Agent for preventing or improving lifestyle-related diseases

Abstract

To provide an agent for preventing and improving lifestyle-related diseases.SOLUTION: The prophylactic / ameliorating agent for lifestyle-related diseases contains 1-kestose and galactooligosaccharide as active ingredients.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an agent for preventing and ameliorating lifestyle-related diseases, which contains 1-kestose and galactooligosaccharides as active ingredients. [Background technology]

[0002] In recent years, research into the gut microbiota using metagenomic analysis technology has become increasingly popular. One of the reasons for this is the clear association between the gut microbiota and disease. For example, it has been reported that the gut microbiota is associated with metabolic diseases such as obesity and diabetes, allergic diseases such as bronchial asthma and atopic dermatitis, and cancer.

[0003] Representative intestinal bacteria present in the intestines of animals such as humans include the Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria phyla. In a healthy intestinal flora, bacteria belonging to the Bacteroidetes phylum are dominant, and bacteria belonging to the Firmicutes phylum are inferior. However, when the balance of the intestinal flora is disrupted due to a change in diet to a high-fat diet, for example, bacteria belonging to the Bacteroidetes phylum decrease and bacteria belonging to the Firmicutes phylum increase, which is known to result in increased insulin resistance and the like (Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2017-538702 [Patent Document 2] Korean Patent Publication No. 10-2013-0021920 [Patent Document 3] Japanese Patent Publication No. 2022-41955 [Non-patent literature]

[0005] [Non-Patent Document 1] Ley RE et al., Obesity alters gut microbial ecology. Proc Natl Acad Sci 102:11070-11075, 2005 [Non-patent document 2] Turnbaugh PJ et al., Anti obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444;1027-1031, 2006 [Non-patent document 3] Tao Yang et.,al. Gut Dysbiosis Is Linked to Hypertension, Hypertension, 65: 1331-1340, 2015 [Non-patent document 4] Yang JY, Lee YS, Kim Y, Lee SH, Ryu S, Fukuda S, Hase K, Yang CS, Lim HS, Kim MS, Kim HM, Ahn SH, Kwon BE, Ko HJ, Kweon MN. Gut commensal Bacteroides acidifaciens prevents obesity and improves insulin sensitivity in mice. Mucosal Immunol. 2017 Jan;10(1):104-116. doi: 10.1038 / mi.2016.42. Epub 2016 Apr 27. PMID: 27118489. [Non-Patent Document 5] Dao MC, Everard A, Aron-Wisnewsky J, Sokolovska N, Prifti E, Verger EO, Kayser BD, Levenez F, Chilloux J, Hoyles L; MICRO-Obes Consortium; Dumas ME, Rizkalla SW, Dore J, Cani PD, Clement K. Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology. Gut. 2016 Mar;65(3):426-36. doi: 10.1136 / gutjnl-2014-308778. Epub 2015 Jun 22. PMID: 26100928. Non-Patent Document 6 Yanlong Cui, Leshan Zhang, Xin Wang, Yanglei Yi, Yuanyuan Shan, Bianfang Liu, Yuan Zhou, Xin Lu, Roles of intestinal Parabacteroides in human health and diseases, FEMS Microbiology Letters, Volume 369, Issue 1, 2022, fnac072, https: / / doi.org / 10.1093 / femsle / fnac072 Non-Patent Document 7 Wu TR, Lin CS, Chang CJ, Lin TL, Martel J, Ko YF, Ojcius DM, Lu CC, Young JD, Lai HC. Gut commensal Parabacteroides goldsteinii plays a predominant role in the anti-obesity effects of polysaccharides isolated from Hirsutella sinensis . Gut. 2019 Feb;68(2):248-2 doi: 10.1136 / gutjnl-2017-315458. Epub 2018 Jul 14. PMID:30007918.

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[0006] An object of the present invention is to provide an agent for preventing and ameliorating lifestyle-related diseases that can improve the intestinal bacterial flora and effectively prevent or ameliorate lifestyle-related diseases.

[0007] As a result of extensive research to solve the above-mentioned problems, the inventors have found that administering 1-kestose and galactooligosaccharides in combination reduces the ratio of Firmicutes bacteria to Bacteroidetes bacteria (F / B ratio) in the intestinal flora compared to administering either compound alone, and increases the proportion of bacteria involved in lifestyle-related diseases such as obesity and diabetes, such as Akkermansia muciniphila, thereby completing the present invention. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A preventive and ameliorative agent for lifestyle-related diseases containing 1-kestose and galactooligosaccharides as active ingredients. [2] The agent for preventing and improving lifestyle-related diseases according to [1], which is a food or drink. [3] A pharmaceutical agent for preventing and improving lifestyle-related diseases according to [1]. [4] An agent for improving the intestinal flora containing 1-kestose and galactooligosaccharides as active ingredients. [5] The agent for improving the intestinal flora according to [4], which reduces the ratio of bacteria of the phylum Firmicutes to bacteria of the phylum Bacteroidetes (F / B ratio). [6] The intestinal flora improving agent according to claim 4, which increases the proliferation of one or more bacteria selected from the group consisting of Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii. [7] An intestinal immune enhancer containing 1-kestose and galactooligosaccharides as active ingredients. [8] A therapeutic agent for inflammatory diseases, comprising 1-kestose and galactooligosaccharides as active ingredients. [Effects of the Invention]

[0009] The agent for preventing and improving lifestyle-related diseases of the present invention reduces the ratio of Firmicutes bacteria to Bacteroidetes bacteria (F / B ratio) in the intestinal flora, and increases Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii, thereby effectively preventing and improving lifestyle-related diseases, inflammatory diseases, etc., and further activating intestinal immunity. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the results of the alpha diversity analysis of the intestinal microbiota. Figure 1(A) shows the Chao1 index. Figure 1(B) shows the Shannon index. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's test for multiple comparisons. "*", "**", and "***" indicate significant differences (*: p<0.05, **: p<0.01, ***: p<0.001). [Figure 2] Figure 2 shows the results of β-diversity (Unifrac distance) analysis of the intestinal microbiota. [Figure 3] Figure 3(A) shows the relative abundance at the phylum level. Figure 3(B) shows the Firmicutes / Bacteriodetes ratio (F / B ratio). Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's test for multiple comparisons. "***" indicates a significant difference (***: p<0.001). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below. The following description of the present invention may be based on preferred embodiments of the present invention, but the present invention is not limited to such embodiments. In this specification, the use of "to" to indicate a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0012] In the present invention, 1-kestose and galactooligosaccharides are used as active ingredients. 1-kestose is a fructan formed by dehydration polymerization of D-fructose attached to the fructose side of sucrose via a β-(2→1) bond, and is represented by GF2 (G: glucose, F: fructose, n: fructose).

[0013] 1-kestose may be commercially available or synthesized by a known method. Examples of commercially available 1-kestose include iKes Crystal, iKes75 (manufactured by Itochu Sugar Co., Ltd.), Kestose 75, and Kestose 95 (manufactured by Bussan Food Science Co., Ltd.), and examples of the synthesis method include a method of reacting sucrose with the hydrolytic enzyme β-fructofuranosidase.

[0014] Galactooligosaccharide is a general term for oligosaccharides having at least one galactose residue in the molecule, such as sugars in which 2 to 9 monosaccharides are linked together. Examples of galactooligosaccharides include those in which galactose is linked together via β1-2, β1-3, β1-4, and β1-6 bonds.

[0015] Galactooligosaccharides may be commercially available products or those synthesized by known methods. Commercially available products include Cup Oligo (registered trademark). Synthetic methods include, for example, reacting lactose with β-galactosidase.

[0016] The intestinal microbiota is composed of phyla such as Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. In an obese state, the proportion of bacteria belonging to the phylum Bacteroidetes is low and the proportion of bacteria belonging to the phylum Firmicutes is high, leading to increased insulin resistance and other conditions. However, as weight loss progresses, the proportion of bacteria belonging to the phylum Bacteroidetes becomes relatively high and the proportion of bacteria belonging to the phylum Firmicutes becomes relatively low (Non-Patent Documents 1 and 2). Therefore, metabolic diseases such as obesity and diabetes can be prevented or ameliorated by reducing the ratio of bacteria belonging to the phylum Firmicutes to Bacteroidetes (F / B ratio). Furthermore, reducing the F / B ratio can prevent or ameliorate hypertension (Non-Patent Document 3). In the present invention, the synergistic action of 1-kestose and galactooligosaccharides can reduce the ratio of Firmicutes bacteria to Bacteroidetes bacteria (F / B ratio) compared to the administration of either compound alone.

[0017] Furthermore, in the present invention, the combined administration of 1-kestose and galactooligosaccharides promotes the proliferation of Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii in the intestinal flora, increasing their occupancy rate, due to the synergistic action of the two, compared with the use of either compound alone. These bacteria are known to be involved in metabolic diseases such as obesity, diabetes, and dyslipidemia (Non-Patent Documents 4 to 7, Patent Document 1), and increasing their occupancy rate can provide preventive and ameliorative effects.

[0018] Bacteroides acidifaciens promotes IgA production in the large intestine and enhances intestinal immunity (Non-Patent Document 8). It also has therapeutic and preventive effects against inflammatory diseases such as gastritis and Crohn's disease (Patent Document 2), and liver disorders such as autoimmune hepatitis, viral hepatitis, alcoholic liver injury, and drug-induced liver injury (Non-Patent Document 9).

[0019] Akkermansia muciniphila also promotes IgA production in the large intestine and enhances intestinal immunity (Non-Patent Document 10). It also degrades mucin secreted from intestinal cells to produce short-chain fatty acids (Non-Patent Documents 11-12). Short-chain fatty acids regulate the intestinal immune response, and increasing their occupancy may prevent or improve inflammatory diseases such as inflammatory bowel disease (ulcerative colitis and Crohn's disease) and irritable bowel syndrome (Patent Documents 2-3).

[0020] Parabacteroides goldsteinii has anti-inflammatory properties and is effective in treating and preventing chronic obstructive pulmonary disease (COPD) (Non-patent Document 13).

[0021] The combined intake of 1-kestose and galactooligosaccharides has been shown to improve the intestinal microbiota, including reducing the ratio of Firmicutes bacteria to Bacteroidetes bacteria (F / B ratio) and promoting the proliferation of Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii, thereby preventing and improving lifestyle-related diseases in animals and humans. Lifestyle-related diseases are closely related to lifestyle factors such as diet, exercise, rest, smoking, and alcohol consumption, and include obesity, diabetes, cerebrovascular disorders (e.g., stroke, arteriosclerosis), heart disease (e.g., myocardial infarction), dyslipidemia, hypertension, nephropathy (e.g., chronic nephropathy), liver disease, and hyperuricemia.

[0022] Obesity refers not only to being overweight, but also to the state of excessive accumulation of body fat, and is classified into visceral fat obesity and subcutaneous fat obesity. The degree of obesity is determined by the international index BMI (Body Mass Index) = [weight (kg)] ÷ [height (m) 2 ] is used, and obesity is defined as "a state in which excess fat accumulates in adipose tissue and a BMI of 25 or greater." Obesity is considered to be a cause of the aforementioned diseases such as diabetes, dyslipidemia, hypertension, and hyperuricemia.

[0023] Diabetes is a syndrome that manifests as persistent hyperglycemia and other metabolic abnormalities due to a lack of insulin, and is classified into insulin deficiency and insulin resistance according to its mechanism of action. Symptoms of diabetes include excessive drinking and urination and weight loss, and as the condition progresses to ketoacidosis, symptoms such as loss of appetite, weakness, vomiting, and may even lead to coma.

[0024] The preventive and ameliorative agent for lifestyle-related diseases of the present invention uses 1-kestose and galactooligosaccharides as active ingredients. The mass ratio of 1-kestose to galactooligosaccharides is not particularly limited, but is preferably in the range of 1:10 to 10:1, more preferably 1:5 to 5:1, and even more preferably 1:2 to 2:1 (1-kestose:galactooligosaccharide mass ratio).

[0025] The agent for preventing and improving lifestyle-related diseases of the present invention can be formulated by combining the active ingredient with a pharmaceutically acceptable carrier, if necessary. 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.

[0026] The agent for preventing and ameliorating lifestyle-related diseases of the present invention can be applied to mammals including humans, such as humans, dogs, cats, cows, pigs, chickens, sheep, and horses, with humans being preferred. The dosage (total of 1-kestose and galactooligosaccharides) is not particularly limited, but for example, the daily dosage for an adult is about 0.01 to 5 g per kg of body weight, preferably about 0.05 to 1 g per kg of body weight. For dogs, cats, and the like, the dosage is about 0.01 to 5 g per kg of body weight, preferably about 0.05 to 1 g per kg of body weight. The dosage may be, for example, once a day or in multiple divided doses. The dosage interval may be, for example, every day or every few days.

[0027] The preventive and ameliorating agent for lifestyle-related diseases of the present invention can also be in the form of a food or drink, and is prepared by blending the above-mentioned active ingredient with known food additives and / or food ingredients in accordance with a conventional method, and the form is not particularly limited, and examples thereof include starch-based foods such as bread, biscuits, pancakes, noodles, and tablet candy, confectioneries such as gum, candy, and Japanese sweets, meat foods such as ham and sausage, fish foods such as chikuwa and kamaboko, seafood foods, seasonings such as dressing, soy sauce, jam, and furikake, beverages such as tea, juice, soft drinks, and alcoholic beverages, etc. Foods and drinks include foods for specified health uses and foods with functional claims that have been approved or notified to be labeled as being for the prevention and amelioration of lifestyle-related diseases.

[0028] The agent for preventing and ameliorating lifestyle-related diseases of the present invention can be in the form of feed or pet food, and is prepared according to a conventional method by appropriately blending the above-mentioned active ingredient with known feed ingredients, gelling agents, shape-retaining agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc. The form is not particularly limited, and any form such as pellets, flakes, or mash can be exemplified, and the agent can be used, for example, as feed for livestock such as cows, pigs, chickens, sheep, and horses, or as pet food for dogs, cats, etc.

[0029] The intestinal microbiota-improving agent of the present invention can reduce the ratio of bacteria belonging to the phylum Firmicutes to bacteria belonging to the phylum Bacteroidetes (F / B ratio) or can promote the proliferation of one or more bacteria selected from the group consisting of Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii. Based on these actions, the agent can enhance intestinal immunity by promoting IgA production in the large intestine, and provide therapeutic and preventive effects against inflammatory diseases such as gastritis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), and irritable bowel syndrome, autoimmune hepatitis, viral hepatitis, liver disorders such as alcoholic liver injury and drug-induced liver injury, and chronic obstructive pulmonary disease (COPD). The active ingredients, formulation methods, dosages, and other aspects of the intestinal microbiota-improving agent of the present invention are the same as those of the agent for preventing and improving lifestyle-related diseases.

[0030] The intestinal immunity enhancer of the present invention can enhance the intestinal immune mechanism that eliminates orally invaded pathogenic microorganisms, etc., and specifically can activate intestinal immunity by strengthening the intestinal barrier function, promoting the production of IgA secreted into the intestinal lumen and mucus layer, etc. The active ingredient, formulation method, dosage, etc. of the intestinal immunity enhancer of the present invention are the same as those of the above-mentioned agent for preventing and ameliorating lifestyle-related diseases. [Example]

[0031] The present invention will be specifically described below using examples. However, the present invention is not limited to these examples. The examples were conducted in accordance with the ARRIVE guidelines and the Nagoya University of Arts and Sciences animal experiment guidelines. The examples were also conducted in accordance with the standards of the Ministry of the Environment and with the approval of the Nagoya University of Arts and Sciences Ethics Committee.

[0032] [Materials and rearing conditions] Twenty-four 6-week-old female BALB / c mice weighing 15–20 g were purchased from SCL Japan, Inc. They were housed three per cage at 24°C ± 2°C under a 12-hour light / dark cycle with free access to water and food (AIN-93G; CLEA Japan, Inc.). Each cage was arranged so that environmental factors would not affect the mice.

[0033] [Administration schedule] After one week of pre-breeding under the above conditions, 7-week-old mice were randomly divided into a control group (Ctrl group; 6 mice), a 1-kestose-treated group (KES group; 6 mice), a galactooligosaccharide-treated group (GOS group; 6 mice), and a 1-kestose and galactooligosaccharide-treated group (KES + GOS group; 6 mice). Each group was fed the appropriate diet for 4 weeks. The control group was fed a diet (AIN-93G; CLEA Japan, Inc., containing 5% cellulose). The KES and GOS groups were fed a diet in which 5% cellulose was replaced with 5% 1-kestose (Nissin Sugar Co., Ltd.) or 5% galactooligosaccharides (Nissin Sugar Co., Ltd.). The KES + GOS group was fed a diet in which 5% cellulose was replaced with 2.5% 1-kestose and 2.5% galactooligosaccharides.

[0034] Feces were collected for two hours per day for three days, starting four days before the end of the administration period. At the end of the administration period, mice from each group were dissected, and serum, cecal contents, and colon tissue were collected. Serum was stored at -20°C, and feces and each organ at -80°C.

[0035] [Intestinal microbiota analysis] For intestinal microbiota analysis, DNA was extracted from feces using the following method. Frozen feces were thawed on ice, and 100 mg of each sample was suspended in a solution containing 4 M guanidinium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA. The suspension was disrupted with zirconia beads using a FastPrep FP100A (MP Biomedicals). DNA was extracted from the bead-treated suspension using a Magtration System 12GC and a GC series MagDEA DNA 200.

[0036] The resulting DNA was used as a template to amplify the 16S rRNA V3-V4 region by PCR using primers (Pro341F and Pro805R). The resulting PCR products were analyzed using the Illumina MiSeq platform and MiSeq Reagent Kit v3 (both manufactured by Illumina) by paired-end sequencing. The resulting sequences were assigned taxonomically using the EzBioCloud (https: / / www.ezbiocloud.net / ) database (Ver. 20230823) as a reference. The gut microbiota of each group was then analyzed for alpha diversity, beta diversity, phylum-level relative abundance, and intergroup comparison (LEfSe) analysis.

[0037] (alpha diversity analysis) Alpha diversity analysis can analyze the diversity of the intestinal microbiota within each group. In this example, the Chao1 Index and Shannon Index were used as indices. The higher the index, the higher the diversity of the intestinal microbiota. As shown in Figures 1(A) and 1(B), it was confirmed that the species diversity was significantly reduced in the KES+GOS-treated group compared to the control group.

[0038] (beta diversity analysis) Beta diversity analysis can analyze differences in the diversity of the intestinal microbiota between groups. In this example, the analysis was performed using a combination of UniFrac principal coordinate analysis and nonparametric multivariate analysis of variance. The greater the distance between the plots, the greater the difference in the composition of the intestinal microbiota. As shown in Figure 2, it was confirmed that the distance between the plots of the control group and the KES + GOS-treated group was significantly greater.

[0039] (Relative abundance at the phylum level) The relative abundance at the phylum level was calculated for each group and compared. As shown in Figure 3(A), the relative abundance of the Firmicutes phylum decreased and the relative abundance of the Bacteriodetes phylum increased in the KES+GOS-treated group compared to the control group. Based on the results in Figure 3(A), the Firmicutes / Bacteriodetes ratio (F / B ratio) was calculated. As shown in Figure 3(B), the F / B ratio was significantly decreased in the KES+GOS-treated group compared to the control group.

[0040] (Intergroup comparison analysis (LEfSe analysis)) Characteristic bacterial species were identified in each group using intergroup comparison analysis (|LDA score|>4.0, p<0.05, mean relative abundance>1%). As shown in Table 1 below, the KES+GOS group showed higher occupancy rates for Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii than the KES and GOS groups.

[0041] [Table 1]

Claims

1. An agent for preventing and improving lifestyle-related diseases, which contains 1-kestose and galactooligosaccharides as active ingredients.

2. The agent for preventing and improving lifestyle-related diseases according to claim 1, which is a food or drink.

3. The agent for preventing and improving lifestyle-related diseases according to claim 1, which is a pharmaceutical product.

4. An agent for improving intestinal flora, comprising 1-kestose and galactooligosaccharides as active ingredients.

5. 5. The agent for improving intestinal flora according to claim 4, which reduces the ratio of bacteria of the phylum Firmicutes to bacteria of the phylum Bacteroidetes (F / B ratio).

6. The intestinal flora improving agent according to claim 4, which increases the proliferation of one or more bacteria selected from the group consisting of Bacteroides acidifaciens, Akkermansia muciniphila, and Parabacteroides goldsteinii.

7. An intestinal immunity enhancer containing 1-kestose and galactooligosaccharides as active ingredients.

8. A therapeutic agent for inflammatory diseases, comprising 1-kestose and galactooligosaccharides as active ingredients.

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