non-patent Document 12 = Shimadzu review, 57, 121-132 (2000)]. Specifically, it is as follows.

PYF medium (Peptone-Yeast Extract-Field solution broth; Tripty case (BBL) 10 g; Yeast extract (Difco) 5. Og; Fields solution (digested blood) 40 ml; Salt solution 40 ml; L-cysteine · HCl · H ₂ O 0.5 g; purified water 920 ml, adjusted to pH 7.2) and various test sugars (eg, L-arabinose; D-xylose; D-glucose; D-mannose; D 1-fructose; D-galactose; maltose; sucrose; lactose; trehalose; D-sorbitol; D-mannitol; inositol; glycerin; starch, etc.), sterilized, and used as a medium for testing sugar utilization properties. The strain was treated with BLB medium (B iggs Liver Broth; Tomato Juice Solution (Tomato Juice (Del Monte) dissolved in an equal volume of water, pH adjusted to 7.0 with NaOH and filtered through filter paper) 400.0 ml; Neopeptone (Difco) 15.Og Yeast extract (Difco) 6.0 g; Liver extract (Liver extract) (10 g liver powder in 170 ml water, maintained at 50 ° C. to 60 ° C. for 1 hour, boiled for 5 minutes, pH 7) 75.0 ml; glucose 20.0 g; soluble starch 0.5 g; NaC1 5.0 g; Tween 80 1.0 g; L-cysteine · HCl · H ₂ O 0.2 g; distilled water 525.0 ml, pH adjusted to 6.8) at 37 ° C. for 48 hours, and then inoculated one platinum loop into this BLB medium and again at 37 ° C. for 24 hours. Incubate for a period of time, inject 0.3 ml of this into BLB medium dispensed 9 ml each into a centrifuge tube, and again culture at 37 ° C. for 24 hours. Subsequently, the cells are centrifuged, the supernatant is discarded, and the concentration is twice that of the original culture with sterile saline containing 0.1% L-cysteine / HCl / H ₂ O and 0.1% sodium thioglycolate. Use the resuspended material as the inoculum. This bacterial solution is added to the above-described medium for examining sugar utilization properties, cultured for one week in an anaerobic culture apparatus, and the utilization of sugar is determined from the change in pH of the medium.

Here, in particular, the intestinal bacterium of the genus Bifidobacterium of the present invention preferably has the following conditions:
A) Growth under aerobic conditions:-
B) Catalase:-
C) Gelatin liquefaction:-
D) Indole production: −
E) Nitric acid reducing ability:-
In addition, preferred enteric bacteria of the present invention have the following mycological properties.

Grows well at the following temperatures: 37 ° C-43 ° C
Grows well at the following pH: 7.2-7.5
Anaerobic / Aerobic classification: Anaerobic Gram positive / negative: Gram positive bacteria urease −
The presence or absence of acid and gas generation from sugars Esculin +
Glycerin −
D-xylose +
D-Mannose +
Mannitol +.

In another preferred embodiment, the enterobacteria isolated by the method of the present invention produce acids and gases as shown below for the following sugars:
A) L-one arabinose (+)
B) Erythritol (-)
C) Cellobiose (+)
D) D-Raffinose (+)
E) D-fructose (+)
F) Galactose (+)
As a physiological property.
Such properties may be possessed by, but are not limited to, Bifidobacterium adolescentis species.
The bifidobacteria of the present invention has an ability to improve daidzein assimilation ability of human intestinal microflora usually by at least 38% or more. Ability to improve at least 500% or more. A conventional bacterium that improves daidzein assimilation ability by 500% or more has not been found, and can be said to be a bacterium that could be identified for the first time by the method of the present invention.
In the present specification, Bifidobacterium that contributes to the improvement of the ability to produce equol by assimilating daidzein of the gut microbiota according to the present invention is a number of Bifidobacterium strains isolated from feces of infants, adults, etc. Separated from the inside. A representative example of this strain is an independent administrative corporation National Institute of Advanced Industrial Science and Technology patent biological deposit center (〒 305-8565 Tsukuba City Ibaraki Pref. 1-1-1 Chuo No. 6), receipt number FERM AP-20325 (reception date 2004) December 16th; Deposit number FERM P-20325).

  Identification of the isolated Bifidobacterium strain was carried out in accordance with the sugar fermentation test method of Mitsuoka et al. (World of Enterobacteria-Isolation and Identification of Anaerobic Bacteria).

  In the most preferred embodiment, the Bifidobacterium adolescentis TM-1 strain of the present invention has the following bacteriological properties and has therefore been identified as a strain belonging to Bifidobacterium adolescentis: Gram positive spore-free bacilli. Microscopic observation also shows that it was branched by a rod-shaped rod. Growth under aerobic conditions (-), catalase (-), gelatin liquefaction (-), hydrogen sulfide production (-), indole production (-), nitrate reduction (-). On the BL agar plate, it is much larger than the settlement on the EG agar plate, and some of the cells are swollen, curved, thin at one end, and branched. Growth on BS agar is good.

  In one embodiment, the Bifidobac terium adolescentis TM-1 strain of the present invention typically has sugar fermentability as follows.

L-arabinose (+), D-xylose (+), D-fructose (+), galactose (+), maltose (+), cellobiose (+), lactose (+), D-raffinose (+), glucose ( +), Erythritol (-), melibiose (+), trehalose (-), mannitol (+), sorbitol (-), mannose (+), rhamnose (-).

  Identification of the isolated genus Slackia was carried out according to Rapid ID32 A API (manufactured by Nihon Biomelieu Co., Ltd.) using an automatic bacteria identification test apparatus and a bacterial strain classification method by 16S rRNA gene analysis. Moreover, the glucose fermentability test and the bile tolerance test were also examined.

  The other Slackia genus of the human intestinal bacterium of the present invention is a Gram-positive rod and is a so-called anaerobic bacterium that does not grow under aerobic conditions. In EG and BL media, milky white microcolonies are formed. The colony shape rises in a dome shape, and in the microscopic observation of the present bacterium grown on the BL medium, short gonococcus to long gonococcus are mixed, and show polymorphism often observed in so-called bifidobacteria and the like. Glucose is fermented slightly and does not grow in the presence of 10% and 20% bile acids.

  The Slackia sp. TM-30 strain of the present invention has the following biochemical properties (+: positive, −: negative) urease (−), arginine dihydrase (+), α-galactosidase (−), β -Galactosidase (-), β-galactosidase-6-phosphate (-), α-glucosidase (-), β-glucosidase (-), α-arabinosidase (-), β-glucuronidase (-), β- N-acetylglucosaminidase (−), D (+) mannose fermentation (−), raffinose fermentation (−), nitrate reduction (−), indole production (−), alkaline phosphatase (−), arginine allylamidase (+), Proline allylamidase (-), leucylglycine allylamidase (-), phenylalanine allylamidase (-), leucine Sine allylamidase (-), pyroglutamate allylamidase (-), tyrosine allylamidase (-), alanine allylamidase (-), glycine allylamidase (-), glutamate decarboxylase (+), alpha-fucosidase (-), Histidine allylamidase (-), glutamyl glutamate allylamidase (-), serine allylamidase (-).

  In the phylogenetic classification method by 16S rRNA gene analysis of Shinoda et al. Used to identify the genus Slackia, the bacterial genome was extracted with an instagene matrix (Nippon Bio-Rad) and 27f (5'-AGAGTTGATCCTGCTCCAG-3 '( PCR was performed with a universal primer combination of SEQ ID NO: 1)) and 1492r (5′-GGCTACTCTGTTACGACTT-3 ′ (SEQ ID NO: 2), and 16SrDNA was amplified, and 16SrDNA was purified with a Qiagen spin column. PCR using the sequence primer and BigDyeTerminatorVer1.1, gene analysis with ABI310 (Applied Biosystems), and partial base sequence of 16S rDNA of the genus Slackia It was boss.

In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used. In the present specification, the primer can be used as a marker detection means.

  Examples of nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides. At least 13 contiguous nucleotides, at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 At least 19 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 Nucleotides long that connection, at least 50 contiguous nucleotides in length, may be a nucleic acid sequence. Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).

  As used herein, the term “probe” refers to a substance that serves as a search means used in biological experiments such as screening in vitro and / or in vivo. For example, a nucleic acid molecule containing a specific base sequence or a specific nucleic acid molecule Examples include, but are not limited to, peptides containing amino acid sequences. In this specification, the probe is used as a marker detection means.

  Examples of nucleic acid molecules that are usually used as probes include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is homologous or complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides. At least 13 contiguous nucleotide lengths, at least 14 contiguous nucleotide lengths, at least 15 contiguous nucleotide lengths, at least 20 contiguous nucleotide lengths, at least 25 contiguous nucleotide lengths, at least 30 It can be at least a nucleic acid sequence of at least 40 contiguous nucleotides, at least 50 contiguous nucleotides long, of contiguous nucleotides. Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included.

  As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)).

  In the present specification, the “nucleotide” may be natural or non-natural. “Nucleotide derivative” or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-type nucleic acids (PNA). Not.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like. As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction.

  As used herein, “homology” of genes refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.

  In the present specification, comparison of similarity, homology and identity of amino acid and base sequences is calculated using BLAST, which is a tool for sequence analysis, using default parameters. The identity search can be performed using, for example, NCBI BLAST 2.2.9 (issued 2004.5.12). In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  As used herein, “polynucleotide that hybridizes under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A PR Applical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. Thus, any factor in a polypeptide used in the present invention is encoded by a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule encoding a polypeptide specifically described in the present invention. Polypeptides are also included.

  As used herein, “detection” or “quantification” of polypeptide expression can be accomplished using any suitable method, including, for example, measuring mRNA and immunological methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method. Examples of the quantitative method include an ELISA method and an RIA method.

  The partial base sequence (1402 bp) of 16S rDNA of the Slackia genus bacteria TM-30 strain of the present invention is shown.

Partial base sequence (1402 bp) of 16S rDNA of the genus Slackia TM-30
CACATGCAAGTCGAACGAGTAAGACGCCTTCGGGCGTGGATAGAGTGGCGAACGGGTGAGTAACACGTGACCAACCTGCCCCCTCCTCCGGGACAACCTCGGGAAACCGAGGCTAATACCGGATACTCCGGGCCCCCCGCATGGGGAGCCCGGGAAAGCCCTGGCGGGAGGGGATGGGGTCGCGGCCCATCAGGTAGACGGCGGGGTAACGGCCCACCGTGCCTACTACGGGTAGCCGGGCTGAGAGGCCGATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGCGCAATGGGGGCAACCCT ACGCAGCGACGCCGCGTGCGGGACGAAGTCATTCGTGACGTAAACCGCTTTCAGCGAGGAAGAACCATGACGGTACTCGCAGAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTATCCGGAATCATTGGGCGTAAAGCGCGCGCAGGCGGGCTTTCAAGCGGCGGCGTCGAAGCCGGGGGCTCAACCCCCGGAAGCGCCCCGAACTGGAAGCCTCGGATGCGGCAGGGGGAGGCGGAATTCCCGGTGTAGCGGTGAAATGCGCAGATATCGGGAAGAACACCGACGGCGAAGGCAGCCTCCTGGGC GGCATCGACGCTGAGGCGCGAAAGCTGGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCCAGCCGTAAACGATGGACGCTAGGTGTGGGGGGAACATCCCTCCGTGCCGAAGCCAACGCATTAAGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGAAGCAACGCGAAGAACCTTACCAGGGCTTGACATACGGGTGAAGCCGGGAGAGATCCGGTGGCCGAGAGGAGCCCGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGT GTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCGCCGCGTGTTGCCAGCATTCAGTTGGGCACTCACGCGGGACTGCCGGCGTCAAGCCGGAGGAAGGCGGGGACGACGTCAAGTCATCATGCCCCTCATGCCCTGGGCCGCACACGTGCTACAATGGCCGGCACAACGGGTTGCCACCCCGCGAGGGGGAGCGGATCCCAAAAGCCGGCCCCAGTTCGGATCGCAGGCTGCAACCCGCCTGCGTGAAGCCGGAGTTGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACCCGA TCGTCTGCACCCGAAGCCGCCGGCCGAACCCCGAAAGGGGGCGGAGGCGTCGAAGGTGTGGAGGGTGA (SEQ ID NO: 3).

  As a result of searching for a highly homologous bacterium using blastn in the DDBJ nucleic acid database for the above base sequence, 1269 out of 1365 16S rDNA genes of Slackia exigua (Accession no: AF101240) matched the strain of the present invention. (92%). Therefore, this bacterium is considered to belong to the genus Slackia. The Slackia genus strain TM-30 has the following in vitro daidzein assimilation ability.

  Here, when the ratio of the equol concentration after 24 hours of reaction to the concentration of daidzein decreased after the reaction from before the reaction is calculated as the daidzein assimilation ability, one representative strain of the present invention has a daidzein utilization of 1.6% or more. It can be said that it has the ability.

  In this specification, “daidzein assimilation ability” refers to the addition of 1 μl daidzein (20 mg / ml), followed by incubation at 37 ° C. for 24 hours in an anaerobic culture apparatus Aneropack. Add 4 times the amount of methanol / acetic acid mixture (methanol: acetic acid = 100: 5), vigorously vortex for 60 seconds, centrifuge the supernatant obtained by filtration and filter the filtrate electrochemically Analyzes of isoflavones by analysis with HPLC equipped with a mechanical detector (available from JASCO, etc.) to confirm the presence or absence of equol productivity and that equol is produced (ie, daidzein is assimilated) It can be confirmed by confirming.

  As used herein, “human flora mouse” refers to a mouse in which human flora is transplanted into the intestine. Used as an experimental model for human flora.

  In the present specification, “the effect of imparting equol productivity to non-equol-producing human flora mice” refers to the ability to confer the ability to produce equol when administered to human flora mice that do not normally produce equol. Specifically, it is exemplified in Example 3 herein.

  In the present specification, the “effect of improving equol productivity relative to equol-producing human flora mice” refers to the ability of the equol-producing brain to be enhanced when administered to human flora mice that normally produce equol. Specifically, this is exemplified in Example 4 of the present specification.

(Useful composition)
In another aspect, the present invention provides a composition comprising the intestinal bacterium of the present invention for improving the ability to produce equol. Such a composition can be used as food, feed, health food, specified medical insurance food, quasi-drug, pharmaceutical product, and the like. In particular, since the present invention is a human enteric bacterium, it can be used as a human probiotic.
The composition according to the invention can preferably contain further active ingredients. Examples of such active ingredients include, but are not limited to, bifidobacteria growth promoting substances such as soybean oligosaccharides, fructooligosaccharides, and galactooligosaccharides.

  The general preparation method of the pharmaceutical composition of this invention is shown below. In addition, veterinary drug compositions, quasi drugs, marine drug compositions, food compositions, cosmetic compositions and the like can also be produced by known preparation methods.

  The intestinal bacterium or composition of the present invention is mixed with a pharmaceutically acceptable carrier, and is a solid preparation such as a tablet, capsule, granule, powder, powder, suppository, or syrup, injection, suspension. It can be administered orally or parenterally as a liquid preparation such as an agent, solution or spray. As described above, the pharmaceutically acceptable carrier includes an excipient, a lubricant, a binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, an adsorbent, a humectant, a solubilizing agent in a solid preparation, Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like can be mentioned. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than the polynucleotide of this invention, polypeptide, etc. with the composition of this invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal. When intestinal bacteria are administered, it is preferably in a form that can be delivered to the intestine.

  Preferably, when formulating, the lyophilized powder of the intestinal bacterium of the present invention is mixed with excipients such as starch and hydroxypropylcellulose as necessary.

  Examples of the excipient in the solid preparation include glucose, lactose, sucrose, D-mannitol, crystalline cellulose, starch, calcium carbonate, light anhydrous silicic acid, sodium chloride, kaolin and urea.

  Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, boric acid powder, colloidal silicic acid, talc, and polyethylene glycol.

  Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mannitol, crystalline cellulose, dextrin, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, starch solution, gelatin solution, polyvinylpyrrolidone, Examples include calcium phosphate, potassium phosphate, and shellac.

  Examples of the disintegrant in the solid preparation include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan Examples include, but are not limited to, fatty acid esters, sodium lauryl sulfate, starch, stearic acid monoglyceride, lactose, and calcium calcium glycolate.

  Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter, and hardened oil.

  Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

  Examples of the adsorbent in the solid preparation include, but are not limited to, starch, lactose, kaolin, pentonite, and colloidal silicic acid.

  Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

  Examples of the solubilizer in the solid preparation include, but are not limited to, arginine, glutamic acid, aspartic acid and the like.

  Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin and lactose.

  When preparing tablets, pills and the like as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Tablets include tablets with a conventional coating as necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When forming into the form of a suppository, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides and the like can be added in addition to the additives listed above, but are not limited thereto.

  Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. It is not limited to.

Preferable examples of the suspending agent in the liquid preparation include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, pentethonium chloride, and glyceryl monostearate, such as polyvinyl alcohol, Examples include, but are not limited to, hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.
Preferred examples of isotonic agents in liquid preparations include sodium chloride, glycerin, D-
Although mannitol etc. are mentioned, it is not limited to them.
Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate and citrate.
Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferred examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoates, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferred examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When preparing as an injection, it is preferable that the solution and suspension are sterilized and isotonic with blood. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericidal agent, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

Furthermore, if necessary, the pharmaceutical composition comprises a colorant, a preservative, a fragrance, a flavoring agent, a sweetener, etc.
As well as other drugs.

The medicament of the present invention may be a physiologically acceptable carrier, excipient or stabilizer as necessary (Japanese Pharmacopoeia 14th edition, its supplement or its latest edition, Remington's pharmaceutical Sciences, 18th Edition, A (See R. Gennaro, ed., Mack Publishing Company, 1990, etc.) and a composition having the desired degree of purity can be prepared and stored in the form of a lyophilized cake or aqueous solution. .

  Methods for lyophilizing cells are also known. Examples of such a method include a method of storing cells at 4 to 25 ° C. after the cells are put in dimethylformamide or the like, and the ampoule is treated with a freeze dryer so as to be in a vacuum state. In addition to this, liquid nitrogen method: Concentrated bacteria are suspended in a 10% glycerol-added storage medium, which is stored in a dedicated ampoule and stored in a liquid nitrogen tank (−150 to −196 ° C.).

  The amount of the composition of the present invention to be administered depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, etc. Can be easily determined. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration once a few months every day (for example, once a week to once a month). It is preferable to administer once a week to one month while observing the course.

  As used herein, “patient” refers to an organism to which the treatment of the present invention is applied, and is also referred to as “subject” or “subject”. The patient may preferably be a human.

  The present invention provides methods of treatment, inhibition and prevention by administration of an effective amount of a composition of the present invention to a patient. In preferred aspects, the compositions of the present invention can be substantially purified (eg, substantially free of substances that limit their effects or cause undesirable side effects).

  The present invention is also a method for producing a pharmaceutical comprising equol, comprising the step of mixing the intestinal bacterium of the present invention with daidzein, and the conditions under which the intestinal bacterium assimilate the mixed mixture. A method is provided comprising the step of incubating with.

  Here, the Slackia intestinal bacterium of the present invention has an ability to assimilate daidzein into equol, so that it is possible to efficiently produce a medicine containing equol. It is understood that the mixed mixture can be incubated under any conditions that allow the enteric bacteria used to assimilate daidzein to equol.

In this specification, the conditions for assimilating daidzein to equol can be appropriately determined by those skilled in the art using methods known in the art. As such conditions, for example, 37 g of Brain Heart Infusion, 1 g of agar, 0.5 g of L-cysteine-hydrochloric acid, and 4 g of sodium carbonate are heated and dissolved in 1 liter of distilled water, and oxygen-free carbon dioxide gas is blown in Dispense into test tubes, stopper with butyl rubber, and in 0.2 ml of the reaction solution prepared by autoclave sterilization, so that the concentration of Slacia bacteria is about 10 ⁹ / ml in the sample. Although suspended, and added with 1 μl of daidzein (20 mg / ml), incubation with a simple anaerobic culture apparatus Aneropack at 37 ° C. for 24 hours can be mentioned, but is not limited thereto.

The produced equol can be purified or separated as necessary. Such purification and separation methods can be performed using any method known in the art, such as chromatography, batch, HPLC, extraction, and the like.

  Accordingly, the present invention is also produced by a method comprising the steps of mixing the enterobacteria of the present invention with daidzein, and incubating the mixed mixture under conditions that the enterobacteria of the present invention assimilate. A composition is provided. Since such a composition has an estrogenic effect such as equol or is rich in strong isoflavones, it is used for administration in anticipation of anticancer (breast cancer, prostate cancer prevention) action, heart disease prevention effect, etc. obtain.

  In another aspect, the present invention also provides a method for improving the ability to produce equol. Ecole-producing ability refers to the ability to convert daidzein to equol in the intestine when used in animals such as humans. When the ability to produce equol is high, the effect of isoflavones on plasma cholesterol and plasma triglyceride lowering is significant, and anticancer effects and the like are also expected. Therefore, raising the ability to produce equol plays a role in improving the quality of health.

  This method comprises the step of administering an enteric bacterium of the present invention or a composition of the present invention to a subject in need of treatment. Such administration can be performed using methods known in the art.

  As used herein, “administering” means giving a pharmaceutical composition of the present invention or the like or a pharmaceutical composition containing the same to a host intended for treatment alone or in combination with other therapeutic agents. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes presentation where the combined drugs are administered together as a therapeutic mixture, and the procedure where the combined drugs are administered separately but simultaneously (eg, through separate mucosa to the same individual) Including. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

(Intestinal bacteria production method of the present invention)
In another aspect, the present invention relates to a method for producing a bacterium having a daizein-assimilating ability for assimilating daidzein to produce equol and having an effect of improving equol production ability of bacteria and intestinal flora, A method comprising culturing a sample expected to contain EG medium using EG medium is provided.

In a preferred embodiment, the present invention provides a method for producing human intestinal bacteria having the ability to assimilate daidzein to produce equol, comprising A) a sample comprising human intestinal derived material or feces. Step: B) Incubating the sample at 37 ° C. using EG medium; C) 37 g of Brain Heart Infusion, 1 g of agar, 0.5 g of L-cysteine-hydrochloric acid, 4 g of sodium carbonate, 1 liter of distilled water Dissolve in water and dispense into an oxygen-free carbon dioxide gas while dispensing into a test tube, plug a butyl rubber stopper, and perform autoclave sterilization. There were suspended at approximately ¹⁰ 9 / ml, was added daidzein 1μl (20mg / ml), 37 ℃ in simplified anaerobic culture device Anaeropack, 24 hours in Providing a method comprising the steps of: D) analyzing isoflavones in the reaction solution; and E) selecting and producing enterobacteria capable of assimilating daidzein to produce equol. .

  Although not wishing to be bound by theory, the bacteria such as the present invention have not been able to be identified so far because there are many types of anaerobic culture media, and EG media are not commercially available. Since it is necessary to prepare and produce, it is thought that it was normal to search for the bacteria which have the ability to utilize daidzein using not the EG medium but another commercially available medium. The metabolic activity of microorganisms may vary greatly depending on the medium. In the present invention, the identification of daidzein-assimilating bacteria has been made possible for the purpose of searching and identifying daidzein-assimilating bacteria using only an EG medium that has never been used in the world so far. In the embodiment, it is considered that the anaerobic reaction solution is also a series of novel devices in which a reaction solution having a high anaerobic degree is combined.

  In the above method, the present invention preferably uses a sample derived from healthy human feces as a sample. In particular, it is more preferable to use feces of a healthy person with high equol production ability.

(use)
The present invention contemplates the use of the enteric bacteria of the present invention to produce a medicament containing equol or to produce a medicament for improving the ability to produce equol. It is understood that such an enteric bacterium can adopt any form described in the above description regarding (enteric bacterium).

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples. Reagents used in the examples of 0 or less are Sigma (St. Louis) with exceptions. , USA), Wako Pure Chemical (Osaka, Japan), LC Laboratories, Woburn, USA) and the like.
(Example 1: Separation of enterobacteria having daidzein-assimilating ability and enterobacteria improving daidzein-assimilating ability)
In this example, bacteria were isolated using EG medium. The EG medium was prepared as follows.
The EG medium used in the present specification is prepared as follows.

Composition: Horse meat leachate 930mL
Proteose pettone No 3 (Difco) 10g
Yeast extract (Difco) 5g
Na ₂ HPO ₄ 4g
Glucose 1.5g
L-cystine (preliminarily dissolved in a small amount of 0.1N HCl) 0.2 g
Antifoaming agent (Torre Silicon 10% solution) 5mL
L-cysteine / HCL / H ₂ O 0.5 g
Horse blood 50mL
pH 7.6-7.8
Production method: L-Cysteine · HCl · _{H 2} O and heating the components other than equine blood were dissolved, correct the pH, 115 ° C., cooled to 50 ° C. After 20 minutes sterilization, L- cysteine · HCl · _H 2 O Add horse blood and dispense into a petri dish to make a flat plate. The horse leachate uses 500 g of horse leachate per 1000 ml of medium.

  Place fresh human stool in a sterile homogenizer, dilute stepwise 10-fold with anaerobic diluent, inoculate 0.05 ml of each dilution step into EG medium using a sterile pipette and spread with a sterile congeal stick The cells were cultured in a simple anaerobic culture apparatus at 37 ° C. for 48 hours. After completion of the culture, colonies were picked up one by one with platinum ears, spread on another EG medium, and cultured again in a simple anaerobic culture apparatus at 37 ° C. for 48 hours. After completion of the culture, 200 μl of anaerobic reaction solution for each platinum loop (37 g brain heart infusion, 1 g agar, 0.5 g L-cysteine hydrochloride, 4 g sodium carbonate, dissolved in 1 liter of distilled water with heating, free of oxygen The sample was dispensed into a test tube while blowing carbon dioxide gas, stoppered with a butyl rubber stopper, suspended in an autoclave-sterilized reaction solution), 1 μl of daidzein (20 mg / ml) was added, and then a simple anaerobic culture apparatus Incubation with aneropack was performed at 37 ° C. for 24 hours. After completion of the reaction, a 4-fold amount of a mixed solution of methanol and acetic acid (methanol: acetic acid = 100: 5) was added to the reaction solution 1 and vigorously stirred for 60 seconds by vortexing. Next, the supernatant obtained by centrifugation is filtered through a filter, and the filtrate is analyzed by HPLC equipped with an electrochemical detector (available from JASCO, etc.) to analyze isoflavones, resulting in equol productivity. The presence or absence was confirmed. Next, in order to increase the number of bacteria, intestinal bacteria with recognized equol productivity were inoculated into an EG medium and cultured in a simple anaerobic culture apparatus at 37 ° C. for 48 hours. After completion of the culture, colonies are picked up one by one with a platinum loop and suspended in 200 μl of the anaerobic reaction solution. After adding 1 μl of daidzein (20 mg / ml), there is no equol productivity previously enriched in EG medium. One platinum loop of intestinal bacteria was inoculated, and culture and equol were analyzed in the same manner as described above to examine whether equol productivity of equol-producing bacteria would improve.

  As a result, it was confirmed that equol productivity was improved.

(Example 2: assimilation ability of separated intestinal bacteria)
The in vitro utilization of daidzein of the genus Slackia TM-30 was examined by the following method. Slackia spp. That had been anaerobically cultured at 37 ° C. for 48 hours in EG medium were suspended in an anaerobic diluent, 1 μl of daidzein (20 mg / ml) was added to 200 μl of the suspension, and incubation was performed at 37 ° C. for 24 hours. Slackia final concentration (about 10 ⁹ / ml). After completion of the reaction, 4 times the amount of methanol and acetic acid mixture (methanol: acetic acid = 100: 5) was added to reaction solution 1, vigorously vortexed for 60 seconds, filtered through a filter, and the filtrate was equipped with an electrochemical detector. The isoflavones were analyzed by HPLC.

  It can be said that the representative strain of the present invention has a daidzein assimilation ability of 1.6% or more.

(Example 3: Effect of improving the ability to produce equol in vitro by the genus Slackia on non-equol-producing human flora mice)
Sterilization in which healthy human feces are homogenized using an anaerobic transport solution in an anaerobic glove box (nitrogen: carbon dioxide: hydrogen = 85: 10: 5), and this fecal dilution is kept in a vinyl isolator Mice (IQI) were orally administered to ICR mature male mice to produce human flora mice having human intestinal bacterial flora. After this human flora mouse was bred with a gamma-ray sterilized AIN 93M feed for 19 months, the stool was collected in a sterile test tube, diluted 35-fold per stool with the above diluent, and daidzein (20 mg / 20 mg / day) was diluted with 0.2 ml of the diluted solution. ml) 1 μl was added and incubated at 37 ° C. for 24 hours with a simple anaerobic culture apparatus Aneropack. The equol production ability was examined. As a result, a single human flora mouse with very low equol production ability was obtained. Therefore, stool of this human flora mouse is collected in a sterilized test tube, diluted 35-fold per stool with the above diluent, 1 μl of daidzein (20 mg / ml) is added to 0.2 mL of this diluent, and 37 EG is further added. The bacteria of the genus Slackia that had been anaerobically cultured for 48 hours at 0 ° C. were suspended in an anaerobic diluent with a platinum loop, and 20 μl of the bacterial solution was added to the stool dilution. Slackia final concentration (10 ⁷ / ml). For the reaction, incubation was carried out at 37 ° C. for 24 hours in a simple anaerobic culture apparatus Aneropack. As a control, 20 μl of an anaerobic diluent was added instead of 20 μl of the bacterial solution. After completion of the reaction, 4 times the amount of methanol and acetic acid mixture (methanol: acetic acid = 100: 5) was added to reaction solution 1, vigorously vortexed for 60 seconds, filtered through a filter, and the filtrate was equipped with an electrochemical detector. The isoflavones were analyzed by HPLC.

  It can be said that the representative strain of the present invention has an effect of imparting equol production ability to non-equol-producing human flora mice.

(Example 4: Effect of improving the ability to produce equol in vitro by the genus Slackia on equol-producing human flora mice)
Sterilization in which healthy human feces are homogenized using an anaerobic transport solution in an anaerobic glove box (nitrogen: carbon dioxide: hydrogen = 85: 10: 5), and this fecal dilution is kept in a vinyl isolator Mice (IQI) were orally administered to ICR mature male mice to produce human flora mice having human intestinal bacterial flora. The human flora mouse was bred with gamma ray sterilized AIN93M feed for 19 months, and then the stool of the human flora mouse was collected in a sterilized test tube, diluted 35 times per stool with the above diluent, and 0.2 mL of this diluted solution. Was added with 1 μl of daidzein (20 mg / ml), and incubated at 37 ° C. for 24 hours with an anaerobic culture apparatus Aneropack, and equol production ability was examined. As a result, human flora mice having equol production ability were obtained in two mice. It was. Therefore, stool of this human flora mouse is collected in a sterilized test tube, diluted 35-fold per stool with the above diluent, 1 μl of daidzein (20 mg / ml) is added to 0.2 mL of this diluent, and 37 EG is further added. The bacteria of the genus Slackia that had been subjected to anaerobic culture for 48 hours at 0 ° C. were suspended in an anaerobic diluent with a platinum loop, and 4 μl of the bacterial solution was added to the fecal diluent. Slackia final concentration (10 ⁷ / ml). For the reaction, incubation was carried out at 37 ° C. for 24 hours in a simple anaerobic culture apparatus Aneropack. As a control, 4 μl of anaerobic diluent was added instead of 4 μl of the bacterial solution. After completion of the reaction, isoflavones were analyzed by the same method as described above.

  It can be said that the representative strain of the genus Slackia of the present invention has an effect of improving equol productivity by 2.1 times or more with respect to equol-producing human flora mice.

(Example 5: Effect of improving the ability to produce equol in vitro by Bifidobacterium strain TM-1 against the genus Slackia having equol production ability)
Slackia spp. And Bifidobacterium TM-1 which were anaerobically cultured at 37 ° C. for 48 hours in EG medium were suspended in an anaerobic diluent separately with platinum ears, and 200 μl of Slackia spp. 10 μl of Fidobacterium TM-1 strain and 1 μl of daidzein (20 mg / ml) were added and incubated at 37 ° C. for 24 hours. As a control, 10 μl of an anaerobic dilution was added instead of 10 μl of the Bifidobacterium solution. The final concentration of the genus Slackia in the reaction solution was about 10 ⁹ / ml, and the final concentration of Bifidobacterium TM-1 strain was about 10 ⁶ / ml. After completion of the reaction, isoflavones were analyzed by the same method as described above.

  It can be said that the Bifidobacterium TM-1 strain of the present invention has an effect of improving equol production ability five times or more with respect to bacteria having equol production ability.

(Example 6: Effect of improving the ability to produce equol from daidzein glycosides in vitro by Bifidobacterium TM-1 strain against Sackia spp. Having equol production ability)
The bacteria of the genus Slackia and Bifidobacterium TM-1 which were anaerobically cultured at 37 ° C. for 48 hours in EG medium were suspended in an anaerobic diluent separately with platinum ears, and the following three types of reactions were performed. A solution was prepared and the utilization of daidzein glycosides was examined.

(1) Addition of 10 μl of Bifidobacterium TM-1 strain and 1 μl of daidzin (40 mg / ml) to 200 μl of Slackia spp. (The final concentration of Slackia spp. In the reaction solution is about 10 ⁹ / ml, Bifidobacterium spp. (Final concentration about 10 ⁹ / ml)
(2) Addition of 1 μl of daidzin (40 mg / ml) to 200 μl of genus Slackia (the final concentration of genus Slackia in the reaction solution is 1.12 × 10 ⁹ / ml)
(3) Add 1 μl of daidzin (40 mg / ml) to 200 μl of Bifidobacterium TM-1 strain (the final concentration of Bifidobacterium in the reaction solution is about 10 ¹⁰ / ml

The above three types of reactions were incubated at 37 ° C. for 24 hours in a simple anaerobic culture apparatus Aneropack. After completion of the reaction, 4 times the amount of methanol and acetic acid mixture (methanol: acetic acid = 100: 5) was added to reaction solution 1, vigorously vortexed for 60 seconds, filtered through a filter, and the filtrate was equipped with an electrochemical detector. The isoflavones were analyzed by HPLC.

  The representative genus Slackia of the present invention cannot produce daidzein or equol from daidzein glycosides, but the Bifidobacterium TM-1 strain of the present invention hydrolyzes glucose from daidzein glycosides. By producing daidzein, it can be said that it contributes to the production of equol from glycosides of daidzein of the genus Slackia.

(Example 7: Effect of improving the ability to produce equol in vitro by Bifidobacterium TM-1 strain on equol-producing human flora mice)
Sterilization in which healthy human feces are homogenized using an anaerobic transport solution in an anaerobic glove box (nitrogen: carbon dioxide: hydrogen = 85: 10: 5), and this fecal dilution is kept in a vinyl isolator Mice (IQI) were orally administered to ICR mature male mice to produce human flora mice having human intestinal bacterial flora. The human flora mouse was bred with gamma ray sterilized AIN93M feed for 19 months, and then the stool of the human flora mouse was collected in a sterilized test tube, diluted 35 times per stool with the above diluent, and 0.2 mL of this diluted solution. Was added with 1 μl of daidzein (20 mg / ml), and incubated at 37 ° C. for 24 hours with an anaerobic culture apparatus Aneropack, and equol production ability was examined. As a result, human flora mice having equol production ability were obtained in two mice. It was. Therefore, stool of this human flora mouse is collected in a sterilized test tube, diluted 35-fold per stool with the above diluent, 1 μl of daidzein (20 mg / ml) is added to 0.2 mL of this diluent, and 37 EG is further added. The Bifidobacterium TM-1 strain which was anaerobically cultured at 48 ° C. for 48 hours was suspended in an anaerobic diluent using a platinum loop, and 8 μl of the bacterial solution was added to the fecal diluent. Bifidobacterium final concentration (about 10 ⁸ / ml). For the reaction, incubation was carried out at 37 ° C. for 24 hours in a simple anaerobic culture apparatus Aneropack. As a control, 8 μl of an anaerobic diluent was added instead of 8 μl of the bacterial solution. After completion of the reaction, isoflavones were analyzed by the same method as described above.

  It can be said that the representative Bifidobacterium strain of the present invention has an effect of improving equol production ability of 38% or more against equol-producing human flora mice.

(Example 8: In vitro equol productivity improvement effect of Bifidobacterium TM-1 strain on equol-producing ICR mice)
The feces of two ICR male mice that have been confirmed to be capable of producing equol in advance were collected, diluted 25-fold per stool with the above diluent, and 1 μl of daidzein (20 mg / ml) was added to 0.2 mL of this diluent. Further, Bifidobacterium TM-1 which was anaerobically cultured at 37 ° C. for 48 hours in EG medium was suspended in an anaerobic diluent using a platinum loop, and 4 μl of the bacterial solution was added to the fecal diluent. Bifidobacterium final concentration (about 10 ⁸ / ml). As a control, 4 μl of anaerobic diluent was added instead of 4 μl of the bacterial solution. After completion of the reaction, isoflavones were analyzed by the same method as described above.

  It can be said that the representative Bifidobacterium TM-1 strain of the present invention has an effect of improving equol productivity of 25% or more with respect to equol-producing conventional mice.

(Example 9: In vitro equol productivity improvement effect of Bifidobacterium TM-1 strain on human having equol production ability)
A healthy male adult whose equol production ability has been confirmed in advance and who has been confirmed to have never taken antibiotics within the past two months is collected and diluted 30-fold per stool with the above diluent. Then, 1 μl of daidzein (20 mg / ml) was added to 0.2 mL of this diluted solution, and Bifidobacterium that had been anaerobically cultured in EG medium at 37 ° C. for 48 hours was suspended in an anaerobic diluted solution with a platinum loop. It became cloudy and 4 μl of the bacterial solution was added to the stool dilution. Bifidobacterium final concentration (about 10 ⁸ / ml). As a control, 4 μl of anaerobic diluent was added instead of 4 μl of the bacterial solution. After completion of the reaction, isoflavones were analyzed by the same method as described above.

  It can be said that the representative Bifidobacterium TM-1 strain of the present invention has an effect of improving equol productivity by 20% or more with respect to equol-producing humans.

(Example 10: In vitro equol productivity improvement effect of Bifidobacterium breve JCM1273 strain on human having equol production ability)
A healthy male adult whose equol production ability has been confirmed in advance and who has been confirmed to have never taken antibiotics within the past two months is collected and diluted 30-fold per stool with the above diluent. Then, 1 μl of daidzein (20 mg / ml) was added to 0.2 mL of this diluted solution, and Bifidobacterium breve JCM1273 bacteria that had been anaerobically cultured at 37 ° C. for 48 hours in a BL medium was further anaerobically diluted with a platinum loop. And 4 μl of the bacterial solution was added to the stool dilution. Bifidobacterium breve final concentration (about 10 ⁷ / ml). As a control, 4 μl of anaerobic diluent was added instead of 4 μl of the bacterial solution. After completion of the reaction, isoflavones were analyzed by the same method as described above.

It can be said that the Bifidobacterium breve JCM1273 strain has an effect of improving equol productivity by 18% or more with respect to equol-producing humans.

(Example 11: Estimation of species of genus Slackia based on 16S rRNA gene analysis and acquisition of species-specific primers for detection of equol-producing species of genus Slackia)
Bacterial genomic DNA was extracted with an instagene matrix (manufactured by Bio-Rad) from the genus Slackia that had been anaerobically cultured at 37 ° C. for 48 hours in EG medium. Bacterial strain classification method [Non-patent document 8 = Shimazu review, 57, 121-132 (2000)] of this genomic DNA was analyzed by 16S rRNA gene analysis of Shinoda et al. In this bacterium, 1269 out of 1365 16S rDNA genes of Slackia exigua (Accession No: AF101240) matched (92%). Therefore, although this bacterium is considered to belong to the genus Slackia, there is a possibility of a completely new bacterium at the species level. Therefore, an attempt was made to create a species-specific primer that specifically detects this bacterium. Using DDBJ's nucleic acid database and CLUSTAL W program [Nucleic Acids Res 22, 4673-4680 (1994)], a species-specific primer for detection of equol-producing Slackia species was obtained.
Species-specific primers for detection of Slaccia species:
Forward SLCf 5′AAGCCCTCGGATGCGGCAG3 ′ (SEQ ID NO: 4)
Reverse SLCr 5′TCTCGGCCACCGGATCT3 ′ (SEQ ID NO: 5)
Product size (bp) 383 bp
PCR product (sequence) with Slackiasp.TM-30 specific primer: 383 bp
AAGCCTCGGATGCGGCAGGGGGAGGCGGAATTCCCGGTGTAGCGGTGAAATGCGCAGATATCGGGAAGAACACCGACGGCGAAGGCAGCCTCCTGGGCCGGCATCGACGCTGAGGCGCGAAAGCTGGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCCAGCCGTAAACGATGGACGCTAGGTGTGGGGGGAACATCCCTCCGTGCCGAAGCCAACGCATTAAGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGAAGCAACGCGAAGAACCTTACCAGGGCTTGACATACGGGTGAAGCCGGGAGAGATCCGGTGGCCGAGA (951 from position 569 of SEQ ID NO: 3)
(Example 12: Formulation example)
The Bifidobacterium TM-1 strain identified as described above was inoculated into Bifidobacterium medium JCM catalog medium No19) and then subjected to static resuscitation at 37 ° C. for 18 to 24 hours. After completion of the culture, centrifugation was performed at 7,000 rpm for 15 minutes to obtain 1/100 amount of concentrated bacterial cells of the culture solution. Next, the same amount as the dispersion medium containing 5% (by weight) sodium glutamate, 5% (by weight) soluble starch, 5% (by weight) sucrose and 1% (by weight) magnesium sulfate heptahydrate in each concentrated cell. After mixing and correcting to pH 7.0, the sample was frozen at -40 ° C. or lower and lyophilized. The obtained freeze-dried bacterial powder is pulverized by tightening with 60 mesh to prepare a dried bacterial powder of the strain. 2 mg of this dry fungus (number of bacteria, equivalent to 5 × 10 ⁸ ), 61 mg of lactose (JP), 116,2 mg of starch (JP), 20 mg of polyvinylpyrrolidone K25 (JP) as binder, stearic acid as lubricant Magnesium (JP) 0.8mg is added and mixed uniformly, and compression molding is performed with a key press to make 200mg uncoated tablets. Further, hydroxypropylcellulose is used for film coating to produce a white film coating. Pills are produced.

  Such tablets can be administered as a health food or a medicament.

(Example 13: Preparation of equol formulation)
Reaction solution (37g brain heart infusion, 1g agar, 0.5g L-cysteine monohydrochloric acid, 0.5g sodium carbonate, sodium carbonate, soyaflavone HG (Fuji Oil, soy ethanol extract, infrabon content about 40%) 4 g is heated and dissolved in 1 liter of distilled water, dispensed into a test tube while blowing oxygen-free carbon dioxide, put into a butyl rubber stopper, and added to a reaction solution subjected to autoclave sterilization. About 10 ⁹ / ml Slackia genus TM-30 was added to ⁹ ml of this reaction solution, and static culture was performed at 37 ° C. for 24 hours to generate equol. This is lyophilized to a powder. This equol-containing powder can be used for an equol-containing food by mixing with the equol preparation and other foods.

(Example 14: Identification of another strain)
In the same manner as in the above example, a new strain is obtained that assimilate daidzein and produces 1.6% or more equol. About the strain to be identified, it is possible to identify bacteria belonging to Slackia. Such a bacterium can be identified as a bacterium belonging to Slackia that has daidzein assimilation ability.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention can be manufactured and used in industries such as health foods, foods, and medicines. Therefore, the present invention has applicability in various fields.

Sequence number 1: Universal primer 27f 5'-AGAGTTGATCCTGCTCAG-3 '
Sequence number 2: Universal primer 1492r 5'-GGCACTCTTGTACGACTT-3 '
SEQ ID NO: 3: Partial base sequence (1402 bp) of 16S rDNA of genus Slackia TM-30
CACATGCAAGTCGAACGAGTAAGACGCCTTCGGGCGTGGATAGAGTGGCGAACGGGTGAGTAACACGTGACCAACCTGCCCCCTCCTCCGGGACAACCTCGGGAAACCGAGGCTAATACCGGATACTCCGGGCCCCCCGCATGGGGAGCCCGGGAAAGCCCTGGCGGGAGGGGATGGGGTCGCGGCCCATCAGGTAGACGGCGGGGTAACGGCCCACCGTGCCTACTACGGGTAGCCGGGCTGAGAGGCCGATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGCGCAATGGGGGCAACCCT ACGCAGCGACGCCGCGTGCGGGACGAAGTCATTCGTGACGTAAACCGCTTTCAGCGAGGAAGAACCATGACGGTACTCGCAGAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTATCCGGAATCATTGGGCGTAAAGCGCGCGCAGGCGGGCTTTCAAGCGGCGGCGTCGAAGCCGGGGGCTCAACCCCCGGAAGCGCCCCGAACTGGAAGCCTCGGATGCGGCAGGGGGAGGCGGAATTCCCGGTGTAGCGGTGAAATGCGCAGATATCGGGAAGAACACCGACGGCGAAGGCAGCCTCCTGGGC GGCATCGACGCTGAGGCGCGAAAGCTGGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCCAGCCGTAAACGATGGACGCTAGGTGTGGGGGGAACATCCCTCCGTGCCGAAGCCAACGCATTAAGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGAAGCAACGCGAAGAACCTTACCAGGGCTTGACATACGGGTGAAGCCGGGAGAGATCCGGTGGCCGAGAGGAGCCCGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGT GTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCGCCGCGTGTTGCCAGCATTCAGTTGGGCACTCACGCGGGACTGCCGGCGTCAAGCCGGAGGAAGGCGGGGACGACGTCAAGTCATCATGCCCCTCATGCCCTGGGCCGCACACGTGCTACAATGGCCGGCACAACGGGTTGCCACCCCGCGAGGGGGAGCGGATCCCAAAAGCCGGCCCCAGTTCGGATCGCAGGCTGCAACCCGCCTGCGTGAAGCCGGAGTTGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACCCGA TCGTCTGCACCCGAAGCCGCCGGCCGAACCCCGAAAGGGGGCGGAGGCGTCGAAGGTGTGGAGGGTGA
SEQ ID NO: 4: Species-specific primer for detection of genus Slackia: Forward SLCf 5′AAGCCCTCGGATGCGGCAG3 ′
SEQ ID NO: 5: Species-specific primer for detection of genus Slackia: Reverse SLCr 5′TCTCGGCCACCCGATCT3 ′

Claims (14)

A human enteric bacterium that has the ability to assimilate daidzein to produce equol, and has the accession number FERM P-20729 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Slacia sp., Deposited on December 2, 2005). Enterobacteria which is TM-30 strain . A combination of the intestinal bacterium according to claim 1 and another enteric bacterium . To improve the Ecole producing ability, the composition comprising a combination according to Enterobacteriaceae or claim 2 of claim 1. The composition according to claim 3 , wherein the composition is food or medicine. The composition according to claim 4 , further comprising another active ingredient. The composition according to claim 4 , wherein the enteric bacterium is provided by being dissolved in a liquid. The composition according to claim 4 , wherein the enteric bacterium is provided in a solid form. The composition according to claim 4 , wherein the enteric bacterium is provided by being lyophilized. 5. The composition of claim 4 , further comprising a component that protects the enteric bacteria from gastric juice. A method for improving the Ecole producing ability in non-human subjects, intestinal bacteria according to claim 1, composition according to the combination or any one of claims 3-9 according to claim 2 Administering the product to a non-human subject in need of treatment. A method for producing or medicament for improving the Ecole productivity including equol, and combinations described in Enterobacteriaceae or claim 2 of claim 1, step mixing the daidzein, and mixed And incubating the mixture under conditions that the enteric bacteria assimilate. The method comprises a combination according to Enterobacteriaceae or claim 2 wherein the step of mixing the daidzein, and the mixed mixture the intestinal bacteria is the step of incubating under conditions assimilated to claim 1 Produced by the composition. For the manufacture of a medicament comprising equol, enteric bacterium according to claim 1, or the use of a combination according to claim 2. For producing a medicament for improving the Ecole producing ability, enteric bacterium according to claim 1, or the use of a combination according to claim 2.