JP2022156305A - Composition for promoting erythropoietin production - Google Patents

Abstract

To provide an ingredient capable of promoting EPO production, particularly an orally ingestible ingredient.SOLUTION: The ingredient includes Bifidobacterium bacterium as an active ingredient for promoting erythropoietin (EPO) production and is preferably used for preventing and/or improving anemia due to shortage of EPO production amount such as renal anemia.SELECTED DRAWING: None

Description

The present invention relates to compositions for promoting erythropoietin production.

Erythropoietin (hereinafter also referred to as “EPO”) is one of the hematopoietic factors that promote the production of erythrocytes, composed of 165 amino acids. EPO is produced primarily in the peritubular interstitial cells of the kidney, with ancillary production in the liver. EPO is known to act on erythroid progenitor cells in the bone marrow to promote differentiation and proliferation into erythrocytes, and an insufficient amount of EPO causes anemia. Since most of EPO is produced in the kidney, the amount of EPO produced decreases when renal function declines, such as chronic renal failure. Therefore, deterioration of renal function causes renal anemia.
Recombinant human EPO preparations are indicated for the treatment of renal anemia, and their efficacy and QOL have been improved (Non-Patent Document 1). On the other hand, the high cost and intravenous administration by injection impose a considerable burden on both patients and medical institutions. Therefore, there is a demand for an ingredient that can be orally ingested and is effective in treating renal anemia.

As mentioned above, EPO is also produced in the liver, and hypoxia-inducible factor-1α (HIF-1α) controls the expression of EPO in the liver as a master regulator. HIF-1α has the property of being rapidly degraded in the presence of oxygen. are known to be improved (Non-Patent Documents 2-4).

In recent years, Bifidobacterium genus bacteria (so-called Bifidobacterium), which is a type of intestinal bacteria, have been found to have various effects useful for the health of humans and the like, and are utilized in foods, beverages, and pharmaceuticals.
For example, certain Bifidobacterium bacteria are known to be useful for prevention and treatment of iron deficiency anemia (Patent Document 1).
However, it is unknown whether Bifidobacterium is involved in the prevention or treatment of anemia caused by insufficient EPO production, such as renal anemia, or in EPO production.

WO2011/114916

S. V. Bhoopalan et al., F1000Research 2020, 9. https://doi.org/10.12688/f1000research.26648.1 Lacombe et al., Am J Kidney Dis, 18(4), 14-19 (1991) Vazquez-Valls et al., J Neuroimmunol, 238(1-2), 12-18(2011) Jiang et al., Cell Growth Differ, 12(7), 363-369 (2001)

An object of the present invention is to provide a component capable of promoting the production of EPO, particularly an orally ingestible component.

As a result of intensive studies aimed at solving the above problems, the present inventors have found that bacteria of the genus Bifidobacterium have an effect of promoting the production of EPO, and have completed the present invention.

That is, the present invention is a composition for promoting EPO production containing bacteria of the genus Bifidobacterium.
In a preferred embodiment, said Bifidobacterium bacterium is Bifidobacterium breve.
In a preferred embodiment, said Bifidobacterium breve is Bifidobacterium breve MCC1274 (FERM BP-11175).
In a preferred embodiment, the Bifidobacterium bacterium is contained in the composition of the present invention as a heat-killed body.
The composition of the present invention is preferably used for preventing and/or improving anemia caused by insufficient EPO production.
The promotion of EPO production in the present invention may be by Akt activation.
The composition of the present invention is preferably food or drink.
The compositions of the invention are preferably pharmaceuticals.

According to the present invention, EPO production can be promoted. In addition, it is possible to prevent and/or improve anemia caused by insufficient EPO production. Since the composition of the present invention can be orally ingested, it can be conveniently used in the form of foods, beverages, and pharmaceuticals.

The present invention will now be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.

The composition of the present invention contains Bifidobacterium. In the present invention, Bifidobacterium bacteria may be contained singly or in combination of two or more.

Bifidobacterium bacteria include, but are not limited to, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium infantis, and Bifidobacterium infantis. Bacterium longum subspecies infantis), Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium catenulatum), Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium lactis, and Bifidobacterium pseudolongum).

Bifidobacterium breve more specifically includes Bifidobacterium breve MCC1274. Bifidobacterium breve MCC1274 was established on August 25, 2009 by the Independent Administrative Institution National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (currently Independent Administrative Institution National Institute of Technology and Evaluation Patent Organism Depositary Center (NITE-IPOD) ( It has been internationally deposited under the Budapest Treaty under the accession number FERM BP-11175 at Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818.
Further, more specific examples of Bifidobacterium breve include Bifidobacterium breve M-16V. Bifidobacterium breve M-16V was approved on January 26, 2018 by the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (NPMD) (2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818). 8 122) under the accession number NITE BP-02622 under the Budapest Treaty. A commercially available product, for example, “Bifidobacterium breve M-16V” manufactured by Morinaga Milk Industry Co., Ltd. may be used.

More specifically, Bifidobacterium longum includes Bifidobacterium longum BB536. Bifidobacterium longum BB536 has been deposited with NPMD under the Budapest Treaty on January 26, 2018 under the accession number NITE BP-02621. The same bacterium, Bifidobacterium longum subsp. longum ATCC BAA-999 (number: ATCC BAA-999), is available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Virginia, USA, 20110). Manassas, VA 20110, United States of America)) as ATCC BAA-999 (see, for example, JP-A-2012-223134).

Bifidobacterium infantis more specifically includes Bifidobacterium longum subspecies infantis M-63. Bifidobacterium longum subspecies infantis M-63 has been deposited under the Budapest Treaty with the NPMD under the accession number NITE BP-02623 on January 26, 2018. Synonymous with Bacterium infantis M-63.

Among these bacteria of the genus Bifidobacterium, Bifidobacterium breve is more preferred, and Bifidobacterium breve MCC1274 (accession number: FERM BP-11175) is particularly preferred.

In addition, each strain of the Bifidobacterium genus bacteria described above is not limited to the strain itself that has been deposited or registered with a predetermined institution under the name of the bacterium (hereinafter also referred to as "deposited strain" for convenience of explanation). , and substantially equivalent strains thereof (also referred to as "derivative strains" or "derived strains"). With respect to bacteria, a “substantially equivalent strain” to the deposited strain belongs to the same species as the deposited strain, and has a genome sequence similarity (Average Nucleotide Identity value) with the deposited strain of preferably 99. It refers to a strain having an identity of 0.0% or more, more preferably 99.5% or more, still more preferably 100%, and preferably having the same mycological properties as the above-deposited strain. For bacteria, a strain that is substantially equivalent to the above deposited strain may be, for example, a derivative of the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that arise naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. be done. Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Such strains include mutants naturally occurring by culturing (eg, subculturing) the deposited strain. Derivative strains may be constructed with one modification, or may be constructed with two or more modifications.

The Bifidobacterium bacterium to be contained in the composition of the present invention may be a commercially available product, may be obtained by appropriately producing it, or may be obtained by culturing the above-mentioned bacteria. may be used.
The culture method is not particularly limited as long as the bacteria of the genus Bifidobacterium can grow. As a culture method, for example, a method commonly used for culturing Bifidobacterium bacteria can be used as it is or after being modified as appropriate. The culture temperature may be, for example, 25-50°C, preferably 35-42°C. Culturing is preferably carried out under anaerobic conditions, for example, while passing anaerobic gas such as carbon dioxide. Cultivation can also be performed under microaerobic conditions such as liquid stationary culture. Culturing can be carried out, for example, until the bacteria have grown to the desired extent.

The medium used for culture is not particularly limited as long as the Bifidobacterium bacteria can grow. As the medium, for example, a medium commonly used for culturing the bacteria can be used as it is or after being appropriately modified. That is, as carbon sources, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, blackstrap molasses, etc. can be used depending on the assimilation. . As the nitrogen source, for example, ammonia, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic ingredients such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may also be used. In addition, as the medium usually used for culturing the bacteria, specifically, reinforced Clostridial medium, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium), TOSP medium (TOS propionate medium), TOSP Mup medium (TOS propionate mupirocin medium), GAM (Gifu Anaerobic Medium) medium, YCFA (Yeast Extract-casein Hydrolysate Acid) medium, and the like.

The Bifidobacterium bacterium to be contained in the composition of the present invention may be in the form of bacterial cells, bacterial cultures, or treated bacteria.
The cells may be, for example, viable cells, dead cells (including damaged cells), or a mixture of viable cells and dead cells. Body is more preferred.
As the bacterial culture, for example, a culture obtained by culturing may be used as it is, the culture may be diluted or concentrated and used, or the cells collected from the culture may be used.
As the processed bacterium, microbial cells or cultures subjected to crushing, heating, freeze-drying, or spray-drying, and dilutions, dried products, or fractions thereof can be used.

The form of Bifidobacterium bacterium to be contained in the composition of the present invention is particularly preferably heat-sterilized. The heat-sterilized product is obtained by heat-treating bacteria belonging to the genus Bifidobacterium, specifically, for example, at 70-100°C for 10-40 minutes, or at 90-150°C for 5-30 seconds. It is obtained by Note that pressure may be applied during the heat treatment. During the heat treatment, the temperature does not necessarily have to be constant, and it is sufficient that the temperature is within the above temperature range for a predetermined period of time.
The heat-sterilized Bifidobacterium bacterium may be used as it is after the heat treatment, or may be used after being subjected to treatment such as crushing, drying by heating, freeze-drying, or spray-drying.

The content of the ^{Bifidobacterium} bacterium in the composition of the present invention is not particularly limited. Preferably, it contains 1.0×10 ⁷ cfu or more of the cells. In addition, cfu refers to a colony forming unit. In this specification, for example, the value obtained when cultured at 38° C. in a solid medium containing 10% by mass of reconstituted skim milk can be used. Also, when the cells are dead cells, the cfu can be replaced with cells.
These may be in the range of contents when they are usually distributed as a composition for promoting EPO production.

The composition of the present invention has an effect of promoting EPO production. Here, the term “production enhancement” means that the amount or production rate of EPO increases after application of the composition of the present invention compared to before application, or when the composition of the present invention is applied compared to when it is not applied. , means to increase.
As shown in the examples below, the composition of the present invention activates (phosphorylates) Akt in the liver, thereby enhancing the level of HIF-1α, one of EPO expression regulators. It is believed that the composition of the invention promotes EPO production in the liver through such a mechanism. That is, the EPO production promoting effect of the composition of the present invention may be due to Akt activation.
The EPO production-promoting action of the composition of the present invention may be mediated by a mechanism other than Akt activation. It is also not excluded that EPO production in tissues other than liver, such as kidney, is enhanced.

EPO promotes the differentiation of erythroid progenitor cells in the bone marrow into erythrocytes and proliferates erythrocytes. Therefore, the composition of the present invention can prevent and/or improve anemia caused by insufficient EPO production. An example of such anemia is renal anemia. Renal anemia is known to be caused mainly by insufficient EPO production due to decreased renal function.
Here, "prevention" refers to the prevention or delay of the occurrence of a disease or symptom in an applicable subject, or a reduction in the risk of a disease or symptom in an applicable subject, and "improvement" refers to the alleviation or alleviation of a disease or symptom in an applicable subject. Suppression or delay of exacerbation.

Another aspect of the present invention is the use of Bifidobacterium in the manufacture of a composition for promoting EPO production.
Another aspect of the invention is the use of Bifidobacterium in promoting EPO production.
Another aspect of the present invention is a Bifidobacterium bacterium used for promoting EPO production.
Another aspect of the invention is a method of promoting EPO production comprising administering Bifidobacterium to a subject.

Another aspect of the present invention is the use of Bifidobacterium in the production of a composition for preventing and/or improving anemia caused by insufficient EPO production.
Another aspect of the present invention is the use of Bifidobacterium in the prevention and/or amelioration of anemia caused by insufficient EPO production.
Another aspect of the present invention is a Bifidobacterium bacterium used for preventing and/or improving anemia caused by insufficient EPO production.
Another aspect of the present invention is a method for preventing and/or ameliorating anemia caused by insufficient EPO production, comprising administering Bifidobacterium to a subject.

Subjects to whom the composition of the present invention is administered (administered person) and subjects to be ingested (consumer) are not particularly limited as long as they are animals, but are usually humans. Also, it may be an adult, a child, an infant, a newborn, or the like. Moreover, sex is not specifically limited.

In the present specification, "administering a Bifidobacterium bacterium to a subject" may be synonymous with "making a subject ingest a Bifidobacterium bacterium." The intake is usually voluntary (free intake), but may be forced (forced intake). Specifically, the administration step may be, for example, a step of mixing the Bifidobacterium bacterium into a food, drink or feed and supplying it to the subject, thereby allowing the subject to freely ingest it.

The timing of ingestion (administration) of the composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the subject of ingestion (administration).

The intake (administration) amount of the composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the intake (administration) target.
The intake (administration) amount of the composition of the present invention is, for example, 1×10 ⁷ to 1×10 ¹² cfu/day for an adult as the intake (administration) amount of Bifidobacterium bacteria according to the present invention. , more preferably 1×10 ⁸ to 1×10 ¹¹ cfu/day, even more preferably 1×10 ⁹ to 1×10 ¹¹ cfu/day.
The composition of the present invention can be ingested (administered) once a day or divided into multiple times a day, regardless of the amount and duration of ingestion (administration).

The intake (administration) period of the composition of the present invention is not particularly limited. In addition, there is no particular upper limit for the intake (administration) period, and continuous long-term intake (administration) is possible.

The intake (administration) route of the composition of the present invention may be oral or parenteral, but oral is preferred. In addition, parenteral intake (administration) includes percutaneous, intravenous, rectal administration, inhalation, and the like.

When the composition of the present invention is to be orally ingested (administered), it is preferably in the form of a food or drink.
As food and drink, the form and properties are not particularly limited as long as they can be orally ingested (administered) without impairing the effects of the present invention. It can be manufactured by a normal method using the raw materials obtained.

Food and drink, regardless of the form such as liquid, paste, gel-like solid, powder, etc. Wheat flour products such as fried flour and bread crumbs; instant noodles, cup noodles, retort/cooked foods, cooked canned foods, microwave oven foods, instant soups/stews, instant miso soups/soups, canned soups, freeze-dried foods, other instant foods, etc. Instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (processed grains) and other processed agricultural products; canned marine products, fish hams and sausages, fish paste products, marine delicacies Processed marine products such as tsukudani; Canned livestock products, pastes, processed livestock products such as meat hams and sausages; Processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams, cream, and other dairy products Oils and fats such as butter, margarines, and vegetable oils; Basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, and vinegar; Cooking mixes, curry ingredients, and sauces Compound seasonings and foods such as dressings, noodle soups, spices, and other compound seasonings; Ingredients Frozen foods such as frozen foods, half-cooked frozen foods, and cooked frozen foods; caramels, candies, chewing gums, chocolates , cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; , fruit drinks with fruit, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, other favorite drinks, baby food, Other commercial foods such as furikake and ochazuke seaweed; nutritional compositions such as supplements and formula milk (including powdered milk, liquid milk, etc.); enteral nutritional foods; is mentioned.

Moreover, it can also be set as feed as one aspect|mode of food-drinks. Examples of the feed include pet food, livestock feed, fish feed, and the like.
The form of the feed is not particularly limited, and in addition to bacteria of the genus Bifidobacterium, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean meal, rapeseed meal, coconut oil meal, and linseed meal. brans such as wheat bran, wheat bran, rice bran, and defatted rice bran; manufacturing lees such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim milk powder, whey, yellow grease, and tallow; torula yeast, Yeasts such as brewer's yeast; mineral feeds such as tribasic calcium phosphate and calcium carbonate; oils and fats; simple amino acids;

When the composition of the present invention is in the form of a food or drink (including feed), it is provided and sold as a food or drink labeled with uses such as promotion of EPO production and prevention/improvement of anemia caused by insufficient EPO production. It is possible to be In addition, the Bifidobacterium bacterium according to the present specification can be used for producing these foods and drinks.

Such "display" acts include all acts for informing consumers of the above-mentioned use. Regardless of the object, medium, etc. to be displayed, all of them fall under the act of "display" in the present invention.
In addition, it is preferable that the "display" be performed in an expression that allows the consumer to directly recognize the use. Specifically, the act of transferring, handing over, displaying for the purpose of transfer or delivery, importing products related to food and beverages or product packaging that describes the above-mentioned use, advertisements related to products, price lists or transaction documents Examples include the act of displaying or distributing information with the above-mentioned use described, or providing information containing such information with the above-mentioned use by electromagnetic means (Internet, etc.).

On the other hand, the content of the display is preferably a display approved by the government (for example, a display that is approved based on various systems established by the government and performed in a manner based on such approval). In addition, it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, POP and other advertising materials at sales sites, other documents, and the like.

In addition, "labeling" includes health foods, functional foods, enteral nutrition foods, foods for special dietary uses, foods with health claims (foods for specified health uses, foods with nutrient function claims, foods with function claims), nutritional supplements, and pharmaceuticals. Display as an off-the-shelf item is also possible. Among these, in particular, the labeling approved by the Consumer Affairs Agency, for example, the labeling approved by the system related to food for specified health use, food with nutrient function claims, or food with function claims, or similar system. Specifically, labeling as a food for specified health use, labeling as a food for specified health use with certain conditions, labeling to the effect that it affects the structure and function of the body, labeling to reduce the risk of disease, labeling for functionality based on scientific evidence. Labeling, etc. can be mentioned, more specifically, the Cabinet Office Ordinance Concerning Permission for Special Use Labeling as stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) A typical example is labeling as a food for specified health use (especially labeling for health use) and similar labeling.
Examples of such indications include indications such as “for promoting erythropoietin production”, “increasing red blood cells”, “improving anemia due to erythropoietin deficiency”, and “improving renal anemia”.

The compositions of the invention can also be in the form of pharmaceuticals.
The route of administration of pharmaceuticals may be either oral or parenteral, but oral is preferred. In addition, parenteral intake (administration) includes percutaneous, intravenous, rectal administration, inhalation, and the like.
As for the pharmaceutical form, it can be appropriately formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, solid preparations such as powders, granules, tablets and capsules; and liquid preparations such as solutions, syrups, suspensions and emulsions can be formulated. In the case of parenteral administration, it can be formulated into suppositories, ointments, injections, and the like.
For formulation, in addition to the Bifidobacterium bacterium, ingredients such as excipients, pH adjusters, colorants, corrigents, and the like, which are usually used for formulation, can be used. In addition, it is also possible to use other medicinal ingredients, ingredients known or to be discovered in the future that have EPO production-enhancing action, therapeutic agents for renal anemia, and the like.
In addition, formulation can be appropriately carried out by a known method depending on the dosage form. For formulation, a carrier that is usually used for formulation may be blended as appropriate to form the formulation. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.

Excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate;

Examples of binding agents include gelatin, polyvinylpyrrolidone, macrogol, etc., in addition to the excipients described above.

Examples of disintegrants include, in addition to the excipients described above, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

Lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gairou; ; carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid anhydride and silicic acid hydrate; be done.

Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride;

Flavoring agents include, for example, sweeteners, acidulants, flavoring agents, and the like.
In addition, solvents such as water and the like can be used as carriers for liquid formulations for oral administration.

The timing of taking the drug of the present invention is not particularly limited, such as before meals, after meals, between meals, and before bedtime.

EXAMPLES The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

(1) Preparation of dosage product Bifidobacterium breve FERM BP-11175 lyophilized powder was diluted with physiological saline to a concentration of 5×10 ⁸ cfu/mL, and then heated at 90° C. for 30 minutes to obtain bifidobacteria. A product administered with a bacterium heat-sterilized product (hereinafter also referred to as a product administered with a heat-sterilized product) was prepared.

(2) Animal test
Using Crl:CD(SD) rats (Charles River Japan: healthy rats) as experimental animals,
The effects of 4-week continuous ingestion of heat-sterilized products of Bifidobacterium breve FERM BP-11175 were evaluated.
Rats received at the age of 7 weeks were given Labo MR stock feed (manufactured by Nosan Kogyo Co., Ltd.) and tap water ad libitum. After acclimatization for 1 week, the rats were divided into 2 groups (13 rats in each group), and the rats in the control group were orally administered 2 mL/rat of physiological saline using an oral probe once a day for 4 weeks, followed by heat sterilization. The rats in the body administration group were orally administered 2 mL/rat of the heat-sterilized body-administered product prepared in (1) once a day for 4 weeks using an oral probe.
One hour after the end of the final administration, the experimental animals were treated under sevoflurane anesthesia, blood was collected from the abdominal posterior vena cava, and the serum was recovered by centrifugation. The amount of blood erythropoietin (EPO) in the collected serum was quantified using Rat Erythropoietin ELISA Kit (RayBiotech).

In addition, the liver was extracted from the rat after blood collection, and the expression levels of EPO in the liver were analyzed by real-time PCR, and the expression levels of HIF-1α and active Akt were analyzed by Western blotting.
RNA was extracted from a portion of the excised liver using RNeasy Mini Kit (Qiagen), and a complementary DNA (cDNA) library was prepared using PrimeScript RT Reagent Kit (Takara Bio). Using the obtained cDNA library and SYBR Premix Ex Taq2 (Takara Bio Inc.), the expression level of EPO mRNA level was quantified by real-time PCR. At this time, the expression level of EPO was corrected with the expression level of GAPDH.
Proteins were extracted from a portion of the excised liver using RIPA buffer (CST), and quantitatively analyzed for phosphorylated Akt (activated Akt) and HIF-1α by Western blotting. At that time, the amount of phosphorylated Akt was corrected with the amount of total Akt, and the amount of HIF-1α was corrected with the amount of β-actin.

(3) Results Table 1 shows the amount of EPO in blood in each group. Administration of heat-killed bifidobacterium was confirmed to significantly increase the amount of EPO in the blood compared to the control. Specifically, a significant increase of 12.70% was confirmed in the heat-sterilized body administration group compared to the control group.

Table 2 shows the expression level of EPO mRNA in the liver as a relative amount when the control is set to 1. It was confirmed that administration of the heat-killed Bifidobacterium breve FERM BP-11175 significantly increased the EPO expression level compared to the control. Specifically, it was confirmed to increase 1.43 times compared to the control.

Table 3 shows the amounts of active Akt and HIF-1α in the liver as relative amounts when the control is set to 1. Administration of the heat-killed Bifidobacterium breve FERM BP-11175 was confirmed to increase the amount of active Akt and HIF-1α compared to the control. Specifically, it was confirmed that the amount of active Akt and the amount of HIF-1α were significantly increased by 1.66 times and 1.19 times, respectively, in the heat-killed body administration group, compared to the control.

The above results suggest that administration of the heat-killed Bifidobacterium breve FERM BP-11175 promotes the production of EPO, increases the amount of EPO in the blood, and as a result can prevent or improve anemia symptoms. was done. As a mechanism for promoting EPO production, it was suggested that administration of the heat-killed body increases the amount of HIF-1α due to activation of Akt in the liver, thereby increasing the expression level of EPO.

Claims (8)

A composition for promoting erythropoietin (EPO) production, containing bacteria of the genus Bifidobacterium. 2. The composition of claim 1, wherein said Bifidobacterium bacterium is Bifidobacterium breve. 3. The composition of claim 2, wherein said Bifidobacterium breve is Bifidobacterium breve MCC1274 (FERM BP-11175). The composition according to any one of claims 1 to 3, wherein the Bifidobacterium bacterium is contained as a heat-sterilized body. The composition according to any one of claims 1 to 4, which is used for preventing and/or improving anemia caused by insufficient EPO production. The composition according to any one of claims 1 to 5, wherein the promotion of EPO production is due to Akt activation. The composition according to any one of claims 1 to 6, which is a food or drink. A composition according to any one of claims 1 to 6, which is a medicament.