Novel synbiotic preparation

Abstract

To provide an agent for improving the intestinal environment and an agent for suppressing chronic renal failure.SOLUTION: The present invention discloses agents comprising, as main components: polyhydroxybutyrate (PHB), which functions as a prebiotic; and spores of Clostridium butyricum (CB), which function as a probiotic.SELECTED DRAWING: None

Description

[Technical field]

[0001] [Background technology]

[0002] Gut-renal correlation: There is a close correlation between renal function and intestinal flora (gut-renal correlation). As a result of the deterioration of the intestinal flora, toxic substances such as indoxyl sulfate induce chronic renal failure (Patent Document 1, Non-Patent Document 1). Conversely, chronic renal failure can be improved by improving the intestinal flora (Non-Patent Document 2). Since butyric acid bacteria are the key to the intestinal flora, a preparation that proliferates butyric acid bacteria is necessary (Non-Patent Document 3). This technology focuses on polyhydroxybutyrate (PHB), which induces a butyric acid bacteria-dominant intestinal flora in a short period of time, and provides an inhibitor of chronic renal failure (Patent Documents 2-3).

[0003] Polyhydroxybutyrate (PHB): Polyhydroxybutyrate (PHB) offers a revolutionary new method for prebiotics (Patent Documents 4-6). PHB (chemical formula 1) is a compound in which ketone bodies are polymerized with ester bonds. These ester bonds cannot be hydrolyzed by mammalian esterases. PHB is hydrolyzed by intestinal bacteria in the large intestine, and provides ketone bodies (β-hydroxybutyrate: 3HB) to intestinal bacteria, activating butyric acid bacteria. This prebiotic is revolutionary in that it starts with providing 3HB to intestinal bacteria, and is called "ketobiotics" (Non-Patent Documents 4-7). "Chemical formula 1:PHB" TIFF2025048680000001.tif37147

[0004] Clostridium butyricum: Clostridium butyricum has been commercialized under the trade names BioThree (Toa Shinyaku) and Miya BM (Miyarisan Pharmaceutical), and has been highly evaluated in clinical practice as an effective intestinal regulator. In particular, it is frequently used in nephrology clinical practice for patients with chronic renal failure (Patent Document 1, Non-Patent Document 8).

[0005] Polyhydroxybutyrate (PHB) and Clostridium butyricum: This technology demonstrated a synergistic effect when polyhydroxybutyrate (PHB) and Clostridium butyricum spores were used in combination, contributing to the suppression of chronic renal failure (Figure 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-181409 [Patent Document 2] Patent 2018-567965 [Patent Document 3] Patent 2022-525258 [Patent Document 4] Patent 2005-513522 [Patent Document 6] Patent No. 2022-177817 [Non-patent literature]

[0007] [Non-Patent Document 1] Rysz J, et al.The Impact of CKD on Uremic Toxins and Gut Microbiota.Toxins(Basel).2021 Mar 31;13(4):252. [Non-Patent Document 2] Nanto-Hara F,et al.The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.Nephrol Dial Transplant.2020 Feb 1;35(2):250-264. [Non-Patent Document 3] Li YJ, et al. Dietary Fiber Protects against Diabetic Nephropathy through Short-Chain Fatty Acid-Mediated Activation of G Protein-Coupled Receptors GPR43 and GPR109A. J Am Soc Nephrol. 2020 Jun;31(6):1267-1281.

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

[0008] The number of patients with chronic renal failure in Japan is estimated at 13.3 million, and the number of stage 5 patients who require dialysis has exceeded 300,000. There is a need for a formulation that will prevent chronic renal failure patients from becoming so severe that they require dialysis. This technology creates synbiotics and provides an inhibitor of chronic renal failure. [Means for solving the problem]

[0009] We have created a new "synbiotic" by combining polyhydroxybutyric acid (PHB), which acts as a "prebiotic," and spores of Clostridium Butyricum, which acts as a "probiotic," and provide an intestinal regulator, an inhibitor of ulcerative colitis, and an inhibitor of chronic renal failure. (1) A butyric acid release agent whose main components are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (2) A lactic acid release agent whose main components are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (3) An acetic acid release agent whose main components are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (4) A propionic acid release agent whose main components are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (5) A pH lowering agent whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (6) An intestinal regulator whose main ingredients are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (7) An inhibitor of ulcerative colitis, the main components of which are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (8) A suppressant for chronic renal failure, the main components of which are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum (9) An intestinal regulator whose main ingredients are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and live, dead or spore-form "good bacteria" (10) An inhibitor of ulcerative colitis, the main components of which are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and live, dead or spore-forming "good bacteria" (11) A chronic renal failure inhibitor consisting mainly of poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and live, dead or spore-form "good bacteria" (12) The “good bacteria” described in (9) to (11) are species selected from the following species: Streptococcus: Streptococcus thermophilus Bifidobacterium: Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium mongoliense, Bifidobacterium longum, Bifidobacterium adolescentis Lactococcus: Lactococcus lactis Genus Lactobacillus: Lacticaseibacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lacticaseibacillus fructivorans, Lacticaseibacillus hilgardii, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibachillus paracasei, Lacticaseibacillus plantarum, Lacticaseibacillus lactis Genus Enterococcus: Enterococcus faecalis, Enterococcus faecium Genus Lentilactobacillus: Lentilactobacillus otakiensis Genus Secandiractobacillus: Secundilactobacillus odoratitofui Genus Lactycaseibacillus: Lacticaseibacillus casei Genus Akkermansia: Akkermansia muciniphila Genus Bacillus: Bacillus polyfermenticus, Bacillus subtilis, Bacillus uniflagellatus, Bacillus globigii, Bacillus natto, Bacillus coagulans, Bacillus clausii, Bacillus halodurans, Bacillus coahuilensis, Bacillus pumilus, Bacillus licheniformis

Effects of the Invention

[0010] This technology creates a new "synbiotic" consisting of polyhydroxybutyrate (PHB), which acts as a "prebiotic," and spores of Clostridium butyricum, which acts as a "probiotic." This technology can effectively increase the number of butyricum bacteria contained in the bacterial groups belonging to groups IV, XIVa, and XVIII of the Clostridium cluster, such as Eubacterium, Roseburia, Coprococcus, Faecalibacterium, Ruminococcus, Lachnospira, and Clostridium.

[0011] This technology is expected to have two effects. 1. A new agent for improving intestinal environment 2. New drug for preventing chronic renal failure It is possible to prevent humans from falling into dialysis due to the worsening of diabetic nephropathy. [Brief description of the drawings]

[0012] Synbiotics of PHB and spores in an in vitro culture system of Clostridium butyricum Figure 1: Acetic acid, Figure 2: Butyric acid, Figure 3: Propionic acid, Figure 4: pH Clostridium butyricum produces a certain amount of organic acids even without stimulating factors, but the coexistence of PHB and spores enhances the ability to release organic acids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Synbiotics: Synbiotics are a combination of probiotics and prebiotics. By ingesting live bacteria (probiotics) that are beneficial to the human intestine, such as butyric acid bacteria and lactic acid bacteria, together with prebiotics that promote the growth of beneficial bacteria, such as PHB, the functions of both can be exerted synergistically.

[0014] Clostridium butyricum: Clostridium butyricum is a spore-forming obligate anaerobic bacterium, a gram-positive rod-shaped bacterium. It is widely distributed in the environment and in the intestines of humans and animals, and is a type of commensal bacterium isolated from 10-20% of human feces. In Japan, several strains have been approved as pharmaceuticals for intestinal regulation and have been used for many years. Numerous reports have been published about the effectiveness of this bacterium against intestinal infections (Patent Document 1; Non-Patent Document 8).

[0015] Clostridium butyricum spores: Clostridium butyricum is a butyric acid bacterium and also forms spores. The spores survive in conditions that would normally kill bacteria. When the spores are placed in an environment suitable for bacterial growth, they germinate to produce bacterial cells with normal growth and metabolic capabilities (Patent Document 1; Non-Patent Document 8).

[0016] Hydrolysis of PHB: Chemically, PHB is a polyester of ketone body (3-hydroxybutyric acid: 3HB). Mammalian digestive enzymes (esterase and lipase) cannot hydrolyze the ester bond of PHB. An extracellular enzyme called PHB depolymerase can hydrolyze this ester bond. PHB changes the intestinal flora and provides prebiotics (Non-Patent Document 4).

[0017] Ketobiotics proposal: It was found that when microminipigs were fed a diet containing 2% PHB, the concentrations of SCFAs such as acetate, propionate, and butyrate in the feces increased significantly. In addition, the pH level decreased significantly in response to the release of SCFA. This shows that PHB improves the intestinal environment. (Non-patent Document 4)

[0018] Microbiome: Microbiome analysis was performed to demonstrate that PHB induces a butyric acid bacteria-dominated intestinal environment. Analysis was performed at the "phylum" level as a criterion for indicators of improvement of the intestinal flora. The Firmicutes phylum increased, while the Proteobacteria phylum decreased. Within the Firmicutes phylum, analysis was performed at the genus level. The genera Clostridium, Roseburia, and Ruminoclostridium showed a statistically significant increase. (Non-Patent Document 6).

[0019] Roseburia-dominant intestinal environment: PHB showed a significant increase in the Roseburia genus of butyric acid bacteria. This butyric acid bacteria has been linked to healthy longevity in humans. Many papers have suggested that Roseburia butyric acid bacteria are linked to obesity, diabetes, and dementia in human microbiome analyses, and it is the bacteria that has attracted the most attention as an indicator of fecal (intestinal bacteria) transplantation (Non-patent Document 6).

[0020] Suppression of colon cancer: Since a deterioration of the intestinal environment is the starting point for colon cancer, inducing a butyric acid-dominant intestinal environment suppresses colon cancer. In mice fed a diet containing 10% PHB, butyric acid and propionic acid in the cecum increased, and the number of polyps and the area of ​​cancer decreased. In other words, PHB suppressed colon cancer (Fernandez J, Saettone P, Franchini MC, Villar CJ, Lombo F. Antitumor bioactivity and gut microbiota modulation of polyhydroxybutyrate (PHB) in a rat animal model for colorectal cancer. Int J Biol Macromol. 2022 Apr 1; ​​203: 638-649.).

[0021] Suppression of ulcerative colitis: We found that PHB suppresses ulcerative colitis and increases Treg in a mouse dextran sulfate (DSS) model. This is because PHB increases butyric acid, activates Treg, and suppresses inflammation (Non-Patent Document 5).

[0022] Suppression of weaning diarrhea: Weaning diarrhea is a major problem in the pig farming industry. If this problem can be solved, it will contribute greatly to the pig farming industry. Feeding with 0.5% PHB increased polysaccharides in the mucosa and suppressed weaning diarrhea. (Ma N, Guo P, Chen J, Qi Z, Liu C, Shen J, Sun Y, Chen X, Chen GQMa X. Poly-β-hydroxybutyrate alleviated diarrhea and colitis via Lactobacillus johnsonii biofilm-mediated maturation of sulfomucin. Sci China Life Sci. 2023 Jul; 66(7): 1569-1588.). EXAMPLES

[0023] Clostridium butyricum was cultured in 100 mL of PYG medium (1.5 g peptone, 1.0 g yeast extract, 5 g glucose, 1 L water, pH 7.0) in an anaerobic chamber until it reached the logarithmic growth phase (10 6 ~10 8 / mL). 5 / mL (total number of bacteria 10 7 ) butyric acid bacteria were added and cultured anaerobically for 72 hours in the following four groups: 1. Control 2. +PHB2% 3.+10 spores 6 / mL 4. +PHB2%+spores10 6 / mL The concentrations of organic acids (acetic acid, butyric acid, and propionic acid) in the culture solution were then measured using high performance liquid chromatography. The pH of the solution was also measured using a pH electrode. In Figures 1 to 4, the amount of organic acid produced by each group, with the control taken as 1, is shown at the top of the column. Additionally, * indicates the result of a significant difference test from the control (student t-test, <0.05).

[0024] Comparison of acetate (Figure 1) and butyrate (Figure 2) production in each group

[0025] "PHB" produced 2.7 times more acetic acid than the control. Spores produced 1.2 times more acetic acid than the control. "PHB + spores" produced 15.6 times more acetic acid than the control This was 4.2 times the amount of acetic acid produced by "PHB" + "spores" (3.7 (2.7 + 1.2) times).

[0026] "PHB" produces 2.8 times more butyric acid than the control Spores produced 2.4 times more butyric acid than the control "PHB + spores" produced 10.6 times more butyric acid than the control This was 2.0 times the butyric acid production of ``PHB'' + ``spores'', which was 5.2 (2.8 + 2.4) times. TIFF2025048680000002.tif87162

[0027] Comparison of the amount of propionic acid (Figure 3) produced and the solution pH (Figure 4) of each group

[0028] "PHB" produced 3.1 times more propionic acid than the control Propionic acid production by "spores" was 2.1 times higher than that of the control. Propionic acid production by "PHB + spores" was 24.0 times higher than that of the control. This was 4.6 times the amount of propionic acid produced by "PHB" + "spores" (5.2 (3.1 + 2.1) times). TIFF2025048680000003.tif89161

[0029] PHB is hydrolyzed by PHB depolymerase on the membrane of butyric acid bacteria, and when there are many spores, ketone bodies generated by hydrolysis of PHB promote the growth of the spores. Furthermore, since ketone bodies enhance the production of acetic acid and butyric acid, the production amounts of acetic acid, butyric acid, and propionic acid in the "PHB + spores" group are greater than the results of adding the "PHB" group and the "spores" group together. In other words, it should be considered that a synergistic effect has been expressed in the "PHB + spores" group. This is the synergistic effect (synbiotics) of adding PHB (prebiotics) and spores (probiotics). The pH of the solution was greatly reduced in the "PHB + spores" group in response to this synbiotic.

Claims

1. A butyric acid release agent whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum.

2. A lactic acid release agent whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum.

3. An acetic acid release agent whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum.

4. A propionic acid release agent whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum.

5. A pH lowering agent whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum.

6. An intestinal regulator whose main ingredients are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of butyric acid bacteria (Clostridium Butyricum)

7. An inhibitor of ulcerative colitis, mainly composed of poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum

8. A chronic renal failure inhibitor whose main components are poly(R)-3-hydroxybutyric acid (PHB) powder with a weight-average molecular weight of 10,000 or more and spores of Clostridium Butyricum.

9. An intestinal regulator whose main ingredients are poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and live, dead or spore-form "good bacteria"

10. An ulcerative colitis inhibitor that consists mainly of poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and live, dead or spore-form "good bacteria"

11. A chronic renal failure inhibitor that consists primarily of poly(R)-3-hydroxybutyrate (PHB) powder with a weight-average molecular weight of 10,000 or more and live, dead or spore-form "good bacteria"

12. The "good bacteria" described in claims 9 to 11 are one or two species selected from the following species: Streptococcus: Streptococcus thermophilus Bifidobacterium: Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium mongoliense, Bifidobacterium longum, Bifidobacterium adolescentis Lactococcus: Lactococcus lactis Lactobacillus: Lacticaseibacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lacticaseibacillus fructivorans, Lacticaseibacillus hilgardii, Lacticaseibacillus paracasei, Lacticaseibacillus Rhamnosus, Lacticaseibacillus paracasei,Lacticaseibacillus plantarum,Lacticaseibacillus lactis Enterococcus: Enterococcus faecalis, Enterococcus faecium Lentilactobacillus: Lentilactobacillus otakiensis Secundilactobacillus: Secundilactobacillus odoratitofui Lacticaseibacillus: Lacticaseibacillus casei Ackermansia genus: Ackermansia muciniphila Bacillus genus: Bacillus polyfermenticus, Bacillus subtilis, Bacillus uniflagellatus, Bacillus globigii, Bacillus natto, Bacillus coagulans, Bacillus clausii, Bacillus halodurans, Bacillus coahuilensis, Bacillus pumilus, Bacillus licheniformis

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