Chronic kidney disease progression inhibitor

Abstract

This chronic kidney disease progression inhibitor utilizes Bifidobacterium bifidum as an active ingredient. As a result, present invention provides an agent that is more effective by using a bacterial strain that is different from hitherto-reported bacteria that belong to the genus Bifidobacterium and that have been used against chronic kidney diseases.

Description

Drugs that slow the progression of chronic kidney disease

[0001] This invention relates to an agent for inhibiting the progression of chronic kidney disease, in which Bifidobacterium bifidum is the active ingredient.

[0002] Chronic kidney disease (CKD) is a disease characterized by persistent kidney damage and a decline in kidney function, which can progress to end-stage renal failure. It is estimated that there are approximately 13.3 million CKD patients in Japan. Since CKD also increases the risk of cardiovascular disease and death, early diagnosis and appropriate treatment of CKD are crucial.

[0003] It has been found that the production of uremic toxins, one of the factors in CKD, is related to gut bacteria, and it has been proposed to utilize gut bacteria to reduce uremic toxins.

[0004] Examples of bacteria used here include Bifidobacterium breve (Patent Document 1), Bifidobacterium breve etc. (Patent Document 2), Bifidobacterium breve (Patent Document 3), Bifidobacterium longum, Bifidobacterium lactis (Patent Document 4), Bifidobacterium longum (Patent Document 5), Bifidobacterium infantis etc. (Patent Document 6), and others.

[0005] Furthermore, regarding Bifidobacterium breve, it has been reported that it can be used not only alone but also in combination with indigestible oligosaccharides such as lactulose to increase aromatic lactic acid compounds such as 4-hydroxyphenyllactic acid (HPLA), which are associated with uremic toxins, in the culture medium (Patent Document 7).

[0006] However, generally speaking, bacteria of the genus Bifidobacterium differ in properties from species to species, and it is completely unknown whether bacteria other than those specifically mentioned can be used for suppressing CKD, etc.

[0007] Japanese Patent Publication No. 2023-126305, Japanese Patent Publication No. 2023-534702, Japanese Patent No. 7309436, Japanese Patent Publication No. 2021-6562, Japanese Patent Publication No. 2017-536828, Japanese Patent Publication No. 9-110707, Japanese Patent Publication No. 2023-34914

[0008] The objective of this invention was to provide a more effective treatment using a different species of Bifidobacterium bacteria than those previously reported for use in treating chronic kidney disease.

[0009] As a result of diligent research to solve the above problems, the inventors of the present invention discovered that Bifidobacterium bifidum metabolizes indole, a precursor of indoxyl sulfate, one of the uremic toxins, to indole lactic acid, and thus completed the present invention.

[0010] Furthermore, based on the above findings, we discovered that indole-lactic acid can be produced by reacting indole with Bifidobacterium bifidum, thus completing the present invention.

[0011] In other words, the present invention is as follows: [1] A chronic kidney disease progression inhibitor comprising Bifidobacterium bifidum as an active ingredient. [2] The chronic kidney disease progression inhibitor according to [1], further comprising sugars assimilated by Bifidobacterium bifidum. [3] The chronic kidney disease progression inhibitor according to [2], wherein the sugars assimilated by Bifidobacterium bifidum are one or more selected from galactose, galactooligosaccharides, glucose, lactose, and fructose. [4] The chronic kidney disease progression inhibitor according to any one of [1] to [3], wherein Bifidobacterium bifidum is Bifidobacterium bifidum YIT 10347 strain (FERM BP-10613) or Bifidobacterium bifidum YIT 4007 strain (NITE BP-04192). [5] A chronic kidney disease progression inhibitor according to any one of [1] to [4], which reduces the amount of indoxyl sulfate in the blood. [6] A method for inhibiting the progression of chronic kidney disease in a mammal, characterized by administering a chronic kidney disease progression inhibitor according to any one of [1] to [5] to a mammal. [7] A method for producing indole lactic acid, characterized by acting Bifidobacterium bifidum on indole. [8] A method for producing indole lactic acid according to [7], wherein Bifidobacterium bifidum is Bifidobacterium bifidum YIT 10347 strain (FERM BP-10613) or Bifidobacterium bifidum YIT 4007 strain (NITE BP-04192).

[0012] The present invention's chronic kidney disease progression inhibitor uses Bifidobacterium bifidum, which has a long history of being consumed as food, as its active ingredient, allowing for safe and continuous intake and effective suppression of the progression of chronic kidney disease.

[0013] Furthermore, the present invention provides a simple method for producing indole lactic acid, which involves reacting indole with Bifidobacterium bifidum.

[0014] This figure shows the indole-reducing ability and indole-lactic acid production ability of the Bifidobacterium standard strain in Example 1. This figure compares the indole-reducing ability and indole-lactic acid production ability of YIT 10347 and YIT 4007 in Example 2. This figure shows the indole-reducing ability and indole-lactic acid production ability of each formulation in Example 3. This figure shows the indole-reducing ability and indole-lactic acid production ability of YIT 10347 in Example 4. This figure compares the indole-reducing ability of YIT 10347 and YIT 4042 in Example 5.

[0015] The chronic kidney disease progression inhibitor of the present invention (hereinafter referred to as "the inhibitor of the present invention") contains Bifidobacterium bifidum as its active ingredient.

[0016] The inhibitor of the present invention, Bifidobacterium bifidum, can metabolize indole to indole-lactic acid, thereby reducing indole, which is a precursor of indoxyl sulfate. As a result, the inhibitor of the present invention can reduce the amount of indoxyl sulfate in the blood, and thus can suppress the progression of chronic kidney disease. The metabolism of indole to indole-lactic acid by Bifidobacterium bifidum can be confirmed, for example, by the method described in the examples below. Furthermore, the suppression of the progression of chronic kidney disease by the inhibitor of the present invention can be confirmed, for example, by measuring and comparing the amount of urinary protein and glomerular filtration rate before and after administration of the inhibitor of the present invention.

[0017] The Bifidobacterium bifidum used in the inhibitor of the present invention is not particularly limited as long as it falls within this classification, but preferably it has adhesive properties to gastric cells (mucin) and mucin assimilation properties. Adhesion to gastric cells (mucin) and mucin assimilation properties can be confirmed, for example, by the method described in the literature (iScience. 2021 Nov 19; 24(11): 103363.). Examples of Bifidobacterium bifidum strains that exhibit adhesiveness to gastric cells (mucin) and mucin assimilation include Bifidobacterium bifidum YIT 10347 strain (FERM BP-10613), Bifidobacterium bifidum YIT 4007 strain (NITE BP-04192), Bifidobacterium bifidum YIT 4042 strain (ATCC 11863), and Bifidobacterium bifidum YIT 4013 strain (ATCC 15696). These Bifidobacterium bifidum strains can be used individually or in combination of two or more.

[0018] Among these Bifidobacterium bifidum strains, Bifidobacterium bifidum YIT 10347 strain (FERM BP-10613), Bifidobacterium bifidum YIT 4007 strain (NITE BP-04192), and Bifidobacterium bifidum YIT 4042 strain (ATCC 11863) are preferred, and from the viewpoint of efficacy, Bifidobacterium bifidum YIT 10347 strain (FERM BP-10613) and Bifidobacterium bifidum YIT 4007 strain (NITE BP-04192) are more preferred.

[0019] The Bifidobacterium bifidum used in the inhibitor of the present invention includes not only the aforementioned strains, but also strains that have been artificially genetically modified while maintaining the properties of Bifidobacterium bifidum. Furthermore, since Bifidobacterium bifidum is effective in live form, live bacteria are preferred.

[0020] Bifidobacterium bifidum YIT 10347 strain is internationally deposited with the National Institute of Technology and Evaluation (NIITE), the international depositary authority under the Budapest Convention, as Bifidobacterium bifidum YIT 10347 (FERM BP-10613, deposit date: May 29, 2006) at Room 120, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818.

[0021] The Bifidobacterium bifidum YIT 4007 strain was deposited domestically with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE P-04192, provisional deposit date: October 29, 2024), which is the domestic depositary authority under the Patent Law. On August 25, 2025, a request was made to transfer it to international deposit, and the name changed to Bifidobacterium bifidum YIT 4007 (NITE P-04192). It has been internationally deposited under the Budapest Convention as BP-04192 (deposit date: October 29, 2024) with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan), which is the international depositary authority under the Budapest Convention.

[0022] Bifidobacterium bifidum YIT 4042 strain (ATCC 11863) can be purchased as Bifidobacterium bifidum (Tissier) Orla-Jensen 11863 from the ATCC (American Type Culture Collection: 10801 University Boulevard Manassas, Virginia 20110-2209 U.S.A.) web catalog (https: / / www.atcc.org / products / 11863).

[0023] Bifidobacterium bifidum YIT 4013 strain (ATCC 15696) can be purchased from the ATCC web catalog (https: / / www.atcc.org / products / 15696) as Bifidobacterium bifidum (Tissier) Orla-Jensen 15696.

[0024] The inhibitor of the present invention is preferably further enriched with sugars that are utilized by Bifidobacterium bifidum, as this promotes the metabolism of indole to indole-lactic acid by Bifidobacterium bifidum. Whether or not a sugar is utilized by Bifidobacterium bifidum can be confirmed, for example, by its growth rate in a culture medium containing the test sugar.

[0025] The sugars that Bifidobacterium bifidum utilizes are not particularly limited, but examples include galactose, galactooligosaccharides, fructose, glucose, lactose, fructooligosaccharides, 3'-sialyl lactose, 6'-sialyl lactose, 2'-fucosyl lactose, and 3'-fucosyl lactose. These sugars utilized by Bifidobacterium bifidum can be used individually or in combination of two or more.

[0026] Among the sugars that Bifidobacterium bifidum utilizes, galactose, galactooligosaccharides, glucose, lactose, and fructose are preferred, and from the viewpoint of effectiveness, galactose, galactooligosaccharides, lactose, and fructose are more preferred, with galactooligosaccharides being particularly preferred.

[0027] Furthermore, when the inhibitor of the present invention contains sugars that are utilized by Bifidobacterium bifidum, the sugars may be included in an amount of 5 to 100, preferably 25 to 50, per 1 unit mass of Bifidobacterium bifidum.

[0028] The inhibitor of the present invention can be any well-known component in the form described below, as long as it does not impair the effects of the present invention.

[0029] The form of the inhibitor of the present invention is not particularly limited, and examples include foods and beverages such as fermented milk, yogurt, and bacterial powder supplements, and pharmaceuticals such as live bacterial preparations, similar to conventionally known Bifidobacterium bacteria (sometimes called Bifidobacteria). Among these, fermented foods such as fermented milk and pharmaceuticals such as live bacterial preparations are preferred.

[0030] The inhibitor of the present invention described above can safely suppress the progression of chronic kidney disease and related diseases.

[0031] The inhibitor of the present invention can suppress the progression of chronic kidney disease when administered to mammals such as humans, dogs, and cats. In this case, the inhibitor of the present invention contains 10 units of Bifidobacterium bifidum per day. ５ ~10 １０ It should be included in an amount that corresponds to an intake level similar to that of CFU.

[0032] The inhibitor of the present invention can suppress the progression of chronic kidney disease, and therefore can also suppress diseases associated with chronic kidney disease. Examples of such diseases include cardiovascular disease, dialysis due to decreased renal function, and kidney transplantation. The inhibitor of the present invention can prevent the onset of these diseases or prolong the time until they occur.

[0033] Furthermore, the inhibitor of the present invention can be used in a method for suppressing the progression of chronic kidney disease by administering it to mammals such as humans, dogs, and cats, preferably mammals such as dogs and cats (excluding humans). In this case, the dosage is the same as described above.

[0034] The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited in any way to these examples.

[0035] Example 1: Screening of bacterial strains that metabolize indole to indolelactic acid: We investigated whether reference strains of bacterial species belonging to the Bifidobacterium class have the ability to metabolize indole to indolelactic acid. The ability to metabolize indole to indolelactic acid is known to exist in only a few bacterial strains belonging to the Bifidobacterium class.

[0036] (1) The target strains, the reference strains used for medium screening, and the media to be used are shown in Table 1. The composition of mILS broth is shown in Table 2.

[0037] Each reference strain was obtained from ATCC (American Type Culture Collection), JCM (Japan Collection of Microorganisms), DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) or IFO (Institute for Fermentation, Osaka).

[0038]

[0039] (2) Measurement of indole reduction ability and indole lactic acid production ability One strain stored in microbeads was inoculated into 2 mL of medium at one pellet per strain and cultured at 37 °C for 24 hours. 100 μL of the culture solution was inoculated at 1% (v / v) into 10 mL of fresh medium and cultured at 37 °C for 24 hours. The cell culture solution was washed with PBS, and indole (final concentration; 50 μM) was added to 2 mL of medium (0.1 M Bis-Tris, 0.75 mM MgSO ６００ ), 0.5% glucose, pH ≒ 6.5) so that the turbidity (OD ４ ) became 0.3, and the mixture was cultured at 37 °C for 24 hours. The supernatant after culture was collected, and the indole and indole lactic acid concentrations were measured with a high performance liquid chromatography mass spectrometer.

[0040] To 50 μL of the above medium sample, 50 μL of acetonitrile (ACN) containing 1% formic acid (FA) and 100 μL of ACN containing 1% FA (containing 20 ng / mL of the internal standard substance ethyl p-hydroxybenzoate {EHB}) were added and mixed, and then centrifuged (4 °C, 5 minutes, 20,400 g). 150 μL of the supernatant was suction filtered through a Sirocco plate (Waters), and the filtrate was used as the measurement sample.

[0041] Measurement equipment: ACQUITY UPLC I-class (Waters), Xevo TQ-S micro (Waters) Column: ACQUITY UPLC column BEH C18 (2.1 mm I.D. × 50 mm, 1.7 μm, Waters) Column temperature: 40 °C Autosampler temperature: 4 °C Mobile phase: Line A; 0.1% FA; Line B; ACN Gradient: Conditions as shown in Table 3

[0042]

[0043] Injection volume: 1 μL Ionization method: Electrospray ionization (ESI) Scan mode: Multiple Reaction Monitoring Data analysis: ver. 4.1 (Waters) Ion source temperature: 150 °C MS / MS measurement conditions: Conditions shown in Table 4

[0044]

[0045] (3) Results The indole-reducing ability and indole-lactic acid production ability of Bifidobacterium bifidum YIT 10347 were compared with that of reference strains of other Bifidobacterium species. In all strains, the reduced indole was metabolized to indole-lactic acid (Figure 1). Bifidobacterium bifidum YIT 10347 showed a higher indole-reducing ability than any of the reference strains.

[0046] Based on these results, Bifidobacterium bifidum YIT 10347 had higher indole-reducing and indole-lactic acid-producing abilities compared to other type strains of the genus Bifidobacterium.

[0047] Example 2 Confirmation of indole metabolites of other Bifidobacterium bifidum: Indole and indole lactic acid were measured in the same manner as in Example 1, except that Bifidobacterium bifidum YIT 10347 and Bifidobacterium bifidum YIT 4007 were used as the bacterial strains. The results are shown in Figure 2.

[0048] In Bifidobacterium bifidum YIT 4007, indole was also metabolized to indole-lactic acid. Furthermore, Bifidobacterium bifidum YIT 4007 had a higher indole-reducing ability than Bifidobacterium bifidum YIT 10347. Both Bifidobacterium bifidum YIT 10347 and Bifidobacterium bifidum YIT 4007 share the ability to adhere to gastric cells (mucin) and assimilate mucin.

[0049] Example 3: Combination of Bifidobacterium bifidum and galactooligosaccharides: The indole-reducing effect of Bifidobacterium bifidum YIT 4007 was evaluated in a diluted fecal solution from healthy individuals, which is a condition similar to the human intestinal environment.

[0050] (1) The target strain was Bifidobacterium bifidum YIT 4007.

[0051] (2) Measurement of indole reduction ability and indole lactate production ability in the presence of galactooligosaccharides (hereinafter referred to as "GOS") <Feces of healthy adults> Ten healthy adults were used as subjects, and this study was conducted in accordance with the Declaration of Helsinki. The subjects themselves collected the entire amount of fresh stool in a plastic bag, sealed it in an aluminum pouch with Aneropack Kenki (Mitsubishi Gas Chemical), and collected it after maintaining a low temperature with a coolant. The stool was kneaded by hand on the same day to make it uniform, and a portion (about 4 g) was dispensed into a centrifuge tube (capacity 25 mL) and stored at -80 °C until the day of use.

[0052] <Group Composition> The following groups were established: Bifidobacterium bifidum YIT 10347 and no GOS (control group), Bifidobacterium bifidum YIT 10347 alone group, GOS alone group, and Bifidobacterium bifidum YIT 10347 + GOS combination group.

[0053] <Culture Conditions> The indole metabolic capacity of Bifidobacterium bifidum YIT 10347 and GOS in combination in a diluted solution of healthy adult feces was evaluated by adding Bifidobacterium bifidum YIT 10347 and GOS to a suspension of healthy adult feces diluted 10-fold with PBS that had been autoclaved (121 °C, 15 min) before use, and culturing with shaking at 37 °C for 6 hours. The concentrations of indole and indole lactate in the culture solution were then measured. The test was repeated three times.

[0054] The amount of Bifidobacterium bifidum YIT 10347 to add is approximately 2 × 10⁶ per 1 mL of culture medium. ８ The solution was prepared to form cells. 0.25 w / v% GOS was added. Shaking culture was performed by sealing an Eppendorf tube (Eppendorf; #2029-04-28) containing the culture medium in an anero pouch (Mitsubishi Gas Chemical; A-92W) together with an anero pouch kenki (Mitsubishi Gas Chemical; A-13) to maintain anaerobic conditions, and shaking was performed using a small shaking incubator (AS ONE Corporation; PIC-101S) with the shaking speed set to 8. The culture medium was stored at -80 °C until it was used for various measurements.

[0055] <Preparation of bacterial strain suspension> The number of bacteria during the preparation of the Bifidobacterium bifidum YIT 10347 suspension to be added to the culture medium is measured by turbidity (OD). ６００ Based on ), approximately 1 x 10 ９ Prepared to a CFU / mL concentration. OD ６００ Measurements were performed by dispensing each diluent (100 μL) into a 96-well plate and using a plate reader (Molecular Devices; Spectra Max Plus 384).

[0056] <Preparation of Bifidobacterium bifidum YIT 10347 Suspension> 1% lactose mILS medium (2 mL) was added to a test tube (φ13×100 mm) sterilized by autoclaving (121 °C, 15 minutes), and one pellet of Bifidobacterium bifidum YIT 10347 stored in a bead stock was added. After anaerobic culture (37 °C, overnight), it was homogenized using a vortex mixer (preculture solution). 1% lactose mILS (10 mL × 6 tubes) was added to a test tube (φ18×150 mm) sterilized by autoclaving (121 °C, 15 minutes), and the preculture solution (100 μL) was added, followed by anaerobic culture (37 °C, overnight). After homogenizing the culture solution using a vortex mixer, it was combined into one container to obtain a Bifidobacterium bifidum YIT 10347 culture solution (OD ６００ : approximately 0.4). The Bifidobacterium bifidum YIT 10347 culture solution was divided into two (approximately 30 mL × 2 tubes), and each was centrifuged (4 °C, 5 minutes, 8,000 g) to remove the supernatant. The obtained cells were each suspended in PBS (5 mL), centrifuged (4 °C, 5 minutes, 8,000 g), and the supernatant was removed. The cells were suspended together using PBS (9 mL) to prepare a Bifidobacterium bifidum YIT 10347 suspension.

[0057] <Preparation of GOS Solution> GOS was dissolved in 40-fold amount of PBS to prepare a GOS solution (2.5 w / v%). The composition of GOS is oligomate 55N (Yakult Pharmaceutical Industry Co., Ltd.) purified by a known method to have a galactooligosaccharide of 3 to 4 sugars of 97.9% by mass or more (hereinafter referred to as GOS (purified product)).

[0058] <Preparation of 5-fold Diluted Fecal Solution> 4-fold amount of PBS was added to the feces returned to room temperature, and it was vigorously shaken using a vortex mixer. The suspension was filtered through sterilized gauze to remove the residue, and the filtrate was collected to prepare a 5-fold diluted fecal solution.

[0059] <Preparation of Bifidobacterium bifidum YIT 10347 monoculture> Bifidobacterium bifidum YIT 10347 suspension (200 μL), fecal 5-fold dilution (500 μL), and PBS (300 μL) were added to Eppendorf tubes and mixed using a vortex mixer to prepare the sample.

[0060] <Preparation of GOS-only group> GOS solution (100 μL), 500 μL of 5-fold diluted fecal solution (500 μL), and PBS (400 μL) were added to Eppendorf tubes and mixed using a vortex mixer to prepare the sample.

[0061] <Preparation of the Bifidobacterium bifidum YIT 10347 + GOS combination group> Bifidobacterium bifidum YIT 10347 suspension (200 μL), 500 μL of 5-fold diluted fecal solution, GOS solution (100 μL), and PBS (200 μL) were added to Eppendorf tubes and mixed using a vortex mixer to prepare the solution.

[0062] <Preparation of Bifidobacterium bifidum YIT 10347 and GOS-free (control) group> A 5-fold diluted fecal solution (500 μL) and PBS (500 μL) were added to Eppendorf tubes and mixed using a vortex mixer to prepare the samples.

[0063] <Measurement of Indole and Indole-Lactate Concentrations> The culture medium was returned to room temperature and homogenized using a vortex mixer. 20 ng / mL EHB-containing ethanol (300 μM) was added to the culture medium (100 μL) and mixed, then centrifuged (4 °C, 5 min, 20,400 g). 200 μL of the supernatant was filtered by suction using a Sirocco plate (Waters), and the filtrate was used as the measurement sample. The measurement was performed using a high-performance liquid chromatography-mass spectrometer (Waters ACQUITY UPLC I-class and Waters Xevo TQ-S micro) in the same manner as in Example 1. Data analysis was performed using spreadsheet software (Microsoft Office 2013 Excel 2013).

[0064] <PMA-qPCR> The viable cell count and total cell count of Bifidobacterium bifidum YIT 10347 before and after culturing were calculated by PMA-qPCR. Culture media (50 μL) of the Bifidobacterium bifidum YIT 10347-treated group (Bifidobacterium bifidum YIT 10347 alone group, Bifidobacterium bifidum YIT 10347 + GOS combined group) and the control group were diluted with three times the volume of PBS (150 μL). PMAxx (Biotium; #40069) was added to the PMA-treated group for viable cell count measurement to a final concentration of 50 μM. The mixtures were left to stand for 10 minutes under ice cooling in the dark, reacted with an LED Crossinker for 10 minutes, and DNA was extracted by the bead-phenol method. For amplification of Bifidobacterium bifidum YIT 10347, the pBF-1_Fw primer and pBF-1_Rv primer described in the literature (Appl. Environ. Microbiol. 79, 2182-2188 (2013)) were used. For the PCR reaction, a GoTaq qPCR Master Mix (Promega; #A6002) was used, and the reaction was carried out on a QuantStudio 12K Flex (Applied Biosystems) at 94 °C for 2 minutes, followed by 40 cycles of reaction at 94 °C for 20 seconds, 60 °C for 10 seconds, and 72 °C for 50 seconds. The detection limit was 1 x 10⁻⁶. ６ The concentration was set to cells / mL.

[0065] (3) Results <Indole and indole lactate concentrations in the control group at the start of culture> The indole concentration in each culture medium of the control group at the start of culture was 12.5 ± 4.7 μM (5.5–19.5 μM), and individual differences were observed. The indole lactate concentration was 0.6 ± 1.0 μM (0.1–3.3 μM), which was lower than the indole concentration.

[0066] <Indole and indole lactate concentrations at the end of culture> At the end of culture, the indole concentrations in the culture media of the control group, Bifidobacterium bifidum YIT 10347 monotherapy group, GOS monotherapy group, and Bifidobacterium bifidum YIT 10347 + GOS combination group were 25.4 ± 16.2 μM (5.0–54.5 μM), 23.2 ± 16.4 μM (5.0–52.5 μM), 7.2 ± 2.2 μM (5.4–12.6 μM), and 5.5 ± 0.7 μM (5.0–6.8 μM), respectively. Furthermore, the indole-lactic acid concentrations were 1.8 ± 1.2 μM (0.6–4.6 μM), 3.3 ± 1.7 μM (1.4–7.1 μM), 3.8 ± 2.1 μM (1.6–7.8 μM), and 13.4 ± 3.5 μM (7.8–17.5 μM), respectively (Figure 3).

[0067] <Number of viable Bifidobacterium bifidum YIT 10347 cells> The number of viable Bifidobacterium bifidum YIT 10347 cells at the start of culture was 1 x 10⁶ ９．７ The cell count was cells / mL. At the end of the culture, the number of viable Bifidobacterium bifidum YIT 10347 cells in the Bifidobacterium bifidum YIT 10347 monotherapy group and the Bifidobacterium bifidum YIT 10347 + GOS combination group was 1 × 10⁶. １０．１±０．５ cells / mL and 1 × 10 １０．２±０．４ The result was cells / mL.

[0068] Indole concentrations in the Bifidobacterium bifidum YIT 10347 monotherapy group were lower than in the control group (control group: 25.4 ± 16.2 μM, Bifidobacterium bifidum YIT 10347 group: 23.2 ± 16.4 μM). Furthermore, indole lactate concentrations in the Bifidobacterium bifidum YIT 10347 monotherapy group were approximately 1.8 times higher than in the control group (control group: 1.8 ± 1.2 μM, Bifidobacterium bifidum YIT 10347 monotherapy group: 3.3 ± 1.7 μM). The indole concentration in the GOS-only group (7.2 ± 2.2 μM) was lower than that of the control group (25.4 ± 16.2 μM), while the indole-lactate concentration was approximately 2.2 times higher (control group: GOS alone = 1.8 ± 1.2 μM: 3.8 ± 2.1 μM). The addition of Bifidobacterium bifidum YIT 10347 + GOS resulted in a lower indole concentration (5.5 ± 0.7 μM) compared to the control group, while the indole-lactate concentration (13.4 ± 3.5 μM) was higher than that of all other groups, approximately 7.4 times higher than that of the control group. Furthermore, since there was no change in the number of viable Bifidobacterium bifidum YIT 10347 cells with or without GOS, it was considered that the increase in indole-lactic acid was due to the enhanced indole metabolism capacity of Bifidobacterium bifidum YIT 10347 by GOS. There were also no individual differences in this effect.

[0069] The results above show that Bifidobacterium bifidum YIT 10347 or GOS, respectively, have a higher indole reduction effect and indole lactate production than the control group. Furthermore, the combination of Bifidobacterium bifidum YIT 10347 and GOS showed that, although there was little change in the number of viable Bifidobacterium bifidum YIT 10347 cells due to the addition of GOS, the indole reduction effect and indole lactate production were more significant than when each was used alone. These results indicate that Bifidobacterium bifidum YIT 10347 can reduce the amount of indoxyl sulfate in the blood, and this effect is further enhanced when combined with GOS.

[0070] Example 4: Combination of Bifidobacterium bifidum and sugars: The indole-reducing effect of Bifidobacterium bifidum YIT 10347 was evaluated using sugars such as GOS.

[0071] (1) The target substances used were fructose (D-(-)-Fructose: Tokyo Chemical Industry Co., Ltd.), glucose (D(+)-Glucose: Fujifilm Wako Pure Chemical Industries Co., Ltd.), galactose (D-(+)-Galactose Anhydrus: Tokyo Chemical Industry Co., Ltd.), lactose (lactose monohydrate: Kanto Chemical Co., Ltd.), fructooligosaccharides (FOS) (Fructooligosaccharides: Fujifilm Wako Pure Chemical Industries Co., Ltd.), and GOS (refined product).

[0072] (2) Measurement of indole reduction ability: indole (100 μM), Bis-Tris (0.1 M), and MgSO ４ Bifidobacterium bifidum YIT 10347 (1.3 ± 0.4 × 10) was added to a culture medium (Scr medium) containing (0.75 mM). ８ 0.45 (w / v)% of bacterial powder (CFU / mL) and sugars containing GOS (glucose, galactose, lactose, fructose, FOS) were added, and the cultures were incubated statically at 37 °C for 72 hours under anaerobic conditions. The concentrations of indole and indole-lactic acid in the culture supernatant were then measured in the same manner as in Example 1 (Figure 4).

[0073] (3) Results In culture supernatants to which GOS, glucose, galactose, lactose, or fructose were added, the indole-lactic acid concentration increased as the indole concentration decreased. The amount of indole reduction (= indole-lactic acid production) at this time was in the order of GOS ≈ galactose ≈ fructose ≈ lactose > glucose. In addition, in culture supernatants to which FOS was added or no sugar was added, the indole concentration decreased slightly, and the indole-lactic acid concentration increased accordingly.

[0074] From these results, it was found that the indole-reducing effect of Bifidobacterium bifidum YIT 10347 is enhanced not only by GOS but also by all sugars that Bifidobacterium bifidum utilizes. In particular, the indole-reducing effect of Bifidobacterium bifidum YIT 10347 was significantly enhanced by GOS, galactose, fructose, and lactose.

[0075] Example 5 Confirmation of indole metabolites of other Bifidobacterium bifidum: Indole and indole lactic acid were measured in the same manner as in Example 1, except that Bifidobacterium bifidum YIT 10347 or Bifidobacterium bifidum YIT 4042 (ATCC 11863) was used as the bacterial strain. The results are shown in Figure 5.

[0076] Similar to Bifidobacterium bifidum YIT 4042, indole was metabolized to indole-lactic acid in Bifidobacterium bifidum YIT 10347. Furthermore, Bifidobacterium bifidum YIT 10347 had a higher indole-reducing ability than YIT 4042. Like Bifidobacterium bifidum YIT 10347, Bifidobacterium bifidum YIT 4042 also possesses adhesion to gastric cells (mucin) and mucin assimilation capabilities.

[0077] The chronic kidney disease progression inhibitor of the present invention can suppress the progression of chronic kidney disease.

Claims

1. A drug containing Bifidobacterium bifidum as the active ingredient to suppress the progression of chronic kidney disease.

2. The chronic kidney disease progression inhibitor according to claim 1, further comprising sugars utilized by Bifidobacterium bifidum.

3. The chronic kidney disease progression inhibitor according to claim 2, wherein the sugars utilized by Bifidobacterium bifidum are one or more selected from galactose, galactooligosaccharides, glucose, lactose, and fructose.

4. The chronic kidney disease progression inhibitor according to claim 1 or 2, wherein the Bifidobacterium bifidum is Bifidobacterium bifidum YIT 10347 strain (FERM BP-10613) or Bifidobacterium bifidum YIT 4007 strain (NITE BP-04192).

5. The chronic kidney disease progression inhibitor according to claim 1 or 2, which reduces the amount of indoxyl sulfate in the blood.

6. A method for inhibiting the progression of chronic kidney disease in a mammal, characterized by administering the chronic kidney disease progression inhibitor described in claim 1 or 2 to the mammal.

7. A method for producing indole lactic acid, characterized by reacting indole with Bifidobacterium bifidum.

8. The method for producing indole lactic acid according to claim 7, wherein the Bifidobacterium bifidum is Bifidobacterium bifidum strain YIT 10347 (FERM BP-10613) or Bifidobacterium bifidum strain YIT 4007 (NITE BP-04192).

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