Composition for promoting erythropoietin production

Abstract

To provide a novel erythropoietin production promoting composition.SOLUTION: Cells of bacteria belonging to Lactobacillus, Leuconostoc and Pediococcus are used as active ingredients, to provide food and drink, and feed having erythropoietin production promotion action.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for promoting erythropoietin production, which contains lactic acid bacteria cells as an active ingredient. [Background technology]

[0002] Erythropoietin is a hematopoietic factor that promotes the production of red blood cells. Recombinant erythropoietin is available on the market as a treatment for renal anemia, with sales exceeding 80 billion yen in fiscal 2017.

[0003] It has been reported that administering erythropoietin preparations to elderly people who tend to be anemic can improve fatigue (Non-Patent Document 1).

[0004] On the other hand, erythropoietin increases red blood cells, thereby increasing blood hemoglobin levels, which is effective in improving endurance athletic performance. For this reason, administration of erythropoietin has been viewed as a form of doping in athletes.

[0005] Therefore, if it were possible to promote the production of erythropoietin in the body without administering erythropoietin, it would be possible to achieve improved endurance and reduced fatigue, which would not be considered doping.Patent Document 1 discloses a compound that induces erythropoietin production by inhibiting the transcription factor GATA, thereby relieving the inhibition of erythropoietin expression.

[0006] However, there have been no reports to date of easily ingested foods inducing erythropoietin production in the body.

[0007] Incidentally, lactic acid bacteria and Bifidobacterium are bacteria that have been used in the production of fermented foods since ancient times, and recent research has revealed that they contribute to the enhancement of various health functions. Patent Document 2 shows that peptides produced by lactic acid bacteria decomposing mammalian milk are effective in enhancing muscle strength and reducing fatigue. However, whether the lactic acid bacteria themselves are involved in promoting the production of erythropoietin has not been disclosed or suggested in any literature.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a novel composition for promoting erythropoietin production.

Means for Solving the Problems

[0011] As a result of intensive studies to solve the above problems, the present inventors have found lactic acid bacteria having an action of promoting erythropoietin production and completed the present invention. That is, the present invention provides a novel composition for promoting erythropoietin production containing lactic acid bacteria as an active ingredient. The present invention also provides a novel lactic acid bacterial strain that can be industrially utilized. Therefore, the present invention has the following configuration. <1>A composition for promoting erythropoietin production, comprising as an active ingredient the cells of a bacterium belonging to the genus Lactobacillus, Leuconostoc or Pediococcus <2>The composition for promoting erythropoietin production according to <1>, wherein the bacterium belonging to the genus Lactobacillus, Leuconostoc or Pediococcus is one or more selected from Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus vaginalis, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus pentosaceus <3>The composition for promoting erythropoietin production according to <2>, characterized in that the bacterium belongs to the genus Lactobacillus, Leuconostoc or Pediococcus, and is one or more selected from Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus paracasei SBT0228 (NITE P-02805), Lactobacillus plantarum SBT0092 (NITE P-02806), Lactobacillus rhamnosus SBT2490 (NITE P-02807), Lactobacillus vaginalis SBT0337 (NITE P-02808), Leuconostoc mesenteroides subsp. cremoris SBT1395 (NITE P-02809), Leuconostoc pseudomesenteroides SBT0835 (NITE P-02810), Pediococcus pentosaceus SBT3316 (NITE P-02804). <4>A feed composition for promoting erythropoietin production, a pharmaceutical composition for promoting erythropoietin production, or a food composition for promoting erythropoietin production, comprising the composition for promoting erythropoietin production according to any one of <1> to <3>. <5>The novel lactic acid bacterium Lactobacillus mucosae SBT2958, <6>The novel lactic acid bacterium Lactobacillus paracasei SBT0228, <7>The novel lactic acid bacterium Lactobacillus plantarum SBT0092, <8>The novel lactic acid bacterium Lactobacillus rhamnosus SBT2490, <9>The novel lactic acid bacterium Lactobacillus vaginalis SBT0337, <10>The novel lactic acid bacterium Leuconostoc mesenteroides subsp. cremoris SBT1395, <11>The novel lactic acid bacterium Leuconostoc pseudomesenteroides SBT0835, and <12>The novel lactic acid bacterium Pediococcus pentosaceus SBT3316.

Advantages of the Invention

[0012] According to the present invention, it is possible to provide a composition for promoting the production of erythropoietin, which contains, as an active ingredient, cells of lactic acid bacteria belonging to the genus Lactobacillus, Leuconostoc, and Pediococcus. Further, according to the present invention, it is possible to provide a novel lactic acid bacterial strain that can be industrially utilized.

Brief Description of the Drawings

[0013]

Figure 1

Modes for Carrying Out the Invention

[0014] (Lactic acid bacteria) Any lactic acid bacteria classified into the genus Lactobacillus, Leuconostoc, and Pediococcus can be used as the lactic acid bacteria of the present invention. Specifically, examples include, but are not limited to, Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus vaginalis, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus pentosaceus, and the like.

[0015] The lactic acid bacteria of the present invention are preferably Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus paracasei SBT0228 (NITE P-02805), Lactobacillus plantarum SBT0092 (NITE P-02806), Lactobacillus rhamnosus SBT2490 (NITE P-02807), Lactobacillus vaginalis SBT0337 (NITE P-02808), Leuconostoc mesenteroides subsp. cremoris SBT1395 (NITE P-02809), Leuconostoc pseudomesenteroides SBT0835 (NITE P-02810), Pediococcus pentosaceus SBT3316 (NITE P-02804).

[0016] (Preparation of lactic acid bacteria) Lactic acid bacteria can be cultured according to the conventional methods for culturing each bacterium, and the desired amount can be prepared. An example of the preparation is shown below. Culture using MRS medium (Difco), and collect the obtained culture by centrifugation to obtain bacterial cells. The obtained bacterial cells can be used as they are, or bacterial cells that have been subjected to concentration, drying, or freeze-drying treatment can also be used. Dead bacterial cells can also be used. Dead bacterial cells can be prepared, for example, by heat drying, acid treatment, ultrasonic disruption, etc.

[0017] (Novel lactic acid bacterial strain) The present invention relates to novel lactic acid bacterial strains. These novel lactic acid bacterial strains are Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus paracasei SBT0228 (NITE P-02805), Lactobacillus plantarum SBT0092 (NITE P-02806), Lactobacillus rhamnosus SBT2490 (NITE P-02807), Lactobacillus vaginalis SBT0337 (NITE P-02808), Leuconostoc mesenteroides subsp. cremoris SBT1395 (NITE P-02809), Leuconostoc pseudomesenteroides SBT0835 (NITE P-02810), Pediococcus pentosaceus SBT3316 (NITE P-02804). Hereinafter, the same lactic acid bacterial strains may be described as "the lactic acid bacteria of the present invention", "the lactic acid bacterial strains of the present invention", or simply SBT2958, SBT0228, SBT0092, SBT2490, SBT0337, SBT1395, SBT0835, SBT3316 strains. The lactic acid bacteria SBT2958 and SBT0337 of the present invention were isolated from human feces, SBT0228 from alcoholic beverages, SBT0092, SBT2490, SBT1395, and SBT0835 from fermented milk, and SBT3316 from fermented foods. These lactic acid bacterial strains were deposited on October 31, 2018, at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture 292-0818), with the accession numbers of SBT2958 being NITE P-02803, SBT0228 being NITE P-02805, SBT0092 being NITE P-02806, SBT2490 being NITE P-02807, SBT0337 being NITE P-02808, SBT1395 being NITE P-02809, SBT0835 being NITE P-02810, and SBT3316 being NITE P-02804. The lactic acid bacteria of the present invention are not limited to the deposited lactic acid bacterial strains, and may be lactic acid bacterial strains that are substantially equivalent to these deposited lactic acid bacterial strains. Substantially equivalent lactic acid bacterial strains refer to lactic acid bacterial strains belonging to Lactobacillus mucosae, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus buchneri, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides, and Pediococcus pentosaceus, and having an erythropoietin production promoting effect as high as that of the deposited lactic acid bacterial strains. "Lactic acid bacterial strains having an erythropoietin production promoting effect as high as that of the deposited lactic acid bacterial strains" means, for example, lactic acid bacterial strains in which the amount of erythropoietin measured by the following procedures 1) to 4) has no significant difference from the amount of erythropoietin in each deposited lactic acid bacterial strain. 1) Seed the human liver cancer-derived cell line HEP-3B at 6×10 4 cells / well in Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 10% FBS and 1% penicillin-streptomycin (Sigma), and culture for 24 hours. 2) Remove the medium, add 150 μl of the medium in which various heat-killed lactic acid bacteria cells are suspended at 50 μg / ml to the cells, culture for 48 hours, collect the culture supernatant, and centrifuge at 12,000×g for 10 minutes at 4°C to remove cell debris and the like. 3) Measure 50 μl of the undiluted cell culture supernatant obtained in 2) above by Human Erythropoietin DuoSet (registered trademark) ELISA (R&D Systems). Specifically, as the chromogenic reagent, QuantaBlu TM Use the Fluorogenic Peroxidase Substrate Kit (Thermo) to develop the color of the erythropoietin protein in 50 μl of the cell culture supernatant obtained in 2) above. 4) Transfer the colored solution to TM Corning 96-Well Solid White Polystyrene Microplates (Corning), and measure the fluorescence intensity at Ex. 325 nm and Em. 420 nm using a plate reader (Biotek). When the lactic acid bacteria of the present invention are evaluated for the function of promoting erythropoietin production according to the above procedures 1) to 4), preferably, the measured amount of erythropoietin is 1.5 times or more greater than the amount of erythropoietin in the control without the addition of lactic acid bacteria strains. When the lactic acid bacteria of the present invention are evaluated for the function of promoting erythropoietin production according to the above procedures 1) to 4), the measured amount of erythropoietin is preferably 5 mIU / mL, more preferably 10 mIU / mL, still more preferably 15 mIU / mL or more greater than the amount of erythropoietin in the control without the addition of lactic acid bacteria strains. Note that the evaluation method is not limited to the above, and it goes without saying that the function of promoting erythropoietin production can be evaluated by adopting an evaluation method for the action of promoting erythropoietin production known to those skilled in the art. In addition, substantially equivalent lactic acid bacteria strains further have a nucleotide sequence homology of 98% or more, preferably 99% or more, more preferably 100% with the nucleotide sequence of the 16S rRNA gene of the deposited lactic acid bacteria strain, and preferably have the same mycological properties as the deposited lactic acid bacteria strain. Furthermore, the lactic acid bacteria of the present invention may be lactic acid bacteria strains bred from the deposited lactic acid bacteria strain or a lactic acid bacteria strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc., as long as the effects of the present invention are not impaired.

[0018] (Method of use) As described above, since the composition of the present invention can be used as the cells, active ingredients that have been subjected to concentration, drying, and freeze-drying treatments, it can be widely used as a raw material for pharmaceuticals, foods and drinks, and feeds. Dead cells can be prepared, for example, by heat drying, acid treatment, ultrasonic disruption, etc. The administration target of the composition of the present invention is not particularly limited and can be administered to humans, but the administration target may also be animals other than humans (for example, dogs, cats, horses or rabbits, etc.). When the administration target is a human, it can be administered to minors under 20 years old, adults, men and women, or the elderly over 65 years old, etc. The intake amount of the composition of the present invention is determined individually in consideration of the symptoms, age, and gender of the administration target person, etc., but in the case of ordinary adults, the intake amount of the cells per day may be 0.5 - 5000 mg, preferably 0.5 - 500 mg, and most preferably 0.5 - 50 mg.

[0019] The composition of the present invention can be administered to a subject for the purpose of preventing or improving renal anemia and anemia. The composition of the present invention may be administered to a subject already suffering from renal anemia and anemia, or can also be administered to a healthy subject not yet suffering from it for the prevention of renal anemia and anemia. The composition of the present invention can be administered to a subject for improving fatigue, enhancing endurance exercise ability, and / or strengthening muscle strength. As the administration timing, it can be administered before exercise, during exercise, and / or after exercise. Examples of exercise include walking, running, soccer, basketball, volleyball, swimming, cycling, and tennis.

[0020] (Promotion of erythropoietin production) "Promotion of erythropoietin production" means, for example, when evaluating the erythropoietin production promoting function by the above procedures 1) to 4), the measured amount of erythropoietin is significantly higher compared to the amount of erythropoietin in the control, preferably, the measured amount of erythropoietin is 1.5 times or more higher than the amount of erythropoietin in the control without adding lactic acid bacteria.

[0021] (Method for evaluating the ability to promote erythropoietin production) Evaluation can be performed by the method described in the examples. That is, evaluation can be performed by the above procedures 1) to 4).

[0022] Hereinafter, the present invention will be described in more detail based on examples of the present invention, but the present invention is not construed as being limited to such examples. Unless otherwise specified, % indicates weight %.

Examples

[0023] (Example Product 1) Heat-killed lactic acid bacteria Each of the test bacteria in (1) below was cultured using MRS medium (Difco) for lactic acid bacteria belonging to the genus Lactobacillus, Leuconostoc, and Pediococcus, M17 medium (Difco) for lactic acid bacteria belonging to the genus Lactococcus and Streptococcus, and GAM broth (Nissui) containing 1% glucose for Bifidobacterium bifidum belonging to the genus Bifidobacterium. The cultures were washed twice with physiological saline and once with sterilized water to obtain washed bacterial cells. These washed bacterial cells were freeze-dried to obtain cell powders. The cell powders were suspended in sterilized PBS(-) to a concentration of 10 mg / ml and heated at 80°C for 30 minutes to obtain heat-killed bacterial cells. The heat-killed bacterial cells were diluted with DMEM (Sigma) containing 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma) to a concentration of 50 μg / ml.

[0024] (1) Test bacteria The following 11 strains belonging to the genus Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, and Bifidobacterium were used as test bacteria. Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus paracasei SBT0228 (NITE P-02805), Lactobacillus plantarum SBT0092 (NITE P-02806), Lactobacillus rhamnosus SBT2490 (NITE P-02807), Lactobacillus vaginalis SBT0337 (NITE P-02808), Leuconostoc mesenteroides subsp. cremoris SBT1395 (NITE P-02809), Leuconostoc pseudomesenteroides SBT0835 (NITE P-02810), Pediococcus pentosaseus SBT3316 (NITE P-02804), Lactococcus lactis subsp. lactis JCM5805T, Streptococcus thermophilus JCM17834T, Bifidobacterium longum subsp. longum JCM1217T.

[0025] [Test Example 1] Test on the effect of promoting erythropoietin production by lactic acid bacteria Example product 1 was subjected to the following test. Human liver cancer-derived cell line HEP-3B was seeded in a 48-well plate (BD) at 6×10 4 cells / well and cultured in DMEM (Sigma) containing 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma) for 24 hours. After removing the medium, 150 μl of the medium in which various bacterial cells were suspended at 50 μg / ml was added to the test group, and 150 μl of the medium without bacterial cells was added to the control group, followed by culturing for 48 hours. As a positive control, cobalt chloride (CoCl2, Sigma) was added in the same manner at a final concentration of 50 μM. Then, the culture supernatant was collected and centrifuged at 12,000×g at 4°C for 10 minutes to remove cell debris and the like.

[0026] The erythropoietin protein in the cell culture supernatant was measured by Human Erythropoietin DuoSet (registered trademark) ELISA (R&D Systems). 50 μl of the undiluted culture supernatant was used as the measurement sample. QuantaBlu TM Fluorogenic Peroxidase Substrate Kit (Thermo) was used as the chromogenic reagent. The solution after color development was transferred to Corning TM 96-Well Solid White Polystyrene Microplates (Corning), and the fluorescence intensity was measured at Ex. 325 nm and Em. 420 nm using a plate reader (Biotek).

[0027] (Test results) Figure 1 shows the amount of erythropoietin protein produced into the culture supernatant when any one of 11 strains of lactic acid bacteria was added to HEP-3B cells and cultured for 48 hours. As a result, 8 strains of lactic acid bacteria (SBT2958, SBT0228, SBT0092, SBT2490, SBT0337, SBT1395, SBT0835, SBT3316) enhanced erythropoietin production. On the other hand, 3 reference strains of lactic acid bacteria (JCM5805T, JCM17834T, JCM1217T) did not enhance erythropoietin production. From these results, it became clear that lactic acid bacteria belonging to the genera Lactobacillus, Leuconostoc, and Pediococcus enhance the production of erythropoietin.

Industrial applicability

[0028] It is possible to provide a composition for promoting erythropoietin production, which contains the cells of bacteria belonging to the genera Lactobacillus, Leuconostoc, and Pediococcus as an active ingredient.

Deposit number

[0029] <Reference to deposited biological material> (1) Lactobacillus mucosae SBT2958 (i) Name and address of the depository institution with which the biological material was deposited National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu-shi, Chiba 292-0818, Japan (ii) Date on which the biological material was deposited with the depository institution October 31, 2018 (iii) Accession number assigned by the depository institution for the deposit NITE P-02803 (2) Lactobacillus paracasei SBT0228 (i) Name and address of the depository institution with which the biological material was deposited National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu-shi, Chiba 292-0818, Japan (ii) Date on which the biological material was deposited with the depository institution October 31, 2018 (iii) Accession number assigned by the depository institution for the deposit NITE P-02805 (3) Lactobacillus plantarum SBT0092 (i) Name and address of the depository institution with which the biological material was deposited National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu-shi, Chiba 292-0818, Japan (ii) Date on which the biological material was deposited with the depository institution October 31, 2018 (iii) Accession number assigned by the depository institution for the deposit NITE P-02806 (4) Lactobacillus rhamnosus SBT2490 (i) Name and address of the depository institution with which the biological material was deposited National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818) Date on which the biological material was deposited with the depository institution of Roy October 31, 2018 Accession number assigned by the depository institution of Hi for the deposit NITE P-02807 (5) Lactobacillus baguinaris SBT0337 Name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818) Date on which the biological material was deposited with the depository institution of Roy October 31, 2018 Accession number assigned by the depository institution of Hi for the deposit NITE P-02808 (6) Leuconostoc mesenteroides subsp. cremoris SBT1395 Name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818) Date on which the biological material was deposited with the depository institution of Roy October 31, 2018 Accession number assigned by the depository institution of Hi for the deposit NITE P-02809 (7) Leuconostoc pseudomesenteroides SBT0835 Name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Microorganisms Depositary Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818) Date on which the biological material was deposited with the depository institution of Roy October 31, 2018 Deposit number assigned by the depository institution of the present invention for the deposit NITE P-02810 (8) Pediococcus pentosaceus SBT3316 Name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center Room 122, 2-5-8 Kazusa Kamashidzu, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date on which the biological material was deposited with the depository institution of the present invention October 31, 2018 Deposit number assigned by the depository institution of the present invention for the deposit NITE P-02804

Claims

【Claim 1】 A composition for promoting erythropoietin production, comprising as an active ingredient the cells of a bacterium belonging to the genus Lactobacillus, Leuconostoc or Pediococcus, wherein the bacterium belonging to the genus Lactobacillus is one or more selected from Lactobacillus mucosae SBT2958 (NITE P-02803), Lactobacillus paracasei SBT0228 (NITE P-02805), Lactobacillus plantarum SBT0092 (NITE P-02806), Lactobacillus rhamnosus SBT2490 (NITE P-02807) and Lactobacillus vaginalis SBT0337 (NITE P-02808), and the bacterium belonging to the genus Leuconostoc or Pediococcus is one or more selected from Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus pentosaceus; the composition for promoting erythropoietin production. **Claim 2** The composition for promoting erythropoietin production according to claim 1, characterized in that the bacterium belonging to the genus Leuconostoc or Pediococcus is one or more selected from Leuconostoc mesenteroides subsp. cremoris SBT1395 (NITE P-02809), Leuconostoc pseudomesenteroides SBT0835 (NITE P-02810), and Pediococcus pentosaceus SBT3316 (NITE P-02804). **Claim 3** A feed composition for promoting erythropoietin production, a pharmaceutical composition for promoting erythropoietin production, or a food composition for promoting erythropoietin production, comprising the composition for promoting erythropoietin production according to claim 1 or 2. **Claim 4** Novel lactic acid bacterium Lactobacillus mucosae SBT2958 (NITE P-02803). **Claim 5** Novel lactic acid bacterium Lactobacillus paracasei SBT0228 (NITE P-02805). **Claim 6** Novel lactic acid bacterium Lactobacillus plantarum SBT0092 (NITE P-02806). **Claim 7** Novel lactic acid bacterium Lactobacillus rhamnosus SBT2490 (NITE P-02807). **Claim 8** Novel lactic acid bacterium Lactobacillus vaginalis SBT0337 (NITE P-02808). **Claim 9** Novel lactic acid bacterium Leuconostoc mesenteroides subsp. cremoris Leuconostoc mesenteroides subsp. cremoris SBT1395 (NITE P-02809). **Claim 10** Novel lactic acid bacterium Leuconostoc pseudomesenteroides SBT0835 (NITE P-02810). **Claim 11** Novel lactic acid bacterium Pediococcus pentosaceus SBT3316 (NITE P-02804).

Images