The preparation aims to improve vascular endothelial function.

Abstract

The invention relates to a novel method for improving vascular endothelial function. According to one design, the invention relates to a preparation intended to improve vascular endothelial function, comprising bifidobacterium bacteria and water-soluble fiber.

Description

Composition for improving vascular endothelial function

[0001] The present invention relates to a composition for improving vascular endothelial function.

[0002] Cardiovascular disease (CVD) is a leading cause of death worldwide, with a particularly disproportionate impact on low- and middle-income countries. An estimated 17.9 million people died from CVD in 2019, accounting for 32% of all deaths worldwide. Despite current prevention and treatment strategies, mortality from CVD is expected to increase further over the next decade. Atherosclerosis is an inflammatory cardiovascular disease characterized by narrowing of the arterial lumen due to plaque formation, the progression of which directly leads to the development of CVD. Endothelial dysfunction leads to plaque formation and disease progression, leading to the development of atherosclerosis.

[0003] Similarly, many studies have reported on the relationship between CVD and metabolic syndrome. Metabolic syndrome is a clinical condition characterized by the presence of at least three of the following metabolic risk factors: excess visceral fat (abdominal obesity), insulin resistance, hyperglycemia, hypertension, and dyslipidemia (high triglycerides and low high-density lipoprotein (HDL) cholesterol). Each of these metabolic syndrome components is known to impair endothelial function. Furthermore, patients with metabolic syndrome are known to frequently develop vascular endothelial dysfunction. Under these circumstances, the development of new methods to improve vascular endothelial function is eagerly awaited.

[0004] Patent No. 2021-169517 Patent No. 5881801

[0005] J Biosci Bioeng. 2012;113:587-91.Sci Rep. 2017;7:43522Food Sci Nutr. 2019;7:1828-37Scientific Reports. volume 4, Article number: 4548 (2014)Hypertension. 2018;72:1060-71Biosci Microbiota Food Health. 2016;35(4):163-171.Genome Biol. 2022 Apr 14;23(1):95.Microbiome. 2021 Apr 29;9(1):95. doi: 10.1186 / s40168-021-01048-3.Edited by the Japanese Circulation Society and the Japanese Vascular Insufficiency Society “Physiological diagnostic guidelines for vascular insufficiency” Life Science Publishing 2021 Am J Clin Nutr 2018;107:965-83.

[0006] The problem to be solved by the present invention is to provide a new means for improving vascular endothelial function.

[0007] Under these circumstances, the present inventors conducted extensive research and discovered that vascular endothelial function can be improved by combining bifidobacteria with water-soluble dietary fiber. The present invention is based on this novel finding. Accordingly, the present invention provides the following: Item 1. A composition for improving vascular endothelial function, comprising bifidobacteria and water-soluble dietary fiber.

[0008] Item 2. The composition according to Item 1, wherein the improvement in vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1.

[0009] Item 3. A composition for maintaining or inhibiting a decline in vascular flexibility or suppleness, comprising bifidobacteria and water-soluble dietary fiber.

[0010] Item 4. A composition for preventing arteriosclerosis, comprising bifidobacteria and water-soluble dietary fiber.

[0011] Item 5. A composition for improving kidney function, comprising bifidobacteria and water-soluble dietary fiber.

[0012] Item 6. The composition according to any one of Items 1 to 5, wherein the water-soluble dietary fiber comprises inulin.

[0013] Item 7. The composition according to any one of Items 1 to 6, wherein the bifidobacteria have the ability to grow in the intestine.

[0014] Item 8. The composition according to any one of Items 1 to 7, wherein the bifidobacterium has an anti-metabolic syndrome effect.

[0015] Item 9. The composition according to any one of Items 1 to 8, which is a food product.

[0016] Item 10. The composition according to any one of Items 1 to 9, further comprising arginine.

[0017] Item 11. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for improving vascular endothelial function.

[0018] Item 12. The use according to Item 11, wherein the improvement in vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1.

[0019] Item 13. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for maintaining or inhibiting the decline of vascular flexibility or resilience.

[0020] Item 14. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for preventing arteriosclerosis.

[0021] Item 15. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for improving kidney function.

[0022] Item 16. Use of water-soluble dietary fiber for producing a composition for improving vascular endothelial function, comprising bifidobacteria.

[0023] Item 17. The use according to Item 16, wherein the improvement in vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1.

[0024] Item 18. Use of water-soluble dietary fiber for producing a composition containing bifidobacteria for maintaining or inhibiting a decline in vascular flexibility or suppleness.

[0025] Item 19. Use of water-soluble dietary fiber for producing a composition for preventing arteriosclerosis containing bifidobacteria.

[0026] Item 20. Use of water-soluble dietary fiber for producing a composition containing bifidobacteria for improving renal function.

[0027] Item 21. Use of bifidobacteria for producing a composition for improving vascular endothelial function, which contains water-soluble dietary fiber.

[0028] Item 22. The use according to Item 21, wherein the improvement in vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1.

[0029] Item 23. Use of bifidobacteria for producing a composition containing water-soluble dietary fiber for maintaining or inhibiting a decline in flexibility or suppleness of blood vessels.

[0030] Item 24. Use of bifidobacteria for producing a composition for preventing arteriosclerosis containing water-soluble dietary fiber.

[0031] Item 25. Use of bifidobacteria for producing a composition for improving renal function, which contains water-soluble dietary fiber.

[0032] Item 26. The use according to any one of Items 11 to 25, wherein the water-soluble dietary fiber comprises inulin.

[0033] Item 27. The use according to any one of Items 11 to 26, wherein the bifidobacteria have the ability to grow in the intestine.

[0034] Item 28. The use according to any one of Items 11 to 27, wherein the bifidobacterium has an anti-metabolic syndrome effect.

[0035] Item 29. The use according to any one of Items 11 to 28, wherein the composition is a food product.

[0036] Item 30. The use according to any one of Items 11 to 29, wherein the composition further comprises arginine.

[0037] Item 31. A method for improving vascular endothelial function, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof.

[0038] Item 32. The method according to Item 31, wherein the improvement of vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1.

[0039] Item 33. A method for maintaining or inhibiting a decline in vascular flexibility or suppleness, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof.

[0040] Item 34. A method for preventing arteriosclerosis, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof.

[0041] Item 35. A method for improving renal function, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof.

[0042] Item 36. The method according to any one of Items 31 to 35, wherein the water-soluble dietary fiber comprises inulin.

[0043] Item 37. The method according to any one of Items 31 to 36, wherein the bifidobacteria have the ability to grow in the intestine.

[0044] Item 38. The method according to any one of Items 31 to 37, wherein the bifidobacterium has an anti-metabolic syndrome effect.

[0045] Item 39. The method according to any one of Items 31 to 38, wherein the bifidobacteria and water-soluble dietary fiber are ingested as a food product.

[0046] Item 40. The method according to any one of Items 31 to 39, further comprising administering arginine.

[0047] Item 41. A combination of bifidobacteria and water-soluble dietary fiber for use in improving vascular endothelial function.

[0048] Item 42. The combination according to Item 41, wherein the improvement in vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1.

[0049] Item 43. A combination of bifidobacteria and water-soluble dietary fiber for use in maintaining or inhibiting the decline of vascular flexibility or resilience.

[0050] Item 44. A combination of bifidobacteria and water-soluble dietary fiber for use in preventing arteriosclerosis.

[0051] Item 45. A combination of bifidobacteria and water-soluble dietary fiber for use in improving kidney function.

[0052] Item 46. The combination according to any one of Items 41 to 45, wherein the water-soluble dietary fiber comprises inulin.

[0053] Item 47. The combination according to any one of Items 41 to 46, wherein the bifidobacteria have the ability to grow in the intestine.

[0054] Item 48. The combination according to any one of Items 41 to 47, wherein the bifidobacterium has an anti-metabolic syndrome effect.

[0055] Item 49. The combination according to any one of Items 41 to 48, which is a food product.

[0056] Item 50. The combination of any one of Items 41 to 49, further comprising arginine.

[0057] Item 51. A water-soluble dietary fiber for use in combination with bifidobacteria in improving vascular endothelial function.

[0058] Item 52. The water-soluble dietary fiber according to Item 41, wherein the improvement in vascular endothelial function includes a decrease in blood LDL-c concentration and / or a decrease in PAI-1.

[0059] Item 53. A water-soluble dietary fiber for use in combination with bifidobacteria in maintaining or inhibiting the decline of vascular flexibility or resilience.

[0060] Item 54. A water-soluble dietary fiber for use in combination with bifidobacteria in the prevention of arteriosclerosis.

[0061] Item 55. A water-soluble dietary fiber for use in combination with bifidobacteria in improving renal function.

[0062] Item 56. The water-soluble dietary fiber according to any one of Items 51 to 55, wherein the water-soluble dietary fiber comprises inulin.

[0063] Item 57. The water-soluble dietary fiber according to any one of Items 51 to 56, wherein the bifidobacteria have the ability to proliferate in the intestine.

[0064] Item 58. The water-soluble dietary fiber according to any one of Items 51 to 57, wherein the bifidobacterium has an anti-metabolic syndrome effect.

[0065] Item 59. The water-soluble dietary fiber according to any one of Items 51 to 58, which is a food product.

[0066] Item 60. The water-soluble dietary fiber according to any one of Items 51 to 59, further used in combination with arginine.

[0067] Item 61. Bifidobacterium for use in combination with water-soluble dietary fiber in improving vascular endothelial function.

[0068] Item 62. The bifidobacterium according to Item 61, wherein the improvement in vascular endothelial function includes a decrease in blood LDL-c concentration and / or a decrease in PAI-1.

[0069] Item 63. Bifidobacterium for use in combination with water-soluble dietary fiber in maintaining or inhibiting the decline of vascular flexibility or resilience.

[0070] Item 64. Bifidobacteria for use in combination with water-soluble dietary fiber in the prevention of arteriosclerosis.

[0071] Item 65. Bifidobacterium for use in combination with water-soluble dietary fiber in improving renal function.

[0072] Item 66. The bifidobacterium according to any one of Items 61 to 65, wherein the water-soluble dietary fiber comprises inulin.

[0073] Item 67. The bifidobacterium according to any one of Items 61 to 66, which has the ability to grow in the intestine.

[0074] Item 68. The bifidobacterium according to any one of Items 61 to 67, which has an anti-metabolic syndrome effect.

[0075] Item 69. The bifidobacterium according to any one of Items 61 to 68, which is in the form of a food product.

[0076] Item 70. The bifidobacterium according to any one of Items 61 to 69, which is further used in combination with arginine.

[0077] According to the present invention, a new means for improving vascular endothelial function can be provided.

[0078] The flow chart from subject enrollment to analysis in this example is shown in Figure 1. The effect of ingestion of a test food containing GCL2505 and inulin on fecal microbiota in the subgroup analysis population (test food group: n = 22, placebo group: n = 21) is shown. Box plots represent the 5th percentile, 95th percentile, interquartile range (25%-75%), and median. A) α-diversity (Chao1). B) β-diversity (principal component analysis of Bray-Curtis distances at the genus level). C) Relative abundance of B. animalis at week 12. Data were analyzed using LinDA.

[0079] 1. Composition for Improving Vascular Endothelial Function The present invention provides a composition for improving vascular endothelial function, comprising bifidobacteria and water-soluble dietary fiber. In the present invention, the type of bifidobacterium is not particularly limited, and examples thereof include Bifidobacterium animalis, Bifidobacterium adolesentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum, with Bifidobacterium animalis being preferred. Among Bifidobacterium animalis, Bifidobacterium animalis subsp. lactis is preferred. Furthermore, bifidobacteria that have the ability to grow in the intestine are preferred. More specifically, bifidobacteria that can increase the total number of bifidobacteria in the intestine are preferred. Whether or not a bifidobacterium has the ability to grow in the intestine can be determined, for example, by measuring the number of bifidobacteria in a fecal sample using real-time PCR. More specifically, this can be measured and evaluated in accordance with the method described in Non-Patent Document 1. Furthermore, in the present invention, if an increase in the intestine can be confirmed by the above method, the bifidobacterium can be selected as a preferred bifidobacterium of the present invention. Bacteria that can grow to a value measured by the above method that is at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold the number of bacteria ingested into the body are preferred. Furthermore, in the present invention, the bifidobacterium preferably has an anti-metabolic syndrome effect. The anti-metabolic syndrome effect can be determined, for example, by measuring visceral fat area, and more specifically, can be measured and evaluated according to the method described in Non-Patent Document 6. In the present invention, if a reduction in visceral fat area can be confirmed by the above method, the bifidobacterium can be selected as a preferred bifidobacterium of the present invention.In a preferred embodiment of the present invention, bifidobacteria that provide a visceral fat area of ​​0.98 times or less, as measured by the above-mentioned method, are preferred, and bifidobacteria that provide a visceral fat area of ​​0.96 times or less are more preferred. Examples of bifidobacteria include GCL2505, which belongs to Bifidobacterium animalis subsp. lactis. The GCL2505 strain has been deposited under accession number FERM ABP-21918. This strain was deposited on February 17, 2010, in accordance with the Budapest Treaty with the Patent Organism Depositary, National Institute of Technology and Evaluation (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan).

[0080] The number of bifidobacteria in the composition of the present invention is not limited, and can be appropriately set within the range of, for example, 1,000 cells / g, preferably 3,000 cells / g, more preferably 5,000 cells / g, even more preferably 8,000 cells / g or more, and particularly preferably 100 million cells / g or more. There is no particular upper limit, and examples include 10 trillion cells / g or less, 100 billion cells / g or less, and 10 billion cells / g or less. In a typical embodiment, live bifidobacteria are used in the present invention.

[0081] Examples of water-soluble dietary fibers include inulin, indigestible dextrin, water-soluble soybean polysaccharides, polydextrose, sodium alginate, psyllium, fucoidan, laminaran, sodium carboxymethylcellulose, pullulan, curdlan, low-molecular-weight hemicellulose, etc., and inulin is preferred. These water-soluble dietary fibers may be blended alone or in combination.

[0082] The content of water-soluble dietary fiber in the composition of the present invention is not limited, but can be set, for example, in the range of 0.5 to 10% by weight, preferably 1 to 5% by weight, and more preferably 1.5 to 3% by weight, based on the mass of the total composition.

[0083] In the present invention, a combination of bifidobacteria, which are the active ingredients of the present invention, and water-soluble dietary fiber may be used as a composition for improving vascular endothelial function, or this combination may be used as a composition combining various pharmaceutically acceptable carriers that can be added to foods (e.g., isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, excipients, binders, etc.). In embodiments containing various carriers, the total content of bifidobacteria and water-soluble dietary fiber in the composition is not limited, and can be appropriately set within ranges such as 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more.

[0084] Examples of the isotonicity agent include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonicity agents can be used alone or in combination of two or more.

[0085] Examples of the chelating agent include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid, etc. These chelating agents may be used alone or in combination of two or more.

[0086] The stabilizer may, for example, be sodium hydrogen sulfite.

[0087] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusters can be used alone or in combination of two or more.

[0088] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0089] Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants may be used alone or in combination of two or more.

[0090] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0091] Examples of thickeners include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickeners may be used alone or in combination of two or more.

[0092] Examples of excipients include lactose, corn starch, L-cysteine, trehalose, maltitol, sorbitol, etc. These excipients may be used alone or in combination of two or more.

[0093] Examples of binders include crystalline cellulose, starch, sucrose, hydroxypropyl cellulose, gelatin, powdered gum arabic, polyvinylpyrrolidone, pullulan, dextrin, cyclodextrin, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, polyethylene glycol, etc. These binders can be used alone or in combination of two or more.

[0094] Ingestion of the composition of the present invention by a subject (preferably a mammal such as a human) can improve vascular endothelial function. In Example 2 of Patent Document 1, a powder of live bacteria of Bifidobacterium animalis subsp. lactis LKM512 strain (approximately 6 × 10 ９ It is reported that vascular endothelial function (EndoPAT measurement value) was improved when LKM512 strain (100 cfu / packet x 1 pack) was combined with arginine tablets (100 mg / tablet x 3 tablets) to form a set, and the combination was taken twice a day after breakfast and dinner for 8 weeks. However, it is also reported that vascular endothelial function was not improved in a placebo group in which starch was combined with the LKM512 strain instead of the arginine tablets. Therefore, Patent Document 1 states that vascular endothelial function was not improved by using bifidobacteria alone. Furthermore, there have been no reports, including Patent Document 1, that vascular endothelial function can be improved by combining bifidobacteria with water-soluble dietary fiber. Therefore, the effect of the present invention could not be predicted from the prior art.

[0095] Furthermore, Patent Document 2 describes that arginine intake increases the concentration of polyamine (putrescine) in the metabolic products of intestinal bacteria, and Non-Patent Document 4 describes that ingestion of the LKM512 strain in combination with arginine results in upregulation of polyamine (putrescine). Therefore, it is suggested that the increase in EndoPAT measurement value due to inoculation of the LKM512 strain in combination with arginine is due to the production of polyamine (putrescine) through arginine uptake. The effect of combining water-soluble dietary fiber with bifidobacteria of the present invention is not due to the production of polyamine (putrescine), and is therefore useful because it can improve vascular endothelial function through a mechanism completely different from that of arginine administration.

[0096] The intake amount of the composition of the present invention is not limited, but the daily intake of bifidobacteria as an active ingredient can be appropriately set within a range of, for example, 10 million to 10 trillion, preferably 100 million to 1 trillion, more preferably 1 billion to 100 billion, even more preferably 5 billion to 30 billion, and particularly preferably 10 billion to 20 billion. The intake amount of the composition of the present invention based on the weight of bifidobacteria is also not limited, but the daily intake of bifidobacteria as an active ingredient can be appropriately set within a range of, for example, 1 mg to 10 g, preferably 10 mg to 1000 mg, more preferably 40 mg to 300 mg, and even more preferably 200 mg or more.

[0097] In addition to the bifidobacteria, the composition of the present invention may further contain a substance known to have vascular endothelial function. Examples of substances known to have vascular endothelial function include arginine, pine bark-derived procyanidins, black soybean polyphenols, and bonito-derived elastin peptides. These substances may be used alone or in combination of two or more.

[0098] The composition of the present invention is preferably an oral composition. Oral compositions include food and drink compositions, pharmaceutical compositions, etc. In the present invention, the food and drink compositions also include health functional foods (nutrient functional foods, foods for specified health uses, foods with functional claims), etc.

[0099] Examples of food and beverage compositions include beverages such as vegetable juice drinks, fruit juice drinks, mixed vegetable and fruit juice drinks, fermented milk drinks, and almond-containing drinks; and foods such as ice creams, frozen desserts, candies, gummies, almond-containing foods, biscuits (cream sandwich biscuits, etc.), chocolates (including semi-chocolate), and fermented milk foods (yogurt, cheese). Preferred ice creams include lacto ice cream (lacto ice cream containing fermented milk, etc.). Examples of semi-chocolate include corn-containing semi-chocolate. Examples of dairy products include fermented milk, ice creams, and milk drinks. The food and beverage compositions of the present invention also include supplements, etc.

[0100] Other Compositions, etc. Although the present invention has been described above using some embodiments, the present invention is not limited to these embodiments. For example, as shown in the examples described below, according to the present invention, vascular flexibility (typically, the degree of vascular dilation after vascular constriction) can be improved by combining bifidobacteria with water-soluble dietary fiber. In the present invention, the "blood vessel" in terms such as "vascular endothelial function" and "vascular flexibility" is not particularly limited, but includes, for example, blood vessels in the brain and heart. In the present invention, "blood vessel" also includes, but is not limited to, arteries. Furthermore, combining bifidobacteria with water-soluble dietary fiber can improve vascular flexibility. For these reasons, it is expected that the composition will also have a preventative effect against these vascular conditions caused by aging, etc. Therefore, in one embodiment, the present invention provides a composition for maintaining or inhibiting the decline of vascular flexibility or flexibility, comprising bifidobacteria and water-soluble dietary fiber. Vascular flexibility (or flexibility) can be measured, for example, by FMD (Flow-Mediated Dilation) testing. "Maintaining or inhibiting the decline of vascular flexibility (or suppleness)" means, for example, maintaining the FMD value at 4% or higher (preferably 6% or higher, more preferably 7% or higher) or increasing the FMD value from 4% or higher (preferably 6% or higher, more preferably 7% or higher) (Non-Patent Document 5). Furthermore, as shown in the Examples below, the combination of bifidobacteria and water-soluble dietary fiber has been shown to improve vascular endothelial function and reduce blood LDL-c levels. Based on these findings, the present invention can reduce the risk of arteriosclerosis. Therefore, in one embodiment, the present invention provides a composition for preventing arteriosclerosis, comprising bifidobacteria and water-soluble dietary fiber. Furthermore, as shown in the Examples below, the combination of bifidobacteria and water-soluble dietary fiber improves vascular flexibility as measured by FMD testing. Since FMD measurements have also been reported to correlate with renal function, improving vascular endothelial dysfunction may reduce the risk of developing kidney disease. Therefore, the present invention provides a composition for improving renal function, comprising bifidobacteria and water-soluble dietary fiber.In these embodiments, the conditions such as the type of bifidobacteria and water-soluble dietary fiber, the amount used, and other ingredients may be the same as those described in the section "Composition for improving vascular endothelial function."

[0101] In another embodiment, the present invention relates to the use of bifidobacteria and water-soluble dietary fiber for producing a composition for improving vascular endothelial function; the use of bifidobacteria and water-soluble dietary fiber for producing a composition for maintaining or inhibiting the decline of vascular flexibility or resilience; the use of bifidobacteria and water-soluble dietary fiber for producing a composition for preventing arteriosclerosis; the use of bifidobacteria and water-soluble dietary fiber for producing a composition for improving renal function; the use according to Item 21, wherein the improvement of vascular endothelial function includes one or both of a decrease in blood LDL-c concentration and a decrease in PAI-1; the use of bifidobacteria for producing a composition for maintaining or inhibiting the decline of vascular flexibility or resilience, comprising water-soluble dietary fiber; the use of bifidobacteria for producing a composition for preventing arteriosclerosis, comprising water-soluble dietary fiber; the use of bifidobacteria for producing a composition for improving renal function, comprising water-soluble dietary fiber; a method for improving vascular endothelial function, comprising administering an effective amount of bifidobacteria and water-soluble dietary fiber to a subject in need thereof; a method for maintaining or inhibiting the decline of vascular flexibility or resilience, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof; a method for preventing arteriosclerosis, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof; a method for improving renal function, comprising administering effective amounts of bifidobacteria and water-soluble dietary fiber to a subject in need thereof; a combination of bifidobacteria and water-soluble dietary fiber for use in improving vascular endothelial function; a combination of bifidobacteria and water-soluble dietary fiber for use in maintaining or inhibiting the decline of vascular flexibility or resilience; a combination of bifidobacteria and water-soluble dietary fiber for use in the prevention of arteriosclerosis; a combination of bifidobacteria and water-soluble dietary fiber for use in improving renal function; water-soluble dietary fiber for use in combination with bifidobacteria in improving vascular endothelial function; water-soluble dietary fiber for use in combination with bifidobacteria for maintaining or inhibiting the decline of vascular flexibility or resilience; water-soluble dietary fiber for use in combination with bifidobacteria in the prevention of arteriosclerosis;The present invention provides water-soluble dietary fiber for use in combination with bifidobacteria to improve renal function; bifidobacteria for use in combination with water-soluble dietary fiber to improve vascular endothelial function; bifidobacteria for use in combination with water-soluble dietary fiber to maintain or inhibit the decline of vascular flexibility or resilience; bifidobacteria for use in combination with water-soluble dietary fiber to prevent arteriosclerosis; and bifidobacteria for use in combination with water-soluble dietary fiber to improve renal function. In these embodiments, details (type, amount, etc.) of the bifidobacteria, water-soluble dietary fiber, and optional other components, as well as methods of use (medicines, foods and beverages, etc.), and applications (improvement of vascular endothelial function, prevention of arteriosclerosis, improvement of renal function, etc.) are the same as those described above. In the present invention, subjects to which the bifidobacteria and water-soluble dietary fiber are administered (ingested) include, for example, mammals such as humans, mice, rats, and guinea pigs (preferably humans). When the subject is a human, the subject may be, but is not limited to, a human who does not suffer from arteriosclerosis, kidney disease, high blood pressure, etc. The age of the subject is not particularly limited, but examples include age groups in which vascular endothelial function, renal function, etc. generally decline with age. In a non-limiting preferred embodiment, the subject of the present invention includes humans (particularly middle-aged and elderly humans) who have not yet developed arteriosclerosis, kidney disease, hypertension, etc.;

[0102] Specific embodiments of the present invention will be described in more detail below using examples, but the present invention is not limited to such examples.

[0103] To evaluate the potential for the reduction of arteriosclerosis risk by GCL2505 and inulin, which have anti-metabolic syndrome effects, we investigated their effects on vascular endothelial function in a randomized, double-blind, placebo-controlled, parallel-group comparative study.

[0104] Subjects (Analysis Population): In accordance with the Declaration of Helsinki, written informed consent was obtained from all participants in this study. Participants were Japanese men and women aged 40 to 65 years who were prone to impaired vascular endothelial function, met the inclusion criteria, did not fall under the exclusion criteria, and were deemed eligible by the principal investigator. The inclusion criteria were as follows: (1) those who fully understood the significance, content, and purpose of this study and provided written consent to participate in the study; (2) Japanese men and women aged 40 to 65 years at the time of screening; (3) those with high pentraxan 3 (PTX3) levels at the time of screening; and (4) those with a high LDL-cholesterol / HDL-cholesterol ratio (L / H ratio) at the time of screening.The exclusion criteria were as follows: (1) those for whom it was difficult to measure FMD on the right arm; (2) those receiving medical treatment, medication, or lifestyle advice from a doctor due to hypertension, dyslipidemia, or diabetes; (3) those with a history of respiratory problems such as asthma, tuberculosis, or pleurisy; (4) those using a pacemaker or defibrillator; (5) those with serious cerebrovascular disease, heart disease, liver disease, kidney disease, digestive system disease, or notifiable infections; (6) those with a history of major surgery on the digestive system such as gastrectomy, gastrointestinal suture, or intestinal resection; (7) those with significant abnormalities in blood pressure measurement, physical measurements, or blood tests; (8) those with severe anemia; and (9) pre- and post-menopausal women with significant physical conditions. (10) Those who may have allergic symptoms to drugs or foods (especially dairy ingredients); (11) Those who have taken or plan to take antibiotics between 12 weeks before the start of intake and the end of the study; (12) Those who regularly take medicines that affect bowel movements (intestinal regulators, laxatives, anti-diarrheals, etc.); (13) Those who cannot stop taking yogurt, lactic acid bacteria drinks, or health foods with intestinal regulating effects (those containing ingredients such as lactic acid bacteria, bifidobacteria, oligosaccharides, and dietary fiber-enriched foods) during the study period; (14) Those who are smokers, regular alcohol users, or have extremely irregular lifestyles; (15) Those who have taken more than 400mg of lactic acid bacteria within 16 weeks before the start of intake. The study participants included: (1) women who donated 100 mL of blood, men who donated 400 mL of blood within 12 weeks prior to the start of the study, and those who donated 200 mL of blood within 4 weeks or blood components within 2 weeks prior to the start of the study. (2) Pregnant or possibly pregnant women, breastfeeding women. (3) Individuals participating in other clinical trials, observational studies, clinical trials, or home use tests between 4 weeks prior to the start of the study and the end of the study. (4) Individuals deemed ineligible by the investigator. Figure 1 shows a flowchart of the study period. A total of 149 participants were screened for this study. Sixty participants were selected as eligible: 30 were assigned to the test food group and 30 to the placebo group. Three participants withdrew for personal reasons before the end of the study (n = 1 in the test food group and n = 2 in the placebo group). One participant was discontinued due to compliance issues. After the completion of the study, one participant was excluded due to a confirmed illness unrelated to the study, which could have affected the results.Therefore, a total of 55 people, consisting of 27 people in the test food group and 28 people in the placebo group, were selected as the analysis population.

[0105] Participants in the test food group (Active) and placebo group (Placebo) each consumed a beverage with the composition shown in Table 1 below once a day. There were no restrictions on the food menu other than the beverage, but there were some restrictions, such as prohibiting the intake of foods containing lactic acid bacteria. The intake of the above beverage was continued for 84 days.

[0106]

[0107] Table 2 below shows an overview of the subject background and safety assessment for the entire analysis population. Table 3 shows the measurement results of FMD and blood components for the test food group (Active) and placebo group (Placebo) for the entire analysis population.

[0108]

[0109]

[0110] Table 4 below shows the measurement results of FMD and blood components in the test food group (Active) and placebo group (Placebo) in the subject population with an FMD (%) of 4% or more at Week 0.

[0111]

[0112] Safety evaluation: Safety was confirmed in the analysis population (Table 2), and no clinically significant findings were noted. Furthermore, no side effects or medically significant adverse events were observed. Therefore, the milk beverages used in the study were considered safe for consumption.

[0113] FMD FMD was measured on days 0, 56, and 84 of the study using the following method: a UNEX-EF18VG (UNEX Co., Ltd.) was used for the measurements. The right arm was used for the measurements. First, the vascular diameter of the right brachial artery was measured at rest, and then the right forearm was avascularized for 5 minutes. After avascularization was released, the vascular diameter at maximum diastole was measured again. The rate of vascular diameter expansion from rest was expressed as FMD (%). In the group of subjects whose FMD (%) at Week 0 was 4% or higher, an increase in FMD (%) was confirmed in the test food group at Week 12 (0.88% ± 2.23%). Furthermore, there was a statistically significant difference between the change in FMD (%) from week 0 to week 12 in the test food group and that in the placebo group (-0.68% ± 2.81%) (p = 0.046 by Student's t-test) (Table 4). In the analysis population, an increase in FMD (%) was confirmed in the test food group at week 12 (1.05 ± 2.21%). The change in FMD (%) from week 0 to week 12 in the test food group was greater than that in the placebo group (-0.26% ± 2.87%) (p = 0.065 by Student's t-test) (Table 3).

[0114] Fecal Microbiota: Fecal microbiota was examined in subgroup analysis populations at Week 0 and Week 12 using shotgun metagenomics. α-diversity (Chao1) and β-diversity analyses were performed to compare the fecal microbiota of both groups. Interestingly, no differences were observed between or within the two groups at Weeks 0 and 12 (Figures 2A and 2B). However, the relative abundance of B. animalis in the intestines of the test food group increased from 0.061% ± 0.002% at Week 0 to 1.503% ± 0.018% at Week 12. In the placebo group, the abundance was 0.085% ± 0.004% at Week 0 and 0.014% ± 0.001% at Week 12. The difference in abundance between the two groups was assessed using the Linear Model for Differential Abundance (LinDA) method for compositional data, and p-values ​​were corrected using the Benjamini-Hochberg procedure to minimize the false discovery rate (Non-Patent Document 7). At week 12, a statistically significant difference was observed in the relative abundance of B. animalis in the test food group compared to the placebo group (p = 5.62 × 10－９ ) (Figure 2C).

[0115] <Test Method> (Feces Collection) Feces samples at weeks 0, 8, and 12 were collected by participants at home using a feces container containing RNAlater Stabilization Solution (Invitrogen, Carlsbad, CA) from 3 days before the designated examination date until the morning of the day. The feces samples were stored in the refrigerator at home and brought to the examination room on the day of the examination while cooled with ice packs.

[0116] (Fecal DNA Extraction) Bacterial DNA was extracted from a 10-fold diluted fecal sample using the ISOSPIN Fecal DNA Kit (Nippon Gene Co., Ltd., Tokyo, Japan) as previously described (Non-Patent Document 8). Specifically, the sample (here, 200 μL of diluted fecal solution), 700 μL of FE1 buffer, and 10 μL of RNase were added to the attached tube with beads. Cells were disrupted by bead-beating using a FastPrep-24 (MP Biomedicals, Irvine, CA) at a speed of 6 m / s for 1 minute. Bead-beating was repeated three times, with the sample being kept at room temperature for 5 minutes between bead-beatings. Then, 90 μL of FE2 buffer was added, and the sample was centrifuged at 12,000 × g for 15 minutes. The supernatant (up to 500 μL) was collected and mixed with 0.4 volumes of FB buffer and isopropanol. Finally, the sample was loaded onto a spin column and washed according to the manufacturer's instructions. The purified DNA was eluted with 50 μL of Tris-EDTA (pH 8.0) buffer.

[0117] Shotgun Library Construction and Sequencing: Unless otherwise noted, metagenomic sequencing library construction was performed using the QIAseq FX DNA Library Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly, the enzymatic fragmentation reaction (50 μL) contained 10x FX buffer, 10 μL FX enzyme mix, and 500 ng DNA template, and the reaction was incubated at 32°C for 9 minutes. For adapter ligation, 5 μL of adapter, 20 μL of DNA ligase buffer, 10 μL of DNA ligase, and 15 μL of RNase-free HO were added, and the reaction was incubated at 20°C for 15 minutes. Using an Agencourt AMPure XP PCR purification system (Beckman Coulter, Brea, Calif.), the adapter-ligated fragments were purified and size-selected using 1 volume and 0.8 volumes of bead solution sequentially and eluted with 10 mM Tris-HCl buffer.

[0118] (Quality control of metagenomic reads) Quality control of metagenomic reads and trimming of adapter sequences were performed using fastp (version 0.20.0). Reads shorter than 50 bp were excluded from further analysis. The remaining reads were mapped to the human (hg38) and phiX bacteriophage genomes using minimap2 (version 2.17), and mapped reads were excluded. Paired reads excluded in the filter step were also removed.

[0119] (Construction of non-redundant gene set and functional annotation) A non-redundant gene set was constructed based on HMP and population-level metagenomics data. All genes were clustered using cd-hit (version 4.8.1) at a 95% identity threshold. Functional annotation of non-redundant genes was performed using eggNOG-mapper (version 2.1.9) based on the eggNOG orthology database (version 5.0.2). Sequence searches were performed using DIAMOND (version 2.0.15).

[0120] Taxonomic and Functional Profiles of Samples: Taxonomic profiles at the species and genus levels were obtained using a marker gene-based approach using mOTUs2 (version 3.0.3). To quantify gene function, metagenomic reads were mapped to non-redundant genes and the number of mapped reads for each gene was counted using minimap2 with an identity threshold of 95% or higher. The number of multiply mapped reads was distributed to mapped genes based on the ratio of the number of uniquely mapped reads to the gene. Counts were normalized to transcripts per million to form a transcripts per million matrix.

[0121] Physical Parameters: In subjects with an FMD (%) of 4% or higher at Week 0, the test food group experienced a decrease in blood total cholesterol levels (Week 8: -2.75±11.51 mg / dL, Week 12: -3.75±21.09 mg / dL), and the change in blood cholesterol levels from Week 0 to Week 8 in the test food group was greater than that in the placebo group (3.87±13.74 mg / dL) (p=0.081 by Student's t-test). The test food group also experienced a decrease in blood LDL-c levels (Week 8: -4.17±9.18 mg / dL, Week 12: -4.29±15.14 mg / dL). There was a statistically significant difference between the change in blood LDL-c levels in the test food group and the placebo group (3.57±10.93 mg / dL) from weeks 0 to 8 (p=0.012 by Student's t-test). The change in blood LDL-c levels in the test food group from weeks 0 to 12 was greater than that in the placebo group (2.14±9.73 mg / dL) (p=0.092 by Student's t-test). The test food group also had a decrease in blood PAI-1 levels (week 8: -1.83±5.25 ng / mL, week 12: -1.25±4.66 ng / mL). The change in blood PAI-1 concentration in the test food group from week 0 to week 8 was greater than that in the placebo group (0.52±2.61 ng / mL) (p=0.058 by Student's t-test), and the change in blood PAI-1 concentration in the test food group from week 0 to week 12 was greater than that in the placebo group (0.86±3.12 ng / mL) (p=0.076 by Student's t-test) (Table 4). Through the above study, the effect of intake of yogurt containing Bifidobacterium animalis subsp. lactis GCL2505 and inulin on vascular endothelial function in healthy adults was investigated.

[0122] Flow-mediated dilation (FMD) was used in this study. FMD is a vascular endothelial function test that utilizes the flow-dependent vasodilation response that occurs upon release of brachial avascularization. Increased blood flow after release of avascularization places shear stress on the vascular endothelium, resulting in the production of vasodilators, including nitric oxide. These substances act on the vascular smooth muscle adjacent to the vascular endothelium, causing relaxation and eliciting a vasodilation response. The ratio of the maximum dilated vessel diameter to the resting vessel diameter is expressed as FMD (%). Vascular endothelial dysfunction reduces the bioavailability of nitric oxide produced and released from the vascular endothelium, weakening the vasodilation response and resulting in a low FMD (%). FMD is noninvasive, minimally burdensome to patients, and is recognized as a useful test for assessing CVD risk (Non-Patent Document 9).

[0123] In this study, participants took yogurt containing GCL2505 and inulin for 12 weeks, and FMD (%) increased by 0.85% in the test food group. Furthermore, the change in FMD (%) from week 0 to week 12 in the test food group was significantly greater than that in the placebo group.

[0124] Analysis of the subjects' fecal microbiota revealed no effect of GCL2505 or inulin on α-diversity and β-diversity analysis. It has been reported that dietary fiber intervention in healthy humans increases the abundance of certain bacteria but does not affect the diversity of the intestinal microbiota (Non-Patent Document 10). The lack of change in fecal microbiota may be due to the fact that the subjects in this study were healthy and did not have intestinal dysbiosis. The relative abundance of B. animalis increased in the intestine only in the active group subjects, but not in the placebo group. In other words, the ingested GCL2505 reached the intestine alive. On the other hand, the increase in the relative abundance of B. animalis in the intestine may have increased the production of acetic acid, a major SCFA.

[0125] This study confirmed an effect on blood LDL cholesterol (LDL-c) levels in the test food group. LDL-c deposited in the vascular wall becomes oxidized LDL, which stimulates endothelial cell dysfunction and increases the expression of adhesion factors. Oxidized LDL is taken up by macrophages and is involved in foam cell formation, contributing to local and systemic plaque inflammation and sustaining the growth of atherosclerotic lesions. Thus, the improvement in vascular endothelial function observed in this study may have been caused by a decrease in blood LDL-c levels. Furthermore, this study also showed a decrease in blood plasminogen activator inhibitor-1 (PAI-1) levels in the test food group compared to the placebo group (week 8, p = 0.058; week 12, p = 0.076). PAI-1 is a cytokine responsible for regulating fibrinolysis and strongly inhibits clot dissolution by inhibiting tissue plasminogen activator (t-PA), a known thrombolytic agent, on fibrin. Elevated PAI-1 levels are correlated with atherothrombosis, and PAI-1 levels have been reported to be elevated in obese patients with metabolic syndrome and type II diabetes. Therefore, it is possible that the reason that intake of GCL2505 and inulin improved vascular endothelial function is due to a decrease in blood PAI-1 levels via a reduction in visceral fat, thereby activating fibrinolysis. Furthermore, because a positive correlation has been reported between visceral fat and blood LDL-c levels, the test food group in this study, which confirmed a decrease in blood LDL-c levels, may have had an effect on visceral fat mass.

[0126] It is hypothesized that the improvement of vascular endothelial function by GCL2505 and inulin is achieved through a two-step mechanism of action. First, intake of GCL2505 and inulin reduces visceral fat through changes in the intestinal microbiota. Clinical studies have shown that daily consumption of yogurt containing GCL2505 reduces abdominal visceral fat mass. Furthermore, Horiuchi et al. demonstrated using GPR43 knockout mice that GCL2505 administration increases the number of B. lactis in the intestine and plasma acetate levels, which subsequently modulates host energy metabolism in a GPR43-dependent manner (e.g., suppression of body fat accumulation, improvement of insulin sensitivity, and promotion of whole-body fatty acid metabolism). GPR43 is a G protein-coupled receptor known as a host short-chain fatty acid receptor. It has been reported that acetate activates GPR43 in adipocytes, modulating insulin signaling in adipocytes and suppressing fat accumulation. Step 2: The reduction of visceral fat triggers the suppression of vascular endothelial dysfunction through the following steps: 1) Reduction of blood lipid levels through the reduction of visceral fat, resulting in a decrease in blood LDL-c levels: LDL-c is a substance that causes plaque formation, and its synthesis is related to the amount of free fatty acids excreted from visceral adipose tissue. 2) Suppression of plasminogen activator inhibitor-1 (PAI-1) expression through the reduction of visceral fat: PAI-1 is an adipocytokine produced in adipocytes and controls fibrinolysis, which is closely related to arteriosclerosis. This study demonstrated that GCL2505 and inulin contribute to vascular endothelial function through their inhibitory effect on fat accumulation.

[0127] These findings suggest that consuming a synbiotic drink containing GCL2505 and inulin reduces visceral fat and improves vascular endothelial function. Meta-analysis results suggest that a 1% increase in FMD (%) reduces the risk of CVD by approximately 13%, making these findings clinically significant. Furthermore, a correlation between FMD (%) and renal function has been reported, suggesting that improving vascular endothelial dysfunction may also reduce the risk of kidney disease. Therefore, these combinations may reduce the risk of multiple diseases beyond CVD and significantly contribute to health promotion, making these findings clinically significant.

Claims

1. A composition for improving vascular endothelial function comprising bifidobacteria and water-soluble dietary fiber.

2. The composition according to claim 1, wherein the improvement of vascular endothelial function includes one or both of a reduction in blood LDL-c concentration and a reduction in PAI-1.

3. A composition for maintaining or inhibiting the decline of vascular flexibility or resilience, comprising bifidobacteria and water-soluble dietary fiber.

4. A composition for preventing arteriosclerosis comprising bifidobacteria and water-soluble dietary fiber.

5. A composition for improving renal function comprising bifidobacteria and water-soluble dietary fiber.

6. The composition according to any one of claims 1 to 5, wherein the water-soluble dietary fiber comprises inulin.

7. The composition according to any one of claims 1 to 5, wherein the bifidobacteria have the ability to proliferate in the intestine.

8. The composition according to any one of claims 1 to 5, wherein the bifidobacterium has an anti-metabolic syndrome effect.

9. The composition according to any one of claims 1 to 5, which is a food product.

10. The composition according to any one of claims 1 to 5, further comprising arginine.

11. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for improving vascular endothelial function.

12. The use according to claim 11, wherein the improvement of vascular endothelial function includes one or both of a reduction in blood LDL-c concentration and a reduction in PAI-1.

13. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for maintaining or inhibiting the decline of vascular flexibility or resilience.

14. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for preventing arteriosclerosis.

15. Use of bifidobacteria and water-soluble dietary fiber for producing a composition for improving renal function.

16. Use of water-soluble dietary fiber for producing a composition for improving vascular endothelial function, comprising bifidobacteria.

17. The use according to claim 16, wherein the improvement of vascular endothelial function includes one or both of a reduction in blood LDL-c concentration and a reduction in PAI-1.

18. Use of water-soluble dietary fiber for producing a composition containing bifidobacteria for maintaining or inhibiting the decline of vascular flexibility or resilience.

19. Use of water-soluble dietary fiber for producing a composition for preventing arteriosclerosis containing bifidobacteria.

20. Use of water-soluble dietary fiber for producing a composition for improving renal function, comprising bifidobacteria.

21. The use according to any one of claims 11 to 20, wherein the water-soluble dietary fibre comprises inulin.

22. The use according to any one of claims 11 to 20, wherein the bifidobacteria have the ability to proliferate in the intestine.

23. The use according to any one of claims 11 to 20, wherein the bifidobacterium has an anti-metabolic syndrome effect.

24. The use according to any one of claims 11 to 20, wherein the composition is a food product.

25. The use according to any one of claims 11 to 20, wherein the composition further comprises arginine.