Agent for proliferating microorganisms belonging to genus megamonas and agent for inhibiting proliferation of microorganisms belonging to genus escherichia and / or genus shigella

Abstract

The present invention addresses the problem of providing a method which has high safety and is capable of continuous ingestion and by which, on the basis of the proliferation action of useful bacteria or the proliferation inhibitory action of pathogenic bacteria in the intestine, diseases caused by the microorganisms can be prevented and ameliorated.　An agent for proliferating microorganisms belonging to the genus Megamonas and an agent for inhibiting the proliferation of microorganisms belonging to the genus Escherichia and / or the genus Shigella, which solve the problem of the present invention, contain a galactooligosaccharide as an active ingredient.

Description

Growth inhibitor for Megamonas genus microorganisms and growth inhibitor for Escherichia genus and / or Shigella genus microorganisms

[0001] The present invention relates to a growth inhibitor for microorganisms of the genus Megamonas and a growth inhibitor for microorganisms of the genus Escherichia and / or Shigella.

[0002] In recent years, the number of pet companion animals, such as dogs and cats (18.5 million), has far exceeded the population under the age of 15 (approximately 16 million), and they have come to be positioned as members of the family in Japan, where the birthrate is declining and the population is aging. On the other hand, as the average lifespan has increased due to improvements in the environment in which they are kept and advances in medical technology, the risk of them contracting various diseases has also increased.

[0003] The intestinal flora is known to not only contribute to the host's metabolism, but also to directly and indirectly affect the host's physiological functions, such as preventing infection from pathogenic microorganisms and strengthening the immune system.The relationship between intestinal bacteria and certain diseases is also becoming clear in companion animals.

[0004] For example, it has been reported that the intestinal flora of dogs suffering from mitral valve insufficiency or perianal fistula (canine furunculosis ani) shows a decrease in Megamonas bacteria and an increase in Escherichia bacteria (Non-Patent Documents 1 and 2), and that dogs suffering from diabetes or protein-losing enteropathy (Yorkshire terrier enteropathy) show an increase in Escherichia and / or Shigella bacteria (Non-Patent Documents 3 and 4). It has also been reported that Escherichia and / or Shigella bacteria are the causative bacteria of pyometra, and that an increase in Shigella bacteria is associated with the worsening of canine parvovirus infection (Non-Patent Documents 5 and 6).

[0005] When these diseases develop, various medical and surgical treatments are administered depending on the condition and progression, but long-term treatment is often required and complete recovery is difficult. For example, there is no drug that can provide a fundamental cure for mitral valve regurgitation; the goal is merely to alleviate symptoms and extend survival. For perianal fistula (canine furunculosis ani), immunosuppressive therapy is effective, but recurrence is common, necessitating lifelong treatment. Diabetes requires long-term blood glucose control through a combination of insulin and dietary therapy. Protein-losing enteropathy (Yorkshire terrier enteropathy) is treated with immunosuppressants and other medications, but often fails to provide a complete cure. For pyometra, when surgical treatment is difficult due to advanced age or other factors, hormone therapy is often used, but side effects are a concern. Parvovirus infection, in severe cases, has a high mortality rate. Therefore, there is a need for ingredients that can be consumed daily to prevent these diseases and maintain health even in old age.

[0006] Qinghong Li, Eva Larouche-Lebel, Kerry A Loughran, Terry P Huh, Jan S Suchodolski, Mark A Oyama. Gut Dysbiosis and Its Associations with Gut Microbiota-Derived Metabolites in Dogs with Myxomatous Mitral Valve Disease. mSystems. 2021;6(2):e00111-21.Rachel Pilla, Jan S. Suchodolski, The Role of the Canine Gut Microbiome and Metabolome in Health and Gastrointestinal Disease. Front.Vet.Sci.,Volume 6-2019,14 January 2020Pavlos G. Doulidis, Alexandra I. Galler, Bela Hausmann, David Berry, Alexandro Rodriguez-Rojas, Iwan A. Burgener. Gut Microbiome Signatures of Yorkshire Terrier Enteropathy during Disease and Remission. bioRxiv. 2023Nicole L Laia, Patrick C Barko, Drew R Sullivan, Maureen A McMichael, David A Williams, Jennifer M Reinhart. Longitudinal analysis of the rectal microbiome in dogs with diabetes mellitus after initiation of insulin therapy. PLoS One.2022;17(9)Hui-Hua Zheng, Chong-Tao Du, Yu-Zhu Zhang, Chao Yu, Rong-Lei Huang, Xin-Yue Tang, Guang-Hong Xie.A study on the correlation between intrauterine microbiota and uterine pyogenesis in dogs. Theriogenology. 2023;196:97-105.Yun Zhenga, Xiangqi Hao, Xi Lin, Qingxu Zheng, Wenyan Zhang, Pei Zhou, Shoujun Lia. Bacterial diversity in the feces of dogs with CPV infection. Microbial Pathogenesis. 121(2018)70-76.

[0007] The present invention aims to provide a method for preventing and ameliorating diseases caused by these microorganisms, which is highly safe, can be taken continuously, and has the effect of promoting the growth of beneficial bacteria in the intestines or inhibiting the growth of harmful bacteria.

[0008] As a result of extensive research to solve the above-mentioned problems, the inventors have discovered that by feeding companion animals galactooligosaccharides, beneficial microorganisms of the genus Megamonas can be grown in the intestines while the growth of pathogenic microorganisms of the genus Escherichia and / or Shigella can be suppressed, thereby providing preventive and therapeutic effects against diseases caused by these microorganisms, and have completed the present invention.

[0009] That is, the present invention is an agent for growing microorganisms of the genus Megamonas, which contains galactooligosaccharides as an active ingredient.

[0010] The present invention also relates to an inhibitor of the growth of microorganisms of the genus Escherichia and / or Shigella, which contains galactooligosaccharide as an active ingredient.

[0011] The growth agent for Megamonas microorganisms and the growth inhibitor for Escherichia and / or Shigella microorganisms of the present invention are highly safe and can be taken on a daily basis. They can grow beneficial microorganisms of the genus Megamonas in the intestines and inhibit the growth of pathogenic microorganisms of the genera Escherichia and Shigella, thereby enabling simple and effective prevention and treatment of diseases caused by these microorganisms.

[0012] The growth inhibitor for Megamonas microorganisms and the growth inhibitor for Escherichia and / or Shigella microorganisms of the present invention contain galactooligosaccharides as active ingredients. Galactooligosaccharide is a general term for oligosaccharides containing at least one galactose residue in the molecule, and examples thereof include saccharides in which 2 to 9, preferably 3 to 4, monosaccharides are linked together. Galactooligosaccharides include those in which galactose is linked via β1-2, β1-3, β1-4, or β1-6 bonds, with β1-4 and β1-6 bonds being particularly preferred. More preferred galactooligosaccharides include β-1,4 galactosyllactose (Galβ1-4Galβ1-4Glc), β-1,6 galactosyllactose (Galβ1-6Galβ1-4Glc), and β-1,3 galactosyllactose (Galβ1-3Galβ1-4Glc). Galactooligosaccharides containing β-1,4 galactosyllactose as the main component are more preferred, and for example, Oligomate 55N (manufactured by Yakult Pharmaceutical Industry Co., Ltd., galactooligosaccharide content of the solid content is 55% by mass, of which β-1,4 galactosyllactose content is 18.5% by mass) can be used.

[0013] In the present invention, the ingestion of the above-mentioned galactooligosaccharides enables the proliferation of beneficial microorganisms of the genus Megamonas in the intestines. Meanwhile, the proliferation of harmful microorganisms of the genus Escherichia and Shigella can be inhibited, thereby providing excellent preventive and therapeutic effects against diseases caused by these microorganisms. The growth inhibitors of Megamonas microorganisms and Escherichia and / or Shigella microorganisms of the present invention are suitable for use in companion animals, such as dogs, cats, rabbits, and hamsters. Among these, dogs and cats are preferred, with dogs being particularly suitable.

[0014] Diseases associated with a reduction in Megamonas microorganisms and suggested for prevention and treatment using Megamonas microorganisms include mitral valve insufficiency and perianal fistula (canine furunculosis ani). Mitral valve insufficiency occurs when the mitral valve, which repeatedly opens and closes according to the cardiac cycle to allow blood to flow from the left atrium to the left ventricle, thickens due to myxomatous degeneration or other factors, causing the mitral valve leaflets to protrude toward the left atrium, resulting in backflow of blood from the left ventricle to the left atrium. Perianal fistula (canine furunculosis ani) is a disease characterized by the formation of multiple ulcers or fistulas around the anus accompanied by chronic suppurative inflammation, resulting in pus discharge and pain. This disease is most prevalent in middle-aged, medium- to large-sized dogs. Since the occupancy rate of Megamonas microorganisms in the intestinal flora of dogs suffering from mitral valve insufficiency and perianal fistula (canine furunculosis ani) is lower than that of healthy dogs (Non-Patent Documents 1 and 2), these diseases can be prevented and improved by increasing the occupancy rate of Megamonas.

[0015] Diseases associated with the proliferation of Escherichia and / or Shigella microorganisms include the aforementioned mitral valve insufficiency and perianal fistula (canine furunculosis ani), as well as diabetes, protein-losing enteropathy (Yorkshire terrier enteropathy), pyometra, and parvovirus infection. Diabetes is a syndrome characterized by persistent hyperglycemia and other metabolic abnormalities due to insulin deficiency. It is classified into insulin-deficient and insulin-resistant types based on its mechanism of action. Diabetic symptoms include polydipsia and polyuria and weight loss. Progression of the condition to ketoacidosis leads to symptoms such as loss of appetite, weakness, vomiting, and even coma. Protein-losing enteropathy (Yorkshire terrier enteropathy) is a syndrome caused by abnormal leakage of plasma proteins from the intestinal mucosa into the intestinal lumen. Hypoproteinemia results when leakage exceeds the liver's protein synthesis capacity. Symptoms often include chronic diarrhea and weight loss, and severe hypoproteinemia can lead to ascites, pleural effusion, and edema due to decreased intravascular osmotic pressure. Pyometra is a disease characterized by cystic endometrial proliferation (cystic hyperplasia), which leads to inflammation (endometritis) caused by bacterial infection, resulting in the accumulation of pus within the uterus. It is believed to be caused by indigenous bacteria present in healthy individuals entering the uterus via the cervix, resulting in symptoms such as loss of appetite, fever, polydipsia and polyuria, and vomiting. Escherichia and / or Shigella microorganisms have been associated with diabetes, protein-losing enteropathy (Yorkshire terrier enteropathy), and parvovirus infection (Non-Patent Documents 3, 4, and 6), and are also causative agents of pyometra (Non-Patent Document 5). Therefore, reducing the prevalence of Escherichia and / or Shigella microorganisms can prevent and treat these diseases.

[0016] The form of the preventive and therapeutic agent of the present invention is not particularly limited and can be in the form of pet food, pet supplements, pet medicines, etc. As pharmaceuticals, galactooligosaccharides can be formulated as is or, if necessary, in combination with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include glucose, lactose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. Furthermore, conventional additives such as stabilizers, humectants, emulsifiers, binders, isotonicity agents, and excipients can be added as needed. The dosage form is not particularly limited, and examples include liquids, powders, granules, capsules, and tablets, which can be prepared according to conventional methods. The content of galactooligosaccharides in the preventive and therapeutic agent of the present invention is not particularly limited and is, for example, preferably 1 to 100% by mass, more preferably 5 to 95% by mass, and even more preferably 30 to 90% by mass.

[0017] The dose of the prophylactic and / or therapeutic agent of the present invention is not particularly limited, but is, for example, about 0.03 to 0.4 g per kg of body weight per day, preferably about 0.04 to 0.3 g, and particularly preferably about 0.1 to 0.25 g.

[0018] The preventive and therapeutic agent of the present invention can be in the form of pet food or pet supplement, and can be produced by using galactooligosaccharide as it is or, if necessary, by appropriately mixing with the raw materials used in known pet food or pet supplement.Such raw materials include, for example, meats such as beef, chicken, pork, etc., seafood such as bonito, mackerel, scallops, and clams, grains such as corn, barley, wheat, and rye, starches such as corn starch, wheat starch, and rice starch, dairy products such as butter and cheese, vitamins such as vitamin A, B1, B2, D, E, niacin, and pantothenic acid, minerals such as iron, calcium, sodium, and potassium, etc.Additives such as gelling agents, shape-retaining agents, pH adjusters, seasonings, preservatives, and nutritional supplements can also be added.

[0019] Pet food or pet supplements can be prepared by mixing the above ingredients in a conventional manner and can be in any form, such as dry type, wet type, jerky, gum, granules, powder, drink, etc.

[0020] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples in any way.

[0021] Example 1: The effects of galactooligosaccharides (GOS) on the intestinal microflora of dogs were investigated. A total of 26 domestic dogs were studied: 12 small dogs (under 5 kg), 9 medium-sized dogs (5-10 kg), and 5 large dogs (over 10 kg). The sex ratio was 9:17, with an average age of 8.88 years and an average weight of 7.76 kg. Oligomate 55N (galactooligosaccharide liquid sugar; Yakult Pharmaceutical Co., Ltd.) was administered for 8 weeks at doses set as shown in Table 1. A designated amount of Oligomate 55N was administered either orally via a spoon or sprinkled on top of the dog's food.

[0022]

[0023] Feces were collected before GOS ingestion (week 0, sometimes referred to as week), during the ingestion period (weeks 4 and 8), and after the end of ingestion (week 12). Approximately 0.1 g of feces was placed in a screw tube and homogenized (1,048 rpm, 10 min) using a Shake Master Auto (BMS-A20TP, Biomedical Sciences, Inc.) with 9 times the fecal weight of RNAlater and zirconia beads. The homogenized samples were then stored at 4°C. 200 μL of the suspension was mixed with 1 mL of PBS(-) and centrifuged (13,000 G × 5 min, 4°C). The supernatant was discarded. This procedure was repeated once more, and the resulting fecal pellet was stored at −25°C until DNA extraction. DNA was extracted according to the method of Fujimoto et al. (Fujimoto, J., Matsuki, T., Sasamoto, M., Tomii, Y. & Watanabe, K. Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA. Int. J. Food Microbiol. 126, 210-215 (2008)) and dissolved in 0.2 mL of Tris-EDTA buffer (TE) (pH 8.0). The DNA concentration was measured using a microspectrophotometer (DS-11, Scrum Co., Ltd.), adjusted to 5 ng / μL with TE (pH 8.0), and stored at −30°C until analysis.

[0024] The fecal microbiota composition was determined by amplicon sequencing of the 16S rRNA gene. The V4 region of the 16S rRNA gene of the intestinal microbiota was amplified using the forward primer 5′-GTGCCAGCMGCCGCGGTAA-3′ and reverse (806R): 5′-GGACTACHVGGGTWTCTAAT-3′. PCR amplicon libraries were generated according to the Illumina 16S sample preparation guide (16S Sample Preparation Guide, 15044223; Illumina, San Diego, CA, United States). Library quality was assessed using an Agilent 2200 Tapestation (Agilent Technologies, Inc.), and sequencing was performed using the MiniSeq™ Mild Output Reagent Cartridge 300 cycles, 20001311 (Illumina, CA, USA) on a MiniSeq system (Illumina). Each read was processed using QIIME2 (ver. 2022.2) software, and lineages were assigned using the SILVA138 database. The median number of reads for fecal samples was 51,585 (minimum: 11,863, maximum: 68,495). No sample had fewer than 10,000 reads, and all samples underwent bacterial flora analysis.

[0025] Statistical analysis of the occupancy rate of each bacterial species was performed using the "ALDEx2" package, targeting genera with an average occupancy rate of 0.01% or higher. p<0.05 was considered significant, and 0.05≦p<0.1 was considered a significant trend. For bacterial species that showed significant changes before and after GOS intake in the ALDEx2 analysis, the centered logarithmic transformation values ​​of their occupancy rates are shown in Table 2.

[0026]

[0027] As shown in Table 2, the genus Megamonas showed significantly higher occupancy rates at 4 and 8 weeks compared to 0 week, and a tendency for high levels was also observed at 12 weeks, four weeks after the end of GOS intake. Escherichia and Shigella showed a tendency for high levels at 8 weeks, and showed significantly lower levels at 12 weeks, four weeks after the end of GOS intake. These results confirmed that ingestion of galactooligosaccharides increases the proliferation of beneficial bacteria, Megamonas, and suppresses the proliferation of harmful bacteria, Escherichia and Shigella.

[0028] In addition, blood biochemistry data such as total protein, albumin, total bilirubin, blood urea nitrogen, and creatinine were measured by taking samples before galactooligosaccharide intake, and 4 and 8 weeks after the end of intake, and no changes in the data that could be considered adverse events were observed, confirming that there were no safety issues.

[0029] The preventive and therapeutic agent of the present invention is highly safe, can be taken on a daily basis, and has excellent preventive and therapeutic effects against diseases caused by these microorganisms by promoting the growth of Megamonas microorganisms in the intestines of dogs, cats, etc. and suppressing the growth of Escherichia and / or Shigella microorganisms. Therefore, the agent can be used as a medicine, pet food, etc. for companion animals.

Claims

1. A growth agent for Megamonas microorganisms containing galactooligosaccharides as an active ingredient.

2. The growth agent for Megamonas microorganisms according to claim 1, which is used for the prevention and treatment of mitral regurgitation or perianal fistula.

3. An inhibitor of the proliferation of Escherichia and / or Shigella microorganisms, which contains galactooligosaccharide as an active ingredient.

4. The growth inhibitor of Escherichia and / or Shigella microorganisms according to claim 3, which is used for the prevention and treatment of mitral regurgitation, perianal fistula, diabetes, protein-losing enteropathy, pyometra or parvovirus infection.

5. A method for preventing or treating mitral regurgitation, perianal fistula, diabetes, protein-losing enteropathy, pyometra, or parvovirus infection, which comprises administering galactooligosaccharides to a companion animal.

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