WO2023068279A1 - Method for screening bacteria of genus blautia - Google Patents

Abstract

The present invention provides a method for determining whether a Blautia wexlerae strain has a metabolism-improving function, wherein a Blautia wexlerae strain to be examined is determined to have a metabolism-improving function if the production of two or more substances selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid, and lactic acid is detected when the Blautia wexlerae strain to be examined is anaerobically cultured.

Description

Screening method for Blautia bacteria

The present invention relates to a screening method for bacteria belonging to the genus Blautia. More specifically, the present invention provides that Blautia wexlerae species have metabolic improving effects (that is, body weight gain reducing action, adipose tissue reducing action, adipose tissue inflammation suppressing action, fasting blood sugar level lowering action, fasting The present invention relates to a screening method for determining whether or not the present invention has blood insulin-lowering action, insulin resistance-improving action).

In recent years, obesity due to overeating and lack of exercise, especially metabolic syndrome accompanied by accumulation of visceral fat, has become a social problem. Metabolic syndrome is a condition in which two or more of hyperglycemia, hypertension, and dyslipidemia are combined with visceral fat obesity, and arteriosclerosis is likely to occur. It is estimated that 1 in 2 men and 1 in 5 women in Japan aged 40 to 74 have metabolic syndrome or are at risk of developing it. Therefore, the importance of optimizing calorie intake through dietary therapy has been advocated for the purpose of preventing or eliminating lipid accumulation in metabolic syndrome.

Recently, it has become clear that changes in the intestinal flora are involved in the host's energy regulation, nutritional intake, immune function, etc., and have a direct effect on pathological conditions such as obesity and diabetes. In particular, the correlation between obesity and intestinal bacteria has been actively studied by researchers around the world, and obese people have more (or less) specific types of intestinal bacteria than nonobese people It is also known that mice transplanted with intestinal bacteria from obese people tend to become obese. For example, Japanese people, who are said to have a low BMI and longevity, have a higher proportion of Blautia bacteria in their intestinal bacteria than people from other countries such as the United States, Denmark, China, and Venezuela. There is a report (Non-Patent Document 1). Also disclosed are compositions comprising bacterial strains of the genus Brauchia as a means of increasing microbiota diversity for use in the treatment and/or prevention of diseases associated with decreased microbiota diversity such as obesity and type 2 diabetes. It has been proposed (Patent Document 1).

However, what is specifically described in Patent Document 1 is that viable bacterial strains belonging to Blautia hydrogenotrophica have therapeutic activity against autoimmune diseases. No effect of Wexlerae is given. Moreover, even for Brautia hydrogenotrophica, it has not been proved at all whether it has a metabolic improving effect or an effect of killing dead bacteria.

To date, there have been no reports of a direct causal relationship between obesity and Brautia wexlerae. Comprehensive analysis of the bacterial flora in feces revealed that the proportion of Brautia wexlerae in the feces of non-obese Japanese was 11.91%, whereas the proportion of Brautia wexlerae in the feces of obese Japanese was 11.91%. While there is a report that it was significantly lower at 3.79% (Non-Patent Document 2), the proportion of Brautia wexlerae in the feces of lean Japanese is 3.798%, compared with 3.798% in obese Japanese. There is also a report that it was 5.589% in humans (Non-Patent Document 3), and it remains unclear whether there is a correlation between obesity and the amount of Brauchia wexlerae in the intestine. In addition, some strains of Brautia wexlerae are causative of diabetes, and it has been reported that the characteristics of the strains of Brautia wexlerae differ depending on their characteristics (Patent Document 2).

WO2018/109461 WO2019/235447

S. Nishijima et al., DNA Research, 23(2) 125-133 (2016). C. Kasai et al., BMC Gastroenterology, 15:100 (2015). A. Andoh et al., J Clin Biochem Nutr, 59(1) 65-70 (2016)

An object of the present invention is to provide a screening method for the intestinal bacterium, Brautia wexlerae, for determining whether it has the effect of preventing and/or improving metabolic disorders.

The present inventors have conducted intensive research in view of the above problems, and found that Brauchia wexlerae has a body weight gain-reducing action, adipose tissue-reducing action, adipose tissue inflammation-inhibiting action, fasting blood sugar level-lowering action, fasting L-ornithine, S ^- adenosylmethionine, It was discovered that acetylcholine, amylopectin, acetic acid, succinic acid, lactic acid, etc. are produced. The present inventors have succeeded in screening Brautia wexlerae strains having a metabolic improvement effect by using the production amounts of these substances as indices, and have completed the present invention.

That is, the present invention provides the following.
[1] A method for determining whether a strain belonging to Blautia wexlerae has a function of improving metabolism, comprising:
(i) anaerobically culturing the isolated test strain of Brautia wexlerae;
(ii) detecting production of two or more substances selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid in the resulting culture; and (iii) ) A method comprising the step of determining that the test strain has a function of improving metabolism when the production of two or more substances can be detected in step (ii).
[2] The method of [1], wherein two or more strains to be tested are screened for a strain of Brautia wexlerae having a function of improving metabolism.
[3] The method of [1] or [2], wherein the test strain is a strain of Brautia wexlerae present in the sample.
[4] The method of [3], wherein the specimen is human or non-human mammal feces.
[5] A composition for preventing or ameliorating metabolic disorders, comprising as an active ingredient at least one selected from the group consisting of viable, dead, and culture supernatants of Brautia wexlerae, the Brautia wexlerae strain. produces two or more substances selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid when the isolated strain is anaerobically cultured A thing, a composition.
[6] The metabolic disorder is at least one selected from the group consisting of obesity, diabetes, impaired glucose tolerance, hyperinsulinemia, dyslipidemia, increased adipose tissue and fatty liver, according to [5]. Composition.
[7] The composition according to [5] or [6], which is a food or food additive.
[8] The composition of [5] or [6], which is a pharmaceutical.
[9] The composition of [5] or [6], which is a feed or feed additive.
[5A] A method for preventing or improving metabolic abnormality in a subject, comprising having the subject ingest an effective amount of at least one selected from the group consisting of live bacteria, dead bacteria, and culture supernatant of Brautia wexlerae. 2 selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid, when the isolated strain is anaerobically cultured. A method that produces more than one species.
[5B] At least one selected from the group consisting of viable, dead and culture supernatants of Brautia wexlerae for use as a composition for preventing or improving metabolic disorders, said Brautia wexlerae The strain produces two or more substances selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid when the isolated strain is anaerobically cultured. At least one selected from the group consisting of live bacteria, dead bacteria and culture supernatant.

According to the present invention, live bacteria (probiotics) and / or dead bacteria (prebiotics) of the target Brautia wexlerae strain have an inhibitory effect on weight gain, an inhibitory effect on adipose tissue increase, and an adipose tissue inflammation Diseases or pathological conditions accompanied by metabolic abnormalities, such as obesity, diabetes, impaired glucose tolerance, and hyperinsulinemia, can be determined without conducting experiments such as administration to animals, as it can be determined whether they have inhibitory effects and inhibitory effects on elevation of blood sugar levels. , useful strains of Brautia wexlerae for preventing or improving dyslipidemia, increased adipose tissue, fatty liver, etc., can be quickly and easily found. In addition, since it is possible to isolate Brautia wexlerae strains in human feces and to confirm whether the strains are useful for metabolic disorders, it is also a clue to understand the characteristics of the host's intestinal flora. Therefore, the present invention can be used in the field of health industry, and compositions containing strains screened in the present invention can be used in various fields such as pharmaceuticals, foods, and feeds.

Mice were fed with a normal diet (Control diet, CD) and a high fat diet (HFD) for 8 weeks, during which the high-fat diet mice were fed with a viable bacterial culture of Brautia wexlerae (Bw) three times a week. (HFD+Bw) or medium (HFD) as a control was orally administered with a sonde, showing body weight fluctuation results (A) and photographs of mice after 8 weeks (B). The bar in the photograph indicates 1 cm. Mice were fed a normal diet (CD), a high-fat diet (HFD) for 8 weeks, during which time the high-fat diet mice were fed three times a week with live bacterial cultures of Brautia wexlerae (Bw) (HFD+Bw) or control. 8 shows a photograph of the peri-testicular adipose tissue and the weight of the peri-testicular adipose tissue (eAT) of mice after 8 weeks of administration of medium (HFD) by an oral probe. The bar in the photograph indicates 1 cm. *P<0.05 Mice were fed a normal diet (CD), a high-fat diet (HFD) for 8 weeks, during which time the high-fat diet mice were fed three times a week with live bacterial cultures of Brautia wexlerae (Bw) (HFD+Bw) or control. The blood sugar level (A) and blood insulin concentration (B) after 8 weeks are shown when the medium (HFD) was administered as an oral probe. **P<0.01 Mice were fed a normal diet (CD), a high-fat diet (HFD) for 8 weeks, during which time the high-fat diet mice were fed three times a week with live bacterial cultures of Brautia wexlerae (Bw) (HFD+Bw) or control. HOMA-IR, an index of insulin resistance after 8 weeks, is shown when the medium (HFD) was administered as an oral probe. HOMA-IR=fasting insulin level (μU/mL)×fasting blood glucose level (mg/dL)/405. **P<0.01 Mice were fed on a normal diet (CD), high-fat diet (HFD) for 8 weeks, during which time the high-fat diet mice were fed three times a week with live bacterial cultures (HFD + Bw) of Brautia wexlerae JCM17041 strain ^T (Bw). ) or the heat-killed cell suspension (HFD + BwHK) of Brautia wexlerae JCM17041 ^T strain (Bw) or the medium (HFD) as a control was administered with an oral sonde, body weight change result (A) and 8 weeks later (B) of the glucose tolerance test IPGTT in mice. Fig. 2 shows the results of the decrease and increase of components in the medium in which the Brauchia wexlerae JCM17041 ^T strain (Bw) was anaerobically cultured. S-adenosyl in each culture supernatant after anaerobically culturing Brauchia wexlerae JCM17041 ^T strain (Bw), Bacteroides vulgataus (Bv), Prevotella copri (Pc), and Faecalibacterium prausnitzii (Fp) Concentrations of methionine, acetylcholine, and L-ornithine are shown. Brautia wexlerae JCM17041 ^T strain (Bw), Bacteroides vulgataus (Bv), Prevotella copri (Pc), Faecalibacterium prausnitzii (Fp) Amylose and amylopectin contained in each freeze-dried cell after anaerobic culture indicate quantity. FIG. 4 shows concentrations of succinic acid, lactic acid, and acetic acid in the culture supernatant after anaerobic culture of Brautia wexlerae JCM17041 ^T strain (Bw). FIG. 1 shows the concentration of S-adenosylmethionine and L-ornithine in each culture supernatant after anaerobically culturing Brauchia wexlerae JCM17041 ^T strain (Bw 17041) and JCM31267 ^T strain (Bw 31267). Mice were fed on a normal diet (CD), high-fat diet (HFD) for 8 weeks, during which time the high-fat diet mice were fed three times a week with live bacterial cultures (HFD+Bw) of Brautia wexlerae JCM31267 ^T strain (Bw). 31267) or the heat-killed cell suspension (HFD + BwHK 31267) of Brautia wexlerae JCM31267 ^T strain (Bw) or the culture medium (HFD) as a control was administered with an oral sonde (C) and 8 Results (D) of the mouse glucose tolerance test IPGTT after a week are shown.

The present invention provides a method for determining whether a strain of Brautia wexlerae has an effect of preventing and/or improving metabolic disorders, and a method for screening a strain of Brautia wexlerae having a function of improving metabolism (hereinafter collectively referred to as "the strain of the present invention. method”).

1. Brautia wexlerae Brautia wexlerae , which is the test subject in the method of the present invention, belongs to the phylum Gram-positive bacteria (Firmicutes), the class Clostridia, the order Clostridiales, the family Lachnospiraceae, and the genus Blautia. It is classified as a Gram-positive obligate anaerobe. The genus Blautia includes, in addition to Blautia wexlerae, Blautia hydrogenotrophica, Blautia stercoris, Blautia faecis, Blautia coccoides, and Blautia coccoides. (Blautia gluceras), Blautia hansenii, Blautia luti, Blautia producta, Blautia schinkii, and the like exist, each of which assimilates various carbon sources. They differ in potency, final metabolites, 16S rRNA similarity, etc. (see, eg, International Journal of Systematic and eEvolutionary Microbiology (2008), 58, 1896-1902). Brauchia wexlerae is suggested to have a therapeutic effect on autoimmune diseases in the above-mentioned Patent Document 1 (WO2018/109461). Big difference.

Whether or not the bacterium isolated from the isolation source is a strain belonging to Brautia wexlerae can be determined, for example, by PCR-amplifying all or part of the 16S rRNA gene using the genomic DNA extracted from the strain as a template, and determining the nucleotide sequence of the amplified fragment. can be determined and compared to known Brautia wexlerae sequence data and a phylogenetic analysis can be performed. A phylogenetic analysis and a method for constructing a phylogenetic tree can be performed, for example, according to the following procedures.

First, genomic DNA that serves as a template is extracted from bacteria. Methods for extracting DNA from bacteria are known, and any method may be used. In general, a method of treating cells with a cell wall-degrading enzyme such as lysozyme, a method of physical disruption with glass beads, a treatment method of repeated freezing and thawing, and the like are used. A commercially available reagent for DNA extraction can also be used. Genomic DNA does not necessarily have to be extracted in an intact state. Therefore, it is possible to appropriately select a method that has a low possibility of sample contamination, is easy to operate, and can be performed quickly.

Next, the target DNA encoding 16S rRNA is amplified by polymerase chain reaction (PCR). The sequences of the primers used in PCR can be appropriately designed so that target DNAs encoding 16S rRNAs of all known bacteria belonging to at least Brautia wexlerae are amplified, but are usually conserved across species. A primer consisting of a sequence (universal primer; for example, a primer set of 27F and 357R that amplifies about 350 bases of the V1-V2 region, and 342F that amplifies about 460 bases of the V3-V4 region used in the examples described later and 806R primer sets) are used. In addition, PCR conditions are not particularly limited, and can be appropriately selected within a commonly used range. Using commercially available PCR reagents, the reaction can be performed according to the attached instructions.

DNA fragments amplified by PCR are purified using a spin column or the like as necessary, and then their base sequences are determined. Determination of base sequences can be carried out according to standard methods.

The determined nucleotide sequence can be searched for homology with known bacterial 16S ribosomal DNA sequences using an appropriate gene sequence database and homology search program to extract known sequences showing the highest homology. . For example, BLAST and FASTA can be used through the homepage of the DNA Data Bank of Japan (DDBJ). By selecting blastn or fasta as a program, using the determined nucleotide sequence as a query, and selecting 16S rRNA (Prokaryotes) as a search target database, a known sequence exhibiting high homology is extracted and output. Any other gene sequence database can be used as long as it contains a data set of nucleotide sequences of bacterial 16S rRNA genes (for example, RDP (http://rdp.cme.msu.edu), Silva (http:/ /www.arb-silva.de) etc.). A homology search program known per se other than the above can also be used.

In general, for bacteria, 95% or more 16S rRNA gene sequence identity is required for identification at the genus level, and 98% or more for identification at the species level (Science 2005, 307, 1915-1920). . Therefore, when the sequence of the 16S rRNA gene of an isolated bacterium has 98% or more identity with the known sequence of a bacterium belonging to Brautia wexlerae as a result of homology search, the bacterium is regarded as a bacterium belonging to Brautia wexlerae. can be identified.

A known 16S rRNA gene sequence that serves as a reference for discrimination includes, but is not limited to, the sequence derived from Blautia wexlerae JCM 17041 ^T strain (SEQ ID NO: 1) registered in GenBank under accession number LC037229.1. Therefore, when the nucleotide sequence of the PCR-amplified fragment has 98% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, the isolated bacterium can be identified as a bacterium belonging to Brautia wexlerae. .

In addition, it is also possible to estimate the molecular evolutionary tree based on the base sequence of the amplified DNA and identify the taxonomic position of the isolated bacteria. Molecular evolution phylogenetic tree analysis software is open to the public on the Internet, etc., and can be used (CLUSTAL W, etc.). As a result of phylogenetic tree analysis, when the isolated bacterium is positioned within the same cluster as a bacterium belonging to Blautia wexlerae, the bacterium is identified as a bacterium belonging to one of Blautia wexlerae. can do.

In the present invention, the strain of Brautia wexlerae is anaerobically cultured, the medium after culture is analyzed by LC-MS/MS or the like, and an increase in a specific component is detected to metabolize the strain of Brautia wexlerae. It is determined that there is an effect on the anomaly.

Brautia wexlerae can be cultured under known culture conditions and maintained and amplified. Since Brautia wexlerae is an obligatory anaerobe, it is cultured in an anaerobe medium. In addition to the carbon source, nitrogen source and inorganic substances necessary for the growth of bacteria, reducing agents (e.g., cysteine hydrochloride, nitrogen sulfide, titanium citrate, etc.) are added to the anaerobic culture medium to lower the redox potential. Supplemented medium is included. Here, carbon sources include, for example, glucose, dextrin, soluble starch, sucrose, etc.; nitrogen sources include, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato. Inorganic or organic substances such as extracts; inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like, respectively. Furthermore, blood derived from horses, rabbits, and sheep, hemin, vitamin K, and the like can be added to the anaerobic culture medium. The medium may be solid or liquid.

More specifically, Difco ^™ reinforced clostridial medium (#218081; BD Bioscience, San Jose, Calif.) can be used as the medium.

Since Brautia wexlerae is an obligatory anaerobic bacterium, cultivation is carried out under anaerobic conditions (oxygen concentration of 1 ppm or less). For example, it can be cultured in an anaerobic gas chamber under a mixed gas atmosphere of 10% CO ₂ , 10% H ₂ and 80% N ₂ . The culture temperature is about 37°C. The culture period is, for example, 12 to 72 hours, preferably 24 to 48 hours, but is not particularly limited.

2. The method of the present invention After the culture of Brautia wexlerae, the culture supernatant is analyzed for components. Cultivation may be performed under known culture conditions, but anaerobic conditions (oxygen concentration of 1 ppm or less), for example, in an anaerobic gas chamber, under a mixed gas atmosphere of 10% CO ₂ , 10% H ₂ and 80% N ₂ Cultivate in The culture temperature is about 37° C., and the culture period is 12 to 72 hours, preferably 24 to 48 hours. Specific components of the culture supernatant after culturing are measured by LC-MS/MS or HPLC. As for amylopectin produced not only in the medium but also in the cells, the cells collected after culturing are freeze-dried, and the freeze-dried cells are analyzed.

Measurement of L-ornithine, S-adenosylmethionine, and acetylcholine in the culture supernatant by LC-MS/MS L-ornithine, S-adenosylmethionine, and acetylcholine in the culture supernatant of Brautia wexlerae were measured by liquid chromatography-mass spectrometry. It can be analyzed by the method (LC-MS/MS). Although the apparatus used for LC-MS/MS analysis is not particularly limited, for example, LCMS-8050, an ultrafast triple quadrupole LC/MS/MS manufactured by Shimadzu Corporation, can be used.

Measurement of Succinic Acid, Lactic Acid and Acetic Acid in Culture Supernatant by HPLC Succinic acid , lactic acid and acetic acid in the culture supernatant of Brautia wexlerae can be analyzed by high performance liquid chromatography (HPLC). Although the apparatus used for HPLC analysis is not particularly limited, for example, a high-performance liquid chromatograph apparatus manufactured by Shimadzu Corporation or Waters Corporation is used. A fatty acid analysis kit manufactured by YMC Co., Ltd. can be used for labeling fatty acids.

Measurement of Amylopectin in Freeze-dried Cells After Culture After culturing Brautia wexlerae, only the cells are collected by centrifugation, and the collected cells are freeze-dried. The amount of amylopectin in the freeze-dried cells can be measured, for example, by using an amylose/amylopectin analysis kit manufactured by Megazyme.

Determination of whether a Brauchia wexlerae strain has a function of improving metabolism Whether a strain of Brautia wexlerae has a function of improving metabolism can be confirmed by LC-MS/MS analysis, HPLC analysis, and analysis of freeze-dried bacterial cells of the medium. The production of two or more of ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid, and lactic acid can be confirmed to determine that the substance has a function of improving metabolism. The determination is made by confirming the production of preferably 4 or more, more preferably 6 or more. Measurement may be performed for any two or more of the above seven substances as long as production of two or more substances can be confirmed. In one preferred embodiment, all of the above seven substances are measured. The production of L-ornithine, S-adenosylmethionine, acetylcholine, acetic acid, succinic acid and lactic acid by the tested strains is measured, for example, in media not inoculated with microorganisms after incubation under identical anaerobic conditions. It can be confirmed by the amount of each substance in the culture supernatant of the strain to be tested being significantly higher than the amount of each substance. In addition, the production of amylopectin by the strain to be tested can be confirmed, for example, by detecting a significant increase in the amount of amylopectin per cell weight of the strain to be tested before and after anaerobic culture.

3. Determination of whether Brautia wexlerae in a sample has a function of improving metabolism Whether or not a strain of Brautia wexlerae contained in a sample such as feces has a function of improving metabolism can be determined by isolating Brautia wexlerae from a sample. It can be determined by anaerobically culturing the Brautia wexlerae strain and measuring in the same manner. Specimens include not only intestinal flora such as feces, but also oral flora such as saliva, skin flora on the skin surface, vaginal flora, and other living organisms. It can also be applied to environmental specimens such as soil and water. Preferred specimens in the present invention are human and non-human mammal feces.
As a method for isolating Brautia wexlerae from a specimen, for example, the specimen is suspended in sterile phosphate-buffered saline (PBS) or the like, and the resulting suspension is treated with a platinum loop or the like to isolate Brautia wexlerae. Candidates for Brautia wexlerae can be selected from the mycological characteristics (morphological characteristics, biochemical characteristics, etc.) of colonies that have appeared after plating on a plate medium suitable for culture. Identification of a selected colony as a Brautia wexlerae colony can be performed, for example, by the 16S rRNA gene analysis described above.

4. A composition containing Brautia wexlerae determined to have a function of improving metabolism. may be provided). Brautia wexlerae strains determined to have a function of improving metabolism by the method of the present invention (that is, the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid when anaerobically cultured A strain that produces two or more substances selected from) isolated viable bacteria, as long as they are in a living state, can be obtained from the culture (bacteria) as it is, or from the culture by a method known per se, For example, wet cells collected by centrifugation, filtration, magnetic separation, etc., or their washed products (can be washed with sterilized water, medium, PBS, etc.), or their freeze-dried powders, etc. It can be blended in the composition of the present invention in the state of "product".

Killed bacteria of Brautia wexerellae can be prepared by physically and/or chemically treating and sterilizing viable cells according to standard methods. Examples of physical treatment methods include heat treatment (autoclave treatment, pasteurization, high temperature sterilization, etc.), oxygen exposure treatment, drying treatment (heat drying, freeze drying, etc.), electromagnetic wave treatment (ultraviolet sterilization, gamma ray sterilization, etc.), Grinding/crushing treatment (glass bead treatment, French press treatment, ultrasonic treatment, etc.), or a combination thereof. Examples of chemical treatment methods include chemical treatment (formaldehyde treatment, surfactant treatment, acid treatment, alkali treatment), enzymatic treatment (protease treatment, saccharifying enzyme treatment, etc.), and combinations thereof. For example, in the case of heat treatment, dead cells can be prepared by treating live cells of the bacterium of the present invention at a temperature of about 60 to 120° C. for about several seconds to 30 minutes. Killed bacteria of Brautia wexerellae can be obtained directly from the above dead cells (including physically and chemically disrupted cells) or by various known extraction methods (e.g., various solvent extractions, supercritical fluid extractions). It can be blended in the composition of the present invention in the state of "treated dead cells" such as the resulting extract.

As an active ingredient in the composition of the present invention, a culture supernatant obtained by culturing a Brautia wexlerae strain determined to have a function of improving metabolism by the above method can also be used. The culture supernatant can be prepared by removing cells from the culture solution of Brautia wexlerae by a method known per se such as centrifugation or filtration. The culture supernatant can be added to the composition of the present invention as it is or after being concentrated as appropriate.

The Brautia wexlerae strain to be blended in the composition of the present invention may be used singly or in combination of two or more strains. In addition, live bacteria and dead bacteria (including each treated bacterial cell), live bacteria and their culture supernatant (the whole culture may be used), killed bacteria and culture supernatant, and live bacteria, dead bacteria and culture Any combination of supernatants may be used.

Viable or dead bacteria of Brautia wexlerae strains determined to have a function of improving metabolism, their processed products, and their culture supernatants are added alone or pharmaceutically or food or feed processing acceptable additives can be formulated with a product. Alternatively, it can be incorporated into a pharmaceutical composition or food or feed as a pharmaceutical additive or food or feed additive.

When the composition of the present invention is provided as a pharmaceutical or pharmaceutical additive, the pharmaceutical or pharmaceutical containing the additive can be, for example, powders, granules, pills, soft capsules, hard capsules, tablets, chewable tablets, rapidly disintegrating tablets, It can be formulated into tablets, syrups, solutions, suspensions, suppositories, injections and the like.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and capsules (including soft capsules). , syrups, emulsions, suspensions and the like. Such compositions are produced by known methods and may contain additives commonly used in the pharmaceutical field, such as excipients, binders, disintegrants, lubricants, and the like. Examples of excipients include animal and vegetable oils such as soybean oil, safflower oil, olive oil, germ oil, sunflower oil, beef tallow, and sardine oil; polyhydric alcohols such as polyethylene glycol, propylene glycol, glycerin and sorbitol; and sorbitan fatty acid esters. , surfactants such as sucrose fatty acid esters, glycerin fatty acid esters and polyglycerin fatty acid esters, purified water, lactose, starch, crystalline cellulose, D-mannitol, lecithin, gum arabic, sorbitol solution, sugar solution and the like. Binders include, for example, hydroxypropylmethylcellulose, hydroxypropylcellulose, gelatin, pregelatinized starch, polyvinylpyrrolidone, polyvinyl alcohol and the like. Disintegrants include, for example, carmellose calcium, carmellose sodium, croscarmellose sodium, crospovidone, low-substituted hydroxypropylcellulose, corn starch and the like. Lubricants include, for example, talc, hydrogenated vegetable oils, waxes, light silicic anhydride derived from natural products and their derivatives, stearic acid, magnesium stearate, calcium stearate, aluminum stearate and the like.

Sweeteners, coloring agents, pH adjusters, flavoring agents, various amino acids, etc. can also be added to the above composition. Moreover, tablets and granules may be coated by a well-known method. A liquid formulation may be dissolved or suspended in water or other suitable medium at the time of administration.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections can be prepared, for example, by suspending or emulsifying cells or processed cells of Blautia wexlerae in a sterile aqueous or oily liquid commonly used for injections. can. Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like. As the oily liquid, for example, sesame oil, soybean oil, or the like is used. Suppositories used for rectal administration can be prepared by mixing the bacterial cells, treated bacterial cells and/or culture supernatant of Brautia wexlerae with a conventional suppository base.

Furthermore, when provided as a pharmaceutical or pharmaceutical additive, the composition of the present invention may be used in combination with other drugs such as anti-inflammatory drugs, anti-arteriosclerotic drugs, anti-diabetic drugs, etc., depending on the target disease. good. The composition of the present invention and the concomitant drug may be formulated as a single composition (mixture), or may be provided as separate compositions. When provided as separate compositions, the composition of the present invention and the concomitant drug can be administered to a subject at the same time or at different times, by the same route or by different routes.

When the composition of the present invention is provided as a food (or feed) or a food additive (or feed additive), the food (or feed) or the food (or feed) containing the additive may be a solution, suspension, There is no particular limitation as long as it is in an orally ingestible form such as turbidity, powder, or solid molding. Specific examples include supplements (powder, granules, soft capsules, hard capsules, tablets, chewable tablets, rapidly disintegrating tablets, syrups, liquids, etc.), beverages (carbonated drinks, lactic acid drinks, sports drinks, fruit juice drinks, vegetable drinks, soy milk drinks). , coffee drinks, tea drinks, powdered drinks, concentrated drinks, nutritional drinks, alcoholic beverages, etc.), dairy products (yogurt, butter, cheese, ice cream, etc.), confectionery (gummy, jelly, gum, chocolate, cookies, candy, caramel , Japanese sweets, snacks, etc.), instant foods (instant noodles, retort pouch foods, canned foods, microwave oven foods, instant soups/miso soups, freeze-dried foods, etc.), oils, fatty foods (mayonnaise, dressings, cream, margarine, etc.), Wheat flour products (bread, pasta, noodles, cake mixes, bread crumbs, etc.), seasonings (sauces, processed tomato seasonings, flavor seasonings, cooking mixes, sauces, etc.), processed livestock products (livestock meat hams, sausages, etc.) be done.

The above food (or feed) contains various nutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K, etc.), various minerals ( magnesium, zinc, iron, sodium, potassium, selenium, etc.), dietary fiber, dispersants, stabilizers such as emulsifiers, sweeteners, taste ingredients (citric acid, malic acid, etc.), flavors, royal jelly, propolis, agaricus, etc. can be compounded.

The viable cell count of Brautia wexlerae contained in the composition of the present invention is, for example, 10 ⁴ to 10 ¹² colony forming units (cfu), preferably 10 ⁶ to 10 ¹⁰ cfu, as a daily intake. . As the number of dead bacteria, the number of viable bacteria (cfu) before sterilization is used as a guide for the number of dead bacteria, and the intake per day is 10 ⁴ to 10 ¹² cfu before sterilization, preferably 10 ⁶ ~10 ¹⁰ cfu. Also, the culture supernatant can be prepared from a culture containing the above number of viable bacteria per unit volume (amount to be added to the composition).

The composition of the present invention may further contain cells of other useful microorganisms or processed cells thereof. Such other microorganisms include, for example, genus Lactobacillus, genus Streptococcus, genus Leuconostoc, genus Pediococcus, genus Lactococcus, and Enterococcus. ) genus, Bifidobacterium genus, yeast, Bacillus genus, Clostridium butyricum, Aspergillus oryzae, etc., but not limited thereto. These concomitant microorganisms can be added to the composition of the present invention in the form of not only viable cells but also dead cells or crushed cells, cell extracts, cell components, etc., as long as they are effective. You can also
The amount of the microorganism to be used in combination is, for example, 10 ⁴ to 10 ¹² colony forming units (cfu), preferably 10 ⁶ to 10 ¹⁰ cfu, as a daily intake.

5. Uses of the composition of the present invention As described above, the Brautia wexlerae strain determined to have a function of improving metabolism has a metabolic improving effect (that is, weight gain rate reducing effect, adipose tissue reducing effect, adipose tissue inflammation inhibitory action, fasting blood sugar level lowering action, fasting blood insulin lowering action, and insulin resistance improving action), and therefore metabolic function is improved by enriching the bacterial species in the intestinal flora. can do. In addition, dead cells and culture supernatants of Brautia wexlerae can exert a metabolic improving effect as prebiotics in the same manner as viable cells. Therefore, the composition of the present invention is useful as a pharmaceutical or pharmaceutical additive, or a functional food (or feed) or food (or feed) additive for preventing and/or improving metabolic disorders.

As a disease related to the composition for improving metabolic disorder of the present invention, it can also be applied to improvement of lifestyle-related diseases. “Lifestyle-related diseases” refers to a group of diseases in which lifestyle habits such as eating habits, exercise habits, rest, smoking, and drinking are involved in the onset and progression of such diseases. Adult obesity, childhood obesity, malnutrition, anorexia, and stomach cancer , colon cancer, gout, hypertension, arteriosclerosis, kidney stones, myocardial infarction, angina pectoris, gastric ulcer, kidney disease, osteoporosis, periodontitis, alcoholic hepatitis, liver cirrhosis, liver cancer, lung cancer, bronchitis , emphysema, periodontal disease, stroke, cerebral infarction, aneurysm, death from overwork, and insomnia.

Furthermore, the composition for improving metabolism of the present invention is useful for diabetes (type 1 diabetes, type 2 diabetes, gestational diabetes, etc.), diabetic complications (arteriosclerotic disease, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy). etc.), dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, insulin resistance, and prevention or improvement of fatty liver.

The composition of the present invention can be applied to humans or other mammals (e.g., dogs, cats, mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, monkeys, etc.) as described above. The daily intake can be taken orally once or divided into several times a day. Alternatively, it can be administered rectally.

When the composition of the present invention is provided as a food, the food can be sold with a label indicating that it is used for improving metabolic disorders. Here, "indication" means all actions to inform consumers of the above-mentioned use. Regardless of the object, medium, etc. to be displayed, all correspond to the "display" in the present invention. However, it is preferable to display the product in an expression that allows consumers to directly recognize the usage. Specifically, the act of describing the above-mentioned use on the product or product packaging related to the food of the present invention, transferring, delivering, or displaying for transfer or delivery the product or product packaging that describes the above-mentioned use , the act of importing, displaying or distributing the above-mentioned use in advertisements, price lists or transaction documents related to products, or by electromagnetic (Internet, etc.) methods by describing the above-mentioned use in information containing these contents Act of providing, etc. can be exemplified.

On the other hand, as the labeling, it is preferable to be a labeling approved by the government, etc. , catalogs, pamphlets, POP and other advertising materials at sales sites, and other documents.

In addition, for example, labeling as health food, functional food, enteral nutrition food, food for special dietary use, food with nutrient function claims, quasi-drugs, etc. can be exemplified, and other labels approved by the Ministry of Health, Labor and Welfare, such as , food for specified health use, and labeling approved by similar systems. Examples of the latter include labeling as a food for specified health uses, labeling as a conditionally specified health food, labeling to the effect that it affects the structure and functions of the body, and labeling to reduce the risk of disease. In other words, labeling as a food for specified health use (especially labeling for health use) stipulated in the Ordinance for Enforcement of the Health Promotion Law (Ministry of Health, Labor and Welfare Ordinance No. 86 of April 30, 2003), and similar Display is a typical example.

Although the present invention will be described in more detail with reference to the following examples, the examples are merely illustrations of the present invention and do not limit the scope of the present invention.

Example 1 Effect of Improving Metabolism in Mice by Administration of Live or Killed Bacteria of Brautia wexlerae JCM17041 (Materials and Methods)
Animals Mice used were C57BL/6JJmsSlc. All mice were housed in an SPF grade animal facility within the National Institutes of Biomedical Innovation, Health and Nutrition. Animals were allowed food and water ad libitum under a strict 12 hour light cycle. 4-week-old mice were fed a normal diet (Oriental yeast, AIN-93M), a high-fat diet (Oriental yeast, AIN-93G), a high-fat diet + Brautia wexlerae JCM17041 (Blautia) live bacteria, a high-fat diet + Brautia wexlerae JCM17041 (Blautia) killed cells were divided into 4 groups. Blautia wexlerae JCM17041 (Blautia) viable bacteria were cultured to a turbidity (OD600 value) of 1 to 3 in the culture solution, and then ingested with an oral probe at a dose of 0.5 mL three times a week. Brautia wexlerae JCM17041 (Blautia) dead cells were obtained by heat-treating the same number of Brautia wexlerae JCM17041 viable cells at 60° C. for 30 minutes. Experimental animals were used in accordance with the Declaration of Helsinki and the methods described in the "Standards for the Care and Storage of Experimental Animals" established by the Japanese Association for Laboratory Animals. All experiments were approved by the National Institute of Biomedical Innovation, Health and Nutrition (Institutional Approval Nos. DS25-2, DS25-3).

Cultivation and preparation of Brautia wexlerae JCM17041 Brautia wexlerae (JCM17041; Japan Collection of Microorganisms) was cultured anaerobically in Difco ^™ reinforced clostridial medium (#218081; BD Bioscience, San Jose, 3 CA). An anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.) containing 10% _CO2 , 10% hydrogen and 80% nitrogen was used for all anaerobic microbial steps. The cultured Brautia wexlerae was used as a viable cell. As for dead cells, viable cells of Brautia wexlerae were collected by centrifugation, resuspended in Difco ^™ reinforced clostridial medium, treated at 60° C. for 30 minutes, and heat sterilized. Successful heat sterilization was confirmed by plating heat-treated bacteria and no growth.

Evaluation of Weight Gain Rate Each mouse was weighed weekly and evaluated as the weight gain rate from the start of administration.

Evaluation of Peritesticular Adipose Tissue Amount In each mouse, necropsy was performed 10 weeks after the start of administration, and the peritesticular adipose tissue was excised separately on the left and right sides, and the weight thereof was measured.

Fasting Blood Glucose Level, Plasma Insulin Level, Evaluation of Insulin Resistance Blood was collected from each mouse during early morning fasting at week 8 from the start of administration, and fasting blood sugar level (mg/dL) and plasma insulin level (μU/ mL) was measured. Further, from the obtained results, HOMA-IR (fasting insulin level (μU/mL)×fasting blood sugar level (mg/dL)/405) was used as an index of insulin resistance to evaluate. Blood glucose levels were measured from tail vein blood using One Touch Ultraview (manufactured by LifeScan Japan). Plasma insulin levels were obtained by collecting blood from the fundus using hematocrit capillary heparin treatment (manufactured by HIRSCHMANN), and measuring the plasma using a Levis insulin mouse T (manufactured by Fujifilm Wako Shibayagi).

Evaluation of Glucose Tolerance Test IPGTT In each mouse, an intraperitoneal glucose tolerance test IPGTT was performed using 8 weeks after the start of administration. Eight weeks after the start of administration, a 0.2 g/mL glucose solution was injected intraperitoneally at 10 μL per 1 g of mouse body weight during early morning fasting. Blood glucose levels were measured from blood samples before administration and 10, 20, 30, 60, 90, and 120 minutes after the start of administration.

Statistical Analysis Data were expressed as mean±standard error or Box Plot. Student's two-tailed t-test was used to assess significant differences between two groups, and analysis of variance (ANOVA) was used to assess significant differences between multiple groups. For all tests, values of P<0.05 were considered statistically significant.

(result)
Assessment of body weight gain following administration of Brautia wexleraea JCM17041 To determine the effect of Brautia wexleraea JCM17041 on body weight, 4-week-old mice were orally fed with live bacteria of Brautia wexleraea JCM17041 three times per week for 8 weeks. let me Mice that orally ingested live bacteria of Brautia wexlerae JCM17041 had a significantly reduced body weight gain rate compared to high-fat-fed mice that received only the medium as a control (Fig. 1).

Evaluation of adipose tissue by administration of Brautia wexlerae JCM17041 Mice administered live bacteria of Brautia wexlerae suppressed the accumulation of peri-testicular adipose tissue compared to high-fat diet mice administered only medium as a control (Fig. 2 ).

Evaluation of fasting blood glucose level, blood insulin, and insulin resistance by administration of Brautia wexlerae JCM17041 Mice administered with live bacteria of Brautia wexlerae JCM17041 are hungry compared to high-fat-fed mice administered medium only as a control. It significantly suppressed the blood glucose level, blood insulin, and insulin resistance at that time (Figs. 3 and 4).

Evaluation of Weight Gain Rate by Administering Killed Brauchia wexlerae JCM17041 Mice administered with killed Brauchia wexlerae JCM17041 had significantly suppressed weight gain rate compared to high-fat diet mice administered medium only as a control. , the effect was similar to that of the viable B. wexlerae bacteria (Fig. 5A).

Evaluation of glucose tolerance test IPGTT by administration of Brauchia wexlerae JCM17041 Mice administered live or killed Brauchia wexlerae JCM17041 significantly increased blood glucose compared to high-fat diet mice administered medium only as a control The effect was similar between viable and dead bacteria (Fig. 5B).

Example 2 LC-MS/MS analysis of ^culture supernatant of Brautia wexlerae JCM17041 Anaerobically cultured at 37° C. for 48 hours. Water was added to 100 μL of the culture supernatant after culturing to make 200 μL, mixed with 400 μL of a methanol solution containing methionine sulfone as an internal standard, and then 400 μL of chloroform. After centrifugation at 4° C. and 20,000×g for 15 minutes, 200 μL of the supernatant was centrifuged through a 5-kDa cutoff filter (manufactured by Human Metabolome Technology). The filtrate was lyophilized, resuspended in water and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). LC-MS/MS analysis was performed using a Nexera system (manufactured by Shimadzu Corporation) equipped with two LC-40D pumps, a DGU-405 degasser, a SIL-40C autosampler, a CTO-40C column oven, and a CBM-40 control module. , LCMA-8050 quadrupole mass spectrometer (manufactured by Shimadzu Corporation). A pentafluorophenylpropyl column (Discovery HS F5, 150 mm x 2.1 mm, 3 μm; Sigma-Aldrich) was used for the separation of metabolites using the software LabSolutions LCMS with the Shimadzu LC-MS/MS method package. analysis was performed. Fig. 6 shows the results of the decrease and increase of the components in the culture supernatant obtained by anaerobically culturing the Brauchia wexlerae JCM17041 ^T strain (Bw). In addition, S-adenosylmethionine and acetylcholine in the culture supernatant of Bacteroides vulgataus (Bv), Prevotella copri (Pc), and Faecalibacterium prausnitzii (Fp) prepared by culturing in the same manner as Brauchia wexlerae JCM17041 , and L-ornithine concentrations are shown in FIG. S-adenosylmethionine, acetylcholine and L-ornithine can be confirmed in the culture supernatant after culture of Brauchia wexlerae JCM17041, indicating that these substances are produced by culture.

Example 3 Measurement of amylopectin in freeze-dried cells after culture of Brautia wexlerae JCM17041 The cells of Brautia wexlerae JCM17041 cultured in Example 2 after culture were centrifuged at 10,000 x g at 4°C for 10 minutes. Harvested and lyophilized at -80°C. The amounts of amylose and amylopectin in the freeze-dried samples were determined using an amylose/amylopectin assay kit manufactured by Megazyme. Bacteroides vulgataus (Bv), Prevotella copri (Pc), and Faecalibacterium prausnitzii (Fp) were similarly measured. The results are shown in FIG. Amylopectin is highly accumulated in the freeze-dried cells of Brautia wexlerae JCM17041 after culture, indicating that the culture produces amylopectin.

Example 4 Fatty acid analysis of Brautia wexlerae JCM17041 culture supernatant by HPLC After culture of Brautia wexlerae JCM17041 cultured in Example 2, the medium supernatant was labeled with a fatty acid analysis kit manufactured by YMC Co., Ltd., followed by the manufacturer's instructions. , 6.0×250 mm YMC-Pack FA column (manufactured by YMC) was used to measure succinic acid, lactic acid and acetic acid by high performance liquid chromatography (HPLC). Detecting UV spectra were measured at 400 nm. The results are shown in FIG. Succinic acid, lactic acid, and acetic acid can be confirmed in the culture supernatant of Brautia wexlerae JCM17041, indicating that these short-chain fatty acids are produced by cultivation.

From the above results, Brautia wexlerae JCM17041, which was confirmed to have a function of improving metabolism by mouse animal experiments, is L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid in anaerobic culture. , characteristically produces lactic acid, and by confirming the production of these, it can be determined that it has the ability to improve metabolism.

Example 5 Verification with another strain of Brautia wexlerae (JCM31267) For another strain of Brautia wexlerae JCM31267 (Japan Collection of Microorganisms), LC-MS/MS of the culture supernatant after anaerobic culture in the same manner as in Example 2 Analysis was carried out. Brautia wexlerae JCM31267 was cultured in the same manner as JCM17041, and the culture supernatant was prepared. The results are shown in FIG. It was confirmed that Brauchia wexlerae JCM31267 also produced S-adenosylmethionine and L-ornithine in the culture supernatant.
From this result, it was determined that Brautia wexlerae JCM31267 has a function of improving metabolism. The results are shown in FIG. From the weight gain results (C), regardless of whether the bacteria were live or killed, the high-fat diet mice administered with Brautia wexlerae JCM31267 showed a higher fat diet than the high-fat diet mice administered medium only as a control. It can be seen that weight gain is suppressed by In addition, the result (D) of the glucose tolerance test IPGTT also shows that the administration of Brautia wexlerae JCM31267 significantly suppresses the increase in blood glucose in both live and dead bacteria compared to the control.

From the above results, it was shown that it is possible to verify that the strain of Brautia wexlerae has a function of improving metabolism by detecting specific substances produced by anaerobic culture of the strain.

While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments may be varied.
The contents of all publications, including patents and patent applications mentioned herein, are hereby incorporated by reference to the same extent as if expressly set forth in their entirety. .
This application is based on Japanese Patent Application No. 2021-170540 filed in Japan on October 18, 2021, the entire contents of which are incorporated herein by reference.

The method of the present invention can easily determine whether the intestinal bacterium, Brautia wexlerae, is useful for metabolic diseases such as lifestyle-related diseases. Moreover, it is useful also as a medical use using it.

Claims (9)

A method for determining whether a strain belonging to Blautia wexlerae has a function of improving metabolism, comprising:
(i) anaerobically culturing the isolated test strain of Brautia wexlerae;
(ii) detecting production of two or more substances selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid in the resulting culture; and (iii) ) A method comprising the step of determining that the test strain has a function of improving metabolism when the production of two or more substances can be detected in step (ii). The method according to claim 1, wherein a strain of Brautia wexlerae having a function of improving metabolism is screened from two or more strains to be tested. The method according to claim 1 or 2, wherein the strain to be tested is a strain of Brautia wexlerae present in the specimen. The method according to claim 3, wherein the specimen is feces of a human or non-human mammal. A composition for preventing or improving metabolic disorders, comprising as an active ingredient at least one selected from the group consisting of viable, dead, and culture supernatants of Brautia wexlerae, wherein the Brautia wexlerae strain is a single When the isolated strain is anaerobically cultured, it produces two or more substances selected from the group consisting of L-ornithine, S-adenosylmethionine, acetylcholine, amylopectin, acetic acid, succinic acid and lactic acid. ,Composition. The composition according to claim 5, wherein the metabolic disorder is at least one selected from the group consisting of obesity, diabetes, impaired glucose tolerance, hyperinsulinemia, dyslipidemia, increased adipose tissue and fatty liver. The composition according to claim 5 or 6, which is a food or food additive. The composition according to claim 5 or 6, which is a pharmaceutical. The composition according to claim 5 or 6, which is a feed or feed additive.

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