GROWTH INHIBITOR OF MICROORGANISMS BELONGING TO FAMILY Peptostreptococcaceae AND GROWTH PROMOTOR OF MICROORGANISMS BELONGING TO GENUS Slackia

Abstract

The present invention addresses the problem of providing a highly safe and continuously ingestible agent for: preventing or ameliorating diseases attributed to microorganisms belonging to the family Peptostreptococcaceae by inhibiting the growth of these microorganisms in the intestine; or for preventing or ameliorating diseases potentially preventable or ameliorable by a substance produced from microorganisms belonging to the genus Slackia by promoting the growth of these microorganisms in the intestine.　The problem of the present invention has been solved by a growth inhibitor of microorganisms belonging to the family Peptostreptococcaceae or a growth promotor of microorganisms belonging to the genus Slackia, each containing a galactooligosaccharide as an active ingredient.

Description

Growth inhibitor for microorganisms belonging to the Peptostreptococceae family and growth inhibitor for microorganisms belonging to the genus Slackia

[0001] The present invention relates to an inhibitor of the growth of microorganisms belonging to the family Peptostreptococcaceae and an agent for inhibiting the growth of microorganisms of the genus Slackia.

[0002] The number of pet companion animals, such as dogs and cats (approximately 15.9 million), far exceeds the population of people under the age of 15 (approximately 14.1 million), and they have come to be regarded 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 of pets 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 microbiota 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. In humans and companion animals, the relationship between intestinal bacteria and certain diseases, as well as the relationship between specific metabolites produced by intestinal bacteria and diseases, is becoming clear.

[0004] For example, it has been reported that microorganisms belonging to the Peptostreptococcaceae family, which are the main intestinal bacteria in cats, are more prevalent in diabetic and obese cats than in healthy cats (Non-Patent Documents 1 and 2). Microorganisms of the genus Slackia are known to produce equol (equol), which has preventive effects against hormone-dependent diseases such as breast cancer and prostate cancer, using daidzein, a type of isoflavone found in soybeans, as a substrate.

[0005] Bermingham EN, Young W, Butowski CF, Moon CD, Maclean PH, Rosendale D, Cave NJ and Thomas DG (2018) The Fecal Microbiota of the Domestic Cat (Felis catus) Is Influenced by Interactions Between Age and Diet; A Five Year Longitudinal Study. Front. Microbiol. 9:1231. doi: 10.3389 / fmicb.2018.01231Kieler IN, Osto M, Hugentobler L, Puetz L, Gilbert MTP, Hansen T, Pedersen O, Reusch CE, Zini E, Lutz TA, Bjornvad CR. Diabetic cats have decreased gut microbial diversity and a lack of butyrate producing bacteria. Sci Rep. 2019 Mar 18;9(1):4822. doi: 10.1038 / s41598-019-41195-0. PMID: 30886210; PMCID: PMC6423039.Shima T, Amamoto R, Kaga C, Kado Y, Sasai T, Watanabe O, Shiinoki J, Iwazaki K, Shigemura H, Tsuji H. Association of fermented milk intake with stool frequency, defecatory symptoms and intestinal microbiota in healthy Japanese adults. Benef Microbes 10: 841-854.

[0006] An object of the present invention is to provide a highly safe, continuously ingestible agent for preventing and ameliorating diseases caused by microorganisms belonging to the family Peptostreptococcaceae or promoting the growth of microorganisms of the genus Slackia in the intestines, or an agent for preventing and ameliorating diseases that can be expected to be prevented or ameliorated by components produced by these microorganisms, based on its inhibitory effect on the growth of these microorganisms or promoting the growth of microorganisms of the genus Slackia in the intestines.

[0007] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that by feeding companion animals galactooligosaccharides, the growth of microorganisms belonging to the Peptostreptococcaceae family in the intestines can be suppressed, while microorganisms of the genus Slackia can be increased, thereby providing preventive and therapeutic effects against diseases caused by these microorganisms, or preventive and therapeutic effects against diseases that can be expected to be prevented or improved by components produced by these microorganisms, and thus completed the present invention.

[0008] That is, the present invention is a growth inhibitor for microorganisms belonging to the family Peptostreptococcaceae, which contains galactooligosaccharide as an active ingredient.

[0009] The present invention also relates to an agent for growing microorganisms of the genus Slackia, which contains galactooligosaccharides as an active ingredient.

[0010] The growth inhibitor of microorganisms belonging to the Peptostreptococcaceae family and the growth agent for microorganisms of the genus Slackia of the present invention are highly safe and can be taken on a daily basis. They can suppress the growth of microorganisms belonging to the Peptostreptococcaceae family in the intestines and promote the growth of microorganisms of the genus Slackia, and can simply and effectively prevent and treat diseases that are caused by these microorganisms or that can be expected to be prevented or improved by components produced by these microorganisms.

[0011] Figure 1 shows the change in the occupancy of microorganisms belonging to the Peptostreptococcaceae family in cats before galactooligosaccharide ingestion (0 wk), during the ingestion period (4 wk, 8 wk), and 4 weeks after the end of ingestion (12 wk). "***" and "†" indicate significant differences (***: p<0.001, †: p<0.1, Wilcoxon signed rank test (Bonferroni correction)). Figure 2 shows the change in the total bacterial count and the bacterial counts of microorganisms belonging to the Peptostreptococcaceae and Eggerthellaceae families in cats before galactooligosaccharide ingestion (0 wk), during the ingestion period (4 wk, 8 wk), and 4 weeks after the end of ingestion (12 wk). "*", "**", and "***" indicate significant differences (*: p<0.05, **: p<0.01, ***: p<0.001, Wilcoxon signed rank sum test (Bonferroni correction)). Figure 3 shows the changes in the bacterial counts of Slackia microorganisms in cats before galactooligosaccharide ingestion (0 wk), during the ingestion period (4 wk, 8 wk), and 4 weeks after the end of ingestion (12 wk). "***" indicates a significant difference (***: p<0.001, Wilcoxon signed rank sum test (Bonferroni correction)). Figure 4 shows the changes in the occupancy rate of microorganisms belonging to the Peptostreptococcaceae family in dogs before galactooligosaccharide ingestion (0 wk), during the ingestion period (4 wk, 8 wk), and 4 weeks after the end of ingestion (12 wk). "*" and "***" indicate significant differences (*: p<0.05, ***: p<0.001, Wilcoxon signed rank test (Bonferroni correction)).

[0012] The growth inhibitor for microorganisms belonging to the Peptostreptococcaceae family and the growth inhibitor for microorganisms belonging to the genus Slackia of the present invention contain galactooligosaccharides as active ingredients. Galactooligosaccharide is a general term for oligosaccharides having at least one galactose residue in the molecule, and examples thereof include sugars having 2 to 9, preferably 3 to 4, monosaccharides 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. 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 can inhibit the growth of microorganisms belonging to the family Peptostreptococcaceae in the intestines and promote the growth of microorganisms of the genus Slackia, thereby providing excellent preventive and therapeutic effects against diseases caused by these microorganisms or diseases that can be expected to be prevented or improved by components produced by these microorganisms. The subjects to which the growth inhibitor of microorganisms belonging to the family Peptostreptococcaceae and the growth agent of microorganisms of the genus Slackia of the present invention are administered are not particularly limited to mammals including humans, but are preferably used in companion animals, such as dogs, cats, rabbits, and hamsters, with dogs and cats being preferred, and cats being particularly preferred.

[0014] Examples of diseases caused by microorganisms belonging to the Peptostreptococcaceae family include diabetes and obesity. Diabetes is a syndrome that presents with metabolic disorders including persistent hyperglycemia due to insulin deficiency, and is classified into insulin deficiency and insulin resistance according to its mechanism of action. In diabetes, symptoms such as polydipsia and polyuria and weight loss appear, and as the symptoms progress to ketoacidosis, symptoms such as loss of appetite, weakness, vomiting, and even coma may occur. Obesity is not just about being overweight, but also about the excessive accumulation of body fat, and is classified into visceral fat obesity and subcutaneous fat obesity according to its mechanism of action. The degree of obesity is determined in humans using the international standard index BMI (Body Mass Index) = [body weight (kg)] ÷ [height (m)]. ２ ] is used, and obesity is defined as "a state in which fat is excessively accumulated in adipose tissue and a BMI of 25 or higher." For dogs and cats, the Body Condition Score (BCS) is used, which is evaluated on a 5- or 9-point scale based on body shape, appearance, and tactile condition. Obesity is considered to be a cause of lifestyle-related diseases such as diabetes, dyslipidemia, hypertension, angina pectoris, myocardial infarction, and arteriosclerosis. Microorganisms belonging to the Peptostreptococcaceae family, which are the main intestinal bacteria in cats, occupy a higher proportion of the intestinal flora in diabetic and obese cats than in healthy cats (Non-Patent Documents 1 and 2). Therefore, these diseases can be prevented and improved by suppressing the increase in the proportion of microorganisms belonging to the Peptostreptococcaceae family.

[0015] Diseases that can be expected to be prevented or improved by components produced by Slackia microorganisms include, for example, breast cancer, prostate cancer, osteoporosis, arteriosclerosis, dyslipidemia, menopausal disorders, and premenstrual syndrome. Breast cancer is cancer of mammary gland tissue and is classified according to its mechanism of action into luminal A, luminal B (HER2 negative or positive), HER2, and triple negative. Symptoms of breast cancer include breast masses, eczema and sores on the nipple and areola, abnormal nipple discharge, and breast skin depressions. Prostate cancer is cancer of prostate tissue, an organ present in men, and primarily develops in the outer gland (peripheral area) of the prostate. Prostate cancer presents with symptoms similar to those of benign prostatic hyperplasia (e.g., difficulty urinating, residual urine, frequent urination, urinary retention, etc.), and as the cancer progresses, symptoms such as hematuria, hydronephrosis, hematospermia, and lower back pain due to bone metastasis appear. Osteoporosis is a disease that increases the risk of fractures due to decreased bone mass (bone density) and bone quality. It is classified into primary osteoporosis and secondary osteoporosis based on its mechanism of action. Most cases of osteoporosis are primary, and are believed to be caused by factors such as decreased calcium absorption due to aging and decreased estrogen secretion after menopause. Osteoporosis increases the risk of fractures of the spine, hip, and wrist, including femoral neck fractures, vertebral compression fractures, distal radius fractures, and proximal humerus fractures. Arteriosclerosis, a condition in which arteries harden and lose elasticity, is classified into atherosclerosis, arteriosclerosis, and Mönckeberg arteriosclerosis based on its mechanism of action. In atherosclerosis, plaques form in the arterial lining due to the deposition of fat and cholesterol, narrowing the blood vessels and leading to myocardial infarction and cerebral infarction. Arteriolar sclerosis is likely to occur in organs with small arteries, such as the pancreas and kidneys, where hardening of the arterioles reduces blood flow and leads to rupture of the blood vessels. Mönckeberg's arteriosclerosis occurs when calcified calcium deposits in the arterial media, causing hardening of the arteries and leading to rupture of the blood vessels. Dyslipidemia refers to a condition in which lipids such as neutral fats (triglycerides) and cholesterol are higher or lower than a certain standard, and is classified according to its mechanism of action as hyper-LDL (bad) cholesterolemia, hypo-HDL (good) cholesterolemia, and hypertriglyceridemia.In dyslipidemia, lipids such as triglycerides and cholesterol are deposited on the walls of blood vessels, forming plaques, which lead to arteriosclerosis and myocardial and cerebral infarction. Slackia microorganisms have the ability to produce equol, which has the preventive effect of hormone-dependent diseases, from daidzein, an isoflavone found in soybeans. Therefore, increasing the population of Slackia microorganisms may be able to 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 a pharmaceutical product, such as pet food, pet supplements, or pet medicines. Pharmaceutical products can be formulated using galactooligosaccharides as is or, if necessary, in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, 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 0.05 to 0.2 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 known raw materials used in 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 may 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, paste 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: Cats were given galactooligosaccharides (GOS) to examine changes in their intestinal flora. Twenty-five domestic cats (female:male ratio 2:3, average age 5.20 years, average weight 4.65 kg) were studied. Over the eight-week study period, 1.2 g of Oligomate 55N (GOS content 0.5 g) (galactooligosaccharide liquid sugar; Yakult Pharmaceutical Co., Ltd.) was given per day. A predetermined amount of Oligomate 55 was dispensed onto a spoon and given orally or sprinkled on top of the cat's food.

[0022] Feces were collected before galactooligosaccharide ingestion (week 0 (hereinafter 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 dispensed into a screw tube and crushed (1,048 rpm, 10 min) with RNAlater and zirconia beads in an amount 9 times the weight of the feces using a bead-type crusher, Shake Master Auto (BMS-A20TP, Biomedical Science Co., Ltd.), and then stored at 4°C. 200 μL of the suspension was collected and mixed with 1 mL of PBS(-), followed by centrifugation (13,000 G x 5 min, 4°C), and 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. The DNA was extracted according to the method described in "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 TE (pH 8.0). The concentration of the resulting DNA 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.

[0023] 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 following primers: Forward: 5'-GTGCCAGCMGCCGCGGTAA-3' and Reverse (806R): 5'-GGACTACHVGGGTWTCTAAT-3'. PCR amplicon libraries were prepared 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). Reads were processed using QIIME2 (ver. 2022.2) software, and lineages were assigned using the SILVA138 database. The median number of reads per fecal sample was 65,148 (min: 8, max: 323,006). There was one sample with fewer than 10,000 reads, and this sample was excluded from the analysis.

[0024] The change in the occupancy rate of fecal bacterial families was statistically analyzed using the Wilcoxon signed-rank test with Bonferroni correction. The total number of bacteria in each fecal sample was measured using PCR, and for families that showed a significant change in occupancy rate before and after galactooligosaccharide intake, the number of bacteria was calculated by multiplying each occupancy rate by the total number of bacteria (Non-Patent Document 3). The results are shown in Figures 1 and 2.

[0025] The occupancy and number of microorganisms belonging to the Peptostreptococcaceae family were significantly decreased during the galactooligosaccharide intake period (4 weeks and 8 weeks) compared with 0 week (Figs. 1 and 2). On the other hand, the number of microorganisms belonging to the Eggerthellaceae family was significantly increased during the galactooligosaccharide intake period (4 weeks and 8 weeks) compared with 0 week. Furthermore, a Wilcoxon signed-rank test with Bonferroni correction at the genus level confirmed that the number of microorganisms belonging to the genus Slackia was significantly increased during the galactooligosaccharide intake period (4 weeks and 8 weeks) compared with 0 week (Fig. 3).

[0026] In addition, blood samples were taken before galactooligosaccharide intake (0 wk), during the intake period (4 wk, 8 wk), and after the end of intake (12 wk), and blood biochemistry data such as total protein, albumin, total bilirubin, blood urea nitrogen, and creatinine were measured. No changes in the data that could be considered adverse events were observed, confirming that there were no safety issues.

[0027] Example 2: Dogs were administered galactooligosaccharides (GOS) to examine changes in the intestinal microflora. A total of 26 dogs were studied, including 12 small dogs (under 5 kg), 9 medium-sized dogs (5-10 kg), and 5 large dogs (over 10 kg). The male / female ratio was 17:9, the average age was 8.88 years, and the average weight was 7.76 kg. For the 8-week study period, the dogs were fed Oligomate 55N (galactooligosaccharide liquid sugar; Yakult Pharmaceutical Co., Ltd.) in the amounts shown in Table 1 per day. The feeding method was the same as in Example 1.

[0028]

[0029] The occupancy rate was determined for the bacterial species that showed a significant change before and after the intake of galactooligosaccharides using the same method as in Example 1. The results are shown in FIG.

[0030] As shown in Figure 4, the percentage of microorganisms belonging to the Peptostreptococcaceae family was significantly reduced during the ingestion period (4 weeks, 8 weeks) and after the end of ingestion (12 weeks) compared to before ingestion of galactooligosaccharides (0 weeks). This result confirmed that the growth of bacteria belonging to the Peptostreptococcaceae family was suppressed.

[0031] The preventive and therapeutic agent of the present invention is highly safe and can be taken on a daily basis. It inhibits the growth of microorganisms belonging to the Peptostreptococcaceae family in the intestines of dogs, cats, etc., and by promoting the growth of microorganisms of the genus Slackia, it has excellent preventive and therapeutic effects against diseases caused by these microorganisms or diseases that can be expected to be prevented or improved by components produced by these microorganisms. Therefore, it can be used as a medicine, pet food, etc.

Claims

1. A growth inhibitor of microorganisms belonging to the Peptostreptococcaceae family that contains galactooligosaccharides as an active ingredient.

2. A growth inhibitor of microorganisms belonging to the Peptostreptococcaceae family according to claim 1, for use in the prevention and treatment of diabetes or obesity.

3. A method for preventing or treating diabetes or obesity, which comprises administering galactooligosaccharides to a companion animal.

4. A growth agent for Slackia genus microorganisms containing galactooligosaccharides as an active ingredient.

5. The Slackia microorganism proliferation agent according to claim 4, which is used for the prevention and treatment of breast cancer, prostate cancer, osteoporosis, arteriosclerosis or dyslipidemia.

6. A method for preventing or treating breast cancer, prostate cancer, osteoporosis, arteriosclerosis or dyslipidemia, which comprises administering galactooligosaccharides to a companion animal.

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