WO2019092896A1

WO2019092896A1 - Akkermansia muciniphila proliferation material

Info

Publication number: WO2019092896A1
Application number: PCT/JP2018/009869
Authority: WO
Inventors: 睦生市島; 寛人森田
Original assignee: アサヒグループホールディングス株式会社
Priority date: 2017-11-08
Filing date: 2018-03-14
Publication date: 2019-05-16
Also published as: JPWO2019092896A1; TWI772384B; JP7171602B2; TW201918251A

Abstract

The present invention addresses the problem of providing a novel proliferation material which enables the proliferation of Akkermansia muciniphila. An Akkermansia muciniphila proliferation material containing at least one polyphenol component selected from the group consisting of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin and tannic acid.

Description

Akkermansia mushiniphila breeding material

The present invention relates to a propagation material capable of propagating Akkermansia muciniphila.

Akkermansia muciniphila is an obligately anaerobic, gram-negative bacterium with a size of about 0.6 to 1.0 μm, no spores, and no motility (non-patent literature) 1). Akkermansia muciniphila is a eubacteria normally present in the intestine of many mammals including humans.

In recent years, Akkermansia muciniphila has been shown to decrease in the intestines of obese and diabetic people, and administration of Akkermansia muciniphila has been shown to improve fat gain and improve insulin resistance in mice. (Non-Patent Documents 2 and 3), it has been pointed out that Akkermansia muciniphila is associated with a disorder such as obesity. Furthermore, Akkermansia muciniphila is inversely proportional to the severity of appendicitis, and its amount is decreased in the intestine of patients with diseases accompanied by inflammation such as ulcerative colitis and Crohn's disease. It has been reported (Non-patent Documents 4 and 5) and Akkermansia muciniphila is considered to have anti-inflammatory activity.

Therefore, there is a growing interest in Akkermansia muciniphila as a promising tool to prevent or ameliorate these diseases and disorders.

Attempts have been made to increase the number of Akkermansia muciniphila in the intestine in terms of prebiotics. For example, Patent Document 1 describes that the proportion of Akkermansia bacteria has increased in the intestinal flora of a human who receives a composition containing polydextrose. Patent Document 2 describes that Akkermansia bacteria proliferated in the intestine of a mouse administered with xylose. Patent Document 3 describes that the reduction in the amount of Akkermansia muciniphila observed in leptin-deficient obese mice and high-fat fed mice was recovered by intake of fructooligosaccharides. Patent Document 4 describes that the proportion of Akkermansia bacteria has increased in the bacterial flora of the cecal contents of rats that have received water-soluble cellulose acetate. Non-Patent Document 5 describes that the proportion of Akkermansia bacteria has increased in the intestinal flora of mice that have taken high molecular weight procyanidins.

However, new means for propagating Akkermansia muciniphila are still needed in the art.

JP-A-2014-532710 WO 2016/122889 Japanese Patent Application Publication No. 2016-503418 WO 2015/146853

An object of the present invention is to provide a new propagation material capable of propagating Akkermansia muciniphila.

As a result of intensive studies to solve the above problems, the present inventors have found that some polyphenols have an action to propagate Akkermansia muciniphila, and have completed the present invention.

That is, the present invention includes the following inventions.
[1] An Akkermansia muciniphilus growth material comprising one or more polyphenols selected from the group consisting of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid.
[2] The growth of [1], wherein the quercetin glycoside comprises one or more selected from the group consisting of quercetin 3-D-galacside, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside Material.
[3] The growth material of [1] or [2], which comprises quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, or tannic acid.
[4] The propagation material according to any one of [1] to [3], which is food and drink, medicine, feed or feed additive.
[5] Ackerman cultured using a growth material containing one or more polyphenols selected from the group consisting of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid A culture comprising S. muciniphila.
[6] The culture of [5], wherein the quercetin glycoside comprises one or more selected from the group consisting of quercetin 3-D-galacside, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside object.
[7] The culture of [5] or [6], wherein the propagation material comprises quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, or tannic acid.
[8] The culture according to any one of [5] to [7], which is food and drink, medicine, feed or feed additive.
[9] Akkermansia muciniphila, a growth material containing one or more polyphenols selected from the group consisting of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid A method for producing Akkermansia muciniphila and / or a culture thereof, comprising using and culturing.
[10] The production of [9], wherein the quercetin glycoside comprises one or more selected from the group consisting of quercetin 3-D-galacside, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside Method.
[11] The production method of [10], wherein the propagation material comprises quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, or tannic acid.

According to the present invention, it is possible to provide a new propagation material capable of propagating Akkermansia muciniphila.

FIG. 1-1 is a graph showing the results of evaluation of the effect on growth of Akkermansia muciniphila by the addition of quercetin glycoside to the culture medium. The results are shown as relative values (growth ratio [%]) where the growth amount of the control is 100%. n = 4. *: P <0.05, **: p <0.01 (vs control, Dunnett's multiple comparison test) Figure 1-2 is a graph showing the results of evaluation of the effect on growth of Akkermansia muciniphila by the addition of isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, or tannic acid to the medium. It is. The results are shown as relative values (growth ratio [%]) where the growth amount of the control is 100%. n = 4. *: P <0.05, **: p <0.01 (vs control, Dunnett's multiple comparison test) FIG. 2 is a graph showing the results of evaluation of the effects of oral intake of rutin or hesperidin on the growth of Akkermansia muciniphila in vivo. The results show quantitative results of Akkermansia muciniphila DNA in feces collected from the water administration group (control), rutin administration group and hesperidin administration group. n = 4. **: p <0.05, *: p <0.1 (vs control, Mann-Whitney U test)

1. Breeding material The present invention relates to a breeding material of Akkermansia muciniphila.

"Ackermansia muciniphila" is known and described by Muriel Derrien et al., Int J Syst and Evol Microbiol. 54: 1469-1476. (2004) and the like, and is characterized based on known mycological properties as described in (2004). (See above) Gram negative, obligately anaerobic, non-motile, non-spore, oval, size 0.6-1.0 μm, mucin added to the reference strain (strain ATCC BAA-835). It has been described that the medium has characteristics such as growth and capsular formation.

In addition, the nucleotide sequence of the 16S rRNA gene of Akkermansia muciniphila is known, and is disclosed, for example, in a known database such as GenBank, and registered as AY271254, NR_074436. Akkermansia muciniphila can be identified by analyzing 16S rRNA gene using these sequence information. Analysis of 16S rRNA gene is performed based on known methods such as quantitative PCR, DGGE / TGGE, FISH, 16S rDNA cloning library method, T-RFLP method, FISH-FCM method, sequencing method, etc. be able to. For example, according to the quantitative PCR method, a primer specific to the bacterium is prepared based on known nucleotide sequence information of 16S rRNA gene of Akkermansia muciniphila. Perform a PCR reaction using the primers with the DNA extracted from the selected bacteria as a template, and determine whether the bacteria is Akkermansia muciniphila based on the presence or absence of the PCR amplification product of the intended size. can do. Design of specific primers and determination of PCR conditions can be performed according to a standard method (Bio-Experimental Illustrated 3 PCR: Cell Engineering Appendix: Experimental Note Series to be Viewed by the Cell; Hiroki Nakayama, Shujunsha Co., Ltd.).

In the present invention, the "propagation material" refers to a composition having a proliferative effect that supports the growth of Akkermansia muciniphila in vitro or in vivo in a mammal, and can increase the absolute number of Akkermansia muciniphila. means.

The growth material of the present invention comprises quercetin glycoside, isorhamnetin (also referred to as 3'-O-methyl quercetin), cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid. It contains one or more polyphenols selected. The “one or more polyphenols” means one polyphenol selected from the above, or two, three, four, five, six, seven, eight, nine, or the like selected from the above. It means a combination of ten, eleven, twelve, thirteen or fourteen polyphenols.

The quercetin glycoside may be a compound having a structure in which quercetin is glycosidically linked to a sugar (eg, glucose, mannose, galactose, rhamnose, fructose, glucuronic acid, xylose, maltose, lactose, gentiobiose, rutinose etc.), particularly There is no limitation. The quercetin glycoside which can be used in the present invention is not particularly limited. For example, quercetin 3-D-galactide, quercetin 3-O-glucopyranoside, quercetin 3,4-diglucoside, rutin (also referred to as quercetin 3-rutinoside) And pertatoside (also referred to as quercetin 3-O-α-arabinopyranosyl-β-glucopyranoside). Preferably, the quercetin glycoside is one or more (for example, one or two) selected from the group consisting of quercetin 3-D-galacside, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside. Species, three, four or a combination of five). Quercetin 3-D-galactide, quercetin 3-O-glucopyranoside, quercetin 3,4-diglucoside, rutin, and pertatoside are represented by the following structural formulas, respectively.

Isorhamnetin is a methylated form of quercetin and is represented by the following structural formula.

Cyanidin glucoside is a type of cyanidin glycoside and is represented by the following structural formula.

Hesperidin is a type of flavanone glycoside and is represented by the following structural formula. Hesperetin is an aglycone of hesperidin and is represented by the following structural formula.

Apigenin, chrysin, tangeretin, and nobiletin are aglycones of flavone glycosides, respectively, and are represented by the following structural formulas.

Tannic acid is a kind of gallotannin formed by combining a plurality of gallic acids with glucose, and is represented by the following structural formula.

Among these, it is particularly preferable to include one or more polyphenols selected from the group consisting of quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, and tannic acid. Quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, and tannic acid have a high proliferative action as compared to the other polyphenols described above.

In the present invention, the one or more polyphenols may be natural products extracted from plants, or may be chemically synthesized. When it is a natural product, the plant and the extraction method used as its raw material are not particularly limited, and the one or more polyphenols can be used in the form of one isolated from plants or in the form of an extract.

The propagation material is 0.001 to 99% by weight, preferably 0.001 to 90% by weight, more preferably 0.001 to 80% by weight, and still more preferably 0.001 to 70% by weight of the one or more polyphenols. The amount is not particularly limited, and may vary depending on the type and number of polyphenols, and the form and use of the propagation material. When the growth material contains a combination of two or more polyphenols, each polyphenol can be contained in any proportion. The propagation material of the present invention may be in any form suitable for use, such as liquid, semi-solid, solid (powder, granules, etc.), but, for example, when the propagation material has a liquid form, 1 An amount of 5 μg / mL or more, preferably 10 μg / mL or more, more preferably 20 μg / mL or more, still more preferably 30 μg / mL or more can be included per polyphenol of a species. The upper limit is not particularly limited, and can be, for example, 2000 μg / mL or less, preferably 1500 μg / mL or less, more preferably 1000 μg / mL or less, and further preferably 500 μg / mL or less.

The amount of the one or more polyphenols contained in the growth material can be quantified using a known mass spectrometry (for example, liquid chromatography mass spectrometry, high performance liquid chromatography, etc.).

The growth material of the present invention may contain, in addition to the above one or more polyphenols, carbon sources, nitrogen sources, inorganic salts and the like usually used for microbial culture. For example, as a carbon source, glucose, fructose, sucrose, starch, molasses etc. can be used, and as a nitrogen source, for example peptone, yeast extract, tryptone, casein hydrolysate, meat extract, ammonium sulfate etc. As inorganic salts, salts of phosphoric acid, potassium, magnesium, calcium, sodium, iron, manganese and the like can be used. Furthermore, if necessary, agar, gelatin, vitamins, amino acids, surfactants and the like can be included.

In addition, the propagation material of the present invention may contain other components known to have an effect on the growth or increase in intestinal ratio of Akkermansiella. Such components include mucin, glucose, N-acetylglucosamine, N-acetylgalactosamine (Muriel Derrien et al., Supra), polydextrose (Table 2014-532710), xylose (WO2016-122889), fructooligosaccharide ( JP-A-2016-503418), water-soluble cellulose acetate (WO2015 / 146853), high-molecular procyanidins (Masumoto S et al., Sci. Rep., 6, 31208 (2016)), etc., but not limited thereto. .

The growth material of the present invention can be used as a culture medium for culturing Akkermansia muciniphila, or can be added to a basal medium to support the growth of Akkermansia muciniphila in vitro, and for Akkermansia muciniphila You can increase the absolute number.

Alternatively, the propagation material of the present invention can be in the form of a medicine, food or drink, or feed or feed additive, and can be administered or ingested to a mammal.

The propagation material of the present invention can contain additives such as excipients, lubricants, binders, disintegrants and the like that are usually used in the manufacture of medicines and foods and drinks, and the intended administration route and intake It can be manufactured and provided as a medicine or food and drink having a dosage form or form suitable for the method.

Excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, glucose, corn starch, crystalline cellulose, low substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Silica anhydride, synthetic aluminum silicate, magnesium aluminum metasilicate and the like can be mentioned.

As the lubricant, for example, sugar esters such as sucrose fatty acid ester and glycerin fatty acid ester, calcium stearate, magnesium stearate, stearic acid, stearyl alcohol, hydrogenated oils such as powdered vegetable fats and oils, waxes such as white beeswax, Examples include talc, silicic acid, silicon and the like.

As a binder, pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol Pyrrolidone etc. are mentioned.

Disintegrants include lactose, sucrose, starch, carboxymethylcellulose, calcium carboxymethylcellulose, sodium croscarmellose, sodium carboxymethyl starch, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.

In addition, examples of additives that can be used in the present invention and that are generally used in the manufacture of medicines and food and drink include various fats and oils (eg, vegetable oil such as soybean oil, corn oil, safflower oil, olive oil, etc., beef tallow, Animal fats and oils such as sardine oil), herbal medicine (eg royal jelly, ginseng etc), amino acids (eg glutamine, cysteine, leucine, arginine etc), polyhydric alcohols (eg ethylene glycol, polyethylene glycol, propylene glycol, glycerin, sugar alcohol, Examples include sorbitol, erythritol, xylitol, maltitol, mannitol etc., natural polymers (eg gum arabic, agar, water soluble corn fiber, gelatin, xanthan gum, casein, gluten or gluten hydrolysate, lecithin, starch, dextrin etc) , Tamine (eg vitamin C, vitamin B group etc.), mineral (eg calcium, magnesium, zinc, iron etc.), dietary fiber (eg mannan, pectin, hemicellulose etc.), surfactant (eg glycerol fatty acid ester, sorbitan fatty acid ester etc. ), Purified water, diluents, stabilizers, tonicity agents, pH adjusters, buffers, wetting agents, solubilizers, suspending agents, coloring agents, flavoring agents, flavoring agents, fragrances, antioxidants And sweeteners, taste ingredients, acidulants and the like.

The dosage form or form of the medicine or food or drink is not particularly limited. The medicine may be, for example, tablets, capsules, granules, powders, powders, syrups, dry syrups, solutions, suspensions, inhalants, suppositories, etc., preferably an oral preparation. It is. The liquid preparation such as liquid preparation and suspension preparation is provided in a state where it can be lyophilized and stored, and used at the time of use after being dissolved in a buffer solution containing water, embedded saline etc. It may be In addition, those having a solid dosage form such as a tablet may be coated if necessary (for example, sugar coated tablets, gelatin coated tablets, enteric coated tablets, etc.), using known techniques. It may be a controlled release preparation such as a sustained release preparation, a delayed release preparation or an immediate release preparation.

Examples of food and drink include health food and drink having the form of tablets, tablets, chewable tablets, tablets, powders, powders, capsules, granules, drinks etc. Etc), soft drinks, tea drinks, jelly drinks, sports drinks, coffee drinks, carbonated drinks, vegetable drinks, fruit juice drinks, fermented vegetable drinks, fermented fruit juice drinks, fermented milk drinks (yogurt etc.), lactic acid bacteria drinks, milk drinks, powder It may be in the form of a beverage, cocoa beverage, confectionery (eg, biscuits and cookies, chocolate, candy, chewing gum, tablets), jelly, etc. (without limitation).

The food and drink may be health food (including food for specific health (including food for conditional health), nutritive food, functional indication food, health food, beauty food and the like.

In addition, the propagation material of the present invention can be in the form of feed or feed additives such as livestock, race horses, and pets as well as food and drink for humans.

The propagation material of the present invention can be used as a food or drink, medicine or feed or feed additive, in mammals (eg, humans, monkeys, chimpanzees, cows, horses, pigs, sheep, dogs, cats, cats, mice, rats, etc.), preferably humans. It can be administered or ingested, it can support the growth of Akkermansia muciniphila in the living body of the administered or ingested mammal, and can increase the absolute number of Akkermansia muciniphila.

The dose or intake of the propagation material of the present invention may vary depending on factors such as the age and body weight of the subject, administration route, administration / intake frequency, type and number of polyphenols included, and any dose or intake The amount can be adopted. For example, when orally administered or ingested, an amount selected from 0.01 mg / kg to 600 mg / kg per day for one type of polyphenol contained may be one or more times (for example, 2 to 5 times) , Preferably 2-3 times, in divided doses. When a combination of two or more types of polyphenols is included, each polyphenol may be administered or taken in one or more divided doses in an amount selected from the above range per day.

The propagation material of the present invention can be effective in a small amount and in a short time, but can be administered or ingested for a long time. For example, according to the above dosage, the propagation material of the present invention can be continued for a period of 1 week or more, 2 weeks or more, 1 month or more, 2 months or more, 6 months or more, 6 months or more, 1 year or more, or more. It can be administered or ingested.

2. Culture The present invention also relates to a culture of Akkermansia muciniphila and a method for producing the same.

In the present invention, the "culture" is Akkermansia muciniphila cultured using the above-mentioned growth material, and a composition containing a medium component and a metabolite. Media components preferably include the one or more polyphenols described above.

In the present invention, "Ackermansia muciniphila" is isolated from a mammal (eg, human, monkey, chimpanzee, cow, horse, pig, sheep, dog, cat, mouse, rat, etc.), preferably human intestine. The thing can be used. Akkermansia muciniphila is cultured under anaerobic conditions in a medium supplemented with mucin, according to a known method (Muriel Derrien et al., Supra), a sample containing mammalian feces, feces or feces, It can be isolated from the grown cells.

In the present invention, any strain classified into Akkermansia muciniphila, or a mutant or breeding strain thereof can be used. Examples of Akkermansia muciniphila strains which can be used in the present invention include, but are not limited to, ATCC BAA-835 strain, YL44 strain, JCM 30893 strain and the like.

The culture of the present invention can be obtained by culturing Akkermansia muciniphila using the growth material. The propagation material can be used as a culture medium for cultivating Akkermansia muciniphila or added to a basal culture medium. The medium may contain an amount of 5 μg / mL or more, preferably 10 μg / mL or more, more preferably 20 μg / mL or more, and still more preferably 30 μg / mL or more per one polyphenol. The upper limit is not particularly limited, and can be, for example, 2000 μg / mL or less, preferably 1500 μg / mL or less, more preferably 1000 μg / mL or less, and further preferably 500 μg / mL or less. Agar and gelatin may be further added to the medium, if necessary.

The culture can be performed under anaerobic conditions at pH 5.5-8.0, preferably pH 6.5, 20 ° C.-40 ° C., preferably 37 ° C. "Anaerobic conditions" may be any low-oxygen environment to the extent that Akkermansia muciniphila can grow, for example, using an anaerobic chamber, an anaerobic box, a sealed vessel or a culture vessel containing an oxygen scavenger, etc. It can be a condition.

The culture can be performed in any format such as stationary culture, shaking culture, tank culture, etc. The culture time is not particularly limited, and can be, for example, 3 hours to 7 days. The culture may be performed batchwise or continuously.

After the culture, the obtained culture may be used as it is as the culture of the present invention, or a product obtained by concentrating or roughly purifying Akkermansia muciniphila from the culture may be used as the culture of the present invention . Enrichment or crude purification of the cells from the culture can be performed using any means, for example, centrifugation, filtration and the like.

The cultures may be subjected to pasteurisation. Pasteurization of the culture can be carried out by heating at a temperature of 60 ° C. to 80 ° C., preferably 70 ° C., for about 30 minutes.

According to the above culture method, the propagation material can support the growth of Akkermansia muciniphila, the absolute number of Akkermansia muciniphila can be increased, and the culture of Akkermansia muciniphila can be efficiently produced. it can.

The culture of the present invention can contain additives such as excipients, lubricants, binders, disintegrants and the like that are usually used in the manufacture of medicines and foods and drinks, and the intended administration route and intake It can be manufactured and provided as a medicine or food and drink having a dosage form or form suitable for the method. Additives such as excipients, lubricants, binders, disintegrants and the like can use the above-mentioned ones, and obtain the food or drink, medicine or feed or feed additives having the above-mentioned dosage form or form it can.

The culture of the present invention can be used as a food or drink, medicine or feed or feed additive, in mammals (eg, humans, monkeys, chimpanzees, cows, horses, pigs, sheep, dogs, cats, cats, mice, rats, etc.), preferably humans. It can be administered or ingested, and the absolute number of Akkermansia muciniphila can be increased in vivo of the administered or ingested mammal.

The dose or intake of the culture of the present invention may vary depending on factors such as the age and body weight of the subject, route of administration, frequency of administration / intake, etc, and any dose or intake may be adopted. For example, when orally administered or ingested, the number of cells of Akkermansia muciniphila is 1 × 10 ⁵ / kg to 1 × 10 ¹⁵ / kg / day, preferably 1 × 10 6 / day. ^{An amount selected from 6} / kg to 1 × 10 ¹⁴ / kg, more preferably 1 × 10 ⁸ / kg to 1 × 10 ¹³ / kg per day, in one or more times (eg, 2 to 5) It can be administered or taken in divided doses, preferably 2 to 3 times).

The culture of the present invention can be effective in a small amount and in a short period, but can be administered or ingested for a long period of time. For example, according to the above dosage, the culture of the present invention may be continued for a period of 1 week or more, 2 weeks or more, 1 month or more, 2 months or more, 6 months or more, 6 months or more, 1 year or more, or more. It can be administered or ingested.

The culture of the present invention may be administered or taken together with the growth material.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

Example 1 Akkermansia mucinifira proliferation effect by addition of polyphenols 1. Method (1) Strain Akkermansia muciniphila strain was obtained by cloning using a mucin-supplemented medium from a healthy human (adult) fecal sample according to the method described in Muriel Derrien et al., Supra. The nucleotide sequence of the 16S rRNA gene was determined for the strain, and it was confirmed to be Akkermansia muciniphila based on the sequence comparison with the nucleotide sequence of the 16S rRNA gene of Akkermansia muciniphila registered in GenBank (AY 271254) The strain was used.

(2) Preparation of culture medium The culture medium (main culture medium) of Akkermansia muciniphila was prepared by diluting each polyphenol diluted to various concentrations with dimethylsulfoxide (DMSO) in the synthetic medium described in Nature Medicine 23, 107-113, 2017. And 1% by mass of the whole culture medium.

The synthetic medium is 0.4 g KH ₂ PO ₄ , 0.53 g Na ₂ HPO ₄ , 0.3 g NH ₄ Cl, 0.3 g NaCl, 0.1 g MgCl ₂ · 6 H ₂ per liter according to a known method. O, 0.11 g CaCl ₂ , 1 ml alkaline trace element solution (alkaline trace element solution has the following composition: 0.1 mM Na ₂ SeO ₃ , 0.1 mM Na ₂ WO ₄ , 0.1 mM Na ₂ MoO ₄ , and 10 mM NaOH, 1 ml acidic trace element solution (acid trace element solution has the following composition: 7.5 mM FeCl ₂ , 1 mM H ₃ B 0 ₄ , 0.5 mM ZnCl ₂ , 0.1 mM CuCl ₂ , 0.5 mM MnCl ₂ 0.5 mM CoCl ₂ , 0.1 mM NiCl ₂ , and 50 mM HCl), 1 ml vitamin solution (vitamin solution) Has the following composition: 0.02 g / L biotin, 0.2 g / L niacin, 0.5 g / L pyridoxine, 0.1 g / L riboflavin, 0.2 g / L thiamine, 0.1 g / L cyanocobalamin, 0 .1g / L p- aminobenzoic acid, and 0.1 g / L pantothenic acid), 0.5 mg resazurin, _4g NaHCO 3, in a sterile basal medium containing 0.25g Na2S · 7-9H ₂ O, 0.7 % (V / v) sterile purified rumen fluid was added and further prepared by adding 16 g / L soy peptone, 4 g / L threonine, and a mixture of glucose and N-acetylglucosamine (25 mM each).

Thus, any of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, tannic acid, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, A medium containing 250 μg / mL or 500 μg / mL was prepared and used for the following main culture.

As quercetin glycosides, quercetin 3-D-galactide, quercetin 3-O-glucopyranoside, quercetin 3, 4 diglucoside, rutin, pertatoside were used.

Control media were prepared by adding the same amount of DMSO without polyphenols to a known synthetic media.

(3) Culture The strain was inoculated in GAM medium (Gifu Anaerobic Medium) supplemented with 0.4% (v / v) mucin and precultured at 37 ° C. for 24 hours under anaerobic conditions.

After completion of the preculture, a preculture solution (10 μL) was added to the above-mentioned main medium (200 μL), and main culture was performed at 37 ° C. for 90 hours under anaerobic conditions.

The control was similarly cultured using a control medium instead of the main medium.

(4) Measurement of growth ratio After completion of the main culture, cells are recovered from the main culture solution, and DNA is extracted from the cells by the glass bead method according to the method described in Enteric Bacteriology 20: 245-258, 2006. did.

Then, using the obtained DNA as a template, Appl. Environ. Microbiol. December 2007 vol. 73 no. According to the method described in 23 7767-7770, quantitative PCR was carried out using a primer set prepared based on the sequence of the 16S rRNA gene of Akkermansia muciniphila, and the amount of DNA of the recovered cells was measured.

Similarly, for the control, DNA was extracted from the cells, and the amount of DNA of the collected cells (the amount of control DNA) was measured.

The amount of DNA obtained was expressed relative to the amount of control DNA as 100% (n = 4), and the growth of Akkermansia muciniphila in each medium was evaluated.

2. Results With respect to the type and amount of polyphenol added, those showing growth of Akkermansia muciniphila over the control are shown in FIGS. 1-1 and 1-2.

With regard to quercetin glycosides, the addition of the medium to the compounds resulted in an increase in the amount of Akermansia muciniphila DNA exceeding the control (FIG. 1-1). This result suggests that quercetin glycosides can promote the growth of Akkermansia muciniphila regardless of the type of the compound.

In addition, addition of isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, or tannic acid to the medium also increased the amount of Akkermansia muciniphila over control (Figure 1-2). 2.) It was confirmed that the addition of each polyphenol promoted the growth of Akkermansia muciniphila.

In particular, quercetin 3-O-glucopyranoside (31.25 μg / mL, 125 μg / mL), rutin (62.5 μg / mL), cyanidin glucoside (62.5 μg / mL, 125 μg / mL, 250 μg / mL), hesperidin (31) Good growth was observed at 25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL), apigenin (31.25 μg / mL, 62.5 μg / mL), tannic acid (31.25 μg / mL) (Values in parentheses indicate the content in the medium).

From the above results, quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, or tannic acid support the growth of Akkermansia muciniphila, and the absolute number of Akkermansia muciniphila It has been confirmed that it is possible to increase.

Example 2 Examination of Akkermansia muciniphila growth effect The mice were fed a high-fat diet, and the Akkermansia muciniphila growth effect when rutin and hesperidin were administered for 8 weeks was examined.

1. Method (1) Use Animal The high-fat diet D12492 (Research Diets, Inc) was fed to 30 9-week-old mice (C57BL / 6J). Each is divided into water administration group (control), rutin administration group, hesperidin administration group (1 group 10 mice), and in the rutin administration group and hesperidin administration group, the amount to be 600 mg / kg is suspended in water and orally administered daily Administered. This oral administration was performed for 8 weeks from the start of administration.

(2) Test substance Rutin used αG rutin PS (Toyoh Co., Ltd.). The hesperidin used (alpha) G hesperidin PA (Toyo Sangyo Co., Ltd.).

(3) Extraction of DNA 8 weeks after oral administration, feces were collected, and DNA was extracted from about 100 mg of feces by the glass bead method according to the method described in Enteric Bacteriology 20: 245-258, 2006.

(4) Determination of Akkermansia muciniphila
Using the obtained DNA as a template, Appl. Environ. Microbiol. December 2007 vol. 73 no. Quantitative PCR is performed using the primer set prepared based on the sequence of the 16S rRNA gene of Akkermansia muciniphila according to the method described in 23 7767-7770, and the DNA amount of the collected bacterial cells is determined did.

2. Result The quantitative value of DNA of Akkermansia muciniphila in feces collected from a mouse orally administered for 8 weeks is shown in FIG.
In the rutin-administered group and the hesperidin-administered group, it was confirmed that the DNA amount of Akkermansia muciniphila was increased as compared to the control group.

From the above results, oral intake of rutin or hesperidin can support the growth of Akkermansia muciniphila in vivo, and can increase the absolute number of Akkermansia muciniphila in vivo. It was confirmed that there is.

The present specification includes the contents described in the specification and / or the drawings of Japanese Patent Application No. 2017-215558, which is the basis of the priority of the present application.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

An Akkermansia muciniphila growth material comprising one or more polyphenols selected from the group consisting of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid.
The propagation material according to claim 1, wherein the quercetin glycoside comprises one or more selected from the group consisting of quercetin 3-D-galactide, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside. .
The growth material according to claim 1, comprising quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin or tannic acid.
The propagation material according to any one of claims 1 to 3, which is a food or drink, a medicine or a feed or a feed additive.
Akkermansia muciniphila cultured using a growth material containing one or more polyphenols selected from the group consisting of quercetin glycoside, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid Containing the culture.
The culture according to claim 5, wherein the quercetin glycoside comprises one or more selected from the group consisting of quercetin 3-D-galactide, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside. .
6. The culture of claim 5, wherein the propagation material comprises quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, or tannic acid.
The culture according to any one of claims 5 to 7, which is a food or drink, medicine or feed or feed additive.
Akkermansia muciniphila is cultured using a growth material containing one or more polyphenols selected from the group consisting of quercetin glycosides, isorhamnetin, cyanidin glucoside, hesperetin, hesperidin, apigenin, chrysin, tangeretin, nobiletin, and tannic acid A process for producing Akkermansia muciniphila and / or a culture thereof, comprising:
10. The method according to claim 9, wherein the quercetin glycoside comprises one or more selected from the group consisting of quercetin 3-D-galactide, quercetin 3-O-glucopyranoside, quercetin 3,4 diglucoside, rutin, and pertatoside. .
The method according to claim 10, wherein the propagation material comprises quercetin 3-O-glucopyranoside, rutin, cyanidin glucoside, hesperidin, apigenin, or tannic acid.